Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 716545 12 pageshttpdxdoiorg1011552013716545
Research ArticleCharacterization of Thermo- and Detergent Stable AntigenicGlycosylated Cysteine Protease of Euphorbia nivuliaBuch-Ham and Evaluation of Its Ecofriendly Applications
Shamkant B Badgujar12 and Raghunath T Mahajan2
1 Department of Biochemistry National Institute for Research in Reproductive Health (ICMR) Jehangir Merwanji Street ParelMumbai Maharashtra 400 012 India
2 Faculty of Science Department of Biotechnology Moolji Jaitha College North Maharashtra University JalgaonMaharashtra 425002 India
Correspondence should be addressed to Shamkant B Badgujar sham83badgujargmailcom
Received 15 August 2013 Accepted 9 September 2013
Academic Editors A A Guevara-Garcia and C R C Calado
Copyright copy 2013 S B Badgujar and R T Mahajan This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
An antigenic glycosylated cysteine protease has been purified from the latex of Euphorbia nivulia Buch-Ham It exhibits remarkableprotease activity in the presence of metal ions oxidizing agents organic solvents and detergents This enzyme showed potentialrole in leather processing industry due to its dehairing activity for animal hide without hydrolyzing fibrous proteins producingby this way a better quality product The enzyme can also be used for silver recovering from X-ray plates In addition the stability(temperature and surfactants) and hydrolysis of blood stain data also revealed its application in detergent industries Agriculturallythis protease finds application in biocontrol process against the infectious management of root knot nematode Meloidogyneincognita Biologically it shows noticeable wound healing haemostatic and antibacterial activity
1 Introduction
Latex is a milky fluid composed of a liquid serum holdingin suspension or in solution a complex mixture of con-stituents It may contain a variety of cellular componentslike nuclei mitochondria ribosome-like particles and lyso-some analogues Agglomerative low density materials suchas various enzymes terpenes alkaloids vitamins carbohy-drates lipids and free amino acids have been identifiedamong the components [1]The characteristic feature of plantfrom EuphorbiaceaeApocynaceaeMoraceaeAsclepiadaceaeSapotaceaeCaricaceae andConvolvulaceae families has latexsecreting properties Latex has been reported to occur in12000 plant species belonging to 900 genera A numberof proteases from latex bearing species have been isolatedand characterized [2] Additionally in our laboratory wehave studied proteolytic activities of 21 latex bearing plantsbelonging to seven different laticiferous families A commonfeature that can be found in the latex of the Euphorbiaceae is
the presence of noticeable proteolytic activity [3] Euphorbiais a large genus consisting of about 2000 species About 52species have been recorded from India The genus includesherbs shrubs and trees of widely diverse habitats [4 5]The present study was carried out on the proteolytic activityof latex of Euphorbia nivulia Buch-Ham This is a wildthorny xerophytic succulent plant found in boundariesof the agricultural field and also in dry barren areasThe secretion of milky juice is a characteristic propertyof this plant Phytochemical studies have led to the iso-lation of ingol diterpenes (3-acetyl-8-methoxyl-7-angolyl-12-hydroxylingol 312-diacetyl-7-hydroxy-8-methoxylingol312-diacetyl-7-angolyl-8-hydroxylingol 312-diacetyl-8-benzoylingol and 312-diacetyl-7-benzoyl-8-nicotinylingol) alongwith three macrocyclic ingol diterpenes derivatives (3712-triacetyl-8-benzoylingol 312-diacetyl-7-angeloyl-8-methoxyingol and 7-angeloyl-12-acetyl-8-methoxyingol) [6] Thelatex of E nivulia has been cited for its antioxidant immunomodulator cytotoxic anti-inflammatory wound healing
2 The Scientific World Journal
haemostatic and antiproliferative activity [5] During thecourse of screening for biochemical constituents a substan-tial amount of proteolytic and milk clotting activity wasfound in the latex of this plant [7] Recently an attempthas been made on peptide sequencing of 31 kDa Tubulinalpha-1 chain-like protein called Nivulian-I present in thelatex of E nivulia [6] Very recently a comparative accounton proteolytic activity of E nivulia and other three plantsnamely Calotropis procera Carica papaya and Ficus caricawas reported by us Additionally we report the glycosylatedcysteine protease called Nivulian-II of the latex of E nivulia[6] This paper describes the further biochemical character-ization of this cysteine-like protease with some ecofriendlyapplications
2 Materials and Methods
21 Chemicals All chemicals with the highest purity analyti-calHPLCgradewere purchased fromSigmaChemicalsUSAHimedia Laboratories Mumbai SRL Chemicals MumbaiQualigen Fine Chemicals Mumbai Merck Chemicals Indiaand Bangalore Genie India
22 Animals Used Four- to five-week-old albinoWistermalerats (120 to 180 g body weight) were used for antigenicitySwiss albino mice of either sex (50ndash100 g) were used forthe study of wound healing activity The animals weremaintained under standard laboratory conditions in animalhouse approved by Committee for the Purpose of Controland Supervision on Experiments on Animals (CPCSEAregistration number 1062C07CPCSEA24 May 2007)Theexperimental protocol was approved by Institutional AnimalEthics Committee constituted under CPCSEA rules India
23 Animal Hides Freshly flayed wet rat hide was obtainedfrom animal house ofMoolji JaithaCollege NorthMaharash-tra University Jalgaon India Cow hide of healthy animal wascollected from local meat shops of Jalgaon city
24 Blood Sample Fresh blood samples of domestic animalsnamely goat (Capra hircus) buffalo (Bubalus bubalis) andox (Ovibos moschatus) of either sexes were collected underthe supervision of Dr N M Pawar veterinary practitionerof Paldhi unit Jalgaon District Maharashtra India Bloodsample of healthy hen was collected from local chicken shopsof Jalgaon city (MS)
25 Nematode Sample Root-knot nematodes (Meloidogyneincognita) were collected along with soil sample by zig-zag manner from vicinity of Department of AgriculturalEntomology Mahatma Phule Krishi Vidyapeeth RahuriAhmednagar Maharashtra India
26 Microbial Culture The microbial cultures like Staphylo-coccus aureus (ATCC 25923) Escherichia coli (ATCC 25992)Klebsiella pneumoniae (ATCC 23357) Pseudomonas aerug-inosa (ATCC 27853) Proteus vulgaris (NCIM 2027) andBacillus subtilis (NCIM 2063) were procured from National
Collection of Industrial Microorganisms (NCIM) PuneMaharashtra India
27 Plant Material Collection of Latex Preparation of CrudeEnzyme and Purification of Cysteine Protease The detailedinformation about identification collection preservationand preparation of crude enzyme and its proteolytic activityof Euphorbia nivulia latex and its quantification is describedin our previous communication [6] Method of purificationof protease was done using acetone precipitation DEAEcellulose chromatography and dialysis and followed byrechromatography on DEAE cellulose column as describedin our earlier communication [8]
28 Characterization of Cysteine Protease
281 Thermal Stability The thermal behavior of the purifiedenzyme fraction was evaluated by incubating the enzymeat the desired temperatures in the range of 20ndash800∘C for15min in 001M phosphate buffer (pH 66) and an aliquotwas used for the enzyme activity measurement at the sametemperature At each temperature a control assay was carriedout without enzyme
282 Effect of Metal Ions on Proteolytic Activity Impact ofvarious metal ions namely K+ Na+ Zn++ Ag++ Cd++Fe++ Hg++ Mg++ and Mn++ at 5mM concentration onthe enzyme catalytic behaviour was studied The enzymefraction alongwith 001M phosphate buffer (at optimumpH)was preincubated at room temperature for 30 minutes withrespective metal ions separately before enzyme assay Thenthe residual proteolytic activity wasmeasured by using caseinaccording to standard assay procedure relative to control(without metal ion)
283 Effect of Surfactant and Oxidizing Agent on ProteolyticActivity The effect of different surfactants namely sodiumdodecyl sulphate Triton X-100 and Tween 80 and oxidizingagent (H
2O2) at 1 (vv) final concentration on enzyme
activity was studied by preincubating enzyme preparationfor 60min at 37∘C in the above surfactants and oxidizingagent before analysis Then the residual proteolytic activitywas measured by using casein according to standard assayprocedure relative to control (without chemical surfactantsand oxidizing agent) and the resulting activity was taken as100 percent
284 Effect of Organic Solvents on Proteolytic Activity Thestability of enzyme activity in presence of different watermiscible or immiscible organic solvents was investigatedby incubating the enzyme preparation with various organicsolvents (30 50 and 70 vv) at 37∘C for 1 h After incu-bation the residual enzyme activity was determined as perthe previously discussed method with casein The enzymeactivity of a control sample (without solvent) incubatedunder the same conditions was taken as 100 percent [9]For this assay the solvents used were acetone acetophenonebenzyl alcohol benzene butanol chloroform chloroben-zene dichloromethane dimethylformamide diethyl ether
285 Compatibility of Protease with Laundry DetergentsThe compatibility of enzyme preparation in presence ofcommercial solid laundry detergents was examined by incu-bating enzyme preparation for 60min at 37∘C with variousdetergent preparations and the residual enzyme activity wasdetermined as per the method using casein The enzymeactivity of a control sample (without detergent) incubatedunder the same conditions was taken as 100 percent Thesolid detergents used were Ariel Oxy Blue Tide (Procter andGamble Company USA) Fena Ultra New Impact (Fena (P)Ltd New Delhi India) Nirma (Nirma Ltd AhmedabadIndia) Wheel Active Rin Surf Excel (Hindustan LiverLtd India) and Ujala (Jyoti Laboratories Ltd MumbaiIndia) The detergents were diluted in tap water to give afinal concentration of 7mgmL and 10mgmL to simulatewashing conditions The endogenous proteases contained inthese detergents were inactivated by incubating the diluteddetergents at 65∘C for 60min prior to the addition of enzyme[10]
29 Antigenic Property
291 Immunization of Rat and Production of PolyclonalAntisera Theprotein sample (100 120583g in 005mLof phosphatebuffer saline 001M pH 60) thoroughly mixed with equalvolume of Freundrsquos complete adjuvant and injected into malealbino Wister rat (180 to 200 g body weight) subcutaneouslyat multiple sites Two booster doses were administered atweekly intervals with the same concentration but with equalvolume of Freundrsquos incomplete adjuvant After nine daysfrom last booster dose blood was drawn through retroorbitalplexussinus with a glass capillary tube and antisera wasseparated after allowing the blood to coagulate at 8∘C for 24 h
292 Detection of Antibodies and Immunological CrossReactivity The presence of antibodies was confirmed byOuchterlonyrsquos double immunodiffusion assay [11] 1 agarosein phosphate buffer saline containing 002 sodium azidewas solidified on glass plate and appropriate holes (5mmdiameter) were punched into it The desired concentration ofantigen (100 120583g) of protein sample that is enzyme prepara-tion in peripheral wells and 20120583L of antiprotein serum wereloaded in the side wells and left at 28∘C for 24 to 48 h forvisualization of precipitin line Then the antigen antibodyreaction (precipitin line) was verified simultaneously byloading preimmune serum (considered as control)
210 Ecofriendly Applications of Cysteine Protease
2101 Dehairing Studies The fresh fleshed rat and cow hidewere washed with a commercial detergent and cut into 5 times5 cm pieces Eight to twelve grams of hide (usually two tothree pieces) was processed in a flask with crude cysteineprotease (CCP) and 001M phosphate buffer (control) in aproportion of 50mL of liquid enzyme per g of hide At theend of the process the hide pieces were gently scraped withfingers to remove loose hairs This procedure was necessary
because rubbing in this laboratory-scale process was not asvigorous as in industrial drums The skin depilation startedwith 50Umg protein concentrations of enzyme and it wascompleted during 18 h at 70 pH30∘C temperature
2102 Histochemical Studies of Dehaired Skin The tissuesamples of protease-treated hides were put in 10 formolsaline The treated samples were processed in ascendinggrades of alcohol cleaned in xylene and then embeddedin the paraffin wax for making tissue blocks Sections of4mm (5 120583m thick) tissue were obtained using microtomeafter embedding in paraffin wax block and they were stainedusing Hematoxylin and Eosin to examine the histologicalfeatures as per protocol [12 13]
2103 Evaluation of Washing Performance of Crude Cys-teine Protease Application of protease (5Umg proteinminus1) in002mol Lminus1 phosphate buffer pH 74 as a detergent additivewas studied on white cotton cloth pieces (1510158401015840 times1510158401015840) stainedwith blood samples of different animals (ox buffalo goathen and human being) The stained cloth pieces were takenin separate trays The following groups were setup (A) traywith 50mLof 002mol Lminus1 phosphate buffer pH 74 and bloodstained cloth (B) traywith 50mLprotease (5Umg proteinminus1)in 002mol Lminus1 phosphate buffer pH 74 and blood stainedcloth (C) Tray with 50mL detergent (7mgmL) and bloodstained cloth (D) tray with 50mL mixture of detergent(7mgmL) protease (5Umg proteinminus1) and blood stainedcloth [14]These trays were incubated at 30∘C for 25minThecloth pieces were taken out from each set at regular intervalsof 5min rinsed with water dried and visually examinedUntreated cloth pieces stained with blood were taken as con-trol Additionally after washing performance dried cottonpieces were subjected to cutting The resulting small piecesof individual destained cotton cloth piece were suspended innormal saline at 30∘Cand centrifuged at 5000 rpm for 20minThe progress of destaining of blood stain was monitored bymeasuring the absorbance of resulting supernatant at 420 nmThe test cotton fabric pieces stained with egg yolk were alsotreated under similar conditions at 30∘C Stain removal waschecked qualitatively by visualization
2104 Hydrolysis of Gelatin and Release of Silver Used X-ray films were washed with distilled water and wiped withcotton impregnated with ethanol The washed film was driedat room temperature for 30min One g of X-ray film (cutinto 2 times 2 cm pieces) was then incubated with 10mL ofprotease (5Umg proteinminus1) in 002mol Lminus1 phosphate bufferpH 74 (such that the film is completely immersed in theenzyme) at 30∘C with continuous shaking Turbidity of thereaction mixture (hydrolysate) increased with time (as thehydrolysis progressed) and no further increase in turbiditywas observed when hydrolysis was completed Samples wereremoved at 30min intervals and time required for completeremoval of gelatin layer was noted [15]
211 Biological Activities of Cysteine Protease
2111 Wound Healing Activity (Excision Wound Model)Albino mice were divided into three groups of six animals
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in each group Circular wounds of approximately 300 to350mm2 in diameter were inflicted on the shaved skin undermild ether anesthesia Groups II and III were treated withcrude cysteine protease (CCP) and 1 ww framycetin sul-phate IP (Soframycin) respectively Group I was untreatedand considered as controlThe progressive changes in woundarea were recorded in mm2 by tracing the wound boundariesaround it on a transparent paper on every day Woundcontractionwas expressed as percentage reduction of originalwound size [16]
2112 Coagulation Time of Whole Blood Twelve tubes werearranged in a water bath at 37∘C Into six of these tubes(test) 01mL of the crude cysteine protease (CCP)was addedand nothing was added to the remaining six tubes (control)05mL of blood was collected separately from mice by cleanvenipuncture and 05mL was added into each of the tubesthe tubes were observed for clot formation and the clottingtime was recorded using a stop watch The average of theclotting time of the six tubes with protease (test) and the sixtubes without protease (control) were taken as the clottingtime respectively [17]
2113 BleedingClotting Time Test The effect of the proteaseon bleeding from fresh experimentally induced wounds wasevaluated using the bleedingclotting time test in mice [18]After sterilizing the skin with 70 alcohol a puncture wasmade on the tail with a sterile sharp blade Immediately adrop of the CCP (1000 120583gmL) was placed on the cut portionand at the same time a stopwatch was switched on Sterilizedfilter paper was used to absorb blood coming out and timetaken for ceasing bleeding was recorded the average wastaken as bleeding time (test) The procedure was repeated onthe second group ofmice but here after puncturing the tail adrop of protease (CCP)was not applied serving it as a controlgroup of animal
2114 Antimicrobial Activity Antimicrobial activity of differ-ent concentrations of CCP (50 and 100 120583gmL) was studiedagainst Gram positive and Gram negative bacteria by usingdisc diffusion method [19] After overnight incubation at37∘C the zone of inhibitionwasmeasured and comparedwithreference antibiotic (Gentamicin) A control experiment wasset up by using an equal amount of phosphate buffer in placeof protease
212 Agricultural Application Nematicidal Activity TheBhendi (Okra) crops were grouped into eight groups with sixplants in each group
Group I Normal sterile soil plant (control or untreated-uninoculated)
Group II Nematode affected plant (untreated)
Groups III IV V and VI Nematode control by 25 50 75and 100mg of CCP per g soil respectively
Groups VII and VIII Nematode control by carbofuran 3G nematicide and Sanjeevani 1 WP (Trichoderma viride)respectively dose 20mg per g soil
The study was undertaken in 48 earthen pots (15 cmdiameter) Each was filled with 800 g of sterile soil alongwith BioOrganic Fertilizer (1 1) Experimental okra seed wasgrown in each and every pot under greenhouse conditionAfter 15 days of germination the inoculation was achievedby pouring the nematode water suspension containing 2000nematodes through 4 holes (5ndash8 cm depth) around theroot system of experimental crop which were immediatelycovered by sterile soil except Group I pots Exactly after 15days CCP was applied in four doses that is 25 50 75 and100mg per g soil (Groups III IV V and VI) Carbofuran3 G (encapsulated) nematicide (Group VII) and Sanjeevani1 WP (Trichoderma viride) (Group VIII) were also appliedseparately in 20mg per g soil by the same proceduredescribed earlier Then exactly after 20 days we counted thenumber of nematodes to determine the percent mortalitythat is PM (PM= (Number of dead nematodesTotal numberof nematodes) times 100) [20]
3 Results and Discussion
31 Characterization of Cysteine Protease Euphorbia nivuliaBuch-Ham belongs to the Euphorbiaceae family whosemembers are characterized by secretory tissues (laticifers)which frequently include proteolytic and milk clottingenzymes The young stem latex of E nivulia possessesproteolytic and milk clotting enzyme in more quantity ascomparedwith other investigated laticiferous plants of north-ern region of Maharashtra India [6] E nivulia latex containsa thermostable glycosylated cysteine protease presenting anoptimum activity at pH of 66 and temperature of 45∘C amolecular weight of 4342 kDa (analyzed by SDDS-PAGE)is activated by cysteine hydrochloride and inhibited bymercuric chloride [6] According to protease nomenclaturethis protein is designated as Nivulian-II due to previouslyNivulian-I (31486985Da) was already characterized from Enivulia latex [21]
The stability of proteins and enzymes is usually a factorthat limits their usefulness in many applicationsThe thermalstability of this cysteine protease was examined bymeasuringthe residual enzyme activity of aliquots of the enzyme byincubating at different temperatures This cysteine proteasewas stable up to 60∘C At least 80 residual proteolyticactivity of cysteine protease was retained after incubationat 60∘C as shown in Figure 1 After enzyme incubation at60 and 70∘C the enzyme activity was retained about 8743and 5436 percent activity respectively The enzyme wasalmost completely inactivated by heating for 15min at 80∘CSimilar thermal profile of cysteine protease was reported incysteine protease of Araujia hortorum fruits freesia cormsand endopeptidase of Bromelia hieronymi [22ndash24]
The effect of various metal ions at 5mmol litminus1 concen-tration on the proteolytic activity at 37∘C is summarized inTable 1 Enzyme activity was affected by all the examinedmetal cations except Ca++ Mg++ andMn++ Enzyme activitywas affected by (i) 25ndash35 in presence of Fe++ and Na++ions (ii) 40ndash46 in presence of K+ Zn++ Ag++ and Cd++ions and (iii) 89ndash92 in presence of Hg++ ion A similar
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Table 1 Effect of surfactants oxidizing agent and metal ions onproteolytic activity
inhibition profile was also reported with characterizationof a cysteine protease isolated from wheat grain Triticumaestivum [25] The effect of surfactants and oxidizing agentat 1 concentration on the proteolytic activity at 37∘C is alsosummarized in the same table In presence of SDS and H
2O2
protease activity was inhibited up to 4849 and 3182respectively and activitywas unaffected by exposure to TritonX-100 and Tween-80 indicating that the purified proteasecould not be lipoprotein Our results are in good agreementwith the earlier observations reported in the purification ofmetalloprotease of Pseudomonas aeruginosa [26]
Enzymes are generally inactivated in the presence oforganic solvents The partially purified protease retainedits 85 to 100 activity at three concentrations of organicsolvents that is (i) 30 solvents of acetone benzenedimethylformamide ethyleneglycol and propanol (ii) 50solvents of acetone acetophenone benzene chlorobenzene
Table 2 Effect of various organic solvents on proteolytic activity
Srno Solvent
Residual activity ()Concentration of organic solvent (vv)
and dimethylformamide and (iii) 70 solvents of acetoneand benzene but more than 60 inactivation was observedin presence of remaining solvents (Table 2) Amongstthe organic solvents studied dichloromethane and butanolstrongly inhibited protease activity Acetone causes slightlyincreased protease activity The enzyme was stable in thepresence of acetone benzene and ethyleneglycol Our resultsare in good agreement with earlier observations [27]
The high activity and stability of the enzyme preparationin the pH range from 50 to 80 and its relative stabilitytowards surfactants is very useful for its application asdetergent additive To check the compatibility of the enzymepreparation with commercial solid detergents the enzymewas preincubated in the presence of various commerciallaundry detergents for 60min at 37∘C The data presentedin Table 3 illustrates that the enzyme is extremely stable inthe presence of detergents like Tide Ujala and Nirma atboth concentration of 7mgmL and 10mgmL The enzymeretained 90 to 98 of its activity in the presence of Wheeland Ariel and 80 to 90 in presence of Rin Surf ExcelFenna and Ghari at 7mgmL Enzyme preparation was lessstable in presence of Sasa and Impact where it lost up to22 to 33 of its activity The obtained results clearly indicatethat the performance of enzyme in detergents depends on anumber of factors including the detergentsrsquo compounds sincethe proteolytic stability varied with each laundry detergentThementioned compatibility of the enzyme preparation withcommercial solid detergents was in accordance with previousstudies [10 13 27]
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Table 3 Stability of protease enzyme preparation in various localdetergents
Polyclonal antibodies against protein (cysteine protease)were raised in male albino Wister rats The presence ofantibodies in the anti rat serum was checked by Ouchter-lonyrsquos double immunodiffusionmethod Precipitin lines startappearing after 10ndash12 h of incubation at 28∘C and are dis-tinctly visible in about 36 to 42 h (Figure 2) Additionally thepolyclonal antibodies are specific to specific concentrationof glycoprotein This observation confirms that the proteinspresent in the enzyme fractions are distinct It reveals that theglycoprotein has unique antigenic determinants The poly-clonal antibodies raised for glycosylated cysteine proteasewould be of immense importance in detecting and as a ligandfor various future studies Our results are in good agreementwith earlier observations of antigenic property of cysteineprotease named Procerain of Calotropis procera latex [28]Milin a protease of Euphorbia milii latex [29] Cryptolepainof Cryptolepis buchanani latex [30] Indicain latex proteaseof Morus indica [31] and a novel cysteine protease namedProcerain B of Calotropis procera latex [32]
32 Ecofriendly Applications of Cysteine Protease Enzymaticdehairing process is gaining importance as an alternativechemical methodology in present day scenario This processis significant in reduction of toxicity in addition to improve-ment of leather quality [33] The experimental dehaired peltsof cow and rat hide showed complete removal of fine hairs(Figures 3 and 4) with increased brightness and it may bedue to elimination of sulfide in the process Similar results arenoticed by Subba Rao et al [13] with thermostable protease ofBacillus circulans Cysteine protease could remove hairs of ratand cow hides after 18 h incubation with hide at 30∘C easilycompared to each control with no observable damage on thecollagenTherefore the dehaired skin exhibits clean hair poreand clear grain structure
Histological sections of dehaired pelts stainedwith hema-toxylin and eosin revealed the removal of epidermis glandu-lar structures hair shafts bulb and follicles (Figures 4(c) and4(e)) Complete absence of the previous structural features
along with opening up of collagen fibre structure was seenwith samples (hide) treated for more than 12 h (Figure 4(e))On the other hand partial and moderate removal of hairis observed with 12 h incubation (Figure 4(c)) The datadepicted that there was no apparent damage to the collagenfibres in dehaired pelts (Figure 4(e)) The histological studiesfurther strengthened the easy removal as the hair follicleswere found to be empty (Figure 4(e)) The skin with noprotease treatment showed hair follicles in the dermis withintact hair (Figure 4(a)) and hair was also observed comingout of the epidermal layer This showed that the proteasewas effective in removing hair from hair follicles Comparingtreated skins with untreated controls we observed that onlyskin epidermis and adnexa (skin appendages) including hairbulbs were digested showing a histological autolytic-likeappearance (Figures 4(c) and 4(e)) as could be expectedfor proper skin depilation Protease enzyme has advantagesin dehairing process as this enzyme effectively unhaired therat and cow hides within 18 h compared to earlier reportswhere proteases from Bacillus cereus and Aspergillus tamariidehaired the goat skin in 21 and 24 h respectively [33 34]indicating its potential application in leather industry forecofriendly economizing the process
The results (Figure 5) of evaluation of enzyme for washingperformance pointed out that the blood stains on the clothpieces remained as they were even after 15min of rinsingin the case of controls and commercial detergents Bloodstain was completely removed from the cloths after rinsingthem with a combination of detergent and crude cysteineprotease (CCP) for a period of 15min whereas it wasremoved after 25min when rinsed with CCP individuallyThese results clearly indicate that the enzyme is fairly stableas an ingredient in the presence of detergents Our results ofwashing performance of CCP are in good accordance withthe earlier observations reported in washing performance ofprotease of Pseudomonas aeruginosa [35] and Streptomycesgulbdrgensis [14]
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(a) (b)
Figure 3 Dehairing of cow hide (a) Cow hide control and (b) cow hide after 18 h incubation with CCP
ED
HS
HF
GS HB
(a) (b)
ED
HSDHF
HF DHB
(c)
(d)
DED
EHF
DHB
(e) (f)
Figure 4 Dehairing of rat hide Hematoxylin and eosin staining skin sections from (a) control without treatment (c) partially dehaired peltsof enzymatic process after 12 h and (e) dehaired pelts of enzymatic process after 18 h incubation with CCP Rat hide images from (b) controlwithout treatment (d) after 12 h incubation with CCP and (f) after 18 h incubation with CCP ED epidermis HS hair shaft HF hairfollicles GS glandular structure HB hair bulb DHB degraded hair bulb DED degraded epidermis and EHF empty hair folliclesDHF degraded hair follicles
In order to evaluate the performance of CCP with respectto its capability of removing stains of different blood samplesnamely human being ox buffalo and hen egg yolk stainswere used On incubating several pieces of stained cloth at30∘C for 25min results of these findings are interesting theuse of enzyme alone showed more effective removal of bloodand egg yolk stains (Figure 6) In fact protease facilitatesthe release of proteinaceous materials in a much easier waythan the commercially available detergent Furthermore thecombination of CCP with detergent resulted in completestain removal (Figure 6) A similar study reported on theusefulness of alkaline proteases from Bacillus brevis [36] and
Bacillus pumilus [37] These destaining profiles made clearthe idea about removal of blood stains with minimum useof commercial detergent within 25min Rapid blood stainremoval was noticed with supplementation of commerciallyavailable detergents (Table 4) Similar results of destainingof blood with combination of protease and detergent werenoticed by Subba Rao et al [13]
Treatment of X-ray films with protease resulted in thesliver bound with gelatin being stripped off into the reactionmixture and this results in clean appearance of plastic filmThe loss in weight after the treatment was around 5 (ww)based on initial weight of the film (10 g) The gelatin layer of
8 The Scientific World Journal
(i) (ii) (iii) (iv)
(a)
(i) (ii) (iii) (iv)
(b)
(i) (ii) (iii) (iv)
(c)
(i) (ii) (iii) (iv)
(d)
(i) (ii) (iii) (iv)
(e)
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
haemostatic and antiproliferative activity [5] During thecourse of screening for biochemical constituents a substan-tial amount of proteolytic and milk clotting activity wasfound in the latex of this plant [7] Recently an attempthas been made on peptide sequencing of 31 kDa Tubulinalpha-1 chain-like protein called Nivulian-I present in thelatex of E nivulia [6] Very recently a comparative accounton proteolytic activity of E nivulia and other three plantsnamely Calotropis procera Carica papaya and Ficus caricawas reported by us Additionally we report the glycosylatedcysteine protease called Nivulian-II of the latex of E nivulia[6] This paper describes the further biochemical character-ization of this cysteine-like protease with some ecofriendlyapplications
2 Materials and Methods
21 Chemicals All chemicals with the highest purity analyti-calHPLCgradewere purchased fromSigmaChemicalsUSAHimedia Laboratories Mumbai SRL Chemicals MumbaiQualigen Fine Chemicals Mumbai Merck Chemicals Indiaand Bangalore Genie India
22 Animals Used Four- to five-week-old albinoWistermalerats (120 to 180 g body weight) were used for antigenicitySwiss albino mice of either sex (50ndash100 g) were used forthe study of wound healing activity The animals weremaintained under standard laboratory conditions in animalhouse approved by Committee for the Purpose of Controland Supervision on Experiments on Animals (CPCSEAregistration number 1062C07CPCSEA24 May 2007)Theexperimental protocol was approved by Institutional AnimalEthics Committee constituted under CPCSEA rules India
23 Animal Hides Freshly flayed wet rat hide was obtainedfrom animal house ofMoolji JaithaCollege NorthMaharash-tra University Jalgaon India Cow hide of healthy animal wascollected from local meat shops of Jalgaon city
24 Blood Sample Fresh blood samples of domestic animalsnamely goat (Capra hircus) buffalo (Bubalus bubalis) andox (Ovibos moschatus) of either sexes were collected underthe supervision of Dr N M Pawar veterinary practitionerof Paldhi unit Jalgaon District Maharashtra India Bloodsample of healthy hen was collected from local chicken shopsof Jalgaon city (MS)
25 Nematode Sample Root-knot nematodes (Meloidogyneincognita) were collected along with soil sample by zig-zag manner from vicinity of Department of AgriculturalEntomology Mahatma Phule Krishi Vidyapeeth RahuriAhmednagar Maharashtra India
26 Microbial Culture The microbial cultures like Staphylo-coccus aureus (ATCC 25923) Escherichia coli (ATCC 25992)Klebsiella pneumoniae (ATCC 23357) Pseudomonas aerug-inosa (ATCC 27853) Proteus vulgaris (NCIM 2027) andBacillus subtilis (NCIM 2063) were procured from National
Collection of Industrial Microorganisms (NCIM) PuneMaharashtra India
27 Plant Material Collection of Latex Preparation of CrudeEnzyme and Purification of Cysteine Protease The detailedinformation about identification collection preservationand preparation of crude enzyme and its proteolytic activityof Euphorbia nivulia latex and its quantification is describedin our previous communication [6] Method of purificationof protease was done using acetone precipitation DEAEcellulose chromatography and dialysis and followed byrechromatography on DEAE cellulose column as describedin our earlier communication [8]
28 Characterization of Cysteine Protease
281 Thermal Stability The thermal behavior of the purifiedenzyme fraction was evaluated by incubating the enzymeat the desired temperatures in the range of 20ndash800∘C for15min in 001M phosphate buffer (pH 66) and an aliquotwas used for the enzyme activity measurement at the sametemperature At each temperature a control assay was carriedout without enzyme
282 Effect of Metal Ions on Proteolytic Activity Impact ofvarious metal ions namely K+ Na+ Zn++ Ag++ Cd++Fe++ Hg++ Mg++ and Mn++ at 5mM concentration onthe enzyme catalytic behaviour was studied The enzymefraction alongwith 001M phosphate buffer (at optimumpH)was preincubated at room temperature for 30 minutes withrespective metal ions separately before enzyme assay Thenthe residual proteolytic activity wasmeasured by using caseinaccording to standard assay procedure relative to control(without metal ion)
283 Effect of Surfactant and Oxidizing Agent on ProteolyticActivity The effect of different surfactants namely sodiumdodecyl sulphate Triton X-100 and Tween 80 and oxidizingagent (H
2O2) at 1 (vv) final concentration on enzyme
activity was studied by preincubating enzyme preparationfor 60min at 37∘C in the above surfactants and oxidizingagent before analysis Then the residual proteolytic activitywas measured by using casein according to standard assayprocedure relative to control (without chemical surfactantsand oxidizing agent) and the resulting activity was taken as100 percent
284 Effect of Organic Solvents on Proteolytic Activity Thestability of enzyme activity in presence of different watermiscible or immiscible organic solvents was investigatedby incubating the enzyme preparation with various organicsolvents (30 50 and 70 vv) at 37∘C for 1 h After incu-bation the residual enzyme activity was determined as perthe previously discussed method with casein The enzymeactivity of a control sample (without solvent) incubatedunder the same conditions was taken as 100 percent [9]For this assay the solvents used were acetone acetophenonebenzyl alcohol benzene butanol chloroform chloroben-zene dichloromethane dimethylformamide diethyl ether
285 Compatibility of Protease with Laundry DetergentsThe compatibility of enzyme preparation in presence ofcommercial solid laundry detergents was examined by incu-bating enzyme preparation for 60min at 37∘C with variousdetergent preparations and the residual enzyme activity wasdetermined as per the method using casein The enzymeactivity of a control sample (without detergent) incubatedunder the same conditions was taken as 100 percent Thesolid detergents used were Ariel Oxy Blue Tide (Procter andGamble Company USA) Fena Ultra New Impact (Fena (P)Ltd New Delhi India) Nirma (Nirma Ltd AhmedabadIndia) Wheel Active Rin Surf Excel (Hindustan LiverLtd India) and Ujala (Jyoti Laboratories Ltd MumbaiIndia) The detergents were diluted in tap water to give afinal concentration of 7mgmL and 10mgmL to simulatewashing conditions The endogenous proteases contained inthese detergents were inactivated by incubating the diluteddetergents at 65∘C for 60min prior to the addition of enzyme[10]
29 Antigenic Property
291 Immunization of Rat and Production of PolyclonalAntisera Theprotein sample (100 120583g in 005mLof phosphatebuffer saline 001M pH 60) thoroughly mixed with equalvolume of Freundrsquos complete adjuvant and injected into malealbino Wister rat (180 to 200 g body weight) subcutaneouslyat multiple sites Two booster doses were administered atweekly intervals with the same concentration but with equalvolume of Freundrsquos incomplete adjuvant After nine daysfrom last booster dose blood was drawn through retroorbitalplexussinus with a glass capillary tube and antisera wasseparated after allowing the blood to coagulate at 8∘C for 24 h
292 Detection of Antibodies and Immunological CrossReactivity The presence of antibodies was confirmed byOuchterlonyrsquos double immunodiffusion assay [11] 1 agarosein phosphate buffer saline containing 002 sodium azidewas solidified on glass plate and appropriate holes (5mmdiameter) were punched into it The desired concentration ofantigen (100 120583g) of protein sample that is enzyme prepara-tion in peripheral wells and 20120583L of antiprotein serum wereloaded in the side wells and left at 28∘C for 24 to 48 h forvisualization of precipitin line Then the antigen antibodyreaction (precipitin line) was verified simultaneously byloading preimmune serum (considered as control)
210 Ecofriendly Applications of Cysteine Protease
2101 Dehairing Studies The fresh fleshed rat and cow hidewere washed with a commercial detergent and cut into 5 times5 cm pieces Eight to twelve grams of hide (usually two tothree pieces) was processed in a flask with crude cysteineprotease (CCP) and 001M phosphate buffer (control) in aproportion of 50mL of liquid enzyme per g of hide At theend of the process the hide pieces were gently scraped withfingers to remove loose hairs This procedure was necessary
because rubbing in this laboratory-scale process was not asvigorous as in industrial drums The skin depilation startedwith 50Umg protein concentrations of enzyme and it wascompleted during 18 h at 70 pH30∘C temperature
2102 Histochemical Studies of Dehaired Skin The tissuesamples of protease-treated hides were put in 10 formolsaline The treated samples were processed in ascendinggrades of alcohol cleaned in xylene and then embeddedin the paraffin wax for making tissue blocks Sections of4mm (5 120583m thick) tissue were obtained using microtomeafter embedding in paraffin wax block and they were stainedusing Hematoxylin and Eosin to examine the histologicalfeatures as per protocol [12 13]
2103 Evaluation of Washing Performance of Crude Cys-teine Protease Application of protease (5Umg proteinminus1) in002mol Lminus1 phosphate buffer pH 74 as a detergent additivewas studied on white cotton cloth pieces (1510158401015840 times1510158401015840) stainedwith blood samples of different animals (ox buffalo goathen and human being) The stained cloth pieces were takenin separate trays The following groups were setup (A) traywith 50mLof 002mol Lminus1 phosphate buffer pH 74 and bloodstained cloth (B) traywith 50mLprotease (5Umg proteinminus1)in 002mol Lminus1 phosphate buffer pH 74 and blood stainedcloth (C) Tray with 50mL detergent (7mgmL) and bloodstained cloth (D) tray with 50mL mixture of detergent(7mgmL) protease (5Umg proteinminus1) and blood stainedcloth [14]These trays were incubated at 30∘C for 25minThecloth pieces were taken out from each set at regular intervalsof 5min rinsed with water dried and visually examinedUntreated cloth pieces stained with blood were taken as con-trol Additionally after washing performance dried cottonpieces were subjected to cutting The resulting small piecesof individual destained cotton cloth piece were suspended innormal saline at 30∘Cand centrifuged at 5000 rpm for 20minThe progress of destaining of blood stain was monitored bymeasuring the absorbance of resulting supernatant at 420 nmThe test cotton fabric pieces stained with egg yolk were alsotreated under similar conditions at 30∘C Stain removal waschecked qualitatively by visualization
2104 Hydrolysis of Gelatin and Release of Silver Used X-ray films were washed with distilled water and wiped withcotton impregnated with ethanol The washed film was driedat room temperature for 30min One g of X-ray film (cutinto 2 times 2 cm pieces) was then incubated with 10mL ofprotease (5Umg proteinminus1) in 002mol Lminus1 phosphate bufferpH 74 (such that the film is completely immersed in theenzyme) at 30∘C with continuous shaking Turbidity of thereaction mixture (hydrolysate) increased with time (as thehydrolysis progressed) and no further increase in turbiditywas observed when hydrolysis was completed Samples wereremoved at 30min intervals and time required for completeremoval of gelatin layer was noted [15]
211 Biological Activities of Cysteine Protease
2111 Wound Healing Activity (Excision Wound Model)Albino mice were divided into three groups of six animals
4 The Scientific World Journal
in each group Circular wounds of approximately 300 to350mm2 in diameter were inflicted on the shaved skin undermild ether anesthesia Groups II and III were treated withcrude cysteine protease (CCP) and 1 ww framycetin sul-phate IP (Soframycin) respectively Group I was untreatedand considered as controlThe progressive changes in woundarea were recorded in mm2 by tracing the wound boundariesaround it on a transparent paper on every day Woundcontractionwas expressed as percentage reduction of originalwound size [16]
2112 Coagulation Time of Whole Blood Twelve tubes werearranged in a water bath at 37∘C Into six of these tubes(test) 01mL of the crude cysteine protease (CCP)was addedand nothing was added to the remaining six tubes (control)05mL of blood was collected separately from mice by cleanvenipuncture and 05mL was added into each of the tubesthe tubes were observed for clot formation and the clottingtime was recorded using a stop watch The average of theclotting time of the six tubes with protease (test) and the sixtubes without protease (control) were taken as the clottingtime respectively [17]
2113 BleedingClotting Time Test The effect of the proteaseon bleeding from fresh experimentally induced wounds wasevaluated using the bleedingclotting time test in mice [18]After sterilizing the skin with 70 alcohol a puncture wasmade on the tail with a sterile sharp blade Immediately adrop of the CCP (1000 120583gmL) was placed on the cut portionand at the same time a stopwatch was switched on Sterilizedfilter paper was used to absorb blood coming out and timetaken for ceasing bleeding was recorded the average wastaken as bleeding time (test) The procedure was repeated onthe second group ofmice but here after puncturing the tail adrop of protease (CCP)was not applied serving it as a controlgroup of animal
2114 Antimicrobial Activity Antimicrobial activity of differ-ent concentrations of CCP (50 and 100 120583gmL) was studiedagainst Gram positive and Gram negative bacteria by usingdisc diffusion method [19] After overnight incubation at37∘C the zone of inhibitionwasmeasured and comparedwithreference antibiotic (Gentamicin) A control experiment wasset up by using an equal amount of phosphate buffer in placeof protease
212 Agricultural Application Nematicidal Activity TheBhendi (Okra) crops were grouped into eight groups with sixplants in each group
Group I Normal sterile soil plant (control or untreated-uninoculated)
Group II Nematode affected plant (untreated)
Groups III IV V and VI Nematode control by 25 50 75and 100mg of CCP per g soil respectively
Groups VII and VIII Nematode control by carbofuran 3G nematicide and Sanjeevani 1 WP (Trichoderma viride)respectively dose 20mg per g soil
The study was undertaken in 48 earthen pots (15 cmdiameter) Each was filled with 800 g of sterile soil alongwith BioOrganic Fertilizer (1 1) Experimental okra seed wasgrown in each and every pot under greenhouse conditionAfter 15 days of germination the inoculation was achievedby pouring the nematode water suspension containing 2000nematodes through 4 holes (5ndash8 cm depth) around theroot system of experimental crop which were immediatelycovered by sterile soil except Group I pots Exactly after 15days CCP was applied in four doses that is 25 50 75 and100mg per g soil (Groups III IV V and VI) Carbofuran3 G (encapsulated) nematicide (Group VII) and Sanjeevani1 WP (Trichoderma viride) (Group VIII) were also appliedseparately in 20mg per g soil by the same proceduredescribed earlier Then exactly after 20 days we counted thenumber of nematodes to determine the percent mortalitythat is PM (PM= (Number of dead nematodesTotal numberof nematodes) times 100) [20]
3 Results and Discussion
31 Characterization of Cysteine Protease Euphorbia nivuliaBuch-Ham belongs to the Euphorbiaceae family whosemembers are characterized by secretory tissues (laticifers)which frequently include proteolytic and milk clottingenzymes The young stem latex of E nivulia possessesproteolytic and milk clotting enzyme in more quantity ascomparedwith other investigated laticiferous plants of north-ern region of Maharashtra India [6] E nivulia latex containsa thermostable glycosylated cysteine protease presenting anoptimum activity at pH of 66 and temperature of 45∘C amolecular weight of 4342 kDa (analyzed by SDDS-PAGE)is activated by cysteine hydrochloride and inhibited bymercuric chloride [6] According to protease nomenclaturethis protein is designated as Nivulian-II due to previouslyNivulian-I (31486985Da) was already characterized from Enivulia latex [21]
The stability of proteins and enzymes is usually a factorthat limits their usefulness in many applicationsThe thermalstability of this cysteine protease was examined bymeasuringthe residual enzyme activity of aliquots of the enzyme byincubating at different temperatures This cysteine proteasewas stable up to 60∘C At least 80 residual proteolyticactivity of cysteine protease was retained after incubationat 60∘C as shown in Figure 1 After enzyme incubation at60 and 70∘C the enzyme activity was retained about 8743and 5436 percent activity respectively The enzyme wasalmost completely inactivated by heating for 15min at 80∘CSimilar thermal profile of cysteine protease was reported incysteine protease of Araujia hortorum fruits freesia cormsand endopeptidase of Bromelia hieronymi [22ndash24]
The effect of various metal ions at 5mmol litminus1 concen-tration on the proteolytic activity at 37∘C is summarized inTable 1 Enzyme activity was affected by all the examinedmetal cations except Ca++ Mg++ andMn++ Enzyme activitywas affected by (i) 25ndash35 in presence of Fe++ and Na++ions (ii) 40ndash46 in presence of K+ Zn++ Ag++ and Cd++ions and (iii) 89ndash92 in presence of Hg++ ion A similar
The Scientific World Journal 5
Table 1 Effect of surfactants oxidizing agent and metal ions onproteolytic activity
inhibition profile was also reported with characterizationof a cysteine protease isolated from wheat grain Triticumaestivum [25] The effect of surfactants and oxidizing agentat 1 concentration on the proteolytic activity at 37∘C is alsosummarized in the same table In presence of SDS and H
2O2
protease activity was inhibited up to 4849 and 3182respectively and activitywas unaffected by exposure to TritonX-100 and Tween-80 indicating that the purified proteasecould not be lipoprotein Our results are in good agreementwith the earlier observations reported in the purification ofmetalloprotease of Pseudomonas aeruginosa [26]
Enzymes are generally inactivated in the presence oforganic solvents The partially purified protease retainedits 85 to 100 activity at three concentrations of organicsolvents that is (i) 30 solvents of acetone benzenedimethylformamide ethyleneglycol and propanol (ii) 50solvents of acetone acetophenone benzene chlorobenzene
Table 2 Effect of various organic solvents on proteolytic activity
Srno Solvent
Residual activity ()Concentration of organic solvent (vv)
and dimethylformamide and (iii) 70 solvents of acetoneand benzene but more than 60 inactivation was observedin presence of remaining solvents (Table 2) Amongstthe organic solvents studied dichloromethane and butanolstrongly inhibited protease activity Acetone causes slightlyincreased protease activity The enzyme was stable in thepresence of acetone benzene and ethyleneglycol Our resultsare in good agreement with earlier observations [27]
The high activity and stability of the enzyme preparationin the pH range from 50 to 80 and its relative stabilitytowards surfactants is very useful for its application asdetergent additive To check the compatibility of the enzymepreparation with commercial solid detergents the enzymewas preincubated in the presence of various commerciallaundry detergents for 60min at 37∘C The data presentedin Table 3 illustrates that the enzyme is extremely stable inthe presence of detergents like Tide Ujala and Nirma atboth concentration of 7mgmL and 10mgmL The enzymeretained 90 to 98 of its activity in the presence of Wheeland Ariel and 80 to 90 in presence of Rin Surf ExcelFenna and Ghari at 7mgmL Enzyme preparation was lessstable in presence of Sasa and Impact where it lost up to22 to 33 of its activity The obtained results clearly indicatethat the performance of enzyme in detergents depends on anumber of factors including the detergentsrsquo compounds sincethe proteolytic stability varied with each laundry detergentThementioned compatibility of the enzyme preparation withcommercial solid detergents was in accordance with previousstudies [10 13 27]
6 The Scientific World Journal
Table 3 Stability of protease enzyme preparation in various localdetergents
Polyclonal antibodies against protein (cysteine protease)were raised in male albino Wister rats The presence ofantibodies in the anti rat serum was checked by Ouchter-lonyrsquos double immunodiffusionmethod Precipitin lines startappearing after 10ndash12 h of incubation at 28∘C and are dis-tinctly visible in about 36 to 42 h (Figure 2) Additionally thepolyclonal antibodies are specific to specific concentrationof glycoprotein This observation confirms that the proteinspresent in the enzyme fractions are distinct It reveals that theglycoprotein has unique antigenic determinants The poly-clonal antibodies raised for glycosylated cysteine proteasewould be of immense importance in detecting and as a ligandfor various future studies Our results are in good agreementwith earlier observations of antigenic property of cysteineprotease named Procerain of Calotropis procera latex [28]Milin a protease of Euphorbia milii latex [29] Cryptolepainof Cryptolepis buchanani latex [30] Indicain latex proteaseof Morus indica [31] and a novel cysteine protease namedProcerain B of Calotropis procera latex [32]
32 Ecofriendly Applications of Cysteine Protease Enzymaticdehairing process is gaining importance as an alternativechemical methodology in present day scenario This processis significant in reduction of toxicity in addition to improve-ment of leather quality [33] The experimental dehaired peltsof cow and rat hide showed complete removal of fine hairs(Figures 3 and 4) with increased brightness and it may bedue to elimination of sulfide in the process Similar results arenoticed by Subba Rao et al [13] with thermostable protease ofBacillus circulans Cysteine protease could remove hairs of ratand cow hides after 18 h incubation with hide at 30∘C easilycompared to each control with no observable damage on thecollagenTherefore the dehaired skin exhibits clean hair poreand clear grain structure
Histological sections of dehaired pelts stainedwith hema-toxylin and eosin revealed the removal of epidermis glandu-lar structures hair shafts bulb and follicles (Figures 4(c) and4(e)) Complete absence of the previous structural features
along with opening up of collagen fibre structure was seenwith samples (hide) treated for more than 12 h (Figure 4(e))On the other hand partial and moderate removal of hairis observed with 12 h incubation (Figure 4(c)) The datadepicted that there was no apparent damage to the collagenfibres in dehaired pelts (Figure 4(e)) The histological studiesfurther strengthened the easy removal as the hair follicleswere found to be empty (Figure 4(e)) The skin with noprotease treatment showed hair follicles in the dermis withintact hair (Figure 4(a)) and hair was also observed comingout of the epidermal layer This showed that the proteasewas effective in removing hair from hair follicles Comparingtreated skins with untreated controls we observed that onlyskin epidermis and adnexa (skin appendages) including hairbulbs were digested showing a histological autolytic-likeappearance (Figures 4(c) and 4(e)) as could be expectedfor proper skin depilation Protease enzyme has advantagesin dehairing process as this enzyme effectively unhaired therat and cow hides within 18 h compared to earlier reportswhere proteases from Bacillus cereus and Aspergillus tamariidehaired the goat skin in 21 and 24 h respectively [33 34]indicating its potential application in leather industry forecofriendly economizing the process
The results (Figure 5) of evaluation of enzyme for washingperformance pointed out that the blood stains on the clothpieces remained as they were even after 15min of rinsingin the case of controls and commercial detergents Bloodstain was completely removed from the cloths after rinsingthem with a combination of detergent and crude cysteineprotease (CCP) for a period of 15min whereas it wasremoved after 25min when rinsed with CCP individuallyThese results clearly indicate that the enzyme is fairly stableas an ingredient in the presence of detergents Our results ofwashing performance of CCP are in good accordance withthe earlier observations reported in washing performance ofprotease of Pseudomonas aeruginosa [35] and Streptomycesgulbdrgensis [14]
The Scientific World Journal 7
(a) (b)
Figure 3 Dehairing of cow hide (a) Cow hide control and (b) cow hide after 18 h incubation with CCP
ED
HS
HF
GS HB
(a) (b)
ED
HSDHF
HF DHB
(c)
(d)
DED
EHF
DHB
(e) (f)
Figure 4 Dehairing of rat hide Hematoxylin and eosin staining skin sections from (a) control without treatment (c) partially dehaired peltsof enzymatic process after 12 h and (e) dehaired pelts of enzymatic process after 18 h incubation with CCP Rat hide images from (b) controlwithout treatment (d) after 12 h incubation with CCP and (f) after 18 h incubation with CCP ED epidermis HS hair shaft HF hairfollicles GS glandular structure HB hair bulb DHB degraded hair bulb DED degraded epidermis and EHF empty hair folliclesDHF degraded hair follicles
In order to evaluate the performance of CCP with respectto its capability of removing stains of different blood samplesnamely human being ox buffalo and hen egg yolk stainswere used On incubating several pieces of stained cloth at30∘C for 25min results of these findings are interesting theuse of enzyme alone showed more effective removal of bloodand egg yolk stains (Figure 6) In fact protease facilitatesthe release of proteinaceous materials in a much easier waythan the commercially available detergent Furthermore thecombination of CCP with detergent resulted in completestain removal (Figure 6) A similar study reported on theusefulness of alkaline proteases from Bacillus brevis [36] and
Bacillus pumilus [37] These destaining profiles made clearthe idea about removal of blood stains with minimum useof commercial detergent within 25min Rapid blood stainremoval was noticed with supplementation of commerciallyavailable detergents (Table 4) Similar results of destainingof blood with combination of protease and detergent werenoticed by Subba Rao et al [13]
Treatment of X-ray films with protease resulted in thesliver bound with gelatin being stripped off into the reactionmixture and this results in clean appearance of plastic filmThe loss in weight after the treatment was around 5 (ww)based on initial weight of the film (10 g) The gelatin layer of
8 The Scientific World Journal
(i) (ii) (iii) (iv)
(a)
(i) (ii) (iii) (iv)
(b)
(i) (ii) (iii) (iv)
(c)
(i) (ii) (iii) (iv)
(d)
(i) (ii) (iii) (iv)
(e)
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
285 Compatibility of Protease with Laundry DetergentsThe compatibility of enzyme preparation in presence ofcommercial solid laundry detergents was examined by incu-bating enzyme preparation for 60min at 37∘C with variousdetergent preparations and the residual enzyme activity wasdetermined as per the method using casein The enzymeactivity of a control sample (without detergent) incubatedunder the same conditions was taken as 100 percent Thesolid detergents used were Ariel Oxy Blue Tide (Procter andGamble Company USA) Fena Ultra New Impact (Fena (P)Ltd New Delhi India) Nirma (Nirma Ltd AhmedabadIndia) Wheel Active Rin Surf Excel (Hindustan LiverLtd India) and Ujala (Jyoti Laboratories Ltd MumbaiIndia) The detergents were diluted in tap water to give afinal concentration of 7mgmL and 10mgmL to simulatewashing conditions The endogenous proteases contained inthese detergents were inactivated by incubating the diluteddetergents at 65∘C for 60min prior to the addition of enzyme[10]
29 Antigenic Property
291 Immunization of Rat and Production of PolyclonalAntisera Theprotein sample (100 120583g in 005mLof phosphatebuffer saline 001M pH 60) thoroughly mixed with equalvolume of Freundrsquos complete adjuvant and injected into malealbino Wister rat (180 to 200 g body weight) subcutaneouslyat multiple sites Two booster doses were administered atweekly intervals with the same concentration but with equalvolume of Freundrsquos incomplete adjuvant After nine daysfrom last booster dose blood was drawn through retroorbitalplexussinus with a glass capillary tube and antisera wasseparated after allowing the blood to coagulate at 8∘C for 24 h
292 Detection of Antibodies and Immunological CrossReactivity The presence of antibodies was confirmed byOuchterlonyrsquos double immunodiffusion assay [11] 1 agarosein phosphate buffer saline containing 002 sodium azidewas solidified on glass plate and appropriate holes (5mmdiameter) were punched into it The desired concentration ofantigen (100 120583g) of protein sample that is enzyme prepara-tion in peripheral wells and 20120583L of antiprotein serum wereloaded in the side wells and left at 28∘C for 24 to 48 h forvisualization of precipitin line Then the antigen antibodyreaction (precipitin line) was verified simultaneously byloading preimmune serum (considered as control)
210 Ecofriendly Applications of Cysteine Protease
2101 Dehairing Studies The fresh fleshed rat and cow hidewere washed with a commercial detergent and cut into 5 times5 cm pieces Eight to twelve grams of hide (usually two tothree pieces) was processed in a flask with crude cysteineprotease (CCP) and 001M phosphate buffer (control) in aproportion of 50mL of liquid enzyme per g of hide At theend of the process the hide pieces were gently scraped withfingers to remove loose hairs This procedure was necessary
because rubbing in this laboratory-scale process was not asvigorous as in industrial drums The skin depilation startedwith 50Umg protein concentrations of enzyme and it wascompleted during 18 h at 70 pH30∘C temperature
2102 Histochemical Studies of Dehaired Skin The tissuesamples of protease-treated hides were put in 10 formolsaline The treated samples were processed in ascendinggrades of alcohol cleaned in xylene and then embeddedin the paraffin wax for making tissue blocks Sections of4mm (5 120583m thick) tissue were obtained using microtomeafter embedding in paraffin wax block and they were stainedusing Hematoxylin and Eosin to examine the histologicalfeatures as per protocol [12 13]
2103 Evaluation of Washing Performance of Crude Cys-teine Protease Application of protease (5Umg proteinminus1) in002mol Lminus1 phosphate buffer pH 74 as a detergent additivewas studied on white cotton cloth pieces (1510158401015840 times1510158401015840) stainedwith blood samples of different animals (ox buffalo goathen and human being) The stained cloth pieces were takenin separate trays The following groups were setup (A) traywith 50mLof 002mol Lminus1 phosphate buffer pH 74 and bloodstained cloth (B) traywith 50mLprotease (5Umg proteinminus1)in 002mol Lminus1 phosphate buffer pH 74 and blood stainedcloth (C) Tray with 50mL detergent (7mgmL) and bloodstained cloth (D) tray with 50mL mixture of detergent(7mgmL) protease (5Umg proteinminus1) and blood stainedcloth [14]These trays were incubated at 30∘C for 25minThecloth pieces were taken out from each set at regular intervalsof 5min rinsed with water dried and visually examinedUntreated cloth pieces stained with blood were taken as con-trol Additionally after washing performance dried cottonpieces were subjected to cutting The resulting small piecesof individual destained cotton cloth piece were suspended innormal saline at 30∘Cand centrifuged at 5000 rpm for 20minThe progress of destaining of blood stain was monitored bymeasuring the absorbance of resulting supernatant at 420 nmThe test cotton fabric pieces stained with egg yolk were alsotreated under similar conditions at 30∘C Stain removal waschecked qualitatively by visualization
2104 Hydrolysis of Gelatin and Release of Silver Used X-ray films were washed with distilled water and wiped withcotton impregnated with ethanol The washed film was driedat room temperature for 30min One g of X-ray film (cutinto 2 times 2 cm pieces) was then incubated with 10mL ofprotease (5Umg proteinminus1) in 002mol Lminus1 phosphate bufferpH 74 (such that the film is completely immersed in theenzyme) at 30∘C with continuous shaking Turbidity of thereaction mixture (hydrolysate) increased with time (as thehydrolysis progressed) and no further increase in turbiditywas observed when hydrolysis was completed Samples wereremoved at 30min intervals and time required for completeremoval of gelatin layer was noted [15]
211 Biological Activities of Cysteine Protease
2111 Wound Healing Activity (Excision Wound Model)Albino mice were divided into three groups of six animals
4 The Scientific World Journal
in each group Circular wounds of approximately 300 to350mm2 in diameter were inflicted on the shaved skin undermild ether anesthesia Groups II and III were treated withcrude cysteine protease (CCP) and 1 ww framycetin sul-phate IP (Soframycin) respectively Group I was untreatedand considered as controlThe progressive changes in woundarea were recorded in mm2 by tracing the wound boundariesaround it on a transparent paper on every day Woundcontractionwas expressed as percentage reduction of originalwound size [16]
2112 Coagulation Time of Whole Blood Twelve tubes werearranged in a water bath at 37∘C Into six of these tubes(test) 01mL of the crude cysteine protease (CCP)was addedand nothing was added to the remaining six tubes (control)05mL of blood was collected separately from mice by cleanvenipuncture and 05mL was added into each of the tubesthe tubes were observed for clot formation and the clottingtime was recorded using a stop watch The average of theclotting time of the six tubes with protease (test) and the sixtubes without protease (control) were taken as the clottingtime respectively [17]
2113 BleedingClotting Time Test The effect of the proteaseon bleeding from fresh experimentally induced wounds wasevaluated using the bleedingclotting time test in mice [18]After sterilizing the skin with 70 alcohol a puncture wasmade on the tail with a sterile sharp blade Immediately adrop of the CCP (1000 120583gmL) was placed on the cut portionand at the same time a stopwatch was switched on Sterilizedfilter paper was used to absorb blood coming out and timetaken for ceasing bleeding was recorded the average wastaken as bleeding time (test) The procedure was repeated onthe second group ofmice but here after puncturing the tail adrop of protease (CCP)was not applied serving it as a controlgroup of animal
2114 Antimicrobial Activity Antimicrobial activity of differ-ent concentrations of CCP (50 and 100 120583gmL) was studiedagainst Gram positive and Gram negative bacteria by usingdisc diffusion method [19] After overnight incubation at37∘C the zone of inhibitionwasmeasured and comparedwithreference antibiotic (Gentamicin) A control experiment wasset up by using an equal amount of phosphate buffer in placeof protease
212 Agricultural Application Nematicidal Activity TheBhendi (Okra) crops were grouped into eight groups with sixplants in each group
Group I Normal sterile soil plant (control or untreated-uninoculated)
Group II Nematode affected plant (untreated)
Groups III IV V and VI Nematode control by 25 50 75and 100mg of CCP per g soil respectively
Groups VII and VIII Nematode control by carbofuran 3G nematicide and Sanjeevani 1 WP (Trichoderma viride)respectively dose 20mg per g soil
The study was undertaken in 48 earthen pots (15 cmdiameter) Each was filled with 800 g of sterile soil alongwith BioOrganic Fertilizer (1 1) Experimental okra seed wasgrown in each and every pot under greenhouse conditionAfter 15 days of germination the inoculation was achievedby pouring the nematode water suspension containing 2000nematodes through 4 holes (5ndash8 cm depth) around theroot system of experimental crop which were immediatelycovered by sterile soil except Group I pots Exactly after 15days CCP was applied in four doses that is 25 50 75 and100mg per g soil (Groups III IV V and VI) Carbofuran3 G (encapsulated) nematicide (Group VII) and Sanjeevani1 WP (Trichoderma viride) (Group VIII) were also appliedseparately in 20mg per g soil by the same proceduredescribed earlier Then exactly after 20 days we counted thenumber of nematodes to determine the percent mortalitythat is PM (PM= (Number of dead nematodesTotal numberof nematodes) times 100) [20]
3 Results and Discussion
31 Characterization of Cysteine Protease Euphorbia nivuliaBuch-Ham belongs to the Euphorbiaceae family whosemembers are characterized by secretory tissues (laticifers)which frequently include proteolytic and milk clottingenzymes The young stem latex of E nivulia possessesproteolytic and milk clotting enzyme in more quantity ascomparedwith other investigated laticiferous plants of north-ern region of Maharashtra India [6] E nivulia latex containsa thermostable glycosylated cysteine protease presenting anoptimum activity at pH of 66 and temperature of 45∘C amolecular weight of 4342 kDa (analyzed by SDDS-PAGE)is activated by cysteine hydrochloride and inhibited bymercuric chloride [6] According to protease nomenclaturethis protein is designated as Nivulian-II due to previouslyNivulian-I (31486985Da) was already characterized from Enivulia latex [21]
The stability of proteins and enzymes is usually a factorthat limits their usefulness in many applicationsThe thermalstability of this cysteine protease was examined bymeasuringthe residual enzyme activity of aliquots of the enzyme byincubating at different temperatures This cysteine proteasewas stable up to 60∘C At least 80 residual proteolyticactivity of cysteine protease was retained after incubationat 60∘C as shown in Figure 1 After enzyme incubation at60 and 70∘C the enzyme activity was retained about 8743and 5436 percent activity respectively The enzyme wasalmost completely inactivated by heating for 15min at 80∘CSimilar thermal profile of cysteine protease was reported incysteine protease of Araujia hortorum fruits freesia cormsand endopeptidase of Bromelia hieronymi [22ndash24]
The effect of various metal ions at 5mmol litminus1 concen-tration on the proteolytic activity at 37∘C is summarized inTable 1 Enzyme activity was affected by all the examinedmetal cations except Ca++ Mg++ andMn++ Enzyme activitywas affected by (i) 25ndash35 in presence of Fe++ and Na++ions (ii) 40ndash46 in presence of K+ Zn++ Ag++ and Cd++ions and (iii) 89ndash92 in presence of Hg++ ion A similar
The Scientific World Journal 5
Table 1 Effect of surfactants oxidizing agent and metal ions onproteolytic activity
inhibition profile was also reported with characterizationof a cysteine protease isolated from wheat grain Triticumaestivum [25] The effect of surfactants and oxidizing agentat 1 concentration on the proteolytic activity at 37∘C is alsosummarized in the same table In presence of SDS and H
2O2
protease activity was inhibited up to 4849 and 3182respectively and activitywas unaffected by exposure to TritonX-100 and Tween-80 indicating that the purified proteasecould not be lipoprotein Our results are in good agreementwith the earlier observations reported in the purification ofmetalloprotease of Pseudomonas aeruginosa [26]
Enzymes are generally inactivated in the presence oforganic solvents The partially purified protease retainedits 85 to 100 activity at three concentrations of organicsolvents that is (i) 30 solvents of acetone benzenedimethylformamide ethyleneglycol and propanol (ii) 50solvents of acetone acetophenone benzene chlorobenzene
Table 2 Effect of various organic solvents on proteolytic activity
Srno Solvent
Residual activity ()Concentration of organic solvent (vv)
and dimethylformamide and (iii) 70 solvents of acetoneand benzene but more than 60 inactivation was observedin presence of remaining solvents (Table 2) Amongstthe organic solvents studied dichloromethane and butanolstrongly inhibited protease activity Acetone causes slightlyincreased protease activity The enzyme was stable in thepresence of acetone benzene and ethyleneglycol Our resultsare in good agreement with earlier observations [27]
The high activity and stability of the enzyme preparationin the pH range from 50 to 80 and its relative stabilitytowards surfactants is very useful for its application asdetergent additive To check the compatibility of the enzymepreparation with commercial solid detergents the enzymewas preincubated in the presence of various commerciallaundry detergents for 60min at 37∘C The data presentedin Table 3 illustrates that the enzyme is extremely stable inthe presence of detergents like Tide Ujala and Nirma atboth concentration of 7mgmL and 10mgmL The enzymeretained 90 to 98 of its activity in the presence of Wheeland Ariel and 80 to 90 in presence of Rin Surf ExcelFenna and Ghari at 7mgmL Enzyme preparation was lessstable in presence of Sasa and Impact where it lost up to22 to 33 of its activity The obtained results clearly indicatethat the performance of enzyme in detergents depends on anumber of factors including the detergentsrsquo compounds sincethe proteolytic stability varied with each laundry detergentThementioned compatibility of the enzyme preparation withcommercial solid detergents was in accordance with previousstudies [10 13 27]
6 The Scientific World Journal
Table 3 Stability of protease enzyme preparation in various localdetergents
Polyclonal antibodies against protein (cysteine protease)were raised in male albino Wister rats The presence ofantibodies in the anti rat serum was checked by Ouchter-lonyrsquos double immunodiffusionmethod Precipitin lines startappearing after 10ndash12 h of incubation at 28∘C and are dis-tinctly visible in about 36 to 42 h (Figure 2) Additionally thepolyclonal antibodies are specific to specific concentrationof glycoprotein This observation confirms that the proteinspresent in the enzyme fractions are distinct It reveals that theglycoprotein has unique antigenic determinants The poly-clonal antibodies raised for glycosylated cysteine proteasewould be of immense importance in detecting and as a ligandfor various future studies Our results are in good agreementwith earlier observations of antigenic property of cysteineprotease named Procerain of Calotropis procera latex [28]Milin a protease of Euphorbia milii latex [29] Cryptolepainof Cryptolepis buchanani latex [30] Indicain latex proteaseof Morus indica [31] and a novel cysteine protease namedProcerain B of Calotropis procera latex [32]
32 Ecofriendly Applications of Cysteine Protease Enzymaticdehairing process is gaining importance as an alternativechemical methodology in present day scenario This processis significant in reduction of toxicity in addition to improve-ment of leather quality [33] The experimental dehaired peltsof cow and rat hide showed complete removal of fine hairs(Figures 3 and 4) with increased brightness and it may bedue to elimination of sulfide in the process Similar results arenoticed by Subba Rao et al [13] with thermostable protease ofBacillus circulans Cysteine protease could remove hairs of ratand cow hides after 18 h incubation with hide at 30∘C easilycompared to each control with no observable damage on thecollagenTherefore the dehaired skin exhibits clean hair poreand clear grain structure
Histological sections of dehaired pelts stainedwith hema-toxylin and eosin revealed the removal of epidermis glandu-lar structures hair shafts bulb and follicles (Figures 4(c) and4(e)) Complete absence of the previous structural features
along with opening up of collagen fibre structure was seenwith samples (hide) treated for more than 12 h (Figure 4(e))On the other hand partial and moderate removal of hairis observed with 12 h incubation (Figure 4(c)) The datadepicted that there was no apparent damage to the collagenfibres in dehaired pelts (Figure 4(e)) The histological studiesfurther strengthened the easy removal as the hair follicleswere found to be empty (Figure 4(e)) The skin with noprotease treatment showed hair follicles in the dermis withintact hair (Figure 4(a)) and hair was also observed comingout of the epidermal layer This showed that the proteasewas effective in removing hair from hair follicles Comparingtreated skins with untreated controls we observed that onlyskin epidermis and adnexa (skin appendages) including hairbulbs were digested showing a histological autolytic-likeappearance (Figures 4(c) and 4(e)) as could be expectedfor proper skin depilation Protease enzyme has advantagesin dehairing process as this enzyme effectively unhaired therat and cow hides within 18 h compared to earlier reportswhere proteases from Bacillus cereus and Aspergillus tamariidehaired the goat skin in 21 and 24 h respectively [33 34]indicating its potential application in leather industry forecofriendly economizing the process
The results (Figure 5) of evaluation of enzyme for washingperformance pointed out that the blood stains on the clothpieces remained as they were even after 15min of rinsingin the case of controls and commercial detergents Bloodstain was completely removed from the cloths after rinsingthem with a combination of detergent and crude cysteineprotease (CCP) for a period of 15min whereas it wasremoved after 25min when rinsed with CCP individuallyThese results clearly indicate that the enzyme is fairly stableas an ingredient in the presence of detergents Our results ofwashing performance of CCP are in good accordance withthe earlier observations reported in washing performance ofprotease of Pseudomonas aeruginosa [35] and Streptomycesgulbdrgensis [14]
The Scientific World Journal 7
(a) (b)
Figure 3 Dehairing of cow hide (a) Cow hide control and (b) cow hide after 18 h incubation with CCP
ED
HS
HF
GS HB
(a) (b)
ED
HSDHF
HF DHB
(c)
(d)
DED
EHF
DHB
(e) (f)
Figure 4 Dehairing of rat hide Hematoxylin and eosin staining skin sections from (a) control without treatment (c) partially dehaired peltsof enzymatic process after 12 h and (e) dehaired pelts of enzymatic process after 18 h incubation with CCP Rat hide images from (b) controlwithout treatment (d) after 12 h incubation with CCP and (f) after 18 h incubation with CCP ED epidermis HS hair shaft HF hairfollicles GS glandular structure HB hair bulb DHB degraded hair bulb DED degraded epidermis and EHF empty hair folliclesDHF degraded hair follicles
In order to evaluate the performance of CCP with respectto its capability of removing stains of different blood samplesnamely human being ox buffalo and hen egg yolk stainswere used On incubating several pieces of stained cloth at30∘C for 25min results of these findings are interesting theuse of enzyme alone showed more effective removal of bloodand egg yolk stains (Figure 6) In fact protease facilitatesthe release of proteinaceous materials in a much easier waythan the commercially available detergent Furthermore thecombination of CCP with detergent resulted in completestain removal (Figure 6) A similar study reported on theusefulness of alkaline proteases from Bacillus brevis [36] and
Bacillus pumilus [37] These destaining profiles made clearthe idea about removal of blood stains with minimum useof commercial detergent within 25min Rapid blood stainremoval was noticed with supplementation of commerciallyavailable detergents (Table 4) Similar results of destainingof blood with combination of protease and detergent werenoticed by Subba Rao et al [13]
Treatment of X-ray films with protease resulted in thesliver bound with gelatin being stripped off into the reactionmixture and this results in clean appearance of plastic filmThe loss in weight after the treatment was around 5 (ww)based on initial weight of the film (10 g) The gelatin layer of
8 The Scientific World Journal
(i) (ii) (iii) (iv)
(a)
(i) (ii) (iii) (iv)
(b)
(i) (ii) (iii) (iv)
(c)
(i) (ii) (iii) (iv)
(d)
(i) (ii) (iii) (iv)
(e)
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
in each group Circular wounds of approximately 300 to350mm2 in diameter were inflicted on the shaved skin undermild ether anesthesia Groups II and III were treated withcrude cysteine protease (CCP) and 1 ww framycetin sul-phate IP (Soframycin) respectively Group I was untreatedand considered as controlThe progressive changes in woundarea were recorded in mm2 by tracing the wound boundariesaround it on a transparent paper on every day Woundcontractionwas expressed as percentage reduction of originalwound size [16]
2112 Coagulation Time of Whole Blood Twelve tubes werearranged in a water bath at 37∘C Into six of these tubes(test) 01mL of the crude cysteine protease (CCP)was addedand nothing was added to the remaining six tubes (control)05mL of blood was collected separately from mice by cleanvenipuncture and 05mL was added into each of the tubesthe tubes were observed for clot formation and the clottingtime was recorded using a stop watch The average of theclotting time of the six tubes with protease (test) and the sixtubes without protease (control) were taken as the clottingtime respectively [17]
2113 BleedingClotting Time Test The effect of the proteaseon bleeding from fresh experimentally induced wounds wasevaluated using the bleedingclotting time test in mice [18]After sterilizing the skin with 70 alcohol a puncture wasmade on the tail with a sterile sharp blade Immediately adrop of the CCP (1000 120583gmL) was placed on the cut portionand at the same time a stopwatch was switched on Sterilizedfilter paper was used to absorb blood coming out and timetaken for ceasing bleeding was recorded the average wastaken as bleeding time (test) The procedure was repeated onthe second group ofmice but here after puncturing the tail adrop of protease (CCP)was not applied serving it as a controlgroup of animal
2114 Antimicrobial Activity Antimicrobial activity of differ-ent concentrations of CCP (50 and 100 120583gmL) was studiedagainst Gram positive and Gram negative bacteria by usingdisc diffusion method [19] After overnight incubation at37∘C the zone of inhibitionwasmeasured and comparedwithreference antibiotic (Gentamicin) A control experiment wasset up by using an equal amount of phosphate buffer in placeof protease
212 Agricultural Application Nematicidal Activity TheBhendi (Okra) crops were grouped into eight groups with sixplants in each group
Group I Normal sterile soil plant (control or untreated-uninoculated)
Group II Nematode affected plant (untreated)
Groups III IV V and VI Nematode control by 25 50 75and 100mg of CCP per g soil respectively
Groups VII and VIII Nematode control by carbofuran 3G nematicide and Sanjeevani 1 WP (Trichoderma viride)respectively dose 20mg per g soil
The study was undertaken in 48 earthen pots (15 cmdiameter) Each was filled with 800 g of sterile soil alongwith BioOrganic Fertilizer (1 1) Experimental okra seed wasgrown in each and every pot under greenhouse conditionAfter 15 days of germination the inoculation was achievedby pouring the nematode water suspension containing 2000nematodes through 4 holes (5ndash8 cm depth) around theroot system of experimental crop which were immediatelycovered by sterile soil except Group I pots Exactly after 15days CCP was applied in four doses that is 25 50 75 and100mg per g soil (Groups III IV V and VI) Carbofuran3 G (encapsulated) nematicide (Group VII) and Sanjeevani1 WP (Trichoderma viride) (Group VIII) were also appliedseparately in 20mg per g soil by the same proceduredescribed earlier Then exactly after 20 days we counted thenumber of nematodes to determine the percent mortalitythat is PM (PM= (Number of dead nematodesTotal numberof nematodes) times 100) [20]
3 Results and Discussion
31 Characterization of Cysteine Protease Euphorbia nivuliaBuch-Ham belongs to the Euphorbiaceae family whosemembers are characterized by secretory tissues (laticifers)which frequently include proteolytic and milk clottingenzymes The young stem latex of E nivulia possessesproteolytic and milk clotting enzyme in more quantity ascomparedwith other investigated laticiferous plants of north-ern region of Maharashtra India [6] E nivulia latex containsa thermostable glycosylated cysteine protease presenting anoptimum activity at pH of 66 and temperature of 45∘C amolecular weight of 4342 kDa (analyzed by SDDS-PAGE)is activated by cysteine hydrochloride and inhibited bymercuric chloride [6] According to protease nomenclaturethis protein is designated as Nivulian-II due to previouslyNivulian-I (31486985Da) was already characterized from Enivulia latex [21]
The stability of proteins and enzymes is usually a factorthat limits their usefulness in many applicationsThe thermalstability of this cysteine protease was examined bymeasuringthe residual enzyme activity of aliquots of the enzyme byincubating at different temperatures This cysteine proteasewas stable up to 60∘C At least 80 residual proteolyticactivity of cysteine protease was retained after incubationat 60∘C as shown in Figure 1 After enzyme incubation at60 and 70∘C the enzyme activity was retained about 8743and 5436 percent activity respectively The enzyme wasalmost completely inactivated by heating for 15min at 80∘CSimilar thermal profile of cysteine protease was reported incysteine protease of Araujia hortorum fruits freesia cormsand endopeptidase of Bromelia hieronymi [22ndash24]
The effect of various metal ions at 5mmol litminus1 concen-tration on the proteolytic activity at 37∘C is summarized inTable 1 Enzyme activity was affected by all the examinedmetal cations except Ca++ Mg++ andMn++ Enzyme activitywas affected by (i) 25ndash35 in presence of Fe++ and Na++ions (ii) 40ndash46 in presence of K+ Zn++ Ag++ and Cd++ions and (iii) 89ndash92 in presence of Hg++ ion A similar
The Scientific World Journal 5
Table 1 Effect of surfactants oxidizing agent and metal ions onproteolytic activity
inhibition profile was also reported with characterizationof a cysteine protease isolated from wheat grain Triticumaestivum [25] The effect of surfactants and oxidizing agentat 1 concentration on the proteolytic activity at 37∘C is alsosummarized in the same table In presence of SDS and H
2O2
protease activity was inhibited up to 4849 and 3182respectively and activitywas unaffected by exposure to TritonX-100 and Tween-80 indicating that the purified proteasecould not be lipoprotein Our results are in good agreementwith the earlier observations reported in the purification ofmetalloprotease of Pseudomonas aeruginosa [26]
Enzymes are generally inactivated in the presence oforganic solvents The partially purified protease retainedits 85 to 100 activity at three concentrations of organicsolvents that is (i) 30 solvents of acetone benzenedimethylformamide ethyleneglycol and propanol (ii) 50solvents of acetone acetophenone benzene chlorobenzene
Table 2 Effect of various organic solvents on proteolytic activity
Srno Solvent
Residual activity ()Concentration of organic solvent (vv)
and dimethylformamide and (iii) 70 solvents of acetoneand benzene but more than 60 inactivation was observedin presence of remaining solvents (Table 2) Amongstthe organic solvents studied dichloromethane and butanolstrongly inhibited protease activity Acetone causes slightlyincreased protease activity The enzyme was stable in thepresence of acetone benzene and ethyleneglycol Our resultsare in good agreement with earlier observations [27]
The high activity and stability of the enzyme preparationin the pH range from 50 to 80 and its relative stabilitytowards surfactants is very useful for its application asdetergent additive To check the compatibility of the enzymepreparation with commercial solid detergents the enzymewas preincubated in the presence of various commerciallaundry detergents for 60min at 37∘C The data presentedin Table 3 illustrates that the enzyme is extremely stable inthe presence of detergents like Tide Ujala and Nirma atboth concentration of 7mgmL and 10mgmL The enzymeretained 90 to 98 of its activity in the presence of Wheeland Ariel and 80 to 90 in presence of Rin Surf ExcelFenna and Ghari at 7mgmL Enzyme preparation was lessstable in presence of Sasa and Impact where it lost up to22 to 33 of its activity The obtained results clearly indicatethat the performance of enzyme in detergents depends on anumber of factors including the detergentsrsquo compounds sincethe proteolytic stability varied with each laundry detergentThementioned compatibility of the enzyme preparation withcommercial solid detergents was in accordance with previousstudies [10 13 27]
6 The Scientific World Journal
Table 3 Stability of protease enzyme preparation in various localdetergents
Polyclonal antibodies against protein (cysteine protease)were raised in male albino Wister rats The presence ofantibodies in the anti rat serum was checked by Ouchter-lonyrsquos double immunodiffusionmethod Precipitin lines startappearing after 10ndash12 h of incubation at 28∘C and are dis-tinctly visible in about 36 to 42 h (Figure 2) Additionally thepolyclonal antibodies are specific to specific concentrationof glycoprotein This observation confirms that the proteinspresent in the enzyme fractions are distinct It reveals that theglycoprotein has unique antigenic determinants The poly-clonal antibodies raised for glycosylated cysteine proteasewould be of immense importance in detecting and as a ligandfor various future studies Our results are in good agreementwith earlier observations of antigenic property of cysteineprotease named Procerain of Calotropis procera latex [28]Milin a protease of Euphorbia milii latex [29] Cryptolepainof Cryptolepis buchanani latex [30] Indicain latex proteaseof Morus indica [31] and a novel cysteine protease namedProcerain B of Calotropis procera latex [32]
32 Ecofriendly Applications of Cysteine Protease Enzymaticdehairing process is gaining importance as an alternativechemical methodology in present day scenario This processis significant in reduction of toxicity in addition to improve-ment of leather quality [33] The experimental dehaired peltsof cow and rat hide showed complete removal of fine hairs(Figures 3 and 4) with increased brightness and it may bedue to elimination of sulfide in the process Similar results arenoticed by Subba Rao et al [13] with thermostable protease ofBacillus circulans Cysteine protease could remove hairs of ratand cow hides after 18 h incubation with hide at 30∘C easilycompared to each control with no observable damage on thecollagenTherefore the dehaired skin exhibits clean hair poreand clear grain structure
Histological sections of dehaired pelts stainedwith hema-toxylin and eosin revealed the removal of epidermis glandu-lar structures hair shafts bulb and follicles (Figures 4(c) and4(e)) Complete absence of the previous structural features
along with opening up of collagen fibre structure was seenwith samples (hide) treated for more than 12 h (Figure 4(e))On the other hand partial and moderate removal of hairis observed with 12 h incubation (Figure 4(c)) The datadepicted that there was no apparent damage to the collagenfibres in dehaired pelts (Figure 4(e)) The histological studiesfurther strengthened the easy removal as the hair follicleswere found to be empty (Figure 4(e)) The skin with noprotease treatment showed hair follicles in the dermis withintact hair (Figure 4(a)) and hair was also observed comingout of the epidermal layer This showed that the proteasewas effective in removing hair from hair follicles Comparingtreated skins with untreated controls we observed that onlyskin epidermis and adnexa (skin appendages) including hairbulbs were digested showing a histological autolytic-likeappearance (Figures 4(c) and 4(e)) as could be expectedfor proper skin depilation Protease enzyme has advantagesin dehairing process as this enzyme effectively unhaired therat and cow hides within 18 h compared to earlier reportswhere proteases from Bacillus cereus and Aspergillus tamariidehaired the goat skin in 21 and 24 h respectively [33 34]indicating its potential application in leather industry forecofriendly economizing the process
The results (Figure 5) of evaluation of enzyme for washingperformance pointed out that the blood stains on the clothpieces remained as they were even after 15min of rinsingin the case of controls and commercial detergents Bloodstain was completely removed from the cloths after rinsingthem with a combination of detergent and crude cysteineprotease (CCP) for a period of 15min whereas it wasremoved after 25min when rinsed with CCP individuallyThese results clearly indicate that the enzyme is fairly stableas an ingredient in the presence of detergents Our results ofwashing performance of CCP are in good accordance withthe earlier observations reported in washing performance ofprotease of Pseudomonas aeruginosa [35] and Streptomycesgulbdrgensis [14]
The Scientific World Journal 7
(a) (b)
Figure 3 Dehairing of cow hide (a) Cow hide control and (b) cow hide after 18 h incubation with CCP
ED
HS
HF
GS HB
(a) (b)
ED
HSDHF
HF DHB
(c)
(d)
DED
EHF
DHB
(e) (f)
Figure 4 Dehairing of rat hide Hematoxylin and eosin staining skin sections from (a) control without treatment (c) partially dehaired peltsof enzymatic process after 12 h and (e) dehaired pelts of enzymatic process after 18 h incubation with CCP Rat hide images from (b) controlwithout treatment (d) after 12 h incubation with CCP and (f) after 18 h incubation with CCP ED epidermis HS hair shaft HF hairfollicles GS glandular structure HB hair bulb DHB degraded hair bulb DED degraded epidermis and EHF empty hair folliclesDHF degraded hair follicles
In order to evaluate the performance of CCP with respectto its capability of removing stains of different blood samplesnamely human being ox buffalo and hen egg yolk stainswere used On incubating several pieces of stained cloth at30∘C for 25min results of these findings are interesting theuse of enzyme alone showed more effective removal of bloodand egg yolk stains (Figure 6) In fact protease facilitatesthe release of proteinaceous materials in a much easier waythan the commercially available detergent Furthermore thecombination of CCP with detergent resulted in completestain removal (Figure 6) A similar study reported on theusefulness of alkaline proteases from Bacillus brevis [36] and
Bacillus pumilus [37] These destaining profiles made clearthe idea about removal of blood stains with minimum useof commercial detergent within 25min Rapid blood stainremoval was noticed with supplementation of commerciallyavailable detergents (Table 4) Similar results of destainingof blood with combination of protease and detergent werenoticed by Subba Rao et al [13]
Treatment of X-ray films with protease resulted in thesliver bound with gelatin being stripped off into the reactionmixture and this results in clean appearance of plastic filmThe loss in weight after the treatment was around 5 (ww)based on initial weight of the film (10 g) The gelatin layer of
8 The Scientific World Journal
(i) (ii) (iii) (iv)
(a)
(i) (ii) (iii) (iv)
(b)
(i) (ii) (iii) (iv)
(c)
(i) (ii) (iii) (iv)
(d)
(i) (ii) (iii) (iv)
(e)
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
inhibition profile was also reported with characterizationof a cysteine protease isolated from wheat grain Triticumaestivum [25] The effect of surfactants and oxidizing agentat 1 concentration on the proteolytic activity at 37∘C is alsosummarized in the same table In presence of SDS and H
2O2
protease activity was inhibited up to 4849 and 3182respectively and activitywas unaffected by exposure to TritonX-100 and Tween-80 indicating that the purified proteasecould not be lipoprotein Our results are in good agreementwith the earlier observations reported in the purification ofmetalloprotease of Pseudomonas aeruginosa [26]
Enzymes are generally inactivated in the presence oforganic solvents The partially purified protease retainedits 85 to 100 activity at three concentrations of organicsolvents that is (i) 30 solvents of acetone benzenedimethylformamide ethyleneglycol and propanol (ii) 50solvents of acetone acetophenone benzene chlorobenzene
Table 2 Effect of various organic solvents on proteolytic activity
Srno Solvent
Residual activity ()Concentration of organic solvent (vv)
and dimethylformamide and (iii) 70 solvents of acetoneand benzene but more than 60 inactivation was observedin presence of remaining solvents (Table 2) Amongstthe organic solvents studied dichloromethane and butanolstrongly inhibited protease activity Acetone causes slightlyincreased protease activity The enzyme was stable in thepresence of acetone benzene and ethyleneglycol Our resultsare in good agreement with earlier observations [27]
The high activity and stability of the enzyme preparationin the pH range from 50 to 80 and its relative stabilitytowards surfactants is very useful for its application asdetergent additive To check the compatibility of the enzymepreparation with commercial solid detergents the enzymewas preincubated in the presence of various commerciallaundry detergents for 60min at 37∘C The data presentedin Table 3 illustrates that the enzyme is extremely stable inthe presence of detergents like Tide Ujala and Nirma atboth concentration of 7mgmL and 10mgmL The enzymeretained 90 to 98 of its activity in the presence of Wheeland Ariel and 80 to 90 in presence of Rin Surf ExcelFenna and Ghari at 7mgmL Enzyme preparation was lessstable in presence of Sasa and Impact where it lost up to22 to 33 of its activity The obtained results clearly indicatethat the performance of enzyme in detergents depends on anumber of factors including the detergentsrsquo compounds sincethe proteolytic stability varied with each laundry detergentThementioned compatibility of the enzyme preparation withcommercial solid detergents was in accordance with previousstudies [10 13 27]
6 The Scientific World Journal
Table 3 Stability of protease enzyme preparation in various localdetergents
Polyclonal antibodies against protein (cysteine protease)were raised in male albino Wister rats The presence ofantibodies in the anti rat serum was checked by Ouchter-lonyrsquos double immunodiffusionmethod Precipitin lines startappearing after 10ndash12 h of incubation at 28∘C and are dis-tinctly visible in about 36 to 42 h (Figure 2) Additionally thepolyclonal antibodies are specific to specific concentrationof glycoprotein This observation confirms that the proteinspresent in the enzyme fractions are distinct It reveals that theglycoprotein has unique antigenic determinants The poly-clonal antibodies raised for glycosylated cysteine proteasewould be of immense importance in detecting and as a ligandfor various future studies Our results are in good agreementwith earlier observations of antigenic property of cysteineprotease named Procerain of Calotropis procera latex [28]Milin a protease of Euphorbia milii latex [29] Cryptolepainof Cryptolepis buchanani latex [30] Indicain latex proteaseof Morus indica [31] and a novel cysteine protease namedProcerain B of Calotropis procera latex [32]
32 Ecofriendly Applications of Cysteine Protease Enzymaticdehairing process is gaining importance as an alternativechemical methodology in present day scenario This processis significant in reduction of toxicity in addition to improve-ment of leather quality [33] The experimental dehaired peltsof cow and rat hide showed complete removal of fine hairs(Figures 3 and 4) with increased brightness and it may bedue to elimination of sulfide in the process Similar results arenoticed by Subba Rao et al [13] with thermostable protease ofBacillus circulans Cysteine protease could remove hairs of ratand cow hides after 18 h incubation with hide at 30∘C easilycompared to each control with no observable damage on thecollagenTherefore the dehaired skin exhibits clean hair poreand clear grain structure
Histological sections of dehaired pelts stainedwith hema-toxylin and eosin revealed the removal of epidermis glandu-lar structures hair shafts bulb and follicles (Figures 4(c) and4(e)) Complete absence of the previous structural features
along with opening up of collagen fibre structure was seenwith samples (hide) treated for more than 12 h (Figure 4(e))On the other hand partial and moderate removal of hairis observed with 12 h incubation (Figure 4(c)) The datadepicted that there was no apparent damage to the collagenfibres in dehaired pelts (Figure 4(e)) The histological studiesfurther strengthened the easy removal as the hair follicleswere found to be empty (Figure 4(e)) The skin with noprotease treatment showed hair follicles in the dermis withintact hair (Figure 4(a)) and hair was also observed comingout of the epidermal layer This showed that the proteasewas effective in removing hair from hair follicles Comparingtreated skins with untreated controls we observed that onlyskin epidermis and adnexa (skin appendages) including hairbulbs were digested showing a histological autolytic-likeappearance (Figures 4(c) and 4(e)) as could be expectedfor proper skin depilation Protease enzyme has advantagesin dehairing process as this enzyme effectively unhaired therat and cow hides within 18 h compared to earlier reportswhere proteases from Bacillus cereus and Aspergillus tamariidehaired the goat skin in 21 and 24 h respectively [33 34]indicating its potential application in leather industry forecofriendly economizing the process
The results (Figure 5) of evaluation of enzyme for washingperformance pointed out that the blood stains on the clothpieces remained as they were even after 15min of rinsingin the case of controls and commercial detergents Bloodstain was completely removed from the cloths after rinsingthem with a combination of detergent and crude cysteineprotease (CCP) for a period of 15min whereas it wasremoved after 25min when rinsed with CCP individuallyThese results clearly indicate that the enzyme is fairly stableas an ingredient in the presence of detergents Our results ofwashing performance of CCP are in good accordance withthe earlier observations reported in washing performance ofprotease of Pseudomonas aeruginosa [35] and Streptomycesgulbdrgensis [14]
The Scientific World Journal 7
(a) (b)
Figure 3 Dehairing of cow hide (a) Cow hide control and (b) cow hide after 18 h incubation with CCP
ED
HS
HF
GS HB
(a) (b)
ED
HSDHF
HF DHB
(c)
(d)
DED
EHF
DHB
(e) (f)
Figure 4 Dehairing of rat hide Hematoxylin and eosin staining skin sections from (a) control without treatment (c) partially dehaired peltsof enzymatic process after 12 h and (e) dehaired pelts of enzymatic process after 18 h incubation with CCP Rat hide images from (b) controlwithout treatment (d) after 12 h incubation with CCP and (f) after 18 h incubation with CCP ED epidermis HS hair shaft HF hairfollicles GS glandular structure HB hair bulb DHB degraded hair bulb DED degraded epidermis and EHF empty hair folliclesDHF degraded hair follicles
In order to evaluate the performance of CCP with respectto its capability of removing stains of different blood samplesnamely human being ox buffalo and hen egg yolk stainswere used On incubating several pieces of stained cloth at30∘C for 25min results of these findings are interesting theuse of enzyme alone showed more effective removal of bloodand egg yolk stains (Figure 6) In fact protease facilitatesthe release of proteinaceous materials in a much easier waythan the commercially available detergent Furthermore thecombination of CCP with detergent resulted in completestain removal (Figure 6) A similar study reported on theusefulness of alkaline proteases from Bacillus brevis [36] and
Bacillus pumilus [37] These destaining profiles made clearthe idea about removal of blood stains with minimum useof commercial detergent within 25min Rapid blood stainremoval was noticed with supplementation of commerciallyavailable detergents (Table 4) Similar results of destainingof blood with combination of protease and detergent werenoticed by Subba Rao et al [13]
Treatment of X-ray films with protease resulted in thesliver bound with gelatin being stripped off into the reactionmixture and this results in clean appearance of plastic filmThe loss in weight after the treatment was around 5 (ww)based on initial weight of the film (10 g) The gelatin layer of
8 The Scientific World Journal
(i) (ii) (iii) (iv)
(a)
(i) (ii) (iii) (iv)
(b)
(i) (ii) (iii) (iv)
(c)
(i) (ii) (iii) (iv)
(d)
(i) (ii) (iii) (iv)
(e)
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
Polyclonal antibodies against protein (cysteine protease)were raised in male albino Wister rats The presence ofantibodies in the anti rat serum was checked by Ouchter-lonyrsquos double immunodiffusionmethod Precipitin lines startappearing after 10ndash12 h of incubation at 28∘C and are dis-tinctly visible in about 36 to 42 h (Figure 2) Additionally thepolyclonal antibodies are specific to specific concentrationof glycoprotein This observation confirms that the proteinspresent in the enzyme fractions are distinct It reveals that theglycoprotein has unique antigenic determinants The poly-clonal antibodies raised for glycosylated cysteine proteasewould be of immense importance in detecting and as a ligandfor various future studies Our results are in good agreementwith earlier observations of antigenic property of cysteineprotease named Procerain of Calotropis procera latex [28]Milin a protease of Euphorbia milii latex [29] Cryptolepainof Cryptolepis buchanani latex [30] Indicain latex proteaseof Morus indica [31] and a novel cysteine protease namedProcerain B of Calotropis procera latex [32]
32 Ecofriendly Applications of Cysteine Protease Enzymaticdehairing process is gaining importance as an alternativechemical methodology in present day scenario This processis significant in reduction of toxicity in addition to improve-ment of leather quality [33] The experimental dehaired peltsof cow and rat hide showed complete removal of fine hairs(Figures 3 and 4) with increased brightness and it may bedue to elimination of sulfide in the process Similar results arenoticed by Subba Rao et al [13] with thermostable protease ofBacillus circulans Cysteine protease could remove hairs of ratand cow hides after 18 h incubation with hide at 30∘C easilycompared to each control with no observable damage on thecollagenTherefore the dehaired skin exhibits clean hair poreand clear grain structure
Histological sections of dehaired pelts stainedwith hema-toxylin and eosin revealed the removal of epidermis glandu-lar structures hair shafts bulb and follicles (Figures 4(c) and4(e)) Complete absence of the previous structural features
along with opening up of collagen fibre structure was seenwith samples (hide) treated for more than 12 h (Figure 4(e))On the other hand partial and moderate removal of hairis observed with 12 h incubation (Figure 4(c)) The datadepicted that there was no apparent damage to the collagenfibres in dehaired pelts (Figure 4(e)) The histological studiesfurther strengthened the easy removal as the hair follicleswere found to be empty (Figure 4(e)) The skin with noprotease treatment showed hair follicles in the dermis withintact hair (Figure 4(a)) and hair was also observed comingout of the epidermal layer This showed that the proteasewas effective in removing hair from hair follicles Comparingtreated skins with untreated controls we observed that onlyskin epidermis and adnexa (skin appendages) including hairbulbs were digested showing a histological autolytic-likeappearance (Figures 4(c) and 4(e)) as could be expectedfor proper skin depilation Protease enzyme has advantagesin dehairing process as this enzyme effectively unhaired therat and cow hides within 18 h compared to earlier reportswhere proteases from Bacillus cereus and Aspergillus tamariidehaired the goat skin in 21 and 24 h respectively [33 34]indicating its potential application in leather industry forecofriendly economizing the process
The results (Figure 5) of evaluation of enzyme for washingperformance pointed out that the blood stains on the clothpieces remained as they were even after 15min of rinsingin the case of controls and commercial detergents Bloodstain was completely removed from the cloths after rinsingthem with a combination of detergent and crude cysteineprotease (CCP) for a period of 15min whereas it wasremoved after 25min when rinsed with CCP individuallyThese results clearly indicate that the enzyme is fairly stableas an ingredient in the presence of detergents Our results ofwashing performance of CCP are in good accordance withthe earlier observations reported in washing performance ofprotease of Pseudomonas aeruginosa [35] and Streptomycesgulbdrgensis [14]
The Scientific World Journal 7
(a) (b)
Figure 3 Dehairing of cow hide (a) Cow hide control and (b) cow hide after 18 h incubation with CCP
ED
HS
HF
GS HB
(a) (b)
ED
HSDHF
HF DHB
(c)
(d)
DED
EHF
DHB
(e) (f)
Figure 4 Dehairing of rat hide Hematoxylin and eosin staining skin sections from (a) control without treatment (c) partially dehaired peltsof enzymatic process after 12 h and (e) dehaired pelts of enzymatic process after 18 h incubation with CCP Rat hide images from (b) controlwithout treatment (d) after 12 h incubation with CCP and (f) after 18 h incubation with CCP ED epidermis HS hair shaft HF hairfollicles GS glandular structure HB hair bulb DHB degraded hair bulb DED degraded epidermis and EHF empty hair folliclesDHF degraded hair follicles
In order to evaluate the performance of CCP with respectto its capability of removing stains of different blood samplesnamely human being ox buffalo and hen egg yolk stainswere used On incubating several pieces of stained cloth at30∘C for 25min results of these findings are interesting theuse of enzyme alone showed more effective removal of bloodand egg yolk stains (Figure 6) In fact protease facilitatesthe release of proteinaceous materials in a much easier waythan the commercially available detergent Furthermore thecombination of CCP with detergent resulted in completestain removal (Figure 6) A similar study reported on theusefulness of alkaline proteases from Bacillus brevis [36] and
Bacillus pumilus [37] These destaining profiles made clearthe idea about removal of blood stains with minimum useof commercial detergent within 25min Rapid blood stainremoval was noticed with supplementation of commerciallyavailable detergents (Table 4) Similar results of destainingof blood with combination of protease and detergent werenoticed by Subba Rao et al [13]
Treatment of X-ray films with protease resulted in thesliver bound with gelatin being stripped off into the reactionmixture and this results in clean appearance of plastic filmThe loss in weight after the treatment was around 5 (ww)based on initial weight of the film (10 g) The gelatin layer of
8 The Scientific World Journal
(i) (ii) (iii) (iv)
(a)
(i) (ii) (iii) (iv)
(b)
(i) (ii) (iii) (iv)
(c)
(i) (ii) (iii) (iv)
(d)
(i) (ii) (iii) (iv)
(e)
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
Figure 3 Dehairing of cow hide (a) Cow hide control and (b) cow hide after 18 h incubation with CCP
ED
HS
HF
GS HB
(a) (b)
ED
HSDHF
HF DHB
(c)
(d)
DED
EHF
DHB
(e) (f)
Figure 4 Dehairing of rat hide Hematoxylin and eosin staining skin sections from (a) control without treatment (c) partially dehaired peltsof enzymatic process after 12 h and (e) dehaired pelts of enzymatic process after 18 h incubation with CCP Rat hide images from (b) controlwithout treatment (d) after 12 h incubation with CCP and (f) after 18 h incubation with CCP ED epidermis HS hair shaft HF hairfollicles GS glandular structure HB hair bulb DHB degraded hair bulb DED degraded epidermis and EHF empty hair folliclesDHF degraded hair follicles
In order to evaluate the performance of CCP with respectto its capability of removing stains of different blood samplesnamely human being ox buffalo and hen egg yolk stainswere used On incubating several pieces of stained cloth at30∘C for 25min results of these findings are interesting theuse of enzyme alone showed more effective removal of bloodand egg yolk stains (Figure 6) In fact protease facilitatesthe release of proteinaceous materials in a much easier waythan the commercially available detergent Furthermore thecombination of CCP with detergent resulted in completestain removal (Figure 6) A similar study reported on theusefulness of alkaline proteases from Bacillus brevis [36] and
Bacillus pumilus [37] These destaining profiles made clearthe idea about removal of blood stains with minimum useof commercial detergent within 25min Rapid blood stainremoval was noticed with supplementation of commerciallyavailable detergents (Table 4) Similar results of destainingof blood with combination of protease and detergent werenoticed by Subba Rao et al [13]
Treatment of X-ray films with protease resulted in thesliver bound with gelatin being stripped off into the reactionmixture and this results in clean appearance of plastic filmThe loss in weight after the treatment was around 5 (ww)based on initial weight of the film (10 g) The gelatin layer of
8 The Scientific World Journal
(i) (ii) (iii) (iv)
(a)
(i) (ii) (iii) (iv)
(b)
(i) (ii) (iii) (iv)
(c)
(i) (ii) (iii) (iv)
(d)
(i) (ii) (iii) (iv)
(e)
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
Figure 5 Evaluation of protease (CCP) for washing of goatrsquos blood stains from cloth (a) Control (b) 5min treated (c) 10min treated(d) 15min treated (e) 25min treated (i) Buffer treated (ii) Detergent treated (iii) CCP treated and (iv) CCP + Detergent treated group
Cotton Buffer DetergentCCP CCP + detergent
Figure 6 Removing blood and egg yolk stains from cloth by the application of protease (CCP) and detergent after 25min First columnUntreated blood stained cloth (control) second column blood stained cloth washed (BSCW) by buffer third column BSCW by CCP fourthcolumn BSCW by detergent fifth column BSCW by detergent with CCP First second third and fourth row cloth stained by blood sampleof human being ox buffalo and hen respectively Fifth row cloth stained by egg yolk
The Scientific World Journal 9
(a) (b)
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
Figure 7 Decomposition of gelatin layer of X-ray film by protease (a) Control (b) experimental X-ray film after 3 h exposure to protease
Table 4 Destaining profile of different blood stains after 25min oftreatment
Sr no Treatment Destaining efficacy ()lowast
A B C D E01 Control 1189 869 1591 1566 87402 Protease 062 080 074 097 07103 Detergent 118 151 109 116 07904 Prot + Det 024 012 026 010 024lowastThe intensity if blood stain at 0min treatment was taken as 100 for allblood samplesA human B ox C buffalo D goat and E hen blood stained spot
the X-ray film was completely removed after 3 h of enzymaticexposure as compared with that of the control (Figure 7)Earlier studies reported similar observations [26] in bacterialspecies
33 Biological Activities of Cysteine Protease The tropicalapplication CCP significantly reduces the bleeding and clot-ting time in mice up to 8 and 14 seconds respectively ascompared with control values which were found to be 57and 60 seconds Also the cysteine protease significantlydecreased the coagulation time of whole blood in mice(119875 lt 0001) A significantly improved wound healing activityhas been observed in mice treated by cysteine protease ascompared to that of reference standard (Soframycin) andcontrol groups of animal (Table 5) The study reveals thatin all three groups of animal wound area was reducedprogressively However on 16th postwounding day Group Ianimals have 9202 wound contraction (which may be dueto self-immunity of the animals) whereas in Groups II andIII animals exhibit 9343 and 9329 effect by Soframycinand CCP respectively An enzyme treated group of animalsshowed significant reduction in wound contraction area(119875 lt 0001) A similar type of wound healing activity wasreported in few Euphorbiaceae members that is Jatrophacurcas [38] and Euphorbia neriifolia [39] and a memberof Asclepiadaceae family that is Calotropis procera [40]Our results agreed with the findings of earlier researchers
Present result fully justifies the folkloric use of E nivuliafor healing wound [41 42] Evaluation of the potentials ofE nivulia in wound management showed that the cysteineprotease of plant latex has haemostatic and wound healingpropertiesThe protease arrested bleeding from fresh woundsby reducing bleedingclotting and whole blood coagulationtimewhich are important indices of haemostatic activity Ourresults are in good agreement with the earlier observationsof haemostatic activity of stem latex of another memberof Euphorbiaceae family that is Jatropha gossypifolia [43]The reduction in coagulation time of whole blood by theprotease (CCP) indicates that it may also interfere with theblood coagulation pathways Thus this shrub is a promisinghaemostatic agent and wound healing promoter
The results obtained from the disc diffusion assay(Table 6) showed that there was an increasing effect onbacterial growth inhibition with increasing concentration ofprotease (CCP) It showed comparable inhibitory activityon almost all the bacteria tested Protease had a strongantibacterial activity against Escherichia coli and Staphylo-coccus aureus moderate against Pseudomonas aeruginosaand lowest activity against Klebsiella pneumoniae Proteusvulgaris and Bacillus subtilis The result concludes that theprotease has exhibited a potent antibacterial activity
The final population of Meloidogyne incognita inuntreated pots and pots treated with CCP (10mgg)Trichoderma viride (Sanjeevani) and carbofuran (standardnematicide) was 3800 227 85 and 160 per pot respectivelyas against the initial population of 2000 (Table 7) Thepercent mortality of nematode was raised with increasingconcentration of protease (CCP) The moderate dose ofprotease (5mg) was equivalent to the 1mg of standardnematicide
The results in Table 8 summarize the morphologicalobservation of experimental plants (dose level of CCPis 10mgg of rhizosphere soil) and standard nematicides(2mgg of soil) in the form of seed index whole plant heightspad value and dry biomass of okra plants Seed index is theratio of weight of seed to weight of fresh fruit Highest valueof seed index was found in the CCP exposing crop plant
10 The Scientific World Journal
Table 5 Effect of protease on excision wound parameter in mice
PWD Wound areamdashpercentage of wound contraction is in parenthesesGroup I control Group II soframycin Group III CCP
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
PE 2368 plusmn 103 1987 plusmn 117 2034 plusmn 137PWD postwounding days values are expressed as mean plusmn SD 119899 = 6 animals in each group PE period of epithelialization (days) lowast119875 lt 0001 as comparedto control
AP aerial part of plant UGP underground part of plant lowastbiomass without fruit +10mg CCP per g of soil ++concentration of carbofuran and T viride was2mg per g of soil and data are represented in average values plusmn SD of 119899 = 6 experiment
that is 00015 followed by Sanjeevani exposing crop plantwhile in nematode infected plant and control it was recordedas 0005 and 0008 respectively The weight of root (g) forthe corresponding treatment was 0863 1032 and 0872(carbofuran exposing plant) respectively as against 0640for untreated-uninoculated control Spad value indicates theintensity of chlorophyll contents The lower the spad valuethe less the chlorophyll intensity or vice versa Chlorophyllcontent of leaves of experimental plantwasmeasured digitallyin terms of spad value with the help of spad chlorophyll meter
or as simply called Spadometer which is a small electronicpunching machine with chlorophyll detecting sensor [44]Spad values for the treatment of carbofuran Sanjeevaniand protease were 3304 3409 and 3012 respectively ascompared to 3785 for the control while whole plant heightwas 5195 5520 and 5715 respectively as compared to 5802for the control
The results showed that the reproduction ofMeloidogyneincognita in plants grown in soil treated with protease of plantlatex was significantly suppressed Protease treated plants
The Scientific World Journal 11
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
showed more significant increase in the whole plant heightthan that of untreated-uninoculated soil plant Howevernematode population is declined only in pots treated withprotease and standard nematicide whereas in untreated-inoculated pots it has almost doubled It is therefore rea-sonable to believe that plants grown with protease developcertain degree of resistance against nematode attack It maybe due to the absorption of substances liberated duringdecomposition of protein by soil microbial flora [45]
In conclusion characterization and environmentalfriendly potential application of antigenic glycosylated stablecysteine protease of Euphorbia nivulia latex were studied forthe first time The enzyme revealed excellent stability andcompatibility towards temperature metal ions detergentsoxidizing agents surfactants and organic solvents Studiesindicated its utility for blood stain removal and detergentand dehairing properties The physical properties of theexperimental ratrsquos pelt revealed effective dehairing of finehairs completely within 18 h without sodium sulfateindicating its ecofriendly nature in dehairing Hematoxylinand eosin staining revealed the removal of epidermisglandular structures hair shafts and follicles with completeopening of collagen fiber structure Medicinally it showsnoticeable wound healing and haemostatic activityAgriculturally it finds application in biocontrol processagainst the infectious management of root knot nematode
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
The authors especially thank Dr Gajanan Sapkal ScientistB National Institute of Virology ICMR Pune India for hiskind support during immunological study and for the gift ofFreundrsquos complete and incomplete adjuvants The scientificassistance of Dr M Z Chopda Dr Pankaj Salunkhe Dr NM Pawar and animal house staff is gratefully acknowledged
References
[1] S R Morcelle S A Trejo F Canals F X Aviles and N SPriolo ldquoFunastrain c II a cysteine endopeptidase purified fromthe latex of Funastrum clausumrdquo Protein Journal vol 23 no 3pp 205ndash215 2004
[2] N D Rawlings D P Tolle and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 32 pp D160ndashD164 2004
[3] C Sequeiros M J Torres S A Trejo J L Esteves C LNatalucci and L M I Lopez ldquoPhilibertain g I the most basiccysteine endopeptidase purified from the latex of Philibertiagilliesii hook et Arn (Apocynaceae)rdquo Protein Journal vol 24no 7-8 pp 445ndash453 2005
[4] S K Basak P Bakshi S Basu and S Basak ldquoKeratouveitiscaused by Euphorbia plant saprdquo Indian Journal of Ophthalmol-ogy vol 57 no 4 pp 311ndash313 2009
[5] R T Mahajan and S B Badgujar ldquoBioprospecting of Euphorbianivulia BuchHamrdquo International Journal of Phytopharmacol-ogy vol 2 no 2 pp 37ndash42 2011
[6] S B Badgujar and R T Mahajan ldquoComparison of cysteineproteases of four laticiferous plants and characterization ofEuphorbia nivulia BuchHam latex glycosylated cysteine pep-tidaserdquo Indian Journal of Natural Products and Resources vol 3no 2 pp 152ndash160 2012
[7] S B Badgujar and R T Mahajan ldquoCharacterization of milkclotting cysteine protease of Euphorbia nivulia BuchHamlatexrdquo Green Farming vol 1 no 6 pp 645ndash648 2010
[8] S B Badgujar Proteolytic enzymes of some latex bearing plantsbelonging to Khandesh region of Maharashtra [PhD thesis]North Maharashtra University Jalgaon India 2011
[9] E Quiroga N Priolo J Marchese and S Barberis ldquoStability ofaraujiain a novel plant protease in different organic systemsrdquoActa Farmaceutica Bonaerense vol 24 no 2 pp 204ndash208 2005
[10] A Haddar A Bougatef R Agrebi A Sellami-Kamoun andM Nasri ldquoA novel surfactant-stable alkaline serine-proteasefrom a newly isolated Bacillus mojavensis A21 Purification andcharacterizationrdquo Process Biochemistry vol 44 no 1 pp 29ndash352009
[11] O Ouchterlony and L A Nilsson ldquoImmunodiffusion andimmunoelectrophoresisrdquo in Handbook of Experimental Immu-nology DMWeir Ed vol 1 of Immunohistochemistry pp 321ndash3250 Blackwell Scientific Publications Oxford UK 1986
[12] R K Jaswal G S Kocher H S Banga and R S Brar ldquoAppli-cation of Bacillus alkaline protease for dehairing of buffaloskin and its histological studiesrdquo Indian Journal of VeterinaryPathology vol 32 no 1 pp 26ndash29 2008
[13] C Subba Rao T Sathish P Ravichandra and R S PrakashamldquoCharacterization of thermo- and detergent stable serine pro-tease from isolated Bacillus circulans and evaluation of eco-friendly applicationsrdquo Process Biochemistry vol 44 no 3 pp262ndash268 2009
[14] N Vishalakshi K Lingappa S Amena M Prabhakar and ADayanand ldquoProduction of alkaline protease from Streptomycesgulbargensis and its application in removal of blood stainsrdquoIndian Journal of Biotechnology vol 8 no 3 pp 280ndash285 2009
[15] S Shankar S V More and R S Laxman ldquoRecovery of silverfrom waste X-ray film by alkaline protease from Conidioboluscoronatusrdquo Kathmandu University Journal of Science Engineer-ing and Technology vol 6 no 1 pp 60ndash69 2010
[16] F Sadaf S Rubeena A Muhammad I A Syed and Navaid-ul-Zafar ldquoHealing potential of cream containing extract of Sphaer-anthus indicus on dermal wounds in Guinea pigsrdquo Journal ofEthnopharmacology vol 107 no 2 pp 161ndash163 2006
[17] J V Dacie and S M Lewis Practical Haematology ChurchillLivingstone London UK 4th edition 1970
[18] C O Okoli P A Akah and A S Okoli ldquoPotentials of leaves ofAspilia africana (Compositae) in wound care an experimentalevaluationrdquoBMCComplementary andAlternativeMedicine vol7 article 24 2007
[19] J Annapurna I P Chowdary G Lalitha S V Ramakrishnaand D S Iyengar ldquoAntimicrobial activity of Euphorbia nivulialeaf extractrdquo Pharmaceutical Biology vol 42 no 2 pp 91ndash932004
[20] M A Rizvi and M Shameel ldquoIn vitro nematicidal activitiesof seaweed extracts from Karachi coastrdquo Pakistan Journal ofBotany vol 38 no 4 pp 1245ndash1248 2006
12 The Scientific World Journal
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
[21] S B Badgujar and R T Mahajan ldquoCharacterization of 31-kDa Tubulin alpha-1 chain like protein of Euphorbia nivuliaBuchHam latexrdquo Biochemistry an Indian Journal vol 5 no 4pp 258ndash262 2011
[22] N Priolo S M del Valle M Cecilia Arribere L Lopez andN Caffini ldquoIsolation and characterization of a cysteine proteasefrom the latex of Araujia hortorum fruitsrdquo Protein Journal vol19 no 1 pp 39ndash49 2000
[23] M A Bruno M F Pardo N O Caffini and L M I LopezldquoPurification of a new endopeptidase isolated from fruits ofBromelia hieronymi Mez (Bromeliaceae)rdquo Acta FarmaceuticaBonaerense vol 21 no 1 pp 51ndash56 2002
[24] T Uchikoba S Fukumoto M Onjo M Okubo K Arima andH Yonezawa ldquoThe development of cysteine proteases in freesiacorms during responses to chillingrdquo Journal ofThermal Biologyvol 28 no 8 pp 555ndash562 2003
[25] A S Fahmy A A Ali and S A Mohamed ldquoCharacterizationof a cysteine protease from wheat Triticum aestivum (cv Giza164)rdquo Bioresource Technology vol 91 no 3 pp 297ndash304 2004
[26] U K Patil Studies on Novel Thermostable and Solvent-TolerantAlkaline Protease from Bacteria [PhD thesis] NorthMaharash-tra University Jalgaon India 2010
[27] L V A Reddy Y-J Wee and H-W Ryu ldquoPurification andcharacterization of an organic solvent and detergent-tolerantnovel protease produced by Bacillus sp RKY3rdquo Journal ofChemical Technology and Biotechnology vol 83 no 11 pp 1526ndash1533 2008
[28] V Kumar Dubey and M V Jagannadham ldquoProcerain a stablecysteine protease from the latex of Calotropis procerardquo Phyto-chemistry vol 62 no 7 pp 1057ndash1071 2003
[29] S C Yadav M Pande and M V Jagannadham ldquoHighlystable glycosylated serine protease from the medicinal plantEuphorbia miliirdquo Phytochemistry vol 67 no 14 pp 1414ndash14262006
[30] M Pande V K Dubey S C Yadav and M V Jagannadham ldquoAnovel serine protease cryptolepain from Cryptolepis buchananipurification and biochemical characterizationrdquo Journal of Agri-cultural and Food Chemistry vol 54 no 26 pp 10141ndash101502006
[31] V K Singh A K Patel A J Moir and M V JagannadhamldquoIndicain a dimeric serine protease fromMorus indica cv K2rdquoPhytochemistry vol 69 no 11 pp 2110ndash2119 2008
[32] K A Singh R Kumar G R K Rao and M V JagannadhamldquoCrinumin a chymotrypsin-like but glycosylated serine pro-tease fromCrinum asiaticum purification and physicochemicalcharacterisationrdquo Food Chemistry vol 119 no 4 pp 1352ndash13582010
[33] S Sivasubramanian B Murali Manohar A Rajaram and RPuvanakrishnan ldquoEcofriendly lime and sulfide free enzymaticdehairing of skins and hides using a bacterial alkaline proteaserdquoChemosphere vol 70 no 6 pp 1015ndash1024 2008
[34] A Dayanandan J Kanagaraj L Sounderraj R GovindarajuandG Suseela Rajkumar ldquoApplication of an alkaline protease inleather processing an ecofriendly approachrdquo Journal of CleanerProduction vol 11 no 5 pp 533ndash536 2003
[35] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005
[36] U C Banerjee R K SaniW Azmi and R Soni ldquoThermostablealkaline protease from Bacillus brevis and its characterization as
a laundry detergent additiverdquo Process Biochemistry vol 35 no1-2 pp 213ndash219 1999
[37] B Jaouadi S Ellouz-Chaabouni M Rhimi and S BejarldquoBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS withhigh catalytic efficiencyrdquo Biochimie vol 90 no 9 pp 1291ndash13052008
[38] L K Nath and S K Dutta ldquoWound healing response of theproteolytic enzyme curcainrdquo Indian Journal of Pharmacologyvol 24 pp 114ndash115 1992
[39] A M Rasik A Shukla G K Patnaik B N Dhawan D KKulshrestha and S Srivastava ldquoWound healing activity of latexof Euphorbia neriifolia linnrdquo Indian Journal of Pharmacologyvol 28 no 2 pp 107ndash109 1996
[40] A M Rasik R Raghubir A Gupta et al ldquoHealing potential ofCalotropis procera on dermal wounds in Guinea pigsrdquo Journalof Ethnopharmacology vol 68 no 1ndash3 pp 261ndash266 1999
[41] K R Kirtikar and B D Basu Indian Medicinal Plants vol 1ndash4International Book Distributors Dehradun India 1995
[42] R T Mahajan and S B Badgujar ldquoEthnomedicinal values oflaticiferous plants used by tribal people of North MaharashtraIndiardquo Research Link vol 557 no 8 pp 20ndash23 2008
[43] T Oduela G B Popoola O G Avwioro T A Oduola A AAdemosun and M O Lawal ldquoUse of Jatropha gossypifolia stemlatex as a haemostatic agent how safe is itrdquo Journal ofMedicinalPlants Research vol 1 no 1 pp 14ndash17 2007
[44] K Lal R L Meena S K Gupta D S Bundela R K Yadav andG Singh Conjunctive of Canal and Groundwater Central SoilSalinity Research Institute Karnal India 2008
[45] S B Badgujar and R T Mahajan ldquoEvaluation of nematicidalproperties of some laticiferous plantsrdquo Green Farming vol 2no 10 pp 680ndash684 2009
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