31 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014 Chandhini Rajendran, Femina Carolin Christopher, Nithyakalyani Ramu, Vivek Pazhamalai, Nithyananthi Mohankumar and Sumathi Ethiraj*, Studies on Phytochemical Screening, Antioxidant Activity and Extraction Active Compound (2-Hydroxy 4-Methoxy Benzaldehyde) From Rhizome Extract of Decalepis Hamiltonii Wight and Arn, Int.J.Curr.Biotechnol., 2014, 2(4):31-39. Studies on Phytochemical Screening, Antioxidant Activity and Extraction Active Compound (2-Hydroxy 4-Methoxy Benzaldehyde) From Rhizome Extract of Decalepis Hamiltonii Wight and Arn Chandhini Rajendran 2 , Femina Carolin Christopher 2 , Nithyakalyani Ramu 2 , Vivek Pazhamalai 2 , Nithyananthi Mohankumar 2 and Sumathi Ethiraj 1 * 1 Poonga Biotech Research Centre, Plant Biotechnology Division, Choolaimedu, Chennai - 600 094. Tamil Nadu, India. 2 Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala Engineering College Avadi, Chennai- 600 062. ARTICLE INFO ABSTRACT Article History: Received 31 March 2014 Received in revised form 15 April 2014 Accepted 20 April 2014 Available online 30 April 2014 Key words: Decalepis hamiltonii, Antioxidant activity, Phytochemical screening, HPLC analysis, Antibacterial activity. The present study was performed to investigate the phytochemical screening, total fla- vonoid, antioxidant activity, HPLC analysis of 2-hydroxy 4-methoxy benzaldehyde (2H4MB) and antibacterial activity from the rhizome of Decalepis hamiltonii. The phytochemical analysis revealed the presence of active ingredients such as steroids, saponins, phenols, flavonoids, terpenoids, alkaloids and tannins in the rhizome extract of D. hamiltonii followed by others. Total flavonoid content was quantitatively estimated which recorded maximum in Kanchipuram accession (5.25 mg Quercetin Equivalents (QE) /g). The rhizome extracts were evaluated for antioxidant activities by DPPH (1, 1– Diphenyl -2- picryl - hydrazyl) radical scavenging assay. Among the three accessions with different solvents extractions, maximum antioxidant activity was found in the ethanolic rhizome extract (90.9%) of D. hamiltonii followed by others. HPLC analysis of these extracts showed that the main components of the active principles namely 2H4MB were present in the rhizome extract of D. hamiltonii. Different concentrations of ethanolic rhizome extract were tested using the agar disc diffusion technique for the activity against Bacillus cereus, Pseudomonas aeruginosa, and Bacillus subtilis. It was found to be inactive against Staphylococcus aureus and Escherichia coli. Introduction Medicinal plants have been used in traditional treatments for numerous human diseases for thousands of years and they continue to be an important therapeutic aid for alleviating the ailments of human kind (Momin and Kadam, 2011). The therapeutic benefits are generally traced to specific plant compounds; but are specifically due to the active constituents of the plants (Mary et al., 2012). Phytochemical screening of various plants has been reported by many workers (Mojab et al., 2003; Parekh and Chanda, 2008). These studies have revealed the presence of numerous chemicals including alkaloids, flavonoids, steroids, phenols, glycosides and saponins. The phenolic compounds are one of the largest and most ubiquitous groups of plant metabolites (Hagerman et al., 2008). A number of studies have focused on the biological activities of phenolic compounds which are antioxidants and free radical scavengers (Evans et al., 1995; Cespedes et al., 2008; Reddy et al., 2008). The flavonoids are a category of natural substances belonging to the family of polyphenols. The flavonoids are also widely distributed in plants which have been reported to exert multiple biological benefits, including antioxidant, free radical scavenging abilities, anti- inflammatory and anti-carcinogenic (Miller, 1996). The crude extracts of herbs, spices and other plant materials, rich in phenolics and flavonoids are of increasing interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food (Chu et al., 2000). Free radicals (superoxide, hydroxyl radicals and nitric oxide) and other reactive species (hydrogen peroxide, hypochloric acid and peroxynitrite) produced during aerobic metabolism in the body, can cause oxidative damage of amino acids, lipids, proteins and DNA (Gutteridge, 1995; Halliwell, 1995). It has been established that oxidative stress is among the major causative factors in the induction of many chronic and degenerative diseases including atherosclerosis, ischemic heart disease, ageing, diabetes mellitus, cancer, immunosuppression, neurodegenerative diseases and others (Gulcin et al., 2002 ; Devasagayam et al., 2004). The screening of plant products for antibacterial activity has shown that the higher plants represent a potential source of novel antibiotic prototypes (Afolayan, 2003). There has been an increasing incidence of multiple resistances in human pathogenic microorganism in recent years (Okunji et al., 1999). Decalepis hamiltonii Wight & Arn belonging to the family Asclepiadaceae is commonly known as Magali Kizhangu in Tamil. It is a monogeneric climbing shrub native of the Deccan Peninsula and forest areas of *Corresponding author. Email address: [email protected]International Journal of Current Biotechnology Journal Homepage : http://ijcb.mainspringer.com ISSN: 2321 - 8371
Chandhini Rajendran, Femina Carolin Christopher, Nithyakalyani Ramu, Vivek Pazhamalai, Nithyananthi Mohankumar and Sumathi Ethiraj*, Studieson Phytochemical Screening, Antioxidant Activity and Extraction Active Compound (2-Hydroxy 4-Methoxy Benzaldehyde) From Rhizome Extract of DecalepisHamiltonii Wight and Arn, Int.J.Curr.Biotechnol., 2014, 2(4):31-39.
Studies on Phytochemical Screening, Antioxidant Activity and Extraction Active Compound(2-Hydroxy 4-Methoxy Benzaldehyde) From Rhizome Extract of Decalepis Hamiltonii Wightand Arn
1Poonga Biotech Research Centre, Plant Biotechnology Division, Choolaimedu, Chennai - 600 094. Tamil Nadu,India.2Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala Engineering College Avadi, Chennai- 600 062.
A R T I C L E I N F O A B S T R A C T
Article History:Received 31 March 2014Received in revised form 15 April 2014Accepted 20 April 2014Available online 30 April 2014
The present study was performed to investigate the phytochemical screening, total fla-vonoid, antioxidant activity, HPLC analysis of 2-hydroxy 4-methoxy benzaldehyde (2H4MB)and antibacterial activity from the rhizome of Decalepis hamiltonii. The phytochemical analysisrevealed the presence of active ingredients such as steroids, saponins, phenols, flavonoids,terpenoids, alkaloids and tannins in the rhizome extract of D. hamiltonii followed by others.Total flavonoid content was quantitatively estimated which recorded maximum in Kanchipuramaccession (5.25 mg Quercetin Equivalents (QE) /g). The rhizome extracts were evaluated forantioxidant activities by DPPH (1, 1– Diphenyl -2- picryl - hydrazyl) radical scavengingassay. Among the three accessions with different solvents extractions, maximum antioxidantactivity was found in the ethanolic rhizome extract (90.9%) of D. hamiltonii followed byothers. HPLC analysis of these extracts showed that the main components of the activeprinciples namely 2H4MB were present in the rhizome extract of D. hamiltonii. Differentconcentrations of ethanolic rhizome extract were tested using the agar disc diffusion techniquefor the activity against Bacillus cereus, Pseudomonas aeruginosa, and Bacillus subtilis. Itwas found to be inactive against Staphylococcus aureus and Escherichia coli.
IntroductionMedicinal plants have been used in traditional treatmentsfor numerous human diseases for thousands of yearsand they continue to be an important therapeutic aid foralleviating the ailments of human kind (Momin andKadam, 2011). The therapeutic benefits are generallytraced to specific plant compounds; but are specificallydue to the active constituents of the plants (Mary et al.,2012). Phytochemical screening of various plants hasbeen reported by many workers (Mojab et al., 2003;Parekh and Chanda, 2008). These studies have revealedthe presence of numerous chemicals including alkaloids,flavonoids, steroids, phenols, glycosides and saponins.The phenolic compounds are one of the largest and mostubiquitous groups of plant metabolites (Hagerman et al.,2008). A number of studies have focused on the biologicalactivities of phenolic compounds which are antioxidantsand free radical scavengers (Evans et al., 1995; Cespedeset al., 2008; Reddy et al., 2008).
The flavonoids are a category of natural substancesbelonging to the family of polyphenols. The flavonoidsare also widely distributed in plants which have beenreported to exert multiple biological benefits, includingantioxidant, free radical scavenging abilities, anti-
inflammatory and anti-carcinogenic (Miller, 1996). Thecrude extracts of herbs, spices and other plant materials,rich in phenolics and flavonoids are of increasing interestin the food industry because they retard oxidativedegradation of lipids and thereby improve the qualityand nutritional value of food (Chu et al., 2000). Freeradicals (superoxide, hydroxyl radicals and nitric oxide)and other reactive species (hydrogen peroxide,hypochloric acid and peroxynitrite) produced duringaerobic metabolism in the body, can cause oxidativedamage of amino acids, lipids, proteins and DNA(Gutteridge, 1995; Halliwell, 1995). It has been establishedthat oxidative stress is among the major causative factorsin the induction of many chronic and degenerativediseases including atherosclerosis, ischemic heartdisease, ageing, diabetes mellitus, cancer,immunosuppression, neurodegenerative diseases andothers (Gulcin et al., 2002 ; Devasagayam et al., 2004).The screening of plant products for antibacterial activityhas shown that the higher plants represent a potentialsource of novel antibiotic prototypes (Afolayan, 2003).There has been an increasing incidence of multipleresistances in human pathogenic microorganism in recentyears (Okunji et al., 1999).
Decalepis hamiltonii Wight & Arn belonging to thefamily Asclepiadaceae is commonly known as MagaliKizhangu in Tamil. It is a monogeneric climbing shrubnative of the Deccan Peninsula and forest areas of
Western Ghats of India and also found in the dry andmoist deciduous forests of Karnataka, Andhra Pradeshand Tamil Nadu. D. hamiltonii rhizome has been used asan appetizer, blood purifier and for the treatment ofvarious physiological disorders (Nagarajan et al., 2007).Hence, the present study was performed to investigatethe phytochemical screening, total flavonoid content,antioxidant activity, HPLC analysis of 2-hydroxy 4-methoxy benzaldehyde (2H4MB) and antibacterialactivity from the rhizome of Decalepis hamiltonii.
Material and MethodsCollection of plant materialThe healthy rhizome of Decalepis hamiltonii werecollected from three different regions of Tamil Nadunamely Kanchipuram, Salem and Trichy district. Thecollected rhizome were brought to the laboratory andmaintained at Poonga Biotech Research Centre, Plantbiotechnology division, Chennai- 600094, Tamil Nadu,India.
Preparation of the plant extractPreparation of the extracts was done according to acombination of the methods used by Pizzale et al. (2002)and Lu and Foo, (2001). About 15g of dried rhizome finepowder of Decalepis hamiltonii plant materials wereextracted with 150 ml acetone, ethanol (75%), chloroform,petroleum ether and aqueous extract for 1 min using anUltra Turax mixer (13,000 rpm) and soaked overnight atroom temperature. The sample was then filtered throughWhatman No.1 paper in a Buchner funnel. The filteredsolution was evaporated under vacuum in a rota-evatorat 40°C to a constant weight and then dissolved inrespective solvents. The concentrated extracts werestored in airtight container in refrigerator below 10ºC.
Phytochemical Screening from rhizome extracts ofDecalepis hamiltonii:The phytochemical screening of rhizome extracts wereassessed by standard method as described by Brinda etal., (1981); Siddiqui and Ali (1997) and Savithramma etal., (2011). Phytochemical screening was carried out onthe rhizome extracts using different solvents to identifythe major natural chemical groups such as tannins,saponins, flavonoids, phenols, terpenoids, alkaloids,glycosides, cardiac glycosides, coumarins and steroids.General reactions in these analyses revealed the presenceor absence of these compounds in the rhizome extractstested.
Estimation of Total Flavonoid Content from rhizomeextracts Decalepis hamiltoniiTotal flavonoids content in the ethanolic rhizome extractswas determined by the aluminium chloride colorimetricmethod (Mervat et al., 2009). 0.5ml of rhizome extracts ofDecalepis hamiltonii at a concentration of 1mg/ ml weretaken and the volume was made up to 3ml with ethanol.Then 0.1ml AlCl3 (10%), 0.1ml of potassium acetate and2.8 ml distilled water were added sequentially. The testsolution was vigorously shaken. Absorbance wasrecorded at 415 nm after 30 minutes of incubation. Astandard calibration plot was generated at 415nm usingknown concentrations of quercetin. The concentrationsof flavonoid in the test samples were calculated from thecalibration plot and expressed as mg quercetin equivalent/g of sample.
Qualitative analysis of Antioxidant activity of DecalepishamiltoniiThe antioxidant activity of rhizome extracts of Decalepishamiltonii was determined by following the method as
described by George et al. (1996). 50ìl of rhizome extractsof Decalepis hamiltonii were taken in the microtiter plate.100ìl of 0.1% methanolic DPPH was added over thesamples and incubated for 30 minutes in dark condition.The samples were then observed for discoloration; frompurple to yellow and pale pink were considered as strongand weak positive respectively. The antioxidant positivesamples were subjected for further quantitative analysis.
Quantitative analysis of Free radical scavenging activityof Decalepis hamiltoniiThe antioxidant activities were determined using DPPH,(Sigma-Aldrich) as a free radical. Rhizome extract of 100ìlwere mixed with 2.7ml of methanol and then 200ìl of 0.1 %methanolic DPPH was added. The suspension wasincubated for 30 minutes in dark condition. Initially,absorption of blank sample containing the same amountof methanol and DPPH solution was prepared andmeasured as a control (Lee et al., 2003). Subsequently, atevery 5 min interval, the absorption maxima of the solutionwere measured using a UV double beam spectra scan(Chemito, India) at 517nm. The antioxidant activity of thesample was compared with known synthetic standard of(0.16%) of Butylated Hydroxy Toluene (BHT). Theexperiment was carried out in triplicate. Free radicalscavenging activity was calculated by the followingformula:Inhibition = [(Absorbance of control (Ac 517) –Absorbance of sample (As517) X 100(Absorbance control (Ac517)]
High performance liquid chromatography analysis of 2-hydroxy 4-methoxy benzaldehyde (2H4MB)The fine powder of the rhizome biomass was extractedwith 75% of ethanol and then the extract was evaporated.The residue of extract was mixed with n-butanol and water(2:1) and both the upper layer of n-butanol and lowerlayer of water were separated and evaporated undervacuum. The residues were washed with petroleum etherto remove fatty components and then extracted withmethanol. The concentrated extract in methanol wasseparated and analyzed using high performance liquidchromatography as per standard method (Shimizu et al.,1997).
The extracts were filtered through sartorius RC-membranesyringe filter (0.20 m) and 20 µl of filtrate was injected into the HPLC. Chromatography was performed usingShimadzu HPLC (Model SPD-10A UV-VIS Detector) andsupelcosil LC-18 column (25 cm x 4.6 mm, 5 m) with mobilephase consisting of acetonitrile, water and acetic acid(50:50:0.1). Flow rate was maintained at 1.0ml/minute witha back pressure of 250 psi and the compounds were readat 210 nm using a UV detector. The total run time was 20min but preferably it was extended up to 40min (Shimizuet al., 1997). The results were compared with standard.
Antibacterial activity from rhizome extract of Decalepishamiltonii The ethanol rhizome extracts from mother plant ofDecalepis hamiltonii plant were used for antibacterialstudy (ozkan et al., 2004; Janarthanam and Sumathi, 2010).Different concentration (10, 20 and 30 mg/ml) of theconcentrated ethanol rhizome extracts was tested for itsantimicrobial strain such as Bacillus cereus, Bacillussubtilis, Staphylococcus aureus, Pseudomonasaeruginosa and Escherichia coli. The bacterial cultureswere grown in Mueller Hinton Agar and Mueller Hintonbroth (Himedia) (Lopez et al., 2001).
Antibacterial activity was measured using thestandard method of diffusion disc plates on agar(Erturk et al., 2003). Then 0.1ml of each culture ofbacteria was spread on agar plate surfaces. Forantibacterial assay, all bacterial strains were grownin Mueller Hinton Broth Medium (Hi media) for 24hours at 37°C and plated on Mueller Hinton Agar (Himedia) for agar diffusion experiments. Paper disc (6mmin diameter) were placed on the agar medium to load20µl of different concentrations of ethanol rhizomeextracts of Decalepis hamiltonii were tested.Inhibition diameters were measured after incubationfor 24 - 48 hours at 37°C.
Results and DiscussionIn the present study, phytochemical screening wasperformed with ethanol, chloroform, petroleum ether,acetone and aqueous rhizome extracts of Decalepishamiltonii. The ethanolic rhizome extract of Decalepishamiltonii were rich in terpenoids, quinones, Cardiacglycosides, flavonoids, steroids, phenols, tannins andsaponins followed by other extracts (Table 1,2 and 3).
Phytochemical constituents such as tannins, flavonoids,alkaloids and several other aromatic compounds orsecondary metabolites of plants serve as defensemechanism against predation by many micro-organisms,insects and herbivores. The curative properties ofmedicinal plants are perhaps due to the presence ofvarious secondary metabolites such as alkaloids,flavonoids, glycosides, phenols, saponins, steroids, etc(Britto and Sebastian, 2011). Thus the preliminaryscreening test may be useful in the detection of thebioactive principles and subsequently may lead to thedrug discovery and development (Doss, 2009). Thepresence of alkaloids and saponins in the rhizome extract,the biological function of alkaloids and their derivativesare very important and are used in analgesic,antispasmodic and bactericidal activities (Stary, 1998).Saponins have properties of precipitating and coagulatingred blood cells and they also have cholesterol bindingproperties, formation of foams in aqueous solutions andhaemolytic activity (Sodipo et al., 2000) and traditionallysaponins have been extensively used as detergents andmolluscicides, in addition to their industrial applicationsas foaming and surface active agents and also havebeneficial health effects (Shi et al., 2004). Plant steroidsare known important for their cardiotonic activities andalso used in nutrition, herbal medicine and cosmetics.Thus the preliminary screening studies may be useful inthe detection of the bioactive principles, which leads todrug discovery and development.
Flavonoids are regarded as one of the most widespreadgroups of natural constituents found in plants. Thevalues of flavonoid content varied among plants. It hasbeen recognized that flavonoids show antioxidant activityand their effects on human nutrition and health areconsiderable. The mechanisms of action of flavonoidsare through scavenging or chelating process (Kessler etal., 2003; Cook and Samman, 1996). The results of thepresent study showed that the flavonoid contents of theethanolic rhizome extracts in terms of quercetin equivalent(QE) were found to be maximum in Decalepis hamiltonii(Kanchipuram) 5.25 mg QE/g followed by Salem( 3.9 mg QE /g) and Trichy ( 2.3 mg QE/g) (Table 4).
Scavenging activity for free radicals of DPPH has widelyused to evaluate the antioxidant activity of naturalproducts from plant and natural sources. Free radicalshave broad range of effects in biological systems. It has
been proved that these mechanisms may be important inthe pathogenesis of certain diseases and ageing. Manysynthetic antioxidant components have shown toxic and/or mutagenic effects, which have shifted the attentiontowards the naturally occurring antioxidants (Galvez etal., 2005; Tepe et al., 2005; Mammadov, 2011).
Wild accessions of Decalepis hamiltonii rhizome sampleswere used for antioxidant studies. Analysis on differentextraction of acetone, ethanol (75%), petroleum ether,chloroform and aqueous extract showed the presence ofantioxidants. 100ìl of rhizome extracts were estimated forfree radical scavenging activity using DPPH assay. Thesamples were observed for the colour change from purpleto yellow and pale pink were considered as strongpositive and weak positive respectively (Table 5). Amongthe three wild accessions and five different solventextracts of D. hamiltonii, the ethanolic rhizome extractcollected from Kanchipuram district recorded the mosteffective DPPH radical scavenging activity (90.98%)followed by Salem (67.3%) and Trichy district (40.98%)(Fig. 2). Decalepis hamiltonii (Kanchipuram) value beingvery close to synthetic antioxidant (BHT) as positivecontrol (98.36%). In all accessions, ethanolic rhizomeextracts recorded higher percentage of free radicalscavenging activity followed by acetone, aqueous,chloroform and petroleum ether.
HPLC analysis of 2H4MB compound2-hydroxy 4-methoxy benzaldehyde (2H4MB) compoundfrom D. hamiltonii eluted through HPLC analysis andbased on standard retention time 11.03 min.
The Decalepis hamiltonii rhizome extract was used forHPLC analysis, showed a similar Retention time (Rt) inboth rhizome extract (11.14 min) and 2H4MB compoundStandard (11.03min). Thus confirming the presence of2H4MB compound in rhizome extract of Decalepishamiltonii (Fig. 3).
The data presented in Table 6, indicate that the rhizomeextracts of Decalepis hamiltonii inhibit the growth ofsome microorganism to various concentration. Theconcentrations of 10mg/ml - 30mg/ml ethanolic extractshowed antimicrobial activity against Staphylococcusaureus, Bacillus cereus, Bacillus subtilis, Pseudomonasaeruginosa and inactivity against Escherichia coli (Fig.4). The maximum clear zone of inhibition was found at30mg/ml of 75% ethanolic rhizome extract of Decalepishamiltonii. In rhizome extract, there is no zone ofinhibition was found in lower concentration 10mg/ml.Similar results were obtained on ethanol extracts fromrhizome of Sida acuta and Acalypha wilkesiana whichexhibited antibacterial activity (Oboh et al., 2007; Gotepet al., 2010). The antimicrobial activities of ethanol extractmay be due to the presence of tannins, triterpenoids andflavonoids (Mamtha et al., 2004). Thus from our findings,it is concluded that the 75% ethanolic extracts from drypowdered rhizome of Decalepis hamiltonii had superiorlevel of antimicrobial activity.
In conclusion, phytochemical composition, totalflavonoid, antioxidant activity and antibacterial activityof medicinal plants are very important in identifying newsources of therapeutically and industrially importantcompounds. It is imperative to initiate an urgent step forscreening of plants for secondary metabolites. Thepresent communication attempts to assess the status ofphytochemicals, total flavonoid, antioxidant activity,extraction of compound and antibacterial activity, in therhizome extract of Decalepis hamiltonii to improve thehealth status of people and also to use it in thenutraceuticals products of commercial importance. Theresults indicate that the plant material may become animportant source of natural drug compounds with healthprotective potential and natural antioxidants andantibacterial of significant impact on the status of humanhealth and disease prevention.
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