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Industrial Crops and Products
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lpinia nigra seeds: A potential source of free radical scavengernd antibacterial agent
udipta Ghosha, Guillermo F. Padilla-Gonzálezb, Latha Rangana,∗
Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781039, IndiaQuality Control, Naturcol Laboratories S.A., Calle 17A No 68D-60, Bogotá, Colombia
a r t i c l e i n f o
rticle history:eceived 21 January 2013eceived in revised form 16 April 2013ccepted 5 May 2013
eywords:
a b s t r a c t
The radical scavenging activity and the antibacterial properties of different solvent (hexane, ethyl acetateand methanol) extracts of Alpinia nigra seeds were investigated in the present study. All the extractswere used to assess their potential antioxidant activities using methods for scavenging of 2,2-diphenyl-1-picrylhydrazyl radical. Ethyl acetate and methanol extracts exhibited effective free radical scavengingactivities compared to the standard antioxidant butylated hydroxyl toluene. The efficacy of A. nigra seed
extracts was tested against three gram positive and four gram negative bacteria. Flow cytometry and fieldemission scanning electron microscopy study reveals and confirms the bacterial cell membrane damage,pore formation and membrane depolarization when treated with different solvent extracts. Bacterialcell membrane damage and releasing of cytoplasmic content (nucleic acids) was monitored using UV/visspectrophotometer at 260 nm. Current investigation highlights the antimicrobial potential of A. nigra
l anti
seed extracts and its tota
. Introduction
Plant derived compounds are the most sought after resourcesn the world toward the discovery of various life saving drugsnd other promising agents like cosmetics, natural antioxidants,ood preservatives, etc. Plant extracts possessing high antioxidantotential (Heim et al., 2002) includes a wide range of organic com-ounds such as alkaloids, flavonoids, tannins and phenolics whichould be exploited to treat various chronic as well as communica-le diseases (Siddhuraju and Manian, 2007). Antioxidant moleculesre the key factors which help to cope up with the oxidativetress (Krishnaih et al., 2007) by defending against reactive oxygenpecies (ROS) (Lan et al., 2007) generated in the body. However,se of synthetic antioxidant like butylated hydroxyanisole (BHA)nd butylated hydroxytoluene (BHT) in food products may leado damage of liver and eventually other carcinogenic effect dueo the accumulation in the body (Valentao et al., 2002). On theontrary, antioxidants derived from natural sources could protecthe human heath from cancer, cardiovascular disease and manyther diseases (Shui and Leong, 2006) and also much preferredhan the chemosynthetic agents in the food industries toward
he consumers safety and low toxicity as preservative in the foodroducts (Weerakkody et al., 2010). Therefore, attention towardhe use of alternative antimicrobials and naturally occurring
antioxidants from plant and other natural sources has beenincreased notably in food and pharmaceutical industries in recentyears.
Zingiberaceae, a well recognized family in the plant kingdom,has been investigated by many academic and industrial researchwings due to its versatile nature and high medicinal impact. Thefamily distributed worldwide with about 50 genera and 1300diverse species primarily dispersed in south and southeast Asia (Wuand Larson, 2000). India harbors nearly 22 genera and 178 speciesfrom north eastern and peninsular region (Jain and Prakash, 1995),whereas 19 genera and about 88 diverse species were reportedfrom north east region alone (Prakash and Mehrotra, 1995). Theethnomedical practices of tribal communities toward the uses ofZingiberaceae members from North East India were studied exten-sively and documented for further investigation (Tushar et al.,2010). The largest genus of the family, Alpinia, constitutes about230–250 species distributed through tropical and subtropical cli-mates of Asia and the Pacific (Kress et al., 2005). Diverse membersof the genus, Alpinia, possess significant biomedical potentials andfuture therapeutic values (Ghosh and Rangan, 2012). Alpinia nigra(Gaertn.) B. L. Burtt., member in the genus Alpinia distributed pri-marily in China, Thailand and other Southeast Asian countriesincluding north-eastern part of India (Wu, 1981). Ethnomedicallyimportant but less explored A. nigra known to be used for the treat-
ment of dyspepsia, gastric diseases, insect bites, trematocidal etc.in folk medications (Roy et al., 2009). However, the effectivenessof A. nigra for both antioxidant and antimicrobial activity has notbeen investigated yet.
In the current study, the antibacterial efficacy of three differentolvent extracts (hexane, ethyl acetate and methanol) isolated from. nigra seeds against diverse range of pathogenic bacteria has beenhoroughly investigated. The change of bacterial cell morphologyfter treating with each extract was also observed by field emis-ion scanning electron microscopy (FESEM). We also evaluated thentioxidant potential, which was assessed by the scavenging effectn 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals of all the testedeed extracts.
. Materials and methods
.1. Plant material
The seeds of A. nigra were collected from IIT GuwahatiIITG) campus (26◦12.476 ′N to 91◦41.965 ′E) during the period ofovember 2011–January 2012. The mature fruits (black color) werearvested and seeds were taken out for further processing. Theleaned seeds were dried in shed for 10–15 days. The collectedlants along with the rhizome and flowers were properly taggednd maintained in the departmental green house of IITG and botan-cal garden of Gauhati University (GU). Hooker (1875) and Petersen1889) were used as reference for identification of the plants. Theoucher specimen is maintained as herbarium for future referencet IITG herbarium repository. The botanical name was written asn IPNI database [N.C. Malakar, field No. 109, Herbarium accessionumber: 11500].
.2. Preparation of the organic extracts
Soxhlet extraction gives significantly better yield, total phe-olic content and tannins with economic investments (Aspé andernández, 2011). The shade dried seeds (200 g) were groundednto fine powder and subjected for solvent extraction for 5 h using
Soxhlet apparatus. The extraction for the same fixed amount of theample (200 g) was done according to the polar strength of solvent300 ml each) starting with non-polar (n-hexane) to polar (ethylcetate and methanol) respectively. After each solvent extraction,he same samples were dried properly to remove the trace of pre-ious solvent so as to reduce the carry over effect of the previousolvent and again subjected for next polar solvent. The extractsere subjected for vacuum drying in rotary evaporator (BUCHI,-210, Switzerland).
.3. Determination of DPPH radical scavenging activity
The free radical scavenging activity of seed extracts of A. nigraere determined using DPPH according to the method of Shimada
t al. (1992). Briefly, 0.1 mM solution of DPPH in 99.99% ethanolas prepared and 100 �l of this solution was mixed with 200 �l
f the ethanolic solutions of n-hexane, ethyl acetate and methanolxtracts from A. nigra seeds at concentrations ranging from 10 to00 �g/ml. The mixture was shaken vigorously and allowed to incu-ate at room temperature in dark for 30 min. Butylated hydroxyloluene (BHT) (Sigma–Aldrich, USA) was used as positive referencehile ethanol was used as negative reference. The absorbance waseasured at 517 nm using multimode microplate reader (Tecan,
nfinite M-200, Switzerland). The DPPH radical concentration wasalculated using the following equation:
PPH scavenging effect(%) = 100 −[(
A0 − A1
A
)× 100
],
0
here A0 was the absorbance of the control reactionDPPH + ethanol) and A1 was the absorbance in the presencef the sample (DPPH + sample in ethanol).
Each sample extract (1 mg) were mixed with dry KBr powdersand respective pellets were prepared. The absorption spectra of thesamples were obtained with a Perkin-Elmer FTIR spectrophotome-ter (Norwalk, Connecticut) in the range of 450–4000 cm−1.
The 1H spectra were recorded on a Varian 400 MHz FTNMR. Eachextract sample (25 mg/0.5 ml) were dissolved in CDCl3, and the sol-vent signal was used for spectral calibration. The spectral width was6389.8 Hz, acquisition time 1.998 s, number of scans 4 and the timedomain size was 32 K with relaxation delay for 1 s. The chemicalshift of each spectrum were recorded as ı values.
2.5. Determination of total soluble phenolics (TSP)
Total soluble phenolic compound content (TSP) in A. nigra seedextracts were estimated according to the Folin–Ciocalteu (FC) col-orimetric assay (Singleton and Rossi, 1965), using gallic acid asstandard. To generate the standard curve, different concentrations(10–500 �g/ml) of gallic acid were used. Three different dilu-tions (1:1000, 1:100 and 1:10) were prepared from each extracts(1 mg/ml) and were used for the assay. Both the standard and sam-ples were diluted in ethanol. BHT (125 �g/ml) and ethanol wereused as positive and solvent control (blank), respectively. About100 �l of 1 N Folin–Ciocalteu’s phenol reagent was added to allthe samples, standards and controls and incubated at room tem-perature for 5 min. After incubation, 300 �l of sodium-carbonate(75 g/l) was added and further incubated for 2 h at room tempera-ture. Finally, 300 �l was taken from each samples and transferredto 96-well microtitre plate and measured absorbance at 765 nm.Quantification of TSP in each extracts was determined from thegallic acid standard curve and the values were represented as mil-ligrams of gallic acid equivalents (GAE) per g dry weight of plantextracts. All the samples were analyzed in triplicates and the wholeexperiment was repeated twice.
2.6. Antibacterial activity
2.6.1. MicroorganismsThe antibacterial activities of all the extracts were tested against
seven bacteria, namely Staphylococcus aureus (ATCC6538), Bacil-lus ceresus (ATCC 11778), Listeria monocytogenes (ATCC 19115),Escherichia coli (ATCC 25922), Salmonella paratyphi A (MTCC 735),Escherichia coli enterotoxic (MTCC 723), Yersinia enterocolitica(MTCC 859). The bacteria were grown and maintained on nutrientagar (NA).
2.6.2. Determination of antimicrobial activity by agar holemethod
The antibacterial effect of the all the extracts was investigatedusing the agar hole method (Southwell et al., 1993). NA plates wereseeded with 8 h broth culture of seven tested bacteria. Four wells(5 mm) were made in each agar plate using a sterile cork borer.Twenty microlitres of three different concentrations (5, 10 and25 mg/ml) of extracts prepared in ethanol was added in each welland the same volume of ethanol was used as negative control. Theplates were incubated at 37 ◦C for 18–24 h. Each test was carried outin triplicate and the experiment was repeated twice. The antibacte-rial activity of the extracts was determined and recorded as meandiameter (mm) of the minimal zone of inhibition (ZOI).
The minimal inhibitory concentration (MIC) was determinedby using the microdilution method, in 96 well microtitre plates
3 ps and
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(wTtt
2c
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tawlgac
50 S. Ghosh et al. / Industrial Cro
Camporese et al., 2003). Twofold serial dilutions of extracts pre-ared in ethanol (10–0.08 mg/ml) added in each well containingacterial suspension approximately 106 cells/ml. Ethanol was useds a negative control. The plate was incubated at 37 ◦C for 18 h andhen was examined with multimode microplate reader (Tecan, Infi-ite M-200, Switzerland) at 620 nm. The lowest concentration oflant extract, at which bacterial growth was inhibited, has beenonsidered as MIC. The experiment was carried out in triplicatesnd MIC was recorded as the mean concentration of triplicate val-es.
In order to determine the minimum bactericidal concentrationMBC), 10 �l of broth medium from each well of MIC tested plateas spreaded on nutrient agar plate and incubated at 37 ◦C for 24 h.
he least concentration showing no visible growth on plate wasaken as MBC value. The MBC was recorded as the mean concen-ration of triplicates.
.6.4. Determination of antibacterial activity using flowytometry (FC)
The mode of action of all the extracts from A. nigra seeds againsteven tested bacteria were investigated using flow cytometry (FC)echnique. To study the potentiality of three extracts, each bacte-ial culture was treated with individual extract at their respectiveIC and incubated for 12 h. Heat killed bacteria (70 ◦C for 30 min),
thanol treated bacteria and untreated bacteria were considereds positive control, negative control and control for the experi-ents. Each bacterial suspension was centrifuged at 10000 rpm for
0 min at room temperature followed by washing of bacterial cellellet thrice with PBS (phosphate buffer saline, 50 mM, pH 7.0).inally, the cells were resuspended in PBS and adjusted to cell con-entration of approximately 106 cells/ml. The cells were incubatedith 100 �g/ml of propidium iodide (PI) (Sigma–Aldrich, USA) for
5 min in dark. The FC analysis of the cell samples were performedsing FACS Flow solution (Becton-Dickinson) as sheath fluid andlowJo software (Tree Star, Stanford, USA) was used for histogramlot analysis. The instrument is equipped with an argon ion laser488 nm) and the cytometer was adjusted to count 50,000 fluores-ent events for each sample. The FL-2 channel was used to detecthe red fluorescence of PI stained bacterial cells. The antibacterialffect of extracts were determined according to the fluorescencentensity of PI which correlates with the damage of bacterial cell
embrane (Paparella et al., 2008).
.6.5. FESEM analysisMorphological examinations of the bacterial cells before and
fter the exposure of all the extracts were performed using fieldmission scanning electron microscope (Carl Zeiss, Ultra 55).ESEM studies were carried out on two most susceptible bacte-ia viz. S. aureus and Y. enterocolitica, treated with all the extractst their respective MICs. Bacterial cells without extract treatmentere used as control. The bacterial samples were washed gentlyith 50 mM phosphate buffer solution (pH 7.2), fixed with 2.5%
lutaraldehyde in PBS and rinsed with the same buffer solution.he specimen was dehydrated using sequential exposure for eachthanol concentrations ranging from 30% to 100%. Finally, the spec-mens were coated with gold and analyzed with FESEM.
.6.6. Effect of extracts on bacterial cell membraneThe damage of bacterial cell membrane after treating with all
he extracts were further studied by monitoring the loss of 260 nmbsorbing materials, releasing from bacterial cells. The experimentas carried out for S. aureus and Y. enterocolitica, whose morpho-
ogical damage were previously examined using FESEM. Overnightrowth of bacterial suspension was harvested by centrifugationt 10,000 rpm for 10 min and resuspended in 0.9% sterile sodiumhloride solution. Then each bacterial culture was treated with
Products 49 (2013) 348– 356
individual extract at their respective MIC and incubated for 0, 4, 8and 16 h. After incubation, samples were centrifuged at 10,000 rpmfor 5 min in order to separate out the additional extracts and thebacterial cells from the low molecular weight metabolites suchas nucleotides, amino acids and inorganic ions which are knownto leak from cells after membrane damage. Finally, the level ofreleased material from the bacterial cell was determined by mea-suring optical density (OD) of the supernatant at 260 nm usingUV/vis spectrophotometer (Varian Carry 50, USA).
3. Results and discussion
3.1. Preparation of the organic extracts
In particular, organic solvents are frequently used for the extrac-tion of phenolic compounds from the plant samples to be used asantioxidants (Pokorny and Korczak, 2001). Moreover, the extrac-tion yield and biological activity of the extracts have a strongrelationship with the solvent employed, mainly due to the differ-ent polarity of the chemical compounds obtained (Moure et al.,2001). Therefore, selection of the most appropriate solvent is adeterminant factor on extract properties and due to the diversestructure and composition of the sample (Al-Farsi and Lee, 2008).Here, we have fractionated the chemical components of the seedsduring subsequent steps of extraction by using gradient polarityof solvent system. Initially, defatting of seed samples was donewith non-polar n-hexane which yielded with principally non-polarcompounds and little fraction of polar compounds during the con-tinuous extraction process. The same seeds when subjected toethyl acetate and later methanol, yielded with fractions contain-ing more polar compounds. After each extraction approximately230–250 ml of solvent was recovered using rotary evaporator andwas subsequently used for further extraction process. Therefore,only 100–150 ml of total amount of solvents were used to ratio-nally fractionate the chemical components of the sample duringthe extraction process. The solvent extraction process yielded 0.7,1.8 and 3.7 g for S-Hex, S-EtAc and S-Met, respectively. In orderto use the extracts for biological studies, the stock solutions wereprepared from the dried samples of each extract by dissolving inethanol to make the final concentration as 10 mg/ml.
3.2. DPPH free radical scavenging activity
DPPH is a stable free radical, which loses its purple color onaccepting an electron from an antioxidant molecule (Zou et al.,2004). It is evidently based on the reduction of DPPH in alcoholicsolution in the presence of a hydrogen-donating antioxidant dueto the formation of the non-radical form DPPH-H in the reaction.Lower absorbance of the reaction mixture indicates higher free rad-ical scavenging activity. Therefore, DPPH radical scavenging is acommonly used method to evaluate free radical scavenging activ-ity of plant extracts (Chung et al., 2006). The various concentrationsof hexane, ethyl acetate and methanol fractions of seed extract(10–100 �g/ml) showed antioxidant activities in a dose dependentmanner in the DPPH radical scavenging assay (Fig. 1). In our inves-tigation, a pattern of sharp increase of % inhibition was observedwith initial concentration of extracts, gradually saturating after60 �g/ml concentration depending on the availability of free OHgroup of the chemical constituents in each extracts. Similar reportsdescribed the saturation nature of tested sample after certainthreshold concentration (Xu et al., 2009). In general, plant extracts
having polyphenolic compounds could serve as an attractive sub-stitute of synthetic food antioxidants (Martinez-Tome et al., 2001).According to our recent study, plant extracts derived from vari-ous members of Alpinia genus were found to possess remarkable
S. Ghosh et al. / Industrial Crops and Products 49 (2013) 348– 356 351
FSr
apwaicvetap
3
icpbnatgia1�s
ig. 1. DPPH free radical scavenging activity of three different seed extracts (S-Hex,-EtAc and S-Met) of A. nigra. BHT used as positive control at varying concentrationanging from 10 to 100 �g/ml). Values represent means ± SE.
ntioxidant potential (Ghosh and Rangan, 2012). However, in theresent study, it was found that hexane extracts showed veryeak DPPH radical-scavenging activity. On contrary, ethyl acetate
nd methanol extracts exhibited significant free radical scaveng-ng activity in comparison with standard drug BHT. The inhibitoryoncentration 50% (IC50) was determined for each sample. The IC50alue of the DPPH radical by the hexane, ethyl acetate and methanolxtracts were >100, 27.22 ± 0.45, and 25.81 ± 0.94 �g/ml, respec-ively. Notable free radicals scavenging activity observed in S-EtAcnd S-Met extracts possibly due to the presence of high level ofolyphenolic moieties in the extracts.
.3. FTIR and NMR spectral analysis
Both the infrared and NMR spectrum of a crude plant extracts like a fingerprint, and the nature of the principal compoundsan be interpreted from its spectrum. FTIR spectral analyses wereerformed by correlating absorbance bands with a wide range ofonds and their functional groups. FTIR spectrum can illustrate theature of the functional groups present in the complex mixturend set the individual fingerprints for each extract. In our study,he acquired data were plotted as percent transmittance using Ori-in 5.0 software. Two sharp peaks at 2930.16 and 2844.03 cm−1
ndicates the presence of alkanes (C H stretch) and aldehyde (C H
ldehyde stretch), whereas, strong peaks at 1726.74, 1682.91 and638.88 cm−1 refers to the signature of aldehyde (C O stretch), �,-unsaturated aldehydes/ketones (C O stretch) and alkenes (C Ctretch, conjugated) in the extracts (Fig. 2). Similarly, sharp peaks
Fig. 2. FTIR spectra of A. nigra seed extracts. (a) S-
Fig. 3. 400 MHz 1D 1H NMR of crude seed extracts from seeds of A. nigra. Charac-teristic fingerprints were showed for S-Hex (A), S-EtAc (B) and S-Met (C) extracts.
at 1110.55 and 1060.88 cm−1 revealed the presence of ether link-age (C O C stretch, diaryl) and C N stretch of aliphatic amines,respectively. Peaks from 759 to 770 cm−1 refers to the presence ofaromatic moieties (C H bond, ortho) and 3432–3356 cm−1 indi-cates the existence of alcohols and phenols (O H stretch) in all theextracts irrespectively (Prabhakaran et al., 2012) (Fig. 2).
In case of NMR spectral analysis, all the spectra were comparedwith the standard reference chart and possible mixture of com-pounds were determined (Silverstein et al., 2005). The spectra of
each extract showed many peaks at 0–5 ppm with varying inten-sity and respective signatures (Fig. 3). Comparison of those peaksrevealed the presence of various group of molecules, viz. 0–2 ppmfor aliphatic acyclic compounds, 1–2 represents beta substituted
Hex, (b) S-EtAc and (c) S-Met, respectively.
352 S. Ghosh et al. / Industrial Crops and Products 49 (2013) 348– 356
Table 1The zone of inhibition (ZOI) of tested bacteria against seed extracts of A. nigra.
S-Hex: hexane extract; S-EtAc: ethyl acetate extract; S-Met: methanol extract from A. nigra seeds; a, b and c refers to concentration of each extract as 5, 10 and 25 mg/ml.E in (30m
asppooavafp(piagnn(eot1Seavt
3
eiartrsa(Cepips(
thanol (20 �l/well) used as negative control. Standard antibiotic used gentamiceans ± SE.
liphatic compounds and 2–5 ppm describes the presence of monoubstituted aliphatic compounds which is well in agreement withrevious reports (Patra et al., 2012). Beside these, characteristiceaks at 9.4 and 9.7 ppm in all the extracts revealed the presencef two aldehydes which are also convincing from the FTIR spectraf each extracts. Similarly, two triplets at 6.6 and 6.8 ppm char-cteristic to alkene which was almost absent in S-Hex whereas,ery prominent in case of S-EtAc and S-Met (Fig. 3). Except S-Hex,nother two prominent doublets at 2.3 and 2.5 ppm possibly comesrom the alkene adjacent to COR and CHO, respectively. A sharpeak was observed at 3.4 ppm which could be from either etherHC–OR) or alcohols (HC–OH). It was clearly visible that spectraossess a resemblance with one another as have been extracted
n gradient fractionation. Moreover, due to higher polarity S-EtAcnd S-Met extracts possess a wide range of compounds whichives clear and characteristic NMR fingerprints compared to theon-polar compounds from S-Hex fraction. Apart from ployphe-olic compounds, few other labdane type diterpene molecules, viz.E)-labda-8(17),12-diene-15,16-dial and (E)-8�, 17-epoxylabd-12-ne-15, 16-dial were reported from various species of Alpinia andther members of zingiberaceae, which showed promising antibac-erial, antifungal and anticancerous activities (Morita and Itokawa,988; Ayafor et al., 1994; Kubo et al., 2001; Abe et al., 2002;ingh et al., 2010). In our study, NMR and FTIR spectra of seedxtracts showed the presence of characteristic peaks similar tobove two compounds which possibly present in the extracts inarying amount and conferring the observed antibacterial activi-ies.
.4. Estimation of phenolic content
In plants, phenolic compounds are present as ether- and/orster-linked molecules. Many phenolic bioactive compounds weredentified from the various members of the genus Alpinia (Ghoshnd Rangan, 2012). In the current study, hexane fraction showedelatively low bactericidal and free radical scavenging activityhan other two fractions, which are well in accordance with theesults of estimated TSPs in each extract. Here, methanolic extracthowed highest TSP (3533.91 ± 23.66 mg GAE/g S-Met), than ethylcetate (2427.58 ± 24.15 mg GAE/g S-EtAc) and hexnane extracts530.46 ± 38.89 mg GAE/g S-Hex). The current assay based on Folin-iocalteu reagent, determines the reducing capacity of each samplextracts relative to its content of TSP as compared with standardhenolic acid (Wang et al., 2010). The notable antibacterial activ-
ty revealed by S-EtAc and S-Met extracts might be attributed byolyphenolic moieties in the extracts which are interlinked foruch bactericidal activities conferred by different plant extractsEscuredo et al., 2012).
�g/well). All the values represent inhibition zone size in mm. Values represent
3.5. Antimicrobial activities of extracts
There are several reports published on the activities of crudeplant extracts against various pathogenic bacteria till date. Previ-ous studies revealed effective antibacterial activity against a broadspectrum of pathogenic bacteria using various solvent extractsfrom several Alpinia species (Habsah et al., 2000; Mayachiewand Devahastin, 2008). In the current investigation, bactericidalefficacy of A. nigra seed extracts were qualitatively and quanti-tatively assayed by the presence or absence of inhibition zones,zone diameters, MIC and MBC values. The mean diameters of thegrowth inhibition zones of all the seed extracts against seventested pathogenic bacteria measured by using agar hole methodare presented in Table 1. The data revealed that the mean diam-eter of inhibitory zone (mm) against those bacteria varied from6 to 18 mm. Among the bacterial strains tested, it was observedthat the diameter of ZOI increased as the extract concentration wasincreased (Table 1) which signifies the dose dependant antibacte-rial activity of the seed extracts. Moreover, there was significantvariation of ZOI diameter observed between all the extracts (S-Hex, S-EtAc and S-Met) of A. nigra seeds. However, the methanolextract showed significantly higher overall inhibition against sevenbacteria tested, whereas hexane extract found to be less sensitivetoward all the tested gram negative bacteria (Table 1). Although,ZOI of S-EtAc showed comparatively higher values than S-Hex andpromisingly active similar to S-Met extract. Polarity index of ethylacetate and methanol are 4.4 and 5.1, respectively whereas, hex-ane (0.1) stands far below. The observed activity of the extractsmight have resulted due to the nature of existing chemical com-ponents and their relative quantity in each extract respectivelydepending on the solvent of choice. Among all the extracts, S-Met showed promising antibacterial activity against all the testedbacteria. Specifically, among all the tested gram-positive bacteria S.aureus and among gram negative bacteria Y. enterocolitica recordedas most sensitive toward all the tested crude extracts of A. nigra.Although, it would generally be expected that the extracts wouldbe more active against gram positive bacteria compared to thegram-negative (Mc Cutcheon et al., 1992), in contrast, accordingto the present study, S-EtAc and S-Met showed remarkable activityagainst both. Therefore, the result signifies that both the extractsS-Met and S-EtAc might be having the broad spectrum antibioticcompounds.
All the extracts from A. nigra seeds were used to study MIC andMBC by using the broth dilution method. The results of the MICand MBC values of all the extracts were represented in Table 2.
The MIC and MBC values for microbial strains were in the rangeof 0.16–5.00 mg/ml. Moreover, it was clearly observed that thesolvent type used in the extraction had a significant impact onMIC and MBC. The methanol extract showed relatively lower MIC
S. Ghosh et al. / Industrial Crops and
Table 2The minimum inhibitory concentration (MIC) and minimum bactericidal concentra-tion (MBC) values (mg/ml) of A. nigra seed extracts against selected gram-positiveand gram-negative bacteria.
nd MBC values compared to other extracts against all the testedacteria (<0.62 mg/ml). Furthermore, it was observed that amongll the tested bacteria S. aureus (MIC and MBC 0.16 mg/ml) and
. enterocolitica (MIC and MBC 0.31 mg/ml) were most sensitiveo the methanol extract among all the gram positive and gramegative bacteria, respectively. However, antibacterial activity of
ig. 4. Flow cytometric histograms of PI-stained seven tested bacteria at their respectivoftware (Tree Star, Stanford, USA) and plotted as PI fluorescence (FL2-H) against toal celMFI) of PI (FL2-H) for S. aureus (SA), B. ceresus (BC), L. monocytogenes (LM), E. coli (EC),
untreated bacteria (control), N bacteria treated with ethanol (negative control), HK hextract, ethyl acetate extract and methanol extract, respectively. Significant increase in Maximum effect was obtained with S-MET extracts irrespective of gram negative and gra
Products 49 (2013) 348– 356 353
hexane extract remained low irrespectively against all the testedbacteria.
3.6. FC investigation
Traditional methods for assaying antimicrobial activity arebased on the visualization of bacterial growth in a qualitative man-ner. However, in our study, along with the qualitative assays, thequantification of individual cells in the heterogeneous populationhas also been carried out by FC. Moreover, the sensitivity of flowcytometric analysis is significantly higher as the detectable changeof the fluorescence histogram of this method can directly corre-late the bacterial cell membrane damage. In order to provide someinsights into the mechanisms of action of all the extracts of A.nigra on seven tested bacterial cells, the multiparametric FC tech-nique was exploited. The evolution of the physiological states ofthe tested bacterial cells was monitored using the hexane, ethylacetate and methanol fractions of A. nigra seeds at their MIC values.
cell populations were stained with PI, a nucleic acid stain not takenup by intact live cells. Flow cytometric histograms and medianfluorescence intensity (MFI) of PI-stained bacteria are shown in
e MIC values for each extracts. Histogram analysis has been done by using FlowJol counts. (A)–(G) represents overlay histograms and median fluorescence intensityS. paratyphi A (SP), E. coli enterotoxic (EE), and Y. enterocolitica (YE), respectively.at killed bacteria, S-HEX, S-EtAc and S-MET are bacteria treated with seed hexaneFI and peak shifting was clearly observed in each case with respective treatments.m positive characteristics of bacterial population.
354 S. Ghosh et al. / Industrial Crops and Products 49 (2013) 348– 356
F erocolt .nigra
Fowts(dwct
ig. 5. Field emission scanning electron micrographs of S. aureus (A)–(D) and Y. entreatment with S-Hex (B and F), S-EtAc (C and G) and S-Met (D and H) extracts of A
ig. 4. Here, the negative controls (N-cell populations in presencef respective solvents) showed the minimum relative fluorescenceith respect to control cell populations (Fig. 4A–G). Conversely,
he positive control (HK-heat killed bacterial population) showedignificant increase in relative fluorescence in all tested bacteriaFig. 4A–G) and confirms the major cell populations as damaged or
ead. The rightward shifting of fluorescence peak in the histogramas observed when the bacterial cells were treated with extracts as
ompared to control populations (Fig. 4). The histogram peak shif-ing occurs due to high PI fluorescence intensity from the treated
itica (E)–(H). (A and E) untreated bacterial cells, (B–D and F–H) bacterial cells afterseeds at their respective MIC.
and heat killed cells. Untreated cell population has its characteristicunaltered cell membrane whereas, treated and heat killed cell pop-ulation has compromised cell membrane which allows PI to enterinto the cell and stain the nucleic acids. The extent of damaged anddead cells were estimated on the basis of MFI with reference tohistogram peak shifting. In the current study, the result allowed us
to understand the impact of all the extracts on bacterial cell dam-age. We observed that the response of the extracts varied amongthe seven tested bacteria. Interestingly it was observed that shif-ting of fluorescence peak in the histograms (toward right) and MFI
S. Ghosh et al. / Industrial Crops and Products 49 (2013) 348– 356 355
F cells (a
wem
3
tcusSuabaoaoaTtc
3
ncaoitvacot
4
uEibm
ig. 6. Absorbance of the cell materials contents at 260 nm releasing from S. aureus
nd S-Met) at 0, 4, 8 and 16 h. The data are expressed as means ± standard errors.
ere maximum when the cells were treated with S-EtAc and S-Metxtracts which indicates significant damage and depolarization ofost of the tested bacterial cytoplasmic membrane (Fig. 4).
.7. FESEM study
Membrane damage is found to be the key mechanism by whichhe plant extracts rich in phenolic compounds exerts their antimi-robial activities (Hammer and Heel, 2012). However, in order tonderstand the mode of action of A. nigra seed extracts on mostensitive gram positive and gram negative pathogenic bacteria,. aureus and Y. enterocolitica was further examined by FESEM tonveil the changes in bacterial cell morphology after treating withll the solvent extracts of A. nigra seeds. FESEM study of untreatedacteria revealed characteristic morphological features (Fig. 5And E), however shrinking and degradation of the cell walls werebserved in bacterial cells treated with seed extracts (Fig. 5B–Dnd F–H). Similar type of abnormalities of bacterial cell morphol-gy as an evidence of the disruption of membrane structure waslso reported previously (Koyama et al., 1997; Shin et al., 2007).hese findings indicate that A. nigra seed extracts possess antibac-erial activity and they cause lysis of bacteria by degrading bacterialell walls and damaging cytoplasmic membrane proteins.
.8. Effect of extracts on bacterial cell membrane
The FC and FESEM results illustrate that all the extracts from A.igra seeds can damage the bacterial cells by rupturing the outerell membrane of all the tested bacteria. In order to determine themount of leakage of bacterial cytoplasmic membrane, the releasef cell materials such as nucleic acid, metabolites and ions was mon-tored by the absorbance of the suspension at 260 nm. Fig. 6 showshe different amount of cell leakage which is represented as OD260alue after treating with all the seed extracts at MIC concentrationt 0, 4, 8 and 16 h, respectively. Our results demonstrate that theell leakage increases with time of exposure and significant releasef cellular material was observed in case of S-Met irrespective ofhe tested bacteria.
. Conclusions
A. nigra, an important member of Zingiberaceae family, is widelysed in ethnomedical practices of the tribal communities of North
ast India. Present study explored the promising bactericidal activ-ties and free radical scavenging potential of A. nigra seed extractsy introducing an economically viable and effective extractionethod. Among three different solvent extracts, methanol extract
A) and Y. enterocolitica (B) after treatment with A. nigra seed extracts (S-Hex, S-EtAc
found to be most active against all tested pathogenic bacteria. Bac-terial membrane damage and subsequent cell leakage were foundto be the key mechanism underlying the efficacy of seed extracts.Therefore, A. nigra seed extracts could be considered as effectivebactericides and also as natural antioxidants which need moreattention in the future as these efforts will also significantly con-tribute to a better understanding of the use of non-food crop andits application in pharmaceutical industries.
Acknowledgments
SG thanks Department of Information Technology (DIT), Gov-ernment of India for fellowship and GFP thanks to Maria CamilaRomero, Technical Director of Colfrigos Laboratories (Bogotá– Colombia), for experimental laboratory facilities and themicroorganisms. LR acknowledges funding by the Department ofInformation Technology, Ministry of Information Technology, Gov-ernment of India (DIT Grant No. DIT No: 0526/T/IITG/014/0809/38).
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