journa l h om epage: www.elsev ier .com/ locate / indcrop
hemical composition and bioactivity studies of Alpinia nigrassential oils
udipta Ghosha,∗, Temel Ozekb, Nurhayat Tabancac, Abbas Ali c, Junaid ur Rehmanc,khlas A. Khanc,d, Latha Rangana,∗
Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781039, IndiaDepartment of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskisehir, TurkeyNational Center for Natural Products Research, The University of Mississippi, University, MS 38677, USADepartment of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
r t i c l e i n f o
rticle history:eceived 24 August 2013eceived in revised form1 December 2013ccepted 16 December 2013
eywords:edes aegypti
a b s t r a c t
Free radical scavenging, bactericidal and bitting deterrent properties of Alpinia nigra essential oils(EOs) were investigated in the present study. Chemical composition of the EOs was analyzed usingGC–MS/GC–FID which revealed the presence of 63 constituents including �-caryophyllene as major com-ponent. All the EOs were found to possess notable antioxidant activities as determined using methodsfor scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Rhizome essential oil (REO) exhibitedbest effective free radical scavenging activities among other EOs compared to the standard antioxidant,butylated hydroxyl toluene. The efficacy of A. nigra EOs was tested against three Gram positive and four
Gram negative bacteria. Flow cytometry, field emission scanning electron microscopy and transmissionelectron microscopy studies revealed the bacterial cell membrane damage and disintegration when theyare treated with REO. Further, all EOs showed weak biting deterrent and larvicidal activity against theblood-feeding female adults and 1 day old Aedes aegypti larvae except flower essential oil which wasinactive at 125 ppm. Current investigation highlights the detailed chemical composition and bioactivepotential of A. nigra EOs for the first time.
. Introduction
Essential oils (EOs) are aromatic oily liquids which are basicallyecondary metabolites by nature and play a vital role in the pro-ection of the plants against various biotic factors (Bakkali et al.,008). Essential oils and their active components are gaining atten-ion from pharmaceutical and perfume industry due to their herbalature, versatile uses and wide acceptance (Ormancey et al., 2001;awamura, 2000). Usually EOs from plants are considered non-hytotoxic and highly active against various microbes (Devi et al.,013). Increasing bacterial resistance to antibiotics lead to the alter-ative approach where EOs and plant derived compounds were
nvestigated for antibacterial efficacy toward the use as food preser-ative and infectious disease control (Bakri and Douglas, 2005).dditionally, due to the natural occurrence of various phenolic
ompounds in plants, they get many attentions as source of antiox-dant molecules and flavoring ingredients (Sacchetti et al., 2005).
oreover, uses of these products in the form of food, vegetable and
flavoring agent would generally assumed to lower the risk asso-ciated with free radical and other infectious diseases (Young andWoodside, 2001).
In continuation to our previous study of ethnomedical practicesof tribal communities toward the uses of Zingiberaceae membersfrom North East India (NEI) (Tushar et al., 2010), we were currentlytargeted on the traditional diverse but less explored plant, Alpinianigra (Gaertn.) B. L. Burtt. This plant is locally known as “Tora” inAssam (India) and widely distributed in China, Thailand and otherSoutheast Asian countries (Wu, 1981). Folk uses of this plant aremany which widely used against many diverse health problems likeintestinal parasitic infection, gastric ulcers, irregular menstruation,bone weakness and jaundice in different states of NEI (Roy et al.,2012). Recently, the plant is also reported for its uses as a vegetablediet and also most popular uses as food flavoring agents by tribalpeople from different parts of NEI (Roy et al., 2012). Food industrynow use plant derived food flavoring agents and sometimes theyfacilitate to control the food spoilage due to their innate antimi-crobial efficacy. It has been observed that consumer preferences
have been turned toward the herbal products from the syntheticones due to its safety issues and less toxicity as preservative in thefood products (Weerakkody et al., 2010; Srivastava et al., 2014).Moreover, plant essential oils from diverse species of Alpinia could
rotect the human health from deadly microbes, cancer, cardiovas-ular disease, insects and parasitic infections (Ghosh and Rangan,013). According to our recent review on the genus Alpinia, it haseen found that the genus has tremendous antimicrobial activ-
ty along with other therapeutic potential which has triggered ournterest to investigate the essential oil composition and bioactiv-ty studies of A. nigra toward its probable candidature as futureood preservative and other pharmaceutical needs. Therefore, theresent study was conducted to investigate the effectiveness of A.igra essential oils against the food borne and other pathogenicacteria and further explored these oils for larvicidal and bitingeterrent activity against Aedes aegypti L.
. Materials and methods
.1. Plant material
Various parts of A. nigra (leaf, flower, rhizome and seeds) wereollected from Indian Institute of Technology Guwahati (IITG) cam-us (26◦12.476′ N to 91◦41.965′ E) during the period of November011–January 2012. The botanical name was written according to
PNI database and Hooker (1875) and Petersen (1889) were useds reference for identification of the plants. Live specimens of thelant are maintained in the departmental green house of IITG andotanical garden of Gauhati University (GU). The voucher speci-ens are also deposited as herbarium for future reference at IITG
The isolation of essential oil was carried out by following therocedure described in European Pharmacopoeia (Pharmacopoeia,005). The air dried leaves, flowers, seeds and rhizomes (25 g each)ere separately subjected to hydrodistillation for 3 h using a Cle-
enger type apparatus. This type of apparatus is made up of glassnd is used for extraction of essential oil from plant materials byydrodistillation method (Walton and Brown, 1999). The oil sam-les were collected and anhydrous Na2SO4 was used to removehe traces of water leftover. The oil yields were estimated on dryeight basis in each case. All the oil samples were kept in air tight
ials at 4 ◦C until GC/MS, GC–FID analyses and other bioactivitytudies. Each oil was diluted in n-hexane (10%, v/v) to carry outhromatographic determination of its composition.
.3. Gas chromatography–mass spectrometry (GC/MS)
The GC/MS analysis was performed with an Agilent 5975 GC-SD system (Agilent, USA; SEM Ltd., Istanbul, Turkey). HP-Innowax
SC column (60 m × 0.25 mm, 0.25-�m film thickness, Agilent, Walt Jennings Scientific, Wilmington, DE, USA) was used with a heliumarrier gas at 0.8 mL min−1. GC oven temperature was kept at 60 ◦Cor 10 min and programmed to 220 ◦C at a rate of 4 ◦C min−1, keptonstant for 10 min at 220 ◦C, and then programmed to increaset a rate of 1 ◦C min−1 to 240 ◦C. The oil (1 �L-10% in hexane) wasnalyzed with a split ratio of 40:1. The injector temperature was50 ◦C. Mass spectra were taken at 70 eV and the mass range wasrom m/z 35 to 450. All the oil samples were analyzed by GC–FIDnd GC/MS techniques prior to biological studies.
.4. Gas chromatography (GC)
The GC–FID analysis was carried out with capillary GC using angilent 6890N GC system (SEM Ltd., Istanbul, Turkey). The temper-ture was set at 300 ◦C for FID in order to obtain the same elution
Products 53 (2014) 111– 119
order with GC/MS. Simultaneous injection was performed using thesame column and appropriate operational conditions.
2.5. Biological assays
2.5.1. Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH)radical scavenging activity
The free radical scavenging efficacy of all the isolated EOs ofA. nigra was estimated using DPPH assay according to the methoddescribed by Shimada et al. (1992). DPPH is known as stable freeradical and strong scavenger for other radicals, which loses its pur-ple color on accepting an electron from an antioxidant moleculeavailable in a reaction system (Zou et al., 2004). DPPH free radi-cal scavenging activity of the oil samples can be determined usingcolorimetric assay. Briefly, 100 �L of DPPH solution (0.1 mM DPPHin absolute ethanol) was mixed with 200 �L of EO samples. Theethanol solutions of SEO (seed essential oil), LEO (leaf essential oil),FEO (flower essential oil) and REO (rhizome essential oil) were usedfor DPPH assay at concentrations ranging from 10 to 100 �L mL−1.The EO samples and DPPH solution were mixed thoroughly andincubated for 30 min in dark at 25 ◦C. Butylated hydroxyl toluene(BHT) (Sigma Aldrich, USA) and ethanol were used as positive con-trol and solvent control for the experiment. The absorbance wasrecorded at 517 nm in multimode microplate reader (Tecan, InfiniteM-200, Switzerland). The DPPH radical concentration was calcu-lated using the following equation:
DPPH scavenging effect (%) = 100 −[(
A0 − A1
A0
)× 100
]
where A0 was the absorbance of the control reaction(DPPH + ethanol) and A1 was the absorbance in the presenceof the sample (DPPH + sample in ethanol). Here samples are BHTand EOs.
2.5.2. Antibacterial activity2.5.2.1. Bacterial strains. The effect of A. nigra EOs were testedagainst Staphylococcus aureus (ATCC 6538), Bacillus cereus (ATCC11778), Listeria monocytogenes (ATCC 19115), Escherichia coli (ATCC25922), Salmonella paratyphi A (MTCC 735), E. coli enterotoxic(MTCC 723) and Yersinia enterocolitica (MTCC 859) bacterial strains.Nutrient agar (NA) was used to maintain and grow the tested bacte-ria.
2.5.2.2. Determination of zone of inhibition (ZOI), minimal inhibitoryconcentration (MIC) and minimum bactericidal concentration (MBC).Activity of the essential oils was evaluated against seven testedbacteria using the agar hole method as previously described bySouthwell et al. (1993). Petri plates were prepared with 8 h brothculture of each bacterial strain properly mixed in NA. Plates wereallowed to solidify and dry in vertical laminar flow for 15 min. Ineach agar plate, five holes (5 mm diameter) were made using ster-ile cork borer. For each EO, three fixed concentrations (2.5, 5 and10 �L mL−1) were prepared in ethanol and 20 �L each was addedin respective well. The standard antibiotic (gentamicin) and equalvolume of ethanol were used as positive control and vehicle con-trol, respectively. The plates were incubated at 37 ◦C for 18–24 h.Individual tests were performed in triplicate and were repeatedtwice. The effect of EOs on bacterial strains were determined andrecorded as mean diameter (mm) of the minimal zone of inhibition(ZOI) according to the previously published method (Ghosh et al.,2013a,b).
The antibacterial activities of EOs were determined based on
broth microdilution method as described by Camporese et al.(2003). Serial two fold dilutions of each EO sample were preparedin ethanol with concentrations ranging from 100 to 0.78 �L mL−1
and 10 �L of each concentration was added to individual wells
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ccording to its respective serial dilution. Equal volume of ethanolas used as vehicle control for the experiments. The bacterial sus-ension was adjusted to approximately 106 CFU mL−1 and added90 �L) to each well. The plate was incubated for 18 h at 37 ◦C andubsequently analyzed with multimode microplate reader (Tecan,nfinite M-200, Switzerland) at 620 nm. The lowest concentrationf each EO sample inhibiting the bacterial growth has been consid-red as MIC. The experiment was carried out in triplicates and MICas recorded as the mean concentration of triplicate values.
To determine the MBC, 10 �L of broth medium from each wellf MIC tested plate was spread on nutrient agar plate and incubatedor 24 h at 37 ◦C. The least concentration showing no visible growthn plate was considered as MBC value. The MBC was recorded ashe mean concentration of triplicates.
.5.2.3. Flow cytometry (FC) analysis. The effect of EO on bacterialells was estimated using multiparametric FC technique. The modef action of the most active EO was investigated against sevenested bacteria. Each bacterial culture was treated with the REOample at their respective MICs and incubated for 12 h. Heat killed70 ◦C for 30 min) bacteria, ethanol treated bacteria and untreatedacteria were considered as positive control, vehicle control andontrol for the experiments. Treatment of bacterial cells and furtherrocessing for FC analysis were performed as described previouslyy Ghosh et al. (2013b). Briefly, the FC analysis of the bacte-ial cells was performed using BD FACS Calibur (BD Biosciences,SA) and FlowJo software (Tree Star, Stanford, USA) was used foristogram plot analysis. The cytometer was set to count 50,000uorescent events for each sample and the FL-2 channel (585/42and pass) was used to detect the red fluorescence of propid-
um iodide (PI) stained bacterial cells. The antibacterial effect ofEO sample was determined according to the median fluorescence
ntensity (MFI) of PI which significantly correlates with the damagef bacterial cell membrane as described earlier by Paparella et al.2008).
.5.2.4. Field emission scanning electron microscopy (FESEM) andransmission electron microscopy (TEM) analysis. FESEM studiesere carried out on most susceptible bacteria, Y. enterocolitica
reated with REO at its MIC values. Bacterial cells without treatmentere taken as control. FESEM was used to visualize the alteration
n the surface morphology of the bacterial cells after the treat-ent with the REO sample. Control and treated bacterial samplesere gently washed with 50 mM phosphate buffer solution (pH
.2), fixed with 2.5% glutaraldehyde in PBS. The fixed bacterial sam-les were dehydrated using gradient ethanol solutions (30–100%).he specimens were subsequently coated with gold and analyzedhrough FESEM (Carl Zeiss, Ultra 55) as described previously (Ghosht al., 2013a,b).
The same bacterial samples were subjected to transmissionlectron microscopy (TEM) using JEOL 2100 UHR-TEM. For TEMnalysis, overnight grown cells of Y. enterocolitica were washedwice in PBS and resuspended in the same buffer. Bacterial cellsreated with REO at its MIC values and untreated cells resuspendedn PBS are considered as test and control samples, respectively.oth the samples were washed once with PBS and once with ster-
le MilliQ grade water which were finally resuspended in MilliQrade water. Two microlitre of ultrasonically dispersed bacterialamples were spotted on carbon coated TEM grid (Pacific Grid, USA)nd air-dried in laminar hood. The treated and control samplesere examined in a transmission electron microscope operating
t 100 kV and their images were recorded.
.5.3. Mosquito bioassays
.5.3.1. Insects. Ae. aegypti used in larvicidal and biting deter-ence bioassays were from a laboratory colony maintained at
Products 53 (2014) 111– 119 113
the Mosquito and Fly Research Unit at the Center for Medical,Agricultural and Veterinary Entomology, United States Departmentof Agriculture, Agriculture Research Service, Gainesville, Floridasince 1952 using standard procedures (2009). We received theeggs and stored these in our laboratory (Biological Field Station,The University of Mississippi, Abbeville, MS 38601) until needed.Mosquitoes were reared to the adult stage by feeding the larvaeon a larval diet of 2% slurry of 3:2 Beef Liver powder (now Foods,Bloomingdale, IL) and Brewer’s yeast (Lewis Laboratories Ltd.,Westport, CT). The eggs were hatched and the larvae were heldovernight in the same cup. These larvae were then transferred into500-mL cups (about 100 larvae per cup) filled with water. Larvaldiet was added every day until pupation, and the mosquitoeswere kept in an environment controlled room at a temperatureof 27 ◦C ± 2 ◦C and 60 ± 10% RH in a photoperiod regimen of 12:12(L:D) h. The adults were fed on cotton pads moistened with 10%sucrose solution placed on the top of screens of 4-L cages.
2.5.4. Mosquito biting bioassayBioassays were conducted using a six-celled in vitro Klun & Deb-
boun (K & D) module bioassay system developed by Klun et al.(2005) for quantitative evaluation of biting deterrent propertiesof candidate compounds. Term deterrent refers to a chemical thatinhibits feeding when present in a place where the insects feedin its absence and the repellent is a chemical that causes insectsto make oriented movement away from its source (Dethier et al.,1960). The K & D system consists of a six-well reservoir with eachof the 4 cm × 3 cm wells containing 6 mL of feeding solution. Asdescribed by Ali et al. (2012), we used the CPDA-1 + ATP solutioninstead of human blood. CPDA-1 and ATP preparations were freshlymade on the day of the test and contained a red dye that allowedfor identification of mosquitoes that had fed on the solution (seebelow). DEET (97% purity N,N-diethyl-meta-toluamide) was usedas a positive control. Molecular biology grade ethanol (Fisher Sci-entific Chemical Co., Fairlawn, NJ) was used as solvent control.Stock and dilutions of all essential oils and DEET were preparedin ethanol. All essential oils were evaluated at dosages of 100 and10 �g cm−2 treatments and DEET was tested at a concentrationof 25 nmol cm−2. Treatments were prepared fresh at the time ofbioassay.
During the bioassay, temperature of the solution in the reser-voirs covered with a collagen membrane was maintained at 37.5 ◦Cby circulating water through the reservoir with a temperature-controlled circulatory bath. The test compounds and controls wererandomly applied to six 4 cm × 3 cm marked portions of nylonorgandy strip, which was positioned over the six, membrane-covered wells. A Teflon separator was placed between the treatedcloth and module. A six-celled K & D module containing five10–18 d-old females per cell was positioned over the six wells,trap doors were opened and mosquitoes allowed access for a 3 minperiod, after which they were collected back into the module.Mosquitoes were squashed and the presence of red dye (or not) inthe gut was used as an indicator of feeding. A replicate consisted ofsix treatments: four oils, DEET (a positive control) and 95% ethanolas solvent control. Five replicates were conducted per day usingnew batches of mosquitoes in each replication. Bioassays wereconducted between 13:00 and 16:00 h and 13 replications wereconducted for each treatment.
2.5.5. Larval bioassaysBioassays were conducted by using the bioassay system
described by Pridgeon et al. (2009) to determine the larvicidal activ-
ity of essential oils of various parts of A. nigra against Ae. aegypti.Eggs were hatched and larvae were held overnight in the hatchingcup in a temperature-controlled room maintained at a temperatureof 27 ± 2 ◦C and 60 ± 10% RH. Five 1-d larvae were transferred in
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ach of 24-well tissue culture plates in 30–40 �L droplet of water.ifty microlitre of larval diet (2% slurry of 3:2 Beef Liver powdernd Brewer’s yeast and 1 mL of deionized water were added toach well by using a Finnpipette stepper (Thermo Fisher, Vantaa,inland). All essential oils to be tested were diluted in ethanol.fter the treatment, the plates were swirled in clock-wise andounter clockwise motions and front and back and side to side fiveimes to ensure even mixing of the chemicals. Larval mortality wasecorded 24 h post treatment. Larvae that showed no movement inhe well after manual disturbance by a pipette tip were recordeds dead. A series of 3–5 dosages were used in each treatment to get
range of mortality. Treatments were replicated 15 times for eachil.
.5.6. Statistical analysesFor DPPH and antibacterial studies statistical analysis was car-
ied out using SPSS Statistics 17.0. MFI values obtained from FCata were subjected to analysis of variance (ANOVA) followed byukey’s test (post-hoc analysis) to determine the significant dif-erence between the treatments and vehicle control for testedacterial strain. Differences were considered significant at a valuef p < 0.05.
Proportion not biting (PNB) was calculated using the followingormula:
NB = 1 −(
Total number of females bitingTotal number of females
)
Proportion not biting data were analyzed using SAS Proc ANOVASAS Institute, 2007), and means were separated using Duncan’s
ultiple Range Test.
. Results and discussion
.1. Composition of the oil
The current study revealed a detailed description of the com-osition and biological activity of the essential oil of A. nigra. Theil of A. nigra was analyzed by means of GC–FID and GC/MS tech-iques in order to unveil its qualitative and quantitative profiles.ydrodistillation of the different parts, seeds, flowers, leaves and
hizomes of A. nigra, yielded transparent oil for seeds and flowers,ellowish oil for leaves and reddish brown oil for rhizomes with
characteristic odour. About 0.76%, 0.06%, 0.23% and 0.18% yieldsere recorded for seeds, flowers, leaves and rhizomes of A. nigra,
espectively in dry weight basis. Detailed list of all the detectedompounds with their relative retention indices (RRI), chemicalames and percentages of each was given in Table 1 accordingo their elution on the HP-Innowax FSC column. GC/FID andC/MS analysis of the oil revealed the presence of 63 constituents
epresenting 96.4, 98.3, 97.9 and 98.2% of the leaf, flower, rhizomend seed oil. Principal components in A. nigra essential oils foundo be �-caryophyllene (47.7–49.0%), �-pinene (13.7–14.4%), �-umulene (7.5–7.8%), �-pinene (6.3–6.6%), caryophyllene oxide4.3–4.5%) and (E)-nerolidol (3.6–3.7%).
Monoterpene hydrocarbons, oxygenated monoterpenes,esquiterpene hydrocarbons and oxygenated sesquiterpenes werehe main groups present in the oil. Monoterpene hydrocarbonsere the most abundant among these groups representing 62.2%,
ollowed by oxygenated monoterpenes 6.6%, and the sesquiter-enes were presented in scarce amounts (1.4 and 2.2%). Previously,bout 18 components were reported for leaf and rhizome essentialil of A. nigra (Kanjilal et al., 2010) which prone to variation
epending on the various factors like time of collection of theamples, chromatographic column and reaction condition used. Inur study, 1,8-cineole is found as moderate to low in abundancehere it was found as major constituents in the previous report.
Fig. 1. DPPH free radical scavenging activity of four different EOs of A. nigra. BHTused as positive control at varying concentration ranging from 10 to 100 �g mL−1.Values represent means ± SE.
Conversely, �-caryophyllene was found as major component inthe present study whereas, it was remains undetected in earlierpublished report (Kanjilal et al., 2010). The comparative evaluationof the current study with earlier report of Kanjilal et al. (2010)clearly showed a variable composition of EOs which might beresulted due to primarily for different ecotypes and seasonalvariations as well.
3.2. DPPH free radical scavenging activity
Modern theory of free radical biology and medicine are inter-linked where reactive oxygen species (ROS) are known to involveas key factors in several diseases. The ROS related health prob-lems can be reduced by a suitable dietary habit including naturalantioxidants (Balasundram et al., 2006). Therefore, several investi-gations have been carried out in order to assess the antioxidantpotential of various plant materials including the genus Alpinia(Ghosh and Rangan, 2013). A wide variety of methods have beendeveloped for the estimation of antioxidant potential (Prior et al.,2005). Among all the methods, DPPH method is extensively useddue to its stability, simplicity and its simple reaction system whichinvolves only the direct reaction between the radical and an antiox-idant.
Free radical scavenging activity by DPPH assay is consideredas an important method to understand the potentiality of theplant materials toward its bioactivity. In the present study, variousconcentrations (10–100 �g mL−1) of all the EO samples showedradical scavenging activities in a dose dependent manner in theDPPH assay (Fig. 1). The inhibitory concentration 50% (IC50) wasdetermined for each oil sample and also for the positive control,BHT. It was found that all the samples were similarly effectiveas BHT (IC50 = 36.8218 �g mL−1) for DPPH radical scavengingactivity, however, REO was found little better than other oilsamples (IC50 = 38.6019 �g mL−1) under investigation. The DPPHfree radical scavenging activity of the EOs was not significantlydifferent from each other (p > 0.05, Tukey’s post hoc test). TheIC50 values of the DPPH radical by the SEO, LEO and FEO weredetermined as 40.1138, 42.1378, 43.4058 �g mL−1, respectively.This method is very common toward the evaluation of free radicalscavenging activity of plant essential oil (Chung et al., 2006). Itis based on the reduction of DPPH in alcoholic solution in the
presence of a hydrogen-donating antioxidant due to the formationof the non-radical form DPPH-H in the reaction. Lower absorbanceof the reaction mixture indicates higher free radical scavengingactivity. Previously, Cavalcanti et al. (2012) showed antioxidant
S. Ghosh et al. / Industrial Crops and Products 53 (2014) 111– 119 115
Table 1The composition of the essential oils of Alpinia nigra.
, Alpinia nigra leaf oil; B, Alpinia nigra flower oil; C, Alpinia nigra rhizome oil; D, Alpinrom FID data; tr, trace (<0.1%).
otential of LEO from Alpinia zerumbet using DPPH assay andhe also found significant dose dependent increase of scavengingctivity of LEO. Previously, various Alpinia species has been inves-
igated toward isolation of EOs and organic solvent extracts andound as highly active antioxidant agent (Ghosh and Rangan, 2013).otably, the seeds of A. nigra were investigated before and founds a source of natural free radical scavenger (Ghosh et al., 2013a,b).
96.4 98.3 97.9 98.2
ra seed oil; RRI, relative retention indices calculated against n-alkanes. % Calculated
3.3. Antibacterial activities of EOs
The antibacterial activity of four different oil samples of A. nigra
was evaluated by the presence or absence of inhibition zones, zonediameters, MIC and MBC values. The mean diameters of the growthinhibition zones of all the oil samples against the tested bacteriawere measured by agar hole method and presented in Table 2. The
116 S. Ghosh et al. / Industrial Crops and
Tab
le
2Th
e
zon
e
of
inh
ibit
ion
(ZO
I)
of
test
ed
bact
eria
agai
nst
fou
r
dif
fere
nt
esse
nti
al
oils
of
A. n
igra
.
Test
ed
bact
eria
SEO
LEO
FEO
REO
Eth
anol
An
tibi
otic
s
a
b
c
a
b
c
a
b
c
a
b
c
Gra
m
(+)v
eS.
aure
us6
±
0.5
8
±
0.3
10
±
0.5
7
±
0.7
8
±
0.4
11
±
0.5
6
±
0.8
7
±
1.4
11
±
0.8
8
±
0.2
10
±
1.3
12
±
0.9
5.2
± 0.
3
24
±
0.42
B.
cere
us
6
±
0.3
8
±
0.8
9
±
0.7
6
±
0.8
8
±
0.16
10
±
0.8
6
±
0.9
7
±
0.9
9
±
0.8
7
±
0.23
9
±
1.2
11
±
0.6
5.0
±
0.1
26
±
1.08
L.
mon
ocyt
ogen
es8
±
0.7
9
±
0.3
11
±
0.4
8
±
0.8
9
±
0.6
12
±
0.3
8
±
1.4
9
±
0.8
10
±
0.3
8
±
0.24
9
±
0.7
11
±
0.9
5.0
±
0.6
28
±
1.32
Gra
m
(−)v
eE.
coli
6
±
0.2
7
±
0.9
9
±
0.4
6
±
1.2
8
±
0.4
10
±
1.2
6
±
0.8
8
±
0.3
10
±
0.5
7
±
0.3
9
±
0.8
11
± 0.
2 5.
2
±
0.3
29
±
0.74
S.
para
typh
i
6
±
1.4
7
±
0.6
8
±
0.12
6
±
0.8
7
±
0.4
9
±
0.9
6
±
0.4
8
±
0.5
9
±
0.8
6
±
1.5
8
±
0.9
10
± 0.
3
5.3
±
0.5
24
±
0.23
E.
coli
ente
roto
xic
6
±
1.1
6
±
0.9
8
±
0.4
6
±
0.5
7
±
0.8
9
±
0.4
6
±
0.8
8
±
1.7
9
±
0.8
7
±
0.2
9
±
0.2
11
± 0.
4
5.1
±
0.12
30
±
1.24
Y.
ente
roco
litic
a
6
±
0.8
7
±
0.4
9
±
1.2
6
±
0.4
7
±
0.6
10
±
0.4
6
±
0.2
7
±
0.1
8
±
0.4
8
±
0.8
9
±
0.4
12
±
1.4
5.1
±
0.1
25
±
0.22
SEO
, see
d
esse
nti
al
oil;
LEO
, lea
f ess
enti
al
oil;
FEO
, flow
er
esse
nti
al
oil;
REO
, rh
izom
e
esse
nti
al
oil f
rom
A. n
igra
.a,
b
and
c
refe
rs
to
con
cen
trat
ion
of
each
extr
act
as
2.5,
5
and
10
�L
mL−1
.Et
han
ol
(20
�L/
wel
l)
use
d
as
neg
ativ
e
con
trol
.St
and
ard
anti
biot
ic
use
d
gen
tam
icin
(30
�g/
wel
l).
All
the
valu
es
rep
rese
nt
inh
ibit
ion
zon
e
size
in
mm
. Val
ues
rep
rese
nt
mea
ns
±
SE.
Products 53 (2014) 111– 119
mean diameter of inhibitory zone (mm) against tested bacteriavaried from 6 to 12 mm. Among the bacterial strains tested, it wasobserved that in most of cases the ZOI diameter extended with theincreasing EO concentrations (Table 2) which signifies the dosedependant antibacterial property of the oil samples. In the presentstudy REO showed significantly higher overall inhibition againstall the seven bacteria, whereas rest of the oil samples were beingmoderately less effective compared to REO (p < 0.05, Tukey’s posthoc test) except in case of L. monocytogenes at higher doses of SEOand REO. Furthermore, the results also indicated that all the EOsamples showed more or less equal effectiveness against all thetested bacteria irrespective of their gram positive or gram negativecharacteristics. Among all the bacteria analyzed, Y. enterocoliticawas found highly susceptible to REO treatment compared to otherEOs under study.
MIC and MBC were determined for the seven bacteria using var-ious EO samples from A. nigra. The results of the MIC and MBCvalues of respective EO samples were represented in Table 3. MICand MBC for the tested bacterial strains were found in the rangeof 3.12–6.25 �L mL−1. Moreover, it was also clearly observed thatthe EOs isolated from different parts of the plant had no signifi-cant variation on MIC and MBC of tested bacterial samples. The FEOsample showed relatively lower MIC and MBC values compared toother EOs against all the tested bacteria (6.25 �L mL−1). Similarly,it was also observed that REO had lowest MIC among all the testedbacteria (1.56–3.12 �L mL−1) except S. paratyphi (6.25 �L mL−1). S.paratyphi was found less susceptible to all the EOs under study.Here, REO has considered as most active against Y. enterocolitica(MIC 1.56 �L mL−1) among all the tested gram positive and gramnegative bacteria.
3.4. FC investigation
Flow cytometry is a laser-based and advanced biophysical tech-nology used in diverse field of cell biology to sort and counting offluorescent labeled cells of various characteristic features. In thepresent study, effect of REO on bacterial cells was monitored usingmultiparametric FC technique. The bacterial cells were treated withREO at their respective MIC values. All the control and treated bac-terial cells were stained with PI to analyze the effect of REO onbacterial cells. Flow cytometric histograms and respective medianfluorescence intensity (MFI) of PI-stained bacteria are shown inFig. 2. Here, the vehicle controls (N) and untreated bacterial cells(C) showed minimum relative fluorescence which are not signifi-cantly different (Fig. 2A–G). But, the positive control (HK) showedsignificant increase (p < 0.01, Tukey’s post hoc test) in relative flu-orescence in all tested bacteria with respect to vehicle control(Fig. 2A–G) and confirmed the major cell populations as damagedor dead. In the histogram, the rightward shifting of fluorescencepeaks was observed when the bacterial cells were treated withREO as compared to vehicle control (Fig. 2). Irrespective of theirGram positive and Gram negative characteristics, all the testedbacteria affected significantly by the REO compared to the vehi-cle control (p < 0.001, Tukey’s post hoc test). The result allowed usto understand the impact of essential oil on bacterial cell damage.We observed that the response of the REO varied among the seventested bacteria. Interestingly it was observed that shifting of fluo-rescence peak in the histograms (toward right) and MFI was max-imum when the cells were subjected to heat treatment (HK) andincubation with oil sample (R), indicating significant damage anddepolarization of most of the tested bacterial cell membrane (Fig. 2).
3.5. FESEM and TEM study
Changes in bacterial cell morphology after treating with REOwere studied in order to understand and visualize the antibacterial
S. Ghosh et al. / Industrial Crops and Products 53 (2014) 111– 119 117
Table 3The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values (�L mL−1) of essential oils of A. nigra against selected Gram-positiveand Gram-negative bacteria.
ction. The morphological alteration of Y. enterocolitica after thereatment with REO was examined by using FESEM and TEMFig. 4). FESEM study of untreated bacteria revealed characteristic
orphological features (Fig. 3A and B), however shrinking andegradation of the cell walls were observed in bacterial cellsreated with REO (Fig. 3B). These findings indicate that A. nigra REOossesses antibacterial activity and they cause lysis of bacteria byegrading bacterial cell walls and effecting cytoplasmic membrane.
The TEM images also clearly revealed the effect of REO onhe Y. enterocolitica exhibiting cell membrane damage, clearing ofnternal cellular materials and deformed cellular characteristics
ompared to the untreated cell (Fig. 3C and D). Untreated controlacteria showed the integrity of the membrane and characteris-ic morphology (Fig. 3C) where the TEM image of REO treatedacteria clearly indicated the alteration in outer membrane’s
ig. 2. Flow cytometric histograms of PI-stained seven tested bacteria at their respective Muorescence intensity (MFI) of PI for S. aureus (SA), B. cereus (BC), L. monocytogenes (LMespectively. C, untreated bacteria (control); N, bacteria treated with ethanol (vehicle conotable increase in MFI and peak shifts was clearly observed in each case with respective
3.12 6.25 6.25 3.12 3.123.12 6.25 6.25 1.56 3.12
ntial oil from A. nigra.
integrity with cell membranes being disrupted and damaged(Fig. 3D).
3.6. Mosquito results
Leaf, rhizome and seed essential oils of A. nigra showed larvicidalactivity (Fig. 4) against 1 day old Ae. aegypti larvae. In screeningbioassays, all the oils showed 100% mortality at the dose of 125 ppmexcept flower oil which was totally inactive. Among different plantparts, essential oil of rhizome produced lower mortality at 62.5 ppmthan other leaf and seed oils. These results indicate that A. nigra
essential oils have a weak larvicidal activity.
The A. nigra essential oils showed biting deterrent activity higherthan solvent control (Fig. 5) against female Ae. aegypti. Biting deter-rent effects of the essential oils at 10 �g cm−2 was lower than DEET
IC values for each essential oils. (A)–(G) represent overlay histograms and median), E. coli (EC), S. paratyphi (SP), E. coli enterotoxic (EE), and Y. enterocolitica (YE),trol), HK heat killed bacteria, R, bacteria treated with rhizome essential oil (REO).
treatments.
118 S. Ghosh et al. / Industrial Crops and Products 53 (2014) 111– 119
Fig. 3. Field emission scanning electron micrographs (A and B) and transmission electron mcells, and (B) and (D) are the bacterial cells after treatment with REO at its MIC. Arrows in
Fig. 4. Percent mortality (±SE) of essential oils of A. nigra against 1-d-old Aedesaegypti larvae.
Fig. 5. Biting deterrent effects of essential oils from various parts of A. nigra at10 �g cm−2 and DEET at 4.8 �g cm−2 against Aedes aegypti. Ethanol was used assolvent control.
icrographs of Y. enterocolitica (C and D). (A) and (C) showed the untreated bacterialdicate the damage and pores in the bacterial cells.
at 4.8 �g cm−2. The proportion not biting (PNB) of different plantparts of A. nigra EOs ranged between 0.49 and 0.62. The essentialoil of seed was found more effective (PNB = 0.62) while flower oilwas the least effective (PNB = 0.49).
4. Conclusions
Current investigation highlights the detailed chemical compo-sition of EOs extracted from various parts of A. nigra and theirbioactive potential. All the EOs showed strong free radical scaveng-ing activity similar to BHT in DPPH assay. These EOs also showedbactericidal effect and damage of bacterial cell membrane whichwere confirmed by FC, FESEM and TEM analysis. Beside these, ourfindings also indicate that A. nigra seed essential oil may have someactive biting deterrent components against Ae. aegypti and furtherstudies should be aimed to look at the individual lead compoundtoward bactericidal and antibiting properties of A. nigra essentialoil.
Acknowledgments
SG thanks Department of Information Technology (DIT), Gov-ernment of India for fellowship. LR acknowledges funding bythe Department of Information Technology, Ministry of Infor-mation Technology, Government of India (DIT grant no. DIT no:0526/T/IITG/014/0809/38). This study was supported in part byUSDA-ARS grant no. 56-6402-1-612, Deployed War-Fighter Pro-tection Research Program Grant funded by the U.S. Departmentof Defense through the Armed Forces Pest Management Board.The authors wish to thank Dr. James J. Becnel, Mosquito and FlyResearch Unit, Center for Medical, Agricultural and VeterinaryEntomology, USDA-ARS, Gainesville, for supplying Ae. aegypti eggs.
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