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Chemical Composition, and Antioxidant and

Antimicrobial Activities of Alpinia nutans Rosc.
a a a b
Sushil Joshi , Om Prakash , Anil K. Pant & C. S. Mathela
a
Department of Chemistry , G.B.P.U.A. & T. , Pantnagar, 263145, Uttarakhand, India
b
Department of Chemistry , Kumaun University , Nainital, Uttarakhand, India
Published online: 09 Dec 2011.

To cite this article: Sushil Joshi , Om Prakash , Anil K. Pant & C. S. Mathela (2010) Chemical Composition, and
Antioxidant and Antimicrobial Activities of Alpinia nutans Rosc., Journal of Essential Oil Research, 22:1, 85-90, DOI:
10.1080/10412905.2010.9700270

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 A. nutans

Chemical Composition, and Antioxidant and

Antimicrobial Activities of Alpinia nutans Rosc.

Sushil Joshi, Om Prakash and Anil K. Pant,*

Department of Chemistry, G.B.P.U.A. & T., Pantnagar-263145, Uttarakhand, India
C.S. Mathela,
Department of Chemistry, Kumaun University, Nainital, Uttarakhand, India

Abstract
The essential oils of the aerial parts and flowers of Alpinia nutans Rosc. were analyzed by GC and GC/MS. Oil from
aerial parts was rich in monoterpenoids (85.7%) with sabinene (27.8%), 1,8-cineole (17.4%), terpinen-4-ol (14.9%),
p-cymene (5.2%), g-terpinene (5.1%), cis-sabinene hydrate (1.3%) and linalool (3.3%) as the major components iden-
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tified, and with b-caryophyllene (3.9%) and caryophyllene oxide (1.3%) as sesquiterpenoids. Terpinen-4-ol (25.1%),
g-terpinene (19.4%), sabinene (14.2%), 1,8-cineole (10.8%), linalool (1.6%) and caryophyllene oxide (1.0%) were
the major components characterized in the flower oil. The oils were evaluated for DPPH radical scavenging ability,
chelation of Fe+2, and reducing power activities. Further, these oils were also examined for their antimicrobial activi-
ties against Pasteurella multocida, Escherichia coli, Salmonella enterica enterica, Shigella flexneri and Staphylococcus
aureus. The aerial parts oil was found to be more active than the flower oil in all the cases.

Key Word Index

Alpinia nutans, Zingiberaceae, essential oil composition, sabinene, 1,8-cineole, terpinen-4-ol, g-terpinene,
antioxidant activity, antimicrobial activity.

Introduction and uses in various ailments in traditional system of medicine,

an attempt has been made to investigate A. nutans for phyto-
Alpinia Linn (Zingiberaceae), represented worldwide by
chemical composition, antioxidant activities, and antimicrobial
180 species, are characterized by elongate, leafy stems and
activities.
horizontal root stocks, leaves oblong or lanceolate; flowers in
terminal racemes or panicles, bracteolate large, sometimes
Experimental
enveloping the buds; fruit globose, dry or fleshy, usually de-
hiscent; seed globose or angled (1). Alpinia species are used Plants: Alpinia nutans Rosc. was collected from Pant-
widely in folk medicine for gastric lesions (2), indigestion, colic nagar. Systematic botanical identification was confirmed by
disorders (3), and as a diuretic (4). In the recent years, scientific Y.P.S. Pangty, Taxonomist, Kumaun University, Nainital, and
studies on ethnomedicinal plants have received considerable the voucher specimen was deposited in the Herbarium of the
importance due to growing interest in plant-based medicines. Chemistry Department, CBSH, GBPUA&T, Pantnagar.
Alpinia species have been extensively studied for various phar- Isolation of essential oils: Aerial parts and flowers of A.
macological activities viz. antimicrobial (5), antioxidant (6), nutans (1 kg each) were subjected to separate hydrodistillation
anti-inflammatory (7), myorelaxant and antispasmodic (8), and using Clevenger-type apparatus for 8 h. Distillates were then
antitumor effects (9). extracted with diethyl ether, followed by drying over anhydrous
Alpinia nutans Rosc. (syn. Alpinia speciosa K. Schum or Na2SO4. The oil yield from the fresh flowers and aerial parts
Alpinia zerumbet (Pers.) Burtt. et Smith) is an aromatic herb were 0.51% and 0.76% (v/w), respectively.
distributed widely in tropical and sub-tropical regions of the GC and GC/MS analyses: The essential oils were ana-
world (10). Its use in folk medicine is widespread, predomi- lyzed on Nucon 5765 GC Rtx-5MS column (30 m × 0.32 mm,
nantly in the treatment of indigestion, intestinal disorder and 0.25 μm film thickness, FID), and ThermoQuest Trace GC
hypertension (3,11). Based on the pharmacological potential 2000 coupled with Finnigan Mat PolarisQ ion trap mass

*Address for correspondence Received: January 2008

Revised: April 2008
1041-2905/10/0001-085$14.00/0­—© 2010 Allured Business Media Accepted: August 2008

Vol. 22, January/February 2010 Journal of Essential Oil Research/85

 Pant et al.

spectrometer fitted with Rtx-5 MS fused silica capillary col- DPPH solution was freshly prepared daily, stored in a flask,
umn (30 m × 0.25 mm; 0.25 µm film thickness). The column covered, and kept in the dark at 4°C between the measure-
temperature was programmed for 60–210°C at 3°C/min with ments. The control and standard were subjected to the same
He as carrier gas at 1 mL/min and the injector temperature procedure except for the control, where there was no addition
at 210°C. MS were recorded under EI condition (70 eV) for of sample and the standard 5, 10, 15, 20 and 25 μL of sample
a mass range of 40–450 amu, injection volume of 0.1 µL with were replaced with 5, 10, 15, 20 and 25 mg of BHT, gallic acid
split mode of 1:40. The compounds were identified based on
retention indices, co-injections with authentic samples, mass
spectral library searches (NIST-Wiley), and comparing the
spectra with literature data (12).
Table I. Compositions (%) of the oils of the aerial and flower
Antioxidant Activity parts of Alpinia nutans
Compounds RI (%) in oil Detection
Antioxidant potential of the oils was evaluated in terms
of 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging Aerial part Flower
ability, chelation of Fe+2 and the reducing power in comparison a-thujene 931 1.5 1.2 A, B
with the standard antioxidants viz. butylated hydroxytolu- a-pinene 938 1.1 2.5 A, B
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ene (BHT), gallic acid and catechin. All determinations were camphene 952 - 0.3 A, B
performed in triplicate. sabinene 976 27.8 14.2 A, B, C
Reducing power activity: The reducing power of the b-pinene 980 1.3 0.3 A, B
myrcene 989 0.1 - A, B, C
essential oils was determined according to the methods of
a-phellandrene 1005 - 0.1 A, B
Singh et al. (13). Different amounts of the oils (5, 10, 15, 20 a-terpinene 1018 1.5 - A, B
and 25 μL) were mixed with 2.5 mL of the phosphate buf- p-cymene 1026 5.2 - A, B
fer (200 mM, pH 6.6) and 2.5 mL of 1% potassium ferricya- limonene 1031 0.3 - A, B
nide. The mixtures were incubated at 50°C. After incubation, 1,8-cineole 1035 17.4 10.8 A, B, C
g-terpinene 1060 5.1 19.4 A, B
2.5 mL of 10% trichloroacetic acid was added to the mixtures,
cis-sabinene hydrate 1069 1.3 1.8 A, B
followed by centrifugation at 650 g for 10 min. The upper terpinolene 1088 1.2 - A, B
layer (5 mL) was mixed with 5 mL of distilled water and 1 mL linalool 1098 3.3 1.6 A, B, C
of 0.1% ferric chloride and the absorbance of the resultant so- cis-p-menth-1-en-8-ol 1123 0.8 - A, B
lution were measured at 700 nm using vis-spectrophotometer trans-sabinol 1140 0.7 0.6 A, B
(Speteronic 20). The control and standard were subjected to terpinen-4-ol 1176 14.9 25.1 A, B, C
the same procedure expect for the control, where there was a-terpineol 1189 1.9 3.8 A, B, C
neral 1270 - - A, B
no addition of the sample and for the standard 5, 10, 15, 20
bornyl acetate 1286 0.3 - A, B
and 25 μL of the sample were replaced with 5, 10, 15, 20 and a-copaene 1378 0.1 2.2 A, B
25 mg of the BHT, gallic acid and catechin. Absorbance at b-caryophyllene 1419 3.9 0.3 A, B
700 nm is plotted against the different concentrations of the aromadendrane 1459 0.2 - A, B
oil. Increase is absorbance indicates increase in reducing power. (Z)-b-farnesene 1445 0.2 - A, B
Chelation of Fe+2: The chelating activity of essential oils a-himachalene 1447 0.1 - A, B
a-humulene 1454 0.5 - A, B
on ferrous ions (Fe+2) was measured according to the method
g-gurjunene 1473 0.1 0.3 A, B
of Decker and Welch (14). Different amounts of essential oil (5, germacrene D 1480 0.1 - A, B
10, 15, 20 and 25 μL) were first mixed in 1 mL of methanol then ar-curcumene 1483 0.2 0.6 A, B
mixed with 3.7 mL of deionized water. The mixtures were left b-selinene 1485 0.1 - A, B
for reaction with FeCl2 (2 mM, 0.1 mL) and ferrozine (5 mM, a-selinene 1493 0.3 - A, B
0.2 mL) for 10 min at room temperature and then absorbance b-curcumene 1512 0.5 0.9 A, B
g-cadinene 1513 0.5 - A, B
was measured at 562 nm using vis-spectrophotometer. A lower cubebol 1514 0.3 0.3 A, B
absorbance indicates a higher chelating power. The chelating (E)-nerolidol 1564 0.8 - A, B
activity of the oil on Fe+2 was compared with that of EDTA caryophyllene oxide 1582 1.3 1.0 A, B
at a level of 0.01 mM and citric acid at a level of 0.025 M and globulol 1583 0.4 - A, B
calculated according to the equation: humulene epoxide II 1607 0.1 0.3 A, B
10-epi-g-eudesmol 1619 0.3 - A, B
chelating activity (%) = [1 – (At/Ao)] × 100
epi-cubenol 1629 0.4 - A, B
where At is the absorbance of the sample at 562 nm and g-eudesmol 1630 - 0.5 A, B
Ao is the absorbance of the control at 562 nm. b-eudesmol 1648 0.4 0.8 A, B
DPPH radical scavenging activity: The scavenging a-eudesmol 1652 0.3 - A, B
effect on the DPPH radical was determined according to the a-cadinol 1654 0.2 - A, B
methods reported earlier (13). Various amounts of essential khusinol 1674 0.1 - A, B
Total identified (%) - 97.1 88.9 -
oil (5, 10, 15, 20 and 25 μL) were mixed with 5 mL of 0.004%
methanolic solution of DPPH. Each mixture was incubated Detection: A = KI on Rtx-5 capillary column, B = GC/MS, C = Co-injection with
standards.
for 30 min in the dark and the absorbance of the sample was
read at 515 nm using the UV-visible spectrophotometer. The

86/Journal of Essential Oil Research Vol. 22, January/February 2010

 A. nutans

and catechin. A lower absorbance indicated a higher radical test tubes of small size were taken and marked from 1–11. Using
scavenging power and was calculated according to the equation: aseptic technique, 0.5 mL of the nutrient broth was poured into
DPPH scavenging activity (%) = [1 – (At/Ao)] x 100 each tube, from the second through the eleventh of the series.
where At is the absorbance of the sample at 515 nm and Into the first and second tubes, 0.5 mL of the working solu-
Ao is the absorbance of the control at 515 nm. tion was poured. The contents of the second tube were mixed
well and 0.5 mL from it was transferred into tube no. 3, after
Antibacterial Assay proper mixing 0.5 mL was transferred to tube no. 4, continu-
ing this procedure to tube no. 10; the eleventh tube received
The oils of A. nutans were separately tested against no oil and served as control. To all the tubes then, 0.5 mL of
pathogenic microorganisms including four Gram-negative the inoculum, containing approximately 105 organisms/mL,
[Pasteurella multocida (MTCC 1148), Escherichia coli (MTCC was added. This was prepared by making a 1:1000 dilution in
443), Salmonella enterica enterica (MTCC 3223), Shigella nutrient broth of an overnight stock culture of the organism
flexneri (MTCC 1457)] and one Gram-positive [Staphylococ- tested. All tubes were then incubated at 37°C for 18–24 h
cus aureus (MTCC 737)] bacteria. Standard, pure, identified and examined microscopically for evidence of the growth (if
cultures of these test bacteria were procured from the Institute any). Pure essential oils were used as the working solution.
of Microbial Technology (IMTECH), Chandigarh, India as The lowest concentration of the essential oil preventing the
Microbial Type Culture Collection (MTCC) and maintained growth (manifested by turbidity) was taken as MIC of that oil.
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in the laboratory by regular subculturing on to nutrient agar.

Disc diffusion method: Antibacterial screening of the
Results and Discussion
oils was done by the disc diffusion method (13). The bacterial
suspension of 0.1 mL of (diluted 10 times) was added to the The results of GC and GC/MS analysis of the oils of A.
previously prepared nutrient agar plate and bacterial strain was nutans (aerial parts and flowers) are given in Table I. Forty-two
thoroughly spread over the agar surface using bent rod. The compounds were identified in the aerial parts’ oil contributing
sterilized Whatman filter paper no. 1 disc (5 mm in diameter) to 97.1% of the oil. The oil was found to be rich in monoterpe-
was thoroughly soaked with the oils (50 μL) and placed in noids (85.7%), whereas sesquiterpenoids contributed to 11.2%
the inoculated plates. These plates were incubated at 37°C of the oil. Sabinene (27.8%), 1,8-cineole (17.4%) and terpinen-
overnight to observe the zone of inhibitions (mm). The results 4-ol (14.9%) were characterized as the major components
were compared with the standard antibiotics. of the oil along with p-cymene (5.2%), g-terpinene (5.1%),
Assessment of minimum inhibitory concentrations linalool (3.3%) and b-caryophyllene (3.9%).
(MICs): MIC levels of the essential oils were determined by Twenty-three components were identified in the flower
tube dilution method (15). Eleven clear, sterile, cotton-plugged oil, contributing to 88.9% of the oil. Terpinen-4-ol (25.1%),

Table II. Antibacterial activity of the essential oils of Alpinia nutans by Disc Diffusion method against standard microorganisms
Essential Oil Amount of oil (μL/disc) Zone of inhibition (mm)
SA SF PM EC SE
A. nutans flower 50 18 13 19 13 22
A. nutans aerial parts 50 20 12 20 10 20
Reference used Conc. (μg/disc)
Amikacin 30 8 22 15 20 24
Ciprofloxacin 5 36 3.5 20 34 41
Ampicillin 10 19 21 21 20 25
Gentamicin 30 22 22 22 19 23
Tetracycline 30 11 14 12 26 12
SA = Staphylococcus aureus; SF = Shigella flexneri; PM = Pasturela multocida; EC = Escherichia coli; SE = Salmonella enterica enterica.

Table III. Minimum Inhibitory Concentrations (MICs) of the effective essential oils against standard microorganisms
Essential oil Minimum Inhibitory Concentrations (μL/mL)
SA SF PM EC SE
A. nutans flower 0.97 15.62 0.97 7.81 0.97
A. nutans aerial parts 0.97 15.62 0.97 31.25 0.97
SA = Staphylococcus aureus; SF = Shigella flexneri; PM = Pasturela multocida; EC = Escherichia coli; SE = Salmonella enterica enterica.

Vol. 22, January/February 2010 Journal of Essential Oil Research/87

 Pant et al.
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Figure 1. Reducing power of the oils of the Alpinia nutans

g-terpinene (19.4%), sabinene (14.2%), 1,8-cineole (10.8%) and The oils exhibited good Fe+2 chelating activity affording
linalool (1.6%) were the major monoterpenoids identified in the protection against oxidative damage. It either chelated metal
oil. Sesquiterpene hydrocarbons included a-copaene (2.2%) ions or suppressed reactivity by occupying all coordination
and b-caryophyllene (0.3%). Caryophyllene oxide (1.0%) sites of the metal ion (16).
and b-eudesmol (0.8%) were among oxygenated terpenoids DPPH scavenging activity: The radical scavenging activity of
identified in the oil. the test oils of A. nutans is shown in Figure 3. The aerial parts
Antioxidant activity: oil exhibited a higher activity than the corresponding flower oil.
Reducing power activity: The variation in the reducing Antimicrobial activity: The zones of inhibition (mm) of
power of the flower and aerial parts of A. nutans is shown in the essential oils against different bacterial strains are presented
Figure 1. The oil of the aerial parts exhibited higher reducing in Table II. Both the oils were found to be effective against
power than the flower oil. The reducing powers of these oils S. aureus. The flower oil was most effective against S. enterica
were, however, less than the standards. enterica, whereas the aerial parts oil was equally effective against
Chelation of Fe+2: Both the oils showed chelating activity S. aureus, S. enterica enterica and P. multocida.
on Fe+2 in a dose-dependent manner (Figure 2). Chelating Assessment of minimum inhibitory concentrations: The
activity of aerial parts oil was higher than that of EDTA at minimum inhibitory concentrations (MICs) of the oils are
0.01 mM and citric acid at 0.025 M (34.8 ± 1.1% and 30.8 ± expressed in Table III. These range from 0.97 to 62.5 μL/mL.
2.3%, respectively). Both the oils exhibited higher activity than The oils were most effective against S. enterica enterica and
EDTA and citric acid at all the tested levels. S. aureus.

88/Journal of Essential Oil Research Vol. 22, January/February 2010

 A. nutans
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Figure 2. Effect of the oils of the Alpinia nutans on the chelating activity of the Fe (II) ions

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Figure 3. Radical scavenging ability of the oils of Alpinia nutans on 2,2’-diphenyl-1- picrylhydrazyl radical

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