assessment of antimicrobial activity of alpinia … · international journal of research article...

INTERNATIONAL JOURNAL OF RESEARCH ARTICLE PHARMACEUTICAL INNOVATIONS ISSN 2249-1031

1 | P a g e Volume 4, Issue 1, January ₋ February 2014 http://www.ijpi.org

ASSESSMENT OF ANTIMICROBIAL ACTIVITY OF Alpinia Calcarata

Roscoe. - A VALUABLE MEDICINAL PLANT

Silvy Mathew*, S. John Britto, Sinjumol Thomas

The Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph’s College

(Autonomous), Tiruchirappalli -620002, Tamilnadu (St), India

ABSTRACT

The present study was designed to investigate the anti-microbial activity of four solvent

extracts (Petroleum ether, Dichloromethane, Acetone and Methanol) of rhizome of Alpinia

calcarata Rosce. The rhizome of this plant is an important antimicrobial agent and a

digestive stimulant. The plant extracts showed considerable activity against Ten tested strains

viz., Pseudomonas aeroginosa, Escherichia coli, Enterobacter aerogens, Bacillus subtilis,

Staphylococcus aureus, Streptococcus faecalis, Vibrio cholerae, Salmonella paratyphi,

Klebsiella pneumoniae and Proteus vulgaris by using agar disc diffusion assay. This study

reveals that Alpinia calcarata has antimicrobial activity against gram positive and gram

negative bacteria. Methanol extract showed inhibition against eight strains and showed

highest inhibition zone (15±0.3) against Bacillus subtilis. The minimum inhibitory

concentration (MIC) of the Alpinia calcarata was ranging from 0.39 mg mL-1

to 0.65 mg

mL-1

.

Keywords: Disc diffusion assay, Organic extracts, Rhizome, Gram positive, Gram negative,

minimum inhibitory concentration.

INTRODUCTION

From ancient time many medicinal plants

had been used as antimicrobial agents

(Mahesh and satheesh, 2008). In recent

years researchers have focused on the

higher plants for the search of

antimicrobial substances (Trakranrungsie

et al. 2004; Udomkusonsri et al. 2007).

Medicinal plants have been a major

sources of cure for human diseases since

time immemorial. It is no wonder that one-

fourth of world population i.e. 1.42 billion

people are dependent on traditional

medicines for the treatment of various

ailments (Reddy 2004). This practice

continues inspite of advances in the field

of allopathy during 20th

century. Thus

plants still remain one of the major sources

of drug in the modern as well as traditional

system of medicine throughout the world.

Many plants contain microbial inhibitors

(Voravuthinkuchai 2006) and they are

used as traditional medicines and as

sources of wide range of substances which

can be useful to treat infectious or other

serious diseases (Yineger H, 2009).

Zingiberaceae as one of the largest

Corresponding Author*

Silvy Mathew



families of the plant kingdom include 53

genera and over 1300 species (Kai Larsen,

K 1980) and well known for a number of

medicinal properties (Kumar et al. 2006,

Prajapathi et al. 2005). Alpinia is the

largest genus of the family with more than

200 species. Albuquerque (2004) recently

detected a broad spectrum of antibacterial

and antifungal compounds in this genus

(Ibrahim, 2009). Alpinia calcarata

commonly known as snap ginger is an

important cultivated medicinal crop of

India and it is known for its antimicrobial

activity (George et al., 1949, Robinson et

al., 2009). It is widely distributed in

warmer part of Asia and India (Kritikar

2003). It is a well known hailed official

drug throughout the country and also as a

holistic gift of nature with varied

medicinal, culinary and cosmetic uses.

This species has various therapeutic

activities, viz. thermogenic, aromatic,

nervinetonic, stimulant, revulsive,

carminative, stomachic, disinfectant,

aphrodisiac, expectorant, broncho-dilator,

antifungal, febrifuge, tonic and many more

(Warrier et al. 1993). The rhizomes of

Alpinia calcarata are anti-inflammatory

(Asolkar, et al. 1992).

MATERIALS AND METHODS

Collection of Plant Material and

Extraction

The rhizomes were collected in the month

of December 2012 from the botanical

garden of St. Joseph’s College,

Mannanam, Kottayam, Kerala and a

voucher specimen has been deposited in

Rapinat Herbarium, Tiruchirappalli for

future reference (Fig.1).

Fig.1. Alpinia calcarata - inflorescence

The rhizomes were washed thoroughly in

tap water, air – dried, powdered and stored

in an air – tight container for further use.

80 g of powdered rhizomes were soaked in

300 ml of Petroleum ether,

Dichloromethane, Acetone and Methanol

separately in Soxhlet apparatus for 48h at

310 C until complete extraction of

materials. Extracts were filtered using a

buckner fnnnel using Whatman No.1 filter

paper and filtrates were concentrated at

room temperature in order to reduce the

volume into 50ml. The concentrated

extract was stored in pre-weighed screw

capped bottle and kept in refrigerator at

40C.

Bacterial Strains

The antibacterial acitivities of the plant

extracts were studied against three pure

cultures of gram positive bacteria viz.,

Bacillus subtilis MTCC # 441 (BS),

Staphylococcus aureus MTCC # 3163

(SA), Streptococcus faecalis MTCC # 459

(SF) and seven pure cultures of gram

negative viz., Escherichia coli MTCC #

119 (EC), Enterobacter aerogenes MTCC

# 2990 (EA), Klebsiella pneumoniae

MTCC # 3040 (KP), Pseudomonas



aeruginosa MTCC # 2474 (PA),

Salmonella paratyphi MTCC # 734 (SP),

Vibrio cholerae ATCC # 14104 (VC),

Proteus vulgaris # MTCC 1771(PV).The

purity and viability of cultures were

checked by culturing on nutrient agar

slants and incubated at 370C for 24 h. The

isolates were subcultured in nutrient broth

and stored in 40C.

Antibacterial activity

A modified agar diffusion method was

adopted to determine antibacterial activity.

Around 6mm diameter of discs of

whatman filter paper (No.1) were

impregnated with 10µl of crude extracts

using sterile automatic pipettes. The discs

were allowed to dry at room temperature

for 2h and were placed at equidistance in

each of the plates using a sterile forceps.

The reference antibiotic (RA) discs

containing Ciproflaxin were prepared at

appropriate concentrations to serve as the

positive controls for gram positive and

gram negative bacteria respectively. Each

plate then was poured with melt soft agar

containing 105CFU/ml of the bacteria. The

air dried discs were placed on inoculated

agar plates. The plates were incubated at

370C for 24 hr. The area of the zone of

inhibition was measured. The antibacterial

activity was expressed as the mean zone of

inhibition diameters (mm) produced by the

plant extract. Studies were performed in

triplicates and mean and standard errors

(mean ± S.E.) were calculated.

Minimum inhibitory Concentration

(MIC)

Bacterial strains that were sensitive to

rhizome extracts of dried Alpinia calcarata

by the agar disc diffusion method were

further tested to determine minimum

inhibitory concentrations (MICs). The

Minimum inhibitory Concentrations of the

methanol extract was tested against the

Bacillus subtilis MTCC # 441 (BS),

Staphylococcus aureus MTCC # 3163

(SA), Streptococcus faecalis MTCC # 459

(SF), Escherichia coli MTCC # 119 (EC),

Klebsiella pneumoniae MTCC # 3040

(KP), Salmonella paratyphi MTCC # 734

(SP), Vibrio cholerae ATCC # 14104

(VC), Proteus vulgaris # MTCC 1771(PV)

cultures in a 96 – well microtiter plate.

The tested sample was first of all dissolved

in DMSO (stock - 200mg mL-1

) and the

solution obtained was added to NBPG

(Nutrient broth containing 0.05% phenol

red and supplemented with 10% glucose)

to a final concentration of 4 mg mL-1

(0.1

mL of stock solution to 5mL of NBPG) for

each crude extract. 100µL of each

concentration was added in a well

containing 95 µL of NBPG and 5 µL of

standard inoculums. The plates were

covered with a sterile plate scale, and

incubated at 350C for 24h. The assay was

repeated twice. Microbial growth was

determined by observing the change of

colour in the wells (red when there is no

growth and yellow when there is

growth).The lowest concentration that

does not permit any visible growth when

compared to the control (no colour change)

was considered as the Minimum inhibitory

Concentration (MIC).

RESULTS AND DISCUSSION

Petroleum ether, Dichloromethane,

Acetone, methanol extracts of the rhizome

powder were screened for their

antibacterial effects. Methanolic extracts

of rhizomes showed a wide spectrum of

activity against test bacteria (Table.1).



Table.1. Antimicrobial profile of different extracts of rhizomes of Alpinia calcarata against

human pathogenic bacteria (Zones if inhibition in mm)

Extraction B.

subtilis

S.

aureus

S.

faecalis

E.

coli

E.

aerogenes

K.

pneumoniae

P.

aeruginosa

S.

paratyphi

V.

cholera

P.

vulgaris

Petroleum

ether R R R 7 ±

0.3 R R R R R R

Dichloromet

hane R R R R R R R R R R

Acetone 11 ±

0.2

R R 8 ±

0.4 14 ± 0.3 10 ± 0.3 13 ± 0.6 R 9 ± 0.4 7 ± 0.3

Methanol 15 ±

0.3

10 ± 0.3

10 ± 0.4

12 ±

0.4

R 8 ± 0.6 R 7 ± 0.5 9 ± 0.3 10 ± 0.4

Reference

Antibiotic

(Ciproflaxin)

16±

0.5

11 ± 0.2 15 ±

0.5

- 11 ± 0.4 - 17 ± 0.2 15 ± 0.4 14 ±

0.6

7 ± 0.6

Values are mean of three trials ± standard error. R = resistance to the extract

This suggests that methanol is a good

solvent for extracting bioactive compounds

extract from Alpinia calcarata against

bacteria. The diameter of inhibition zone

for each of the organisms was compared

with standard antibiotic. It was noted that

the inhibition zone of the organisms to be

either less than or greater than or equal to

standard antibiotic. Higher inhibition zone

of (15 ± 0.3 mm) was recorded for the

methanol extracts of rhizomes against

Bacilus subtilis. Methanol extracts have

MIC values of 0.39mg mL-1

for Bacilus

subtilis (Fig. 3 & 4).

CONCLUSION

Antimicrobial properties are useful tools in

the control of microorganisms especially

in the treatment of infections and food

spoilage. Based on these results, we may

conclude that Alpinia calcarata showed

antibacterial activity against all tested

organisms and had large inhibition against

B.subtilis. The promissory extracts of

Alpinia calcarata open the possibility of

finding new clinically effective

antimicrobial compounds. The MIC is a

helpful parameter used to assess

bacteriostatic activity under similar

conditions.



Fig.3. MIC plate shows the antibacterial effects of the methanolic rhizome extract of

Alpinia calcarata

Fig.4. MIC values of the methanolic rhizome extract of Alpinia calcarata

mg

mL-

1

Bacteria



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