Indian Journal of Natural Products and Resources

Vol. 2(4), December 2011, pp. 504-507

Short Commucications

In vitro antimicrobial activity of aerial

parts extracts of Aconitum heterophyllum

Wall. ex Royle

Nidhi Srivastava1, Vikas Sharma

1, Kriti Saraf

1,

Anoop Kumar Dobriyal2, Barkha Kamal

1 and

Vikash Singh Jadon1*

1Plant Molecular Biology Laboratory, Department of

Biotechnology, Sardar Bhagwan Singh (P.G.) Institute of

Biomedical Sciences & Research Balawala, Dehra Dun-248 161,

Uttarakhand, India

2Department of Zoology & Biotechnology, H.N.B.Garhwal

University, Campus Pauri, Pauri Garhwal-246 001, Uttarakhand

Received 7 September 2010; Accepted 30 June 2011

Antimicrobial activity of extracts of aerial parts of the

plant samples of Aconitum heterophyllum Wall. ex Royle was

evaluated against different bacterial and fungal strains. It was

observed that methanolic extract (50 mg/well) was the most

potent extract which showed significant inhibition of the growth

of Gram positive bacteria, Staphylococcus aureus and Bacillus

subtilis. Antifungal activity was shown by two extracts against

Candida albicans and Aspergillus flavus. Methanolic extract

showed considerable antifungal activity while feeble antifungal

activity was obtained with ethyl acetate extract even at higher

concentrations.

Keywords: Aconitum heterophyllum, Antimicrobial, Atis, Methanolic

extract, Ranunculaceae.

IPC code; Int. cl. (2011.01) A61K 36/714, A61K 135/00,

A61P 31/04, A61P 31/10

Introduction Even though pharmacological industries have

produced a number of new antibiotics in the last three decades; resistance to these drugs by microorganisms

has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs,

which are utilized as therapeutic agents. The problem

of microbial resistance is growing and the outlook for the use of antimicrobial drugs in the future is still

uncertain. The use of plant extracts and phytochemical, both with known antimicrobial

properties can be of great significance in therapeutic use in such case. World Health Organization has

reported that 80% of the world’s population rely

chiefly on traditional systems of medicines and a major part of therapies involve use of plant extracts or

their active constituents and the organization is also

encouraging, promoting and facilitating the effective use of herbal medicines in developing countries for

health-care programmes. Many of these medicinal plants show antimicrobial activity that can kill or

inhibit the growth of microorganism such as bacteria, fungi or protozoa as well as destroying viruses.

Therefore, they represent a rich source from which

novel antibacterial, antifungal and chemotherapeutic agents may be obtained. The ongoing recognition of

medicinal plants is due to several reasons, including escalating faith in herbal medicines. Allopathic

medicines may cure a wide range of diseases;

however its high price and side-effects are causing many people to return to herbal medicine. Herbal

medicines have a major advantage associated with them that they have better acceptability by the body,

as our body rarely treats them as foreign material because they are always associated with natural and

biological entities, viz. protein, carbohydrate, lipids,

etc. India is perhaps the largest producer of medicinal herbs and is rightly called the botanical garden of the

world. There are very few medicinal herbs of commercial importance which are not found in this

country. India officially recognizes over 3000 plants

for their medicinal value. It is generally estimated that over 6000 plants in India are in use in traditional, folk

and herbal medicine, representing about 75% of the medicinal needs of the Third World countries

1. The

country has about 2500-3000 species of medicinal plants and our dependence on medicinal plants has in

no way minimized by the use of modern systems of

synthetic drug whose use are not without side-effects. Phytochemical contents of the genus Aconitum

(Family Ranunculaceae) have been bestowed with the number of medicinal compassion which includes

antibacterial, antioxidant antiproliferative, enzyme

inhibition activities, etc.2-7

. Aconitum heterophyllum Wall. ex Royle commonly known as Atis is one of the

most important medicinal plants of family Ranunculaceae. The parts used are dried tuberous

roots. The alkaloids from the roots have been reported to have considerable antibacterial and enzyme

inhibition activities8,9

. Although the main activity of ——————

*Correspondent author: E-mail: jadon@rediffmail.com

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A. heterophyllum is seen in the root, but in the present study attempts have been made to see the

antimicrobial activity of crude plant extracts of aerial parts of the plant. This is because the drugs derived

from the root extract are quite expensive as well as

with this we can utilize this golden plant wholly without any slaying.

Materials and Methods

Plant collection and preparation of extracts

Vegetative plants of A. heterophyllum were

procured from Forest Nursery, Munsiyari (Plate 1).

One plant specimen of A. heterophyllum was

submitted to Botanical Survey of India, Dehra Dun

for authentication and Accession No. 113341 was

obtained. The samples were washed and dried for

30-45 days in shade. Plants were weighed before and

after drying for calculating moisture content. Then the

aerial parts of the plant samples were crushed and

loaded to the Soxhlet Extractor. Solvents were used

on the basis of their increasing polarity (petroleum

ether, chloroform, ethyl acetate, methanol, aqueous)

and the temperature of the mantle was regulated

according to the boiling point of the respective

solvent. The crude extracts were dissolved in 1% (v/v)

DMSO for screening antimicrobial activity.

Test microorganisms

Bacterial and fungal strains were procured from

Microbial Test Culture Collection (MTCC),

Chandigarh, Punjab. The accession no. of Gram

positive bacterial strains i.e., Staphylococcus aureus,

Bacillus subtilis and Streptococcus pyrogenes are:

MTCC-3160, MTCC-441 and MTCC-442,

respectively; for Gram negative bacterial strains

Escherichia coli, Klebsiella pneumonia, Pseudomonas

aeruginosa and P. fragi are: MTCC-614, MTCC-

4030, MTCC-2453 and MTCC-2458, respectively.

The accession no. of fungal strains Candida albicans,

Aspergillus niger, A. fumigatus, A. flavus are:

MTCC-183, MTCC-961, MTCC-2584 and MTCC-2456,

respectively.

Antimicrobial activity

The antibacterial activity was determined by Agar

Well Diffusion Method10

using Mueller Hinton Agar

(MHA). One loop full of 24 h old cultures (obtained

from MTCC) containing approximately 104-10

6 CFU

was spread on the surface of the medium. Wells were

dug in the medium with the help of sterile metallic

cork borer. Stock solutions of the test samples were

prepared by dissolving 100 mg of each extract in 1 ml

of 1% (v/v) DMSO, and 100-500 µl (10-50 mg/well)

of each samples were added in the respective wells

and incubated for 24-48 h at 35°C. The antibacterial

activity was compared with the two standard

antibacterial antibiotics Amoxicillin and Cefuroxime.

For the evaluation of antifungal activity, slants were

prepared from Sabourauds dextrose agar (SDA) and

inoculated with sample solution (250-500 µl). The

fungal cultures were then inoculated on the slants and

incubated for 5-7 days at 27°C and growth inhibition

was compared with standard Nystatin. 1% (v/v)

DMSO was used as control in both the case. These

tests heavily depend on variable experimental

conditions such as media composition, temperature,

pH and inoculum size, etc. So, all the possible

precautions and precessions were taken in the entire

course of study to get correct experimental results

without much variation. Minimum Inhibitory

Concentration (MIC) was determined by using the

different concentrations of methanolic extracts against

bacterial cultures. These test organisms were also

subjected to increasing concentrations of methanolic

extract (5, 10, 20, 40 and 60 mg/ml) for estimating

surviving fractions.

Results and Discussion

Moisture content of green plant samples of A.

heterophyllum collected from Forest Nursery,

Deoban-Chakrata, was found to be 35%. The

percentage yield of each extract was determined,

which revealed the maximum yield of aqueous

extract. Among all the extracts tested, methanolic

extract was found to be the most potent extract against

both bacteria and fungi.

Results of antibacterial and antifungal activity have

been tabulated in Table 1 and 2, respectively (Plate 2).

At higher concentration of methanolic extracts

(50mg/well) growth in both the groups of bacteria

Plate 1 Aconitum heterophyllum Wall.

INDIAN J NAT PROD RESOUR, DECEMBER 2011

506

was inhibited and the results were comparable to

standard antibiotics Amoxicillin and Cefuroxime

(100µg/well) only to some extent. It showed fair

activity against S. aureus and B. subtillis. Same

results were also obtained when essential oils

obtained from A. tanguticum (Maxim.) Stapf. were

used and it showed activity against methicillin

resistant S. aureus3. The antifungal activity of all the

extracts revealed that methanolic extract at higher

concentration inhibited the growth of Candida

albicans and Aspergillus flavus firmly but inhibited

the growth of other strains gauzily. Ethyl acetate

extract also showed inhibition against some strains

but was very weak even at higher concentration.

However, these results were not comparable with the

standard antifungal Nystatin 30µg (15-25mm). These

results are in a little proximity with the results

obtained with crude methanolic and ethyl acetate

extracts of A. chasmanthum Stapf. ex Holmes against

bacterial and fungal strains2.

Minimum Inhibitory Concentration (MIC) of the

methanolic extract was found to be 40mg/well against

B. subtilis and for S. aureus, it was 50mg/well.

Determination of the surviving fractions of strains

against the increasing concentrations of methanol

revealed that there was a considerable decrease in the

surviving fractions with the increasing concentration

of the extracts. Least surviving fractions (0.0047)

were found in case of B. subtilis, while maximum

were in case of Pseudomonas aeruginosa (0.543),

Table 1 Antibacterial activity of crude methanolic extract of Aconitum heterophyllum

Zone of Inhibition (mm)

Crude Methanolic Extract (mg/well) Standard Antibiotics (µg/well)

Amoxicillin Cefuroxime

Groups

10 20 30 40 50 100 500 100 500

Gram positive

Staphylococcus aureus - - - 7 13 17 25 16 25

(MTCC-3160)

Bacillus subtilis - - 6 10 16 16 24 10 15

(MTCC-441)

Streptococcus pyogenes - - 6 6 7 16 25 16 23

(MTCC-442)

Gram negative

E. coli (MTCC-614) - - - - 9 17 23 17 30

Klebsiella pneumonia - - - - 7 11 14 11 16

(MTCC-4030)

Pseudomonas aeruginosa - - - - - - - - 16

(MTCC-2453)

Pseudomonas fragi - - - - - - 11 - 16

(MTCC-2458)

Table 2 Antifungal activity of crude methanolic extract of

Aconitum heterophyllum.

Inhibition Intensity

Crude Methanolic Extract Nystatin

(mg/well)

Name of

organisms

10 20 30 40 50 30 µg

Candida albicans - - + ++ +++ 25 mm

(MTCC-183)

Aspergillus niger - - - - + 12 mm

(MTCC-961)

A. fumigatus - - - - + 13 mm

(MTCC-2584)

A. flavus - - + ++ +++ 15 mm

(MTCC-2456)

+++ (considerable inhibition): ++ (average inhibition): + (weak

inhibition): - (no inhibition)

Plate 2 Antimicrobial activity of methanolic extracts of

Aconitum heterophyllum aerial parts: a- Antibacterial activity

against Staphylococcus aureus, numbers 1-5 indicates 10-50 mg

of extracts; b- Antifungal activity against Candida albicans.

a b

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when the concentration of extracts was 60mg/ml.

Other species of Aconitum are also restrained with

various biological activities, e.g., A. orochryseum

Stapf. contain bioactive alkaloids which are

potential antimalarial drug leads. It could also have

antiviral activities since the plant was indicated

ethno-medically for treating cold and flu. From

A. orochryseum 11 alkaloids were obtained11

. Thus, this

genus provides lots of species which are manifested

with different biological and pharmacological

activities. But still this area needs to be explored more

scientifically and prudently to utilize these golden

herbs more efficiently.

Conclusion

In the present study it has been observed that

methanolic extract of aerial parts of A. heterophyllum

was the more potent extract. Thus, this protocol can

be efficiently used for combating the various

microbial diseases caused by the strains studied after

some standardization and enhancement of extraction

method of active constituent. This can only be

achieved by integrated techniques including

cultivation and conservation, adequate processing,

formulations and marketing.

Acknowledgements

The authors are thankful to Management, Sardar

Bhagwan Singh (P.G.) Institute of Biomedical Sciences

& Research Balawala, Dehra Dun, Uttarakhand, India

for providing necessary research facilities. The

Uttarakhand Council of Science and Technology

(UCOST) is thanked for financial assistance.

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