Antimicrobial Studies on Selected Medicinal Plants in Khandesh Region, Maharashtra, India

 

M.A. Chaudhari1*, S.M. Sarode2, Y.A. Chaudhari3, G.Vidyasagar2 and M.K. Kale1

 

1Department of Pharmacology, K.Y.D.S.C.T’s College of Pharmacy, Sakegaon- 425201

2Veerayatan Institute of Pharmacy, Bhuj, Gujarat.

3TVES’S Hon’ble L. M. C. College of Pharmacy, Faizpur

 

 

ABSTRACT:

Medicinal plants contribute in human health care system. Most of the plants utilized by village peoples as a folk medicine. Now we are turned in to medicinal plant analysis of active compounds and conservation aspect. In the present study we had select the four important medicinal plants in the Khandesh region. Such plants are widely used in this region for ayurvedic purpose by villagers and local herbal health masters. We have colleted four medicinally important medicinal plants such as Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri for antimicrobial studies. The experiment carried out in the selected medicinal plants leaves and roots. The results are discussed with the available literature.

 

 

 

INTRODUCTION:

In India, the use of different parts of several medicinal plants to cure specific ailments has been in vague from ancient times. The indigenous system of medicine namely Ayurvedic, Siddha and Unani have been in existence for several centuries. These systems of medicine cater to the needs of nearly seventy percent of our population residing in the villages. In Homeopathy system, 70% of the medicines are prepared from plants. As Homeopathy originated in Europe naturally, majority of the drugs prepared from plants are of exotic origin1. The plants supply us with large number of excellent “chemicals” which form sources for different types of drugs. The present trend in modern medicine is towards a change from the use of cellulose coated medicinal pills to extracts of plant supplied either in pure forms or in synthetic versions for curing many human ailments. Thus plants have provided the blue prints for the modern medicine.

 

From over 3, 00,000 species of higher plants to occur in nature, only about 2 percent have been screened so for. Extract of plants from 157 families have been reported to be active against microorganisms2.

 

Plants are rich in alkaloids and other phytochemical contents and many of them are effectively used to cure a wide range of ailments. The alkaloids and phytochemical contents may be present in all organs of the plant including roots, stems, buds, leaves, flowers and fruits.

 

All plant parts syntheses some chemicals with themselves which metabolite their physiological activities. These phytochemicals were used to cure the disease in herbal and homeopathic medicines. Now a day most of the peoples like to use the traditional methods to cure general diseases. Plant based to


control major diseases and the need to discover new molecular structures as lead compounds from the plant kingdom3. This worldwide interest in medicinal plants reflects recognition of the validity of many traditional claims regarding the value of natural products in health care and the development of microbial resistance to the available antibiotics has led the authors to investigate the antimicrobial activity of medicinal plants.

 

MATERIALS AND METHODS:

Plant materials are collected from the Revenue Village of Pal, Jalgaon District, Khandesh region. Plants are identified and confirmed with the authentic. Plant selected for the present study was Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri. Fresh leaves and roots are collected and shade dried under room temperature. The dried leaves and roots are grained into a coarse powder and used for further investigations.

 

The dried powdered leaves and roots were macerated by using mortar and pestle. The collected Sam herbarium specimen available in the Botany Department of D.N. College, Faizpur was analyzed for photochemical analysis by quantitative analysis.

 

Extraction: A soxhlet apparatus were used for the extracting antimicrobial active compounds from the plant leaves and roots. The collected plant leaves and roots were shade dried and powdered separately. 20 gm of dried powder was packed with thimble and then subjected to extraction with the water and ethanol separately. The collected extracts were concentrated by evaporation under room temperature. The collected extracts were then chosen for antibacterial activity.

 

Bacterial Inoculum Preparation: Bacterial cultures used in this study were obtained from MTCC, Chandigar. Bacterial cultures included in this study were Escherichia coli, Proteus vulgaris, Staphylococcus aureus and Bacillus subtilis. All the cultures were grown in Muller - Hilton agar medium. The inoculum was used for antibacterial assay.

 

Antibacterial Assay: The media and the test bacterial cultures were poured into dishes. [Muller - Hilton agar media]. The test strain (0.2 ml) was inoculated into the media to inoculum size (108cells/ml) when the temperature reached 40-42°C. Care was taken to ensure proper homogenization. The plant extracts were tested for antibacterial activity in the agar well diffusion assay, against Escherichia coli, Proteus vulgaris, Staphylococcus aureus and Bacillus subtilis

 

Agar Well Diffusion Method: The antibacterial activity was tested against (Ethanol, Aqueous) leaves and roots of Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri. The inoculation of microorganism was prepared from bacterial culture4. About 15-20 ml of Muller-Hilton agar medium was poured in the sterilized petridish and allows solidifying. One drop of bacterial strains was spread over the medium by a rod. Wells of 6nm in diameter and about 2 cm apart punctured in the culture medium using sterile Cork borers. About 100 ml of plant extracts was added to the wells. Plates were incubated in air at 37°C for 24 hours. Antibacterial activities were evaluated by measuring inhibition zone diameters.

 

RESULTS:

Medicinally important plant species viz., Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri were selected for screening of secondary metabolites. During this investigation, an attempt has been made to decipher the effect of these secondary metabolites towards its antibacterial activities. Ethanol and aqueous extracts from the leaf and root of Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri exhibit antibacterial activity against Escherichia coli, Proteus vulgaris, Staphylococcus aureus and Bacillus subtilis.

 

Antibacterial Activity: The effect of different extracts of four test plants on Escherichia coli was shown in Tables 1 and 2. The results clearly showed that plant extracts were specific in action against the growth of bacteria. Ethanol extract was most effective followed by aqueous extract. Escherichia coli were more sensitive for ethanol extract of roots and leaves of all the tested plants. Aqueous extracts did not show much inhibition against the tested organism compared to ethanolic extract. The extracts of leaf of Phyllanthus niruri exhibit relatively higher zone of inhibition followed by Cassia auriculata, Eclipta alba and Acalypha indica.

 

 

Table 1: Effect of ethanolic extract of root and leaf samples of certain medicinal plants on Escherichia coli (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

2.4

4.8

0.5

1.9

3.8

Cassia auriculata

0.5

1.9

3.8

0.5

2.2

4.4

Eclipta alba

0.5

2.6

5.2

0.5

1.8

3.6

Phyllanthus niruri

0.5

2.5

5.0

0.5

2.2

4.4

 

 

 

 

 

 

 

 

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

 

 

 

Table 2: Effect of aqueous extract of root and leaf samples Of certain medicinal plants on Escherichia coli (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

1.3

2.6

0.5

1.4

2.8

Cassia auriculata

0.5

1.6

3.2

0.5

2.1

4.2

Eclipta alba

0.5

1.6

3.2

0.5

1.6

3.2

Phyllanthus niruri

0.5

1.9

3.8

0.5

2.2

4.4

 

 

 

 

 

 

 

 

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

 

Tables 3 and 4 showed the antibacterial activity of ethanolic and aqueous extracts (root and leaf) of the tested plants on the selected bacteria Proteus vulgaris. The results showed inhibition diameters ranging from 1.2 cm to 2.1 cm. Aqueous leaf extract from all the four plants were found to have a higher antibacterial activity compared to ethanolic leaf extracts. Except Eclipta alba aqueous extract of all the three plants exhibit relatively higher zone of inhibition compared to ethanolic extract. The susceptibility of the test organism Staphylococcus aureus to the test plants was given in Tables 5 and 6. Ethanolic extracts (leaf and root) were most effective followed by aqueous extracts. Maximum inhibition was recorded in ethanolic root extract of Cassia auriculata. This was followed by ethanolic leaf extracts of Phyllanthus niruri, Eclipta alba and Cassia auriculata. The test organism was resistant to the aqueous root extracts of Acalypha indica and Phyllanthus niruri.

 

Table 3: Effect of ethanolic extract of root and leaf samples of certain medicinal plants on Proteus vulgaris (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

1.1

2.2

0.5

1.2

2.4

Cassia auriculata

0.5

1.3

2.6

0.5

1.9

3.8

Eclipta alba

0.5

1.4

2.8

0.5

1.5

3.0

Phyllanthus niruri

0.5

1.3

2.6

0.5

1.7

3.4

 

 

 

 

 

 

 

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

 

Table 4: Effect of aqueous extract of root and leaf samples of certain medicinal plants on Proteus vulgaris (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

1.7

3.4

0.5

1.4

2.8

Cassia auriculata

0.5

1.6

3.2

0.5

1.6

3.2

Eclipta alba

0.5

2.1

4.2

0.5

1.2

2.4

Phyllanthus niruri

0.5

2.1

4.2

0.5

1.4

2.8

 

 

 

 

 

 

 

 

 

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

 

Table 5: Effect of ethanolic extract of root and leaf samples of certain medicinal plants on Staphylococcus aureus (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

1.2

2.4

0.5

1.9

3.8

Cassia auriculata

0.5

1.9

3.8

0.5

2.4

4.8

Eclipta alba

0.5

1.9

3.8

0.5

1.8

3.6

Phyllanthus niruri

0.5

2.1

4.2

0.5

1.7

3.4

 

 

 

 

 

 

 

 

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

 

Table 6: Effect of aqueous extract of root and leaf samples of certain medicinal plants on Staphylococcus aureus (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

1.7

3.4

0.5

-

-

Cassia auriculata

0.5

1.9

3.8

0.5

1.7

3.4

Eclipta alba

0.5

1.8

3.6

0.5

1.7

3.4

Phyllanthus niruri

0.5

1.9

3.8

0.5

-

-

 

 

 

 

 

 

 

 

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

The effect of aqueous and ethanolic (root and leaf) extracts against Bacillus subtilis were depicted in Tables 7 and 8. The highest antibacterial potentials were observed from the ethanolic root extracts of Acalypha indica, Cassia auriculata and Eclipta alba which inhibited 2.6, 2.5 and 2.3 cm of the tested microorganism.

 

Table 7: Effect of ethanolic extract of root and leaf samples of certain medicinal plants on Bacillus subtilis (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

1.9

3.8

0.5

2.6

5.2

Cassia auriculata

0.5

2.3

4.6

0.5

2.5

5.0

Eclipta alba

0.5

2.1

4.2

0.5

2.3

4.6

Phyllanthus niruri

0.5

1.7

3.4

0.5

1.8

3.6

 

 

 

 

 

 

 

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

 

Table 8: Effect of aqueous extract of root and leaf samples of certain medicinal plants on Bacillus subtilis (RMI-cm2)

Name of Medicinal Plant

Leaf

Root

A1

A2

RMI

A1

A2

RMI

Acalypha indica

0.5

-

-

0.5

1.6

3.2

Cassia auriculata

0.5

1.7

3.4

0.5

1.8

3.6

Eclipta alba

0.5

1.2

2.4

0.5

2.7

5.4

Phyllanthus niruri

0.5

1.8

3.6

0.5

2.1

4.2

A1 = Area of well in cm2; A2 = Area of Zone of inhibition in cm2 (including area of well); RMI = A2/A1

 

Cassia auriculata and Eclipta alba ethanolic leaf extracts exhibit more inhibition than Acalypha indica and Phyllanthus niruri. Aqueous leaf extract of Acalypha indica did not respond for its antibacterial activity against Bacillus subtilis. Aqueous root extract of Eclipta alba exhibit more relative magnitude of inhibition [RMI] value compared to the other three plants studied.

 

The growth of bacteria was suppressed by phytochemical compounds of ethanol and aqueous extracts of all the plants with different magnitudes. Cassia auriculata possess higher antibacterial activity among the tested plants. This was followed by Eclipta alba, Phyllanthus niruri and Acalypha indica.

 

The growth of Escherichia coli was inhibited more with ethanolic extracts of Eclipta alba, followed by Phyllanthus niruri and Acalypha indica. Proteus vulgaris showed maximum inhibition with Eclipta alba and Phyllanthus niruri aqueous leaf extracts. Staphylococcus aureus was more sensitive to ethanolic root extract of Cassia auriculata. Bacillus subtilis was resistant to aqueous leaf extract of Acalypha indica compared to all the other three plants studied.

 

DISCUSSION:

In the present study, four medicinal plant species viz., Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri were screened to detect the presence or absence of several bioactive compounds which are reported to cure different diseases and ailments. It was confirmed that the presence of antimicrobial substances in all the tested plants in one form (or) other. The antimicrobial compounds may be found as anthroquinone, alkaloids, catachols, flavonoids, phenolic compounds, saponins, steroids, tannins and triterpenoids, whose presence may be attributed to the medicinal properties of plants5-8.

 

In the present study, Escherichia coli was sensitive to all the four plant extracts and exhibited more or less same Relative magnitude of inhibition (RMI). This is in consonance with the earlier report9 in the seed extract of Citrullus vulgaris and Eclipta alba, in Allium sativum, Citrullus vulgaris and Eclipta alba and in Melia azadirachta against Escherichia coli10-13. On contrary, Escherichia coli was more resistant to acetone extract of Acorus calamus, Zingiber officinal, Cinnamomum zeylanicum, Moringa olifera and Ocimum sanctum14. Proteus vulgaris was sensitive with ethanolic extracts of Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri. The results obtained in this study indicated a considerable difference in antibacterial activity between extracts obtained with ethanol and water. Aqueous leaf extract was more active than the ethanol extract. The widest zone of inhibition was observed with aqueous leaf extracts of Eclipta alba and Phyllanthus niruri. It is in corroboration with the earlier study in the aqueous extract of neem against Salmonella weltevreden15. Reported water extracts of oriental herbal medicines were more antibacterial against Staphylococcus aureus and Bacillus subtillis. On contrary, Bacteria and yeast were more resistant to aqueous extract of Micromeria nerosa16.

 

In the present investigation the ethanolic extract of all the four plants exhibited higher Relative magnitude of inhibition (RMI) against staphylococcus aureus. This credit to ethanol extraction was supposed to be because ethanol is an organic solvent and will dissolve organic compounds better than aqueous extract and also liberate the active component required for antibacterial activity. The volatile components of acetone extracts of Ocimum sanctum were more effective against all the tested microorganisms viz., Staphylococcus aureus, Escherichia coli and Klebsiella speciesVarious organic extracts of Eclipta prostrata against Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shigella dysenteriae and Bacillus subtilis and Cassia elata leaf extract against Staphylococcus aureus and Escherichia coli17-19.

 

Ethanol and aqueous extracts of Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri against Bacillus subtilis revealed that ethanolic root extract seem to be more active compared to aqueous extract. Seed extracts of Citrullus vulgaris, Eclipta alba, Sida acuta and Achyranthus aspera on Bacillus subtilis, Escherichia coli and Salmonella typhimurium20.

It was clear from this study that the solvent of extraction and method of extraction affected the degree of antimicrobial activity. Other factors such as the environmental and climatic conditions of the plants also affected the degree of antimicrobial activity. Successful prediction of botanical compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Traditional healers use primarily water as the solvent but in our studies we found that plant extract in organic solvent provided more consistent antimicrobial activity compared to those extracted in water.

Many substances may be antimicrobial, but only a few of them will be potential therapeutic agents for the simple reason that mammalian cells are more sensitive to chemical inhibition than microbial cells21. Moreover, emphasized the need for toxicity testing of drugs derived from medicinal plants because the crude products obtained from such cheaper sources are often associated with a large number of compounds that have discomforting abilities22. Hence the herbal drugs have to be subjected to extensive pharmacological, toxicological and clinical tests to conform the prescribed status. Thus the ethnobotanical approach will be like a search for molecular diversity subjecting a wide variety of new molecules from plant sources and testing them with as many different tests as possible22.

 

The present study has shown a spectrum of antibacterial activities, which provides a support to some traditional uses of these few medicinal plants. But the effective biomolecules which act as antibacterial have to be identified isolated and subjected to extensive scientific and pharmacological screening that can be used as sources for new drugs.

 

ACKNOWLEDGEMENT:

The authors are gratefully acknowledged The Secretary, Principal and The Head, Department of Botany, D. N. College, Faizpur. and College of Pharmacy, Sakegaon. Maharashtra, India and who are encourage and given their valuable suggestion during the study period.

 

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Received on 24.06.2010

Accepted on 07.07.2010        

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(5): Sept.-Oct. 2010, 386-390