Antimycobacterial and Antimicrobial Activity of Leaf Extracts of Vitex negundo Linn.

 

 

PL Ladda1* ,NS Naikwade1 and CS Magdum2

1Appasaheb Birnale College of Pharmacy, south-Shivaji nagar, Sangli-Miraj road,Sangli.416416(MS)

2Ashokrao Mane College of Pharmacy, Pethwadgaon., (M.S.) India.

 

ABSTRACT:

Vitex negundo belonging to the family verbanaceae has been reported to posses many medicinal properties according to indigenous system of medicine. The present work deals with antimycobacterial and antimicrobial activity of vitex negundo leaves. The different solvent extracts of the leaves prepared were studied for their antimycobacterial activity by Conventional proportion method and Nitrate reductase assay method. The activity was compared with standard drugs rifampicin and isoniazid. The ethanolic, successive ethanolic and methanolic extracts showed highest antitubercular activity, while petroleum ether extract and chloroform extract was moderately active as compared to other extracts of Nirgundi. Aqueous, benzene, acetone extract does not shows any antitubercular activity

Antimicrobial activity of all extracts was screened by Paper Disc method against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The benzene extract showed significant antimicrobial activity compared to petroleum ether and chloroform extract. Preliminary phytochemicals of all extracts were also investigated.

Conclusion:

In conclusion nitrate reductase assay was found to be rapid, inexpensive and easy to perform. It also showed good concordance between conventional proportion method and nitrate reductase assay.

All these findings justified the claim made in the indigenous system of medicine for the use of the plant vitex negundo against tuberculosis and many other micro-organisms.

 

KEYWORDS: Antimicrobial, antitubercular, nirgundi, NRA etc.

 

 

INTRODUCTION:

Tuberculosis remains an important public health problem worldwide, causing the deaths of about 1.6 million people each year. According to WHO it is estimated that 8.8 million new TB cases occurred in 20071. Despite the improvements in chemotherapy, TB control is severely affected by the development of multidrug resistant M. tuberculosis strains. Natural products and/or their semi-synthetic derivatives can lead to compounds and may play important roles in the treatment of TB2-5.

 

Plants used in traditional medicine represent a priceless tank of new bioactive molecules. Vitex negundo is commonly known as Nirgundi. Vitex negundo is one of the important plant from traditional system of medicine found all over the world. This plant is large aromatic shrub distributed throughout India. It has been reported to possess potent pharmacological properties like antinflammatory, antirhumatic, hepatoprotective antioxidant and anti-allergic activities6. The leaves of Nirgundi are anti-parasitical, alternative used as aromatic, vermifuge, pain reliever, anti-inflammatory etc. The various chemical constituents like flavonoids, flavone glycosides, volatile oils, triterpenes, tannins and lignin etc. were identified in this plant7. Despite its popular use as a medicinal plant, No data have been published on the antitubercular activity of vitex negundo.

 

In this present work, we have reported the phytochemicals and screening of antimycobacterial and antimicrobial activities of various extracts of Nirgundi leaves.


MATERIAL AND METHODS:

                      Collection of plant material-

Fresh leaves of Vitex negundo were collected from Sangli and Miraj areas. It was authenticated by Dr. Yadav. U. S, Botany department of Willingdon college.

 

          Extraction procedure-8

The leaves were washed and were shade dried to obtain coarse powder. This powder was subjected to different extraction procedures.

 

1] Aqueous extract--- The air dried powdered plant material macerated with 10% chloroform water for 7 days with occasional shaking.

 

2] Sucessive solvent extraction --- The air dried powdered plant material extracted in soxhlet extraction assembly with petroleum ether, benzene, chloroform, acetone, ethanol and methanol. Each time before extracting with the next solvent, the powdered material was dried in hot air oven below 50C.

 

3] Ethanolic extract--- The powdered plant material was extracted in soxhlet extraction assembly with ethanol.

The solubility of thick paste extracts was checked in different solvents. Some dissolved in H2O, some in DMF and DMSO. DMF and DMSO showed no any antimicrobial activity against the test micro organism during this work.

 

                      Phytochemical investigation of Nirgundi leaves

Each extract was subjected to preliminary phytochemicals investigation by chemical tests9.

 

Sr. No.

Name of extract

Phytochemicals investigated.

1

Aqueous

Carbohydrates, proteins, alkaloids, phenolic compounds, tannins, volatile oils. sapponins and irridoid glycosides, flavonoids, vitamin-c etc.

2

Petroleum ether

Steroids, gum, mucilage, fats andoils etc.

3

chloroform

Fats andoils, phenolic compounds, tannins, flavonoids etc.

4

Benzene

Carbohydrates, steroids etc.

5

Acetone

Carbohydrates, fats andoils, polyphenolic compounds, tannins, alkaloids, flavonoids etc.

6

Successive ethanolic

Carbohydrates, polyphenolic compounds, tannins, steroids, flavonoids etc.

7

Methanolic

Carbohydrates, fats andoils, polyphenolic compounds, tannins, alkaloids, flavonoids, glycosides, steroids, gums, mucilage etc.

8

Ethanolic

Carbohydrates, fats andoils, polyphenolic compounds, tannins, alkaloids, flavonoids, irridoid glycosides, steroids,volatile oils, gums, mucilage,sapponins,vit.c etc.

 

 

Preparation of Mycobacterium tubercle inoculum:-

By using pure culture of standard strain of Mycobacterium tuberculosis H37RV procured from Government Medical College, Miraj was inoculated in solid media Loweinstein-Jensen media in macartney bottles.

 

 

Conventional Proportion method:10,11

Antitubercular activity of all these extracts of Nirgundi was screened by proportion method against Mycobacterium tuberculosis std. strain H37RV.Different concentrations (50,100,150 and 200 g/ml) of each extract ,0.2 g/ml of isoniazide and 40 g/ml of rifampicin were prepared and added to the Loweinstein-Jensen media. The media was distributed as 6-8ml into sterile macartney bottles in triplicate and inspissated .the control media was prepared at the same time as he drug contain media. An inoculum as 10-2 was prepared from standard suspension of tubercle bacilli and the turbidity was compared with the turbidity of macfarland standard. A loopful of each diluted inoculam inoculated on drug contains media and control media that becomes 10-4 all bottles were incubated and results were obtained after 28 days and confirmatory on 42nd day the same procedure was repeated. The results were calculated by using formula ,the ratio between average number of colonies on the drug contain slopes and control slopes multiplied by 100 is the proportion of resistant bacilli existing in the strain.

The critical resistance considered resistant 1% for rifampicin and isoniazide.

 

Nitrate reductase assay (NRA) for drug susceptibility:

NRA was performed as described by Golyshevskaia et al and Angeby et aln. The following critical concentration were used 0.2g/ml for INH, 40g/ml for rifampicin Briefly, fresh subculture(1l loops of bacteria) from isolates of M. tuberculosis grown on LJ medium was taken and vortexed in 3ml phosphate buffer saline(PBS, pH7.4) and turbidity was adjusted according to Mcfarland standard no.1. Part of the suspension was diluted 1:10 in PBS. For each isolate , 0.2ml of suspension was inoculated into the tubes containing LJ medium with potassium nitrate ( KNO3) and the antitubercular drugs; 0.2ml of the 1:10 dilution was inoculated into drug free media(LJ media) containing KNO3 which served as growth controls. Tubes in triplicate were incubated at 370c.for 14 days and 0.5ml of a mixture of three reagents (25 l of concentrated HCl, 50 l of 2%sulphanilamide and 50 l of 1% n-1-napthyl-ethylenediamine dihydrochloride) was added to one drug-free control tube after 7 days of incubation. If its colour changes to pink then tubes with drugs were tested. An drug sample was considered resistant if there was colour changes (pink or deep red to violet) in the drug tube in question greater than in the 1:10 diluted growth control on the same day. If the tubes did not show any colour change and remains the same, these were further incubated for 10 days and 14 days as described by Angeby et al12,13.

 

Preparation of suspension for test bacteria for antimicrobial activity:-

18 to 24 hours old culture of both gram positive and gram negative growing bacteria were used for preparation of the suspension of the test bacteria in sterile normal saline. Satphylococcus aureus (ATCC 3752), Pseudomonas aeruginosa and Candida albicans were used as gram +ve, gram -ve and fungi respectively. The turbidity of the suspension was adjusted to the turbidity of the solution of the macfarland standard14

 

Antimicrobial activity testing by Paper disc plate method:

During this work different media were used for testing antimicrobial activity. The dried extracts were dissolved in the (DMF, DMSO) and sterilized by filtration using 0.45 um Millipore filters.


Table showing the effect of all extracts of Nirgundi on Mycobacterium tuberculosis std. strain H37RV.

Sr. no.

Drug

Conventional proportion method

Nitrate reductase assay method

1

Control without drug

+++

+++

2

Rimpicin and isoniazide

--

--

3

Ethanol extract (150 and 200 g/ml)

--

--

4

Petroleum ether extract (200 g/ml)

--

--

5

Benzene extract and acetone, extract ( 200ug/ml)

++

++

6

Chloroform extract (200 g/ml)

--

--

7

Successive ethanol extract (150 and 200 g/ml)

--

--

8

Aqueous extract (150 and 200 g/ml)

+++

+++

9

Methanol extract (150 and 200 g/ml)

--

--

-- indicates no growth, +++ indicates severe growth, ++ indicates moderate growth, + indicates mild growth.

 

 

Table showing the effect of some extracts of Nirgundi on microorganisms:

Sr. No.

Name of Microorganism

Media Used

Petroleum ether extract

Chloroform extract

Benzene extract

Ethanol extract

Methanol extract

Aqueous extract

1

Staphylococcus aureus

Yoghel johnson agar

14

15

20

--

8

--

2

Pseudomonas aerugionsa

Cetrimide agar

15

10

20

11

13.3

12.3

3

Candida albicans

Sabouraud dextrose agar

15

10

20

12

10

--

Zone of inhibition in mm, -- indicates no zone of inhibition

 


The antimicrobial activity was then evaluated by paper disc plate method15. Plates were previously inoculated with the suspension of the test micro-organisms.

 

Small sterile paper of 4mm diameter discs impregnated with 10mg/10l of each extract were placed upon the surface of an inoculated plate. Negative controls were prepared using the same solvents employed to dissolve the plant extracts. A standard disc containing 25g of cotrimazole was also placed on the agar surface as positive reference standards. The inoculated plates were then incubated at 37C for bacteria and 250c for fungi. After 24 hours incubation, their potency was quantitatively assessed by the presence or absence of inhibition zones and zone diameters 16.

 

RESULTS AND DISCUSSION:

The antitubercular activity of Vitex negundo studied by two methods i.e. conventional proportion method and nitrate reductase assay. The ethanol, methanol and successive ethanolic extracts of Vitex negundo all showed better significant antitubercular activity at concentration 150 and 200 g/ml. At higher concentration 200 g/ml only chloroform and petroleum ether extract showed moderate antitubercular activity while aqueous, benzene, acetone extract does not shown any antitubercular activity.

 

The antimicrobial activity of Vitex negundo was studied by Paper Disc Method. The results of this antimicrobial activity indicated that benzene extract shows more significant antimicrobial activity and petroleum ether, methanol and chloroform extract shows moderate antimicrobial activity against all microorganisms. Aqueous extract was shows least activity only against Pseudomonas aerugionsa and no activity against other microorganisms.

 

CONCLUSION:

In conclusion nitrate reductase assay was found to be rapid, inexpensive and easy to perform. It also showed good concordance between conventional proportion method and nitrate reductase assay.

 

All these findings justified the claim made in the indigenous system of medicine for the use of the plant vitex negundo against tuberculosis and many other micro-organisms.

 

ACKNOWLEDGEMENT:

The authors are thankful to Principal, D. D. Chougule of Appasaheb Birnale College of Pharmacy, Sangli, for providing facility to carry out this work.

 

REFERENCES:

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11.   Strong BE, Kubica GP. Isolation and identification of Mycobacterium tuberculosis: A guide for the level II laboratory. Atlanta, Georgia: centers for Disease Control, US department of Health and Human Services.

12.   Golysheyskaia VI, et al. New microbiological techniques in diagnosis of tuberculosis. Probl Tuberk 1996; 6: 22-5.

13.   Angeby K A, Klintz L, Hoffner SE. Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitate reductase assay. J. clinical Microbiology 2002;40:1553-5.

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15.   Murray P.R. Baron E. J., Pfaller M.A., Tenover F. C. and Yolke R.H. Manul of Clin Microbiol., 1995,6,45-46.

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Received on 07.01.2010

Accepted on 15.03.2010

A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 2(2): March -April 2010, 166-168