INVITRO SCREENING OF ANTIFUNGAL ACTIVITY

ISSN 0973 – 6921 ; E – ISSN 2319 – 5983 Journal of Environmental Research And Development

J. Environ. Res. Develop. Vol. 12 No. 04, April-June 2018

INVITRO SCREENING OF ANTIFUNGAL ACTIVITY OF LEAF EXTRACT OF Vitex negundo Dassani S.*, Sharma T. K. and Pandey M. M. Department of Botany and Industrial Microbiology, Bipin Bihari College, Jhansi, U.P. (INDIA) Received September 29, 2017

Accepted March 03, 2018

ABSTRACT Vitex negundo is a medicinal plant of family Verbenaceae which is commonly found throughout India and is used in traditional medicines. The present study was undertaken to screen potential antifungal activity of the extract of leaves of V. negundo against three dermatophytes i.e. Epidermophyton, Microsprorum and Trichophyton. The leaf extract was prepared by crushing the leaves in methanol, ethanol and distilled water as a solvent. These extracts were evaluated for their antifungal activity against Epidermophyton, Microsporum and Trichophyton on Sabouraud Dextrose Agar media (SDA) plates by using agar well diffusion method. The plates were incubated at 35±20C for 24 hrs. Then diameter of zone of inhibition was measured. Best antifungal activity was observed against Trichophyton with ethanol as a solvent and the least activity was observed against Epidermophyton with distilled water as a solvent.

Key Words : Epidermophyton, Trichophyton, Microsporum, Vitex negundo, Antifungal activity

INTRODUCTION V. negundo is commonly known as Chinese chaste tree, five leaved chaste tree, horse shoe vitex or nirgundi is a large aromatic shrub with quadrangular densely whitish, tomentose branchlets. It has analgesic, antibacterial, antifungal and anti-inflammatory properties used by folk as medicines. It is the plant which is very widely grown and flourished. It is a woody aromatic shrub with tri or pentafoliate leaves that gives rise to bluish purple color plant in branched cymes1. It prefers to grow in humid places or along water courses in wasteland and makes open forest in India, Afghanistan, Srilanka, Pakistan, Thailand and Malaysia. it is used by local people for various diseases and treatments such as arthritis, rheumatism, malaria, toothache, bodyache and menstrual problems, premenstrual dysphoric disorder (PMDD), inflammatory conditions, diarrhea and flatulence2-6. The ethanobotanical approach is to study the role of some medicinal plants such as Neem, Tulsi, Nirgundi, and Ginger etc, which are used for cure of various diseases. The practice of using these medicinal plants is not having particular prescribed or written source but these practices are followed *Author for correspondence

at traditional level from ancient times till date. The tribes of Jhansi also use these plants and their parts as a substitute of medicines and heal themselves. Now a days global trend of interest in traditional systems of medicines have increased manifold times7. India is very rich in medicinal plants. The forest in India is the principal repository of large number of medicinal and aromatic plants which are largely collected as raw materials for manufacture of drugs and perfumery products. Treatment of medicinal plants is considered very safe as there are no or minimal side effects8. Use of herbal treatment is independent of any age groups and the sexes. Based on all these benefits, we decided to get the knowledge of some locally used medicinal plants9. Vitex negundo also have medicinal value for prevention of skin diseases as it is reported as an antidermatophytic agent. The dermatophytes are a group of closely related fungi that have the capacity to invade keratinized tissue (spleen, hair and nails) of human and other animals to produce infection called dermatophytosis commonly referred to as ringworm. They are classified in three anamorphic genera: Epidermophyton, Microsporum and Trichophyton10. Antidermatophytic activity of V. negundo was checked against these three

474



dermatophyes Epidermophyton, Microsporum and Trichophyton. Though all parts of V. negundo are used but leaves and barks are the most important ones in field of medicines11. Leaves of V.negundo are considered as tonic, vermifused and are given with long pepper in caterrhal fever12-15. Out of stem, root and leaf extract of Vitex negundo for antibacterial activity, best results were reported for leaf extract16.

MATERIAL AND METHODS Collection of plant sample Fresh leaves of the plant V. negundo were collected from Notghat in Baruasagar, Jhansi district during October - November. Leaves were washed properly under running tap water then air dried, homogenized to fine powder and stored in air tight bottles at 40C. Preparation of leaf extract Leaves extract were prepared by sequential cold maceration method17. Using methanol, ethanol and distill water as a solvent. Five gram of V. negundo leaves powder was soaked in 50 ml of every solvent and incubated for 48 h at room temperature, and then it was filtered by whatman filter paper no-1 and stored at 40C for further use. (Fig. 1)

Microbial culture Above genera were used as test cultures. They were grown on Sabouraud Dextrose Broth (Himedia) at 30±20C for 48h and were maintained at SDA media slants (peptone- 10.0g, dextrose-40.0g, agar-15.0g, distill water-1000 ml, pH-5.6). These plates were then incubated at 30 ±20C for 48h to 72h. Assay for antifungal activity To check the antifungal activity, stock cultures of above genera were revived. The agar plates of SDA media were prepared (peptone- 10.0g, dextrose-40.0g, agar-15.0g, distill water-1000ml, pH-5.6). Each plate was inoculated with 0.5 ml of fungal suspension which was spread uniformly on the plate. Wells of 5 mm diameter were made in plates by using sterile cork borer.18,19 Test samples of every solvent were filled in wells and all the tests were carried out in triplicates. All plates were incubated at 35 0C for 3-4 days and then the diameter of zone of inhibition was measured in millimeters (mm). (Fig. 1)

RESULTS AND DISCUSSION Epidermophyton floccosum It is a dermatophyte that produces mycoses to human being

b

a Symptoms of Epidermophyton

Epidermophyton observed under microscope(×45,×10)

c Fig. 1 : Antifungal activity of Epidermophyton with extracts of Vitex negundo in different solvents a=ethanol inhibition, b= methanol inhibition, c= distilled water inhibition 475



Microsporum canis it is pathogenic asexual fungus which infects dead layers of skin of human (Fig. 2)

a Symptoms of Microsporum

b Microsporum observed under microscope(×45,×10)

c Fig. 2 : Antifungal activity of Microsporum with extracts of Vitex negundo in different solvents a=ethanol inhibition, b=methnol inhibition, c= distilled water inhibition Trichophyton rubrum it is a dermatophytic fungus that colonizes upper layer of skin and causative agent of atheletes foot, nail infection, ring worm etc. (Fig. 3)

b

a Symptoms of Trichophyton

Trichophyton observed under microscope (×45,×10)

c Fig. 3 : Antifungal activity of Trichophyton with extracts of Vitex negundo in different solvents a= ethanol inhibition, b=methnol inhibition, c = distilled water inhibition 476



Antifungal activity of V. negundo The level of antifungal activity of leaves extract of V. negundo in different solvents against test dermatophytes is mentioned in table as observed by measuring the zone of inhibition in petri plates. The values given in Table 1 are the analysis of triplicate data (mean value). Zone of inhibition against Epidermophyton with solvents: distill water, methanol and ethanol is observed as 3.0 mm, 13.0 mm and 14.0 mm respectively. Microsporum growth was inhibited maximum (13.0 mm) by using ethanol as a solvent and minimum (5.0 mm) by distill water as a solvent. Zone of inhibition in case of Trichophyton was minimum (4.0 mm) with distill water and maximum (16.0 mm) with ethanol as a solvent.

Best antifungal activity was observed against Trichophyton with ethanol as a solvent and the least activity was observed against Epidermo phyton with distill water as a solvent. 20

CONCLUSION It is concluded that V. negundo leaves can be used as good antidermatophytic agent as it has given positive results in all conditions. But the best action is in presence of ethanol solvent. Whereas the least activity was observed against Epidermophyton with distilled water as solvent. Therefore Vitex negundo leaves can be used as natural antifungal agent and be a good treatment against skin infection ringworms and mail infections.

Table 1 : Antifungal activity of leaf extract of V.negundo on dermatophytes (Inhibition Zone in mm) Strains Solvents Distilled water Methanol Ethanol

Epidermophyton floccosum

Microsporum canis

Trichophyton rubrum

3.0 13.0 14.0

5.0 11.0 13.0

4.0 11.0 16.0

REFERENCES 1. Panda S. K., Thatoi H. N. and Dutta S. K., Antibacterial activity and phytochemical screening of leaf and bark extracts of Vitex negundo L. from similipal biosphere reserve, Orissa, J. Med. Plant. Res., 3(4), 294-300, (2009). 2. Ono M., Yamasaki T., Konoshita M., Ikeda T., Okawa M. and Kinjo J., Five new diterpenoids, viteagnusins A-E, from the fruit of Vitex agnus-castus. Chem Pharm Bull (Tokyo)., 56, 1621–4, (2008). 3. Costa M. R., Ribeiro C. G., Santos-Filho S. D., Neves R. F., Fonseca A. S. and Bernardo-Filho M., An aqueous extract of Vitex agnus-castus alters the labeling of blood constituents with Technetium99m, Braz Arch Biol Technol., 50, 183–8, (2007). 4. Azarnia M., Ejtemaei-Mehr S., Shakoor A. and Ansari A., Effects of Vitex agnuscastus on mice fetus Development, Acta Med Iran., 45, 264–70, (2007). 5. Carmichael A.R., Can Vitex agnus castus be used for the treatment of mastalgia? What is

6.

7.

8.

9.

10.

477

the current evidence? Evid Based Comple ment Alternat Med., 5, 247–50, (2008). Dugoua J.J., Seely D., Perri D., Koren G. and Mills E., Safety and efficacy of chastetree (Vitex agnus-castus) during pregnancy and lactation, Can J. Clin Pharmacol., 15, 74–9, (2008). Duraipandiyan V., Ayyanar M. and Ignacimuthu S., Antimicrobial activity of some ethno medicinal plants used by Paliyar tribe from Tamil Nadu, India, BMC complementary and alternative medicine., 6(1), 35, (2006). Bandow J. E., Brotz H., Leichert L. I. O., Labischinski H. and Hecker., Proteomic approach to understanding antibiotic action, Amicrob. Agents Chemother., 47, 948-955, (2003). Gupta R. K. and Tandon V. K., Anti nociceptive activity of Vitex negundo Linn leaf extract, Ind. J. Physiol. and pharmacol., 49(2), 163, (2006). Singh P. S. and Vidyasagar G. M., Antifungal screening of 61 folkloric medicinal plant extracts against dermatophytic fungi Trichophyton rubrum J.


11.

12.

13.

14.

15.

Appl. Pharmaceut. Sci., 5(05), 038-044, (2015). Chandramu C., Manohar R. D., Krupadanam D. G. and Dashavantha R.V., Isolation, characterization and biological activity of betulinic acid and ursolic acid from Vitex negundo L, Phytotherapy Res., 17(2), 129134, (2003). Jignesh P., Samir S., Shrikalp D and Gaurang S., Evaluation of the antiasthmatic activity of leaves of Vitex negundo, Asian J. Pharm. Clin. Res., 2(1), 81–6, (2009). Bansod M. S. and Harle U. N., Vitex negundo l: Phytochemical costituets, traditioal uses ad pharmacological properties: Comprehesive review. Pharmacol. online., 1, 286–02, (2009). Tandon V. R. and Gupta R. K., Vitex negundo Linn. (VN) leaf extract as an adjuvant therapy to standard antiinflammatory drugs, Indian J Med Res., 124, 447–50, (2006). Gautam L. N., Shrestha S. L., Wagle P. and Tamrakar B. M., Chemical constituents from Vitex negundo (Linn.) of Nepalese origin, Sci World., 6, 27–32, (2008).


16. Dassani S., Sharma T.K., Singh R. and Pandey M. M., Antibacterial activity of flavonoids isolated from Vitex negundo., Int. J. Adv. Res., 4(11), 150-153, (2016). 17. Dharajiya D., Patel P., Patel M. and Moitra N., In vitro antimicrobial activity and qualitative phytochemical analysis of Withania somnifera (L.) Dunal extracts, Int. J. Pharma. Sci. Rev. Res., 27(2), 349-354, (2014). 18. Mahendra K. Gupta, Shrivastava A. and Singhal P. K., Production of bioactive compounds from mutant strain of Bacillus sp. against fungi, J. Environ. Res. Develop., 11(03), 511-518, (2017). 19. Shrivastava A., Patel A. and Dwivedi S., Micropropagation of endangered medicinal herb Chlorophytum borivilianum sant. et fernand, J. Environ. Res. Develop., 10(03), 486-491, (2017). 20. Patel A. Shrivastava A. and Dwivedi S., Mycorrhizal inoculation enhences growth and saponin accumulation in micropro pagated Chlorophytum borivilianum, J. Environ. Res. Develop., 11(01), 486-491, (2017).

478