Ankita Sharma et al. / Journal of Pharmacy Research 2011,4(5),1313-1314 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Assay of Antifungal Activity of Lawsonia inermis Linn. and Eucalyptus citriodora Hook. Ankita Sharma* and Kanika Sharma Microbial Research Laboratory, Department of Botany, MLS University, Udaipur- 313001, Rajasthan, India. Received on: 05-01-2011; Revised on: 14-02-2011; Accepted on:09-03-2011 ABSTRACT Lawsonia inermis Linn. and Eucalyptus citriodora Hook. leaf extracts were evaluated against 10 plant pathogenic and 2 human pathogenic fungal species viz. Alterneria solani, Drechslera halodes (Helminthosporium halodes), Rhizoctonia solani (ITCC no. 4574), Fusarium solani, Curvularia lunata, Dreschlera graminae, Fusarium moniliformae (ITCC no. 2927), Aspergillus flavus (Navjot 4 NSt), A. parasiticus var. globosus (MTCC No. 411), Trichophyton rubrum (MTCC 296), Aspergillus fumigatus (MTCC 2550) and Candida albicans (MTCC 227). The dried and powdered leaves were successively extracted with petroleum ether, benzene, chloroform, acetone, ethanol and water using soxhlet assembly. The antifungal activity assay was done by poison food technique. Acetone extract of L. inermis leaves and Petroleum ether extract of E. citriodora leaves showed highest activity against all tested fungi. The inhibitory effect was significant and better than the synthetic fungicides used as most of the strains showed resistance against flucanazole and amphotericin B.

Key words: L. inermis, E. citriodora, plant pathogenic, human pathogenic, solvent extracts INTRODUCTION In recent year’s considerable research is prompted for development of novel antifungal agents thus obtained were stored at 4 °C in airtight bottles for further studies. Percent extractive values with diverse mechanism of action due to continuing increase in incidences of life threatening were calculated by the following formula and are listed in table-1. and invasive fungal infections, limited number of antimycotics, their narrow activity spec- Weight of dried extract Percent extractive = ´ 100 trum, fungistatic rather than fungicidal activity and resistance development in fungal species. Weight of dried plant material There are 4 groups of antitherepeutics used (as still no licensed fungal vaccines are available) i.e. azoles derivatives (miconazole and fluconazole), polyene macrolides (amphotericine B, Table 1Percent Extractive of Different Fractions of Lawsonia inermis Leaf and E. AmB), echinocandins (Caspofungin) and fluorinated pyrimidines (flucytosine) and each group citriodora leaf extracts has similar mode of action which raising the incidences of development of resistant species of S.No. Solvent Percent Extractive Value pathogenic fungi(1) for example Candida and Aspergillus species display resistance to azoles(2- L. inermis E. citriodora 3). Therefore, the development of alternative antimicrobial drug from medicinal plants with 1. PE fraction 6.9 6.45 broad fungicidal activity has become necessary. The herbal wealth of india and its medicinal 2. Benzene fraction 0.67 2.87 properties have a long tradition as referred in ancient literature. 3. Chloroform fraction 6.45 3.52 4. Acetone fraction 3.52 5.82 5. Methanol fraction 18.62 15.85 In the present investigation antifungal activity of Lawsonia inermis Linn and Eucalyptus 6. Aqueous fraction 4.3 4.3 citriodora Hook, two economically important genera of order Myrtales was studied. Lawsonia inermis Linn belongs to family Lythraceae and is commonly known as Heena or Mehndi. It is Test Fungi native plant of North Africa and south-west Asia. The various plant parts of L. inermis were Plant pathogenic fungi namely Alterneria solani, Dreschlera halodes (Helminthosporium used traditionally for different medicinal as well as cosmetic uses. The leaves and roots were halodes), Rhizoctonia solani (ITCC no. 4574), Fusarium solani, Curvularia lunata, used in ulcers, cough, bronchitis, diarrhea, leucoderma, boils, anaemia, fever, falling of hair Dreschlera graminae, Fusarium moniliformae (ITCC no. 2927), Aspergillus flavus (Navjot (4) and greyness of hair . The plant also has great medicinal importance due to its antituberculostatic 4 NSt), A. parasiticus var. globosus (MTCC No. 411) and three human pathogenic fungi (5) (6) (7) activity , intestinal antisplasmodic and uterine sedative effects hepatoprotective activity , i.e. Trichophyton rubrum (MTCC 296), Aspergillus fumigatus (MTCC 2550) and Can- (8) (9) cytotoxic activity , anti-inflammatory, antipyretic, and analgesic activities , antibacterial dida albicans (MTCC 227) were used for experimental work. activity(10-11) and fungitoxic activity against different fungi(12). Eucalyptus citriodora is an evergreen tree of 24-40 m in height with tall straight trunk belongs to family Myrtaceae. It is Antifungal assay commonly known as lemon scented tree due to lemon type smell of aromatic substances in The antifungal activity of prepared extracts was tested using poison food technique (18). 100 leaves and gum. Eucalyptus oil has great medicinal values due to its anti-inflammatory, mg of extract was dissolved in 10 ml acetone to prepare 10mg/ml concentration of stock (13) antispasmodic, decongestant and antiseptic properties . In addition, it is also has anti- solution. 1 ml of stock solution was mixed with 9 ml molten sterile potato dextrose agar (14) diabetic activity . Antibacterial and antifungal properties of Eucalyptus extract and oil are medium and this mixture was poured into pre-sterilized petri-plates (9 cm diameter) and (15-17) also reported by some authors . Further screening of these medicinal plants may lead to allowed to solidify at room temperature. Thus prepared petri plates were inoculated asepti- discovery of novel antifungal compounds. In the present investigation different organic solvent cally with 5 mm disc of test fungi. The plate were incubated then 28±2°C for seven days. extracts of L. Inermis and E. citriodora were evaluated for their antifungal potential against Antifungal activity was measured as a function of increase in growth of 5 mm disc of various plant pathogenic and human pathogenic fungi. inoculum. For each fungal strain a negative control was maintained where acetone without extract was used. Standard antifungal agents such as fluconazole, amphotericin, bavistin and MATERIALS AND METHODS mancozeb were also maintained as positive control. The experiment was carried out three times and mean values are presented. Collection of plant material Fresh leaves of L. inermis and E. citriodora were collected from the campus of University Results College of Science, MLS University Udaipur in October 2008. The plants were identified by In the present study L. inermis and E. citriodora leaf extracts prepared in various organic Dr. Roop Singh Rathore Incharge Herbarium, Department of Botany, University of Rajasthan, solvents were screened for antifungal activity. The inhibitory activity of the extract was Jaipur, India and were given voucher specimen nos. RUBL 20427 (Lawsonia inermis) and compared with standard antifungals like fluconazole, amphotericin, bavistin and mancozeb. RUBE 21256 (Eucalyptus citriodora) respectively. Results are presented in table no. 2 and 3.

Extraction of plant material The percent extractive values of partially purified leaf and seed extracts of L. inermis and E. 40 g dried leaf powder was successively extracted with 240 ml. each of organic solvents in the citriodora were also obtained with different organic solvents. Results are given in table no. following series, viz., petroleum ether — benzene— chloroform — acetone— ethanol — water 1. Acetone extracts of L. inermis leaf gave maximum percent extractive value i.e. 18.62%, using soxhlet apparatus. Each time before extracting with next solvent the residual plant while methanol extracts of E. citriodora leaf gave maximum percent extractive value i.e. material was dried in an oven at 50 °C and used for extraction with next solvent. After complete 15.85%. Minimum percent extractive value was obtained with benzene fractions which were extraction, respective solvents were evaporated under reduced pressure and the dried extracts 0.67% and 2.87 % for L. inermis and E. citriodora respectively.

Results of antifungal assay of L. inermis leaves extract (table no. 2, Fig. 1) suggests that of *Corresponding author. all the extracts assayed acetone extract was the most effective as it showed significant Ankita Sharma inhibition of all test fungi which was comparable with that of standards used. Methanol Microbial Research Laboratory, extract also showed significant activity against all plant pathogenic as well as human Department of Botany, MLS University, pathogenic fungi except A. flavus and A. parasiticus, against which petroleum ether and Udaipur- 313001, Rajasthan, India. benzene extract was more effective. Significant inhibition of Dreschlera graminae, Curvularia lunata, Aspergillus fumigatus and Candida albicans were observed with petroleum ether and Journal of Pharmacy Research Vol.4.Issue 5. May 2011 1313-1314 Ankita Sharma et al. / Journal of Pharmacy Research 2011,4(5),1313-1314 Table 2 - Antifungal activity of leaf extracts of L. inermis Test Fungi Growth zone (mm) after 7 days (extracts) Standard Antifungals PE BE CH AC ME AQ FU AM BA MN

A. solani 21.66 ± 0.0 20.22 ± 0.2 18.23 ± 0.2 11.21 ± 0.4 15.66 ± 0.0 19.17 ± 0.6 - - 22.27 ± 0.4 18.23 ± 0.5 D. halodes 22.20 ± 0.3 18.66 ± 0.2 19.99 ± 0.5 10.23 ± 0.7 17.66 ± 0.0 19.67 ± 0.2 - - 26.03 ± 0.1 17.0 ± 0.0 R. solani 32.12 ± 0.0 31. 13 ± 0.1 29. 67 ± 0.4 20.11 ± 0.4 23.34 ± 0.3 28.77 ± 0.2 - - 23.00 ± 0.6 28.13 ± 0.4 F. solani 17.67 ± 0.3 15.90 ± 0.2 14.55 ± 0.6 10.11 ± 0.4 12.23 ± 0.0 14.65 ± 0.2 - - 22.53 ± 0.7 24.97 ± 0.8 C. lunata, 13. 23 ± 0.5 12.11 ± 0.2 12.00 ± 0.0 9.08 ± 0.3 10.14 ± 0.1 13.23 ± 0.8 - - 21.63 ± 1.3 NA D. graminae 10.3 ± 0.0 11.0 ± 0.1 16.0 ± 0.5 7.0 ± 0.1 10.01 ± 0.5 22.1 ± 0.2 - - 23.30 ± 0.7 23.10 ± 0.8 F. moniliformae 13.4 ± 0.0 17.3 ± 0.1 23.0 ± 0.5 9.4 ± 0.1 13.9 ± 0.5 26.0 ± 0.1 - - 26.0 ± 0.05 22.17 ± 0.0 A. flavus 16.2 ± 0.2 16.86 ± 0.1 15.33 ± 0.5 13.9 ± 0. 17.00 ± 0.1 15.6 ± 0.1 - - 13.22 ± 0.2 15.44 ± 0.2 A. parasiticus 14.9 ± 0.1 15.93 ± 0.1 20.06 ± 0.5 14 ± 0.1 19.96 ± 0.5 21.0 ± 0.1 - - 11.20 ± 0.1 14.65 ± 0.2 T. rubrum 20.0 ± 0.0 28.00 ± 0.1 15.2 ± 0.0 10.0 ± 0.1 12.0 ± 0.0 NA NA 18.0 ± 0.1 - - A. fumigatus 11.4 ± 0.1 12.0 ± 0.1 9.0 ±0.0 8.0 ± 0.5 13.2 ± 0.1 NA NA NA - - C. albicans 14.0 ± 0.0 13.1 ± 0.0 13.0 ±0.5 8.6 ± 0.5 12.8 ± 0.1 NA NA 14.0 ± 0.1 - - All values are mean of three replicates. PE= Petroleum ether, BE = Benzene, CH= chloroform, AC= acetone, ME = methanol, AQ= aqueous, FU = fluconazole, AM= Amphotericin B, BA= Bavistin, MN = Mancozeb, NA = Not active

Fig. 2 Antifungal activity of E. citriodora leaves Fig. 1 Antifungal activity of L. Inermis leaves benzene fractions of the leaf. Aqueous extract of L. inermis was not Table 3 - Antifungal activity of leaf extracts of E. citriodora effective against human pathogenic fungi, whereas in the presence of Test Fungi Growth zone (mm) after 7 days (extracts) acetone extract significant inhibition of T. rubrum, A. fumigatus and C. PE BE CH AC ME AQ albicans was observed. All extracts were found to be significant inhibi- Alterneria solani 4.10 ± 0.10 17.00 ± 0.0 6.00 ± 0.01 8.40 ± 0.10 9.20 ± 0.07 11.20 ± 0.30 tors of human pathogenic fungi as compared to plant pathogenic fungi. Drechslera halodes 4.10 ± 0.67 18.00 ± 0.5 11.78 ± 0.00 8.27 ± 0.10 9.19 ± 0.00 14.67 ± 0.23 Rhizoctonia solani 9.6 ± 0.15 14.1 ± 0.05 14.0 ± 0.45 15.0 ± 0.05 16.8 ± 0.05 16.61 ± 0.21 Results of antifungal potential of E. citriodora leaf are presented in table Fusarium solani 10.4 ± 0.10 11.34 ± 0.5 13.9 ± 0.41 16.78 ± 0.05 15.4 ± 0.07 26.62 ± 0.03 no. 3 (Fig. 2). Petroleum ether fraction of leaves showed significant Curvularia lunata, 10.42 ± 0.02 13.45 ± 0.6 11.12 ± 0.32 12.13 ± 0.06 13.23 ± 0.06 12.23 ± 0.04 Dreschlera graminae 6.68 ± 0.45 12.10 ± 0.5 7.23 ± 0.31 8.23 ± 0.23 9.23 ± 0.07 13.40 ± 0.23 inhibition of all test fungi. Benzene fraction showed comparable inhibi- Fusarium moniliformae 8.45 ± 0.41 18.96 ± 0.2 15.66 ± 0.21 10.23 ± 0.22 9.13 ± 0.06 11.00 ± 0.21 tion of A. solani and D. graminae. Aqueous extract was ineffective against Aspergillus flavus 6.23 ± 0.25 11.2 ± 0.84 11.59 ± 0.09 11.55 ± 0.25 10.47 ± 0.29 12.54 ± 0.38 F. solani. A reasonable inhibition of R. solani, C. lunata, A. flavus and A. parasiticus 10.18 ± 0.60 16.5 ± 0.52 10.53 ± 0.27 15.25 ± 0.14 13.61 ± 0.34 14.09 ± 0.08 Trichophyton rubrum 11. 23 ± 0.7 15.66 ± 0.2 12.00 ± 0.2 13.45 ± 0.3 11.80 ± 0.31 15.90 ± 0.03 C. albicans was observed with almost all extracts. E. citriodora extracts Aspergillus fumigatus 10.22 ± 0.87 17.88 ± 0.3 13.89 ± 0.03 14.99 ± 0.2 13.23 ± 0.22 13.45 ± 0.04 was found more effective in comparison to L. inermis extracts. Candida albicans 11.23 ± 0.04 12.23 ± 0.2 13.12 ± 0.6 14.23 ± 0.23 12.11 ± 0.11 12.11 ± 0.02 All values are mean of three replicates. PE= Petroleum ether, BE = Benzene, CH= chloroform, AC= acetone, ME = methanol, AQ= aqueous DISCUSSION On the basis of the results obtained it can be suggested that L. Inermis and E. citriodora has 4. Vaidyaratnam, P.S., Indian Medicinal plants. A compendium of 500 species, 1995, 3:303-304. a broad spectrum antifungal activity against various plant pathogenic and human pathogenic 5. Sharma, K., Tuberculostatic activity of heena (Lawsonia inermis Linn.). Tubercle, 1990, 71: 293- 295. fungi. As compared to aqueous extracts organic solvent extracts was found to be more effective. 6. Shihata, I.M., hassan, A.G. and Mayah, G.Y., Pharmacological effects of Lawsonia inermis leaves Antimicrobial activity of plants is due to the presence of secondary metabolites which are (el-Heena). Egypt. J. Vet. Sci., 1978, 15:31 extracted in organic solvents due to their solubility (19) and each secondary metabolite has 7. Anaad K. K., Singh B., Chand D., and Chandon B. K., An evaluation of Lawsonia alba extract as hepatoprotective agent. Planta Med. 1992, 58: 22-25 specific affinity for specific solvent, thus a organic solvent extract had differential activity for 8. Ali M. and Grever M. R., A cytotoxic naphtho- quinone from Lawsonia inermis. Fitoterapia 1998, different organisms. Of all the extracts assayed acetone extract of L. Inermis and petroleum ether LXIX (2): 1810-1813. extract of E. citriodora showed maximum activity. Alkaloids, steroids, quinines and tannins 9. Ali B. H., Bashir A. K., and Tanira M. O., Anti- inflammatory, antipyretic, and analgesic effects are highly soluble in these solvents (20). Significant activity of these solvent extracts suggests of Lawsonia inermis L. (henna) in rats. Pharmacology 1995, 51:356-63. 10. Bhuvaneswari, K, Poongothai, S.G., Kuruvilla, A., Raju, B.A. Inhibitory concentrations of l. Inermis that the antifungal activity of these plants may be due to the presence of secondary metabolites. dry powder for urinary pathogens. Indian journal of Pharmacology, 2002, 34: 260-263. The antifungal activity of the extracts was found superior than the synthetic fungicides used. 11. Sudharameshwari, K. And Radhika, J. Antibacterial screening of Aegle marmelos, Lawsonia inermis and Albizzia libbek. African journal of traditional, complementary and alternative medicines. 2007, 4(2): 199-204. Despite the great success of pharmaceutical industries in developing new antibiotics finding 12. Tripathi, R.D., Srivastava, H.S. and Dixit, S.N., A fungitoxic principle from the leaves of Lawsonia new broad spectrum antifungal agents is still a priority due to the resistance development of inermis Lam. Cellular and Molecular Life Sciences, 1977, 34(1): 51-52. fungal strains as well as absence of licensed antifungal vaccines. Extract used in present study 13. Ramezani, S., Ramezani, F., Rasouli, F., Ghasemi, M. and Fotokian, H. Diurnal variation of the essential oil of four medicinal plant species in central region of Iran. Research Journal of biological were partially purified fractions, hence contain very small amount of active molecules. 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