2012 International Conference on Environment, Energy and Biotechnology IPCBEE vol.33 (2012) (2012) IACSIT Press, Singapore Sensitization of Solvent Extract of Spirulina platensis (Geitler) against Some Dermatophytes and Related Fungi Vinay Kumar, A.K. Bhatnagar and J.N. Srivastava + Department of Botany, Faculty of Science, Dayalbagh Educational Institute, Dayalbagh, Agra Abstract. In the present investigation Spirulina platensis was tested for antifungal activity for in vitro at different concentration against three clinical isolates of pathogenic fungi i.e., (Candida albicans MTCC-227, Microsporum canis MTCC-3270, and M. fulvum MTCC-7675) by reduction in mycelial weight. Extract showed maximum m inhibition of (57.29%) at third day of incubation at 6000ppm concentration and least inhibition of (32.69%) against C.albicans at a concentration of 6000ppm on ninth day of incubation period. The study concludes that extract from of Spirulina platensis possesses tremendous antifungal activity against pathogenic fungi used in this study. Keywords: Antifungal activity, Acetone extracts, Spirulina platensis, Pathogenic fungi 1. Introduction Pharmaceutical drug discoveries, for past 140 years depended largely on the process of empirical screening of large number of pure compounds. Algal organisms are rich source of structurally novel and biologically active metabolites. Secondary or primary metabolites produced by these organisms may be potential bioactive compounds of interest in the pharmaceutical industry (Ely et al., 2004). Dermatophytes constitute an important public health problem as yet unresolved. In most African countries, traditional phycomedicines are used to control the disease. Fungal infection may be communicated from person to person by combs and towels etc. These infections include ringworm, athlete s foot, jockey s itch etc. Dermatophytes cause infection to the skin, hair and nail due to their ability to obtain nutrients from keratinized material. The organisms colonize in the keratinized tissues and cause inflammation by host s response to the metabolic byproducts. Screening of cyanobacteria for antibiotics and other pharmacologically active compounds has recently received considerable attention Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many based on their uses in traditional medicine. Spirulina platensis produce a diverse range of bioactive molecules, making them a rich source of different types of medicines. Pathogen resistance to synthetic drugs and antibiotics already in use makes search for plants with antimicrobial activity more important, as they can substitute for synthetic antibiotics and drugs. Phycochemistry is a new term first used by Shameel (1990), which is actually the study of natural products and chemical constituents occurring within algal thallus from a biological point of view. It primarily investigates the distribution of secondary metabolites in different body parts of algae under different seasons and variety of habitat conditions. All over the world phycologists studied the different types of natural products occurring within marine algae. A variety of fatty acids (both saturated and unsaturated), sterols, terpenes and sugars have been isolated from them. A very limited amount of phycochemical knowledge is available about freshwater algae, in comparison with the detailed work carried out on seaweeds, which includes not only the isolation of fatty acids but also a complete phycochemical analysis showing the types of sterols, terpenes, glycosides, polyols, halogenated compounds as well as new and novel metabolites. + Corresponding author, Tel.: 09456433062 E-mail: janendra.srivastava@gmail.com 270
2. Material and methods 2.1. Isolation of Spirulina platensis Spirulina platensis culture was obtained from Microbiology Lab, Department of Botany, Dayalbagh Educational Institute, Dayalbagh, and Agra. The culture would be picked up from the stock culture with the help of needle and transferred to petriplates and culture tube containing CFTRI medium, and incubated at 28 C for 30 days with (600-1600 lux) with a continuous light, 12hrs per day. Identification will be done using morphological variation, studies and taxonomical approaches according to (Desikachary, 1959). 2.2. Isolation of Microbial cultures Microbial cultures (C. albicans, M. canis, M. fulvum) used will be obtained from the Microbiology Lab, Department of Botany,Dayalbagh Educational Institute, Dayalbagh, Agra. Fungus cultures would be picked up from the stock cultures with the help of needle and transferred to the petridishes containing SDA medium directly and incubated at 28 ±2 C for 3-5 days. In petridish, when fungal colonies appeared on SDA medium, it would be transferred to other dishes or slants for experiment. Confirmation of fungi would be carried out using manual (A colour atlas of pathogenic fungi (Frey et al., 1986) from Department of Botany, Dayalbagh Educational Institute, Agra. 2.3. Assessment of antifungal by Reduction in mycelial biomass (broth assay) only for fungi by the method of (Kunert, 1972). The antifungal bioassay of acetone extract of Spirulina platensis was tested in different concentration (250ppm-7000ppm) with different incubation periods (3,6,9 days) at 27±2. For antifungal bioassay different concentrates of solvent extract were taken in Erlenmeyer s flasks in which 40 ml Sabouraud s broth was added and autoclaved at 121 C. Therefore, the flasks were inoculated with 6mm disc of 15 days culture colony. The experiment was carried out in triplicate. Percentage inhibition of mycelial growth in each case was calculated by using following formula: % inhibition = 100 (C-T/) C Where C= Fungal mycelial biomass / dry weight in control. T= Fungal mycelial biomass / dry weight in control in various test concentration. 3. Results Spirulina platensis extract was tested to control biomass of dermatophytic and related fungi i.e., C.albicans, M.canis, M. fulvum. The validity of experimental results of percentage inhibition obtained at different test concentrations was checked by various statistical parameters and tabulated in Table-1. Statistically significant increase has been recorded in the percentage inhibition of the target fungal species with increasing the test concentration (250ppm-7000ppm) of acetone extract. It was observed that among all the fungi, maximum inhibition was caused by the mycelial growth of M.canis and minimum of that by C. albicans. In case of M.fulvum, acetone extract showed the maximum inhibition of (57.29%) at third day of incubation at 6000ppm concentration. It was followed by M.canis (54.2%) and that of C. albicans (51.80%) at the same concentration and exposure time. After nine day of incubation rate of inhibition there was a decline in the rate of inhibition. 4. Discussion The antifungal activity of algal compounds extracted from algae depends upon the type of solvent used for extraction. It was hypothesized that lipids kill microorganisms by leading to disruption of the cellular membrane as well as bacteria, fungi and yeasts because they can penetrate the extensive meshwork of peptidoglycan in the cell wall without visible changes and reach the membrane leading to its disintegration This observation clearly indicates that the polarity of antifungal compounds make them more readily extracted by organic solvents and using organic solvent does not negatively affect their bioactivity against antifungal species. In different studies, the antimicrobial effect of Fischerella sp. (Asthana et al., 2006), Oscillatoria anguistisima and Calothrix parietina (Issa, 1999), Anabaena, Oscillatoria, Pseudoanabaena, 271
Synechocystis, Nostoc (Bloor and England, 1989), Phormidium (Fish and Codd, 1994), and Fischerella ambigua (Ghasemi et al., 2004) extracts on some pathogenic micro-organism have been reported. This is in agreement with our findings, on S. platensis extract which shows similar effects on the microbes. Several authors (Ozdemir et al., 2004: Mundt and Teusher, 1998) have attributed the antifungal activity of cyanobacteria due to the presence of -linolenic acid. 5. Conclusion This concludes from the study that extracts of algal strain used in the present investigation showed better antifungal activity against pathogens used. But further research to be made to identify and purify natural product against antifungal. The enhanced antifungal activity expressed in sequential extraction might be due to the fact that both hydrophobic and hydrophilic bioactive compounds were extracted. An improved knowledge of the composition, analysis, and properties of Spirulina platensis with respect to antifungal compounds would assist in efforts for the pharmaceutical application of these Cyanobacteria. 6. Acknowledgements We are thankful to the Prof VG Das, Director and to Prof D.S. Rao, Head, Department of Botany, Dayalbagh Educational Institute, Dayalbagh, Agra, for providing necessary help. We are thankful to Prof. Pushpa Shrivastava of Jaipur University for providing Spirulina platensis cultures. Financial assistance by the UGC to one of us is acknowledged. 7. References [1] R.K. Asthana, A. Srivastava, A.P. Singh, S.P. Singh, G. Nath, R. Srivastava and B.S. Srivastava. Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree. J. Applied Phcology, 18(1): 33-39 (2006). [2] S. Bloor, and R.R. England. Antibiotic production by the cyanobacterium Nostoc muscorum. J.Appl. Phycol. 1989, 1:367-372. [3] T.V. Desikachary. Cyanophyta. Indian council of Agricultural Research. New Delhi.1959. [4] Ely, R, Supriya and C.G. Naaik. Antimicrobial activity of marine organisms collected from the coast of South East India. J. Exp. Biol. Ecol. 2004, 309:121-127. [5] S.A. Fish, and G.A. Codd, GA. Bioactive compound production by thermophilic and thermotolerant cyanobacteria (blue green algae). World J. Microbial Biotech.1994, 10:338-341. [6] D. Frey, R.S. Oldfield, and R.C. Bridger. A colour Atlas of pathogenic fungi. Wolfe Medical Publication Ltd. 1986, pp.168. [7] Y.Ghasemi, M.T Yazdi, A, Shafiee, M, Amini, S, Shokravi, and G, Zarrini Parsiguine, A Novel Antimicrobial substance from Sischerella ambigua. Pharm biol. 2004, 42, (4-5): 318-322. [8] A.A. Issa. Antibiotic production by the cyanobacteria Oscillatoria anguisitissima and Calothrix parietina. Env. Toxic Pharm. 1999, 8: 33-37. [9] J. Kunert. Keratin decomposition by dermatophytes: evidence of the sulphitolysis of the protein. Experintia.1972, 28:1025-1026. [10] S. Mundt, and E. Teusher. Blue-green algae as a source of pharmacologically-active compound. Pharmazie.1998, 43:809-815. [11] G. Ozdemir, G, Karabayn, U, Dalay, M and Pazarbasi, B: Antibacterial activity of volatile component and various extracts of Spirulina platensis. Phyto. Res. 18(9), 754-757 (2004). [12] M, Shameel. Phycochemical studies on fatty acids from certain seaweeds. Bot. Mar. 1990, 33: 429-432. 272
Table-1. Fungicidal effiect of Acetone leaf extract of S. platensis against pathogenic fungi Incubation period (in days) Three Six Nine Fungi Conc. (ppm) Mean±S.E. SD % Inh. Mean±S.E. SD % Inh. Mean±S.E. SD % Inh. 7000 19.1±0.5 0.7 41.7 21.0±0.6 0.84 53.43 26.5±0.4 0.56 46.3 6000 15.0±0.2 0.28 54.2 22.1±0.8 1.13 50.99 25.8±0.1 0.14 48.0 5000 15.9±0.9 1.27 51.5 27.9±0.2 0.28 38.13 28.0±0.35 0.49 42.9 4000 18.3±0.2 0.28 44.2 29.0±0.4 0.56 35.69 28.7±0.2 0.20 41.90 3000 19.0±0.9 1.27 42.0 32.0±0.1 0.14 29.00 33.5±0.3 0.42 32.38 M.canis 2000 19.9±0.6 0.84 39.3 32.7±0.2 0.28 27.4 36.0±0.7 1.01 27.12 1000 21.6±0.7 0.98 34.1 35.6±0.6 0.84 21.0 42.0±0.3 0.42 7.38 500 23.4±0.5 0.70 28.6 35.9±0.3 0.49 19.51 42.0±0.6 0.93 7.38 250 26.0±0.2 0.70 20.7 40.0±0.7 0.98 11.30 45.7±0.08 0.11 7.48 Control 32.8±0.6 0.84 45.1±0.5 0.7 49.4±0.2 0.28 7000 42.6±0.2 0.28 33.80 68.5±0.5 0.70 38.06 98.5±0.7 0.98 27.73 6000 27.5±0.3 0.42 57.29 64.4±0.3 0.42 41.34 82.5±0.8 1.13 39.47 5000 33.0±0.2 0.28 48.75 73.5±0.6 0.84 33.06 88.8±0.6 0.84 34.84 4000 33.5±0.5 0.70 47.98 76.3±0.4 0.56 30.51 88.4±0.3 0.3 35.14 3000 38.8±0.6 0.84 39.75 80.4±0.2 0.28 26.77 96.1±0.1 0.14 29.49 2000 40.5± 0.5 0.70 37.11 80.4±0.4 0.56 26.77 109.3±0.6 0.84 19.80 M.fulvum 1000 42.6±0.8 1.13 33.85 82.8±0.1 0.14 24.59 116.4±0.4 0.4 14.60 500 48.8±0.6 0.84 24.22 90.7±0.2 0.28 17.39 121.2±0.2 0.28 11.07 250 53.8±0.6 0.84 16.45 90.8±0.3 0.42 17.30 133.1±0.5 0.70 2.34 Control 64.4±0.8 1.13 109.8±0.6 0.84 136.3±0.2 0.28 273 7000 88.8±0.1 0.25 19.71 104.5±0.3 0.49 22.30 120.5±0.5 0.74 17.40 6000 53.3±0.3 0.42 51.80 89.4±0.1 0.14 33.53 98.2±0.5 0.73 32.69 5000 61.8±0.8 1.13 44.12 91.7±0.4 0.56 31.82 109.3±0.09 0.12 26.08 4000 64.5±0.2 0.28 41.68 96.7±0.5 0.70 28.1 110.6±0.3 0.50 24.19 3000 76.3±0.6 0.84 31.01 96.9±0.8 1.13 27.95 114.2±0.2 0.28 21.72 Candida albicans 2000 81.6±0.3 0.42 26.22 99.8±0.1 0.14 25.79 116.6±0.9 1.17 20.08 1000 85.4±0.2 0.28 22.78 110.9±0.1 0.26 17.54 121.0±0.3 0.42 17.06 500 91.6±0.5 0.70 17.17 114.0±0.5 0.72 15.25 122.5±0.3 0.52 16.03 250 98.7±0.1 0.14 10.75 115.9±0.1 0.24 13.82 136.9±0.07 0.09 6.16 Control 110.6±0.3 0.42 134.5±0.3 0.42 145.9±0.6 0.84
Fig:1 Photographs showing fungicidal effect of Acetone leaf extract of S. platensis against pathogenic fungi for 3,6,9 days 3 RD DAY Inhibition of C.albicans 6 TH DAY Inhibition of C.albicans 9 TH DAY Inhibition of C.albicans 3 RD DAY Inhibition of M. canis 6 TH DAY Inhibition of M.canis 9 TH DAY Inhibition of M.canis 3 RD DAY Inhibition of M. fulvum 6 TH DAY Inhibition of M. fulvum 9 TH DAY Inhibition of M.canis 274