Article ID: WMC00687 ISSN 2046-1690

Antifungal activity of isolated from Cassia fistula L. flower

Corresponding Author: Dr. S Ignacimuthu, Director, Entomology Research Institute, Loyola College, Nungambakkam, Chennai, 600 034 - India

Submitting Author: Dr. V Duraipandiyan, Scientist, Division of Ethnopharmacology, Entomology Research Institute, Loyola College, 600 034 - India

Article ID: WMC00687 Article Type: Research articles Submitted on:20-Sep-2010, 11:14:49 AM GMT Published on: 20-Sep-2010, 05:16:23 PM GMT Article URL: http://www.webmedcentral.com/article_view/687 Subject Categories:PHARMACOLOGY Keywords:Antifungal, Cassia fistula, MIC, Rhein How to cite the article:Duraipandiyan V, Ignacimuthu S. Antifungal activity of Rhein isolated from Cassia fistula L. flower . WebmedCentral PHARMACOLOGY 2010;1(9):WMC00687 Competing Interests: We have no competing interest

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Antifungal activity of Rhein isolated from Cassia fistula L. flower

Author(s): Duraipandiyan V, Ignacimuthu S

Abstract amount of alkaloids have also been reported in the flowers; traces of triterpenes have been observed in both flowers and fruits [8,9]. Our preliminary evaluation of ethyl acetate extract Antifungal activity of rhein (1, 8- from Cassia fistula flowers showed significant dihydroxyanthraquinone- 3carboxylic acid) isolated antifungal activity [10]. In the present work, we report from the ethyl acetate extract of Cassia fistula flower the separation and identification of rhein from C. fistula was studied. Rhein inhibited the growth of many fungi flowers and its antifungal effect. such as Trichophyton mentagrophytes (MIC 31.25 µg/ml), Trichophyton simii (MIC 125 µg/ml), Materials and Methods Trichophyton rubrum (MIC 62.5 µg/ml) and Epidermophyton floccosum (MIC 31.25 µg/ml). Introduction Plant material Cassia fistula flowers were collected from Loyola College Campus, Chennai, India. It was authenticated by Dr. S. Amerjotthy, Department of Botany, Plants used in traditional medicine usually constitute Presidency College, Chennai, India. A voucher an important source of new biologically active specimen (ERIC-D-73) is deposited at the herbarium compounds. Numerous useful drugs have been of Entomology Research Institute, Loyola College, discovered from higher plants by following up Chennai. ethnomedical uses [1]. During the past several years, Preparation of crude extract there has been an increasing incidence of fungal The extracts were taken using cold percolation method. infections due to a growth in immunocompromised Fresh flowers were collected (9kg) and shade dried at population such as organ transplant recipients, cancer room temperature and ground in a manual mill. The and HIV/AIDS patients. This fact coupled with the powder (1kg) was extracted with 3 ltr (1:3 w/v) of resistance to antibiotics and toxicity during prolonged hexane for 48 hours. The extract was filtered through treatment with several antifungal drugs, has been the a Buchner funnel with Whatman number 1 filter paper. reason for an extended search for newer drugs to treat The filtrate was evaporated to dryness under reduced opportunistic fungal infections [2,3]. pressure using rotary evaporator at 40ºC. The Cassia fistula L., (Caesalpiniaceae), a semi-wild Indian remains of the plant material were extracted with Labernum (also known as the Golden Shower), is chloroform (11g), ethyl acetate (17g), methanol (20) distributed in various countries including Asia, South and water (13g) sequentially in a similar manner. The Africa, Mexico, China, West Indies, East Africa and crude extracts were stored at 4ºC until further use. Brazil. It is an ornamental tree with beautiful bunches Isolation of active compound of yellow flowers. C. fistula exhibited significant The crude ethyl acetate extract (10 g) was subjected antifungal activity and showed properties that support to column chromatography over silica gel (200 folkloric use in the treatment of some diseases as g-acme’s 100–200 mesh) and eluted with hexane broad-spectrum antifungal agent [4]. The whole plant followed by the combination of hexane: ethyl acetate is used to treat diarrhea; seeds, flowers and fruits are ranging from 95:5 to 100. 117 fractions were collected used to treat skin diseases, fever, abdominal pain and in a 200ml conical flask. After checking TLC, the leprosy by traditional people [5]. This plant has a fractions were combined in to 24 fractions. Fraction 10 strong tendency to contain derivatives. showed a crystal which was subjected to From the genus Cassia many quinone derivatives crystallographic analysis and identified reported.10 such as Kaempferol have also been isolated; a Fraction 18 showed single spot on TLC (Rf = 0.36) proanthocyanidin has been isolated from the acetone and yielded 210 mg; this fraction was eluted using extract of the flower [6]. A bianthraquinone glycoside, hexane: ethyl acetate (10:9) as mobile phase solvent fistulin, together with kaempferol and rhein have been system. The spot turned pink on exposure to isolated from ethanol extracts of C. fistula flowers [7]. ammonia vapor; it indicated the presence of Besides phenolics and their derivatives, a certain

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. The compound was subjected to MHz, dimethyl sulfoxide (DMSO)] δ ppm: 161.2 (C-1), spectroscopic analysis. 124.2 (C-2), 165.6 (C-3), 119.0 (C-4), 124.7 (C-5), Spectroscopic analysis 138.5 (C-6), 124.7 (C-7), 161.5 (C-8), 181.2 (C-9), IR, 1H-NMR, 13C NMR and MASS were taken from 181.2 (C-10), 130.0 (C-4a), 118.9 (C-8a), 118.7 Nicholas Primal Pvt. Ltd. Ennore, India and used to (C-9a), 133.3 (C-10a), 191.4 (3-COOH) which identify the isolated compound. corresponded to those reported [12]. The active Fungi compound was identified as Rhein Fungi, Trichophyton rubrum, T. rubrum 57/01, T. (1,8-dihydroxyanthraquinone-2-carboxylic acid) (Fig Mentagrophytes, T. simii, Epidermophyton floccosum, 1). The same compound was previously reported from Scopulariopsis sp., Aspergillus niger, Botyritis cinerea, some other plants. Sun et al. [14] chromatographed Curvularia lunata and Candida albicans MTCC 227 and purified , chrysophanol and rhein from were used for the experiments. All cultures were Rheum officinale extract. Wang et al. [15] separated obtained from the Department of Microbiology, and determined active anthraquinone components Christian Medical College, Vellore, Tamil Nadu, India physcion, chrysophanol, aloe-emodin, emodin, and Assay for antifungal activity rhein from the Chinese herb Polygonum multiflorum. The antifungal activity of the isolated compound was The isolated compound (rhein) was tested for determined using standard method [11]. The antifungal activity and results are shown in table 1. compound was tested by micro broth two-fold serial Rhein showed activity against T. mentagrophytes (MIC dilution technique. The crude extract and compound 31.25 µg/ml), T. simii (MIC 125 µg/ml), T. rubrum (62.5 were dissolved in water + 2% dimethyl sulfoxide µg/ml), T. rubrum 57(MIC 62.5 µg/ml), E. floccosum (DMSO). The initial concentration of extract was 1mg (MIC 31.25 µg/ml), Scopulariopsis sp (MIC 250 µg/ml), /ml; the initial concentration of the compound was C.lunata (MIC 250 µg/ml) and A. niger, M .grisea, B. 250µg/ml. The initial test concentrations were serially cinerea and C. albicans were not inhibited. diluted two-fold. Each well was inoculated with 5 ml of suspension containing 104 spore/ml of fungi. Discussion Fluconazole and Ketoconazole were included in the assays as positive controls. The plates were incubated for 24, 48 or 72 h at 27 ° C up to 9 days. MIC was Fungal diseases have increased dramatically in recent defined as the lowest extract concentration showing years. The treatment of mycoses has lagged behind no visible fungal growth after incubation time. and fewer antifungal than antibacterial substances are available. Therefore, a search for new antifungal drugs Results is extremely necessary [16]. Many plants are now used to treat various infectious diseases. In this study we examined the antifungal activity of C.fistula flower The present study deals with the antifungal activity of ethyl acetate extract and isolated compound rhein. crude ethyl acetate extract and an isolated compound Traditional uses of this plant favor its use as antifungal from C. fistula flower. The crude ethyl acetate extract drug. Ethyl acetate extract of C.fistula showed inhibited the growth of fungi T. mentagrophytes (MIC promising antifungal activity. This indicated that there 250 µg/ml), T. simii (MIC 1000 µg/ml), T. rubrum (1000 may be some active compound involved. So we µg/ml), T.rubrum 57 (MIC 500 µg/ml), E. floccosum selected ethyl acetate extract for isolation of active (MIC 500 µg/ml), Scopulariopsis sp (MIC 500 µg/ml). compound. Isolated active compound was confirmed The structural identification of compound was carried by comparing the IR, 1H NMR, 13C NMR, MASS and out using IR, MS, 1H NMR and 13C NMR spectra as m.p., data with that of the known compound rhein follows: the EI-MS: m/z 284, 267, 256, 239, 228, 211, Previously the same compound was reported against 183, 155, 142, 126. It showed the molecular ion at m/z Botrytis cinerea [17]; Candida albicans, Trichophyton 284, which was corresponding to the molecular mentagrophytes [18]. However, rhein was not yet formula C15H8O6 of rhein; its melting point was at tested against T. rubrum, T. rubrum 57/01, T. simii, E. 321ºC [12]. The 1H NMR spectrum revealed five floccosum, Scopulariopsis sp., A. niger, C. lunata aromatic protons of which two were broad singlets due and M. grisea. Rhein inhibited the growth of T.rubrum, to meta coupling. 1H NMR (75 MHz, DMSO): 11.9 (1H, T. rubrum 57, T. simii, E.floccosum and Scopulariopsis brs, C1–OH), 11.5 (1H, brs, C8–OH), 8.13 (1H,brs, sp. The anthraquinone derivative rhein is significantly C2–H), 7.40 (1H, d, J = 8.5 Hz, C5–H), 7.73 (1H, m, antiseptic and is especially toxic to the pathogen [19]. C6–H), 7.75 (1H,brs, C4–H), 7.81(1H, d, J = 8.0 Hz, Trichophyton rubrum is the most prevalent pathogenic C7–H) corresponded to rhein [13]. 13C NMR [(300 fungus; worldwide it represents 80% of clinical isolates

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[20]. In our findings rhein inhibited the growth of T. 7. Kumar A, Pande CS, Kaul RK. Chemical rubrum at 62.5 µg/ml. examination of Cassia fistula flowers. Ind J Chem Generally anthraquinone derivatives, which include 1966;4: 460. emodin, chrysophanol, rhein, aloe-emodin, physcion, 8. Asseleih LMC, Hernandez OH, Sanchez JR. and their glucosides, and important active components Seasonal variation in the content of Sennosides in with various pharmacological actions such as leaves and pods of two Cassia fistula populations. purgation, antibacterial, antifungal and antitumor Phytochem 1990; 29: 3095–3099. activity [21]. Antifungal activities of anthraquinones 9. Guri-Fakim A, Gueho J, Sewraj MD, Dulloo E. and napthoquinones isolated from natural sources Plantes Medicinales de lile Maurice, Editions de have been reported [22-25]. L’Ocean Indien, Mauritius 1994; 580. Rhein was isolated from the leaves of Cassia 10. Duraipandiyan V, Ignacimuthu S. Antibacterial and reticulata and tested against Neisseria gonorrhoeae. It antifungal activity of Cassia fistula L.: An exhibited significant inhibitory activities [26]. Novel ethnomedicinal plant. J Ethnopharmacol 2007; 112: anthraquinone 590-594. (3,4-dihydroxy-1-methoxyanthraquinone- 11. National Committee for Clinical Laboratory 2-carboxaldehyde) was isolated from ethanolic extract Standards. Reference method for broth dilution of Saprosma fragrans and tested against T. antifungal susceptibility testing of filamentous fungi. mentagrophytes (12.5 µg/ml) [27]. Our results Approved standard M38-A. Wayne, PA: National showed that the isolated compound significantly Committee for Clinical Laboratory Standards, 2002. inhibited the growth of T.mentagrophytes at 31.25 12. Wei Y, Zhang T, Ito Y. Preparative separation of µg/ml and E. floccosum 31.25 µg/ml. rhein from Chinese traditional herb by repeated Kanokmedhakul et al. [28] have isolated seven high-speed counter-current chromatography. J anthraquinone and triterpenoids from Prismatomeris Chromatogr A 2003; 1017: 125-130. fragrans and also reported antifungal activity for 13. Liu R, Li A, Sun A. Preparative isolation and isolated compounds. Previously rhein was also purification of hydroxyanthraquinones and cinnamic reported to inhibit oral bacteria [29]. acid from the Chinese medicinal herb Rheum officinale Baill by high-speed counter-current chromatography. J Conclusion Chromatogr A 2004; 1052: 217-221. 14. Sun M, Sakakibara H, Ashida H, Danno G, Kanazawa K. Cytochrome P4501A1-Inhibitory action Rhein can be used as antifungal agent. The present of antimutagenic anthraquinones-activity Relationship. findings show that rhein exhibited good activity against Biosci Biotech Biochem 2000; 64: 1373-1378. fungi. 15. Wang D, Yang G, Engelhardt H, Liu H, Zhao J. Separation by Capillary Zone Electrophoresis of the References active anthraquinone components of the Chinese herb Polygonum multiflorum Thunb. Chromatographia 2001; 53: 185-189. 1. Fabricant DS, Farnsworth NR. The value of plants 16. Fortes TO, Alviano DS, Tupinamba G, Padron TS, used in traditional medicine for drug discovery. Antoniolli AR, Celuta Sales Alviano CS, Seldin L. Environmental Health Perspectives 2001; 109:69–75. 2008. Production of an antimicrobial substance 2. Giordani R, Trebaux J, Masi M, Regli P. Enhanced against Cryptococcus neoformans by Paenibacillus antifungal activity of ketoconazole by Euphorbia brasilensis Sa3 isolated from the rhizosphere of characias latex against Candida albicans. J Kalanchoe brasiliensis. Microbiol Res 163,200-207. Ethnopharmacol 2001; 78:1–5. 17. Sosa KG, Alvarez NV, Lubben P, Pena-Rodriquez 3. Fostel J, Lartey P. Emerging novel antifungal LM. Chrysophanol, an antimicrobial anthraquinone agents. Drug Discover Today 2000;5: 25–32. from the root extract of Colubrina greggii. J Mex Chem 4. Prashanth Kumar V, Chauhan NS, Padh H, Rajani Soc 2006; 50: 76-78. M. Search for antibacterial antifungal agents from 18. Agarwal SK, Singh SS, Verma S, Kumar S. selected Indian medicinal plants. J Ethnopharmacol Antifungal activity of anthraquinone derivatives from 2006; 107: 182-188. Rheum emodi. J Ethnopharmacol 2000; 72: 43-46. 5. Perry LM. Medicinal plants of East and South East 19. Brown JR, Imam SH. Prog Mag Chem 1984; 21: Asia. MIT Press, Cambridge, 1980. 196. 6. Narayanan V, Seshadri TR. Proanthocyanidins of 20. Chan MMY. Antimicrobial effect of resveratrol on Cassia fistula. Ind J Chem 1972; 10: 379-381. dermatophytes and bacterial pathogens of the skin.

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Biochem Pharmacol 2002; 63: 99-104. 21. Dictionary of Traditional Chinese Medicine, first ed., New Medical College of Jiangsu, People’s Published of Shanghai, 1997: 102. 22. Fuzellier MC, Morter F, Lectard P. Antifungal activity of Cassia alata L. Annals of Pharmacology, France. 1982; 40: 357–363. 23. Inamori Y, Kato Y, Kubo M, Kamiki T, Takemoto T, Nomoto K. Studies on metabolites produced by Aspergillus terreus var. aureus. I. Chemical structures and antimicrobial activities of metabolites isolated from culture broth. Chemical and Pharmaceutical Bulletin. 1983; 31: 4543-4548. 24. Cyong JC, Matsumoto T, Arakawa K, Kiyohara H, Yamada H, Otsuka Y. Antibacteriosides Fragilus substance from rhubarb. J Ethnopharmacol 1987; 19: 279-283. 25. Harvey HE, Waring JM. Antifungal and other compounds isolated from the roots of New Zealand flax plants (The genus Phormium). J Nat Prod 1987; 50: 767–776. 26. Robbins W J, Kavanagh F, Thayer JD. Bull Torrey Bot Club 1947; 74: 287. 27. Barlocco D. A novel antifungal anthraquinone from Saprosma fragrans. Drug Discovery Today 2007; 12, No-5/6(doi:10.1016/j.drudis.2007.01.001). 28. Kanokmedhakul K, Kanokmedhakul S, Phatchana R. Biological activity of Anthraquinones and Triterpenoids from Prismatomeris fragrans. J Ethnopharmacol 2005; 100:284-288. 29. Didry N, Dubreuil L, Pinkas M. Activity of anthraquinonic and naphthoquinonic compounds on oral bacteria. Pharmazie 1994; 49: 681-683.

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Illustrations Illustration 1

Figure 1: Rhein (1, 8-dihydroxyanthraquinone-3-carboxylic acid) isolated from ethyl acetate extract

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Illustration 2

Table 1: Antifungal activity of Rhein (MIC µg/ml)

Tested Fungi EA C Fl Ket

Trichophyton mentagrophytes 66/01 250 31.2 25 <12.5

T. simii 110/02 1000 125 <12.5 <12.5

T. rubrum MTCC 296 1000 62.5 <12.5 <12.5

T. rubrum 57/01 500 62.5 25 <12.5

Epidermophyton floccosum 73/01 500 31.2 12.5 <12.5

Scopulariopsis sp. 101/01 500 250 <12.5 <12.5

Aspergillus niger MTCC 1344 >1000 >250 100 <12.5

Curvularia lunata 46/01 >1000 >250 <12.5 <12.5

Magnaporthe grisea >1000 >250 nt nt

Botrytis cinerea >1000 >250 nt nt WebmedCentral > Research articles Page 7 of 8 Candida albicans MTCC 227 >1000 >250 >100 25 EA- Ethyl acetate (Crude extract) C- Rhein; Fl – Fluconazole (antifungal agent); Ket- Ketoconazole (antifungal agent); nt- not test WMC00687 Downloaded from http://www.webmedcentral.com on 19-Jul-2012, 08:06:11 AM

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