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Antifungal activity of anthraquinone derivatives from Rheum emodi
Journal of Ethnopharmacology 72 (2000) 43 – 46 www.elsevier.com/locate/jethpharm
Antifungal activity of anthraquinone derivatives from Rheum emodi S.K. Agarwal *, Sudhir S. Singh, Sushma Verma, Sushil Kumar Central Institute of Medicinal and Aromatic Plants, P.O.-CIMAP, Lucknow 226 015, India Received 13 July 1999; received in revised form 7 February 2000; accepted 22 February 2000
Abstract Rhein, physcion, aloe-emodin and chrysophanol isolated from Rheum emodi rhizomes exhibited antifungal activity against Candida albicans, Cryptococcus neoformans, Trichophyton mentagrophytes and Aspergillus fumigatus (MIC 25–250 mg/ml). © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rheum emodi; Polygonaceae; Revand-chini; Rhein; Physcion; Chrysophanol; Aloe-emodin; Antifungal activity
1. Introduction Rheum emodi (Polygonaceae), commonly known as revand-chini, is the Himalayan species of Indian rhubarb found wild at an altitude of 4000 – 12 000 feet in Kashmir, Nepal, Sikkim and Bhutan. Rhubarb has been successfully grown in certain parts of Assam (Nadkarni, 1954). Roots of the Indian rhubarb is darker, inferior in aroma, is a well known stomachic, bitter and cathartic, and used all over the world (Thakur et al., 1989). There have already been many reports about antibacterial and antifungal activities of the anthraquinones, the napthoquinones isolated from natural sources (Agarwal et al., 1976; Fuzellier et al., 1982; Inamori et al., 1983; Cyong et al., 1987; Harvey and Waring, 1987). In addition several other biological activities such as laxative, diuretic, and in vivo inhibitory effect towards P388 leukemia in mice are also reported (Lu and Chen, 1989;
Oshio and Kawamura, 1985; Zhou and Chen, 1988). The present communication deals with naturally occurring antifungal principles from R. emodi rhizomes. A bioassay guided isolation, characterisation and evaluation of their methanolic extracts exhibited antifungal activities which resulted in four anthraquinone derivatives (1– 4) as active components.
* Corresponding author. Fax: +91-522-342666. 0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 9 5 - 1
44
S.K. Agarwal et al. / Journal of Ethnopharmacology 72 (2000) 43–46
2. Methods
2.1. Plant material The rhizomes of Rheum emodi were purchased from a local market and identified by Dr S.P. Jain, Botany Department of CIMAP, Lucknow, India, where a voucher specimen has been deposited.
2.2. Analytical material and methods Column chromatography was carried out using silica gel 60–120 mesh (Ranbaxy) and TLC silica gel G (Ranbaxy). IR spectra were recorded with KBr pellets on a Perkin-Elmer 1710 FT-IR spectrophotometer. 1H NMR spectra were obtained on a Bruker DRX (200 MHz) instrument with TMS as internal standard while EI-MS was recorded at 70 eV on a JEOLJMSD 3000 spectrometer.
2.3. Preparation of the extract The dried, ground rhizomes (2.0 kg) were extracted with methanol (3.5 l) at room temperature thrice and the total combined extract was evaporated in vacuo to dryness as a dark brown mass (132 g). A portion of this methanolic extract (30 g) was chromatographed on a silica gel (600 g) column and eluted successively with hexane, hexane–CHCl3 mixtures ((3:1), (2:1), (1:2) and (1:4) v/v), CHCl3 and CHCl3 – MeOH ((9:1), (8:2), (7:3) and (1:1) v/v). The eluates (100 ml each) were monitored by TLC and grouped into 11 fractions.
2.4. Isolation of anthraquinone deri6ati6es Fraction no. 5 (1.05 g) was subjected to column chromatography over silica gel (60 – 120 mesh, 30 g) and eluted with hexane, hexane – CHCl3 ((4:1), (3:2), (2:3), and (1:4)) mixtures yielding compound (1, mp 321o C, 124 mg) and (2, mp 207o C, 86 mg). Further, the fraction no. 7 (2.10 g) was also rechromatographed over silica gel (45 g) and eluted with hexane, hexane – CHCl3 ((3:1), (2:1), (1:1), (1:2), and (1:3) v/v)
and CHCl3 yielding compound 3 (m.p. 223°C, 146 mg) and 4 (m.p. 196°C, 67 mg), respectively.
2.5. Spectroscopic analysis These anthraquinone derivatives were identified as rhein (1, Nawa et al., 1961), physcion (2, Kalidhar, 1989), aloe-emodin (3, Muhatadi and Moss, 1969) and chrysophanol (4, Ayyangar et al., 1961) by comparing the m.p., IR, 1H NMR and MS with reported known compounds.
2.6. Bioassay 2.6.1. Antifungal acti6ity The antifungal activity of the crude methanolic extract as well as the pure compounds was determined by a routine in vitro procedure (Shadomy and Espinel-Ingroff, 1974). Briefly, the compound was tested by macrobroth 2-fold serial dilution technique. The product was dissolved in a minimum quantity of DMSO (10 mg/ml) to get a stock solution. Fungi Candida albicans, Cryptococcus neoformans, Sporotrichum schenckii, Trichophyton mentagrophytes and Aspergillus fumigatus seeded broth (105 cfu/ml) were prepared in Sabouraud dextrose broth and nutrient broth, respectively. Drug dilutions were made serially. The tubes were incubated at 289 1°C and the minimal inhibitory concentration (MIC mg/ml) was recorded after 72–96 h (mycelial fungi) post-incubation. Suitable controls: broth control (without infections), growth controls (with infections), solvent (DMSO) and drug controls of both test product(s) and standard drug were set under identical conditions. The last tube with no apparent growth of the organism represented the MIC of the compound. 3. Results The antifungal screening of crude MeOH extract and anthraquinone derivatives (1–4) showed the MIC to be 25–250 mg/ml. The pure compounds were more active than the crude extract (see Table 1).
S.K. Agarwal et al. / Journal of Ethnopharmacology 72 (2000) 43–46
45
Table 1 Antifungal activity of methanolic extract and anthraquinone derivatives (1–4, MIC mg/ml compared to ketoconazole)a Fungi C. albicans (CA) Cry. neoformans (CN) S. schenckii (SS) T. mentagrophytes (TM) A. fumigatus (AF)
MeOH extract 250 – – 250 250
1
2 50 50 – 25 –
50 50 – 25 –
3 50 – – 50 –
4 50 50 – 25 50
Ketoconazole 0.1 0.4 0.1 2.5 0.7
a –, Not active upto 1000 mg/ml. Sources: CA, Smith Kline & French (SKF); CN and TM, Instl. Pasteur, France; SS and AF, Culture Collection (CDRI), India.
4. Discussion The dark brown methanolic extract of R. emodi rhizomes was subjected to column chromatography. The methanolic extract exhibited potent antifungal activities in our primary screening programme. Chromatographic purification of the methanol extract on silica gel produced rhein (1), physcion (2), aloe-emodin (3) and chrysophanol (4). Their identities were established by comparing the m.p., IR, 1H NMR and MS data with that of the known compounds. The antifungal screening of crude MeOH extract (MIC 250 mg/ml) and anthraquinone derivatives (1–4) showed the MIC to be 25 – 50 mg/ml (Table 1). The pure compounds were more active than the crude extract and it also appears that the substituents at C6 do not affect the relative degree of antifungal activities. Anthraquinone derivatives physcion, chrysophanol, and emodin are reported to exhibit antifungal activity (MIC 90 – 100 mg/ml) and the inhibition effect depends on the presence of substituents (OH, OMe, and OAc at C1 and C8). These also have antimicrobial properties (Manojlovic et al., 1998).
Acknowledgements The authors are grateful to the Head, RSIC, at CDRI, Lucknow, for obtaining the spectral data.
References Agarwal, J.S., Rastogi, R.P., Srivastava, O.P., 1976. In vitro
toxicity of constituents of Rumex maritimus Linn. to ring worm fungi. Current Science 45 (17), 619 – 620. Ayyangar, N.R., Bapat, D.S., Joshi, B.S., 1961. Anthraquinones and anthrone series: Part XXVI — a new synthesis of chrysophanol, rhein, islandicin, emodin and physcion. Journal of Scientific and Industrial Research 20B, 493 – 497. Cyong, J.C., Matsumoto, T., Arakawa, K., Kiyohara, H., Yamada, H., Otsuka, Y., 1987. Antibacteriosides Fragilus substance from rhubarb. Journal of Ethnopharmacology 19, 279 – 283. Fuzellier, M.C., Morter, F., Lectard, P., 1982. Antifungal activity of Cassia alata L. Annals of Pharmacology, France 40 (4), 357 – 363. Harvey, H.E., Waring, J.M., 1987. Antifungal and other compounds isolated from the roots of New Zealand flax plants (The genus Phormium). Journal of Natural Products 50 (4), 767 – 776. Inamori, Y., Kato, Y., Kubo, M., Kamiki, T., Takemoto, T., Nomoto, K., 1983. 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 31 (12), 4543 – 4548. Kalidhar, S.B., 1989. Location of glycosylation and alkylation sites in anthraquinones by 1H NMR. Phytochemistry 28 (9), 2455 – 2458. Lu, M., Chen, Q.H., 1989. Biochemical study of Chinese rhubarb. XXIX. Inhibitory effects of anthraquinone on P388 leukemia in mice. Journal of the China Pharmacology University 20, 155 – 157. Manojlovic, N.T., Solujic, S., Sukdolak, S., Krstic, L.J., 1998. Anthraquinones from the lichen Xanthoria parietina. Journal of the Serbian Chemical Society 63 (1), 7 – 11. Muhatadi, F.J., Moss, M.J.R., 1969. A synthesis of aloeemodin 1,8-di-b-D-glucoside. Tetrahedron Letters 4B, 3751 – 3752. Nadkarni K.M. (1954). Rheum emodi. The Indian Materia Medica, A.K. Nadkarni (Ed.), 3rd edn., Pub. Dhootapapeshwar, Prakashan Ltd., Panvel, Bombay, India, 2113. Nawa, H., Uchibayashi, M., Matsuoka, T., 1961. Structure of rhein. Journal of Organic Chemistry 26, 979 – 980.
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S.K. Agarwal et al. / Journal of Ethnopharmacology 72 (2000) 43–46
Oshio, H., Kawamura, N., 1985. Determination of the laxative compounds in rhubarb by high performance liquid chromatography. Shoyakugaku Zasshi 39, 131– 138. Shadomy, S., Espinel-Ingroff, A., 1974. In: Lennett, E.H., Spaulding, E.H., Truant, J.P. (Eds.), Manual of Clinical Microbiology. American Society of Microbiology, Washington, DC, pp. 569 –573.
Thakur, R.S., Puri, H.S., Hussain, A., 1989. Rheum australe. Major Medicinal Plants of India. CIMAP, Lucknow, pp. 443 – 447. Zhou, X.M., Chen, Q.H., 1988. Biochemical study of Chinese rhubarb. XXII. Inhibitory effect of anthraquinone derivatives on sodium-potassium-ATPase of rabbit renal medulla and their diuretic action. Acta Pharmacologia Sinica 23, 17 – 20.
.
Antifungal activity of anthraquinone derivatives from Rheum emodi S.K. Agarwal *, Sudhir S. Singh, Sushma Verma, Sushil Kumar Central Institute of Medicinal and Aromatic Plants, P.O.-CIMAP, Lucknow 226 015, India Received 13 July 1999; received in revised form 7 February 2000; accepted 22 February 2000
Abstract Rhein, physcion, aloe-emodin and chrysophanol isolated from Rheum emodi rhizomes exhibited antifungal activity against Candida albicans, Cryptococcus neoformans, Trichophyton mentagrophytes and Aspergillus fumigatus (MIC 25–250 mg/ml). © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rheum emodi; Polygonaceae; Revand-chini; Rhein; Physcion; Chrysophanol; Aloe-emodin; Antifungal activity
1. Introduction Rheum emodi (Polygonaceae), commonly known as revand-chini, is the Himalayan species of Indian rhubarb found wild at an altitude of 4000 – 12 000 feet in Kashmir, Nepal, Sikkim and Bhutan. Rhubarb has been successfully grown in certain parts of Assam (Nadkarni, 1954). Roots of the Indian rhubarb is darker, inferior in aroma, is a well known stomachic, bitter and cathartic, and used all over the world (Thakur et al., 1989). There have already been many reports about antibacterial and antifungal activities of the anthraquinones, the napthoquinones isolated from natural sources (Agarwal et al., 1976; Fuzellier et al., 1982; Inamori et al., 1983; Cyong et al., 1987; Harvey and Waring, 1987). In addition several other biological activities such as laxative, diuretic, and in vivo inhibitory effect towards P388 leukemia in mice are also reported (Lu and Chen, 1989;
Oshio and Kawamura, 1985; Zhou and Chen, 1988). The present communication deals with naturally occurring antifungal principles from R. emodi rhizomes. A bioassay guided isolation, characterisation and evaluation of their methanolic extracts exhibited antifungal activities which resulted in four anthraquinone derivatives (1– 4) as active components.
* Corresponding author. Fax: +91-522-342666. 0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 9 5 - 1
44
S.K. Agarwal et al. / Journal of Ethnopharmacology 72 (2000) 43–46
2. Methods
2.1. Plant material The rhizomes of Rheum emodi were purchased from a local market and identified by Dr S.P. Jain, Botany Department of CIMAP, Lucknow, India, where a voucher specimen has been deposited.
2.2. Analytical material and methods Column chromatography was carried out using silica gel 60–120 mesh (Ranbaxy) and TLC silica gel G (Ranbaxy). IR spectra were recorded with KBr pellets on a Perkin-Elmer 1710 FT-IR spectrophotometer. 1H NMR spectra were obtained on a Bruker DRX (200 MHz) instrument with TMS as internal standard while EI-MS was recorded at 70 eV on a JEOLJMSD 3000 spectrometer.
2.3. Preparation of the extract The dried, ground rhizomes (2.0 kg) were extracted with methanol (3.5 l) at room temperature thrice and the total combined extract was evaporated in vacuo to dryness as a dark brown mass (132 g). A portion of this methanolic extract (30 g) was chromatographed on a silica gel (600 g) column and eluted successively with hexane, hexane–CHCl3 mixtures ((3:1), (2:1), (1:2) and (1:4) v/v), CHCl3 and CHCl3 – MeOH ((9:1), (8:2), (7:3) and (1:1) v/v). The eluates (100 ml each) were monitored by TLC and grouped into 11 fractions.
2.4. Isolation of anthraquinone deri6ati6es Fraction no. 5 (1.05 g) was subjected to column chromatography over silica gel (60 – 120 mesh, 30 g) and eluted with hexane, hexane – CHCl3 ((4:1), (3:2), (2:3), and (1:4)) mixtures yielding compound (1, mp 321o C, 124 mg) and (2, mp 207o C, 86 mg). Further, the fraction no. 7 (2.10 g) was also rechromatographed over silica gel (45 g) and eluted with hexane, hexane – CHCl3 ((3:1), (2:1), (1:1), (1:2), and (1:3) v/v)
and CHCl3 yielding compound 3 (m.p. 223°C, 146 mg) and 4 (m.p. 196°C, 67 mg), respectively.
2.5. Spectroscopic analysis These anthraquinone derivatives were identified as rhein (1, Nawa et al., 1961), physcion (2, Kalidhar, 1989), aloe-emodin (3, Muhatadi and Moss, 1969) and chrysophanol (4, Ayyangar et al., 1961) by comparing the m.p., IR, 1H NMR and MS with reported known compounds.
2.6. Bioassay 2.6.1. Antifungal acti6ity The antifungal activity of the crude methanolic extract as well as the pure compounds was determined by a routine in vitro procedure (Shadomy and Espinel-Ingroff, 1974). Briefly, the compound was tested by macrobroth 2-fold serial dilution technique. The product was dissolved in a minimum quantity of DMSO (10 mg/ml) to get a stock solution. Fungi Candida albicans, Cryptococcus neoformans, Sporotrichum schenckii, Trichophyton mentagrophytes and Aspergillus fumigatus seeded broth (105 cfu/ml) were prepared in Sabouraud dextrose broth and nutrient broth, respectively. Drug dilutions were made serially. The tubes were incubated at 289 1°C and the minimal inhibitory concentration (MIC mg/ml) was recorded after 72–96 h (mycelial fungi) post-incubation. Suitable controls: broth control (without infections), growth controls (with infections), solvent (DMSO) and drug controls of both test product(s) and standard drug were set under identical conditions. The last tube with no apparent growth of the organism represented the MIC of the compound. 3. Results The antifungal screening of crude MeOH extract and anthraquinone derivatives (1–4) showed the MIC to be 25–250 mg/ml. The pure compounds were more active than the crude extract (see Table 1).
S.K. Agarwal et al. / Journal of Ethnopharmacology 72 (2000) 43–46
45
Table 1 Antifungal activity of methanolic extract and anthraquinone derivatives (1–4, MIC mg/ml compared to ketoconazole)a Fungi C. albicans (CA) Cry. neoformans (CN) S. schenckii (SS) T. mentagrophytes (TM) A. fumigatus (AF)
MeOH extract 250 – – 250 250
1
2 50 50 – 25 –
50 50 – 25 –
3 50 – – 50 –
4 50 50 – 25 50
Ketoconazole 0.1 0.4 0.1 2.5 0.7
a –, Not active upto 1000 mg/ml. Sources: CA, Smith Kline & French (SKF); CN and TM, Instl. Pasteur, France; SS and AF, Culture Collection (CDRI), India.
4. Discussion The dark brown methanolic extract of R. emodi rhizomes was subjected to column chromatography. The methanolic extract exhibited potent antifungal activities in our primary screening programme. Chromatographic purification of the methanol extract on silica gel produced rhein (1), physcion (2), aloe-emodin (3) and chrysophanol (4). Their identities were established by comparing the m.p., IR, 1H NMR and MS data with that of the known compounds. The antifungal screening of crude MeOH extract (MIC 250 mg/ml) and anthraquinone derivatives (1–4) showed the MIC to be 25 – 50 mg/ml (Table 1). The pure compounds were more active than the crude extract and it also appears that the substituents at C6 do not affect the relative degree of antifungal activities. Anthraquinone derivatives physcion, chrysophanol, and emodin are reported to exhibit antifungal activity (MIC 90 – 100 mg/ml) and the inhibition effect depends on the presence of substituents (OH, OMe, and OAc at C1 and C8). These also have antimicrobial properties (Manojlovic et al., 1998).
Acknowledgements The authors are grateful to the Head, RSIC, at CDRI, Lucknow, for obtaining the spectral data.
References Agarwal, J.S., Rastogi, R.P., Srivastava, O.P., 1976. In vitro
toxicity of constituents of Rumex maritimus Linn. to ring worm fungi. Current Science 45 (17), 619 – 620. Ayyangar, N.R., Bapat, D.S., Joshi, B.S., 1961. Anthraquinones and anthrone series: Part XXVI — a new synthesis of chrysophanol, rhein, islandicin, emodin and physcion. Journal of Scientific and Industrial Research 20B, 493 – 497. Cyong, J.C., Matsumoto, T., Arakawa, K., Kiyohara, H., Yamada, H., Otsuka, Y., 1987. Antibacteriosides Fragilus substance from rhubarb. Journal of Ethnopharmacology 19, 279 – 283. Fuzellier, M.C., Morter, F., Lectard, P., 1982. Antifungal activity of Cassia alata L. Annals of Pharmacology, France 40 (4), 357 – 363. Harvey, H.E., Waring, J.M., 1987. Antifungal and other compounds isolated from the roots of New Zealand flax plants (The genus Phormium). Journal of Natural Products 50 (4), 767 – 776. Inamori, Y., Kato, Y., Kubo, M., Kamiki, T., Takemoto, T., Nomoto, K., 1983. 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 31 (12), 4543 – 4548. Kalidhar, S.B., 1989. Location of glycosylation and alkylation sites in anthraquinones by 1H NMR. Phytochemistry 28 (9), 2455 – 2458. Lu, M., Chen, Q.H., 1989. Biochemical study of Chinese rhubarb. XXIX. Inhibitory effects of anthraquinone on P388 leukemia in mice. Journal of the China Pharmacology University 20, 155 – 157. Manojlovic, N.T., Solujic, S., Sukdolak, S., Krstic, L.J., 1998. Anthraquinones from the lichen Xanthoria parietina. Journal of the Serbian Chemical Society 63 (1), 7 – 11. Muhatadi, F.J., Moss, M.J.R., 1969. A synthesis of aloeemodin 1,8-di-b-D-glucoside. Tetrahedron Letters 4B, 3751 – 3752. Nadkarni K.M. (1954). Rheum emodi. The Indian Materia Medica, A.K. Nadkarni (Ed.), 3rd edn., Pub. Dhootapapeshwar, Prakashan Ltd., Panvel, Bombay, India, 2113. Nawa, H., Uchibayashi, M., Matsuoka, T., 1961. Structure of rhein. Journal of Organic Chemistry 26, 979 – 980.
46
S.K. Agarwal et al. / Journal of Ethnopharmacology 72 (2000) 43–46
Oshio, H., Kawamura, N., 1985. Determination of the laxative compounds in rhubarb by high performance liquid chromatography. Shoyakugaku Zasshi 39, 131– 138. Shadomy, S., Espinel-Ingroff, A., 1974. In: Lennett, E.H., Spaulding, E.H., Truant, J.P. (Eds.), Manual of Clinical Microbiology. American Society of Microbiology, Washington, DC, pp. 569 –573.
Thakur, R.S., Puri, H.S., Hussain, A., 1989. Rheum australe. Major Medicinal Plants of India. CIMAP, Lucknow, pp. 443 – 447. Zhou, X.M., Chen, Q.H., 1988. Biochemical study of Chinese rhubarb. XXII. Inhibitory effect of anthraquinone derivatives on sodium-potassium-ATPase of rabbit renal medulla and their diuretic action. Acta Pharmacologia Sinica 23, 17 – 20.
.