Antimalarial activity of anthothecol derived from Khaya anthotheca (Meliaceae).

Abstract

Antimalarial activity of anthothecol, a limonoid of Khaya anthotheca (Meliaceae) against Plasmodium falciparum was tested using a [[.sup.3] H]-hypoxanthine and 48 h culture assay in vitro. Anthotechol showed potent antimalarial activity against malaria parasites with [IC.sub.50] values of 1.4 and 0.17 [micro]M using two different assays. Also, gedunin had antimalarial activity with [IC.sub.50] values of 3.1 and 0.14[micro]M. However, the citrus limonoids, limonin and obacunone did not show any antimalarial activity. The antimalarial activities were compared with the three currently used antimalarial medicines quinine, chloroquinine and artemisinin.

(C) 2007 Elsevier GmbH. All rights reserved.

Keywords: Khaya anthotheca; Anthothecol; Gedunin; Antimalarial activity

Introduction

In many tropical and sub-tropical countries, folk medicines from the Meliaceae plants have traditionally been used to cure malarial disease (Phillipson and O'Neill, 1986; Bray et al., 1990; MacKinnon et al., 1997). The increased incidence of malaria in recent years has been attributed to the development of resistance of the malarial protozoa (Plasmodium falciparum) to chloroquine (Zhang et al., 2004) and to the development of resistance of the vector mosquitoes to insecticides (Du et al., 2005). All species of Khaya used in the German timber and furniture industry contribute to malarial diseases in Africa (Hausen, 1981; Khalid et al., 1986). These plants contain a variety of limonoids (Adesogan et al., 1970; Adesida et al., 1971) and they possess antimalarial activities (Weenen et al., 1990; .Bickii et al., 2000). Limonoids are the most representative class of secondary metabolites in the order Rutales, which includes the families Rutaceae, Meliaceae, and Simaroubaceae. They are tetranortriterpenoids with 4,4,8-trimethyl-17-furanylsteroidal skeleton, bearing several oxygenated functions (Ruberto et al., 2002). Several limonoids of angolensates, khivorins, mexicanolides, and fissinolides have been isolated from different parts of Khaya anthotheca (Adesida et al., 1971). They are reported to be new sources of biologically active natural products with beneficial properties such as low mammalian toxicity, lack of neurotoxic mode of action, low persistence in the environment and biodegradability (Gonzalez-Coloma et al., 1997; Singh et al., 1997). Herein, we report that anthothecol, a limonoid isolated from K. anthotheca, showed potent antimalarial activity using a [(3)H]-hypoxanthine and 48h culture assay in vitro, when compared with other natural limonoids and currently used antimalarial drugs. Also, the complete structure elucidation of anthothecol was performed by spectroscopic techniques (Fig. 1).

[FIGURE 1 OMITTED]

Materials and methods

Authentic compound of gedunin was purchased from Microsource Discovery Systems, Inc. (Gaylordsville, CT, USA). Two citrus limonoids, limonin and obacunone, were kindly supplied by Dr. Shin Hasegawa, WRRC, USDA-ARS, Albany, CA. Chloroquine, quinine and artemisinin were purchased from Aldrich Chemical Co. (Seoul, South Korea). All other chemicals were of reagent grade.

The air-dried whole plants of K. anthotheca (1 kg) were cut and extracted with MeOH under reflux conditions on a steam bath for 5h ( x3). The MeOH extract (280 g) was suspended in water (3L) and then partitioned sequentially with equal volumes of C[H.sub.2]C[l.sub.2], EtOAc, and n-BuOH. The EtOAc extract (3.5 g) was fractionated by column chromatography (6.3 x 85 Cm) on silica gel (70-230 mesh, Merck, Darmstadt, Germany) using n-hexane: EtOAc (95: 1 [right arrow] 0: 100, v/v) in a stepwise solvent system. The fractions were pooled into 7 major fractions (Frs. E1-E7) based on their TLC profiles. The fraction, E4 (1.8 g), was further separated by column chromatography (3.5 x 65 cm) on silica gel using n-hexane: M[e.sub.2]CO (50: 1 [right arrow] 1: 1, v/v) and gel filtration column chromatography (Sephadex LH-20, 60 g, 2.4 x 65 cm, MeOH: [H.sub.2]O = 70: 30, v/v), yielding anthothecol (1, 39 mg, yellowish powder, [R.sub.f]: A = 0.25, B = 0.54). TLC was performed on precoated Silica gel 60 [F.sub.254] and RP-18 [F.sub.254s] plates, which were developed with n-hexane: M[e.sub.2]CO = 5: 1 (A, v/v) and MeOH: [H.sub.2]O = 80: 20 (B, v/v). The plates were sprayed with 10% [H.sub.2]S[O.sub.4] reagent (in EtOH) and heated for detection. The melting point was determined using a Fisher Scientific melting point apparatus and was uncorrected. NMR spectra were recorded on a Varian Unity Inova 500 spectrometer. The EI-MS (70 EV) spectra were recorded on a JEOL JMS-AX 505H mass spectrometer. The chemical properties of compound 1 isolated form K. anthotheca as follow, yellowish powder; m.p. 225[degrees]; EI-MS: m/z 480 [[M].sup.+]. Also, (1) H-NMR (DMSO-[d.sub.6]) spectral data were compared with previously described report (Bevan et al., 1963).

Two antimalarial assays were carried out for each compound according to procedures described in the literature (Rosenthal et al., 1996). All experiments were with W2-strain (P. falciparum, chloroquine-resistant) parasites, cultured in human erythrocytes using standard methods. In brief, for [H]-hypoxanthine uptake assays, ring-stage parasites were incubated with multiple concentrations of compounds (from 100 x stocks in DMSO) for 24h, [[.sup.3] H]-hypoxathine was added for an additional 18 h, and cells were harvested and counts representing incorporated hypoxanthine were quantified. For development assays, parasites were incubated with different concentrations of compounds for 48 h, beginning at the ring stage. At the completion of the incubation, Giemsa-stained smears were evaluated microscopically, and ring-stage parasites per 1000 erythrocytes were counted. Both assays were performed twice for each inhibitor; each [[.sup.3]H]-hypoxanthine assay was performed in triplicate at each concentration. All values were normalized to percent control (1% DMSO) activity and 50% inhibitory concentrations ([IC.sub.50]) were calculated using the Prism 3.0 program (GraphPad Software).

Results and discussion

The ethyl acetate-soluble fraction of the MeOH extract of the K. anthotheca was fractionated by repeated column chromatography on silica gel and Sephadex LH-20 and the structure performed by spectroscopic techniques 1, anthothecol.

The antimalarial activity (50% inhibitory concentration, [IC.sub.50]) of four naturally occurring limonoids and two antimalarial agents, chloroquine and quinine, was determined against P. falciparum using two in vitro antiparasitic assays (Rosenthal et al., 1996). The results are summarized in Table 1. Among the tested limonoids, anthothecol and gedunin had potent antimalarial activity against P. falciparum parasites with [IC.sub.50] values of 1.4 and 3.1 [micro]M, respectively. However, they were less effective against malaria parasites than two widely used antimalarial drugs, chloroquine ([IC.sub.50] = 0.19 [micro]M) and quinine ([IC.sub.50] = 0.24 [micro]M), as determined by an assay based on inhibition of [(3)H]-hypoxanthine uptake. According to the result of an assay of development that measures the formation of new ring-stage parasites after 48h of incubation with inhibitors, anthothecol, gedunin, limonin, and obacunone exhibited antimalarial activity with [IC.sub.50] values of 0.17, 0.14, 2.7, and 1.6[micro]M, respectively.

Table 1. Antimalarial activities ([IC.sub.50]) of naturally occurring
limonoids with two in vitro antiparasitic assays

Compounds        [3.sup.]H-hypoxanthine  Parasite development
                     uptake ([mu]M)          ([mu]M)

Anthothecol (1)            1.4                 0.17
Gedunin (2)                3.1                 0.14
Limonin (3)              >10                   2.7
Obacuone (4)             >10                   1.6
Chloroquine                0.19                0.036
Quinine                    0.24                0.074
Artemisinin                0.0021              0.0032

Potential new targets for treatment of malarial diseases include parasite enzymes that degrade hemoglobins in host blood (Semenov et al., 1998). Several sulfones have been known as inhibitors of erythocytic malaria parasites on cysteine and aspartic proteases (Rosenthal et al., 1996). A synergy effect has been occurred when mixtures of cysteine and aspartic protease inhibitors were treated to control malarial parasite, P. falciparum (Semenov et al., 1998). Herein, anthothecol showed its potent antimalarial activity when compared with gedunin, which has been known as a potent component of folk medicines in Africa, with lower uptake of [(3)H]-hypoxanthine and inhibitory effect on the formation of new ring-stage of malarial parasite. These results were similar to the previous study on the evaluation of sulfone inhibitors (Semenov et al., 1988). Thereofe, these limonids may partially inhibit the cysteine and aspartic proteases of the malarial parasites. Further studies may provide the inhibitory action of mode of limonoides on the protease activity. In conclusion, anthothecol, a limonoid derived from K. anthotheca, showed 10-100 times less effective against malaria parasites compared with currently used antimalarial drugs.

Acknowledgements

This work was partially supported by a grant from GRRC-project 3-1.

References

Adesida, G.A., Adesogan, E.K., Okorie, D.A., Taylor, D.A.H., Styles, B.T., 1971. The limonoid chemistry of the genus Khaya (Meliaceae). Phytochemistry 10, 1845-1853.

Adesogan, E.K., Okorie, D.A., Taylor, D.A.H., 1970. Limonoids from Khaya anthotheca (Welw) C.DC. J. Chem. Soc. (C) 205-211.

Bevan, C.W.L., Rees, A.H., Taylor, D.A.H., 1963. West African timbers Part VII. Anthothecol - an extractive from Khaya anthotheca. J. Chem. Soc. 983.

Bickii, J., Njifutie, N., Foyere, J.A., Basco, L.K., Ringwald, P., 2000. In vitro antimalarial activity of limonoids from Khaya grandifoliola C.D.C. (Meliaceae). J. Ethnopharamcol. 69, 27-33.

Bray, D.H., Warhurst, D.C., Connolly, J.D., O'Neill, M.J., Phillipson, J.D., 1990. Plants as sources of antimalarial drugs. Part 7. Phytother. Res. 4, 29-35.

Du, W., Awolola, T.S., Howell, P., Koekemoer, L.L., Brooke, B.D., Benedict, M.Q., Coetzee, M., Zheng, L., 2005. Independent mutations in the Rdl locus confer dieldrin resistance to Anopheles gambiae and An. arabiensis. Insect Mol. Biol. 14, 179-183.

Gonzalez-Coloma, A., Gutierrez, C., Carbrera, R., Reina, M., 1997. Silphinene derivatives: their effects and modes of action on Colorado potato beetle. J. Agric. Food Chem. 45, 946-950.

Hausen, B., 1981. Woods Injurious to Human Health. De Gruyter, Berlin, New York.

Khalid, S.A., Farouk, K., Geary, T.G., Jensen, J.B., 1986. Potential antimalarial candidates from African plants. J. Ethnopharmacol. 15, 201-209.

MacKinnon, S., Durst, T., Arnason, J.T., Angerhofer, C., Pezzuto, J., Sanchez-Vindas, P.E., Poveda, L.J., Gbeassor, M., 1997. Antimalarial activity of tropical Meliaceae extracts and gedunin derivatives. J. Nat. Prod. 60, 336-341.

Phillipson, J.D., O'Neill, M.J., 1986. Novel antimalarial drugs from plants? Parasitol. Today 2, 355-358.

Rosenthal, P.J., Olson, J.E., Lee, G.K., Palmer, J.T., Klaus, J.L., Rasnick, D., 1996. Antimalarial effects of vinyl sulfone cysteine proteinase inhibitors. Antimicob. Agents Chemother. 40, 1600-1603.

Ruberto, G., Renda, A., Tringali, C, Napoli, E.M., Sim-monds, M.S.J., 2002. Citrus limonoids and their semisynthetic derivatives as antifeedant agents against Spodoptera frugiperda larvae. A structure activity relationship study. J. Agric. Food Chem. 50, 6766-6774.

Singh, M., Khokhar, S., Malik, S., Singh, R., 1997. Evaluation of neem (Azadirachta indica A. Juss) extracts against American bollworm, Helicoverpa armigera (Hubner). J. Agric. Food Chem. 45, 3262-3268.

Semenov, A., Olson, J.E., Rosenthal, P.J., 1998. Antimalarial synergy of cysteine and aspartic protease inhibitors. Antimicob. Agents Chemother. 44, 2254-2258.

Weenen, H., Nkunya, M.H.H., Bray, D.H., Mwasumbi, L.B., Kinabo, L.S., Kilimali, V.A.E.B., 1990. Antimalarial activity of Tanzanian medicinal plants. Planta Med. 56, 368-370.

Zhang, H., Paguio, M., Roepe, P.D., 2004. The antimalarial drug resistance protein Plasmodium falciparum chloroquine resistance transporter binds chloroquine. Biochemistry 43, 8290-8296.

Sung-Eun Lee (a), Mi-Ran Kim (b), Jeong-Han Kim (b), Gary R. Takeoka (c), Tae-Wan Kim (d), Byeoung-Soo Park (e) *

(a) Nanotoxtech. Inc., 301 Myung Jin Building, 234-27 Nonhyeon dong, Gangnam-gu, Seoul 153-010, Republic of Korea

(b) School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea

(c) US Department of Agriculture, Western Regional Research Center, Agricultural Research Service, Albany, CA 94710, USA

(d) School of Plant Life and Environmental Science, Hankyong National University, Ansung-City, Kyonggi-Do 456-749, Republic of Korea

(e) Institute of Ecological Phytochemistry, Hankyong National University, Ansung-City, Kyonggi-Do 456-749, Korea

Received 3 April 2007; accepted 8 August 2007

* Corresponding author. Tel./fax: + 82316705081.

E-mail address: [e-mail] (B.-S. Park).

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doi: 10.1016/j.phymed.2007.08.001