2004 hiv 1 inhibition by extracts of clusiaceae species from mexico

2004 hiv 1 inhibition by extracts of clusiaceae species from mexico

• 1.
  916 Notes Biol.Pharm. Bull. 27(6) 916—920 (2004) Vol. 27, No. 6 HIV-1 Inhibition by Extracts of Clusiaceae Species from Mexico Maira HUERTA-REYES,a Maria del Carmen BASUALDO,b Lucio LOZADA,c Manuel JIMENEZ-ESTRADA,a Carmen SOLER,b and Ricardo REYES-CHILPA*,a a Department of Natural Products, Institute of Chemistry, National University of Mexico; b HIV/AIDS Section, Institute of Biomedical Sciences, National University of Mexico; and c Faculty of Sciences, National University of Mexico; Ciudad Universitaria, 04510, Mexico D. F., Mexico. Received November 28, 2003; accepted February 7, 2004 The organic plant extracts of 21 species of Clusiaceae from Mexico were screened for anti HIV-1 reverse transcriptase activity in a non-radioactive immuno colorimetric assay. The extracts of 5 species (23.8%) exhib-ited significant inhibition (70%) of HIV-1 RT activity; of these, only 4 extracts showed reduced toxicity to human lymphocytic MT2 cells and were further tested as inhibitors of HIV-1 IIIb/LAV replication in a cellular system. The best extracts were Calophyllum brasiliense (hexane) and Clusia quadrangula (CH2Cl2–MeOH) which inhibited HIV-1 RT (IC5029.6m g/ml and 42m g/ml), and showed an EC5092.5m g/ml and 91m g/ml, respectively, on MT2 cells. However, only Calophyllum brasiliense hexane extract showed significant inhibition on viral repli-cation (ED5037.1m g/ml), while Clusia quadrangula was less active (ED50124m g/ml). These results support the idea that plant extracts represent a valuable source of potential anti HIV compounds. Key words Clusiaceae; HIV-1; reverse transcriptase; HIV-1 inhibition; Calophyllum brasiliense HIV has been identified as the etiological agent of AIDS.1,2) The disease is characterized by opportunistic pathogen infections, accompanied by a drastic decrease in the number of CD4 T cells. It is now accepted that there are two types of HIV: HIV-1 and HIV-2. Most AIDS worldwide is caused by HIV-1.3) The replicative cycle of HIV has been extensively studied to determine specific processes that can serve as drug targets. One of the most critical steps is the re-verse transcription of viral RNA into a cDNA copy, which is catalyzed by enzyme reverse transcriptase (RT).4) Many strategies have focused on the search for inhibitors against this viral enzyme; these compounds should also be non-toxic to uninfected cells.5) However, in spite of the number and va-riety of anti-HIV drugs, the rapid development of HIV resis-tance necessitates continuation of the search for new drugs including synthetic and natural products.6) In this context, highly active anti-retroviral therapy (HAART) has proved to be very beneficial to HIV infected patients, especially triple-drug combinations (3 RT in-hibitors; 2 RT inhibitors1 protease inhibitors) over mono and bi-drug combinations. HAART has provided a sustained reduction of the plasma viral load, increased the CD4 T cells counts, and also delayed disease progression to AIDS.7) However, these benefits are limited by important risks associ-ated with prolonged use of this treatment, such as metabolic disturbances, cross pharmacological reactions with other drugs, and low adherence to long or permanent treatment; these could subsequently induce the rapid development of viral resistance, as well as deep toxicity effects recognized on the cardio-vascular system, liver, kidney, brain, pancreas and skin.7,8) For all these reasons and the fact that RT inhibitors are always used in triple-drug therapy, new substances with possible different toxic or resistant induction sites could offer alternative options in HIV RT therapeutics. In the search for HIV-1 RT inhibitors, screening of plant extracts is a valuable method to discover new compounds po-tentially useful in therapeutics.9,10) Coumarins and benzophe-nones with these antiviral properties have been isolated from several plant species of the Clusiaceae family through biogu-ided fractionation. Among the most important coumarins able to inhibit HIV-1 RT with low cytotoxicity on uninfected cells are: ()-calanolide A, ()-calanolide B,11,12) () ino-phyllum B and P,13,14) soulattrolide;15) ()-cordatolide A and ()-cordatolide B.16) All these substances were isolated from Calophyllum species. The most outstanding compound is ()-calanolide A, which showed potent and specific anti HIV-1 RT activity in in vitro as well as in in vivo pharmaco-logical tests.17—20) This compound is currently in Phase I/II clinical trials.21) In the case of benzophenones, guttiferone A isolated from Garcinia livingstonei, guttiferone E and isox-anthochymol isolated from Garcinia ovalifolia22) showed anti HIV-1 RT activity in vitro, but their cytotoxic effects on human lymphocytic cell lines precluded further studies. Since species from the Clusiaceae family represent a po-tential source of new chemical identities able to inhibit HIV- 1 RT,23) we have carried out a screening of Clusiaceae species that thrive in Mexico. In this study we report the evaluation of organic extracts of 21 species in their capacity for HIV-1 RT inhibition in vitro, their toxicity effects on human lymphocytes and their inhibition of HIV-1 replication in a cellular system. MATERIALS AND METHODS Plant Material To establish which species should be in-cluded in this study, a preliminary taxonomic review was car-ried out with herbaria specimens. Clusiaceae samples were collected by us or obtained by donation from collectors or herbaria. Collections were performed in the states of Guer-rero, Veracruz and Oaxaca in Mexico. Vouchers are de-posited at the IMSS Medicinal Herbarium (IMSSM), Na-tional University of Mexico (MEXU), the Faculty of Sci-ences (FCME) and the Institute of Ecology (XAL). Taxo-nomical identity of each species was confirmed by one of the authors, Lucio Lozada. Preparation of Extracts Plant material, preferably leaves, was dried at room temperature, powdered and ex-tracted with CH2Cl2–MeOH (1 : 1). The extracts were evapo-rated to dryness. In the case of Calophyllum brasiliense, hexane, acetone and methanol extracts were also prepared. ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2004 Pharmaceutical Society of Japan
• 2.
  June 2004 917 Biological Assays Antiviral activities of Clusiaceae ex-tracts were evaluated by three successive assays. HIV-1 RT inhibition was first screened, and those extracts that showed inhibition over 70% were selected for the next bioassay. Cy-totoxic effect of the extracts was examined on human lym-phocyte cell lines. Non-toxic extracts were selected for the last bioassay, which consisted of determining inhibition of HIV-1 IIIb/LAV replication. Concentration-dependent activ-ity of the most active extracts was performed in the three bio-logical assays. Nevirapine, a non-nucleoside reverse tran-scriptase inhibitor (NNRTI) was used as positive control. HIV-1 RT Inhibition HIV-1 RT inhibition by extracts was evaluated by a non-radioactive immuno and colorimetric assay (Lenti RT Activity Assay, Cavidi Tech).24) Assay was performed according to the protocol provided by the manu-facturer. Each extract was dissolved in dimethyl sulfoxide (DMSO) and tested at 50m g/ml. Toxic Effect on Human Lymphocytes Cell Line The cytotoxic effect of anti HIV-1 RT active extracts was deter-mined on human lymphocytic MT2 cells. The assay was per-formed as follows: MT2 cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 0.25m g/ml of streptomycin and 100m g/ml of penicillin G, in the pres-ence of the plant extracts. Culture was maintained at 37 °C under 5% CO2 humidified atmosphere. Plant extracts were dissolved in DMSO and tested at 50m g/ml. After 48 h, cellu-lar death was assessed by direct microscopic examination of trypan blue stained cells. Results were compared with a con-trol of MT2 free of extract. Inhibition of HIV-1 IIIb/LAV Replication Inhibition of viral replication by plant extracts was measured in a cocul-ture of IIIb/LAV-Molt4 cells and non-infected MT2 cells in the presence of the plant extracts. Concentration of extracts and conditions of culture were the same as those described for the toxicity assay. After 48 h inhibition of viral replication was measured by a p24 antigen enzyme immunoassay (Ge-netic Systems HIV-1Ag EIA, Bio-Rad) performed in the cul-ture supernatant. RESULTS Plant Material Clusiaceae from Mexico was reviewed for the last time eighty years ago,25) and 4 genera and 11 species were reported. Our taxonomic investigation in herbaria and in the field revealed that Clusiaceae in Mexico is now composed of 8 genera and 21 species (Table 1). Ex-tracts of all the species of Clusiaceae were prepared and tested at least in the HIV-1 RT screening assay. Although the species Marila laxiflora is present in Mexico, it was not pos-sible to collect it and bioassays were performed with leaves of this species collected in Peru (South America). HIV-1 RT Inhibition Extracts prepared from the leaves of 21 species of Clusiaceae were screened for anti-HIV-1 RT activity (Table 1). Five species (23.8%) showed a high anti HIV-1 RT (70% inhibition), while 7 species (33.3%) were moderately active (50 to 70% inhibition) and 9 species showed less than 50% inhibition (Table 1). The three most active extracts belong to the same species, Calophyllum brasiliense: hexane (77.9%), acetone (81.3%) and methanol (83.3%). Extracts of Clusia massoniana and Vismia mexi-cana produced a 72.9% inhibition of the enzyme, while Clu-sia guatemalensis and Vismia camparaguey showed 70.8% inhibition. The anti-HIV-1 RT activity of extracts from other plant organs is also shown in Table 1. In this case, the highest inhibitory activity found was 64.1% inhibition for the flower extract of Marila laxiflora. Toxic Effect on Human Lymphocytes Cell Line The lymphocyte toxicity assay was performed only with the most active extracts (70%) and C. quadrangula. Only five of the nine selected extracts were non-toxic to lymphocytes MT2: Calophyllum brasiliense (hexane, methanol and CH2Cl2–MeOH), Vismia mexicana and Clusia quadrangula (Fig. 1). Non-toxicity criteria were established based on no difference in cell number between control and treatment (ANOVA p0.05). Inhibition of HIV-1 IIIb/LAV Replication The hexane extract of Calophyllum brasiliense inhibited HIV-1 IIIb/LAV replication in 74.5%, while the methanol extract of Calophyl-lum brasiliense, Vismia mexicana and Clusia quadrangula extracts exhibited less than 52% inhibition (Fig. 2). Concentration-Dependent Effects Since Calophyllum brasiliense hexane extract was the best in the 3 biological as-says, its concentration-dependent effects were determined. The range of concentrations tested was 10—120m g/ml. The HIV-1 RT IC50 was 29.6m g/ml, while ED50 for HIV-1 IIIb/LAV replication was 37.1m g/ml. EC50 on MT2 cells was observed at 92.5m g/ml. For comparison with C. brasiliense hexane extract, concentration-dependent assay was also per-formed for Clusia quadrangula extract. The IC50 (42m g/ml) and EC50 (91m g/ml) were similar to Calophyllum brasiliense hexane extract, but ED50 was 3 times lower (ED50 124m g/ml) (Fig. 3). Nevirapine control showed EC50 200m M and ED500.03m M; these values were very similar to those reported in the literature for this compound with MT4 cells and HIV-1 IIIb.26) DISCUSSION A number of anti HIV-1 plant extract screenings have been performed during the last years and several active plants and secondary metabolites have been recognized, most of them able to act specifically on enzymatic targets.27,28) Neverthe-less, none of these substances is completely innocuous.29) Therefore, evaluation of cytotoxicity is necessary towards the development of effective anti-HIV drugs. Several screenings include HIV-1 RT inhibition and cytotoxicity, but few also involve inhibition of viral replication in cellular systems.30,31) This study offers an evaluation of these three parameters for selected extracts. The present screening comprised the 8 genera and 21 species belonging to Clusiaceae that thrive in Mexico. Twelve species (57.1%) showed high to moderate anti HIV-1 RT activity, supporting the idea of Clusiaceae as a valuable source of HIV inhibitory natural product leads.23) In this study Calophyllum brasiliense hexane extract showed the best performance in the three bioassays. Another interesting extract, Clusia quadrangula overcame the two former tests. Thus we performed concentration–response experiments with both extracts and contrasted them with published data (Fig. 3). Regarding the HIV-1 RT inhibitory properties of plant ex-tracts, some authors have pointed out that an IC5050m g/ml
• 3.
  918 Vol. 27,No. 6 Table 1. Screening of CH2Cl2–MeOH Extracts (50m g/ml) of HIV-1 RT Inhibition by Clusiaceae that Thrive in Mexico Species Collector State Herbarium Part used % inhibition of HIV-1 RT Calophyllum brasiliense R. Reyes-Chilpa s/n Veracruz IMSSM Leaf 70.33.1 CAMBESS. 10 Sept 1999 (Los Tuxtlas) 77.90.5a) 81.31.9b) 83.31.0c) Chrysochlamys guatemaltecana T. Went et al. 2642 Veracruz MEXU Leaf 60.41.0 DONNELL-SMITH Chrysochlamys nicaraguensis B. Ortiz and Veracruz FCME Leaf 58.70.5 (OERSTED, PLANCHON TRIANA) Martiniano 170 Flower 31.02.0 HEMSLEY Clusia flava JACQ. R. Reyes-Chilpa s/n Veracruz IMSSM Leaf 1.00.4 Clusia guatemalensis HEMSL A. Mendez 9593 Chiapas MEXU Leaf 70.81.2 Clusia lundellii STANDL. L. Lozada 2424 Oaxaca IMSSM Leaf 48.01.5 Peel of fruit 59.41.6 Seed 50.01.0 Stem 27.83.4 Clusia massoniana LUNDELL E. Ramirez 414 Oaxaca FCME Leaf 72.91.4 Flower 53.62.4 Clusia minor L. T. P. Ramamoorthy 2395 Oaxaca FCME Leaf 56.11.1 Clusia pringlei LUNDELL J. Panero 3923 Guerrero MEXU Leaf 37.60.6 Clusia quadrangula BARTLETT L. Lozada 2428 Oaxaca IMSSM Leaf 65.61.2 Peel of fruit 51.91.2 Seed 56.60.6 Stem 59.71.9 Clusia rosea JACQ. C. Beutelspacher Veracruz MEXU Leaf 16.92.0 15 March 1969 Clusia salvinii DONN. SM. L. Lozada 2427 Oaxaca IMSSM Leaf 43.61.5 Peel of fruit 53.21.6 Seed 47.31.9 Stem 52.42.4 Clusia tetra-trianthera MAGUIRE D. E. Breedlove 28177 Chiapas MEXU Leaf 39.62.0 Garcinia intermedia (PITTIER) C. Gallardo 326 Guerrero FCME Leaf 55.13.3 HAMMEL Garcinia macrophylla MART. T. Wendt et al. 3304 Oaxaca MEXU Leaf 22.26.7 Mammea americana L. R. Reyes-Chilpa Veracruz IMSSM Leaf 11.23.8 27 February 1999 Marila laxiflora RUSBY J. Schunke 6389 Peru XAL Leaf 64.71.3 Flower 64.11.6 Symphonia globulifera L. f. M. A. Magaña and Tabasco FCME Leaf 17.14.2 S. Zamudio 266 Vismia baccifera (L.) TRIANA L. Lozada 2435 Oaxaca IMSSM Leaf 57.81.9 PLANCHON Stem 62.40.2 Vismia camparaguey SPRAGUE A. Campos 2573 Oaxaca MEXU Leaf 70.81.5 RILEY Vismia mexicana SCHLTDL. E. Martinez 18999 Chiapas FCME Leaf 72.91.1 Flower 56.82.8 a) Hexane extract. b) Acetone extract. c) Methanol extract. Values are meansS.D., n3. Fig. 1. Effect of Clusiaceae Extracts (50m g/ml) on MT2 Cell Viability (A) acetone, (M) methanol, (D–M) dichloromethane–methanol, (H) hexane.
• 4.
  June 2004 919 is considered significant.28,31) Therefore, Calophyllum brasiliense hexane extract (IC5029.6m g/ml) and Clusia quadrangula (IC5042m g/ml) can be included in this cate-gory (Fig. 3A). In relation to inhibition of HIV-1 replication in cellular systems by extracts, it was reported that Crinum asiaticum var. japonicum (Amaryllidaceae) showed signifi-cant anti HIV-1 activity (ED5012.5m g/ml).31) Our data show that Calophyllum brasiliense hexane extract required an ED5037.1m g/ml, that still can be considered as signifi-cant. In contrast, Clusia quadrangula required a concentra-tion almost 10 times higher (ED50124m g/ml). Concerning cytotoxicity, it has been indicated that an EC50200m g/ml is a low cytotoxic profile.31) In our study, Calophyllum brasiliense hexane extract and Clusia quadrangula have an EC5092.5m g/ml and 91m g/ml, respectively; in conse-quence, their performance in this parameter was poor (Fig. 3B). Our data suggest that Calophyllum brasiliense hexane ex-tract possesses anti-HIV activities. Curiously, this species was omitted in previous studies of the evaluation of anti HIV properties by McKee et al.32) These authors performed a chemotaxonomic study of Calophyllum species, mainly col-lected in Malaysia, in order to search for dipyranocoumarins with HIV-1 RT inhibitory properties. Their method included a TLC test for detecting dipyranocoumarins by a characteris-tic deep blue spot, using ()-calanolide B and soulattrolide as standards, and positive extracts were further fractionated. Calophyllum brasiliense was not included as a candidate be-cause of the absence of dipyranocoumarins on TLC. We re-cently detected the existence of two chemotypes of Calophyl-lum brasiliense in Mexico. The first one is included in this study and showed high inhibitory value for hexane, acetone and methanol extracts in HIV-1 RT assay, suggesting that they may contain HIV-1 inhibitory substances. Bioguided fractionation of these extracts is currently in progress to de-termine the active constituents. The second chemotype con-tains mammea type coumarins, all of them inactive against HIV-1 RT, and these will be reported in an independent paper at a later date. Acknowledgments This research was supported by grant IN207301 from DGAPA-UNAM and a CONACyT fel-lowship to Maira Huerta. The authors are grateful to Teresa Ramirez Apan for laboratory facilities and are particularly indebted to Herbaria curators Mario Sousa (MEXU), Fran-cisco Lorea (XAL) and Abigail Aguilar (IMSSM) for dona-tions. REFERENCES AND NOTES 1) Barré-Sinoussi F., Cherman J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauguet C., Axler-Blin C., Vézinet-Brun F., Rouzioux C., Rozenbaum W., Montagnier L., Science, 220, 868—871 (1983). 2) Gallo R. C., Salahuddin S. Z., Popovic M., Shearer G. M., Kaplan M., Haynes B. F., Palker T. J., Redfield R., Oleske J., Safai B., White G., Foster P., Markham P. D., Science, 224, 500—503 (1984). 3) Janeway C. A., Travers P., Walport M., Shlomchick M., “Immunobiol-ogy,” 5th ed., Chap. 11, Garland Publishers, New York, 2001. 4) Turner B. G., Summers M. F., J. Mol. Biol., 285, 1—32 (1999). 5) Borkow G., Barnard J., Nguyen T. N., Belmonte A., Wainberg M. A., Parniak M. A., J. Virol., 71, 3023—3030 (1997). 6) Raulin J., Progr. Lipid Res., 41, 27—65 (2002). 7) De Clerq E., “Antivirals against AIDS,” Chap. 4, ed. by Unger R. E., Fig. 2. Effect on HIV-1 IIIb/LAV Replication by Clusiaceae Extracts (50m g/ml) (M) methanol, (H) hexane. Fig. 3. Concentration–Response Curve of Calophyllum brasiliense Hexane and Clusia quadrangula Extracts (A) Inhibition rates on HIV-1 RT. (B) Cell viability of lymphocytes MT2. (C) Inhibi-tion of HIV-1 IIIb/LAV replication rates. Each point represents the mean of quadrupli-cate determinations. S.D. of the respective means.
• 5.
  920 Vol. 27,No. 6 Kreuter J., Rübsamen-Waigmann H., Marcel Dekker, Inc., N. Y., 2000, pp. 107—150. 8) Centro Nacional para la Prevención y Control del VIH/SIDA CEN-SIDA.: http://www.salud.gob.mx/conasida/medicos/guias/medica/ guia2004.pdf., Secretaría de Salud, Gobierno de México, 20 January 2004. 9) Vanden D. A., Vlietinck A. J., “Screening Methods for Antibacterial and Antiviral Agents from Higher Plants,” Vol. 6, ed. by Dey P. M., Harborne J. B., Academic Press, San Diego, 1991, pp. 47—69. 10) Lednicer D., Narayan V. L., “Acquisition and Screening of Natural Products as Potential Anticancer and AIDS Antiviral Agents,” ed. by Colegate S. M., Molyneux R. J., CRC Press, N. W. Boca Raton, Florida, 1993, pp. 160—172. 11) Kashman Y., Gustafson K. R., Fuller R. W., Cardellina J. H. II, McMa-hon J. B., Currens M. J., Buckheit R. W., Jr., Hughes S. H., Cragg G. M., Boyd M. R., J. Med. Chem., 35, 2735—2743 (1992). 12) Galinis D. L., Fuller R. W., McKee T. C., Cardellina J. H. II, Gu-lankowski R. J., McMahon J. B., Boyd M. R., J. Med. Chem., 39, 4507—4510 (1996). 13) Patil A. D., Freyer A. J., Eggleston D. S., Haltiwanger R. C., Bean M. F., Taylor P. B., Caranfa M. J., Breen A. L., Bartus H. R., Johnson R. K., Hertzberg R. P., Westley J. W., J. Med. Chem., 36, 4131—4138 (1993). 14) Spino C., Dodier M., Sotheeswaran S., Bioorg. Med. Chem. Lett., 8, 3475—3478 (1998). 15) Pengsuparp T., Serit M., Hughes S. H., Soejarto D. D., Pezzuto J. M., J. Nat. Prod., 59, 839—842 (1996). 16) Dharmaratne H. R. W., Wanigasekera W. M. A., Mata-Greenwood E., Pezzuto J. M., Planta Med., 64, 460—461 (1998). 17) Currens M. J., Gulakowski R. J., Mariner J. M., Moran R. A., Buckheit R. W., Jr., Gustafson K. R., McMahon J. B., Boyd M. R., J. Pharma-col. Exp. Ther., 279, 645—651 (1996). 18) Currens M. J., Mariner J. M., McMahon J. B., Boyd M. R., J. Pharma-col. Exp. Ther., 279, 652—661 (1996). 19) Xu Z., Hollingshead M. G., Borgel S., Elder C., Khilevich A., Flavin M. T., Bioorg. Med. Lett., 9, 133—138 (1999). 20) Creagh T., Ruckle J. L., Tolbert D. T., Giltner J., Eiznhamer D. A., Dutta B., Flavin M. T., Xu Z., Antimicrob. Agents Chemother., 45, 1379—1386 (2001). 21) Sarawak MediChem Pharmaceuticals, Inc.: http://www.sarawak-medichem. com/cala/dev.htm., Advances Life Sciences, Inc. and Sdn Bhd Web, 21 November 2003. 22) Gustafson K. R., Blunt J. W., Munro M. H. G., Fuller R. W., McKee T. C., Cardellina J. H. II, McMahon J. B., Cragg G. M., Boyd M. R., Tetrahedron, 48, 10093—10102 (1992). 23) Fuller R. W., Westergaard C. K., Collins J. W., Cardellina J. H. II, Boyd M. R., J. Nat. Prod., 62, 67—69 (1999). 24) Shao X., Ekstrand D. H. L., Bhikhabhai R., Kallander C. F. R., Gronowitz J. S., Antivir. Chem. Chemother., 8, 149—159 (1997). 25) Standley P. C., Contribut. Unit. Stat. Nat. Herb., 23, 824—827 (1923). 26) Merluzzi V. J., Hargrave K. D., Labadia M., Grozinger K., Skoog M., Wu J. C., Shih C. K., Eckner K., Hattox S., Adams J., Rosehthal A. S., Faanes R., Ecner R. J., Koup R. A., Sullivan J. L., Science, 250, 1411—1413 (1990). 27) Tan G. T., Pezzuto J. M., Kinghorn A. D., J. Nat. Prod., 54, 143—154 (1991). 28) El-Mekkawy S., Meselhy M. R., Kusumoto I. T., Kadota S., Hattori M., Namba T., Chem. Pharm. Bull., 43, 641—648 (1995). 29) Bedoya L. M., Sanchez-Palomino S., Abad M. J., Bermejo P., Alcami J., J. Ethnopharmacol., 77, 113—116 (2001). 30) Hnatyszyn O., Broussalis A., Herrera G., Muschietti L., Coussio J., Martino V., Ferraro G., Font M., Monge A., Martínez-Irujo J. J., San-román M., Cuevas M. T., Santiago E., Lasarte J. J., Phytother. Res., 13, 206—209 (1999). 31) Min B. S., Kim Y. H., Tomiyama M., Nakamura N., Miyashiro H., Otake T., Hattori M., Phytother. Res., 15, 481—486 (2001). 32) McKee T. C., Covington C. D., Fuller R. W., Bokesch H. R., Young S., Cardelina J. H. II, Kadushin M. R., Soejarto D. D., Stevens P. F., Cragg G. M., Boyd M. R., J. Nat. Prod., 61, 1252—1256 (1998).