PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 13, 75–78 (2002) DOI: 10.1002/pca.626 Quantitative Determination of Cytotoxic Friedo-nor-oleanane Derivatives from Five Morphological Types of Maytenus ilicifolia (Celastraceae) by Reverse-phase Highperformance Liquid Chromatography Waldemar Buffa Filho,1 Joaquim Corsino,1† Vanderlan da Silva Bolzani,1 Maysa Furlan,1* Ana Maria S. Pereira2 and Suzelei Castro França2 1 Núcleo de Bioensaio, Biossı́ntese e Ecofisiologia de Produtos Naturais, Instituto de Quı́mica, Universidade Estadual Paulista, CP. 355, 14801-970, Araraquara-SP, Brazil 2 Departamento de Biotecnologia, UNAERP, 14096-380, Ribeirão Preto-SP, Brazil Five different morphological types of Maytenus ilicifolia of the same age and harvested under the same conditions showed distinct accumulations of some friedo-nor-oleananes. A rapid, sensitive and reliable reverse-phase HPLC method (employing an external standard) was used for the determination of the cytotoxic triterpenoids, 20a-hydroxymaytenin, 22b-hydroxymaytenin, maytenin, celastrol and pristimerin in each of the five types. Well resolved peaks with good detection response and linearity in the range 1.0–100 mg/mL were obtained. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: Reverse-phase high-performance liquid chromatography; quantitative determination; friedo-nor-oleanane derivatives; root bark; Maytenus ilicifolia; Celastraceae; morphological types. INTRODUCTION The friedo-nor-oleanane derivatives are members of a small group of natural products, known as the quinonemethides (Gunatilaka, 1996), which are typical of the closely-related families Celastraceae and Hippocrateaceae, and are accumulated in the bark and root barks of some species of both families (Corsino et al., 2000). Biological studies on some quinonemethide triterpenes, including 20a-hydroxymaytenin (1), 22b-hydroxymaytenin (2), maytenin (3), celastrol (4) and pristimerin (5) have revealed potential anti-tumour and anti-microbial activities (Bhatnagar and Divekar, 1951; Bavovada et al., 1990; Ferreira de Santana et al., 1971). A previous study of the binding of maytenin to DNA suggests a possible mode of cellular interaction which may elicit anti-tumour activity (Campanelli et al., 1980). The in vitro evaluation of the cytotoxic activity of 1–4 on a variety of human cancer cell lines showed that these quinonemethides exhibited strong in vitro cytotoxic activity, but with no selective activity (Bavovada et al., 1990; Ngassapa et al., 1994). An early study (Gonçalves de Lima et al., 1972) demonstrated that celastrol had a higher antibiotic * Correspondence to: M. Furlan, Núcleo de Bioensaio, Biossı́ntese e Ecofisiologia de Produtos Naturais, Instituto de Quı́mica, Universidade Estadual Paulista, CP 355, 14801-970 Araraquara-SP, Brazil. Email: maysaf@iq.unesp.br †Permanent address: Departamento de Morfologia, CCBS, UFMS, CP. 649, 79070-900 Campo Grande-MS, Brazil. Contract/grant sponsor: FAPESP. Copyright # 2002 John Wiley & Sons, Ltd. activity than pristimerin, and a chemical investigation of Maytenus ilicifolia revealed the occurrence of maytenin, which showed in vitro inhibitory activity against micro-organisms (Gonçalves de Lima et al., 1969, 1972). As part of our on-going investigations concerning the quinonemethide triterpenoids (Corsino et al., 1998, 2000), it has been necessary to select some Brazilian species (mainly Maytenus and Salacia) of Celastraceae and Hippocrateaceae as putative matrices in order to develop biosynthetic studies. As part of this selection, chemical analysis of the hexane-soluble portions of the ethanolic extract obtained from the root barks of various species has been performed. This paper reports the development of a reverse-phase HPLC method for the quantification of the quinonemethide derivatives of five Brazilian types of M. ilicifolia which showed distinct morphological differences. EXPERIMENTAL Materials. The standard compounds 1–5 were isolated from Maytenus aquifolium and Salacia campestris as described elsewhere (Corsino et al., 2000). HPLC-grade methanol was purchased from Mallinckrodt (Baker SA de C.V., 55320 Xalostoc, Mexico); all solvents and samples were filtered through a 0.2 mm nylon membrane. Nanopure water (>18MOhm) was obtained using a Millipore, (Bedford, MA, USA) purifier. Received 6 November 2000 Revised 30 April 2001 Accepted 5 May 2001 76 W. BUFFA FILHO ET AL. Table 1. Yield of dried extracts obtained from 17.0 g of root bark of each of the morphological types I–V of Maytenus ilicifolia Yields of extract (g) Morphological type Ethanol Hexane Chloroform Ethyl acetate I II III IV V 7.76 10.75 6.20 3.93 6.76 0.33 0.40 0.31 0.25 0.34 2.50 2.40 2.15 1.00 2.63 1.02 3.71 1.48 1.53 1.22 Plant material. Root bark of five morphological types (I–V; Fig. 1) of M. ilicifolia were collected in Ribeirão Preto (São Paulo, Brazil) and identified by Dr. Rita Maria de Carvalho, (University of Campinas, Campinas, São Paulo, Brazil). Voucher specimens of I (HPM-BR 0055), II (HPM-BR 0056), III (HPM-BR 0059), IV (HPM-BR 0061) and V (HPM-BR 0062) are deposited in the Herbarium of the University of Campinas, São Paulo, Brazil. followed by equilibration for 10 min; the total analytical run time for each sample was 30 min. Spectral data from the UV detector were collected over 30 min in the 253– 420 nm range; the chromatograms were analysed and plotted at 420 nm. Sample preparation. Dried and powdered root bark of the five types of M. ilicifolia (17.0 g of each) were extracted with ethanol (3  200 mL) overnight at room temperature. The extracts were evaporated to dryness and the residues were dissolved in methanol:water (80:20, v/v). The resulting extracts were dissolved in methanol and partitioned with n-hexane, chloroform and ethyl acetate to give the corresponding fractions (Table 1). The n-hexane fractions were evaporated to dryness and the residues were dissolved in methanol to yield solutions with concentrations of 5.0 mg/mL (extracts of types I, II and V) and of 1.0 mg/mL (extracts of types III and IV): 20 mL aliquots of these solutions were analysed by HPLC. HPLC analysis. A Shimadzu (Tokyo, Japan) model LC10AS chromatograph, coupled to a model SPD-10A UV detector and equipped with a Supelco (Bellefonte, PA, USA) C18 column (150  4.6 mm i.d.; 5 mm) and precolumn (20  4.6 mm i.d.), was employed. Chromatography was carried out under isocratic conditions with methanol:water (80:20, v/v) containing 1% phosphoric acid as the mobile phase at a flow rate of 1 mL/min. The column was purged with the mobile phase for 3 min, The linearity of the detector response and the calibration curve were established for compounds 1–5 by a series of injections of standard solutions within a concentration range of 12.5–100.0 mg/mL (1, 3–5) and 1.0–100.0 mg/mL (2) using a calculated correlation factor for each standard RESULTS AND DISCUSSION Figure 1. Morphological diversity of Maytenus ilicifolia in the Brazilian ¯ora. Copyright # 2002 John Wiley & Sons, Ltd. Some species of the families Celastraceae and Hippocrateaceae seem to be excellent sources of quinonemethide derivatives (Gonçalves de Lima et al., 1972; Martin, 1973; Martinod et al., 1976; Furlan et al., 1990; Itokawa et al., 1991). Root bark of Maytenus ilicifolia accumulates 22b-hydroxymaytenin (2), maytenin (3), and pristimerin (5) (Gonçalves de Lima et al., 1972), together with cangoronine and ilicifoline, which can be biogenetically related to the quinonemethide triterpePhytochem. Anal. 13: 75–78 (2002) QUANTITATIVE DETERMINATION OF CYTOTOXIC DITERPENOIDS Figure 2. HPLC chromatogram (with UV detection at 420 nm) of the standard compounds, 20a-hydroxymaytenin (1), 22bhydroxymaytenin (2), maytenin (3), celastrol (4) and pristimerin (5). (For chromatographic protocol see Experimental section.) 77 noids (Itokawa et al., 1991). Owing to the ability of M. ilicifolia to accumulate quinonemethide derivatives, and its morphological diversity in the Brazilian flora, we have developed a methodology to identify and quantify these compounds in the hexane extracts obtained from each of the five morphological types shown in Fig. 1. Standards of 20a-hydroxymaytenin (1), 22b-hydroxymaytenin (2), maytenin (3), celastrol (4) and pristimerin (5) were obtained in order to isolate and to determine their yield in each extract. For isolation, the crude hexane fractions of the root barks were subjected to column chromatography followed by preparative TLC (Corsino et al., 2000). Compounds 1–5 were identified by UV, IR, MS, 1H- and 13C-NMR spectra (Gunatilaka et al., 1989; Likhitwitaywuid et al., 1993; Nakanishi K et al., 1973), and quantified in plant extracts by analytical reverse-phase HPLC, which gave a good separation of the standards in a run time of 30 min. The chromatogram of a mixture of the standards 1–5 is shown in Fig. 2. The correlation coefficients of the detector responses for Figure 3. HPLC chromatograms (with UV detection at 420 nm) of the root bark extracts of ®ve morphological types (I±V) of Maytenus ilicifolia showing peaks associated with 20a-hydroxymaytenin (1), 22b-hydroxymaytenin (2), maytenin (3), celastrol (4) and pristimerin (5). (For chromatographic protocol see Experimental section.) Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 75–78 (2002) 78 W. BUFFA FILHO ET AL. Table 2. Content of quinonemethide derivatives 1–5 in root bark of each of the morphological types I–V of Maytenus ilicifolia Compositiona of root bark Morphological type 1 2 3 4 5 I II III IV V 35 40 150 95 36 Ð Ð 30 26 Ð 8 13 29 26 8 Ð Ð 141 34 Ð 35 54 70 13 42 a Content expressed in ppm with respect to dry weight of plant material. compounds 1–5 were 0.99996, 39.899; 0.99999, 356.793; 0.99973, 30205.127; 0.99909, 15687.824; 0.99999, 693.364; and 0.99863, 52001.384, respectively, indicating a good linearity and sensitivity of the detector in the measuring range. The detection limits for 1–5 were 18.75, 2.50, 6.25, 18.75 and 22.50 ng, respectively. Chromatograms of the extracts of root bark of M. ilicifolia types I–V (Fig. 3) showed high concentrations of maytenin (3) and pristimerin (5), accounting for more than one half of the triterpene content, together with 22bhydroxymaytenin (2) in low concentration. The extracts prepared from root bark of types III and IV also contained significant amounts of 20a-hydroxymaytenin (1) and celastrol (4) (Table 2). Peaks were characterized by their retention times and further confirmed by comparison of UV spectra with those of the standards. The characteristic retention times for compounds 1–5 under the established conditions were 4.51, 5.41, 7.02, 17.24 and 25.90 min, respectively (Fig. 3). Quantitative analysis of compounds 1–5 was performed using a three or four-point external calibration over the range 0.25– 2.00 mg/20mL (1, 3–5) and 0.02–2.00 mg/20 mL (2), respectively. The demonstration of production of the quinonemethides 1 and 4 in types III and IV of M. ilicifolia shows that these types are promising for the development of culture systems suitable for further biosynthetic and/or enzymatic experiments in order to study the mechanism of formation of these two metabolites which are not accumulated significantly in any other Brazilian Maytenus species. Acknowledgements This work was supported by grants from FAPESP. M. Furlan and V. da S. Bolzani thank CNPq for providing fellowships: W. Buffa Filho and J. Corsino thank FAPESP and CAPES, respectively, for scholarships. J. Corsino is on leave from UFMS to which institution thanks are given. REFERENCES Bavovada R, Blasko G, Shien H-L, Pezzuto JM and Cordell GA. 1990. Spectral assignment and cytotoxicity of 22-hydroxytingenone from Glyptopetalum sclerocarpum. Planta Med 56: 380±382. Bhatnagar and Divekar, 1951. Pristimerin indica, a source of Dulcitol. J Sci Ind Res 10B: 117±118. Campanelli AR, D'Alagni M and Marini-BettoÁlo GB. 1980. Spectroscopic evidence for the interaction of tingenone with DNA. FEBS Lett 122: 256±260. Corsino J, AleÂcio AC, Ribeiro ML, Pereira AMS, FrancËa SC and Furlan M. 1998. Quantitative determination of maytenin and 22b-hydroxymaytenin in callus of Maytenus aquifolium (Celastraceae) by reverse phase highperformance chromatography. Phytochem Anal 9: 245± 247. Corsino J, Carvalho PRF, Kato MJ, Oliveira OMMF, ArauÂjo AR, Bolzani V da S, FrancËa SC, Pereira AMS and Furlan M. 2000. Biosynthesis of friedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifolium and Salacia campestris. Phytochemistry 55: 741± 748. Ferreira de Santana C, Asfora JJ and Cortias CT. 1971. Primeiras observacËoÄes sobre o emprego da maitenina em pacientes cancerosos. Rev Inst AntibioÂticos 11: 37±39. Furlan M, Alvarenga MA and Akizue G. 1990. Triterpenoids and ¯avonoids from Maytenus evonymoides. Rev Latinoamer Quim 21: 72±73. GoncËalves de Lima O, Leoncio D'Albuquerque I, Coehlo JSB, Martins DG, Lacerda AL and Maciel GM. 1969. Antimicrobial and anti-tumoural activity of Maytenin isolated from cortical root zone of Maytenus species. Rev Inst AntibioÂticos 9: 17±25. Copyright # 2002 John Wiley & Sons, Ltd. GoncËalves de Lima O, De Barros Coelho JS, Weighert IL, D'Albuquerque D, De Lima A and De M Souza MA. 1972 Anti-microbial compounds from higher plants: maytenin and pristimerin in root cortex of Maytenus ilicifolia from southern Brazil. Rev Inst Antibioticos 11: 35±38. Gunatilaka AAL. 1996. Triterpenoid quinonemethides and related compounds (Celastroloids). Prog Chem Org Nat Prod 67: 1±123. Gunatilaka AAL, Fernando C, Kikuchi and Tezuka Y. 1989. 1Hand 13C-NMR analysis of three quinonemethides triterpenoids. Magn Res Chem 27: 803±807. Itokawa H, Shirota O, Ikuta H, Morita H, Takeya K and Iitaka Y. 1991. Triterpenes from Maytenus ilicifolia. Phytochemistry 30: 3713±3716. Likhitwitaywuid K, Bavovada R, Lin L-Z and Cordell GA. 1993. Revised structure of 20-hydroxytingenone and 13C-NMR assignments of 22b-hydroxytingenone. Phytochemistry 34: 759±763. Martin JD. 1973. The structure of dispermoquinone, a triterpenoid quinonemethide from Maytenus dispermus. Tetrahedron 29: 2297±3000. Martinod P, Paredes P, Delle Monache F and Marini-Bettolo GB. 1976. Isolation of tingenone and pristimerin from Maytenus chuchuhuasca. Phytochemistry 15: 562±563. Nakanishi K, Gullo VP, Miura I, Govindachari TR and Wisvanathan N. 1973. Structure of two triterpenes. Application of partially relaxed Fourier transform 13Cnuclear magnetic resonance. J Am Chem Soc 95: 6473± 6475. Ngassapa O, Soejarto DD, Pezzuto JM and Farnsworth NR. 1994. Quinonemethide triterpenes and salaspermic acid from Kokoona ochracea. J Nat Prod 57: 1±4. Phytochem. Anal. 13: 75–78 (2002)