A new bioactive steroidal saponin from Sansevieria cylindrica

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PHYTOTHERAPY RESEARCH Phytother. Res. 17, 179–182 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1059

SHORT COMMUNICATION

A New Bioactive Steroidal Saponin from Sansevieria cylindrica Alexandra da Silva Antunes,1 Bernadete Pereira da Silva,1 Jose´ Paz Parente1* and Ana Paula Valente2 1

Nu´cleo de Pesquisas de Produtos Naturiais, Laborato´rio de Quı´mica de Plantas Medicinais, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro, PO Box 68045, CEP 21944-970 Rio de Janeiro, RJ, Brasil Centro Nacional de Ressonaˆncia Magne´tica Nuclear, Departmento de Bioquı´mica Me´dica, Universidade Federal do Rio de Janeiro, CEP 21941, 590 Rio de Janeiro, RJ, Brasil 2

A new steroidal saponin was isolated from the leaves of Sansevieria cylindrica. Its structure was established as (3b,12b,15a,25S)-26-(b-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 12-O- (6-deoxy-a-Lmannopyranosyl)-15-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside. The structural identification was performed using detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR, HMBC and HMQC) and chemical conversions. The steroidal saponin showed no haemolytic effects in the in vitro assays and demonstrated inhibition of the capillary permeability activity. Copyright # 2003 John Wiley & Sons, Ltd. Keywords: Sansevieria cylindrica; steroidal saponin; furostanol tetradesmoside; haemolytic assay; capillary permeability activity.

INTRODUCTION The occurrence of steroidal saponins in the genus Sansevieria is well documented (Wasicky and Hoehne, 1951; Mimaki et al., 1996a; Mimaki et al., 1996b). Some species have an ethnopharmacological background, in particular S. trifasciata which in South Africa and tropical America is used for the treatment of inflammatory conditions and sold as a crude drug in the market to treat victims of snakebite (Morton, 1981). However, a survey of the literature showed that no chemical studies have been carried out on the constituents of Sansevieria cylindrica Boger (Agavaceae), which is native to the subtropical regions of the African continent and is cultivated in Brazil as an ornamental plant. As part of our programme on the chemical investigation of bioactive steroidal saponins, we have now examined the leaves of this plant. As a result a novel steroidal saponin was isolated S. cylindrica, and its haemolytic effects and antiinflammatory properties, evaluated.

MATERIALS AND METHODS

an Electrothermal 9200 micro-melting point and are uncorrected. Optical rotations were measured on a Perkin Elmer 243B polarimeter. IR spectra were measured on a Perkin Elmer 599B, negative LSIMS was carried out using thioglycerol as the matrix and Cs ions accelerated at 35 kV. The acceleration voltage was 8 kV. Mass spectra and GCMS were taken on a VG Auto SpecQ spectrometer. NMR specra were measured in C5D5N (100 mg of 1 in 0.5 mL) at 25 °C with a Bruker DRX-600 NMR spectrometer, with tetramethylsilane (d = 0.00) used as internal standard. 1H NMR spectra were recorded at 600 MHz and 13C NMR spectra at 150 MHz. Silica gel columns (230–400 mesh ASTM, Merck) and Sephadex LH-20 (Pharmacia) were used for CC. TLC was performed on silica gel plates (Kieselgel 60F254, E. Merck) using the following solvent systems: (A) CHCl3–MeOH–H2O (65:35:10, lower phase) for steroidal saponin 1, (B) CHCl3–MeOH (95:5) for sapogenin and (C) n-BuOH–pyridine–H2O (6:4:3) for monosaccharides. Spray reagents were orcinol–H2SO4 for steroidal saponin 1 and monosaccharides, and CeSO4 for sapogenin.

General procedures. Melting poins were determined by

Plant material. S. cylindrica was collected in Rio de Janeiro, 1998. A voucher specimen is maintained in the Laboratory of Chemistry of Medicinal Plants.

* Correspondence to: Dr J. P. Parente, Nu´cleo de Pesquisas de Produtos Naturais, Laborato´rio de Quı´mica de Plantas Medicinais, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro, PO Box 68045, CEP 21944-970, Rio de Janeiro, RJ, Brasil. Contract/grant sponsor: CNPq. Contract/grant sponsor: CAPES. Contract/grant sponsor: FAPERJ. Contract/grant sponsor: FUJB.

Extraction and isolation. The fresh leaves of the plant (3 kg) were extracted with 80% aq EtOH (6 L), followed by concentration to 600 mL and extraction with an equal volume of n-BuOH to give crude material (1.4 g). This was roughly chromatographed on Sephadex LH-20 with MeOH to give crude steroidal glycoside (413 mg). Further purification by chromatography on a silica gel column eluted with CHCl3-MeOH–H2O (70:30:10) afforded one TLC homogeneous compound 1 (253 mg),

Copyright # 2003 John Wiley & Sons, Ltd.

Received 8 May 2001 Accepted 26 June 2001

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A. DA SILVA ANTUNES ET AL.

Table 1. Selected characteristic 1H NMR data (d [ppm]), J[Hz] of 1 in C5d5N Position

1

1

H-3 3.90 m H-6 5.30 br.s H-12 4.18 m H-15 4.80 m H-26 3.55, 3.70 Me-18 0.85 s Me-19 1.10 s Me-21 1.25 d (7.0) Me-27 1.00 d (7.0) Sugar methyl group and anomeric 3-O-Glc-H-1 4.65 d (7.5) 26-O-Glc-H-1 4.85 d (7.5) 12-O-Rha-H-1 5.30 br.s 12-O-Rha-Me 1.80 d (6.5) 15-O-Rha-H-1 6.40 br.s 15-O-Rho-Me 1.40 d (6.5)

H-1H-COSY

H-2, H-4 H-7 H-11 H-14, H-16 H-25 H-20 H-25 protons 3-O-Glc-H-2 26-O-Glc-H-2 12-O-Rha-H-2 12-O-Rho-H-5 15-O-Rha-H-2 15-O-Rha-H-5

R f 0.50 which gave a dark green colour with orcinol– H2SO4. Compound 1. Colourless needles; mp 185 °–188 °C; [a]D25-70 ° (c 0.1, MeOH); IR vmax (KBr): 3389 (OH), 2927, 1633, 1584, 1516, 1453, 1409, 1379, 1316, 1261, 1185, 1130, 1074, 1042 (C-O), 912, 840, 815, 640 cm 1 [(25S)-furostanol, intensity 912 >840]. LSIMS (neg.), m/z 1079 [M-H]; 1H and 13C NMR data (Tables 1 and 2). Acid hydrolysis of 1. Compound 1 (70 mg) was Table 2. 13C NMR data of the aglycone and carbohydrate moieties of 1 in C5D5Na C

1

C

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

37.07 29.50 77.48 38.54 140.36 121.38 31.88 31.22 49.90 36.67 29.77 83.55 40.35 56.16 80.71 79.08 63.23 16.05 18.98 40.35 16.86 110.26 36.67 27.88 33.98 74.69 17.11

26-O-Glc 1 2 3 4 5 6

105.88 74.69 77.75 71.33 77.48 62.15

3-O-glc 1 2 3 4 5 6

102.64 74.71 77.94 70.91 77.50 62.18

12-O-Rha 1 2 3 4 5 6

99.89 72.33 72.35 73.63 69.03 18.20

15-O-Rha 1 2 3 4 5 6

101.59 72.05 72.15 73.68 69.09 18.23

a

The assignments were made on the basis of DEPT, HETCOR, HMBC and HMQC experiments.

Copyright # 2003 John Wiley & Sons, Ltd.

1

H-1H-NOESY

1

H-13C-HMBC

3-O-Glc-H-1

3-O-Glc-C-1

Me-21 Me-18 26-O-Glc-H-1 H-15

12-O-Rha-C-1 15-O-Rha-C-1 26-O-Glc-C-1

H-12 H-3 H-26 H-12

C-3 C-26 C-12

H-15

C-15

hydrolysed with 2 M HCl–1, 4-dioxan (1:1; 10 mL) in a sealed tube for 3 h at 100 °C, and the reaction mixture was extracted with EtOAc (50 mL, twice). The solvent extract was purified by preparative TLC on silica gel plates (CH2Cl2–MeOH 19 : 1, R f 0.51) to yield the aglycone (14.7 mg) as colourless crystals : mp 258 °C, [a]D25 86 ° (c 1.0, CHCl3-MeOH 1 : 1) (Coll et al., 1983). Molar carbohydrate composition and D, L configurations. The molar carbohydrate composition of 1 was determined by GC-MS analyses of its monosaccharides as their trimethylsilylated methylglycosides obtained after methanolysis (0.5 M HCl in MeOH, 24 h, 80 °C) and trimethylsilylation (Kamerling et al., 1975). The configurations of the glycosides were established by capillary GC of their trimethylsilylated ( )-2-butylglycosides (Gerwig et al., 1978). Methylation analysis. Compound 1 was methylated with DMSO–lithium methylsulfinyl carbanion–CH3I (Parente et al., 1985). The methyl ethers were obtained after hydrolysis (4 N TFA, 2 h, 100 °C) and analysed as partially alditol acetates by GC-MS (Fournet et al., 1978). Haemolytic activity. Normal human red blood cell suspension (0.6 mL of 0.5%) was mixed with 0.6 mL diluent containing 5, 10, 20, 30, 40, 50, 100, 250 and 500 mg mL 1 concentrations of compound 1, aluminium hydroxide, and 5–500 mL mL 1 of Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA) in saline solution. Mixtures were incubated for 30 min at 37 °C and centrifuged at 70 g for 10 min. Saline and distilled water were included as minimal and maximal haemolytic controls. The haemolytic percent developed by the saline control was subtracted from all groups. The adjuvant concentration inducing 50% of the maximum haemolysis was considered the HD50 (graphical interpolation). Each experiment included triplicates at each concentration (Santos et al., 1997). Antiinflammatory activity. Antiiflammatory activity was evaluated by measuring acetic acid-induced vascular permeability (Whittle, 1964). Male mice (BALB/c, 15–20 g) in groups of five were dosed orally with comPhytother. Res. 17, 179–182 (2003)

BIOACTIVE SAPONIN FROM SANSEVIERIA CYLINDRICA

pound 1 (100 mg/g body weight) and a positive control, indomethacin (10 mg/g body weight). After injection of the dye, 0.1 N acetic acid (10 mL/g body weight) was injected intraperitoneally. Twenty minutes later, the mice were killed with an overdose of ether and the viscera were exposed after a 1 min period to allow blood to drain away from the abdominal wall. The animal was held by a flap of the abdominal wall and the viscera were irrigated with 10 mL of saline over a petri dish. The washing was filtered through glass wool and transferred to a test tube. To each tube was added 100 mL of 1 N NaOH in order to clear any turbidity due to protein, and the absorbance was read at 590 nm.

181

The fresh leaves of S. cylindrica were extracted with 80% adueous EtOH. After concentration under reduced pressure, the extract was partitioned between water and n-BuOH. Chromatographic separations of the organic phase on Sephadex LH-20 and silica gel gave compound 1 which was detected with orcinol–H2SO4 reagent. Compound 1 was obtained as colourless needles and gave a positive Liebermann–Burchard test for a steroidal saponin. The LSIMS showed an ion peak [M-H] m/z 1079 which, together with 13C NMR spectral data (Table 2), suggested the molecular formula as C51H84O24. In addition to this, the furostanol glycosidic nature of 1 was indicated by the strong absorption bands at 3389 and 1042 cm 1 and a 25S-furostan steroidal structure (815, 840 and 912 cm 1, intensity 912 >840 cm1) in the IR spectrum (Wall et al., 1952) confirmed by 1H and 13C NMR spectra (Tables 1 and 2) (Agrawal et al., 1985; Coll et al., 1983). The 1H NMR spectral data (Table 1) contained signals for an olefinic proton at d 5.30 (br.s), four anomeric protons at d 4.65 (d, J = 7.5 Hz, 3-O-GlcH-1), 4.85 (d, J = 7.5 Hz, 26-O-Glc-H-1), 5.30 (br.s, 12-O-Rha-H-1) and 6.40 (br.s, 15-O-Rha-H-1), four secondary methyl protons at d 1.00 (d, J = 7.0 Hz, Me-27), 1.25 (d, J = 7.0 Hz, Me-21), 1.40 (d, J = 6.5 Hz, 15-O-Rha-Me) and 1.80 (d, J = 6.5 Hz, 12-O-Rha-Me) and two angular methyl protons at d 0.85 and 1.10 (each s) corresponding to Me- 18 and Me-19, respectively. The above 1 NMR spectral data and a comparison of the 13C NMR signals of the aglycone moiety of 1 (Table

2) with those described in the literature (Agrawal et al., 1985; Coll et al., 1983) showed the structure of the aglycone to be (3b,12a,15b,25S)-22-hydroxyfurost-5ene-3,12,15,26-tetraol. In the 13C NMR spectrum of 1, two terminal b-D-glucopyranosyl units and two terminal a-L-rhamnopyranosyl units were clearly observed. In addition to this, the methylation analysis of 1 (Parente et al., 1985; Fournet et al., 1978) furnished 1,5-di-O-acetyl2,3,4,6-tetra-O-methyl glucitol and 1,5-di-O-acetyl2,3,4-tri-O-methyl rhamnitol. These results indicated the presence of four terminal monosaccharides. As shown in Tables 1 and 2, 1H and 13C NMR chemical shift assignments were made by standard ID and 2D NMR techniques. The attachments of the monosaccharides to the aglycone moiety were established by DEPT, COSY, HETCOR, HMBC and HMQC experiments. The HMBC and HMQC spectra displayed long rang couplings between a terminal glucose-H-1 at d 4.65 and aglycone-C-3 at d 77.48, a terminal glucose-H-1 at d 4.85 and aglycone-C-26 at d 74.69, a terminal rhamnose-H-1 at d 5.30 and aglycone-C-12 at d 83.55 and a terminal rhamnose-H-1 at d 6.40 and aglycone-C-15 at d 80.71, indicating that compound 1 is undoubtedly a tetradesmoside steroidal saponin. On acid hydrolysis, 1 gave a pseudosapogenin, glucose and rhamnose. The pseudosapogenin was identified as bahamgenin by direct comparison of TLC, mp, [a]D, IR, 1 H and 13C NMR and EIMS data with the literature (Coll et al., 1983). The molar carbohydrate composition of 1 indicated hte presence of four neutral monosaccharides: glucose:rhamnose (2.0 : 1.8) (the molar response of rhamnose is taken as 1) (Kamerling et al., 1975). Their absolute cofigurations were determined by GC of their trimethylsilylated ( )-2-butylglycosides (Gerwig et al., 1978). D-glucose and L-rhamnose were detected. Consequently, on the basis of IR, 1H and 13C NMR spectroscopy and chemical reactions, the structure of 1 was established as (3b,12b,15a,25S)-26- (b-D-glucopyranosyloxy)-22-hyroxyfurost-5-en-3-yl 12-O-(6-deoxy-a-Lmannopyranosyl)-15-O-(6-deoxy-a-L-mannopyranosyl)b-D-glucopyranoside. According to the literature, steroidal saponins are shown to possess antiinflammatory properties (LacailleDubois and Wagner, 1996). however, this activity is sometimes accomplished by an undesirable haemolytic effect (Oda et al., 2000). In order to evaluate the pharmacological properties of the steroidal saponin 1, it was screened for haemolytic activity in vitro (Santos et

Figure 1. 50% haemolytic dose (mg/mt) of compound 1 and adjuvents.

Figure 2. Antiin¯ammatory property of compound 1. Signi®cantly different from the control group * p < 0.01, ** p < 0.05.

RESULTS AND DISCUSSION

Copyright # 2003 John Wiley & Sons, Ltd.

Phytother. Res. 17, 179–182 (2003)

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A. DA SILVA ANTUNES ET AL.

particular behaviour can be explained easily by the assumption that the saponin 1 possesses sugar units distributed around the aglycone moiety, which considerably reduces its hydrophobicity, resulting in the loss of the amphipathic features. In addition to this, compound 1 at a dose of 100 mg/g inhibited the increase in vascular permeability caused by acetic acid, which is a typical model of first stage inflammatory reaction (Whittle, 1964). The standard drug indomethacin also reduced hte leakage (Fig. 2). The biological results obtained may help explain some biological properties attributed to several steroidal saponins reported in the literature (LacailleDubois and Wagner, 1996). al., 1997) and compared with adjuvants commonly used in animal and human experimental models. Generally, steroidal saponins possess elevated haemolytic activity because steroids have highe affinities for cholesterol on erythrocyte membranes (Oda et al., 2000). Nonetheless, this is not the case for compound 1 (Fig. 1), which demonstrated as absence of haemolytic effects. This

Acknowledgements This work was financially supported by CAPES, CNPq, FAPERJ and FUJB.

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glycosides from Sansevieria trifasciata. Phytochemistry 44: 107±111. Morton JF. 1981. Atlas of Medicinal Plants of Middle America Charles C Thomas Publisher: Illinois, 90. Oda K, Matsuda H, Murakami T, Katayama S, Ohgitani T, Yoshikawa M. 2000. Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biol Chem 381: 67±74. Parente JP, Cardon P, Leroy Y, Montreuil J, Fournet B, Ricart G. 1985. A convenient method for methylation of glycoprotein glycans in small amounts by using lithium methylsul®nyl carbanion. Carbohydr Res 141: 41±47. Santos WR, Bernardo RR, PecËanha, LMT, Palatnik, M, Parente JP, de Sousa CBP. 1997. Haemolytic activities of plant saponins and adjuvants. Effect of Periandra mediterranes saponin on the humoral response to the FML antigen of Leishmania donovani. Vaccine 15: 1024±1029. Wall ME, Eddy CR, McClennan ML, Klump ME. 1952. Detection and estimation of steroidal sapogenins in plant tissue. Anal Chem 24: 1337±1341. Wasicky R, Hoehn W. 1951. The crude saponin content of some Brazilian plants. Anais Faculdade Farm Odontol 9: 17±26. Whittle BA. 1964. The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic analgesics. Br J Pharmacol Chemother 22: 246±253.

Phytother. Res. 17, 179–182 (2003)

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