STEROIDAL AND PHENOLIC COMPOUNDS FROM Sidastrum Paniculatum
(L.) Fryxell AND EVALUATION OF CITOTOXICITY AND ANTI-
INFLAMMATORY ACTIVITIES
José Marcílio Sobral Cavalcante; Tiago Bezerra de Sá de Souza Nogueira; Anna Cláudia de Andrade Tomaz; Paulo Roberto Cavalcanti Carvalho; Silene Carneiro do Nascimento; Teresinha Gonçalves-Silva; Maria de Fátima Vanderlei de Souza*
Laboratório de Tecnologia Farmacêutica “Prof. Delby Fernandes de Medeiros”, Centro de Ciências da Saúde, Universidade Federal da Paraíba, Caixa Postal 5009, 58051-970, João Pessoa, PB, Brasil.
Laboratório de Bioensaios para Pesquisa de Fármacos, Departamento de Antibióticos, Universidade Federal de Pernambuco 50670-901, Recife, PE, Brasil.
STEROIDAL AND PHENOLIC COMPOUNDS FROM Sidastrum Paniculatum
(L.) Fryxell AND EVALUATION OF CITOTOXICITY AND ANTI-
INFLAMMATORY ACTIVITIES
Sidastrum paniculatum (L.) Fryxell belongs to the family Malvaceae and is popularly known as “malva roxa” or “malvavisco”. The phytochemical study of the hexane, CHCl3 and EtOAc phases from the crude extract of S. paniculatum led to the isolation of six compounds: a β-sitosterol and stigmasterol mixture, m- methoxy-p-hydroxy-benzoic acid, m-methoxy-p-hydroxy-benzaldehyde, N-trans- feruloyltyramine and kaempferol-3-O-β-D-(6’’-E-p-coumaroyl) glucoside. The structural identification of these compounds was made on the basis of spectroscopic methods such as IR, NMR 1H and 13C with the add of two- dimensional techniques (HOMOCOSY, HETCOR, HMQC, HMBC and NOESY), besides comparison with literature data.
INTRODUCTION
The species Sidastrum paniculatum (L.) Fryxell is a bush that belongs to the family Malvaceae and is popularly known as “malva-roxa” or “malvavisco”. In Brazil, the northeast region is probably the diversity core of the genus Sidastrum since a great number of species can be found there.1
Previous phytochemical studies on species from Malvaceae have reported the presence of steroids,2,3,4,5 phenols,3,4,6 flavonoids,3,4,5,7,8 triterpenes,2,6 essential oils,9 alkaloids,10 sesquiterpenelactones,11 fatty acids,12,13,14,15 and pheophorbide.3
Aiming at contributing to the chemotaxonomic study of the family Malvaceae and considering the absence of data in literature concerning the chemical constitution of the genus Sidastrum, the species Sidastrum paniculatum was submitted to a phytochemical study to isolate and identify its chemical constituents, through usual chromatographic and spectroscopic methods, besides comparison with literature data.
EXPERIMENTAL General procedures
NMR spectra (HOMOCOSY, HETCOR, HMQC, HMBC and NOESY) were measured in CDCl3, CD3OD and recorded on a Mercury Varian instrument operating at 200 MHz and 50 MHz for 1H and 13C, respectively. The solvent signal was used as internal standard. IR spectra were measured on a Perkin-Elmer, FT- IR-1750 spectrometer in KBr pellets. Chromatography columns were carried out on silica gel (Merck) and Sephadex LH-20 (Merck). TLC were performed on silica gel PF254 plates and the spots were visualized under UV light (254 and 366 nm) and by exposure to the iodine vapor.
Plant material
The whole plant of S. paniculatum was collected in Pedra da Boca, in the city of Araruna, State of Paraíba, on February 2004. The botanical identification was made by Dr. Maria de Fátima Agra from the Botany section of Núcleo de Pesquisa de Produtos Naturais of Laboratório de Tecnologia Farmacêutica (LTF) and a voucher specimen was deposited at the Herbarium Prof. Lauro Pires Xavier (JPB), Universidade Federal da Paraíba, under the code 6051.
Extraction and isolation
The plant material (10 kg) was subjected to dehydration in an oven in a temperature of 40ºC for 72 hours; after that, it was grownded in a mechanical mill, yielding 5.7 kg of a powder which was submitted to maceration with ethanol for three consecutive days. This process was repeated until the maximum extraction of the chemical constituents. The obtained ethanol extractive solution was concentrated in a rotatory evaporator, yielding 500 g of crude ethanol extract (CEE). The latter was suspended in ethanol:H2O (9:1) and successively partitioned with hexane, CHCl3, EtOAc and n-butanol. 6.0 g of the hexane extract were subjected to column chromatography packed with silica gel and eluted with hexane, CHCl3, EtOAc and methanol. 69 fractions of 20 mL each were collected and analysed and combined through analytical thin-layer chromatography (TLC). The sub-fraction 37/42 (1.36 g) was rechromatographed on silica gel column eluted with hexane, CHCl3 and methanol providing 15 sub-fractions which were analysed and joined through analytical TLC. The sub-fraction 11/15 showed itself as crystals, yielding 0.093 g of substance 1. The CHCl3 extract provided a precipitate and a supernatant. 6.0 g of the latter were submitted to
chromatography on a column packed with silica gel and eluted with hexane, EtOAc and methanol, giving 239 fractions that were analysed and joined through analytical TLC. 0.81 g of the sub-fraction 5/15 was rechromatographed on silica gel column utilizing the above eluents, yielding 177 sub-fractions which were combined and analysed through analytical TLC. The sub-fraction 26/57 (0.05 g) showed itself as an amorphous solid, defined as substance 2. The sub-fraction 26/102, on the other hand, was rechromatographed following the previous methodology, providing 96 sub-fractions of which the sub-fraction 43/56 was recrystallized in chloroform and drops of methanol, yielding 0.025 g of the substance 3. 0.15 g of the precipitate was submitted to preparative TLC, utilizing a mixture of hexane:ethyl acetate (1:1) as eluent, resulting on the isolation and purification of 0.021 g of substance 4. 3.0 g of the ethyl acetate extract were subjected to chromatography column packed with Sephadex LH-20 and eluted with methanol, providing 23 sub-fractions that were analysed and combined through analytical TLC. The sub-fraction 12/16 (0.087 g) was rechromatographed according to the above methodology, yielding 14 sub-fractions which were analysed and combined through analytical TLC. The sub-fraction 5/8 (0.032 g) was defined as substance 5.
Cells
The cytotoxicity of β-sitosterol and stigmasterol mixture was tested against NCI-H292 (carcinoma of human lungs), HEp-2 (carcinoma epidermoid of human larynx) and KB (carcinoma of the mouth) all obtained from the Bank of Cells, Rio de Janeiro, Brazil. The cells were grown in DMEM – Minimum Essencial Medium Eagle modified Dulbecco’s- medium supplemented with 10% fetal bovine serum, 2
mM glutamine, 100 µg/ml streptomycin, 100 Uml−1 penicillin at 37°C with a 5% CO2 atmosphere.
Animals
For the tests, were used swiss albino female mice (25–30 g), obtained from the Animal House of Department of Antibiotics of the Federal University of Pernambuco, Brazil. The animals were housed in cages with free access to food and water. All animals were kept under 12h light/dark cycle (lights on at 6:00 am.). The animals were treated according to the ethical principles of animal experimentation of COBEA (Brazilian College of Animal Experiments) Brazil and the norms of the National Institute of Health Guide for Care and Use of Laboratory Animals. The Animal Studies Committee of the Federal University of Pernambuco approved the experimental protocols (number 116/07). Sarcoma 180 and carcinoma of Ehrlich cells are maintained in the peritoneal cavities of the swiss mice in the Laboratory of Experimental Pharmacology and Cancerology of the Federal University of Pernambuco.
Cell Proliferation Assays
The tumor cell growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product (10). A cellular suspension (105 cells/mL) was prepared and distributed in 96-well (225µL/well) and incubated by 24h at 37°C. After that, the β sitosterol and stigmasterol mixture was added. At the end of incubation (72 h), the plate was centrifuged and the medium was then replaced by fresh medium (200 µl) containing 25 µl of MTT. Three hours later, the MTT
formazan product was dissolved in 100 µl DMSO, and the absorbance was measured using a multiplate reader27,28. The drugs effects were quantified as the percentage of control absorbance of reduced dye at 595 nm. The tested concentrations were 10.0, 5.0, 2.5 and 1.25 µg/mL and the experiments were done in triplicate.
Statistical analysis
The experiments were analyzed according to the mean ± standard deviation of the average (DPM) the percentage of inhibition of cell growth using the program GraphPad Prism.
Anti-inflammatory activity
Air pouches were produced on the dorsal cervical region of mice (25-30 g) by subcutaneous injection of 2.5 mL on day 0, followed by a second injection of 2,5 ml of sterile air 3 days later. Only day 6, carrageenan solution 1ml of a 1% (w/v) was injected into the cavity. The compound and indometacin were administered orally 1h before injection of carrageenan. After 6h, mice were sacrificed, pouches were washed with 3 mL of saline solution containing 3 mM EDTA, and number of migrated neutrophils determined, following staining with Turk’s solution (0,01% crystal violet in 3% acetic cid) and counting performed using a Neubauer haemocytometer17.
RESULTS AND DISCUSSION
The structural assignments of compounds 1-3 and 5 were made based on the spectral analysis and are in good agreement with those reported in the literature. Thus, their structures were identified as the mixture of the steroids β- sitosterol and stigmasterol (1),16 m-methoxy-p-hydroxy-benzoic acid (2),4,17 m- methoxy-p-hydroxy-benzaldehyde (3)18 and kaempferol 3-O-β-D-(6’’-E-p- coumaroil) glucopyranoside (5) (tiliroside).3,4,7 All of them are being reported here for the first time in the genus Sidastrum. Compound 5 (tiliroside) was previously isolated from Malvaceae species, for example Sida galheirensis,3 Bakeridesia pickelli,4 Herissantia crispa.5 and Herissantia tiubae.7
The IR spectrum of compound 4 showed a large absorption band at 3300- 3350 cm-1 (N-H bending) and at 1658 cm-1 (C=O bending) suggesting an amide function. The aromatic skeleton was evidenced by the absorptions between 1600 and 1450 cm-1.19 The 1H NMR spectra showed a pair of doublets at δ 6.68 and δ 6.96 (2H, J = 8.40 Hz), characterizing A,A′, B,B′-type aromatic hydrogens.20 It showed yet a doublet at δ 6.74 (1H, J = 8.80 Hz) together with a large doublet at δ 6.93 (1H) and a large singlet at δ 6.90 (1H), characterizing ABX-type aromatic hydrogens.
Those IR and 1H NMR data together with a pair of doublets at δ 6.16 and δ 7.37 (H-8, 7, J = 15.80 Hz) referring to trans olefinic hydrogens 7 and a pair of triplets at δ 2.67 and δ 3.43 (H-7’, 8’), suggested the presence of the coumaroyl and tyramine units, respectively. A methoxyl group in one of the aromatic nuclei was inferred by the signal at δ 3.79.
The 13C NMR spectral data strengthened the information provided by the IR and 1H NMR spectra, emphasizing the presence of the amide function due to the
signal at δ 167.04 (α,β-unsaturated carbonyl) thus reinforcing the coumaroyl unit because of the α,β-unsaturated carbons (C-8 and C-7) at δ 117.47 and δ 140.89, respectively, as well as the tyramine unit whose methylene carbons were shown to be at δ 34.41 (C-7’) and δ 40.89 (C-8’). The absorptions at δ 115.17 and δ 129.53 (2C, C-3’/5’ and C-2’/6’) corroborated the A,A′, B,B′ system, while a methoxyl group was suggested by the signal at δ 55.56. Non-hydrogenated carbons bound to oxygen groups were inferred by the signals at δ 147.20, δ 147.60 and δ 155.12.
The homonuclear correlation spectrum COSY showed coupling between δ 2.67 (H-7’) and δ 3.43 (H-8’) of the tyramine portion, and between at δ 6.16 (H-8) and δ 7.37 (H-7) of the coumaroyl one.
The heteronuclear correlation spectrum HMBC confirmed the suggestion of the α,β-unsaturated amide strengthening the presence of the coumaroyl and tyramine units since it showed, respectively, two- and three-bond correlations (2,3JCH) between C-1’ with H-7’ and H-8’, respectively, and 2JCH and 3JCH correlations between the carbonyl carbon and H-8 and H-7, respectively (Figure 1).
Figure 1: Correlations observed at the HMBC spectrum of 4 CH CH2 CH2 NH CH 1 2 3 4 5 6 7 8 2' 3' 4' 5' 6' 7' 8' O R1 R2 R3 J2 J2 J3 J3
N H
O H
H O H O C H 3
O
The homonuclear correlation spectrum NOESY revealed the space coupling between the methoxyl hydrogens with H-2, thus determining the meta position for the methoxyl group in the coumaroyil portion of the molecule (Figure 2).
The other assignments of carbons and hydrogens were determined based on all spectral data (Table 1) and on comparison with the literature ones,3,4,7 permitting to unambiguously identify substance 4 as being the N-trans- feruloyltyramine (Figure 3), substance already isolated from other vegetal species 21,22,23,24 with studies demonstrating its action against weeds and improvement of seed germination,25,26 being described for the first time in the family Malvaceae.
Figure 3: Substance 4 (N-trans-feruloyltyramine) Figure 2: Coupling observed at the NOESY spectrum of 4
NH 1 2 3 4 5 6 7 8 1' 2' 3' 4' 5' 6' 7' 8' O OH HO OCH3 9
Table 1: 1H and 13C NMR data (δ, CDCl3, 200 and 50MHz) of compound 4 1 H x 13C-HETCOR 1H x 13C- HMBC 1 H x 1H- COSY 1 H x 1H- NOESY C δC δH J2 J3 1 2 3 4 5 6 7 8 9 1’ 2’/6’ 3’/5’ 4’ 7’ 8’ OCH3 126.82 109.70 147.69 147.20 114.91 121.89 140.89 117.47 167.04 129.68 129.53 115.17 155.17 34.41 40.89 55.56 - 6.90(bs) - - 6.74(d, J=8.40Hz) 6.93(bd) 7.37(d, J=15.80Hz) 6.16(d, J=15.80Hz) - - 6.96(d, J=8.40Hz) 6.68(d, J=8.40Hz) - 2.67(t, J=6.9Hz) 3.43(t J=6.9Hz) 3.79(s) - C-3 - - - - - C-9 - - C-1’ C-2’/6’,C-4’ - C-1’, C-8’ C-7’ - - C-6, C-7 - - C-1 - C-9 C-1 - - C-4’ - - C-2’/6’ C-1’ - - - - - - - H-8 H-7 - - - - - H-8’ H-7’ - - OCH3, H-8 - - - H-7 H-6 H-2 - - - - - - - H-2
Citotoxy activity
The in vitro activity of mixture β-sitosterol and stigmasterol against three human tumor cell lines were determined however, the mixture assayed did not show cytotoxicity at the tested concentrations (table 2).
Table 2. CITOTOXY ACTIVITY OF THE MIXTURE OF Β-SITOSTEROL AND STIGMASTEROL Cells lines CI50 (µg/mL) Product KB NCI-H292 HEp-2 Mixture of Β-sitosterol and stigmasterol >10 >10 >10
The mixture of stigmasterol-sitosterolβ isolated from the kind of Sidastrum paniculatum showed no activity cytotoxic in tumor cell lines of human KB, NCI- H293 and Hep-2. According to the protocol of the National Cancer Institute (NCI- USA), for all products from plants are considered active the IC50 should be less than or equal to 30µg/mL.
The compound tested showed IC50 values greater than 10 µgml−1 for all tumor cell lines tested. These values figure show that the product has low toxicity tested cell, which is desirable in the case of anti-inflammatory drugs. It would also appear that in some compounds isolated from the family Malvaceae Hisbiscos taiwanensis also showed no activity cytotoxic in strains of human cells H.549 (carcinoma of the lung) and NC5-7 (breast cancer) to the dose of 20µg/mL28.
The mixture of β-sitosterol of and stigmasterol showed significant antiinflammatory activity when compared to the control group, inhibiting cell migration induced by carrageenan by 30, 56,8 and 81,2% at doses of 10, 30 and 90 mg / kg of weight, respectively (Table 3).
The leukocytes polymorphonuclear of number that migrated to the peritoneal cavity in the group control was 4,53 ± 0,8 and standard dexamethasone was 0,90 ± 0,3 x 106 cells per well (p <0,05 for all doses).
TABLE 3. ATIVIDADE ANTIINFLAMATÓRIA
Product Doses PMN cells ± SD
(x106)/cavity Inhibition (%)a 90 0,85±0,3 81,2 30 1,96±0,3 56,8 Mixture of βββ-sitosterol β and stigmasterol 10 3,17±0,6 30,0 Dexametasona 3 0,90±0,3 80,2 Controle - 4,53± 0,8 -
PMNL = polymorphonuclear leucocytes;. PMNL count is expressed as total cell number by volume of exudate. Activity (%) was calculated considering the control group cells counting as 100%. aValues represent average ± standard desviation.
REFERENCES
1. Fryxell, P. A.; Brittonia 1978, 30, 447-462.
2. Ahmed, Z.; Kazmi, S. N. H.; Walik, A.; J. Nat. Prod. 1990, 1342-1344.
3. Silva, D. A.; Silva, T. M. S.; Lins, A. C. S.; Costa, D. A.; Cavalcante, J. M. S.; Matias, W. N.; Souza, M. F. V.; Quim Nova 2006, 29(6), 1250-1254.
4. Costa, D. A.; Silva, D. A.; Cavalcanti, A. C.; Medeiros, M. A. A.; Lima, J. T.; Cavalcante, J. M. S.; Silva, B. A.; Agra, M. F.; Souza, M. F. V.; Quim. Nova 2007, 30(4), 901-903.
5. Costa, D. A.; Matias, W. N.; Lima, I. O.; Xavier, A. L.; Costa, V. B. M.; Diniz, M. F. F. M.; Agra, M. F.; Batista, L. M.; Souza, M. F. V.; Silva, D. A., in press.
6. Silva; D. A.; Falcão-Silva, V. S.; Gomes, A. Y. S.; Costa, D. A.; Lemos, V. S.; Agra, M. F.; Braz-Filho, R.; Siqueira-Junior, J. P.; Souza, M. F. V., in press.
7. Silva D. A.; Chaves M. C. O.; Costa, D. A.; Moraes, M. R.R.; Nóbrega, F. B. P.; Souza, M. F. V.; Pharmaceutical Biology 2005a, 43, 197.
8. Silva, D. A.; Costa, D. A.; Silva, D. F.; Souza, M. F. V.; Agra, M. F.; Medeiros, I. A.; Barbosa-Filho, J. M.; Braz-Filho, R.; Brazilian Journal of Pharmacognosy 2005b, 15, 23.
9. Ames, J. M.; Macleod, G.; Phytochemistry 1990, 29, 1201.
10. Ghosal, S.; Chauhan, R.; Mehta, R.; Phytochemistry 1975, 14, 830-832. 11. Sharma, P. V.; Ahmad, Z. A.; Phytochemistry 1989, 28, 3525.
12. Carmody, D. R.; Dejong, W.; Smith, T. R.; Oil & Soap 1945, 263. 13. Vickery, J. R.; JAOCS 1980, 87.
14. Schimid, K. M.; Patterson, G. W.; Phytochemistry 1988 27, 2831. 15. Nakatani, M.; Fukunaga, Y.; Hase, T.; Phytochemistry 1986, 25, 449.
16. Kojima, H.; Sato, N.; Hatano, A.; Ogura, H.; Phytochemistry, 1990, 29(7), 2351-2355.
17. Silveira, E. R.; Pessoa, O. D. L.; Constituintes Micromoleculares de Plantas do Nordeste com Potencial Farmacológico, 1 ed., Expressão gráfica e editora: Fortaleza - CE, p. 137-143, 2005.
18. França, V. C.; Tese de Doutorado, Universidade Federal da Paraíba, Brasil, 2002.
19. Silverstein, R. M.; Bassler, G. C.; Identificação Espectrométrica de Compostos Orgânicos, 5 ed.; Editora Guanabara Koogan S.A, 1994.
20. Pavia, D. L.; Lampman, G. M.; Kriz, G.S.; Introduction to Spectroscopy – A guide for students of organic chemistry; Brooks/Cole: U.S.A, 2001
21. Chen, C. Y.; Chang, P. R.; Wu, Y. C.; J. Chin. Chem. Soc. 1997, 44(6), 313- 319.
22. Kim, H. R.; Min, H. Y.; Jeong, Y. H.; Lee, S. K.; Lee, N. S.; Seo, E. K.; Arch. Pharm. Res., 2005, 28(11), 1224-1227.
23. Miyaichi, Y.; Nunomura, N.; Kawata, Y.; Kizu, H.; Tomimori, T.; Watanabe, T.; Takano, A.; Malla, K. J.; Chem. Pharm. Bull., 2006, 54(1), 136-138.
24. Oliveira, P. E. S.; Conserva, L. M.; Lemos, R. P. L.; Biochemical Systematics and Ecology, 2008, 36, 134-137.
25. Lajide, D.; Escoubas, P.; Mizutani, J.; Phytochemistry, 1995, 40(4), 1105- 1112.
26. Cutillo, F.; D’abrosca, B.; Dellagrega, M.; Di Marino, C.; Golino, A.; Previtera, L.; Zarelli, A.; Phytochemistry, 2003, 64, 1381-1387.
27. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer research, 48,589-601, 1998.
28. Berridge, M. V., Tan, A. S., Mccoy, K. D., Wang, R. Biochemica, 4,14-19, 1996.
29. Koster, R.; Anderson, M.; DE BEER, E. J. Fed. Proc., 18, 418-420, 1959. 30. Mossman, T. J. Immunol. Methods, 65: 55-63, 1983.
31. Silva, D. A.; Silva, T. M. S.; Claudio, A.; Costa, D. A.; Cavalcante, J. M. S.; Matias, W. N.; Braz Filho, R.; Souza, M. F. V. Química Nova, v. 29, p. 1250-1254, 2006.
32. Wu, Pei-lin.; Chuang, T. H.; HE, C. X.; WU, T. S. Bioorg Med Chem, 12, 2193- 7, 2004.
7- CONCLUSÃO
Estudo Citotóxico
• Os glicosideos β-sitosterol+estigmasterol glicosilados, diraminosídeo e tirilosídeo não apresentaram atividade citotóxica frente às linhagens celulares KB, NCI-H292 e HEp-2.
Atividade Antitumoral
• O glicosideo tirilosídeo apresentou uma inibição significativa frente ao sarcoma 180 e no carcinoma de Ehrlich, sugerindo, que a atividade seja dose-dependente, sendo ainda confirmada com a administração de outras doses;
• O diraminosídeo não inibiu o crescimento do Sarcoma 180;
• A mistura β-sitosterol+estigmasterol também apresentou uma inibição significativa frente ao sarcoma 180, sugerindo que a atividade seja dose- dependente.
Histopatologia de tumores e órgãos.
• No estudo histológico dos órgãos analisados (fígado e rins) foi verificado um comprometimento hepático pela evidenciação de infiltração linfocitária perivascular e espalhada no parênquima, apresentando um comprometimento renal pela acentuada redução da área de filtração glomerular;
• No sarcoma 180 foi verificado em ambos os grupos (controle e tratado) células com formatos ovóides, tecido adiposo subcutâneo e nos tumores tratados com o tilirosídeo músculo estriado esquelético.
Atividade antiinflamatória
• Os glicosideos tirilosídeo, β-sitosterol+estigmasterol glicosilados e o diraminosídeo apresentaram uma boa inibição na atividade antiinflamatória quando comparados a Indometacina;
• Este estudo revelou que os produtos são uma alternativa promissora a ser explorada para a ação antiinflamatória.
Atividade analgésica
• Os glicosideos tirilosídeo, β-sitosterol+estigmasterol glicosilados e o diraminosídeo apresentaram uma boa inibição na atividade analgésica;
• Este estudo revelou que os produtos são também uma alternativa promissora a ser explorada para a ação analgésica.