Steroidal glycoalkaloids and molluscicidal activity of Solanum asperum Rich. fruits

SHORT REPORT

Steroidal glycoalkaloids and molluscicidal activity of Solanum asperum Rich. fruits

Tania M. S. Silva^I,*; Celso A. Camara^II; Kristerson R. L. Freire^III; Thiago G. da Silva^III; Maria de F. Agra^III; Jnanabrata Bhattacharyya^III

^INúcleo Complexo Produtivo de Saúde, Instituto Multidisciplinar em Saúde, Campus Avançado Anísio Teixeira, Avenida Olívia Flores, 3000, Candeias, 45055-090 Vitória da Conquista-BA, Brazil

^IIDepartamento de Química, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros, s/n, Dois Irmãos, 52171-900 Recife-PE, Brazil

^IIILaboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, CP 5009, 58051-970 João Pessoa-PB, Brazil

ABSTRACT

Bioassay-guided fractionation of the alkaloidal extract of the green fruits of Solanum asperum afforded a new compound, solanandaine along with solasonine and solamargine. The total crude alkaloids as well as the isolated pure alkaloids exhibited significant molluscicidal activity.

Keywords:Solanum asperum, glycoalkaloid, solanandaine, solasonine, solamargine, molluscicidal

RESUMO

O fracionamento bio-monitorado do extrato alcaloídico dos frutos verdes de Solanum asperum forneceu um novo alcalóide esteroidal, denominado solanandaina, juntamente com a solasonina e a solamargina. Tanto o extrato alcaloídico como os glicoalcalóides isolados apresentaram potente atividade moluscicida.

Introduction

Solanum L. (Solanaceae) is distributed mainly throughout the tropical and subtropical regions of the world and is the largest and most complex genus of the family Solanaceae. In Brazil, Solanum is represented by about 350 species.¹ In this region, many species of Solanum are widely used in popular medicine and are commonly known as ‘yubeba', the word derived from Tupi Guarani that refers to the prickles found on the stems of several of the species.¹ Some common and wide-spread Solanum species of Brazil had shown^2,3 considerable molluscicidal activity demonstrated by several members of the genus investigated earlier.^4,5 Thus, we have been studying various species of Solanum growing in our country^3,6-10 with the expectation that the extracts of plants of this genus might be useful in the control of Biomphalaria glabrata, the intermediate host of Schistosoma mansoni, the parasite that causes human schistosomiasis in Brazil. In our previous bioassays, the crude MeOH extract of the unripe fruits of S. asperum Rich showed^3,11 activity in studies with Artemia salina (LC₅₀= 420.5 μg mL^-1) and Biomphalaria glabrata (LC₅₀= 25.5 μg mL^-1). Bioassay-guided fractionation indicated that the activity was concentrated in the alkaloid fraction. Thus, the alkaloid fraction upon CC on Sephadex LH-20 followed by PTLC on Silica gel afforded a new compound, solanandaine, C₄₅H₇₃O₁₆N (1) mp 262-263 °C, [α]_D²⁹-60.0 (MeOH, c = 1.0 mg mL^-1) along with solasonine (2) and solamargine (3). In this work, we wish to report the isolation, characterization and the molluscicidal activity of the alkaloids of S. asperum unripe fruits.

Results and Discussion

Solanum asperum Rich. is popularly known in Brazil as ‘jussara' or ‘coça-coça'. It is a neotropical species belonging to the section Brevantherum with wide distribution in South America. Extract of S. asperum unripe fruits demonstrated significant molluscicidal activity. With the aid of biossay-guided fractionation of the crude alkaloid mixture, solanandaine, solasonine and solamargine were isolated from the green fruits of S. asperum. Solanandaine (1) was obtained from MeOH, mp 262-263 °C; [α]_D²⁹-60.0 (MeOH, c= 1.0 mg mL^-1). The structure of (1) was determined mainly on the basis of positive ion HREIMS and LC-MS along with one and two dimensional ¹H and ¹³C NMR spectral analyses. The assignments of the carbon and proton resonances were made on the basis of HBBD, DEPT, ¹H-¹H COSY, HSQC, HMBC and NOESY experiments. The positive ion HREIMS of solanandaine showed a peak at m/z 884.4964 [M + H]⁺ corresponding to the molecular formula, C₄₅H₇₃O₁₆N (calculated for C₄₅H₇₄O₁₆N, 884.4929). The positive ion LC-MS showed, in addition to the one at m/z 884 (M +1), significant peaks at m/z 738 (M+H - 146), 592 (738 - 146), 430 (592 - 162) and 154. Solanandaine, therefore, contains three hexose units. The sequential loss of 146, 146 and 162 daltons indicate that solanandaine has a rhamnosyl-rhamnosyl-glucosyl side chain attached to an aglycone moiety. There are three typical anomeric proton signals in the ¹H NMR spectrum of solanandaine. The one at δ_H 4.93 (J 7.0 Hz) is certainly due to a β-D-hexose. The ¹H and ¹³C NMR chemical shifts (Table 1) are fully compatible with a β-D-glucose structure for this hexose unit. The other two anomeric signals are broad singlets at δ_H 5.84 and 6.38, typical of α-L-rhamnose unities. The HSQC spectrum shows that the three signals at δ_C 100.6, 102.4 and 103.2 are due to the corresponding anomeric carbons of the three sugars unities in the glycoside chain.

¹H NMR spectrum of solanandaine (Table 1) showed among others the presence of five CH₃ signals, two of which are doublets at δ_H 1.61 (3H, J 6.1 Hz) and 1.75 (3H, J 6.2 Hz), supporting the presence of two deoxyhexose units like rhamnose and other three are 3H signals at δ_H 0.86 (s), 1.04 (s) and 1.08 (d, 7.0 Hz). Thus, the aglycone moiety contains only three CH₃ groups which suggest that it is not solasodine. The ¹H NMR spectrum also showed a signal at δ_H 5.30, characteristic of the CH-6 of Δ⁵-spirosolanes. The proton decoupled ¹³C NMR spectrum of solanandaine (Table 1) shows the presence of 45 signals for 45 carbons in the molecule. In addition to supporting the presence of five CH₃ groups, the spectrum also shows two CH₂ signals (DEPT) at δ_C 61.6 and 66.4, typical of two CH₂OH groups. Therefore, apart from the one in the glucose unit, there must be an additional CH₂OH group in the aglycone moiety of solanandaine. The absence of one CH₃ group compared to solasodine and the appearance of a CH₂OH group instead strongly suggests the aglycone moiety of solanandaine to be solaparnaine.¹² The MS peak at m/z 154.16 daltons more than the corresponding peak in solasodine at m/z 138 is characteristic of an oxygenated ring F, like that in solaparnaine. ¹³C NMR spectrum of solanandaine further shows a signal at δ_C 78.4 for C-3, which is considerably downfield relative to the corresponding shift at ~71.50 in solasodine or solaparnaine (Table 1). This suggests the presence of sugar substitution at that position of the aglycone. This is further supported by the resulting upfield shifts of C-2 and C-4 to δ_C 30.5 and 39.3, respectively, in solanandaine relative to solasodine or solaparnaine.¹

Table 2 shows HMBC and NOESY correlations of the protons and carbons of solanandaine. The proton signal at δ_H 3.87 for H-3 has a cross peak with δ_C 100.6 (C-1') and the signal at δ_H 4.93 (d, J 7.0 Hz) for H-1' shows a cross peak with δ_C 78.4 (C-3) thereby confirming that a β-D-glucose unit is indeed substituted at that position. The NOESY spectrum supports this structure. In addition, it shows cross peaks of glycosidic linkages between δ_H 4.21 (H-2') and δ_H 6.38 (H-1'') as well as between δ_H 4.35 (H-4') and δ_H 5.84 (H-1'''). This is also supported by HMBC correlations (Table 2). Thus, there are two α-L-rhamnose units attached to C-2'and C-4' and an inner β-D-glucose unit which, in turn, is attached to C-3 of the aglycone unit of solanandaine.

Therefore, the structure of solanandaine must be 3-O-[α-L-rhamnosyl-(1→2)-[α-L-rhamnosyl-(1→4)-β-D-glucopyranosyl]-solaparnaine (1). The key HMBC correlations are shown on structure 1. Subsequent acid hydrolysis of solanandaine in the usual way furnished solaparnaine (4). Solasonine (2) and solamargine (3) were identified by comparison of their physical and spectral data with those published in the literature.^13,14

Solanum species are known to produce a great variety of steroidal saponins and glycoalkaloids. The potato glycoalkaloids may have evolved in nature to protect the plant against phytopathogens and other hostile environments.^15,16 In our previous studies we had found that several species of Solanum, including the unripe fruits of S. asperum have potentially significant molluscicidal activity.^2,3 The crude alkaloid fraction obtained from the active total MeOH extract as well as the pure alkaloids isolated were tested for the molluscicidal activity (Table 2). Individually, the glykoalkaloids solanandaine (1) (LC₅₀=73.1 μg mL^-1), solasonine (2) (LC₅₀=47.0 μg mL^-1) and solamargine (3) (LC₅₀=26.3 μg mL^-1), were found to be less active than the total MeOH (LC₅₀=25.5 μg mL^-1) ³ and the crude alkaloidal extracts (LC₅₀=9.7 μg mL^-1) of S. asperum (Table 3). The higher activity of the crude extracts may be attributed to synergistic effects. The results of the bioassay show that solasonine and solamargine, the glycosides of the common aglycone solasodine, possess more molluscicidal effect and solamargine, with lesser polarity of the two shows relatively more activity. On the other hand, solanandaine, which has an aglycone different than solasonine or solamargine, presents the least bioactivity of all three. These results indicate that the activity may be related to both glycosidic and aglycone moieties of the Solanum glycoalkaloids. Earlier, the molluscicidal activity of solasonine and solamargine in a mixture was studied¹⁷ against Lymnaea cubensis and Biomphalaria glabrata. The toxicity was more pronounced for L. cubensis (100% mortality, 10 ppm) and B. glabrata (100% mortality, 25 ppm). In the present work, we studied the activity of the individual glycoalkaloids in the bioassay with B. glabrata upon which the LC₅₀ values were calculated. The molluscicidal activity seen in some Solanum species is generally attributed to the presence of glycoalkaloids, with other classes of secondary metabolites, including alkamines, having litte if any such activity.¹⁷ Besides solasonine and solamargine, others glycoalkaloids of Solanum as tomatine¹⁷ and solamarine¹⁸ presents molluscicidal activity.

Experimental

General

Melting points were determined on a Koefler hot stage and are uncorrected. Optical rotation was measured with a Bellingham & Stanley Ltd., Model ADP220 polarimeter. The infrared absorption spectra were recorded in KBr pellets, using a Bomem/MB-102 spectrophotometer operating in the 4000-400 cm^-1 range. The LC-MS was obtained in positive electrospray mode using a Quattro LC-Micromass (Waters) and HREIMS were obtained by electron impact on a VG Autospec spectrometer. TLC was done using silica gel Kieselgel 60 (E. Merck) and spots were visualized by Dragendorff reagent. ¹H and ¹³C NMR spectra were obtained using a Bruker Advance 500 (500 Hz for ¹H and 125 MHz for ¹³C) Spectrometer as well as a Jeol Eclipse+ 400 spectrometer operating at 400 MHz in pyridine-d₅. Sephadex LH-20 (Sigma) was employed for gel permeation chromatography.

Plant material

Fruits of S. asperum were collected in the State of Paraíba, Brazil, in September 2005 from a secondary vegetation of the Atlantic forest area at the campus of the Universidade Federal da Paraíba, in the municipality of João Pessoa, at 130 to 160 m elevation. The plant was identified by Dr. Maria de Fátima Agra (LTF-UFPB). Voucher specimen (Agra 1243) is deposited at the Prof. Lauro Pires Xavier (JPB) Herbarium, Universidade Federal da Paraíba, João Pessoa, Brazil.

Extraction and isolation

Fresh fruits of S. asperum (740.0 g) were extracted with H₂O:HOAc (8:2) in a blender and filtered through a bed of Celite. The acid aqueous filtrate was basified with NH₄OH and left standing overnight. The gelatinous precipitate (10.1 g) formed was collected by filtration to give a mixture of glycoalkaloids. The alkaloid mixture was then chromatographed over Sephadex LH-20 using MeOH as eluent and fifteen fractions were collected. Fractions 3-10 showed the presence of alkaloids. Fraction 4 (850.0 mg) with three alkaloids was further purified by PTLC in silica gel plates and eluted with CH₂Cl₂:MeOH:NH₃ (9:2:0.5) to furnish solanandaine (1, 120.0 mg), solasonine (2, 450.0 mg) and solamargine (3, 172.0 mg).

Solanandaine (1): white powder (MeOH); mp 262 -263 °C; [α]_D²⁹ -60.0 (MeOH, c= 1.0 mg mL^-1); IR (KBr) 3450, 2940, 1625, 1071, 1045, 980 cm^-1; ¹H NMR and ¹³C NMR (pyridine-d₅, 500 MHz and 125 MHz, respectively), see Tables 1 and 2. Positive-ion HREIMS m/z 884.4964 (calculated for C₄₅H₇₄O₁₆N, [M + H]⁺, 884.4929).

Molluscicidal tests

Molluscicidal activity of the crude alkaloid fraction and the individual glycoalkaloids was measured according to the method described³ using laboratory-bred B. glabrata as the target snails. The samples were dissolved in three drops of Cremophor emulsifier (BASF, Ludwigshafen, Germany) and dechlorinated water to give stock solutions containing 100 μg mL^-1 of each of the samples of crude alkaloid extracts and pure glycoalkaloids. For the preliminary bio-assays, each stock solution was either left undiluted or further diluted with dechlorinated water to give test solutions containing 100, 50 and 10 μg mL^-1 of the samples. Subsequently, five different test solutions, ranging in concentration from 10 to 100 μg mL^-1 were prepared. For each assay, 10 adult snails (measuring 8-12 mm diameter) were exposed to 250 mL of each test solution in a glass beaker for 24 h at room temperature. After this period, each test solution was replaced with dechlorinated water. Snail mortality was then recorded over the following 24 h period, and compared with the positive controls (cupric carbonate at 50 μg mL^-1) and negative controls (extract-free dechlorinated water containing the same amount of Cremophor as the stock solutions). All assays were run in duplicate. The concentrations that kill 90% (LC₉₀), 50% (LC₅₀) and 10% (LC₁₀) of the treated snails (survived in the negative control cultures) were estimated by probit analysis, using the Origin 6.0

Acknowledgments

The authors thank IMSEAR-CNPq, CAPES and PIBIC-UFPB for financial support. JB thanks CAPES for generous support of Visiting Professorship. TMSS thanks Prof. Edilberto R. Silveira and Daniel E. Uchoa (CENAUREM - Centro Nordestino de Aplicação e Uso de RMN) and Prof. Raimundo Braz-Filho (UENF-RJ) for kindly recording the NMR data, and Socrates Golzio (LTF-UFPB) for kindly recording the LC-MS data.

Supplementary Information

Supplementary data of the isolated compounds as ¹³C and ¹H NMR spectra are available free of charge at http://jbcs.sbq.org.br, as PDF file.

Received: June 21, 2006

Web Release Date: April 29, 2008

Supplementary Information

• 1. Agra, M. F.; Bhattacharyya, J. In Solanaceae. IV, Advances in Biology and Utilization; Nee, M.; Symon, D. E.; Lester, R. N.; Jessop, J. P. eds.; Royal Botanic Gardens: Kew, U.K., 1999, pp. 341-343.
• 2. Silva, T. M. S.; Camara, C. A.; Agra, M. F.; Carvalho, M. G.; Frana, M. T.; Brandolini, S. V. P. B.; Paschoal, L. S.; Braz-Filho, R.; Fitoterapia 2006, 77, 449.
• 3. Silva, T. M. S.; Batista, M. M.; Camara, C. A.; Agra, M. F.; Ann. Trop. Med. Parasitol. 2005, 4, 419.
• 4. Hostettmann, K.; Kizu, H.; Tomimori, T.; Planta Medica 1982, 44, 34.
• 5. Marston, A.; Hostettmann, K.; Phytochemistry 1985, 24, 639.
• 6. Silva, T. M. S.; Braz-Filho, R.; Carvalho, M. G.; Agra, M. F.; Biochem. System. Ecol 2002, 30, 1083.
• 7. Silva, T. M. S.; Braz-Filho, R.; Carvalho, M. G.; Agra, M. F.; Biochem. System. Ecol 2002, 30, 479.
• 8. Silva, T. M. S.; Nascimento, R. J. B.; Camara, C. A.; Agra, M. F.; Braz-Filho, R.; Carvalho, M. G.; Biochem. System. Ecol 2004, 32, 513.
• 9. Esteves-Souza, A.; Silva, T. M. S.; Alves, C. C. F.; Carvalho, M. G.; Braz-Filho, R.; Echevarria, A.; J. Braz. Chem. Soc. 2002, 13, 838.
• 10. Silva, T. M. S.; Costa, R. A.; Oliveira, E. J.; Barbosa-Filho, J. M.; Agra, M. F.; Camara, C. A.; J. Braz. Chem. Soc. 2005, 16, 1467.
• 11. Silva, T. M. S.; Nascimento, R. J. B.; Batista, M. M.; Agra, M. F.; Barbosa-Filho, J. M.; Camara, C. A.; Rev. Bras. Farmacogn. 2006, 17, 35.
• 12. Bhattacharyya, J.; Heterocycles 1985, 23, 3111.
• 13. Puri, R.; Wong, T. C.; J. Nat. Prod. 1994, 57, 587.
• 14. Fukuhara, K.; Kubo, I.; Phytochemistry 1991, 30, 685.
• 15. Friedman, M.; Rayburn, J. R.; Bantle, J. A.; Food Chem. Toxicol 1991, 29, 537.
• 16. Friedman, M.; J. Agric. Food. Chem. 2006, 54, 8655.
• 17. Alzerreca, A.; Hart, G.; Toxicol. Lett. 1982, 12, 151.
• 18. Wanyonyi, A. W.; Chlabra, S. C.; Mkoji, G.; Eilert, U.; Njue, W. M.; Phytochemistry 2002, 59, 79.

Publication Dates

• Publication in this collection
  05 Aug 2008
• Date of issue
  2008

History

• Received
  21 June 2006
• Accepted
  29 Apr 2008