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Immunologically active polysaccharides from Centrosema pubescens
Fitoterapia 71 Ž2000. 516᎐521
Immunologically active polysaccharides from Centrosema pubescens Bernadete P. da Silva, Joao ˜ B.F. Tostes, Jose´ P. ParenteU Nucleo de Pesquisas de Produtos Naturais, Uni¨ ersidade Federal do Rio de Janeiro, ´ PO Box 68045, 21944-970 Rio de Janeiro, Brazil
Received 26 February 2000; accepted 13 March 2000
Abstract Two polysaccharides with mean Mr s of 2.0 = 106 and 3.75 = 105, were isolated from powdered seeds of Centrosema pubescens by fractionation on Sephacryl S-300 HR. Chemical and spectroscopic studies indicated that they have a backbone chain composed of -Ž1 ª 4.linked D-galactopyranose residues having branches composed of ␣-Ž1 ª 5.-linked Larabinofuranose residues at position 6 of D-galactose of the backbone chain. The polysaccharides showed reticuloendothelial system-potentiating activity in a carbon clearance test. 䊚 2000 Elsevier Science B.V. All rights reserved. Keywords: Centrosema pubescens; Polysaccharides; Immunomodulatory activity
1. Introduction Centrosema pubescens Benth. ŽFabaceae. is a Brazilian climbing plant widely grown as a fodder crop w1x. It is a common folk medicine used in the treatment of dropsy w2,3x. The chemical study of this plant revealed amino acids w4᎐6x, flavonoids w7x, isoflavonoids w8,9x, cyclohexitols and galactosyl-pinitol w10x and isoflavone U
Corresponding author. Tel.: q55-21-270-2683; fax: q55-21-270-2683. E-mail address: [email protected] ŽJ.P. Parente .. 0367-326Xr00r$ - see front matter 䊚 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 0 . 0 0 2 0 7 - 0
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
517
glycosides w11,12x. Taking into account a galactan isolated from the seeds of Centrosema plumari w13x, we investigated the polysaccharides present in the seeds of C. pubescens. Herein, we report the isolation and chemical characterization of two neutral polysaccharides endowed with immunological activity.
2. Experimental 2.1. Plant material Seeds of C. pubescens were collected at Mangaratiba, Rio de Janeiro in September 1975 and identified by V.P. Barbosa. A voucher specimen ŽNo. 172177. is deposited at the Botanical Garden, Rio de Janeiro. 2.2. General Carbohydrate and protein content were analyzed by the methods of Dubois et al. w25x and Bradford w26x, respectively. The Mr s of Cp1 and Cp2 were estimated from the calibration curve of the elution volume of standard dextrans Žaverage Mr values 2 000 000, 413 000, 282 000, 148 000, 68 000, 37 500, 19 500 and 9500. on Sephacryl S-300 HR Ž5 = 85 cm.. Dialyses were carried out using tubing with a Mr cut-off 12 000. Optical rotations were measured on a Perkin-Elmer 243B polarimeter. 1 H-NMR spectra were obtained on a Varian Gemini 200 NMR spectrometer, in D2 O containing sodium 2,2-dimethyl-2-silapentane-5-sulfonate as an internal standard. Solid state 13 C-NMR spectra were recorded on a Varian VXR-300 spectrometer. GC was carried out with FID, using a glass capillary column Ž0.31 mm = 25 m. SE-30. GC-MS were recorded at 70 eV. TLC of monosaccharides was performed on silica gel coated plates ŽMerck. in n-BuOH᎐pyridine᎐H2 O 6:4:3 and sugars were detected by spraying with orcinol᎐H2 SO4 . The experimental data were tested for statistical differences using the Student’s t-test w27x. 2.3. Extraction Dried and powdered seeds of C. pubescens Ž990 g. were extracted successively with cold CHCl3 , CHCl3 ᎐MeOH 80:20 and hot water Ž4 l. under stirring for 1 h in a boiling water bath. The aqueous extract was filtered and the filtrate centrifuged. By precipitation with two volumes of EtOH Ž12 h stirring and 24 h standing at 4⬚C., a crude polysaccharide fraction ŽCPF. was obtained by centrifugation and subsequent lyophilization Žyield: 9.7 g.. 2.4. Fractionation A sample of CPF Ž100 mg. was dissolved in 0.1 M Tris᎐HCl buffer ŽpH 7.0. and applied to a column Ž5 = 85 cm. of Sephacryl S-300 HR, pre-equilibrated and developed with the same buffer. Fractions of 10 ml corresponding to the peaks Cp1
518
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
and Cp2 were pooled, dialyzed, concentrated and freeze-dried. Each fraction was dissolved in water Ž2 ml. and applied to a column Ž1.5 = 50 cm. of Sephadex G-25. The column was eluted with water, and fractions of 5 ml were collected. The eluates obtained from each fractionation were concentrated and lyophilized to yield Cp1 Ž45 mg. and Cp2 Ž25 mg.. The fractionation procedures were followed by carbohydrate content. 2.5. Molar carbohydrate composition and D,L configurations Monosaccharides were analyzed as their TMS methylglycosides obtained after methanolysis Ž0.5 M HCl in MeOH, 24 h, 80⬚C. and trimethylsilylation w14x. The configurations of the glycosides were established by capillary GC and GC-MS of their TMS Žy.-2-butylglycosides w15x. 2.6. Methylation analysis Polysaccharides Cp1 and Cp2 were methylated with DMSO-lithium methylsulfinyl carbanion-CH3 I w16x. The methyl ethers were obtained after hydrolysis Ž4 N TFA, 2 h, 100⬚C. and analyzed as partially methylated alditol acetates by GC-MS w28x. 2.7. Phagocytic acti¨ ity Male BALBrc mice, weighing 15᎐20 g, were used in groups of five. Cp1, Cp2 and a positive control, zymosan, were each dissolved in physiological saline and dosed intraperitoneally Ž50 grg. once per day, during 5 days. After 48 h, mice were injected via the tail vein with colloidal carbon ŽPelikan drawing ink A. 17 black.. The ink was diluted eight times with phosphate-buffered saline containing 1% gelatin before use, and the amount of the resulting solution used was 10 lrg body weight. Blood samples were drawn from the orbital vein at 0, 3, 6, 9, 12 and 15 min. The blood Ž25 l. was dissolved in 0.1% sodium carbonate Ž2 ml. and the absorbance at 660 nm was determined according to Biozzi et al. w23x.
3. Results and discussion Dried and powdered seeds of C. pubescens were sequentially extracted with cold chloroform, chloroform᎐methanol 80:20 and hot water. The aqueous extract was poured into two volumes of ethanol leading to the isolation of the crude polysaccharide fraction ŽCPF. in a yield of 0.98% which contained 93.5% of carbohydrate, 3.5% of protein and only traces of uronic acid Ž- 1%.. Chromatography of CPF on Sephacryl S-300 HR, equilibrated with buffer Tris᎐HCl, gave polysaccharides Cp1 and Cp2 free of protein ŽFig. 1., followed each one by Sephadex G-25 gel permeation chromatography. Carbohydrate analysis showed that Cp1 was comprised of a major amount of
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
519
Fig. 1. Elution diagram of Cp1 and Cp2 on Sephacryl S-300 HR Ž0.1 M Tris᎐HCl..
galactose Ž91.8%., together with lesser quantities of arabinose Ž6.3%. and rhamnose Ž1.9%.. Cp2 showed a similar composition: galactose Ž92.5%., arabinose Ž4.9%. and rhamnose Ž2.6%.. The absolute configurations of the sugars were determined by GC of their TMS Žy.-2-butylglycosides. D-Galactopyranose, Lrhamnopyranose and L-arabinofuranose were identified by GC-MS of the pertrimethylsilylated methylglycosides w14,15x. 20 Polysaccharides Cp1 and Cp2 showed positive specific rotations, w ␣ xD q 180⬚ Ž c 20 . w x Ž . 0.1, H2 O and ␣ D q 150⬚ c 0.1, H2 O , respectively. The average Mr s of polysaccharides Cp1 and Cp2 were estimated to be 2.0 = 106 and 3.75 = 105, respectively, from the calibration curve which was made by elution volume of standard dextrans by gel filtration on Sephacryl S-300 HR. Polysaccharides Cp1 and Cp2 were methylated by the method of Parente et al. w16x, the fully methylated products were hydrolyzed with acid, and converted into the alditol acetates. The partially methylated alditol acetates were analyzed by GC and GC-MS. Polysaccharides Cp1 and Cp2 furnished 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol, 1,4,5,6-tetra-O-acetyl3,6-di-O-methylgalactitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol, 1,4,5-triO-acetyl-2,3,6-tri-O-methylarabinitol and 1,4-di-O-acetyl-2,3,5-tri-O-methylarabinitol ŽTable 1.. The results of methylation analyses indicated that polysaccharides Cp1 and Cp2 contained mainly Ž1 ª 4. linked galactosyl residues and branching points at O-6 of Ž1 ª 4. linked galactosyl residues. Their molar ratios and structural features are shown in Table 1. 1 H-NMR spectra of polysaccharides Cp1 and Cp2 in D2 O showed signals at ␦ 4.64 Ž d, J s 7.2 Hz., 5.0 Ž s . and 5.10 Ž bs . attributed to anomeric protons of -D-Gal p, ␣-L-Ara f and ␣-L-Rha p residues, respectively. Diagnostic 1 H chemical shift value was observed at ␦ 1.30 Ž d, J s 6.0 Hz. for the methyl group of the rhamnopyranosyl unit. No acetyl signal was observed in polysaccharides Cp1 and Cp2. The essentially similar structural features of Cp1 and Cp2 were confirmed also by the solid state 13 C-NMR spectra, in which the signal due to anomeric carbon at ␦ 106.0 was assigned to Ž1 ª 4. linked -D-galactosyl residues. These
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
520
Table 1 Methylation analysis of Cp1 and Cp2 polysaccharides Methylated alditol acetate derivatives
Relative retention times a
Molar ratios b Cp1
Cp2
Structural features
2,3,5-tri-OMe Ara 2,3,4,6-tetra-OMe Gal 2,3-di-OMe Ara 2,3,6-tri-OMe Gal
1.00 1.39 1.55 1.62
0.7 1.4 2.8 92.7
0.7 0.7 2.1 94.4
Ara 1 ª Gal 1 ª ª 5 Ara 1 ª ª 4 Gal 1 ª
2,3-di-OMe Gal
1.91
1.4
2.1
ª 4 Gal 1 ª 6
a b
Relative to 1,4-di-O-acetyl-2,3,5-tri-O-methyl-L-arabinitol. Calculated from peak areas and molecular weight of derivatives.
results are in accordance with those reported in the literature for several polysaccharides w17᎐20x. Several polysaccharides have been shown to possess reticuloendothelial system potentiating activity w21,22x. In order to investigate the immunological activity of polysaccharides Cp1 and Cp2, the in vivo carbon clearance test was used w23x. As shown in Fig. 2, both polysaccharides demonstrated phagocytosis enhancement, when compared with a positive control, suggesting immunostimulatory properties. These results are in accordance with literature reports, since these substances have a highly substituted backbone with structural similarities to other bioactive polysaccharides w24x.
Acknowledgements The authors are grateful to Eduardo M.B. da Silva, Maria C.P. Lima and Elimar
Fig. 2. Effects of polysaccharides Cp1 and Cp2 on phagocytic activity. control; Student’s t-test.
U
P - 0.05,
UU
P - 0.01 vs.
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
521
T. Brand for obtaining the spectra. They wish to acknowledge Dr Sonia M.C. de Menezes and Boris M.G. Claros from SEQUOPrCENPESrPETROBRAS who performed the solid state magnetic resonance analyses. They express their thanks to CNPq, FINEP and FUJB for financial support.
References w1x w2x w3x w4x w5x w6x w7x w8x w9x w10x w11x w12x w13x w14x w15x w16x w17x w18x w19x w20x w21x w22x w23x w24x w25x w26x w27x
Fevereiro BVP. Rodriguesia 1997;29:159. Carvalho ARA. A cura pelas plantas, 3rd ed. Sao ˜ Paulo: Felco Masucci, 1972:220. Masucci O. As plantas como remedio ´ na cura das doenc¸as. Sao ˜ Paulo: Brasilivros, 1982:404. Rajagopalan N. Curr Sci 1964;33:454. Gaulier R. Rev Elevage Med Vet Pays Trop 1968;21:103. Vangala RR, Menden E. Int Z Vitaminforsch 1969;39:203. Ogbeide ON, Parvez M. J Liq Chromatogr 1992;15:2989. Markham KR, Ingham JL. Z Naturforsch 1980;335C:919. Sukumaran K, Gnanamanickan SS. Ind J Microbiol 1980;20:204. Beveridge RJ, Ford CW, Richards GN. Aust J Chem 1997;30:1583. Tostes JBF, Silva AJR, Parente JP. Phytochemistry 1997;45:1069. Tostes JBF, Silva AJR, Parente JP. Phytochemistry 1999;50:1087. Unrau AM. Can J Chem 1964;42:916. Kamerling JP, Gerwig GJ, Vliegenthart JFG, Clamp JR. Biochem J 1975;151:491. Gerwig GJ, Kamerling JP, Vliegenthart JFG. Carbohydr Res 1978;62:349. Parente JP, Cardon P, Leroy Y, Montreuil J, Fournet B, Ricart G. Carbohydr Res 1985;141:41. Albersheim P, Nevins D, English P, Kaar A. Carbohydr Res 1967;5:340. Wagner H, Jordan E. Phytochemistry 1988;27:2511. Sakurai MH, Matsumoto T, Kiyohara H, Yamada H. Planta Med 1996;62:341. Karacsonyi S, Patoprsty ´ ´ V, Kubakova´ M. Carbohydr Res 1998;307:271. Shimizu N, Asahara H, Tomoda M, Gonda R, Ohara N. Chem Pharm Bull 1991;39:2630. Gonda R, Takeda K, Shimizu N, Tomoda M. Chem Pharm Bull 1992;40:185. Biozzi G, Benacerraf B, Halpern BN. Br J Exp Pathol 1953;34:441. Benencia F, Rodriguez MC, Matulewicz MC, Coulombie ´ FC. Phytochemistry 1999;50:57. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Anal Chem 1956;28:350. Bradford MM. Anal Biochem 1976;72:248. Fisher RA, Yates F. Statistical tables for agricultural, biological and other research workers. Edinburgh: Oliver and Boyd, 1957:157. w28x Fournet B, Dhalluin JM, Leroy Y, Montreuil J, Mayer H. J Chromatogr 1978;153:91.
Immunologically active polysaccharides from Centrosema pubescens Bernadete P. da Silva, Joao ˜ B.F. Tostes, Jose´ P. ParenteU Nucleo de Pesquisas de Produtos Naturais, Uni¨ ersidade Federal do Rio de Janeiro, ´ PO Box 68045, 21944-970 Rio de Janeiro, Brazil
Received 26 February 2000; accepted 13 March 2000
Abstract Two polysaccharides with mean Mr s of 2.0 = 106 and 3.75 = 105, were isolated from powdered seeds of Centrosema pubescens by fractionation on Sephacryl S-300 HR. Chemical and spectroscopic studies indicated that they have a backbone chain composed of -Ž1 ª 4.linked D-galactopyranose residues having branches composed of ␣-Ž1 ª 5.-linked Larabinofuranose residues at position 6 of D-galactose of the backbone chain. The polysaccharides showed reticuloendothelial system-potentiating activity in a carbon clearance test. 䊚 2000 Elsevier Science B.V. All rights reserved. Keywords: Centrosema pubescens; Polysaccharides; Immunomodulatory activity
1. Introduction Centrosema pubescens Benth. ŽFabaceae. is a Brazilian climbing plant widely grown as a fodder crop w1x. It is a common folk medicine used in the treatment of dropsy w2,3x. The chemical study of this plant revealed amino acids w4᎐6x, flavonoids w7x, isoflavonoids w8,9x, cyclohexitols and galactosyl-pinitol w10x and isoflavone U
Corresponding author. Tel.: q55-21-270-2683; fax: q55-21-270-2683. E-mail address: [email protected] ŽJ.P. Parente .. 0367-326Xr00r$ - see front matter 䊚 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 0 . 0 0 2 0 7 - 0
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
517
glycosides w11,12x. Taking into account a galactan isolated from the seeds of Centrosema plumari w13x, we investigated the polysaccharides present in the seeds of C. pubescens. Herein, we report the isolation and chemical characterization of two neutral polysaccharides endowed with immunological activity.
2. Experimental 2.1. Plant material Seeds of C. pubescens were collected at Mangaratiba, Rio de Janeiro in September 1975 and identified by V.P. Barbosa. A voucher specimen ŽNo. 172177. is deposited at the Botanical Garden, Rio de Janeiro. 2.2. General Carbohydrate and protein content were analyzed by the methods of Dubois et al. w25x and Bradford w26x, respectively. The Mr s of Cp1 and Cp2 were estimated from the calibration curve of the elution volume of standard dextrans Žaverage Mr values 2 000 000, 413 000, 282 000, 148 000, 68 000, 37 500, 19 500 and 9500. on Sephacryl S-300 HR Ž5 = 85 cm.. Dialyses were carried out using tubing with a Mr cut-off 12 000. Optical rotations were measured on a Perkin-Elmer 243B polarimeter. 1 H-NMR spectra were obtained on a Varian Gemini 200 NMR spectrometer, in D2 O containing sodium 2,2-dimethyl-2-silapentane-5-sulfonate as an internal standard. Solid state 13 C-NMR spectra were recorded on a Varian VXR-300 spectrometer. GC was carried out with FID, using a glass capillary column Ž0.31 mm = 25 m. SE-30. GC-MS were recorded at 70 eV. TLC of monosaccharides was performed on silica gel coated plates ŽMerck. in n-BuOH᎐pyridine᎐H2 O 6:4:3 and sugars were detected by spraying with orcinol᎐H2 SO4 . The experimental data were tested for statistical differences using the Student’s t-test w27x. 2.3. Extraction Dried and powdered seeds of C. pubescens Ž990 g. were extracted successively with cold CHCl3 , CHCl3 ᎐MeOH 80:20 and hot water Ž4 l. under stirring for 1 h in a boiling water bath. The aqueous extract was filtered and the filtrate centrifuged. By precipitation with two volumes of EtOH Ž12 h stirring and 24 h standing at 4⬚C., a crude polysaccharide fraction ŽCPF. was obtained by centrifugation and subsequent lyophilization Žyield: 9.7 g.. 2.4. Fractionation A sample of CPF Ž100 mg. was dissolved in 0.1 M Tris᎐HCl buffer ŽpH 7.0. and applied to a column Ž5 = 85 cm. of Sephacryl S-300 HR, pre-equilibrated and developed with the same buffer. Fractions of 10 ml corresponding to the peaks Cp1
518
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
and Cp2 were pooled, dialyzed, concentrated and freeze-dried. Each fraction was dissolved in water Ž2 ml. and applied to a column Ž1.5 = 50 cm. of Sephadex G-25. The column was eluted with water, and fractions of 5 ml were collected. The eluates obtained from each fractionation were concentrated and lyophilized to yield Cp1 Ž45 mg. and Cp2 Ž25 mg.. The fractionation procedures were followed by carbohydrate content. 2.5. Molar carbohydrate composition and D,L configurations Monosaccharides were analyzed as their TMS methylglycosides obtained after methanolysis Ž0.5 M HCl in MeOH, 24 h, 80⬚C. and trimethylsilylation w14x. The configurations of the glycosides were established by capillary GC and GC-MS of their TMS Žy.-2-butylglycosides w15x. 2.6. Methylation analysis Polysaccharides Cp1 and Cp2 were methylated with DMSO-lithium methylsulfinyl carbanion-CH3 I w16x. The methyl ethers were obtained after hydrolysis Ž4 N TFA, 2 h, 100⬚C. and analyzed as partially methylated alditol acetates by GC-MS w28x. 2.7. Phagocytic acti¨ ity Male BALBrc mice, weighing 15᎐20 g, were used in groups of five. Cp1, Cp2 and a positive control, zymosan, were each dissolved in physiological saline and dosed intraperitoneally Ž50 grg. once per day, during 5 days. After 48 h, mice were injected via the tail vein with colloidal carbon ŽPelikan drawing ink A. 17 black.. The ink was diluted eight times with phosphate-buffered saline containing 1% gelatin before use, and the amount of the resulting solution used was 10 lrg body weight. Blood samples were drawn from the orbital vein at 0, 3, 6, 9, 12 and 15 min. The blood Ž25 l. was dissolved in 0.1% sodium carbonate Ž2 ml. and the absorbance at 660 nm was determined according to Biozzi et al. w23x.
3. Results and discussion Dried and powdered seeds of C. pubescens were sequentially extracted with cold chloroform, chloroform᎐methanol 80:20 and hot water. The aqueous extract was poured into two volumes of ethanol leading to the isolation of the crude polysaccharide fraction ŽCPF. in a yield of 0.98% which contained 93.5% of carbohydrate, 3.5% of protein and only traces of uronic acid Ž- 1%.. Chromatography of CPF on Sephacryl S-300 HR, equilibrated with buffer Tris᎐HCl, gave polysaccharides Cp1 and Cp2 free of protein ŽFig. 1., followed each one by Sephadex G-25 gel permeation chromatography. Carbohydrate analysis showed that Cp1 was comprised of a major amount of
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
519
Fig. 1. Elution diagram of Cp1 and Cp2 on Sephacryl S-300 HR Ž0.1 M Tris᎐HCl..
galactose Ž91.8%., together with lesser quantities of arabinose Ž6.3%. and rhamnose Ž1.9%.. Cp2 showed a similar composition: galactose Ž92.5%., arabinose Ž4.9%. and rhamnose Ž2.6%.. The absolute configurations of the sugars were determined by GC of their TMS Žy.-2-butylglycosides. D-Galactopyranose, Lrhamnopyranose and L-arabinofuranose were identified by GC-MS of the pertrimethylsilylated methylglycosides w14,15x. 20 Polysaccharides Cp1 and Cp2 showed positive specific rotations, w ␣ xD q 180⬚ Ž c 20 . w x Ž . 0.1, H2 O and ␣ D q 150⬚ c 0.1, H2 O , respectively. The average Mr s of polysaccharides Cp1 and Cp2 were estimated to be 2.0 = 106 and 3.75 = 105, respectively, from the calibration curve which was made by elution volume of standard dextrans by gel filtration on Sephacryl S-300 HR. Polysaccharides Cp1 and Cp2 were methylated by the method of Parente et al. w16x, the fully methylated products were hydrolyzed with acid, and converted into the alditol acetates. The partially methylated alditol acetates were analyzed by GC and GC-MS. Polysaccharides Cp1 and Cp2 furnished 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol, 1,4,5,6-tetra-O-acetyl3,6-di-O-methylgalactitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol, 1,4,5-triO-acetyl-2,3,6-tri-O-methylarabinitol and 1,4-di-O-acetyl-2,3,5-tri-O-methylarabinitol ŽTable 1.. The results of methylation analyses indicated that polysaccharides Cp1 and Cp2 contained mainly Ž1 ª 4. linked galactosyl residues and branching points at O-6 of Ž1 ª 4. linked galactosyl residues. Their molar ratios and structural features are shown in Table 1. 1 H-NMR spectra of polysaccharides Cp1 and Cp2 in D2 O showed signals at ␦ 4.64 Ž d, J s 7.2 Hz., 5.0 Ž s . and 5.10 Ž bs . attributed to anomeric protons of -D-Gal p, ␣-L-Ara f and ␣-L-Rha p residues, respectively. Diagnostic 1 H chemical shift value was observed at ␦ 1.30 Ž d, J s 6.0 Hz. for the methyl group of the rhamnopyranosyl unit. No acetyl signal was observed in polysaccharides Cp1 and Cp2. The essentially similar structural features of Cp1 and Cp2 were confirmed also by the solid state 13 C-NMR spectra, in which the signal due to anomeric carbon at ␦ 106.0 was assigned to Ž1 ª 4. linked -D-galactosyl residues. These
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
520
Table 1 Methylation analysis of Cp1 and Cp2 polysaccharides Methylated alditol acetate derivatives
Relative retention times a
Molar ratios b Cp1
Cp2
Structural features
2,3,5-tri-OMe Ara 2,3,4,6-tetra-OMe Gal 2,3-di-OMe Ara 2,3,6-tri-OMe Gal
1.00 1.39 1.55 1.62
0.7 1.4 2.8 92.7
0.7 0.7 2.1 94.4
Ara 1 ª Gal 1 ª ª 5 Ara 1 ª ª 4 Gal 1 ª
2,3-di-OMe Gal
1.91
1.4
2.1
ª 4 Gal 1 ª 6
a b
Relative to 1,4-di-O-acetyl-2,3,5-tri-O-methyl-L-arabinitol. Calculated from peak areas and molecular weight of derivatives.
results are in accordance with those reported in the literature for several polysaccharides w17᎐20x. Several polysaccharides have been shown to possess reticuloendothelial system potentiating activity w21,22x. In order to investigate the immunological activity of polysaccharides Cp1 and Cp2, the in vivo carbon clearance test was used w23x. As shown in Fig. 2, both polysaccharides demonstrated phagocytosis enhancement, when compared with a positive control, suggesting immunostimulatory properties. These results are in accordance with literature reports, since these substances have a highly substituted backbone with structural similarities to other bioactive polysaccharides w24x.
Acknowledgements The authors are grateful to Eduardo M.B. da Silva, Maria C.P. Lima and Elimar
Fig. 2. Effects of polysaccharides Cp1 and Cp2 on phagocytic activity. control; Student’s t-test.
U
P - 0.05,
UU
P - 0.01 vs.
B.P. da Sil¨ a et al. r Fitoterapia 71 (2000) 516᎐521
521
T. Brand for obtaining the spectra. They wish to acknowledge Dr Sonia M.C. de Menezes and Boris M.G. Claros from SEQUOPrCENPESrPETROBRAS who performed the solid state magnetic resonance analyses. They express their thanks to CNPq, FINEP and FUJB for financial support.
References w1x w2x w3x w4x w5x w6x w7x w8x w9x w10x w11x w12x w13x w14x w15x w16x w17x w18x w19x w20x w21x w22x w23x w24x w25x w26x w27x
Fevereiro BVP. Rodriguesia 1997;29:159. Carvalho ARA. A cura pelas plantas, 3rd ed. Sao ˜ Paulo: Felco Masucci, 1972:220. Masucci O. As plantas como remedio ´ na cura das doenc¸as. Sao ˜ Paulo: Brasilivros, 1982:404. Rajagopalan N. Curr Sci 1964;33:454. Gaulier R. Rev Elevage Med Vet Pays Trop 1968;21:103. Vangala RR, Menden E. Int Z Vitaminforsch 1969;39:203. Ogbeide ON, Parvez M. J Liq Chromatogr 1992;15:2989. Markham KR, Ingham JL. Z Naturforsch 1980;335C:919. Sukumaran K, Gnanamanickan SS. Ind J Microbiol 1980;20:204. Beveridge RJ, Ford CW, Richards GN. Aust J Chem 1997;30:1583. Tostes JBF, Silva AJR, Parente JP. Phytochemistry 1997;45:1069. Tostes JBF, Silva AJR, Parente JP. Phytochemistry 1999;50:1087. Unrau AM. Can J Chem 1964;42:916. Kamerling JP, Gerwig GJ, Vliegenthart JFG, Clamp JR. Biochem J 1975;151:491. Gerwig GJ, Kamerling JP, Vliegenthart JFG. Carbohydr Res 1978;62:349. Parente JP, Cardon P, Leroy Y, Montreuil J, Fournet B, Ricart G. Carbohydr Res 1985;141:41. Albersheim P, Nevins D, English P, Kaar A. Carbohydr Res 1967;5:340. Wagner H, Jordan E. Phytochemistry 1988;27:2511. Sakurai MH, Matsumoto T, Kiyohara H, Yamada H. Planta Med 1996;62:341. Karacsonyi S, Patoprsty ´ ´ V, Kubakova´ M. Carbohydr Res 1998;307:271. Shimizu N, Asahara H, Tomoda M, Gonda R, Ohara N. Chem Pharm Bull 1991;39:2630. Gonda R, Takeda K, Shimizu N, Tomoda M. Chem Pharm Bull 1992;40:185. Biozzi G, Benacerraf B, Halpern BN. Br J Exp Pathol 1953;34:441. Benencia F, Rodriguez MC, Matulewicz MC, Coulombie ´ FC. Phytochemistry 1999;50:57. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Anal Chem 1956;28:350. Bradford MM. Anal Biochem 1976;72:248. Fisher RA, Yates F. Statistical tables for agricultural, biological and other research workers. Edinburgh: Oliver and Boyd, 1957:157. w28x Fournet B, Dhalluin JM, Leroy Y, Montreuil J, Mayer H. J Chromatogr 1978;153:91.