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    Structural Features of A Polysaccharide From Centella Asiatica

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    Hany Dinh
    A polysaccharide, isolated from Centella asiatica, was a complicated arabinogalactan (AG), which contained a little a-(1-4)-linked GalpA and a(1-2)-linked Rhap residues. 1BI was the first polysaccharide isolated from C. Asiatica which had immunostimulating activity in vitro. The structure of 1BI was determined by NMR, ESI-MS and partial acid hydrolysis.

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    Structural Features of A Polysaccharide From Centella Asiatica

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    Hany Dinh
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    A polysaccharide, isolated from Centella asiatica, was a complicated arabinogalactan (AG), which contained a little a-(1-4)-linked GalpA and a(1-2)-linked Rhap residues. 1BI was the first polysaccharide isolated from C. Asiatica which had immunostimulating activity in vitro. The structure of 1BI was determined by NMR, ESI-MS and partial acid hydrolysis.

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    150220-187-03-0014-p4

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    A polysaccharide, isolated from Centella asiatica, was a complicated arabinogalactan (AG), which contained a little a-(1-4)-linked GalpA and a(1-2)-linked Rhap residues. 1BI was the first polysaccharide isolated from C. Asiatica which had immunostimulating activity in vitro. The structure of 1BI was determined by NMR, ESI-MS and partial acid hydrolysis.

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    39 views4 pages

    Structural Features of A Polysaccharide From Centella Asiatica

    Uploaded by

    Hany Dinh
    A polysaccharide, isolated from Centella asiatica, was a complicated arabinogalactan (AG), which contained a little a-(1-4)-linked GalpA and a(1-2)-linked Rhap residues. 1BI was the first polysaccharide isolated from C. Asiatica which had immunostimulating activity in vitro. The structure of 1BI was determined by NMR, ESI-MS and partial acid hydrolysis.

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    Chinese Chemical Letters Vol. 15, No. 2, pp 187 190, 2004 http://www.imm.ac.cn/journal/ccl.

    html

    187

    Structural Features of a Polysaccharide from Centella asiatica

    Xue Song WANG, Jin You DUAN, Ji Nian FANG*


    Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 Abstract: A polysaccharide, isolated from Centella asiatica, was a complicated arabinogalactan (AG), which contained a little -(14)-linked GalpA and -(12)-linked Rhap residues. Based on composition and methylation analyses, partial acid hydrolysis, periodate oxidation, NMR, ESI-MS experiments, it was shown to have a backbone of -(13)-linked D-Galp residues, with heavily branched side chains. Araf residues were linked to O-3 of (13, 6)-linked Galp residues. Keywords: Arabinogalactan, polysaccharide, Centella asiatica, structure.

    Centella asiatica has been used as remedy for sodation, stabilization and against lepra, anabrosis1 in the oriental countries. The low-molecular-weight constituents in Centella asiatica have been investigated1. However, no polysaccharides have been reported. 1BI was the first polysaccharide isolated from C. asiatica, which had immunostimulating activity in vitro. In present study, we report the structural features of 1BI. Experimental Extraction, isolation and purification Dried C. asiatica (19 kg, purchased from Shanghai Medicinal Materials Cooperation, code: 000201), previously defatted with 95% alcohol, was extracted with hot water for 4 h. The solution was extensively dialyzed against water (MWCO 5000 Da). The retentate was concentrated, precipitated with EtOH, washed by acetone and vacuum-dried at 40 C to yield a black powder (JXCA, yield 2.1 %). A portion (8.0 g) of JXCA was fractionated on DEAE-cellulose column (Cl- type, 5.0 50 cm), eluted with H2O, 0.1, 0.3, 0.5, 1.0 mol/L NaCl gradually. From the eluate of 0.1 mol/L NaCl, two fractions, named S1A and S1B (5.1 %, from JXCA), were obtained in turn. S1B was further isolated on DEAE-cellulose column, eluted with 0-0.3 mol/L gradient NaCl to give two fractions (1B-1 and 1B-2). Fraction 1B-1 was purified on a Sephacryl S-300 column ( 2.6 90 cm) to give 1BI, which was homogeneous in HPGPC (Waters HPLC instrument system). The HPLC columns (Ultrahydrogel 2000 and 500 columns) were pre-calibrated using standard T-dextran (T-4T-700) and eluted with 0.003 mol/L NaOAc (0.5 mL/min) at 300.1C.

    E-mail: [email protected]

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    Xue Song WANG et al.

    Sugar composition and methylation analysis Sugars were analyzed by GC after conversion of the hydrolysate into alditol acetates as described2 previously. Methylation was performed according to the Needs method4, and the product was analyzed by GC-MS (Shimadzu QP-5050A, db-1 column), as described2. Reduction and partial acid hydrolysis Reduction was carried out with 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide metho-p- toluenesulphonate (CMC) and NaBH4 as described by Taylor and Conrad3. 1BI (70 mg) was dissolved in 0.05 mol/L TFA (20 mL) and kept at 80C for 30 min. The resulting product was dialyzed and lyophilized to give a degraded polymer (1BI-P1, 62 mg). The dialysate (1BI-O1) was fractionated on a Sephadex G-10 column ( 1.6 90 cm). 1BI-P1 (60 mg) was further hydrolyzed at 100 C in 0.05 mol/L TFA (20 mL) for 20 min. 41.5 mg 1BI-P2 was obtained. 1BI-O2 was the dialysate. Periodate oxidation and Smith degradation 1BI-P2 was dissolved in 0.02 mol/L NaIO4 and kept at 5C in the dark for 7 days. Consumption of NaIO4 was measured by spectrophotometric method5. The resulting mixture was neutralized, reduced, hydrolyzed with 0.2 mol/L TFA at 40 C for 24 h and dialyzed to give a degraded polymer (1BI-P2S), as described2 previously.
    Table 1
    1BI 1.0 0.3 2.1

    Composition analysis of 1BI and its derivativesb (molar ratio)


    1BI-Ra 1.0 0.3 2.4 1BI-P1 1.0 0.4 2.9 1BI-P1Ra 1.0 0.4 3.3 1BI-P2 1.0 7.3 1BI-P2Ra 1.0 8.2

    Ara Rha Gal

    a presence of carboxyl-reduced derivative, brecorded on a Shimadzu-GC-14B apparatus with a 5 % OV225/AW-DMCS-Chromosorb W column ( 3 mm 2.5 m).

    Results and Discussion 1BI was homogeneous in HPGPC [MW=1.9105, [] 20 +18.6 (c 0.296, H2O)]. No D absorbance at 280 nm and a negative response to the Lowry experiment indicated 1BI contained no protein. Sugar composition analysis showed 1BI was composed of Ara, Rha, Gal and GalA in a molar ratio of 1.0:0.3:2.1:0.3 (Table 1). The m-hydroxylbiphenyl method6 showed 1BI contained 9.2 % galacturonic acid. In the 13C NMR spectrum, the signals were listed in Table 3, referring to the previous reports7-8. The residues molar ratio of 1BI and 1BI-R in Table 1 suggested 1BI was an AG that contained Rhap & GalpA residues. With acid hydrolysis of 1BI, 32 % Araf residues were removed and 96 % Galp residues were kept in 1BI-P1 (MW=1.8105). 1BI-O1 contained 80 % (molar %) Ara. 1BI-P2 (MW=1.64105) was obtained with the further acid hydrolysis of 1BI-P1. In the hydrolysis, Araf residues were removed completely, while 84 % Galp residues were kept. 1BI-O2 contained 68 % Ara and 30 % Gal. All the results suggested 1BI, 1BI-P1and 1BI-P2 had the same backbone consisting of Galp residues. Methylation analysis (Table 2)

    Structural Features of a Polysaccharide from Centella asiatica

    189

    showed the ratios of (13, 6)-linked Galp residues decreased and (16)-linked Galp residues increased after hydrolysis, indicating arabinosyl chains were linked to O-3 of (13, 6)-linked Galp residues in 1BI.
    Table 2 Methylation analyses of the derivatives of original and degraded polysaccharides
    Glycosyl residue Araf Deduced glycosyl linkage t 1,3 1,5 1,3,5 t 1,2 1,2,4 t 1,3 1,6 1,3,6 1,4 Molar ratio 1BI-R 3.0 0.6 1.6 2.3 0.6 0.8 1.0 4.3 5.0 2.7 3.7 2.5

    Rhap

    Galp

    1BI 3.0 0.6 1.6 2.3 0.5 0.9 1.0 4.2 5.0 2.7 3.7

    1BI-P2R 0.2 0.9 1.0 4.1 4.9 3.3 3.1 2.0

    The disaccharide and trisaccharide fractions were obtained from 1BI-O1. The disaccharide fraction only contained Ara. The trisaccharide fraction was identified to mainly contain -arabinotriose and -Araf-(15)--Araf-(13)--Galp with ESI-MS and GC-MS experiments. 1BI contained a large amount of terminal residues and (13, 6)-linked Galp residues (Table 2), indicating 1BI had a heavily branched side chains. 1BI-P2S still had (13, 6)-linked Galp residues in methylation analysis (not be listed), indicating the following sequence existed in the branched chain of 1BI.

    Table 3

    13

    C NMR spectral data of acid polysaccharide 1BI in D2O (CH3OH in 49.5 ppm)*
    C-1 109.8 107.9 107.9 107.9 101.3 101.3 101.3 103.2 104.8 104.0 104.5 99.1 C-2 80.8 80.8 80.8 80.8 70.7 77.8 77.8 71.3 71.3 71.3 71.3 70.7 C-3 77.1 82.6 82.6 77.1 70.7 70.7 70.7 73.3 81.8 81.8 73.8 70.6 C-4 84.4 84.4 84.4 84.4 72.3 72.3 77.3 68.9 68.9 69.1 69.1 79.8 C-5 61.5 61.5 69.9 69.9 68.4 68.4 68.4 74.8 75.2 75.6 75.6 75.6 C-6 17.1 17.1 17.3 61.5 61.5 69.9 69.9 176.1

    t--L-Araf -(13)-L-Araf -(13,5)-L-Araf -(15)-L-Araf t--L- Rhap -(12)-L-Rhap -(12,4)-L-Rhap t--D-Galp -(13)-D-Galp -(13,6)-D-Galp -(16)-D-Galp -(14)-D-GalpA

    *Assignment was made up by 2D 1H-1H COSY, HMQC and HMBC experiments with a Brker AM-400 NMR spectrometer

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    Xue Song WANG et al.

    1BI-P2S showed three main peaks: MW=4.6104 (54 %, area %), 3.0104 (27 %) and 2.1104 (15 %) in HPGPC, which confirmed 1BI had heavily branched side chains. The 13 C NMR spectrum of 1BI-P2 was simpler than the spectra of 1BI and 1BI-P1: the signals of Araf residues were lacking and the signals of (16)-Galp residues increased. Those results confirmed Araf was linked to O-3 of Galp residues. In the 1H NMR spectrum of 1BI, the anomeric signals of -L-Araf and -D-Galp occurred at 5.2 and 4.5 ppm, respectively. In conclusion, 1BI had a backbone consisting of (13)-Galp residues and (13, 6)-Galp residues and contained a small amount of (14)-GalpA and (12)-Rhap residues. Its side chains contained arabinosyl chains which were linked to O-3 of (13, 6)-Galp residues, and galactosyl chains. The structure of 1BI was similar to that of the AG reported9, but the difference was the ratio of the side chains. 1BI and 1BI-P2 had immuno-activity in vitro. References and Notes
    1. 2. 3. 4. 5. 6. 7. 8. 9. Jiangsu New Medical College. Dictionary of Chinese Materia Medica, Shanghai Science and Technology Publishers, 1995, p. 1873. Q. Dong, S. W. Ding, X. Yang, J. N. Fang, Phytochemistry, 1999, 50, 81. A. B. Taylor, H. E. Conrad, Biochemistry, 1972, 11, 1385. P. W. Needs, R. R. Selvendran, Carbohydrate Research, 1993, 245, 1. J. S. Dixon, D. Lipkin, Analysis Chemistry, 1954, 26, 1092. N. Blumenkrantz, G. Asboe-Hansen, Analytical Biochemistry, 1973, 54, 484. W. W. Cui, M. N. A. Eskin, C. G. Biliaderis, K. Marat., Carbohydrate Research, 1996, 292, 173. X. F. Bao, Z. Wang, J. N. Fang, X. Y. Li, Planta Medica, 2002, 68, 237. P. Odonamzig, A. Ebringerova, E. Machova, J. Alfordi, Carbohydrate Research, 1994, 252, 317

    Received 3 January, 2003

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