Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000–K2HPO4–KH2PO4–H2O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy (1H, 13C). These results showed AAPS-2 is a polysaccharide with a backbone of (1→3)-linked-β-D-glucopyranosyl and (1→3,6)-linked-β-D-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1→)-β-D-glucopyranosyl at the O-6 position of (1→3,6)-linked-β-D-glucopyranosyl of the main chain.

polysaccharide, structure elucidation, separation, Auricularia polytricha, high-speed countercurrent chromatography

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O[C@@H]2[C@@H](O)[C@H](O[C@@H]3[C@@H](O)[C@H]([*])O[C@H](CO)[C@H]3O)O[C@H](CO[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@H]2O)[C@@H]1O

648.564 g/mol (C₂₄H₄₀R₂O₂₀, R = next and previous repeats, not counted in mol weight)

The new isoflavone glucoside vavain 3'-O-β-D-glucoside (1) and its aglycon, vavain (2), were isolated from the bark of Ceiba pentandra, together with the known flavan-3-ol, (+)-catechin. These novel structures were elucidated by one- and two-dimensional NMR experiments and by MS, IR, and UV spectroscopy as 5-hydroxy-7,4',5'-trimethoxyisoflavone 3'-O-β-D-glucoside (1) and 5,3'-dihydroxy-7,4',5'-trimethoxyisoflavone (2), respectively. The compounds were isolated following bioactivity-directed fractionation, using a cyclooxygenase-1-catalyzed prostaglandin biosynthesis assay in vitro, in which compounds 1 and 2 and (+)-catechin exhibited IC50 values of 381, 97, and 80 μM, respectively (standard: indomethacin, IC50 1.1 μM). When further tested for their inhibitory effects on cyclooxygenase-2-catalyzed prostaglandin biosynthesis, 1 and 2 were found to be inactive (IC50 > 1200 and > 900 μM, respectively).

Ceiba pentandra, catechin, cyclooxygenase 1, cyclooxygenase 2, flavanol derivative, indometacin, isoflavone derivative, plant extract

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12	C13	C14	C15	C16	C17	C18
3'	bDGlcp	103.15	75.22	78.19	71.98	78.31	63.31
	Subst	155.88	124.45	182.16	164.06	99.67	167.47	93.89	159.52	107.50	127.91	113.10	152.02	141.19	154.94	110.21	57.58	61.89	57.27


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9	H10	H11	H12	H13	H14	H15	H16	H17	H18
3'	bDGlcp	4.94	3.36-3.55	3.36-3.55	3.36-3.55	3.36-3.55	3.66-3.81
	Subst	8.12	-	-	-	6.40	-	6.54	-	-	-	7.07	-	-	-	6.9	3.90	3.86	3.88


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9	C10/H10	C11/H11	C12/H12	C13/H13	C14/H14	C15/H15	C16/H16	C17/H17	C18/H18
3'	bDGlcp	103.15/4.94	75.22/3.36-3.55	78.19/3.36-3.55	71.98/3.36-3.55	78.31/3.36-3.55	63.31/3.66-3.81
	Subst	155.88/8.12				99.67/6.40		93.89/6.54				113.10/7.07				110.21/6.9	57.58/3.90	61.89/3.86	57.27/3.88

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

COc1cc(O)c2c(=O)c(-c3cc(OC)c(OC)c(O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)c3)coc2c1

506.46 g/mol (C₂₄H₂₆O₁₂)

High-speed countercurrent chromatography (HSCCC) was applied to the separation of polyphenols from tea leaves (Camellia sinensis L.). The capability of HSCCC to isolate pure tea polyphenols from complex mixtures on a preparative scale was demonstrated for catechins, flavonol glycosides, proanthocyanidins, and strictinin from green and black tea. The purity and identity of isolated compounds was confirmed by (1)H NMR and HPLC-ESI-MS/MS. Gram quantities of polyphenols from tea can be isolated with the procedure described.

high-speed countercurrent chromatography, flavonol glycosides, Camellia sinensis, polyphenols, catechins, green tea, black tea, HPLC-ESI-MS, proanthocyanidins, strictinin

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C[C@@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(-c4ccc(O)cc4)oc4cc(O)cc(O)c4c3=O)[C@H](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@H](O)[C@H]1O

594.522 g/mol (C₂₇H₃₀O₁₅)

High-speed countercurrent chromatography (HSCCC) was applied to the separation of polyphenols from tea leaves (Camellia sinensis L.). The capability of HSCCC to isolate pure tea polyphenols from complex mixtures on a preparative scale was demonstrated for catechins, flavonol glycosides, proanthocyanidins, and strictinin from green and black tea. The purity and identity of isolated compounds was confirmed by (1)H NMR and HPLC-ESI-MS/MS. Gram quantities of polyphenols from tea can be isolated with the procedure described.

high-speed countercurrent chromatography, flavonol glycosides, Camellia sinensis, polyphenols, catechins, green tea, black tea, HPLC-ESI-MS, proanthocyanidins, strictinin

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C[C@@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(-c4ccc(O)c(O)c4)oc4cc(O)cc(O)c4c3=O)[C@H](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@H](O)[C@H]1O

610.521 g/mol (C₂₇H₃₀O₁₆)