The HPLC Fingerprint Of Ophioglossum And Chemical Constituents From Ophioglossum Thermale

Ophioglossum is an old fern which mainly includes Ophioglossum pedunculosum Desv, O. vulgatum Linn. and O. thermale Kom and usually called“Yizhijian”. The plants have many medicinal values which enable to improve blood circulation, reduce fever and have effect on snakebite. The flavonoid constituents of O. thermale were isolated and the HPLC fingerprint for three different producing areas was established by the joint utilization of various chromatography and spectroscopic methods. The oil of three different plants was analyzed by GC-MS.The HPLC fingerprint analysis method of O.pedunculosum, O.vulgatum and O.thermal was established using four reference substances including ophioglonol, 3-O-methylquercetin, ophioglonol4′-O-β-D-glucopyranoside and 3-O-methylquercetin 7-O-β-D-glucopyranosyl-4′-O-β-D-glucopyranoside.Fifteen batches of ophioglossum were classified into three groups with 14 common peaks in the fingerprint.The genetic relationship of O. vulgatum and O. thermal was similar while far away from O. pedunculosum.Four reference substances can be served as chemotaxonomy tag for distinguishing the three species.Seven flavonoids have been isolated from the O. thermale using a variety of chromatographic methods such as silica gel, Sephadex LH-20, ODS and p HPLC as well as the spectroscopic methods including 1H NMR, 13 C NMR, HSQC, HMBC and ROESY. They were 3-O-methylquercetin(1), ophioglonol(2),3-O-methylquercetin7-O-β-D-glucopyranoside(3), 3-O-methylquercetin 7-O-β-D-glucopyranosyl-4′-O-β-D-glucopyranoside(4), kaempferol 3-O-(6-O-caffeoyl)-β-D-glucopyranosyl(1�'2)-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranoside(5), quercetin 3-O-(6-O-caffeoyl)-β-D-glucopyranosyl(1�'2)-α-7-O-L-rhamno pyranoside(6) and 3-O-methylquercetin 4′-O-β-D-glucopyranoside(7). Compound 2 was homflavonoid,compounds 2, 3, 4 and 7 were obtained from this plant for the first time, compounds 5 and 6 were obtained from Ophioglossaceae for the first time.The oil in hexane extract of O. thermal, O. vulgatum and O. pedunculosum were detected by gas chromatography mass(GC-MS), Twenty-eight constituents mainly includes methyl oleate, methyl linoleate,n-Octadecanoic acid methyl ester and methyl hexadecanoate were found from Ophioglossum. Twenty-one kinds of chemical constituents were identified from O. thermal, The overal contents of oil from O. thermal was 2.54%, The main constituents were oleate and linoleate. Thirteen kinds of chemical constituents were identified from O. vulgatum, The overall content of oil from O. vulgatum was 5.60%, The main constituents were methyl oleate and n-Octadecanoic acid methyl ester and 4,7-nonadecadienoic acid.Fourteen kinds of chemical constituents were identified from the hexane extract of O. pedunculosum, The overall content of methylated oil from O. pedunculosum was 5.10%, The main constituents were methyl hexadecanoate, linolenic acid methyl ester and phytol. The content of oil were highest in O. vulgatum but lowest in O. thermal.