Resource And Quality Evaluation Of Genus Hippophae On Qinghai-Tibet Plateau

Sea buckthorn is a deciduous shrub or arbor of Hippophae Linn (Fam.Elaeagnaceae), comprising 7 species and 11 subspecies, distributing widelyin temperate and cold temperate zone of Eurasia. It is a high-valued taxonconsidering the ecological and medicinal benefits. While recorded in ChinesePharmacopoeia 2005â… as dried mature fruits of Hippophae rhamnoidesLinn., sea buckthorn is also a traditional drug in Tibetan and Mongolianmedicines, described in The Four Medical Tantras as early as eighth centuryA.D.. Recently, sea buckthorn attracts increasing attention from all over theworld, and its distinguished therapeutic and nutrient effect against coronaryheart disease and gastric ulcer has been practically proved.In this paper, raw resource of Hippophae on Qinghai-Tibet Plateau andchemical constituents in the berries of H. rhamnoides subsp, sinensis areresearched, with 25 compounds isolated, including 3 new compounds. HPLCfingerprint chromatograph of flavonoids in sea buckthorn is established, and13 major peaks are identified. According to basic principles ofchemotaxonomy, evolvement and taxonomy of Hippophae plants arediscussed by chromatographic fingerprint technology combining withtraditional morphological taxonomy for the first time. The contents of flavonoids in berries of 10 species sea buckthorn were compared. Finally,different products of a preparation made from total flavonoids of Hippophae,Xingdakang Tablet, are compared by qualitative analysis and contentdetermination with HPLC fingerprint chromatograph.The result of raw resource research indicates that there are 7 species and 5subspecies Hippophae distributing in the wide area from HengduanMountains to Qinghai-Tibet Plateau, which contains most abundant rawresource of Hippophae in China, and is considered as the taxa differentiationand origin center of Hippophae. Ten species in this area, H. salicifolia D.Don, H. rhamnoides subsp, sinensis Rousi, H. rhamnoides subsp.yunnanensis Rousi, H. rhamnoides subsp, wolongensis Lian, K. Sun et X. L.Chen, H. litangensis Lian et X. L. Chen ex Swenson et Bartish, H.goniocarpa Lian et al.Swenson et Bartish, H. gyantsensis (Rousi) Lian, H.neurocarpa subsp, stellatopilosa Lian et al.ex Swenson et Bartish, H.neurocarpa subsp. Neurocarpa and H. tibetana Schlecht. are reported,including producing areas, biomass, living environments, external structuresand fruit characters.H. rhamnoides subsp, sinensis Rousi is a special species exclusivelyproduced in China, and also is the main resource of sea buckthorn materialsin our country. To clarify the activity basis and to supply chemical referencestandard for quality evaluation of sea buckthorn crude drug and preparations,chemical constituents in the berries of H. rhamnoides subsp, sinensis, whichwere collected in Xiaojin County, Aba Prefecture, Sichuan Province, werestudied. With multiplicate column chromatographic technology, 25compounds were isolated. Among them, The structures of 22 compoundswere identified by spectral analysis methods such as ¹H-NMR,¹³C-NMR,DEPT,ESI-MS and IR. There are 3 flavonols: isorhamnetin (1), quercetin (7)and kaempferol (13); 15 flavonol glycosides: isorhamnetin-3-O-β-D-glucoside (2), isorhamnetin-3-O-β-D-rutinoside (3), quercetin-3-O-β-D- glucoside-7-O-α-L-rhamnoside (4), isorhamnetin-3-O-β-D-sophoroside-7-O-α-L-rhamnoside (10), isorhamnetin-3-O-β-D-(6-O-trans-sinnapoyl)sophoroside-7-O-α-L-rhamnoside (14), isorhamnetin-3-O-β-D-rutinoside-7-O-α-L-rhamnoside (17), quercetin-3-O-β-D-glucoside (5), quercetin-3-O-β-D-rutinoside (18), quercetin-3-O-β-D-glucoside-7-O-α-L-rhamnoside(6),quercetin-3-O-β-D-sophoroside-7-O-α-L-rhamnoside (9), quercetin-3-O-β-D-(6-O-trans-sinnapoyl)sophoroside-7-O-α-L-rhamnoside (16),kaempferol-3-O-β-D-sophoroside-7-O-α-L-rhamnoside (11),kaempferol-3-O-β-D-(6-O-trans-sinnapoyl)sophoroside-7-O-α-L-rhamnoside(15), kaempferol-7-O-α-L-rhamnoside (12), syringetin-3-O-β-D-rutinoside(8); one sterol:β-sitosterol-3-O-β-D-glucoside (20); one trierpenoid:oleanolic acid (19); 2 phenyl compounds: 1-(4-hydroxy-phenyl)-propan-l-one (21), benzoic acid (22). Of these compounds, 14, 15 and 16are new; 6, 8, 12 and 17 are isolated from Elaeagnaceae plants for the firsttime; 2, 5, 10 and 11 are isolated from berries of H. rhamnoides subsp.sinensis for the first time.Selected H. rhamnoides subsp, sinensis berries of representativeproducing area, living environments, external structures and fruit characterswere determined with RP-HPLC, flavonoids as evaluating marker.Chromatographic data were analyzed by Similarity Evaluation System forChromatographic Fingerprint of Traditional Chinese Medicine andfingerprint of sea buckthorn with satisfying resolution and repeatability wasestablished. 13 chromatographic peaks were identified asquercetin-3-O-β-D-sophoroside-7-O-α-L-rhamnoside, kaempferol-3-O-β-D-sophoroside-7-O-α-L-rhamnoside, isorhamnetin-3-O-β-D-sophoroside-7-O-α-L-rhamnoside, isorhamnetin-3-O-β-D-glucoside-7-O-α-L-rhamnoside,quercetin-3-O-β-D-rutinoside, quercetin-3-O-β-D-glucoside, isorhamnetin-3-O-β-D-rutinoside, syringetin-3-O-β-D-rutinoside, isorhanmetin-3-O-β-D-glucoside, quercetin, kaempferol-7-O-α-L-rhamnoside, kaempferol and isorhamnetin respectively, by compared with reference standards.Flavonoids in berries of 10 species sea buckthorn were compared byHPLC and TLC fingerprint. The result shows that the similarities of samplesfrom same species are relative high, which reflects that same geneticbackground of sea buckthorn from the same species determines theconsistency of their flavonoids in category and content, while the similaritiesof samples from different species are relative low. In the term of evolvement,flavonoids contents in the berries show the trend of decrease with theincrease of evolutional level. Usually, flavonoids of medium and highpolarity are dominant in the berries of primordial species, while flavonoids oflow polarity are dominant in the advanced ones. The changing ofcompounds in different species contributes evidence in study ofdifferentiation and evolvement of Hippophae plants. Combining with basicprinciples of chemotaxonomy, the application of chromatographic technologyin chemotaxonomy of sea buckthorn were preliminarily studied.The contents of total flavonoids in sea buckthorn berries of variousorigins, living environments and collecting times were studied by HPLC. Theresult indicates that total flavonoids contents in three subspecies of H.rhamnoides L., which are H. rhamnoides subsp, sinensis, H. rhamnoindessubsp, yunnanensis and H. rhamnoides subsp, wolongensis, are 5.70±1.62mg/g, 7.00±1.60 mg/g, 5.84±1.05 mg/g respectively. Furthermore, the totalflavonoids content in H. rhamnoides L. is obviously higher than that of otherspecies listed below in order: H. goniocarpa 3.07±0.92 mg/g, H. litangensis2.97±1.18 mg/g, H. neurocarpa 2.43±1.03 mg/g, H. gyantsensis 1.98 mg/g,H. tibetana 1.93±0.84 mg/g, H. salicifolia 1.86±0.32 mg/g and H.neurocarpa subsp, stellatopilosa 1.77±0.42 mg/g.The major component of Xindakang Tablet, a product of Chinesetraditional medicine under protection, is total flavonoids of Hippophae. Torecognize the quality of Xindakang Tablet, the contents of quercetin, kaempferol and isorhamnetin in Xindakang Tablet were determined byRP-HPLC and the content differences of products from variousmanufacturers were compared. The result shows that total flavonoids in theproducts from these manufacturers are significantly different with each other.The highest content is 6.03mg per tablet, which is 120.6% of stated amount,while the lowest is 3.38mg per tablet, which is only 67.6% of stated amount.As for the relative ratio of three major flavonols in Xindakang Tablet, theproducts from these manufacturers also have enormous difference. Moreover,unlike the fact that flavonoids in Hippophae berries mostly occur in flavonols,the total flavonoids of Hippophae determined in Xindakang Tablet are allcomposed of quercetin, kaempferol and isorhamnetin, and flavonols couldbarely be detected.