摘要
藏药“松蒂”是特色藏药“蒂达”(藏茵陈)总名称下的次级分类药物之一为藏医治疗肝胆类(赤巴病)、消化类(赤巴肠病)疾病的常用药物。
前期研究基础(“松蒂”品种、资源及使用现状信息整理总结)
本课题组于2007~2011年期间对“蒂达”类藏药进行了较为全面的资源植物和使用现况实地调研,调查区域包括藏医药主要使用地区的西藏、青海、甘肃、四川、云南等省区。调查结果明确了:“(?)(松蒂)”是“(?)(蒂达)’类藏药总名称下的多个次级分类药物之一。实地踏查中共采集“松蒂”类资源相关标本共47号141份,经分类学鉴定,来源于虎耳草科虎耳草属22种(含变种)植物。收集了藏医医疗机构、门诊、制药企业及市场销售的“松蒂”类商品药材样品8批。
因受藏区药材多来自于“就地自采”的传统模式影响,“松蒂”类藏药存在来源复杂、品种混乱的问题。经基原鉴定,并结合标本分布情况:西藏、青海、四川等藏区使用的“松蒂”主要来源于篦齿虎耳草Saxifraga umbellulata var. pectinata,该品种被《藏药标准》收载,且符合经典藏药文献中对“松蒂”正品的描述,也是目前在藏医院、藏药厂及市场销售的“蒂达(藏茵陈)”四大主流品种之一。但同属的爪瓣虎耳草S. unguiculata(青海玉树州)、青藏虎耳草S.przewalskii和唐古特虎耳草S. tangutica(青海黄南州)等在不同地区也作“松蒂”使用或多种混用。
“松蒂”在整个“蒂达”类药材中疗效突出,偏性明显,在临床上主要应用于肝胆类疾病(“赤巴病”)和消化系统类疾病(“赤巴肠病”),在藏医中是区别于其他“蒂达”使用的,具有不可替代性,在西藏、青海等藏民族聚居地使用量较大,参与组成至少42种经典藏药处方,制成十几种藏成药在藏区上市或医院内部销售,具有可观的发展前景。
“松蒂”正品篦齿虎耳草主要分布在四川西北部、西藏东南部雅鲁藏布江流域海拔3000-4100m的岩壁石隙或山坡裸地上,呈零星分布,植株数量较多,但单株生物量小,加之药用在花期采集全株,严重影响种子的产生,因而资源解体危险度高,被《濒危藏药物种名录》列为一级濒危藏药。
可见藏药“松蒂”类药材的名称、基原、分布等在不同藏区存在显著差异,临床使用量较大,但相关研究较少,质量标准匮乏,无法保证用药准确规范。故本研究采用成分分离纯化、波谱解析、形态组织学比较、仪器分析及聚类分析等方法,重点针对“松蒂”主流品种蓖齿虎耳草S.umbellulata var.petinata开展化学成分分离鉴定、药材鉴别、常规检查、含量分析、指纹图谱比对等研究,按照《中国药典》药材标准研究要求制定了藏药“松蒂”(蓖齿虎耳草)的质量标准草案,并从多方面比较了“松蒂”4种基原植物的差别,取得如下结果:
1蓖齿虎耳草化学成分分离鉴定
利用硅胶色谱、反相色谱等多种分离纯化技术,通过UV、1H-NMR13C-NMR、GC-MS、HPLC-MS等理化方法及光谱分析鉴定各化合物的结构,从蓖齿虎耳草中共分离得到18个化合物,目前鉴定了其中9个化合物,分别为:己降葫芦苦素F(Ⅰ、),虎耳草皂苷A(Ⅱ),虎耳草皂苷B(Ⅲ),虎耳草皂苷C(Ⅳ),山柰酚-3-O-(6”-乙酰基)-D-半乳糖苷(V),2’,4’,5,7-四羟基-5’-甲氧基黄酮(Ⅵ),槲皮素(Ⅶ),芦丁(Ⅷ),绿原酸(Ⅸ)。以上9种成分均为首次从该植物中分离得到,其中虎耳草皂苷A、虎耳草皂苷B、虎耳草皂苷C为新化合物。明确了黄酮和三萜皂苷为蓖齿虎耳草中两类主要成分。
2“松蒂”(蓖齿虎耳草)的质量标准研究
在成分研究基础上,以10批蓖齿虎耳草为材料,首次建立了较为完善的蓖齿虎耳草质量标准。确定了药材的来源、性状鉴别、显微鉴别、薄层鉴别;[检查]项下规定了水分、总灰分、酸不溶性灰分的限量标准;规定了[浸出物]含量;[含量测定]项下采用紫外分光光度法测定了黄酮类成分和皂苷类成分的总量,采用HPLC方法测定多成分指标,综合评价的药材质量,分别规定:按干燥品计算,采用UV法,含总黄酮以芦丁(C27H30016)计,不得少于7.0%;含总皂苷以人参皂苷Rb,(C54H92O23)计算的总量不少于1.8%;采用HPLC法,含绿原酸(C16H1506)不得低于0.02%,含芦丁(C27H30016)和槲皮素(C15H10O7)的总量不得低于0.09%。
在上述基础上,起草了《蓖齿虎耳草质量标准(草案)》及《蓖齿虎耳草质量标准起草说明》,并向藏药质量标准协调工作委员会推荐作为《藏药标准(新版)》收载项目。
3“松蒂”(蓖齿虎耳草)的HPLC指纹图谱
运用高效液相色谱法检测了10批不同来源和不同批次的蓖齿虎耳草药材样品,根据已分离成分标定15个共有色谱峰,指认了其中的9个色谱峰的成分名称。采用“中药色谱指纹图谱相似度评价系统2004A版”软件进行模式分析、共有峰确定及相似度计算,并结合SPSS19.0统计分析软件对所得的高效液相指纹图谱数据进行聚类分析。分析结果表明:各批次样品成分类型基本一致,黄酮和皂苷为蓖齿虎耳草的主要化学成分。但指纹图谱特征峰数量和峰面积受采集地、样品来源方式影响较大,来源于同一地区品种准确性和纯净度较高的样品相似度较高。
4四种“松蒂”虎耳草类基原植物区别研究
从性状鉴别、显微鉴别、薄层鉴别、HPLC指纹图谱等多方面比较了蓖齿虎耳草和其他三种“松蒂”资源植物爪瓣虎耳草、‘唐古特虎耳草、青藏虎耳草的差别。可根据基生叶着生形态、被毛情况、毛茸类型、花序类型、小花数目、茎各组织部位比例、花粉粒形态、三萜皂苷类成分的薄层鉴别以及HPLC指纹图谱等特征的区别有效地鉴别“松蒂”的主流品种篦齿虎耳草及其近似种。
"Songdi" is one of the herbs in the sub-classification of "Dida"(the Tibetan Yinchen) well-known as a traditional Tibetan medicine. It's especially effective in treating diseases related to liver, gallbladder and digestive system.
The basis of preliminary studies (Investigation on the species, sources and uses of "Songdi.")
From2007to2011, a series of comprehensive field research were carried out to investigate the sources and uses of Dida. The surveyed areas, where the Tibetan medicine is widely used, are Tibet, Qinghai, Gansu, Sichuan, Yunnan, etc. The results clearly show Songdi is one of the herbs in the sub-classification of Dida. In the field research,47numbers (171pieces) of specimens related to Songdi were collected, of which22plants (including their varieties) were identified as coming from Saxifragaceae. Meanwhile,8batches of Songdi herbal samples were sourced from the Tibetan medical institutions, clinics, pharmaceutical companies and herbal markets.
As is the traditional practice of Tibetans, the songdi herbs were collected in the neighborhood of their homes. This had inevitably led to a confusing variety of songdi picked from diverse sources. The study of each specimen's origin, identification and source distribution showed that songdi's dominant species is S.umbellulata var. Petinata, which is widely used by Tibetan medical institutions, pharmaceutical companies and sold in herbal markets. This finding is consistent with the description of authentic Songdi recorded in ancient Tibetan materia medica. S.umbellulata var. Petinata is also one of the four dominant species of Dida (the Tibetan Yinchen). In addition, S. unguiculata Engl, S. tangutica Engl, and S. przewalskii are the three most common adulterants or substitutes of "songdi" in most parts of Qinghai and Gansu and parts of Sichuan, Tibet and Yunnan.
"Songdi" has remarkable efficacy among Dida herbs. It's especially effective in treating diseases related to liver, gallbladder and digestive system. That it appears to be irreplaceable in Tibetan medicine, distinguishes it from other Dida herbs. It's one of the most used Tibetan herbs with significant effect and higher usage in the medical health system of Tibetan settlements such as Tibet and Qinghai. It is also a constituent of at least42kinds of traditional Tibetan medicine prescriptions that are readily available in the Tibetan pharmacies or hospitals. This herb has considerable development prospects.
As the authentic origin of "Songdi", S.umbellulata var. Petinata, it is mainly distributed in northwestern Sichuan, Yarlung Zangbo River Basin in southeastern Tibet. It thrives in the rock crevices or slopes with bare ground where altitude is3000-4100m. Despite its large quantity in scattered distribution, its individual biomass is small. As the whole plant is picked at flowering stage, this seriously affects its seed production. Therefore, it's in high risk of source extinction and is listed as a most endangered Tibetan medicine.
This study addresses problems such as significant differences of Songdi's names, origins and clinical uses in different Tibetan areas, and also the absence of a common standard for Songdi which make it nearly impossible to ensure the accurate and standardized use of this drug and other attendant issues. In further research,9chemical compounds are separated and identified after the systematic extraction and separation of Songdi. The quality standards are drafted in accordance with the technical requirement of the herbs standards of "Chinese Pharmacopoeia". In conclusion, the results are as follows:
1The isolation and identification of chemical compounds in S.umbellulata var. Petinata
By using Silica gel column chromatography, reversed phase chromatography and other separation and purification technology, UV,1H-NMR and13C-NMR, GC-MS, HPLC-MS and other physical or chemical methods and spectral analysis,18compounds were separated, of which9compounds were identified. They are as follows:the saxifrage saponin A (I), hexanorcucurbitacin F (II), saxifrage saponin B (III), the saxifrage saponin C (IV), kaempferol-3-O-(6"-acetyl)-D-galactosidase (V),2',4',5,7-tetrahydroxy-5'-methoxyflavone (VI), quercetin (VII), chlorogenic acid (Ⅷ), rutin (IX). All of these components were separated from this plant for the first time, of which3are new compounds.
2Study on the quality standard of Songdi (S.umbellulata var. Petinata)
Based on the study of chemical constituents,10batches of S.umbellulata var. Petinata were tested to establish its quality standards for the first time. In formalizing the standards, Songdi's source, character identification, microscopic identification and TLC identification are specified. The limits of moisture, total ash and acid insoluble ash are regulated in the item of [Check]. The content of extract is shown in the item of [Extract], In the item of [Content determination], in order to evaluate the quality of herbs comprehensively, the total content of flavonoids and saponins are determined by UV spectrophotometer, whereas, HPLC method is set up for the determination of the multi-component indicators. It's specifically noted that for dry herbs, the total content of flavonoids in rutin (C27H30O16) term and saponins in ginsenoside (C54H92O23) term should not be less than7.0%and1.8%respectively when UV method is applied. As for using HPLC method, the content of chlorogenic acid (C16H15O6) should not be less than0.02%,the total content of rutin (C27H30O16) and quercetin (C15H10O7) should not be less than0.09%.
3The HPLC fingerprint of Songdi (S.umbellulata var. Petinata)
10batches of S.umbellulata var. Petinata samples, collected at different time and locations, were tested by HPLC method. This was followed by pattern analysis; definition of common peaks and similarity calculation using the chromatographic fingerprint similarity evaluation system (2004A edition), then combined with the result of clustering analysis using SPSS19.0statistical analysis software.9peaks were identified from the15common peaks calibrated by the compounds separated from S.umbellulata var. Petinata.
Based on the above study,"the quality standards of S.umbellulata var. Petinata (draft)" and "instructions for the quality standards drafting of S.umbellulata var. Petinata" are drafted and recommended to Chinese Pharmacopoeia Commission as a new item to be recorded in the "Drug standard of ministry of public health-standards of Tibetan medicine (new version)"
4Compare the difference of four species of Songdi
Microscopic identification, TLC identification and HPLC fingerprint were used to compare the difference between S.umbellulata var. Petinata and the other three species of Songdi (S. unguiculata Engl, S. tangutica Engl, and S. przewalskii). The above quality control methods are employed to distinguish the difference between these four herbs.
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