基于NMR/UPLC植物代谢组学技术的远志质量评价研究
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摘要
选题依据：远志是远志科植物远志或卵叶远志的干燥根,具有安神益智,祛痰,消肿之功效,是临床常用药物。目前远志资源利用存在以下问题：①远志全草包括根和茎叶,传统认为其药用部位为根,茎叶部分常在根采收后被丢弃；②远志根又由韧皮部(筒)和木质部(心)组成,传统认为木心有“服之令人闷”的副作用,使用时应去掉,以筒入药,而现代毒理研究则表明木心无毒副作用,《中国药典》2010版规定根不用去心,可直接入药；③远志生品有“戟人咽喉”的副作用,一般使用其炮制品,如蜜制品和甘草制品,其临床混用现象严重。由上可知,远志茎叶是否有利用价值,远志根初加工时是否应抽去木心以及远志炮制品的科学性和临床应用这三大问题亟待解决。因此本论文对这些问题进行探究,以期为远志质量评价、资源利用和科学使用提供指导和依据。
目前远志质量评价尚存在以下问题：①对远志不同部位(根和茎叶,筒和木心)以及不同炮制品的质量评价,多以有限成分为指标或进行简单的HPLC-UV(?)旨纹图谱分析,无法对远志代谢组进行全面系统的评价,需要引入更加全面的分析手段；②对远志茎叶和根的化学成分差异研究较少,现有研究只对茎叶中多糖和皂苷含量进行测定,并未对茎叶的化学成分进行全面分析,且远志茎叶的药理作用研究尚未见报道,这不利于茎叶的资源利用。③关于远志皂苷的含量测定,《中国药典》2005版规定其对照品为皂苷酸水解产物远志酸,而2010版对照品则为皂苷碱水解产物细叶远志皂苷。本文选题时中国药品生物制品检定所还没有细叶远志皂苷对照品,因此有必要对其进行分离制备。且目前还没有以细叶远志皂苷为标准对远志不同部位及不同炮制品中皂苷类含量进行评价的研究。
目的：首先,采用NMR和UPLC植物代谢组学技术,结合UPLC技术测定远志中细叶远志皂苷的含量测定方法,对远志不同部位(根和茎叶,筒和木心)及不同炮制品代谢组差异(本研究所涉及远志炮制品和生品均为远志筒)进行分析,并寻找差异性代谢产物。其次,对远志茎叶进行药理作用研究。以期从整体角度对远志进行质量评价,并对远志资源利用、炮制加工及临床使用提供科学依据和指导。
方法：1.建立远志NMR和UPLC植物代谢组学分析方法,并对远志不同部位及不同炮制品(筒)代谢组差异进行分析。首先对远志不同样品提取物进行H NMR和UPLC分析,对所得图谱进行代谢物指认。其次采用相应软件对图谱进行预处理得到大量数据,将数据进行归一化后,采用SIMCA-P软件对数据进行主成分分析(PCA)和最小偏二乘法分析(PLS-DA)。最后根据分析结果,分析样品代谢组差异,并寻找差异代谢产物。
2.分离制备细叶远志皂苷对照品,并采用UPLC技术对远志不同部位及不同炮制品(筒)中细叶远志皂苷含量进行测定。通过对细叶远志皂苷含量进行比较,分析皂苷类成分含量在远志不同部位及不同炮制品(筒)中的差异。
3.采用小鼠悬尾、强迫游泳的抑郁模型及氨水引咳、气管酚红排泌的止咳祛痰模型对远志茎叶提取物的抗抑郁及止咳祛痰活性进行研究。
结果：1.NMR和UPLC植物代谢组学分析表明,远志不同部位及不同炮制品(筒)代谢组明显不同。
远志不同部位PCA分析结果表明,茎叶、根、筒和木心分别聚为一类,且根、筒和木心距离较近,说明茎叶与地下部分代谢组差异很大。与根相比,茎叶中部分初级代谢产物氨基酸类缬氨酸和谷氨酸,有机酸类如琥珀酸和γ-氨基丁酸等,以及次级代谢产物远志口山酮Ⅲ含量较高,而苏氨酸、丙氨酸、天冬氨酸、蔗糖和腺嘌呤以及3,6’-二芥子酰基糖酯、Sibiricose A5、Sibiricose A6、Tenuifoliside A、 Tenuifolise I、Tenuifolise H、远志口山酮Ⅳ、Onjisaponin A和Onjisaponin K等含量较低。同时,远志筒和木心代谢组也存在一定差异,与筒相比,木心中除氨基酸类成分丙氨酸和寡糖酯类Tenuifolise I等外,多数代谢物含量都较低。
远志不同炮制品(筒)PCA分析结果表明,生品、蜜制品和甘草制品分别聚为一类,其代谢组各有不同,说明炮制会对生品代谢组产生一定影响,蜜制使生品中初级代谢产物甲酸和α-葡萄糖,次级代谢产物Tenuifolise I和Onjisaponin A含量升高,而苏氨酸、丙氨酸、果糖和蔗糖以及3,6’-二芥子酰基糖酯、Tenuifoliside A、 Tenuifolise H和远志口山酮Ⅳ含量降低；甘草制使生品中初级代谢产物亮氨酸和酒石酸,次级代谢产物Tenuifolise I、Tenuifolise H及Onjisaponin K含量上升,而苏氨酸、丙氨酸、琥珀酸、富马酸和蔗糖以及3,6’-二芥子酰基糖酯、远志口山酮Ⅳ和Tenuifolise H含量下降。同时,蜜制品和甘草制品代谢组之间也存在差异,与蜜制品相比,甘草制品中α-葡萄糖、甲酸和3,6’-二芥子酰基糖酯含量较低,说明不同炮制方法会对生品代谢组产生不同影响。
2.所分离得到的细叶远志皂苷纯度达到98%以上,可作为对照品使用。
UPLC细叶远志皂苷含量测定结果表明,远志不同部位及不同炮制品(筒)中细叶远志皂苷含量不同。远志不同部位中,茎叶的细叶远志皂苷含量远低于根；而且传统要求抽去的木心中含量远比筒低,含量差异具极显著性,远志筒中细叶远志皂苷含量比根高,含量差异具显著性；远志不同炮制品(筒)中细叶远志皂苷含量也不同,蜜远志中细叶远志皂苷含量与生远志无显著性差异,制远志含量则高于生远志,且含量差异具显著性。
3.远志茎叶药理作用研究表明,远志茎叶无抗抑郁作用,但具有一定的止咳祛痰活性,且止咳活性强于根,祛痰活性弱于根。
结论：本文成功建立了远志NMR和UPLC植物代谢组学分析方法以及远志中细叶远志皂苷UPLC含量测定方法,并对远志皂苷含量测定对照品细叶远志皂苷进行分离制备,同时研究了茎叶的抗抑郁及止咳祛痰活性。研究结果表明：
1.远志茎叶和根代谢组明显不同。与根相比,茎叶中部分初级代谢产物及远志口山酮Ⅲ含量较高,皂苷含量较低。药理研究表明茎叶具有止咳祛痰活性,且止咳活性比远志根强,祛痰活性则较弱,说明远志口山酮Ⅲ可能是远志止咳活性有效成分,而皂苷类不是。茎叶具有一定的药用价值,但其能否被利用,还有待进一步评价。
远志筒和木心代谢组明显不同。与筒相比,木心中除氨基酸类成分丙氨酸和寡糖酯类Tenuifolise等外,多数化合物,特别是皂苷类,含量都较低,说明传统认为远志根使用时应抽去木心具有一定科学依据。
2.远志不同炮制品(筒)代谢组明显不同。蜜制和甘草制对生品(筒)化学成分的改变不同,特别是皂苷类,蜜制使Onjisaponin AⅠ含量升高,但对总皂苷含量影响不大,说明远志中皂苷类成分较为复杂,单一成分并不能代表总皂苷含量变化。而甘草制则使皂苷类含量显著升高,这可能导致蜜制品和甘草制品产生不同药效,因此在临床应用中应根据不同情况进行合理用药。
Rationales:Radix Polygalae, the root of Polygala tenuifolia Willd or Polygala sibirica L., a traditional Chinese herb has long been used as a traditional Chinese medicine and widely used in the vast majority of Chinese medicine prescription for antidepressant, expectorant, antitussive and anti-inflammatory et al. At present, the studies of Polygala tenuifolia Willd mainly concentrated in①the aerial part of P. tenuifolia is discarded usually after the root harvest, resulting in waste of resources.②the xylem is required to be removed traditionally, not required now.③the processed products and so on.
Quality evaluation of P. tenuifolia still have the following deficiencies:①quality evaluation of P. tenuifolia by limited ingredients to cannot do a comprehensive analysi of the chemical composition, and HPLC-UV fingerprints only can do simple chromatographic analysis. It is very important to introduce a more comprehensive analytical tool-metabolomics;②he acid hydrolyzates of saponins cannot be used as reference substance to evaluate saponins content comprehensively. Alkaline hydrolyzates of saponins is single, Chinese Pharmacopoeia2010edition provides tenuifolin to saponin content measured by the standard, there is no report of saponin content evaluation by tenuifloin;③the aerial part of P. tenuifolia is discarded usually after the root harvest, resulting in waste of resources. The xylem is required to be removed traditionally, not required now. And the evalution of different parts of P. tenuifolia only concentrated in a limited composition;④pharmacological studies of the aerial part have not been reported.
Objective:the methods based on NMR/UPLC plant metabolomics research and content of determination of tenuifolin by UPLC have been established. Then the metabolomic profiles of different parts and different processed products of P.tenuifolia have not be evaluated, and the pharmacological efficacies of the aerial part of P. tenuifolia have been studied, providing the scientific guidance from new view for the quality assessment and resource utilization of P. tenuifolia.
Methods:1. The study based on NMR and UPLC plant metabolomics of the root, aerial part, cortex, xylem, Honey-stir-baked Radix Polygalae, Liquorice juice-stir-boiled Radix Polygalae:First, the extracts of samples were determinated by NMR spectrometer, the1H NMR spectras were processed by MestRenova software for large amounts of data, which was analysised for data principal component analysis and the smallest partial squares analysis by SIMC A-P software. Then, part of the metabolites in the NMR spectrum and different metabolites in samples were identified according to the literature and study results, combining with the t-test, the relative concentration of the different metabolites in the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia were determinate, to seek for NMR metabolomics differences among the different medicinal parts and processed products of Polygala tenuifolia Willd.
2. The study based on UPLC plant metabolomics of the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia:First, the extracts of samples were determinate by Ultra High Pressure Liquid Chromatography (UPLC) fingerprint, the peak areas in the chromatograms were integrated manually. Then the datas were analysised for data principal component analysis and the smallest partial squares analysis by SIMCA-P software. Then, part of the metabolites in the UPLC chromatogram and different metabolites in samples were identified according to the literature and study results, combining with the t-test, the relative concentration of the different metabolites in the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia were determinated, to seek for UPLC metabolomics differences among the different medicinal parts and processed products of Polygala tenuifolia Willd.
3. The UPLC content determination of tenuifolin in the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia:The standard substance for UPLC content determination, tenuifolin, was separated by us in the laboratory, the purity more than98%.
4. The antidepressant and antitussive, expectorant effects of the aerial part of Polygala tenuifolia Willd were studied by the classic Mice tail suspension test and Forced swimming test antidepressant model, the classic ammonia induced cough test and racheal phenol red excretion test antitussive, expectorant model.
Results:1. NMR plant metabolomics studies have shown that, there were significant differences among the metabolomic profiles of different parts and processed products, which mainly concentrated in the primary metabolites.①the metabolomes of different parts of Polygala tenuifolia Willd were different. Comparing with the root, the contents of valine, glutamic acid, succinic acid, GAB A, tartaric acid, maleic acid and sinapic acid were higher in the aerial part, and the contents of threonine, alanine, aspartic acid, acetic acid, sucrose, adenine and saponins compounds were lower. Compared with the phloem, the contents of alanine was higher in the xylem, the contents of threonine, aspartic acid, acetic acid, succinic acid, GAB A, tartaric acid, formic acid, choline, fructose and sucrose were lower;②the metabolomes of different processed products of Polygala tenuifolia Willd were different, too. Honey-stir-boiled made the contents of formic acid, and α-glucose increase, the contents of threonine, alanine, fructose and sucrose decreased; Liquorice juice-stir-boiled made the contents of leucine, tartaric acid increased, threonine, alanine, butanedioic acid, fumarate and sucrose decrease; Meanwhile, comparing with H P. tenuifolia, the contents of formic acid, and α-glucose were lower in L P. tenuifolia.
2. UPLC plant metabolomics studies have shown that, there were significant differences among the metabolomic profiles of differert parts and processed products, which mainly concentrated in the secondary metabolites.①the metabolomes of different parts of Polygala tenuifolia Willd, were different. Comparing with the roots, the content of Polygalaxanthone III was higher in the aerial parts, and the contents of3,6'-disinapoylsucrose ester, Sibiricose A5, Sibiricose A6, Tenuifoliside A, Tenuifolise I, Tenuifolise H, PolygalaxanthoneⅣ, Onjisaponin A and Onjisaponin K were lower. Compared with the phloem, the contents of Tenuifolise I was higher in the xylem, the contents of Tenuifoliside A, Tenuifolise I, Tenuifolise H, Onjisaponin A, Polagalasaponin X X X II were lower;②the metabolomes of different processed products of Polygala tenuifolia Willd were different, too. Honey-stir-boiled made the contents of Tenuifolise I Onjisaponin A increase, the contents of3,6'-disinapoylsucrose ester, Tenuifoliside A, Tenuifolise H, Polygalaxanthone IV decrease; Liquorice juice-stir-boiled made the contents of Tenuifolise I, Tenuifolise H, Onjisaponin K increased,3,6'-disinapoylsucrose ester, Polygalaxanthone IV, Tenuifolise H decreased; Meanwhile, comparing with H P. Tenuifolia., the contents of3,6'-disinapoylsucrose ester and Tenuifolise I were lower in L P. Tenuifolia.
3. UPLC content determinations have shown that,①the content of tenuifolin in the aerial part was higher than the root; the xylem which required to remove, the content of tenuifolin was very lower than in cortex, and the content difference was very significant;②In traditionally Preliminary processing of P. Tenuifolia., Honey-stir-boiled made the content of tenuifolin lower, Liquorice juice-stir-boiled made the content higher, and the later the content difference was very significant.
4. Pharmacological studies of the aerial part of P. Tenuifolia. have shown that, the aerial part had low antidepressant activity and strong antitussive, expectorant activities; Comparing with the roots of P. Tenuifolia. and the classic antitussive, expectorant drug carbetapentane and T. farfara, the antitussive effect of the aerial part was stronger, the expectorant effect was lower.
Conclusion:In the paper, the NMR and UPLC plant metabolomics and UPLC content determination of tenuifolin in P. tenuifolia were established, at the same time, the antidepressant and antitussive, expectorant activities of the aerial parts were evaluated. The results showed that the metabolomic profils of the root, aerial part, cortex, xylem, Honey-stir-baked Radix Polygalae, Liquorice juice-stir-boiled Radix Polygalae had significantly differences, and the aerial parts have certain antitussive and expectorant activity, and antitussive acticity is stronger than the root, expectorant activity is weaker. The active compound might be Polygalaxanthone III.
目前远志质量评价尚存在以下问题：①对远志不同部位(根和茎叶,筒和木心)以及不同炮制品的质量评价,多以有限成分为指标或进行简单的HPLC-UV(?)旨纹图谱分析,无法对远志代谢组进行全面系统的评价,需要引入更加全面的分析手段；②对远志茎叶和根的化学成分差异研究较少,现有研究只对茎叶中多糖和皂苷含量进行测定,并未对茎叶的化学成分进行全面分析,且远志茎叶的药理作用研究尚未见报道,这不利于茎叶的资源利用。③关于远志皂苷的含量测定,《中国药典》2005版规定其对照品为皂苷酸水解产物远志酸,而2010版对照品则为皂苷碱水解产物细叶远志皂苷。本文选题时中国药品生物制品检定所还没有细叶远志皂苷对照品,因此有必要对其进行分离制备。且目前还没有以细叶远志皂苷为标准对远志不同部位及不同炮制品中皂苷类含量进行评价的研究。
目的：首先,采用NMR和UPLC植物代谢组学技术,结合UPLC技术测定远志中细叶远志皂苷的含量测定方法,对远志不同部位(根和茎叶,筒和木心)及不同炮制品代谢组差异(本研究所涉及远志炮制品和生品均为远志筒)进行分析,并寻找差异性代谢产物。其次,对远志茎叶进行药理作用研究。以期从整体角度对远志进行质量评价,并对远志资源利用、炮制加工及临床使用提供科学依据和指导。
方法：1.建立远志NMR和UPLC植物代谢组学分析方法,并对远志不同部位及不同炮制品(筒)代谢组差异进行分析。首先对远志不同样品提取物进行H NMR和UPLC分析,对所得图谱进行代谢物指认。其次采用相应软件对图谱进行预处理得到大量数据,将数据进行归一化后,采用SIMCA-P软件对数据进行主成分分析(PCA)和最小偏二乘法分析(PLS-DA)。最后根据分析结果,分析样品代谢组差异,并寻找差异代谢产物。
2.分离制备细叶远志皂苷对照品,并采用UPLC技术对远志不同部位及不同炮制品(筒)中细叶远志皂苷含量进行测定。通过对细叶远志皂苷含量进行比较,分析皂苷类成分含量在远志不同部位及不同炮制品(筒)中的差异。
3.采用小鼠悬尾、强迫游泳的抑郁模型及氨水引咳、气管酚红排泌的止咳祛痰模型对远志茎叶提取物的抗抑郁及止咳祛痰活性进行研究。
结果：1.NMR和UPLC植物代谢组学分析表明,远志不同部位及不同炮制品(筒)代谢组明显不同。
远志不同部位PCA分析结果表明,茎叶、根、筒和木心分别聚为一类,且根、筒和木心距离较近,说明茎叶与地下部分代谢组差异很大。与根相比,茎叶中部分初级代谢产物氨基酸类缬氨酸和谷氨酸,有机酸类如琥珀酸和γ-氨基丁酸等,以及次级代谢产物远志口山酮Ⅲ含量较高,而苏氨酸、丙氨酸、天冬氨酸、蔗糖和腺嘌呤以及3,6’-二芥子酰基糖酯、Sibiricose A5、Sibiricose A6、Tenuifoliside A、 Tenuifolise I、Tenuifolise H、远志口山酮Ⅳ、Onjisaponin A和Onjisaponin K等含量较低。同时,远志筒和木心代谢组也存在一定差异,与筒相比,木心中除氨基酸类成分丙氨酸和寡糖酯类Tenuifolise I等外,多数代谢物含量都较低。
远志不同炮制品(筒)PCA分析结果表明,生品、蜜制品和甘草制品分别聚为一类,其代谢组各有不同,说明炮制会对生品代谢组产生一定影响,蜜制使生品中初级代谢产物甲酸和α-葡萄糖,次级代谢产物Tenuifolise I和Onjisaponin A含量升高,而苏氨酸、丙氨酸、果糖和蔗糖以及3,6’-二芥子酰基糖酯、Tenuifoliside A、 Tenuifolise H和远志口山酮Ⅳ含量降低；甘草制使生品中初级代谢产物亮氨酸和酒石酸,次级代谢产物Tenuifolise I、Tenuifolise H及Onjisaponin K含量上升,而苏氨酸、丙氨酸、琥珀酸、富马酸和蔗糖以及3,6’-二芥子酰基糖酯、远志口山酮Ⅳ和Tenuifolise H含量下降。同时,蜜制品和甘草制品代谢组之间也存在差异,与蜜制品相比,甘草制品中α-葡萄糖、甲酸和3,6’-二芥子酰基糖酯含量较低,说明不同炮制方法会对生品代谢组产生不同影响。
2.所分离得到的细叶远志皂苷纯度达到98%以上,可作为对照品使用。
UPLC细叶远志皂苷含量测定结果表明,远志不同部位及不同炮制品(筒)中细叶远志皂苷含量不同。远志不同部位中,茎叶的细叶远志皂苷含量远低于根；而且传统要求抽去的木心中含量远比筒低,含量差异具极显著性,远志筒中细叶远志皂苷含量比根高,含量差异具显著性；远志不同炮制品(筒)中细叶远志皂苷含量也不同,蜜远志中细叶远志皂苷含量与生远志无显著性差异,制远志含量则高于生远志,且含量差异具显著性。
3.远志茎叶药理作用研究表明,远志茎叶无抗抑郁作用,但具有一定的止咳祛痰活性,且止咳活性强于根,祛痰活性弱于根。
结论：本文成功建立了远志NMR和UPLC植物代谢组学分析方法以及远志中细叶远志皂苷UPLC含量测定方法,并对远志皂苷含量测定对照品细叶远志皂苷进行分离制备,同时研究了茎叶的抗抑郁及止咳祛痰活性。研究结果表明：
1.远志茎叶和根代谢组明显不同。与根相比,茎叶中部分初级代谢产物及远志口山酮Ⅲ含量较高,皂苷含量较低。药理研究表明茎叶具有止咳祛痰活性,且止咳活性比远志根强,祛痰活性则较弱,说明远志口山酮Ⅲ可能是远志止咳活性有效成分,而皂苷类不是。茎叶具有一定的药用价值,但其能否被利用,还有待进一步评价。
远志筒和木心代谢组明显不同。与筒相比,木心中除氨基酸类成分丙氨酸和寡糖酯类Tenuifolise等外,多数化合物,特别是皂苷类,含量都较低,说明传统认为远志根使用时应抽去木心具有一定科学依据。
2.远志不同炮制品(筒)代谢组明显不同。蜜制和甘草制对生品(筒)化学成分的改变不同,特别是皂苷类,蜜制使Onjisaponin AⅠ含量升高,但对总皂苷含量影响不大,说明远志中皂苷类成分较为复杂,单一成分并不能代表总皂苷含量变化。而甘草制则使皂苷类含量显著升高,这可能导致蜜制品和甘草制品产生不同药效,因此在临床应用中应根据不同情况进行合理用药。
Rationales:Radix Polygalae, the root of Polygala tenuifolia Willd or Polygala sibirica L., a traditional Chinese herb has long been used as a traditional Chinese medicine and widely used in the vast majority of Chinese medicine prescription for antidepressant, expectorant, antitussive and anti-inflammatory et al. At present, the studies of Polygala tenuifolia Willd mainly concentrated in①the aerial part of P. tenuifolia is discarded usually after the root harvest, resulting in waste of resources.②the xylem is required to be removed traditionally, not required now.③the processed products and so on.
Quality evaluation of P. tenuifolia still have the following deficiencies:①quality evaluation of P. tenuifolia by limited ingredients to cannot do a comprehensive analysi of the chemical composition, and HPLC-UV fingerprints only can do simple chromatographic analysis. It is very important to introduce a more comprehensive analytical tool-metabolomics;②he acid hydrolyzates of saponins cannot be used as reference substance to evaluate saponins content comprehensively. Alkaline hydrolyzates of saponins is single, Chinese Pharmacopoeia2010edition provides tenuifolin to saponin content measured by the standard, there is no report of saponin content evaluation by tenuifloin;③the aerial part of P. tenuifolia is discarded usually after the root harvest, resulting in waste of resources. The xylem is required to be removed traditionally, not required now. And the evalution of different parts of P. tenuifolia only concentrated in a limited composition;④pharmacological studies of the aerial part have not been reported.
Objective:the methods based on NMR/UPLC plant metabolomics research and content of determination of tenuifolin by UPLC have been established. Then the metabolomic profiles of different parts and different processed products of P.tenuifolia have not be evaluated, and the pharmacological efficacies of the aerial part of P. tenuifolia have been studied, providing the scientific guidance from new view for the quality assessment and resource utilization of P. tenuifolia.
Methods:1. The study based on NMR and UPLC plant metabolomics of the root, aerial part, cortex, xylem, Honey-stir-baked Radix Polygalae, Liquorice juice-stir-boiled Radix Polygalae:First, the extracts of samples were determinated by NMR spectrometer, the1H NMR spectras were processed by MestRenova software for large amounts of data, which was analysised for data principal component analysis and the smallest partial squares analysis by SIMC A-P software. Then, part of the metabolites in the NMR spectrum and different metabolites in samples were identified according to the literature and study results, combining with the t-test, the relative concentration of the different metabolites in the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia were determinate, to seek for NMR metabolomics differences among the different medicinal parts and processed products of Polygala tenuifolia Willd.
2. The study based on UPLC plant metabolomics of the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia:First, the extracts of samples were determinate by Ultra High Pressure Liquid Chromatography (UPLC) fingerprint, the peak areas in the chromatograms were integrated manually. Then the datas were analysised for data principal component analysis and the smallest partial squares analysis by SIMCA-P software. Then, part of the metabolites in the UPLC chromatogram and different metabolites in samples were identified according to the literature and study results, combining with the t-test, the relative concentration of the different metabolites in the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia were determinated, to seek for UPLC metabolomics differences among the different medicinal parts and processed products of Polygala tenuifolia Willd.
3. The UPLC content determination of tenuifolin in the root, aerial part, cortex, xylem, H P. tenuifolia and L P. tenuifolia:The standard substance for UPLC content determination, tenuifolin, was separated by us in the laboratory, the purity more than98%.
4. The antidepressant and antitussive, expectorant effects of the aerial part of Polygala tenuifolia Willd were studied by the classic Mice tail suspension test and Forced swimming test antidepressant model, the classic ammonia induced cough test and racheal phenol red excretion test antitussive, expectorant model.
Results:1. NMR plant metabolomics studies have shown that, there were significant differences among the metabolomic profiles of different parts and processed products, which mainly concentrated in the primary metabolites.①the metabolomes of different parts of Polygala tenuifolia Willd were different. Comparing with the root, the contents of valine, glutamic acid, succinic acid, GAB A, tartaric acid, maleic acid and sinapic acid were higher in the aerial part, and the contents of threonine, alanine, aspartic acid, acetic acid, sucrose, adenine and saponins compounds were lower. Compared with the phloem, the contents of alanine was higher in the xylem, the contents of threonine, aspartic acid, acetic acid, succinic acid, GAB A, tartaric acid, formic acid, choline, fructose and sucrose were lower;②the metabolomes of different processed products of Polygala tenuifolia Willd were different, too. Honey-stir-boiled made the contents of formic acid, and α-glucose increase, the contents of threonine, alanine, fructose and sucrose decreased; Liquorice juice-stir-boiled made the contents of leucine, tartaric acid increased, threonine, alanine, butanedioic acid, fumarate and sucrose decrease; Meanwhile, comparing with H P. tenuifolia, the contents of formic acid, and α-glucose were lower in L P. tenuifolia.
2. UPLC plant metabolomics studies have shown that, there were significant differences among the metabolomic profiles of differert parts and processed products, which mainly concentrated in the secondary metabolites.①the metabolomes of different parts of Polygala tenuifolia Willd, were different. Comparing with the roots, the content of Polygalaxanthone III was higher in the aerial parts, and the contents of3,6'-disinapoylsucrose ester, Sibiricose A5, Sibiricose A6, Tenuifoliside A, Tenuifolise I, Tenuifolise H, PolygalaxanthoneⅣ, Onjisaponin A and Onjisaponin K were lower. Compared with the phloem, the contents of Tenuifolise I was higher in the xylem, the contents of Tenuifoliside A, Tenuifolise I, Tenuifolise H, Onjisaponin A, Polagalasaponin X X X II were lower;②the metabolomes of different processed products of Polygala tenuifolia Willd were different, too. Honey-stir-boiled made the contents of Tenuifolise I Onjisaponin A increase, the contents of3,6'-disinapoylsucrose ester, Tenuifoliside A, Tenuifolise H, Polygalaxanthone IV decrease; Liquorice juice-stir-boiled made the contents of Tenuifolise I, Tenuifolise H, Onjisaponin K increased,3,6'-disinapoylsucrose ester, Polygalaxanthone IV, Tenuifolise H decreased; Meanwhile, comparing with H P. Tenuifolia., the contents of3,6'-disinapoylsucrose ester and Tenuifolise I were lower in L P. Tenuifolia.
3. UPLC content determinations have shown that,①the content of tenuifolin in the aerial part was higher than the root; the xylem which required to remove, the content of tenuifolin was very lower than in cortex, and the content difference was very significant;②In traditionally Preliminary processing of P. Tenuifolia., Honey-stir-boiled made the content of tenuifolin lower, Liquorice juice-stir-boiled made the content higher, and the later the content difference was very significant.
4. Pharmacological studies of the aerial part of P. Tenuifolia. have shown that, the aerial part had low antidepressant activity and strong antitussive, expectorant activities; Comparing with the roots of P. Tenuifolia. and the classic antitussive, expectorant drug carbetapentane and T. farfara, the antitussive effect of the aerial part was stronger, the expectorant effect was lower.
Conclusion:In the paper, the NMR and UPLC plant metabolomics and UPLC content determination of tenuifolin in P. tenuifolia were established, at the same time, the antidepressant and antitussive, expectorant activities of the aerial parts were evaluated. The results showed that the metabolomic profils of the root, aerial part, cortex, xylem, Honey-stir-baked Radix Polygalae, Liquorice juice-stir-boiled Radix Polygalae had significantly differences, and the aerial parts have certain antitussive and expectorant activity, and antitussive acticity is stronger than the root, expectorant activity is weaker. The active compound might be Polygalaxanthone III.
引文
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