Studies on the non-covalent complexes between oleanolic acid and cyclodextrins using electrospray ionization tandem mass spectrometry

Non-covalent inclusion complexes formed between an anti-inflammatory drug, oleanolic acid (OA), and alpha-, beta- and gamma-cyclodextrins (CDs) were investigated by means of solubility studies and electrospray ionization tandem mass spectrometry (ESI-MSn). The order of calculated association constants (K-1:1) of complexes between OA and different CDs in solution is in good agreement with the order of their relative peak intensities and the relative CID energies of the complexes under the same ESI-MSn conditions. These results indicate a direct correlation between the behaviors of solution- and gas-phase complexes. ESI-MS can thus be used to evaluate solution-phase non-covalent complexes successfully. The experimental results show that the most stable 1:1 inclusion complexes between three CDs and OA can be formed, but 2:1 CD-OA complexes can be formed with beta- and gamma-CDs. Multi-component complexes of alpha-CD-OA-beta-CD (1:1:1), alpha-CD-OA-gamma-CD (1:1:1) and beta-CD-OA-gamma-CD (1:1:1) were found in equimolar CD mixtures with excess OA. The formation of 2:1 and multi-component 1:1:1 non-covalent CD-OA complexes indicates that beta- and gamma-CD are able to form sandwich-type inclusion non-covalent complexes with OA. The above results can be partly supported by the relative sizes of OA and CD cavities by molecular modeling calculations.

Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10 μg/mL VALE and 1 μg/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo.

Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors.

In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3' dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC(50): 0.0006 microM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.

The bile acid receptor TGR5 does not interact with β-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid, DCA, taurolithocholic acid, TLCA) and the selective agonists oleanolic acid (OA) and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide (CCDC) stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, assessed by confocal microscopy. DCA, TLCA and OA did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, determined by bioluminescence resonance energy transfer. CCDC stimulated a low level of TGR5 interaction with β-arrestin2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of extracellular signal regulated kinase (ERK1/2). BRET analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS

DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS. Ursolic acid is a natural molecule that presents several pharmacological properties. In this work, an analytical method by RP-HPLC has been developed and validated for quantification of this drug in the solid dispersions, using PEG 6000 and Poloxamer 407 as polymers. The method was specific, linear in the range of 1.0-50.0 mu g mL(-1) (r<0.99), precise (CV < 5% for both inter-and intra-assays), accurate (maximum deviation of +/- 13%), and robust to the parameters evaluated. This method has proved to be simple and useful for ursolic acid determination in solid dispersions, enabling its determination in pharmaceutical dosage form.

四川蜡瓣花、密花樫木、四川溲疏及云南豆腐柴的化学成分研究

本论文对四川蜡瓣花 (Corylopsis willmottiae Rehd. et Wils.)、密花樫木[Dysoxylum densiflorum (Blume) Miq.]、四川溲疏 (Deutzia setchuenensis Franch)及云南豆腐柴 (Premna yunnanensis W. W. Smith)的化学成分进行了研究。通过色谱分离得到44个化合物。主要基于波谱数据鉴定了它们的结构，其中1个为新化合物。 1．从四川蜡瓣花全株的95%乙醇提取物中共分离鉴定了13个化合物，它们是：1-O-(3-O-甲基没食子酸)-岩白菜素(1)、11-O-没食子酰基岩白菜素(2)、 11-O-紫丁香基岩白菜素(3) 、岩白菜素(4)、4-O-没食子酰基岩白菜素(5) 、4,11-O-二没食子酰基岩白菜素 (6)[14]、β-谷甾醇 (7)、acetyl aleuritolic acid (8)、(-)-表没食子儿茶素没食子酸酯(9)、对羟基苯甲酮 (10)、 11-香豆酸酰岩白菜素 (11)[19]、丁香酸 (12)和没食子酸 (13)。其中1为新化合物。 2．从密花樫木根的95%乙醇提取物中共分离纯化了13个化合物，它们是：β-白檀酮(14)、richenone (15)、β-谷甾醇 (7)、cabraleadiol (16)、β-香树脂醇 (17)、龙脑香醇酮 (18)、cabraleadiol monoacetate (19)、cabraleone (20)、3β-hydroxy-5 -pregnen-20-one (21)、3β-hydroxy-5α-pregnan-20-one (22)、cabraleahydroxylactone (23)、川楝子甾醇B (24)、表儿茶素 (25)。 3．从四川溲疏全株95%乙醇提取物中共分离11个化合物，鉴定了其中的9个化合物。它们是：β-谷甾醇 (7)、白桦酯醇(26)、齐墩果酸(27)、hydrangetin (28)、肉桂酸 (29)，齐墩果酸-3-O-β-D-吡喃葡萄糖醛酸苷(30)、β-胡萝卜苷 (31)、齐墩果酸-3-O-（β-D-吡喃葡萄糖醛酸-6-正丁酯）(32)、齐墩果酸-3-O-β-D-吡喃葡萄糖醛酸-28-O-β-D-吡喃葡萄糖苷 (33)。 4．从云南豆腐柴95%乙醇提取物中分离得到12个化合物，分别为白桦脂醇 (25)、7-羟基黄烷酮 (34)、松属素 (35)、2’,4’-羟基查儿酮 (36)、高良姜素-3-甲醚 (37) 、高良姜素-3,7-二甲醚 (38)、异甘草素-4-甲醚 (39)、豆蔻明 (40)、乔松酮 (41)、异甘草素 (42)、arjunolic acid (43)、槲皮素3-O-β-D-木糖苷(44)。 5．综述了1976年以来樫木属植物化学成分和活性研究的概况。 Phytochemical investigation on Corylopsis willmottiae, Dysoxylum densiflorum, Deutzia setchuenensis, and Premna yunnanensis, led to the isolation of 44 compounds, 1 of which was new one. 1. One new compound was isolated from 95% ehanolic extrat of the whole plants of C. willmottiae, identified as 11-O-(3-O-methylgalloyl)-bergenin (1). The twelve known compounds isolated were 11-O-galloylbergenin (2), 11-O-syringylbergenin (3), bergenin (4), 4-O-galloylbergenin (5), 4,11-di-O-galloylbergenin (6), β-sitosterol (7), acetyl aleuritolic acid (8), (-)-epigallocatechin 3-O-gallate (9), 1-(4-hydroxyphenyl) ethanone (10), 11-O-coumaroylbergenin (11), syringic acid (12), gallic acid (13). 2. Thirteen compounds were isolated from 95% ethanol extract from the roots of D. densiflorum and identified as β-amyrenone (14), richenone (15), β-sitosterol (7), cabraleadiol (16), β-amyrin (17), hydroxydammarenone-Ⅱ (18), cabraleadiol monoacetate (19), cabraleone (20), 3β-hydroxy-5-pregnen-20-one (21), 3β-hydroxy-5α-pregnan-20-one (22), cabraleahydroxylactone (23), toosendansterol B (24) and (-)-epicatechin (25). 3. Eleven compounds were isolated from ethanol extract of D. Setchuenensis. Nine were identified as β-sitosterol (7), betulin (26), oleanolic acid (27), hydrangetin (28), cinnamic acid (29), oleanolic acid 3-O-β-D-glucuronopyranoside (30), β-daucosterol (31), oleanolic acid 3-O-β-D-glucuronopyranoside-6-O-butyl ester）(32), oleanolic acid 3-O-β-D-glucuronopyranosyl-28-3-O-β-D-glucopyranoside (33). 4. Twelve compounds were isolated from ethanol extract of P. yunnanensis and identified as betulin (26), 7-hydroxyflavanone (34), pinocembrin (35), 2’,4’-dihydroxychalcone (36), galangin 3-methyl ether (37), galangin 3,7-dimethyl ether (38), isoliquiritigenin 4-methyl ether (39), cardamonin (40), pinostrobin (41), isoliquiritigenin (42), arjunolic acid (43), quercetin 3-O-β-D-lyxosopyranoside (44). 5. Chemical constituents and biological activities of the genus Dysoxylum (Meliaceae) were reviewed during 1976-2009.

野桂花的化学成分研究

首次从野桂花（Osmanthus yunnanensis Fr. P. S. Green）地上部分95%乙醇提取物中通过色谱分离得到20个化合物, 其中化合物20为新化合物。基于波谱数据它们被鉴定为(E)-阿魏酸二十烷基酯(1)、β-谷甾醇(2)、羽扇豆醇(3)、齐墩果酸(4)、7-oxo-β-sitosterol(5)、乙酰齐墩果酸(6)、(6′-O-palmitoyl)-sitosterol 3-O-β-D-glucoside(7)、rotundioic acid(8)、地榆糖甙Ⅱ(9)、27-O-(E)-对羟基肉桂酰-28-齐墩果酸(10)、27-O-(Z)-对羟基肉桂酰-28-齐墩果酸(11)、hycandinic acid ester(12)、绿原酸丁酯(13)、4,5-二咖啡酰奎尼酸丁酯(14)、4,5-dihydroxyprenyl caffeate(15)、28-O-β-D-glucopyranosyl rotundioic acid (16)、4-(6-O-caffeoyl-β-D-glucopyranosyloxy)-5-hydroxyprenyl caffeate (aohada-glycoside C, 17)、 4-β-D-glucopyranosyloxy-5-hydroxy-prenyl caffeate (aohada-glycoside A, 18)、β-胡萝卜甙(19)以及3-[O-β-D-(6-O-咖啡酰吡喃葡萄糖)]-甲基-2-烯-γ-内酯 (20)。化合物13、14、15和17有较强的α-葡萄糖甙酶抑制活性。当浓度为1 mg/ml时，它们对α-葡萄糖甙酶的抑制分别为61.5%、95.5%、72.1%、62.6%，活性高于阿卡波糖。 综述了木犀属植物化学成分及1993年以来苯丙素甙类化合物活性研究进展。 Twenty compounds were isolated from the 95% ethanol extract of the aerial parts of Osmanthus yunnanensis Fr. P. S. Green by chromatography for the first time. On the basis of spectral data, they were identified as (E)-ferulic acid eicosyl ester (1), β-sitosterol (2), lupenol (3), oleanolic acid (4), 7-oxo-β-sitosterol (5), acetyloleanolic acid (6), (6′-O-palmitoyl)-sitosterol 3-O-β-D-glucoside (7), rotundioic acid (8), ziyu glycosideⅡ (9), 3β-hydroxy-27-p-(E)-coumaroyloxy-olean-12-en-28-oic acid (10), 3β-hydroxy-27-p-(Z)-coumaroyloxyolean-12-en-28-oic acid (11), hycandinic acid ester (12), chlorogenic acid butyl ester (13), 4,5-di-O-caffeoylquinic acid butyl ester (14), 4,5-dihydroxyprenyl caffeate (15), 28-O-β-D-glucopyranosyl rotundioic acid (16), 4-(6-O-caffeoyl-β-D-glucopyranosyloxy)-5-hydroxyprenyl caffeate (aohada- glycoside C, 17), 4-β-D-glucopyranosyloxy-5-hydroxyprenyl caffeate (aohada- glycoside A, 18), β-daucosterol(19) and 3-[O-β-D-(6-O-caffeoylglucopyranosyl)]- methyl-2-en-γ-lactone (20). Compound 20 is a new one. Compounds 13, 14, 15 and 17 inhibit α-glucosidase with corresponding inhibitory rate of 61.5%, 95.5%, 72.1% and 62.6% at a concentration of 1 mg/ml, higher than acarbose. The chemical studies on Osmanthus genus and bioactivities of phenylpropanoid glycosides were summarized.

滇金足草、凋缨菊和长喙吴萸的化学成分研究

本论文对滇金足草（Goldfussia yunnanensis）、凋缨菊（Camchaya loloana）和长喙吴萸（Evodia vestia）的化学成分进行了研究，通过色谱分离得到40个化合物。主要基于波谱数据鉴定了它们的结构，其中10个为新化合物。 1．从滇金足草地上枝叶的95%乙醇提取物中共分离鉴定了16个化合物：泽漆内酯A（1）、18-羟基泽漆内酯A（2）、18-氧代泽漆内酯A（3）、18-羟基-3-O-β-D-吡喃葡萄糖-泽漆内酯A（4）、3-O-β-D-吡喃葡萄糖-泽漆内酯A（5）、3-O-β-D-吡喃半乳糖-泽漆内酯A（6）、6-E-肉桂酰哈巴俄苷（7）、E-哈巴俄苷（8）、5，6-异亚丙二氧基哈巴俄苷（9）、β-谷甾醇（10）、β-胡萝卜苷（11）、齐墩果酸（12）、肉桂酸（13）、麦角固醇（14）、硬脂酸（15）和丁二酸（16）。其中2-7为新化合物。5，6-异亚丙二氧基哈巴俄苷（9）以人工产物形式得到。 2．从凋缨菊地上枝叶的95%乙醇提取物中分离并鉴定了13个化合物：凋缨菊内酯A~C (17-19)、1β-乙酰基凋缨菊内酯C(20)、b-谷甾醇（10）、β-胡萝卜苷（11）、羽扇豆醇（21）、桦木醇（22）、桦木酸（23）、芥子醇（24）、紫丁香苷（25）、咖啡酸（26）和熊果酸（27）。其中化合物17-20为桉叶烷内酯类新化合物。化合物17、18、20对细胞株HepG2的GI50依次为7.80、7.08、4.99 µg/mL。 3．从长喙吴萸（E. vestia）地上枝叶的95%乙醇提取物中分离并鉴定了13个化合物：佛手内酯（28）、花椒毒素（29）、异茴芹内酯（30）、七叶内酯（31）、东莨宕素（32）、瑞香素（33）、异紫花前胡内酯(34)、茵芋碱（35）、山刈碱（36）、白鲜碱（37）、黄柏酮（38）、柠檬苦素（39）和对羟基苯甲醛（40）。 4．综述了1990—2007年期间从菊科植物中发现的桉叶烷-12，6内酯的化学结构、生物活性、生物转化及化学合成方面的研究进展。 Phytochemical investigation on Goldfussia yunnanensis, Camchaya loloana, and Evodia vestia, led to the isolation of 40 compounds, 10 of which were new ones. 1. Six new compounds were isolation from 95% ethanolic extract of the aerial parts of G. yunnanensis, and identified as 18-hydroxyhelioscopinolide A (2), 18-oxohelioscopinolide A (3), 18-hydroxy-3-O-β-D-glucopyranosylhelioscopinolide A (4), 3-O-β-D-glucopyranosylhelioscopinolide A (5),3-O-β-D-Galactopyranosyl helioscopinolide A (6), 6-O-trans-cinnamoyl E-harpagoside (7). The known compounds isolated were helioscopinolide A (1), E-harpagoside A (8), 5,6-isopropylidene E-harpagoside A (9), β-sitosterol (10), β-daucosterol (11), oleanolic acid (12), cinnamic acid (13), ergosterol (14), stearic acid (15) and succinic acid (16). Compound 9 was an artifact. 2. Four new compounds, loloanolides A – C (17 - 19) and 1β-acetoxy-loloanolide C (20), were isolation from 95% ethanolic extract of the aerial parts of C. loloana. The known ones were β-sitosterol (10), β-daucosterol (11), lupeol (21), betulin (22), betulinic acid (23), sinapyl (24), syringin (25), caffeic acid (26) and ursolic acid (27). The GI50 values of compounds 17, 18 and 20 to HepG2 cell line were 7.80, 7.08 and 4.99 µg/mL, respectively. 3. Thirteen were isolated from 95% ethanolic extract of the aerial parts of E. vestia for the first time. They were determined to be bergapten (28), xanthotoxin (29), isopimpinellin (30), esculetin (31), scopoletin (32), daphnetin (33), marmesin (34), skimmianine (35), confusameline (36), dictamine (37), obacunone (38), limonin (39) and p-hydroxy phenyl aldehyde (40). 4. The structures, biological activities, biotransformation and chemical syntheses of eudesmane-12, 6-olides from the Asteraceae during 1990-2007 were reviewed.

西藏胡黄连和鸡矢藤的化学成分及黄芪多糖的提取工艺研究

本论文由四部分组成，前三部分为实验论文，第四部分为文献综述。第一、二部分分别报道了中药西藏胡黄连和鸡矢藤的化学成分研究结果。从两种药用植物中共分离和鉴定了32个化学成分，其中3个为新化合物。第三部分为黄芪多糖的提取工艺研究。第四部分概述了近年来植物多糖的研究进展。 第一章为西藏胡黄连化学成分研究。通过正、反相硅胶柱层析等分离方法从药用植物西藏胡黄连（Picrorhiza scrophulariiflora Pennell）的根茎中共分离纯化出7个化合物。运用MS、1H-NMR、13C-NMR、DEPT、HSQC和HMBC等现代谱学方法，结合理化分析对这些化合物的结构进行了分析鉴定。7个化合物中有两个是酚性的葡萄糖苷类成分：西藏胡黄连酚苷D (1)、4-O-β-D-(6-O-vanilloyl glucopyranosyl) vanillic acid (6)；四个苯乙基苷类化合物：plantamajoside (2)、plantainoside D (3)、西藏胡黄连苷A (4) 和西藏胡黄连苷F (5)；一个苯基小分子化合物：香豆酸甲酯 (7)。其中化合物1和5未见文献报道，确定为新化合物；化合物3为首次从该种植物中分到。 第二章为鸡矢藤化学成分研究。从鸡矢藤（Paederia scandense (Lour) Merrill）全草中分离出25个化合物，通过理化常数和波谱数据鉴定了它们的结构。25个化合物中包括一个蒽醌类成分：茜根定-1-甲醚 (1)；两个香豆素：异东莨菪香豆素 (2)和5-羟基-8-甲氧基吡喃香豆素 (3)；两个香豆素-木脂素化合物：臭矢菜素 B (4)和臭矢菜素 D (5)；一个木脂素：异落叶松树脂醇 (6)；两个黄酮：diadzein (7)和蒙花苷 (8)；三个三萜类化合物：齐墩果酸 (9)、乌苏酸 (10)和 3-O-β-D-吡喃葡萄糖基乌苏烷 (11)；三个甾体及其糖苷：b-谷甾醇 (12)、胡萝卜苷 (13)和(24R)-豆甾-4-烯-3-酮 (14)；六个小分子化合物：对羟基苯甲酸 (15)，咖啡酸 (16)，香豆酸 (17)，丁烯二酸 (18)，3,5-二甲氧基-4-羟基苯甲酸(19)，咖啡酸-4-O-β-D-吡喃葡萄糖苷(20)；五个环烯醚萜类化合物：鸡矢藤苷 (21)，鸡矢藤酸 (22)，鸡矢藤酸甲酯 (23)，saprosmoside E (24)和paederoside B (25)。其中化合物25未见文献报道，为新化合物。化合物1～8、11、14、15～20为首次从该化合物中分离得到。同时对鸡矢藤中环烯醚萜类化合物做了高效液相-串联质谱(HPLC-MSn)分析，探讨了这类化合物的质谱裂解规律。 第三章为黄芪多糖的提取工艺研究。首先确定了黄芪多糖含量的测定方法，并进行了方法学验证；其次探讨了黄芪中黄芪多糖的提取工艺，确定以酶法-Sevag法联用来去除黄芪多糖中的蛋白质，可使其提取物中黄芪多糖总含量达到70％以上。 第四章为近年来植物多糖的研究进展。主要包括植物多糖的提取纯化、多糖的定性定量检测方法、多糖的结构分析和多糖的药理活性。 This dissertation consists of four parts. The first and second parts reports the studies on the chemical constituents of medicinal plants of Picrorhiza Scrophulariiflora and Paederia scandens. The third part is about the extract technique of Astragalan Polysaccharide (APS). The last part reviews the progress of the studies on plant polysaccharides. 　 The first chapter is about the chemical constituents of P. Scrophulariiflora which is widely used as an important medicine to treat various immune-related diseases. A new phenyl glycoside, scrophenoside D (1) and a new phenylethyl glycoside, scroside F (5), together with five known compounds, plantamajoside (2), plantainoside D (3), scroside A (4), 4-O-β-D-(6-O-vanilloylglucopyranosyl) vanillic acid (6); and methyl-p-coumarate (7) were isolated from the stems of P. scrophulariiflora. Their structures were elucidated by spectroscopic and chemical methods. The second chapter is about the chemical constituents of medicinal herb of P. scandens. Twenty-five compounds were isolated and purified by normal and reversed phase silica gel column chromatography. By physicochemical properties and spectral analysis, their structures were identified as rubiadin-1-methylether (1), isoscopoletin (2), 5-hydroxyl-8-methoxyl-coumarin (3), cleomiscosin B (4), cleomiscosin D (5), isolariciresinol (6), diadzein (7), linarin (8), oleanolic acid (9), ursolic acid (10), 3-O-β-D-glucopyranosyloxyl-ursane (11), b-sitosterol (12), b-daucosterol (13), (24R)-stigmast-4-ene-3-one (14), p-hydroxyl-benzoic acid (15), caffic acid (16), coumaric acid (17), trans-butenedioic acid (18), 3,5-dimethoxyl-4-hydroxylbenzoic acid (19), caffeic acid 4-O-β-D-glucopyranoside (20), paederoside (21), paederosidic acid (22), paederosidic acid methyl ester (23), saprosmoside E (24), paederoside B (25). Among them, compound 25 is a new compound. Compounds 1～8、11、14、15～20 were isolated from this plant for the first time. Futhermore, we studied the HPLC-MSn analysis and investigation of fragmentation behavior of the sulfur-containing iridoid glucosides. The third chapter is about the extracting process of Astragalan Polysaccharide (APS). The method of the content determination is built. The optimum condition of extraction of polysaccharides from Radix Astragali is defined and the more effective way to remove protein is combined enzyme method with Sevag method, by which the content of polysaccharides extract can be up to 70%. The last part is a review of the research progress of the plant polysaccharides, which includes its extraction, isolation, purification, determination, structure analysis, and pharmacology.

尼泊尔水东哥、水麻、木姜冬青和青荚叶的化学成分研究

活性筛选中发现尼泊尔水东哥 (Saurauia napaulensis DC.) 树皮95%乙醇提取物具有α-淀粉酶抑制活性、水麻(Debregeasia orientalis) 枝叶95%乙醇提取物显示血管紧张素转化酶(ACE)抑制活性、青荚叶(Helwingia japonica (Thunb.) Dieter.) 95%乙醇提取物的中小极性部分显示蛋白酪氨酸磷酸酯酶(PTP)1B抑制活性。为全面了解它们的成分及相关活性成份，主要运用硅胶柱层析方法从这三个植物分离得到39个化合物，通过波谱分析或与已知品对照的方法对其进行了鉴定。对木姜冬青(Ilex litseaefolia Hu et Tang)的成分做了进一步的研究，取得了如下结果。 1. 从尼泊尔水东哥树皮的95%乙醇提取物分离并鉴定12个化合物： auranamide、aurantiamide benzoate、齐墩果酸、β-谷甾醇、β-胡萝卜甙、乌苏酸、2α,3α-二羟基-12-烯-28-乌苏酸、2α,3β,24-三羟基-12-烯-28-乌苏酸、(2S,3S,4R,10E)-2-[(2'R)-2' -hydroxytetracosanoylamino] -10-octadecene -1,3,4-triol、 2α,3α,24-三羟基-12-烯-28-齐墩果酸、2α,3β-二羟基-12-烯-28-乌苏酸和2α,3α,24-三羟基-12-烯-28-乌苏酸。 2. 从水麻枝叶的95%乙醇提取物分离并鉴定了18个化合物：棕榈酸、二十烷酸、二十烷酸甲酯、β-谷甾醇、Monogynol A、桦木酸、Hederagenin、β-胡萝卜甙、18αH-19(29)-烯-3-酮-乌苏烷、3，4-开环-20(30)-烯-乌苏烷-3-酸、Pomolic acid，表儿茶素、儿茶素、槲皮素、槲皮素-3-O-β-D-吡喃葡萄糖苷、紫丁香苷、紫丁香酚苷和山萘酚-3-O-芸香糖。儿茶素、槲皮素和槲皮素-3-O-β-D-吡喃葡萄糖苷为具有ACE抑制活性的成分。 3. 从木姜冬青95%乙醇提取物的乙酸乙酯部分分离并鉴定了5个化合物： 2-O-β-D-吡喃葡萄糖-6,2´-二羟基-4,4´-二香草酰氧甲基-1,1´-二苯醚（冬青苷）和四个已知化合物：七叶内酯、香草酸、3,4-二甲氧基苯乙酸和vanilloylcalleryanin。冬青苷为新化合物。 4. 从青荚叶95%乙醇提取物的中小极性部分分离并鉴定了9个化合物：β-谷甾醇、β-胡萝卜苷、羽扇豆醇、桦木醇、桦木酸、棕榈酸甘油酯、桂皮酸、6αH-4-烯-3-酮-豆甾醇和6βH-4-烯-3-酮-豆甾醇。 5. 对1985-2006年间天然二苯醚类化合物及活性研究进展进行综述. The in vitro test indicated that the 95% ethanolic extract of the barks of Saurauia napaulensis DC showed α-amylase inhibitory activity, the 95% ethanolic extract of the whole plants of Debregeasia. orientalis showed angiotensin converting enzyme (ACE) inhibitory activity and some fractions of the 95% ethanolic extract of the aerial parts of Helwingia japonica showed protein tyrosine phosphatase (PTP)1B inhibitory activity. In order to investigate components and active compounds of the three plants, they were chemically studied mainly using. Thirty-nine compounds were isolated predominantly by column chromatography identified by spectral methods or comparing them with authentic samples. Further investigation of Ilex litseaefolia Hu et Tang was carried out. Major results are as follows: 1. Twelve compounds were isolation from the 95% ethanolic extract of the barks of S. napaulensis DC. They were identified as auranamide, aurantiamide benzoate, oleanolic acid, β-sitosterol, β-daucosterol, ursolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 2α,3β,24-trihydroxyurs-12-en-28-oic acid, (2S,3S,4R,10E)-2-[(2'R)-2'-hydroxytetracosanoyl amino]-10-octadecene-1,3,4-triol, 2α,3α,24 -trihydroxyolean-12-en-28-oic acid, 2α,3β-dihydroxyurs-12-en-28-oic acid, and 2α,3α,24-trihydroxyurs-12-ene-28-oic acid, respectively, by spectral methods or comparing them with authentic samples. 2. Eighteen compounds were isolation from the 95% ethanolic extract of the whole plants of D. orientalis. They were identified as palmitic acid, henicosanoic acid, henicosanoic acid methyl ester, β-sitosterol, monogynol, betulinic acid, hederagenin, β-daucosterol, 18αH-urs-20(30)-en-3-one, 3,4-seco-urs-20(30)-en-3-oic acid, pomolic acid, (-)-epicatechin, (+)-catechin, quercetin, quercetin 3-O-β-D-glucopyranoside, syringin, syringiaresinol digloside and kaempferol-3-O-rutinose. (+)-Catechin, quercetin and quercetin 3-O-β-D-glucopyranoside were the ACE inhibitory active components. 3. Further phytochemical investigation of the ethyl acetate parts of 95% ethanolic extract of the whole plant of I. litseaefolia afforded 2-O-β-D-glucopyranose-4,4´-di-vanilloyloxymethyl-2,6´-dihydroxy-1,1´-diphenyl ether (ilexiside), esculetin, vanillic acid, 3,4-dimethoxybenzylacetic acid and vanilloylcalleryanin. Ilexiside was new compound. 4. Nine compounds were isolation from the 95% ethanolic extract of the whole plant of H. japonica: β-sitosterol, β-daucosterol, lupeol, betulin, betulinic acid, glycerol monopalmitate, cinnamic acid, stignast-4-en-6β-3-one and stignast-4-en-6α-3-one 5.Diphenyl ether compounds from nature between 1985-2006 were summarized.

五枫藤、小驳骨和川西茶藨的化学成分研究

八月瓜属植物五枫藤(Holboellia latifolia Wall.)和驳骨草属植物小驳骨(Gendarussa vulgaris Nees)均为药用植物, 前者化学成分研究不深入, 后者的化学成分未见报道。川西茶藨(Ribes takare D. Don)为茶藨子属植物, 没有化学成分的报道。本论文对三个植物的化学成分和活性成分进行了研究, 主要通过色谱方法分离得到了48 个化合物, 采用波谱分析或与已知标准品对照等手段鉴定了它们的结构, 其中有1 个新的原小檗碱类化合物和3 个新的联苯类化合物,发现了具有细胞毒活性和α-葡萄糖苷酶抑制活性的化合物。1、从五枫藤地上部分的95%乙醇提取物中分离得到了12 个化合物: 五加苷K (1)、hederagenin 3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2)、β-萘乙酸(3) 、3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4) 、3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5) 、3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid 28-O- β-D-glucopyranoside (6)、lup-20(29)-en-3-one (7)、lupeol (8)、β-谷甾醇(9)、齐墩果酸(10)、乌苏酸(11)、β-胡萝卜苷(12)。化合物1 对Lu-06、N-04 和Bre-04 癌细胞株的GI50 分别是0.77µg/mL、1.26 µg/mL 和1.55 µg/mL, 化合物2 对N-04 癌细胞株的GI50 为2.44 µg/mL。2、从小驳骨地上部分的95%乙醇提取物中分离得到了1 个原小檗碱类新化合物13-hydroxyl gusanlung A (25), β-谷甾醇(9)、齐敦果酸(10)、β-胡萝卜苷(12)、棕榈酸(1-)甘油酯(13)、棕榈酸(14)、阿苯哒唑(15)、阿苯哒唑砜(16)、阿苯哒唑亚砜(17)、aurantiamide acetate (18)、华良姜素(19)、芫花素(20)、(-)-丁香树酯醇(21)、gusanlung B (22) 、eupteleasaponinsⅤ acetate (23)、gusanlungA (24)、刺五加苷E (26)、岩白菜素(27)、咖啡酸(28)。化合物25 对肝癌细胞株(HepG2) 的GI50 为2.08 µg/mL。3、从川西茶藨地上部分的95%乙醇提取物中分离鉴定了22 个化合物: β-谷甾醇(9) 、β- 胡萝卜苷(12) 、O-acetyloleanolic aldehyde (29),4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30) 、3', 5-dimethoxy-3, 4-methylenedioxybiphenyl (31) 、桦木醇(32) 、6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33)、3'-hydroxy-5-methoxy-3,4-methylenedioxybiphenyl (34) 、7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35)、桦木醛(36)、没食子酸(37) 、6β- 羟基-4- 烯-3- 酮- 豆甾醇(38) 、5α, 8α-epidioxy-(22E,24R)-ergosta-6, 22-dien-3β-ol (39)、verrucofortine (40)、6-methoxycalpogoniumisoflavone A (41)、2-羟基二苯甲酮(42)、桦木酸(43), 3, 5-二甲氧基苯甲酸-4-O-β-D-吡喃葡萄糖苷(44)、洋芹素(45)、刺槐素(46)、水杨酸(47)、洋芹素-5-O- β-D-葡萄糖苷(48), 化合物30、31 和35 为新的联苯化合物。化合物30的α-葡萄糖苷酶抑制率为10.2% (1.00 mg/mL); 化合物35 的抑制率为17.2% (1.00mg/mL)。4、综述了1960 年以来原小檗碱类化合物药理活性研究进展。 Plants Holboellia latifolia Wall and Gendarussa vulgaris Nees, are used as folkmedicine. Ribes takare D. Don belongs to the genus Ribes. The three plants have notbeen chemically studied in detail. Chemical and bioactive study of three plants led tothe isolation of 48 compounds by chromatography. Their structures were elucidatedon the basis of spectroscopic evidence or comparison with authentic samples. Amongthe 48 componds isolated one protoberberine alkaloid and three biphenyls are newones. Cytotoxic and α-glucosidase inhibitory compounds had been found.1. Twelve compounds were isolated from the 95% ethanol extract of the aerial partof H. latifolia Wall. They were characterized as fellow: eleutheroside K (1),hederagenin-3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2),2-naphthyl acetic acid (3),3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4), 3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5),3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid-28-O- β-D-glucopyranoside (6),lup-20(29)-en-3-one (7), lupeol (8), β-sitosterol (9), oleanolic acid (10), ursolicacid (11), and β-daucosterol (12). Compound 1 showed moderate cytotoxicity againstLu-06 (GI50, 0.77 µg/mL), N-04 (GI50, 1.26 µg/mL) and Bre0-4 (GI50=1.55 µg/mL)and compound 2 showed moderate cytotoxicity against N-04 (GI50=2.44 µg/mL).2. A new protoberberine alkaloid, 13-hydroxyl gusanlung A (25), was isolated fromthe aerial part of Gendarussa vulgaris Nees, together with β-sitosterol (9), oleanolicacid (10), β-daucosterol (12), glycerol monopalmitate (13), palmific acid (14),albendazole (15), albendazole sulphone (16), albendazole sufloxide (17), aurantiamideacetate (18), kumatakenin (19), genkwanin (20), (-)-syringaresinol (21), gusanlung B(22), eupteleasaponinsⅤ acetate (23), gusanlung A (24), eleutheroside E (26),bergenin (27) and caffeic acid (28). Compound 25 showed cytotoxicity against HepG2 cells (GI50, 2.08 µg/mL).3. Phytochemical study of the Ribes takare D. Don led to the isolation of three newbiphenyls, 4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30), 3', 5-dimethoxy-3,4-methylenedioxybiphenyl (31) and 7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35), along with nineteenknown compounds, β-sitosterol (9), β-daucosterol (12), O-acetyloleanolic aldehyde(29), betulin (32), 6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33),3'-hydroxy-5-methoxy-3, 4-methylenedioxybiphenyl (34), betulinic aldehyde (36),gallic acid (37), stigmast-4-en-6β-ol-3-one (38), 5α, 8α-epidioxy-(22E, 24R)-ergosta-6,22-dien-3β-ol (39), verrucofortine (40), 6-methoxycalpogonium isoflavone A (41),2-hydroxybenzophenone (42), betulinic acid (43), 3,5-dimethoxygallic acid-4-O- β-D-glucopryranoside (44), apigenin (45), acacetin (46), salicylic acid (47) andapigenin-5-O- β-D-glucopryranoside (48). α-Glucosidase inhibitory rates ofcompound 30 and 35 were respectively 10.2% and 17.2% at a concentration of 1.00 mg/mL).4. Pharmacological activities of protoberberines were summarized.

齐墩果酸的微生物转化

齐墩果酸（OA）是一个分布广泛、含量丰富的天然三萜化合物，常以皂苷元的形式广泛存在于植物中,具有多种重要生物活性。但是OA许多活性较弱，且生物利用度低，限制了其在临床上的应用。一是OA水溶性差;二是抗癌活性仍与临床应用的抗癌药物相差比较大。 真菌在微生物转化中具有种类多、培养条件比较简单等特点，为了寻找到具有转化OA能力的菌株，采取一步发酵的方法，在18株实验室保藏真菌菌株中筛选到5株目的菌株,TLC分析显示有转化效果。 随后采用二步发酵的方法作为复筛，验证5株菌株转化能力，波谱分析结果表明5株菌株对OA确实有转化作用。 选择5株菌种中代号1F-2 2菌株作为放大实验菌株，分离转化产物，得到OA衍生物108（相对分子量414m/z）和1010（相对分子量340 m/z），分离出的产物用于活性检测。寻找到产物108的RP-HPLC分离条件，质谱得出二者相对分子质量。 为验证OA转化产物抗肿瘤活性，首次研究了OA对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231作用，通过细胞增殖抑制实验、用MTT法检测细胞活性，结果表明齐墩果酸可降低卵巢癌细胞株IGROV1和乳腺癌细胞MDA-MB-231细胞增殖能力并呈剂量依赖性，对肿瘤细胞株的半数有效抑制浓度化IC50 分别为36.58μg/mL和38.8μg/mL (P<0．01)。OA能抑制肿瘤细胞活性，并且OA对卵巢癌细胞株IGROV1抑制活性高于乳腺癌细胞MDA-MB-231。 在此基础上，转化产物108和1010对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231的抑制作用也进行研究，MTT实验结果表明，转化产物对两株癌细胞也有抑制活性（P<0．01）。 总之，本文工作为进一步开展齐墩果酸类化合物结构改造和抗肿瘤活性的研究奠定了基础。 Oleanolic acid (OA) is a triterpenoid widely distributed in the nature which possesses various important bioactivities. OA also serves as aglycon of many natural saponins. However, the relatively weak activities and poor bioavailability hinder its clinical use. Firstly, poor water-solubility results in worse bioavailability. Secondly, compared with clinical antitumor drug, the antitumor effect of OA has a great difference, it is worse. Many fungi have ability to transform nature products into a variety of derivatives, and transformation conditions of fungi are simple. Attempt to obtain fungi strains able to biotransform OA, we carried out the following experiments: To investigate the biotransformation 0f OA by strains supplied firstly, we used one-step fermentation method to screen the aimed strains from 18 fungus strains stored in our laboratory. On the basis of the initial screening experiments, we found 5 aimed strains. The TLC results showed that the 5 fungi strains could transform OA into other components derivatives. Then we used two-step fermentation method as secondly screening. We repeated the five strains to do the experiments, analytical data of the results proved the transformation indeed. In the followed experiments work, we chose 1F-2 2 strain as large-scale transformation fungus from the aimed fungi. We got two biotransformation products of OA by 1F-2 2, and named those derivatives 108 and 1010. We found RP-HPLC separation conditions of product 108. The two products were characterized by ESI-MS. To verify the anti-tumor activity of biotransformation products of OA, we studied the inhibition effect of oleanolic acid on the ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 firstly. With an assay based on a tetrazolium dye (MTT), the effects of various concentrations of oleanolic acid on ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 were studied. MTT method was used to measure the tumor cells viability. Compared with the control group, oleanolic acid can significantly inhibit the viability of the ovarian carcinoma cells IGROV1 and MDA-MB-231 breast cancer cell line (P<0．01), IC50 values were 36.58μg/mL or 38.8μg/mL. Oleanolic acid can inhibit the malignant tumor cells viability, and inhibitory activity of OA to ovarian carcinomas IGROV1 was higher than to breast cancer cell line MDA-MB-231. On this basis, we studied the anti-tumor activity of the two derivatives of OA [called 108 (414 m/z) and 1010(340 m/z)]. It came to the conclusion that the two derivatives also showed potent inhibitory effect on the growth of these tumor cells（P<0．01）. Therefore, the results of studies will benefit the further investigating on the relationships of structures and antitumor activities of OA.

Chemical constituents of marine medicinal mangrove plant Sonneratia caseolaris

Twenty-four compounds including eight steroids (1-8), nine triterpenoids (9-16, 24), three flavonoids (20-22), and four benzenecarboxylic derivatives (17-19, 23) were isolated and identified from stems and twigs of medicinal mangrove plant Sonneratia caseolaris. The structures of the isolated compounds were determined by extensive analysis of their spectroscopic data. Among these metabolites, compounds 1, 4-20 and 22-24 were isolated and identified for the first time from S. caseolaris. In the in vitro cytotoxic assay against SMMC-7721 human hepatoma cells, compound 21 (3',4',5,7-tetrahydroxyflavone) exhibited significant activity with IC50 2.8 mu g/mL, while oleanolic acid (14), 3,3'-di-O-methyl ether ellagic acid (18), and 3,3',4-O-tri-O-methyl ether ellagic acid (19) showed weak activity. None of these compounds displayed significant antibacterial activites.

Analysis of five pharmacologically active compounds from the Tibetan medicine Elsholtzia with micellar electrokinetic capillary chromatography

A high performance capillary electrophoresis method with diode array detector detection for the determination of five bioactive ingredients in Tibetan medicine Elsholtzia, namely quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid, has been developed. The effects of several factors, such as the acidity, concentration of running buffer, separation voltage, temperature, and SDS concentration were investigated. The optimal conditions were 44 mmol/L boric acid running buffer (pH 8.5), 45 mmol/L SDS, 16 KV voltage, 20 degrees C, and 10.0% (V/V) of acetonitrile. Under the optimum conditions, five components could be separated with a good baseline resolution within 17 min. The calibration curves showed good linear relationship over the concentration range of 5 x 10(-4)similar to 0.1 mg/mL for quercetin, rutin, saussurenoside, kaempferol, and 1 x 10(-3) similar to 0.1 mg/mL for oleanolic acid. The average recoveries of the method and RSD were ( 99.2%, 3.2%) for quercetin, (102.1%, 2.1%) for rutin, (99.4%, 1.5%) for saussurenoside, (98.9%, 1.8%) for kaempferol, and (99.0%, 2.9%) for oleanolic acid, respectively. The detection limits (S/N = 3) were 1.1 x 10(-4) mg/mL for quercetin, 2.6 x 10(-4) mg/mL for rutin, 1.8 x 10(-4) mg/mL for saussurenoside, 2.9 x 10(-4) mg/mL for kaempferol, and 6.3 x 10(-4) mg/mL for oleanolic acid, respectively. The method was simple, rapid, and reproducible and could be applied for the determination of quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid in Tibetan medicine Elsholtzia, and the assay results were satisfactory.