云南石仙桃和猴耳环活性成分的研究
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摘要
本论文采用抑制RAW264.7大鼠巨噬细胞一氧化氮(NO)释放的体外活性筛选方法，对兰科石仙桃属植物云南石仙桃(Pholidota yunnanensis Rolfe)和含羞草科猴耳环属植物猴耳环(Pithecellobium clypearia Benth.)进行了活性部位筛选，化合物分离以及结构鉴定的工作。
利用多种现代色谱手段从云南石仙桃干燥全草60％乙醇提取物中分离得到了40个化合物，通过化学和光谱学(IR，MS，NMR)方法鉴定了它们的结梅，分别为：反式-3，3′-二羟基-2′，4′，5-三甲氧基二苯乙烯(1)，反式-3，4′-二羟基-2′，3′，5-三甲氧基二苯乙烯(2)，反式-3，3′-二羟基-2′，5-二甲氧基二苯乙烯(3)，反式-3-羟基-2′，3′，5-三甲氧基二苯乙烯(4)，反式-3，3′，5-三羟基二苯乙烯(5)，反式-3，3′，5-三羟基-2′-甲氧基二苯乙烯(6)，反式-2′，3，3′-三羟基-5-甲氧基二苯乙烯(7)，顺式-3，3′-二羟基-5-甲氧基二苯乙烯(8)，3，3′-二羟基-5-甲氧基联苄(9)，3，4′-二羟基-3′，5-二甲氧基联苄(10)，3，3′，5-三羟基联苄(11)，3，3′，α-三羟基-5-甲氧联苄(12)，正十九烷酸(13)，1，5-二羟基-2，7-二甲氧-9，10-二氢菲(14)，4，7-二羟基-2-甲氧基-9，10-二氢菲(15)，2，4，7-三羟基-9，10-二氢菲(16)，2，7-二羟基-4-甲氧基-9，10-二氢菲(17)，2，7-二羟基-6-甲氧基-9，10-二氢-5H-菲-[4，5-bcd]吡喃(18)，6-[2′-(3，3″-二羟基-5′-甲氧基联苄基)]-4，7-二羟基-2-甲氧基-9，10-二氢菲(19)，6-[6′-(反式-3′，3″-二羟基-5′-甲氧基二苯乙烯基)]-4，7-二羟基-2-甲氧基-9，10-二氢菲(20)，1-[2′-(反式-3′，3″-二羟基-5′-甲氧基二苯乙烯基)]-4，7-二羟基-2-甲氧基-9，10-二氢菲(21)，4，4′，7，7′-四羟基-2，2′-二甲氧基-9，9′，10，10′-四氢-1，1′-二菲(22)，4，4′，7，7′-四羟基-2，2′-二甲氧基-9，9′，10，10′-四氢-1，6′-二菲(23)，1-[(9，10-二氢-4-羟基-2-甲氧基-7-菲基)氧基]-4，7-二羟基-2-甲氧基-9，10-二氢菲(24)，7-O-[6′-(3″，4′-二羟基-5′-甲氧基联苄基)]-4-羟基-2-甲氧基-9，10-二氢菲(25)，2-(4-羟基-3-甲氧基苯基)-3-乙酰氧基甲基-7-甲氧基-2，3，9，10-四氢-菲并[2，3-b]呋喃-5-醇(26)，2-(4-羟基-3-甲氧基苯基)-3-羟甲基-7-甲氧基-2，3，9，10-四氢-菲并[2，3-b]呋喃-5-醇(27)，2-(4-羟基-3，5-二甲氧基苯基)-3-羟甲基-8-甲氧基-2，3，10，11-四氢-菲并[1，2-b]呋喃-5，6-二醇(28)，4，5-氧撑-2-(4-羟基-3，5-二甲氧基苯基)-3-羟甲基-1-甲氧基-2，3，9，10-四氢-菲并[2，3-b]呋喃-7-醇(29)，4，5-氧撑2-(4-羟基-3，5-二甲氧基苯基)-3-(3-羟基-5-甲氧基苯基)-7-甲氧基-2，3，9，10-四氢-菲并[2，3-b)呋喃-8-醇(30)，4′-羟基-3′，7-二甲氧基-5-(3″-羟基苯乙基)黄烷-3-醇(31)，4′-羟基-3′，5′，7-三甲氧-5-(3″-羟基苯乙烯基)黄烷-3-醇(32)，(±)-松脂素(33)，(±)-丁香脂素(34)，(±)-lyoniresinol(35)，(±)-异落叶松树脂醇(36)，(±)-5-甲基-异落叶松树脂醇(37)，4-(3-羟基苯基)-2-丁酮(38)，4-(3-羟基-2-甲氧基苯基)-2-丁酮(39)，(+)-(3R)-Lasiodiplodin(40)。
40个化合物中，有8个二苯乙烯类化合物(化合物1—8)，4个联苄类化合物(化合物9—12)，17个9，10-二氢菲类衍生物(化合物14—30)，2个黄烷类化合物(化合物31、32)，5个木脂素类化合物(化合物33—37)，3个酚酸类化合物(化合物38—40)以及1个脂肪酸类化合物(化合物13)。新化合物共19个，分别为化合物1—4，6—8，19—21，23—30和32；2个(化合物38、39)为首次从植物中分离得到；16个(化合物5、9—16、22、31、33—37)为首次从该属植物中分离得到；2个(化合物17、18)为首次从该种植物中分离得到；并首次报道了9，10-二氢菲并呋喃类化合物的结构及其生物活性。
利用抑制大鼠巨噬细胞NO释放的活性测试方法，对40个化合物进行了活性评价，发现二苯乙烯类、联苄类、9，10-二氢菲类以及9，10-二氢菲并呋喃类化合物显示出了很强的NO释放抑制作用，并具有初步的构效关系。其中化合物2对由脂多糖(LPS)和干扰素-γ(INF-γ)诱导的RAW264.7大鼠巨噬细胞iNOS mRNA的表达具有抑制作用，说明化合物2对NO释放的抑制是通过限制iNOS的基因转录而完成的。
在NO释放抑制活性的基础上，采用MTT法，测定了单体化合物对NCI推荐的三种人肿瘤细胞NCI-H460(人非小细胞肺癌)、SF-268(人神经胶质瘤)和MCF-7(人乳腺癌)，以及HepG2(人肝癌细胞)的细胞毒活性。实验结果表明，部分二苯乙烯类化合物能够选择性地抑制HepG2的生长。化合物2、形成的二聚体的芪族类化合物(stilbenoids)以及9，10-二氢菲并呋喃类化合物，对四种肿瘤细胞都具有生长抑制作用。针对同时具有NO释放抑制活性和细胞毒活性的化合物2进行了较为深入的研究，发现化合物2是通过上调Cyclin B1的表达使得细胞阻滞在G_2／M期，并通过下调Bcl_2的表达引发细胞凋亡，由于PARP蛋白的裂解可知此凋亡可能经过了Caspase3的通路。
针对40个化合物多数具有酚羟基的结构特点，我们还采用了清除1，1-二苯基-2-苦基苯肼(DPPH)自由基的活性测试方法，考察了它们的体外抗氧化能力。结果表明，部分二苯乙烯类化合物以及9，10-二氢菲类衍生物和环木脂素类化合物具有很好的清除DPPH自由基的作用，并具有初步的构效关系。
以上结果提示民间用草药云南石仙桃抗炎作用的物质基础可能为二苯乙烯类、联苄类、9，10-二氢菲类以及9，10-二氢菲并呋喃类化合物；抗癌作用的物质基础可能为二苯乙烯类化合物、形成二聚体的芪族类化合物以及9，10-二氢菲并呋喃类化合物；抗氧化作用的物质基础可能为二苯乙烯类化合物、9，10-二氢菲类衍生物以及环木脂素类化合物。其中该植物富含的二苯乙烯类化合物有可能作为iNOS选择性抑制剂，用作预防和治疗炎症、癌症等疾病。本文为今后对该种中草药的开发与利用，提供了详实的理论和实验依据。
我们采用抑制大鼠巨噬细胞NO释放的体外活性测试方法，确定含羞草科猴耳环属植物猴耳环(Pithecellobium clypearia Benth．)的60％乙醇提取物具有很强的NO释放抑制活性。利用多种现代色谱手段对其中的乙酸乙酯萃取部分进行了初步的分离，从中分离得到了8个化合物，通过化学和光谱学方法鉴定了它们的结构，分别为：没食子酸(1)，没食子酸乙酯(2)，(-)-(2R，3R)-表没食子儿茶素(3)，(-)-5，7，3′，4′，5′-五羟基黄烷(4)，(-)-(2R 3R)-表没食子儿茶素-7-没食子酸酯(5)，(-)-5，3′，4′，5′-四羟基黄烷-7-没食子酸酯(6)，槲皮素-3-O-α-L-吡喃鼠李糖苷(7)，杨梅树皮素-3-O-α-L-吡喃鼠李糖苷(8)。8个化合物中，有2个小分子酚酸类化合物(化合物1、2)，4个黄烷类化合物(化合物3—6)以及2个黄酮苷类化合物(化合物7、8)。化合物6为新化合物，化合物4为首次从植物中分离得到，化合物2、5和8为首次从该属植物中分离得到，化合物3和7为首次从该种植物中分离得到。
我们测试了8个化合物抑制大鼠巨噬细胞NO释放的活性，发现化合物6具有较强的NO释放抑制活性，化合物5和8具有较弱的活性。针对各单体化合物具有多酚羟基的结构特点，我们又采用了DPPH自由基清除能力以及氧自由基清除能力(ORAC)两种活性测试方法，考察了它们的体外抗氧化能力。实验结果表明，8个化合物都具有很强的DPPH自由基清除活性。化合物S、7和8的氧自由基清除能力强于V_C。各单体化合物对于不同的自由基表现出了不同的体外抗氧化能力。此外，我们又采用了ConA刺激小鼠脾细胞增殖的模型，以观察猴耳环中各单体化合物对T淋巴细胞增殖和活化的影响。实验结果显示，化合物1、2、4、6和8具有一定抑制T淋巴细胞增殖的活性，而化合物5具有较强的抑制活性。
本论文通过对猴耳环活性成分的初步研究发现，该植物中的酚类成分，尤其是黄烷类化合物，具有抑制大鼠巨噬细胞NO释放、清除DPPH自由基和氧自由基以及抑制ConA诱导的T淋巴细胞增殖多种生物活性。由此，我们推测猴耳环的抗炎疗效是该植物中所富含的酚类成分潜在的iNOS选择性抑制、抗氧化以及免疫抑制多种作用的综合体现。在上述工作的基础上，建立了NO释放抑制活性部位乙酸乙酯萃取物的指纹图谱，为进一步提高猴耳环的质量标准奠定了基础。
In this dissertation, the bioassay of the inhibitory effects on nitric oxide (NO) production in a murine macrophage-like cell line (RAW 267.4) activated by lipopolysaccharide (LPS) and interferon-γ(IFN-γ) was used to search NO inhibitors from herbs Pholidota yunnanensis Rolfe and Pithecellobium clypearia Benth from which 40, and 8 compounds were isolated respectively. The structures of 48 compounds were elucidated on the basis of chemical evidences and spectral analysis.The structures of 40 compounds isolated from Pholidota yunnanensis are trans-3,3'-dihydroxy-2',4',5-trimethoxystilbene (1), trans-3,4'-dihydroxy-2',3',5-trimethoxystilbene (2), trans-3,3'-dihydroxy-2',5-dimethoxystilbene (3), trans-3-hydroxy-2',3',5-trimethoxystilbene (4), trans-3,3',5-trihydroxystilbene (5), trans-3,3',5-trihydroxy-2'-methoxystilbene (6), trans-2',3,3'-trihydroxy-5-methoxystilbene (7), cis-3,3'-dihydroxy-5-methoxystilbene (8), 3,3'-dihydroxy-5-methoxybibenzyl (batatasin-III) (9), 3,4'-dihydroxy-3',5-dimethoxybibenzyl (gigantol) (10), 3,3',5-trihydroxybibenzyl(11), 3,3',α-trihydroxy-5-methoxybibenzyl (12), n-nonadecanoic acid (13), 1,5-dihydroxy-2,7-dimethoxy-9,10-dihydrophenanthrene (eulophiol) (14), 4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (lusianthridin) (15), 2,4,7-trihydroxy-9,10-dihydrophenan threne (16), 2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene (coelonin) (17), 2,7-dihydroxy-6-methoxy-9,10-dihydro-5H-phenanthro-[4,5-bcd]pyran (imbricatin) (18), 6-[2'-(3',3"-dihydroxy-5'-methoxybibenzy)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (19), 6-[6'-(trans-3',3"-dihydroxy-5'-methoxystilbeny)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (20), 1-[2'-(trans-3',3"-dihydroxy-5'-methoxystilbeny)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (21), 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,1'-biphenanthrene (22), 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,6'-biphenanthrene (23),1 -[(9,10-dihydro-4-hydroxy-2-methoxy-7-phenanthrenyl)oxy]-4,7-dihydroxy-2-methoxy-9,10-dihydrophena nthrene (24), 7-O-[6'-(3",4'-dihydroxy-5'-methoxybibenzy)]-4-hydroxy-2-methoxy-9,10-dihydrophenanthrene (25), 4-hydroxy-3-methoxyphenyl)-3-acetoxymethyl-7-methoxy-2,3,9,10-tetrahydro-phenanthro[2,3-b]furan-5-ol (26), 2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-2,3,9,10-tetrahydro-phenanthro[2,3-b]furan-5-oI (27), 2-(4-hydroxy-3,5-dimethoxy phenyl)-3-hydroxymethyl-8-methoxy-2,3,10,11-tetrahydro-phenanthro[1,2-b]furan-5,6-diol (28), 4,5-epoxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-hydroxymethyl-1-methoxy-2,3,9,10-tetrhydro- phenanthro[2,3-b]furan-7-ol (29), 4,5-epoxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(3-hydroxy-5- methoxyphenyl)-7-methoxy-2,3,9,10-tetrhydro-phenanthro[2,3-b]furan-8-ol (30), 4'-hydroxy-3',7- dimethoxy-5-(3"-hydroxyphenethyl)flavan-3-ol (31), 4'-hydroxy-3',5',7-trimethoxy-5-(3"- hydroxystyryl)flavan-3-ol (32), (±)-pinoresinol (33), (±)-syringaresinol (34), (±)-lyoniresinol (35), (±)-isolariciresinol (36), (±)-5-methoxy-isolariciresinol (37), 4-(3-hydroxyphenyl)-2-butanone (38), 4-(3-hydroxy-2-methoxyphenyl)-2-butanone (39), (+)-(3R)-Lasiodiplodin (40), respectively. All the compounds include eight stilbenes (1—8), four bibenzyls (9—12), seventeen 9,10-dihydrophenanthrene derivatives (14—30), two flavans (31, 32), five lignans (33—37), three phenolic acid compounds (38—40) and one fatty acid (13). Compounds 1—4, 6—8, 19—21, 23—30 and 32 are new compounds, compounds 38 and 39 were isolated from plants for the first time, compounds 5, 9—16, 22, 31and 33—37 were isolated from Pholidota genus for the first time, and compounds 17 and 18 were isolated from Pholidota yunnanensis for the fast time. The structures and bioactivities of dihydrophenanthrofurans were reported for the first time.
The inhibitory effects of all the compounds on NO production were examined. The stilbenes, bibenzyls, 9,10-dihydrophenanthrene and dihydrophenanthrofurans showed strong inhibitory effects on NO production, and the primary structure-activity relationship was discussed. Furthermore, it was demonstrated that compound 2 exhibited inhibitory effects on NO production through inhibiting NO synthase (iNOS) mRNA expression.
MTT method was also used to test the cytotoxic activity of the compounds on NCI-H460, SF-268, MCF-7, and HepG2 cell lines. Partial stilbenes showed selective cytotoxicity on HepG2 cell line, and compound 2, dimeric stilbenoids and dihydrophenanthrofurans exhibited cytotoxicity on all the cell lines. It was further indicated that compound 2 arrested the HepG2 cell cycle in G_2/M phase by up-regulating the protein level of Cyclin B1, and induced apoptosis by down-regulating Bcl2 protein. Compound 2 could cleave the protein PARP which is the target of caspase-3, suggesting the pathway of caspase-3 may induce the apoptosis on HepG2 cells.
We also tested the DPPH free radical scavenging activity of all the compounds. Partial stilbenes, 9,10-dihydrophenanthrene derivatives and cyclolignans were found to have strong antioxidant activity and primary structure-activity relationship.
Those findings suggested that the anti-inflammation components are stilbenes, bibenzyls, 9,10-dihydrophenanthrene and dihydrophenanthrofurans, the anti-cancer components are stilbenes, dimeric stilbenoids and dihydrophenanthrofurans, and stilbenes, 9,10-dihydrophenanthrene derivatives and cyclolignans are the potent free radical scavenger. Stilbenes may be used as the selective iNOS inhibitors for the prevention and treatment of inflammatory and cancer.
The structures of 8 compounds isolated from Pithecellobium clypearia Benth. are gallic acid (1), ethyl gallate (2), (-)-(2R,3R)-epigallocatechin (3), (-)-(2S)-5,7,3',4',5'-pentahydroxyflavan (4), (-)-(2R,3R)-epigallocatechin-7-gallate (5), (-)-(2S)-5,3',4',5'-tetrahydroxyflavan-7-gallate (6), quercitin-3-O-α-L-rhanmpyranoside (quercitrin) (7), myricitin-3-O-α-L-rhamnpyranoside (myricitrin) (8), respectively, which included two phenolic acid compounds (1, 2), four flavans (3-6), and two flavonol glycosides (7, 8). Compound 6 is a new compound, compound 4 was isolated from plants for the first time, and compounds 2, 5 and 8 were isolated from Pithecellobium genus for the first time.
The inhibitory effects and their IC_(50) values of 8 compounds on NO production in murine macrophage activated by LPS and IFN-γwere estimated, and compound 6 showed strong inhibitory effects and the inhibitory activity of compounds 5 and 8 were weak. All the compounds exhibited DPPH free radical and oxygen free radical scavenging effects. The inhibitory effects of all the compounds on T lymphocyte were assayed using mice splenocytes induced to proliferate by mitogens ConA. The results indicated that compound 5 had strong inhibitory effects on T lymphocyte. In summary, the polyphenolic compounds as the potent NO inhibitors, free radical scavengers and immunosuppressors play the important role in the anti-inflammatory effects of Pithecellobium clypearia. Our investigation on the bioactive constituents from Pithecellobium clypearia Benth. provided a reasonable ground for continuous study of this traditional Chinese medicine.
利用多种现代色谱手段从云南石仙桃干燥全草60％乙醇提取物中分离得到了40个化合物，通过化学和光谱学(IR，MS，NMR)方法鉴定了它们的结梅，分别为：反式-3，3′-二羟基-2′，4′，5-三甲氧基二苯乙烯(1)，反式-3，4′-二羟基-2′，3′，5-三甲氧基二苯乙烯(2)，反式-3，3′-二羟基-2′，5-二甲氧基二苯乙烯(3)，反式-3-羟基-2′，3′，5-三甲氧基二苯乙烯(4)，反式-3，3′，5-三羟基二苯乙烯(5)，反式-3，3′，5-三羟基-2′-甲氧基二苯乙烯(6)，反式-2′，3，3′-三羟基-5-甲氧基二苯乙烯(7)，顺式-3，3′-二羟基-5-甲氧基二苯乙烯(8)，3，3′-二羟基-5-甲氧基联苄(9)，3，4′-二羟基-3′，5-二甲氧基联苄(10)，3，3′，5-三羟基联苄(11)，3，3′，α-三羟基-5-甲氧联苄(12)，正十九烷酸(13)，1，5-二羟基-2，7-二甲氧-9，10-二氢菲(14)，4，7-二羟基-2-甲氧基-9，10-二氢菲(15)，2，4，7-三羟基-9，10-二氢菲(16)，2，7-二羟基-4-甲氧基-9，10-二氢菲(17)，2，7-二羟基-6-甲氧基-9，10-二氢-5H-菲-[4，5-bcd]吡喃(18)，6-[2′-(3，3″-二羟基-5′-甲氧基联苄基)]-4，7-二羟基-2-甲氧基-9，10-二氢菲(19)，6-[6′-(反式-3′，3″-二羟基-5′-甲氧基二苯乙烯基)]-4，7-二羟基-2-甲氧基-9，10-二氢菲(20)，1-[2′-(反式-3′，3″-二羟基-5′-甲氧基二苯乙烯基)]-4，7-二羟基-2-甲氧基-9，10-二氢菲(21)，4，4′，7，7′-四羟基-2，2′-二甲氧基-9，9′，10，10′-四氢-1，1′-二菲(22)，4，4′，7，7′-四羟基-2，2′-二甲氧基-9，9′，10，10′-四氢-1，6′-二菲(23)，1-[(9，10-二氢-4-羟基-2-甲氧基-7-菲基)氧基]-4，7-二羟基-2-甲氧基-9，10-二氢菲(24)，7-O-[6′-(3″，4′-二羟基-5′-甲氧基联苄基)]-4-羟基-2-甲氧基-9，10-二氢菲(25)，2-(4-羟基-3-甲氧基苯基)-3-乙酰氧基甲基-7-甲氧基-2，3，9，10-四氢-菲并[2，3-b]呋喃-5-醇(26)，2-(4-羟基-3-甲氧基苯基)-3-羟甲基-7-甲氧基-2，3，9，10-四氢-菲并[2，3-b]呋喃-5-醇(27)，2-(4-羟基-3，5-二甲氧基苯基)-3-羟甲基-8-甲氧基-2，3，10，11-四氢-菲并[1，2-b]呋喃-5，6-二醇(28)，4，5-氧撑-2-(4-羟基-3，5-二甲氧基苯基)-3-羟甲基-1-甲氧基-2，3，9，10-四氢-菲并[2，3-b]呋喃-7-醇(29)，4，5-氧撑2-(4-羟基-3，5-二甲氧基苯基)-3-(3-羟基-5-甲氧基苯基)-7-甲氧基-2，3，9，10-四氢-菲并[2，3-b)呋喃-8-醇(30)，4′-羟基-3′，7-二甲氧基-5-(3″-羟基苯乙基)黄烷-3-醇(31)，4′-羟基-3′，5′，7-三甲氧-5-(3″-羟基苯乙烯基)黄烷-3-醇(32)，(±)-松脂素(33)，(±)-丁香脂素(34)，(±)-lyoniresinol(35)，(±)-异落叶松树脂醇(36)，(±)-5-甲基-异落叶松树脂醇(37)，4-(3-羟基苯基)-2-丁酮(38)，4-(3-羟基-2-甲氧基苯基)-2-丁酮(39)，(+)-(3R)-Lasiodiplodin(40)。
40个化合物中，有8个二苯乙烯类化合物(化合物1—8)，4个联苄类化合物(化合物9—12)，17个9，10-二氢菲类衍生物(化合物14—30)，2个黄烷类化合物(化合物31、32)，5个木脂素类化合物(化合物33—37)，3个酚酸类化合物(化合物38—40)以及1个脂肪酸类化合物(化合物13)。新化合物共19个，分别为化合物1—4，6—8，19—21，23—30和32；2个(化合物38、39)为首次从植物中分离得到；16个(化合物5、9—16、22、31、33—37)为首次从该属植物中分离得到；2个(化合物17、18)为首次从该种植物中分离得到；并首次报道了9，10-二氢菲并呋喃类化合物的结构及其生物活性。
利用抑制大鼠巨噬细胞NO释放的活性测试方法，对40个化合物进行了活性评价，发现二苯乙烯类、联苄类、9，10-二氢菲类以及9，10-二氢菲并呋喃类化合物显示出了很强的NO释放抑制作用，并具有初步的构效关系。其中化合物2对由脂多糖(LPS)和干扰素-γ(INF-γ)诱导的RAW264.7大鼠巨噬细胞iNOS mRNA的表达具有抑制作用，说明化合物2对NO释放的抑制是通过限制iNOS的基因转录而完成的。
在NO释放抑制活性的基础上，采用MTT法，测定了单体化合物对NCI推荐的三种人肿瘤细胞NCI-H460(人非小细胞肺癌)、SF-268(人神经胶质瘤)和MCF-7(人乳腺癌)，以及HepG2(人肝癌细胞)的细胞毒活性。实验结果表明，部分二苯乙烯类化合物能够选择性地抑制HepG2的生长。化合物2、形成的二聚体的芪族类化合物(stilbenoids)以及9，10-二氢菲并呋喃类化合物，对四种肿瘤细胞都具有生长抑制作用。针对同时具有NO释放抑制活性和细胞毒活性的化合物2进行了较为深入的研究，发现化合物2是通过上调Cyclin B1的表达使得细胞阻滞在G_2／M期，并通过下调Bcl_2的表达引发细胞凋亡，由于PARP蛋白的裂解可知此凋亡可能经过了Caspase3的通路。
针对40个化合物多数具有酚羟基的结构特点，我们还采用了清除1，1-二苯基-2-苦基苯肼(DPPH)自由基的活性测试方法，考察了它们的体外抗氧化能力。结果表明，部分二苯乙烯类化合物以及9，10-二氢菲类衍生物和环木脂素类化合物具有很好的清除DPPH自由基的作用，并具有初步的构效关系。
以上结果提示民间用草药云南石仙桃抗炎作用的物质基础可能为二苯乙烯类、联苄类、9，10-二氢菲类以及9，10-二氢菲并呋喃类化合物；抗癌作用的物质基础可能为二苯乙烯类化合物、形成二聚体的芪族类化合物以及9，10-二氢菲并呋喃类化合物；抗氧化作用的物质基础可能为二苯乙烯类化合物、9，10-二氢菲类衍生物以及环木脂素类化合物。其中该植物富含的二苯乙烯类化合物有可能作为iNOS选择性抑制剂，用作预防和治疗炎症、癌症等疾病。本文为今后对该种中草药的开发与利用，提供了详实的理论和实验依据。
我们采用抑制大鼠巨噬细胞NO释放的体外活性测试方法，确定含羞草科猴耳环属植物猴耳环(Pithecellobium clypearia Benth．)的60％乙醇提取物具有很强的NO释放抑制活性。利用多种现代色谱手段对其中的乙酸乙酯萃取部分进行了初步的分离，从中分离得到了8个化合物，通过化学和光谱学方法鉴定了它们的结构，分别为：没食子酸(1)，没食子酸乙酯(2)，(-)-(2R，3R)-表没食子儿茶素(3)，(-)-5，7，3′，4′，5′-五羟基黄烷(4)，(-)-(2R 3R)-表没食子儿茶素-7-没食子酸酯(5)，(-)-5，3′，4′，5′-四羟基黄烷-7-没食子酸酯(6)，槲皮素-3-O-α-L-吡喃鼠李糖苷(7)，杨梅树皮素-3-O-α-L-吡喃鼠李糖苷(8)。8个化合物中，有2个小分子酚酸类化合物(化合物1、2)，4个黄烷类化合物(化合物3—6)以及2个黄酮苷类化合物(化合物7、8)。化合物6为新化合物，化合物4为首次从植物中分离得到，化合物2、5和8为首次从该属植物中分离得到，化合物3和7为首次从该种植物中分离得到。
我们测试了8个化合物抑制大鼠巨噬细胞NO释放的活性，发现化合物6具有较强的NO释放抑制活性，化合物5和8具有较弱的活性。针对各单体化合物具有多酚羟基的结构特点，我们又采用了DPPH自由基清除能力以及氧自由基清除能力(ORAC)两种活性测试方法，考察了它们的体外抗氧化能力。实验结果表明，8个化合物都具有很强的DPPH自由基清除活性。化合物S、7和8的氧自由基清除能力强于V_C。各单体化合物对于不同的自由基表现出了不同的体外抗氧化能力。此外，我们又采用了ConA刺激小鼠脾细胞增殖的模型，以观察猴耳环中各单体化合物对T淋巴细胞增殖和活化的影响。实验结果显示，化合物1、2、4、6和8具有一定抑制T淋巴细胞增殖的活性，而化合物5具有较强的抑制活性。
本论文通过对猴耳环活性成分的初步研究发现，该植物中的酚类成分，尤其是黄烷类化合物，具有抑制大鼠巨噬细胞NO释放、清除DPPH自由基和氧自由基以及抑制ConA诱导的T淋巴细胞增殖多种生物活性。由此，我们推测猴耳环的抗炎疗效是该植物中所富含的酚类成分潜在的iNOS选择性抑制、抗氧化以及免疫抑制多种作用的综合体现。在上述工作的基础上，建立了NO释放抑制活性部位乙酸乙酯萃取物的指纹图谱，为进一步提高猴耳环的质量标准奠定了基础。
In this dissertation, the bioassay of the inhibitory effects on nitric oxide (NO) production in a murine macrophage-like cell line (RAW 267.4) activated by lipopolysaccharide (LPS) and interferon-γ(IFN-γ) was used to search NO inhibitors from herbs Pholidota yunnanensis Rolfe and Pithecellobium clypearia Benth from which 40, and 8 compounds were isolated respectively. The structures of 48 compounds were elucidated on the basis of chemical evidences and spectral analysis.The structures of 40 compounds isolated from Pholidota yunnanensis are trans-3,3'-dihydroxy-2',4',5-trimethoxystilbene (1), trans-3,4'-dihydroxy-2',3',5-trimethoxystilbene (2), trans-3,3'-dihydroxy-2',5-dimethoxystilbene (3), trans-3-hydroxy-2',3',5-trimethoxystilbene (4), trans-3,3',5-trihydroxystilbene (5), trans-3,3',5-trihydroxy-2'-methoxystilbene (6), trans-2',3,3'-trihydroxy-5-methoxystilbene (7), cis-3,3'-dihydroxy-5-methoxystilbene (8), 3,3'-dihydroxy-5-methoxybibenzyl (batatasin-III) (9), 3,4'-dihydroxy-3',5-dimethoxybibenzyl (gigantol) (10), 3,3',5-trihydroxybibenzyl(11), 3,3',α-trihydroxy-5-methoxybibenzyl (12), n-nonadecanoic acid (13), 1,5-dihydroxy-2,7-dimethoxy-9,10-dihydrophenanthrene (eulophiol) (14), 4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (lusianthridin) (15), 2,4,7-trihydroxy-9,10-dihydrophenan threne (16), 2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene (coelonin) (17), 2,7-dihydroxy-6-methoxy-9,10-dihydro-5H-phenanthro-[4,5-bcd]pyran (imbricatin) (18), 6-[2'-(3',3"-dihydroxy-5'-methoxybibenzy)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (19), 6-[6'-(trans-3',3"-dihydroxy-5'-methoxystilbeny)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (20), 1-[2'-(trans-3',3"-dihydroxy-5'-methoxystilbeny)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (21), 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,1'-biphenanthrene (22), 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,6'-biphenanthrene (23),1 -[(9,10-dihydro-4-hydroxy-2-methoxy-7-phenanthrenyl)oxy]-4,7-dihydroxy-2-methoxy-9,10-dihydrophena nthrene (24), 7-O-[6'-(3",4'-dihydroxy-5'-methoxybibenzy)]-4-hydroxy-2-methoxy-9,10-dihydrophenanthrene (25), 4-hydroxy-3-methoxyphenyl)-3-acetoxymethyl-7-methoxy-2,3,9,10-tetrahydro-phenanthro[2,3-b]furan-5-ol (26), 2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-2,3,9,10-tetrahydro-phenanthro[2,3-b]furan-5-oI (27), 2-(4-hydroxy-3,5-dimethoxy phenyl)-3-hydroxymethyl-8-methoxy-2,3,10,11-tetrahydro-phenanthro[1,2-b]furan-5,6-diol (28), 4,5-epoxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-hydroxymethyl-1-methoxy-2,3,9,10-tetrhydro- phenanthro[2,3-b]furan-7-ol (29), 4,5-epoxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(3-hydroxy-5- methoxyphenyl)-7-methoxy-2,3,9,10-tetrhydro-phenanthro[2,3-b]furan-8-ol (30), 4'-hydroxy-3',7- dimethoxy-5-(3"-hydroxyphenethyl)flavan-3-ol (31), 4'-hydroxy-3',5',7-trimethoxy-5-(3"- hydroxystyryl)flavan-3-ol (32), (±)-pinoresinol (33), (±)-syringaresinol (34), (±)-lyoniresinol (35), (±)-isolariciresinol (36), (±)-5-methoxy-isolariciresinol (37), 4-(3-hydroxyphenyl)-2-butanone (38), 4-(3-hydroxy-2-methoxyphenyl)-2-butanone (39), (+)-(3R)-Lasiodiplodin (40), respectively. All the compounds include eight stilbenes (1—8), four bibenzyls (9—12), seventeen 9,10-dihydrophenanthrene derivatives (14—30), two flavans (31, 32), five lignans (33—37), three phenolic acid compounds (38—40) and one fatty acid (13). Compounds 1—4, 6—8, 19—21, 23—30 and 32 are new compounds, compounds 38 and 39 were isolated from plants for the first time, compounds 5, 9—16, 22, 31and 33—37 were isolated from Pholidota genus for the first time, and compounds 17 and 18 were isolated from Pholidota yunnanensis for the fast time. The structures and bioactivities of dihydrophenanthrofurans were reported for the first time.
The inhibitory effects of all the compounds on NO production were examined. The stilbenes, bibenzyls, 9,10-dihydrophenanthrene and dihydrophenanthrofurans showed strong inhibitory effects on NO production, and the primary structure-activity relationship was discussed. Furthermore, it was demonstrated that compound 2 exhibited inhibitory effects on NO production through inhibiting NO synthase (iNOS) mRNA expression.
MTT method was also used to test the cytotoxic activity of the compounds on NCI-H460, SF-268, MCF-7, and HepG2 cell lines. Partial stilbenes showed selective cytotoxicity on HepG2 cell line, and compound 2, dimeric stilbenoids and dihydrophenanthrofurans exhibited cytotoxicity on all the cell lines. It was further indicated that compound 2 arrested the HepG2 cell cycle in G_2/M phase by up-regulating the protein level of Cyclin B1, and induced apoptosis by down-regulating Bcl2 protein. Compound 2 could cleave the protein PARP which is the target of caspase-3, suggesting the pathway of caspase-3 may induce the apoptosis on HepG2 cells.
We also tested the DPPH free radical scavenging activity of all the compounds. Partial stilbenes, 9,10-dihydrophenanthrene derivatives and cyclolignans were found to have strong antioxidant activity and primary structure-activity relationship.
Those findings suggested that the anti-inflammation components are stilbenes, bibenzyls, 9,10-dihydrophenanthrene and dihydrophenanthrofurans, the anti-cancer components are stilbenes, dimeric stilbenoids and dihydrophenanthrofurans, and stilbenes, 9,10-dihydrophenanthrene derivatives and cyclolignans are the potent free radical scavenger. Stilbenes may be used as the selective iNOS inhibitors for the prevention and treatment of inflammatory and cancer.
The structures of 8 compounds isolated from Pithecellobium clypearia Benth. are gallic acid (1), ethyl gallate (2), (-)-(2R,3R)-epigallocatechin (3), (-)-(2S)-5,7,3',4',5'-pentahydroxyflavan (4), (-)-(2R,3R)-epigallocatechin-7-gallate (5), (-)-(2S)-5,3',4',5'-tetrahydroxyflavan-7-gallate (6), quercitin-3-O-α-L-rhanmpyranoside (quercitrin) (7), myricitin-3-O-α-L-rhamnpyranoside (myricitrin) (8), respectively, which included two phenolic acid compounds (1, 2), four flavans (3-6), and two flavonol glycosides (7, 8). Compound 6 is a new compound, compound 4 was isolated from plants for the first time, and compounds 2, 5 and 8 were isolated from Pithecellobium genus for the first time.
The inhibitory effects and their IC_(50) values of 8 compounds on NO production in murine macrophage activated by LPS and IFN-γwere estimated, and compound 6 showed strong inhibitory effects and the inhibitory activity of compounds 5 and 8 were weak. All the compounds exhibited DPPH free radical and oxygen free radical scavenging effects. The inhibitory effects of all the compounds on T lymphocyte were assayed using mice splenocytes induced to proliferate by mitogens ConA. The results indicated that compound 5 had strong inhibitory effects on T lymphocyte. In summary, the polyphenolic compounds as the potent NO inhibitors, free radical scavengers and immunosuppressors play the important role in the anti-inflammatory effects of Pithecellobium clypearia. Our investigation on the bioactive constituents from Pithecellobium clypearia Benth. provided a reasonable ground for continuous study of this traditional Chinese medicine.
引文
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