短柄五加的化学成分及生物活性研究

【作者】 胡浩斌；

【导师】 樊君；

【作者基本信息】 西北大学 ， 化学工程， 2010， 博士

【摘要】 短柄五加(Acanthopanax brachypus)为五加科(Araliaceae)五加属(Acanthopanax)多年生落叶小灌木,在世界范围内的分布范围极其狭窄,主要分布于我国西北黄土高原上的陕西、甘肃、宁夏等地,是我国特有的民间药用植物。其药用部位主要为茎皮和根,具有益气健脾、养心安神、解郁和血等功效。据研究表明：其根茎的提取物除具有治疗神经衰弱、男子性功能障碍、继发性高血压、低血压、白细胞减少等疾病外,还有防癌抗癌功效；其皮的醇提物具有抗炎作用,还可抑制佐剂关节炎及Ⅰ、Ⅱ、Ⅲ型变态反应性炎症。本文首先综述了目前国内外对五加属植物化学成分及生物活性的研究进展,在此基础上分析了五加属植物资源的开发现状及应用前景,并通过试管反应和薄层色谱法对短柄五加茎皮中的化学成分进行了系统预试验,为各类有效成分提取分离方案的设计提供依据。采用水蒸汽蒸馏法分别提取了短柄五加花、叶、茎皮、根和果实中的挥发油,利用气相色谱-质谱联用技术对其化学成分进行了分离和结构鉴定,用面积归一化法测定了各成分的相对百分含量,通过体外抑菌试验研究了各挥发油的抑菌活性。结果表明,从短柄五加不同部位提取的挥发油中,化学成分的类型和相对含量不同,其中花、叶、茎皮和根部的挥发油以单萜烯、倍半萜烯及其氧化物、长链烃及衍生物等成分为主,为β-蒎烯／芳樟醇／吉玛烯-D型,而果实挥发油以脂肪酸(酯)为主。短柄五加挥发油中含有多种抑菌活性成分,对11种供试菌种均有不同程度的抑制作用,尤其是花和叶部位的挥发油对革兰氏菌的抑菌杀菌作用最为显著。以乙醇为提取溶剂,采用超声波法提取了短柄五加茎皮中的非挥发性成分,并通过各种色谱(硅胶制备薄层色谱、硅胶柱色谱、大孔树脂和葡聚糖凝胶柱色谱)和重结晶等分离手段,从短柄五加茎皮中分离得到了52种单体化合物,通过对各种理化性质和波谱数据(包括紫外光谱、红外吸收光谱、核磁共振谱和质谱等)的分析,鉴定了46种化合物的结构,分别属于苯丙素、黄酮、酚酸、三萜及皂苷类等成分,它们分别为：(-)-海松-9(11),15-二烯-19-酸(1)、左旋贝壳杉-16-烯-19-酸(2)、β-谷甾醇(3)、6,7,10-三羟基-8-十八碳烯酸(4)、丁香树脂酚(5)、汉黄芩素(6)、藿香黄酮醇(7)、表儿茶素(8)、异嗪皮啶(9)、香草醛(10)、丹皮酚(11)、没食子酸甲酯(12)、丁二酸(13)、对-香豆酸(14)、咖啡酸(15)、水杨酸(16)、香草酸(17)、丁香酸(18)、胡萝卜苷(19)、扁枝衣二酸(20)、邻甲氧基苯酚-O-β-D-吡喃葡萄糖苷(21)、(E)-3-O-β-D-吡喃葡萄糖基茴香脑(22)、水杨苷(23)、天麻苷(24)、紫丁香苷(25)、地衣酚-O-β-D-吡喃葡萄糖苷(26)、白桦脂酸(27)、绿原酸(28)、山奈酚-7-O-β-D-吡喃葡萄糖苷(29)、奥卡宁-4-甲醚-3′-O-β-D-葡萄糖苷(30)、黄芩苷(31)、金丝桃苷(32)、丁子香酚-O-β-D-吡喃木糖基-(1→6)-β-D-吡喃葡萄糖苷(33)、(E)-白藜芦醇3-O-β-D-吡喃葡萄糖苷(34)、3α-(E)-芥子酰氧基泽兰醇18-O-β-D-吡喃葡萄糖苷(35)、丁香树脂酚-4,4′-二-O-β-D-双葡萄糖苷(36)、牡荆苷(37)、荭草苷(38)、3-O-甲基-(E)-白藜芦醇5-O-β-D-呋喃芹糖基-(1→6)-β-D-[2″-香草酰基]-吡喃葡萄糖苷(39)、3-O-甲基-(E)-白藜芦醇5-O-α-L-吡喃鼠李糖基-(1→6)-β-D-[2″-(E)-阿魏酰基]-吡喃葡萄糖苷(40)、3-O-甲基-(E)-白藜芦醇5-O-β-D-呋喃芹糖基-(1→6)-β-D-[2″-(E)-肉桂酰基]-吡喃葡萄糖苷(41)、3α,11α-二羟基羽扇豆-20(29)-烯-28-酸28-O-α-L-鼠李糖基-(1→4)-β-D-葡萄糖基-(1→6)-β-D-葡萄糖基酯(42)、3β,23,29-三羟基齐墩果-12-烯-28-酸28-O-α-L-鼠李糖基-(1→4)-β-D-葡萄糖基-(1→6)-β-D-葡萄糖基酯(43)、3-O-β-D-吡喃半乳糖基-(1→2)-[β-D-吡喃木糖基-(1→3)]-β-D-葡萄糖醛酸基-2β,3β,16α,23-四羟基齐墩果-12-烯-28-酸28-O-β-D-呋喃芹糖基-(1→3)-β-D-吡喃木糖基-(1→4)-a-L-吡喃鼠李糖基-(1→2)-a-L-阿拉伯糖基酯(44)、3β-O-β-D-葡萄糖基-(1→3)-α-L-鼠李糖基-(1→2)-α-L-阿拉伯糖基金合欢酸28-O-α-L-鼠李糖基-(1→4)-β-D-葡萄糖基-(1→6)-β-D-葡萄糖基酯(45)和3β-O-β-D-葡萄糖基-(1→2)-α-L-阿拉伯糖基齐墩果酸28-O-α-L-鼠李糖基-(1→4)-β-D-葡萄糖基-(1→6)-β-D-葡萄糖基酯(46)。其中35、39、40、41和44为未见文献报道的新化合物,化合物4、6-8、10-14、16、20-22、24、26、29-31、33、34、37和38系首次从该属植物中分离得到,而化合物1、2、5、9、15、17-19、23、27、28、32、42、43、45和46为首次从短柄五加中分离得到。采用超声-微波辅助流动提取技术(UMAFE),分别对短柄五加叶中的总黄酮、总皂苷及茎皮中的多糖类成分进行提取分离。以各类成分的提取率为指标,通过正交试验,考察了各因素对提取率的影响,优选出了最佳提取工艺。研究结果显示,短柄五加中含有丰富的黄酮、皂苷和多糖类成分,其中叶中总黄酮和总皂苷的含量分别为22.95和9.67mg／g,茎皮中多糖的含量为56.37 mg／g。与其它提取方法相比较,用超声-微波辅助流动法来提取短柄五加中的总黄酮、皂苷和多糖类成分,具有提取时间短、提取率高、能耗低、有效成分不易破坏等特点。更多还原

【Abstract】 Acanthopanax brachypus Harms is a small perennial deciduous shrub, which belongs to the genus Acanthopanax (Araliaceae family), and distributed in a narrow geographical area in the world, most in Shanxi, Gansu and Ningxia province on the loess plateau of the Northwest of China. It is a peculiar folk medicinal plant. As medicinal parts, the root and stem bark of this plant have the effect that replenishing qi to invigorate the spleen, raising a heart to calm the nerves, dispelling melancholy, as well as activating the blood and so on. Studies have shown that the rhizome extract have been used for the treatment of neurasthenic, male sexual dysfunction, secondary hypertension, hypotension and leucopenia disease, also have the effect of prevention cancer and anti-cancer. The alcohol extract of bark have anti-inflammatory action, and also inhibit adjuvant arthritis, as well as I, II and III type allergic inflammation.This paper firstly reviewed domestic and international research progress on the chemical constituent and bioactivity of Acanthopanax genus. Based on this, the present situation and exploiting prospect of the resources of Acanthopanax were analyzed. With the methods of tube reaction and thin-layer chromatography, systemic preparative test was used to research the chemical constituent of the bark of Acanthopanax brachypus, to provide the foundation for the designs of extracting and separating scheme on the different active components.The essential oils from the flower, leaves, bark, root and fruit of A. brachypus were individually extracted by hydrodistillation, and their chemical constituents were isolated and characterized by means of gas chromatography-mass spectrum. The relative percentages of the constituent were obtained by peak-area normalization. The in vitro antimicrobial activities of the essential oil were evaluated. The results indicated that the oils from the flower, leaves, bark and root consisted mainly of monoterpene and sesquiterpene hydrocarbons, oxygenated monoterpenes and sesquiterpenes, as well as long chain hydrocarbons and its derivatives and so on, belonged toβ-pinene/linalool/Germacrene-D chemotype, while the fruit oil consisted mainly of fatty acids (esters). The essential oils from the flower and leaves contain various antimicrobial activity constituents, and have the inhibitory effect on 11 tested microorganisms at different degree, especially for gram-bacteria.By the ultrasonic extraction method, non-volatile component was extracted from the ethanol extract of the bark of A. brachypus,52 compounds were isolated by various chroma-tographic (Preparation Thin-Layer chromatography, Silica gel, Macroporous resin and Sephadex column chromatography) and recrystallized methods. On the basis of the analysis of physico-chemical properties and spectral data (UV, IR, NMR and MS), the structure of 46 compounds, which belonging to phenylpropanoid, flavonoid, phenolic acid, triterpene and triterpenoid saponin and so on, were identified as:(-)-pimara-9(11),15-dien-19-oic acid (1), (-)-kaur-16-en-19-oic acid (2),β-sitosterol (3),6,7,10-trihydroxy-8-octadecen oic acid (4), syringaresinol (5), wogonin (6), pachypodol (7), epicatechin (8), isofraxidin (9), vanillin (10), paeonol (11), methyl gallate (12), succinic acid (13), p-coumaric acid (14), caffeic acid (15), salicylic acid (16), vanillic acid (17), syringic acid (18), daucosterol (19), evernic acid (20), 2-methoxyphenyl-O-β-D-glucopyranoside (21), (E)-3-O-β-D-glucopyranosylanethole (22), salicin (23), gastrodin (24), syringin (25), orcinol-O-β-D-glucopyranoside (26), betulinic acid (27), chlorogenic acid (28), kaempferol-7-O-β-D-glucopyranoside (29), okanin 4-methyl ether-3’-O-β-D-glucopyranoside (30), baicalin (31), hyperoside (32), eugenol-O-β-D-xylopyrano syl-(1→6)-β-D-glucopyranoside (33), (E)-resveratrol 3-O-β-D-glucopyranoside (34),3a-(E)-sinapoyloxy-jhanol 18-O-β-D-glucopyranoside (35), (-)-syringaresinol-4,4’-di-O-β-D-glucop yranoside (36), vitexin (37), orientin (38),3-O-methyl-(E)-resveratrol 5-O-β-D-apiofuranosyl-(1→6)-β-D-[2"-vanilloyl]-glucopyranoside (39),3-O-methyl-(E)-resveratrol 5-O-a-L-rhamn opyranosyl-(1→6)-β-D-[2"-(E)-feruloyl]-glucopyranoside (40),3-O-methyl-(E)-resveratrol 5-O-β-D-apiofuranosyl-(1→6)-β-D-[2"-(E)-cinnamoyl]-glucopyranoside (41),3α,11α-dihydro xylup-20(29)-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (42),3β,23,29-trihydroxy-olean-12-en-28-oic acid 28-O-a-L-rham nopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (43),3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucuronopyranosyl-2β,3β,16α,23-tetrahydroxy olean-12-en-28-oic acid 28-O-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl ester (44),3β-O-β-D-glucopyrano syl-(1→3)-a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl acacic acid 28-O-a-L-rhamno pyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (45),3β-O-β-D-gluco pyranosyl-(1→2)-a-L-arabinopyranosyl oleanolic acid 28-O-a-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (46). Among them,35,39,40,41 and 44 are firstly reported as new compounds, compounds 4,6-8,10-14,16,20-22,24,26,29-31,33, 34,37 and 38 were isolated from this genus for the first time, and compounds 1,2,5,9,15, 17-19,23,27,28,32,42,43,45 and 46 were obtained from this plant for the first time.The ultrasonic-microwave assisted-flow extraction (UMAFE) method was used to extract total flavonoid, saponin and polysaccharide from the leaves and bark of A. brachypus, respectively. The various factors were investigated with the extraction efficiency by orthogonal test design. The optimum extracting processes were optimized. The result indicat- ed that A. brachypus contains rich flavonoid, saponin and polysaccharide. The contents of total flavonoid, saponin and polysaccharide were 22.95,9.67 and 56.37 mg/g in the leaves and bark of A. brachypus, respectively. Comparing with the other extraction methods, UMAFE is characteristic of short time, high efficiency, low energy consumption and stable structure.更多还原