陕西槐角的资源、品质、化学成分及其生物活性研究

【作者】 王剑波；

【导师】 曾成鸣；

【作者基本信息】 陕西师范大学 ， 植物学， 2012， 博士

【摘要】 槐角(Sophorae Fructus)为豆科植物(Leguminosae sp.)槐(Sophora japonicaL.)的干燥成熟果实。是我国常用的传统中药之一,具有清热泻火,凉血止血的功效,用于肠热便血,痔肿出血,眩晕目赤等症。为《中华人民共和国药典》历版收载的品种。槐角富含黄酮、异黄酮类等多种成分,药理作用显著,临床应用广泛,具有极好的应用前景和开发前途。陕西为槐角的主产区,槐角多处于自然生长状态,分布广,来源复杂,对槐角相关的生物学特性、资源、品质、成分和生物活性等尚不十分清楚。因此,对陕西境内槐角的综合研究需要在已有基础上,进行更深入的物效基础和生物活性研究,扩大应用范围,为槐角的合理利用提供科学依据。本课题应用植物资源学、植物化学、药理学和分子生物学等多种技术方法,从资源、品质、化学成分和生物活性四个方面对陕西槐角进行了较系统的研究,包括对陕西境内槐角进行了实地资源调查,分析了不同来源槐角的品质,考察了槐角总黄酮栓的抗炎止血活性、槐角苷的提取分离、纯化、结构鉴定及其药代动力学特点、染料木黄酮对海马神经元的保护作用以及槲皮素对胰岛素淀粉样纤维形成的影响,在整体动物、细胞和分子水平上探讨了槐角活性成分的作用及其机制,以期为进一步开发陕西的槐角资源提供实验依据。研究的内容和结果如下：1.槐角资源调查：在查阅相关文献资料和核对标本的基础上,采用走访调查和实地调查相结合的方法进行槐角资源调查。结果显示：槐角的资源树种槐在陕西境内分布较广泛,多处于野生状态,其生长的植被主要为农耕地、荒地、田埂、道沿和部分天然草本群落。伴生植物主要为人工栽培的农作物、以及村镇林木,零星散在苜蓿、铁杆蒿、白羊草等草本。种质资源类型有白槐与黑槐之分,白槐是栽培的主要树种。样品区树龄均>20年,单株平均产量>200Kg,槐角在全省自然蕴藏量估算可达2100吨/年。随着城市化进程加快,槐角的原植物发生多方面的变化,包括成年植株移栽数量巨大、产量锐减(年递减率达到13%)、变种增多,这些都会影响到槐角的品质,因此需要加强野生槐角种质资源的收集、保存,并保护好现有资源。2.槐角品质评价：通过建立UV法和RP-HPLC法,分别测定了陕西不同地区槐角样品中总黄酮和槐角苷的含量,并检查了槐角中灰分和重金属含量,结合小鼠雌激素样作用考察,共同评价槐角的品质。结果显示,采用8倍量65%乙醇回流提取3次,每次1小时,为总黄酮提取的最佳条件,按此条件提取槐角总黄酮,平均得率在10.92-20.18%之间。槐角苷含量在4.21-8.05%之间。不同区域的槐角品质有一定差异,宝鸡陇县槐角中槐角苷含量最高,而杂质及重金属以延安宜川为最低。陕北黄土丘陵沟壑区9月的槐角样品中总黄酮含量较高；渭北黄土高原沟壑区则10-11月槐角中总黄酮含量较高。槐角提取物显示了一定的弱雌激素样作用,与空白对照组相比,在第3天就能促进小鼠阴道的开口数量,第7天达到54%,并能显著增加小鼠卵巢和子宫的重量(*P<0.05或**<0.01),其中延安宜川、渭南蒲城的槐角提取物雌激素样作用较强。3.槐角黄酮栓生物活性评价：槐角黄酮栓在一定程度上可减轻二甲苯所致小鼠耳廓肿胀,其中0.60g/kg及0.30g/kg在给药6d、9d、14d对二甲苯引起的小鼠耳廓肿胀有显著的抑制作用,与模型组相比具有极显著差异(**P<0.01),并表现出一定的量效关系。各剂量组均能明显减轻小鼠滤纸肉芽组织重量(**P<0.01),其中高剂量组抑制率在50%以上,与阳性药物阿司匹林组(0.50g/kg)作用接近。黄酮栓各剂量组对醋酸所致小鼠腹腔毛细血管通透性增高具有明的抑制作用(*P<0.05或**P<0.01),并表现出一定的量效关系,但弱于阿司匹林栓组。槐角黄酮栓能显著缓解角叉菜胶引起的大鼠足肿胀,与模型组比较有统计学意义(*P<0.05或**P<0.01)。从致炎后2h开始即显示有抑制作用,持续到4h时抑制作用更为明显,抑制率在40%以上。槐角黄酮栓也能缩短正常小鼠的出、凝血时间,与空白对照组比较差异明显(*P<0.05或**P<0.01)表明其具有促进止血、加强凝血的作用。槐角黄酮栓能使10%醋酸所致大鼠直肠渗出液减少,溃疡面积缩小,溃疡愈合时间缩短,与模型对照组比较,具有显著性差异(*P<0.05或**P<0.01),证明槐角黄酮栓具有抗直肠溃疡,保护创面的药理作用,有望开发成治疗痔疮的新药。4.槐角苷的分离、纯化、鉴定及药代动力学研究：建立了槐角中槐角苷含量测定的HPLC法,色谱条件为,流动相：甲醇-乙腈-10mL·L-1冰乙酸(40：5：55)；流速：1.0mL/min,检测波长：260nm,进样量为20gL,最低检出浓度为0.015μg/mL。该法灵敏、准确、可靠。采用正交试验法对槐角的提取工艺进行了考察,结果最佳提取条件为95%乙醇8倍量(W/V),提取3次,每次1.5小时。经对三批1000g槐角样品经过醇提、水沉淀、乙酸乙酯除杂、水洗步骤处理后,获得槐角苷的粗品,收率分别为1.326%、1.258%、1.203%；纯度分别为：85.21%、85.08%、84.46%。在此基础上,运用大孔树脂吸附的方法纯化槐角苷粗提物,考察了吸附条件,结果洗脱剂浓度为80%乙醇、流速以2BV/h,上样液质量浓度在1.0mg/mL能使槐角苷粗品的纯度由84.92%提高到了91.98%。采用甲醇热溶,丙酮沉淀、反复析晶的方法,获得了纯度>97%的槐角苷单体,通过理化性质和波谱学研究,证明获得的提取物为5,7-二羟基异黄酮-4’-O-葡萄糖苷,即槐角苷。首次建立HPLC测定大鼠血浆中槐角苷的定量分析方法,并通过尾静脉注射给药的方式来研究血药浓度变化规律,结果各取样时间点雌性大鼠血浆中原形药物的浓度要高于雄性大鼠,Cmax=2.531mg/L(雌)、Cmax=1.648mg/L(雄)。槐角苷雌性大鼠的达峰时间Tmax=10min,雄性大鼠Tmax=7min,雌性大鼠对药物的吸收速度明显要高于雄性大鼠。槐角苷原形药物在血浆中的代谢过程符合一室模型。大鼠槐角苷半衰期为t1/2=55.70min(雌),半衰期t1/2=23.14min(雄),说明槐角昔在雌性大鼠吸收、代谢均比雄性大鼠要慢。5.染料木黄酮对Aβ25-35引起海马神经元损伤的拮抗作用及机制研究：在获得槐角苷单体的基础上,用含10%HCl的50%乙醇在90℃热回流酸水解4h,10mol/L的NaOH溶液调pH为7,乙醚萃取,水洗,80℃干燥,甲醇重结晶,可以得到染料木黄酮单体,通过理化性质和波谱鉴定为5,7,4’-三羟基异黄酮,HPLC检测纯度>98%。以原代培养的大鼠海马神经元为研究对象,应用Aβ25-35处理海马神经元,观察从槐角中提取的染料木黄酮对Aβ25-35毒性的拮抗作用,结果显示：染料木黄酮能够显著改善Aβ25-35诱导引起的海马神经细胞形态的改变,减轻细胞膜的损伤,对抗Aβ引起的海马神经元活性降低,提高细胞的存活率,减少氧化损伤所致的细胞凋亡。与雌二醇相比,两者抗Aβ神经毒性作用的效果类同。染料木黄酮能抑制caspase-3酶活性,提高抑凋亡分子Bcl-2表达,对抗ROS和MDA的改变,提示染料木黄酮发挥神经保护作用可能与其抗氧化作用有关。此外,染料木黄酮还能拮抗Aβ所致的nAChR亚单位α7mRNA、蛋白表达的下调,使得海马细胞正常代谢活动得以维持,从而起到神经保护作用。6.槲皮素对牛胰岛素蛋白淀粉样纤维化的抑制作用研究：采用牛胰岛素作为模式蛋白,通过体外孵育,综合采用ThT荧光检测、透射电子显微镜观察、蛋白沉积率测定、溶血实验和蛋白凝胶电泳分别考察了槐角中提取的槲皮素抗蛋白淀粉样纤维形成的作用及其机制。结果显示,当浓度为25gg/mL或50μg/mL的槲皮素与胰岛素进行共孵育时,ThT荧光强度出现明显的剂量依赖减弱,使胰岛素纤维的ThT荧光特征S曲线消失。说明槲皮素对胰岛素淀粉样蛋白纤维形成具有良好的抑制效果。而用不同浓度(10、25、50μg/mL)的槲皮素处理成熟的胰岛素纤维,可以使胰岛素纤维的形貌受到破坏,电镜下观察为无具体形貌的小颗粒或无定形聚合物。用40μg/mL的槲皮素进行培育时,13%的胰岛素纤维在4h内转化成了无定形沉积物,槲皮素对胰岛素纤维抑制表现的沉积效应呈现出明显的时间和剂量依赖性。当与槲皮素进行同孵育后,由同等剂量胰岛素纤维引起的溶血现象受到抑制,并呈剂量依赖性的下降。凝胶电泳研究证明,胰岛素纤维可诱导血影蛋白质的聚集,而槲皮素减弱了胰岛素纤维诱导的高分子量聚合物的形成。疏水作用、芳香氨基酸重迭以及氢键结合可能是槲皮素发挥其抗淀粉样纤维化和破坏纤维形成作用的驱动力。更多还原

【Abstract】 As one of the most commonly used Chinese traditional medicine, Sophorae Fructus refers to the fruits from Sophora japonica L. which belongs to Leguminosae sp. After being dried, Sophorae fructus has the function of clearing heat-fire and cooling blood and hemostasis, and it is widely used to cure enteric fever hemafecia, bleeding hemorrhoids and vertigo red eye and so on. The fruit, as a kind of medicine, has been written in every edition of Pharmacopoeia of The People’s Republic of China. Containing several ingredients of flavones and isoflavones, the medicine has shown an outstanding pharmacological action and has been widely used in clinic, which indicates its value in future research and application. Shaanxi Province is the main producing area of sophorae fructus. As the plant usually grows in a natural way with features of wide distribution and complicated sources, the related biological characteristics, sources, quality, ingredients and biological activity of the medicine still remain unclear. Therefore, the study of this medicine will be based on the existed results to deepen the research on its Physical effect of foundation and biological activity, so as to enlarge the field of application and provide scientific prove for the rational use of sophorae fructus. This thesis is to apply several technologies and methods including plant resources, phytochemistry, pharmacology, and molecular biology, in order to conduct a systematic research on Shaanxi sophorae fructus from four aspects of source, quality, chemical ingredients and biological activity. The research also includes the investigation on the wild sophorae fructus in Shaanxi. Through analyzing the quality of the plant from different sources, we tested the antiphlogistic hemostatic activity of the sophorae fructus flavonoids suppository, observed the extraction, separation, purification, and structure analysis process of sophoricoside, and found out its pharmacokinetic characteristics, the protection effect of genistein on hippocampal neurons, and the influence of quercetin on insulin fibrils. This thesis will discuss on the overall level of animal, cell and molecular level the function and medicine process of the active constituent of sophorae fructus and finally to provide experimental evidence for further development of Shaanxi’s sophorae fructus.The contents and results are following: 1. Sophorae Fructus Source Investigation:The relevant literature and data of Sophorae Fructus were scrutinized and herbarium specimens were compared, interview and field survey methods were carried out. According to the investigation, the sources of sophorae fructus are widely distributed in Shaanxi Province and remain in wild state, which include natural herbs found mainly in farmland, uncultivated land, balk, or along the roads. Its associated plants are artificial cultivated crops, forest tree in villages, and a few herbs of M.sativa, artemisia gmelinii, and bothriochloa ischaemum. There are white and black Sophora japonica. L divided by their quality type in which the white one is considered the main seeding tree for sophorae fructus. In the sample area, the age of the tree is over twenty years, and the average production of each plant is over200kilogram. The natural reserved quantity of the fruit is estimated to reach2100ton per year. With the speed up of the urbanization, the original source of the fruit has changed in many ways including the transplant of a great amount of adult trees, sharply declining of the production (the annual declining rate is13%), the increasing of the varietas of the tree. These factors are affecting the quality of sophorae fructus and we need to make efforts to collect and preserve the fine quality seed resource of the plant and protect the existing sources.2. Quality Assessment of Sophorae Fructus:the application of the methodology of UV and RP-HPLC makes possible the determination of the total flavonoid content and the sophoricoside content as well as the examination of the ash content and the heavy metal content in the sophorae fructus samples elicited from different regions of Shaanxi province. The results of the determination and examination together with that of the investigation of the estrogen-like effect in mice formed the foundation for the quality assessment of sophorae. The research results show that the optimal process for the extraction of total flavonoids is adding eight-time-amount of ethanol in the reflux extraction for three times (one hour per time). In this case, the average yield of the total flavonoid content ranges from10.92%to20.18%, while the sophoricoside content is in-between4.21%and8.05%. The quality of sophorae fructus varies in different regions. Amongst the samples, the maximum sophoricoside content resides in the sophorae fructus in Bao Ji, while the minimum impurity and heavy metal exists in the sophorae fructus in Yichuan County in Yan’an. Comparatively speaking, higher level of total falvonoid content can be found in the sophorae fructus samples obtained in September from the gully regions of the Loess Plateau in Northern Shaanxi and of the hilly areas to the north of Weihe River, and in those obtained in October and November from the flatland and hilly regions to the north of Weihe River. Compared with the blank control group, the sophorae fructus extract shows certain weak estrogen-like effect in mouse. The number of the mouse’s vaginal opening has been increased in the first three days of the experiment, and the proportion amounted to54%on the seventh day. Meanwhile, the weight of the mouse’s ovary and uterus has also been increased significantly (*P<0.05or**P<0.01). The research has it that the sophorae fructus extract obtained from Yichuan County in Yan’an and Pucheng County in Weinan has a comparatively strong estrogen-like effect.3. Evaluation of Biological Activity of Sophorae Fructus Flavonoids Suppository:Sophorae Fructus Flavonoids Suppository (SFFS) can release on some extent the swelling auricle of the mice caused by xylol. specifically,0.60g/kg and0.30g/kg medicine were given to mice6d,9d,14d and showed an obvious restrain effect on the swelling auricle, and the result was different compared with the model (**P<0.01). It also reflected certain relations between the dosage and the effect. Every dose group caused the lightening of the filter paper granulation tissue (**P<0.01), in which the high dose groups have an inhibition ratio of over50%whose effect is close to positive drug aspirin group (0.50g/kg). The entire SFFS dose groups showed clear restrains effect for capillary permeability caused by acetic acid on mice cavum abdominis (*P<0.05or**P<0.01), and reflected certain dose-effect relationship, which is weaker than the aspirin bolt group. The SFFS in the medicine can release the foot swelling of the rats caused by carrageenin and means valuable in statistics as well compared to model group (*P<0.05or**P<0.01). The experiment took effect in2hours after the inflammation and the restrain effect became more obvious after4hours with an inhibition ratio of over40%. The medicine can also shorten the bleeding and coagulating time of the normal mice. The difference (*P<0.05or**P<0.01) between the group and the blank group indicates the medicine has the effect on stanching bleeding. The bolt can reduce the rectal exudates of rats caused by10%acetic acid, reduce the ulcer area, which showed an obvious difference with the model group (*P<0.05or**P<0.01). These experiments proved that the SFFS has pharmacological action to control intestinal ulcer and protect the wound, therefore, it can be developed as a new medicine for haemorrhoids.4. Separation, purification, testing, and pharmacokinetics study of sophoricoside: We established a method to measure the contents of sophoricoside in sophorae fructus under the chromatographic condition, the mobile phase was composed of methanol-acetonitrile-10mL-L-1glacial acetic acid(40:5:55); at a flow rate of1.0mL/min, The detector wavelength was260nm, and sample size was20μL, the lowest detectable concentration was0.015μg/mL,This method has been proved to be reliable and accurate. Extraction processes were studied by orthogonal design, the optimal process was performed by1.5-hour extraction for three times, with8-fold volume of95%ethanol (W/V), the raw sophoricoside will be obtained after alcohol extraction, water sedimentation, ethyl acetate purification and washing the1000g sophorae fructus in three batches of samples. the extraction rate of sophoricoside was1.326%、1.258%、1.203%, and the purity was85.21%、85.08%、84.46%respectively. Based on this experiment, we applied macroporous resin adsorption to purify the raw sophoricoside and tested the adsorption condition. As a result, the eluant potency is80%ethanol with the flow speed of2BV/h, and the sample solution potency is1.0mg/mL. Under the above condition, the purity of the raw sophoricoside was improved from84.92%to91.98%. Then we used methods of methanol hot dissolve, acetone precipitation, and repeated crystallization to extract sophoricoside monomer with the purity of over97%. Through physicochemical property and spectral identification, the monomer was proved to be5,7-Dihydroxy isoflavones-4’-O-glucoside which is sophoricoside. For the first time we established HPLC, a method of quantitative analysis to measure the amount of sophoricoside in the rats’blood plasma, then found out the rules of blood drug level change through tail intravenous injection. The results indicate that the prototype drug potency in female rats’plasma is higher than that in male rats at every sampling point-Cmax=2.531mg/L (female rats) and Cmax=1.648mg/L (male rats). Tmax of sophoricoside in female rats is in10minutes, while in male rats the time is7minutes. The uptake speed of female rats to the medicine are higher than the male. The metabolic process of prototype drug in blood plasma meets a criterion with one compartment model. The sophoricoside in rats has a half-life period of t1/2=55.70min (female) and t1/2=23.14min (male), which means the absorbing and metabolizing of sophoricoside in female rats are slower than that in male rats.5. The Antagonism and Mechanism Research on Genistein in Fructus Sophorae to Cure Damage of Hippocampus Neurons Caused by Aβ25-35:With obtaining the sophoricoside monomer at first place, hydrolyzing the50%alcohol (with10%HC1 inside) in90℃thermal reflux AW (acid water) for4hours, adjusting the pH of NaOH liquor of10mol/L to7, extracting the alcohol, washing, desiccating in80℃and making methanol recrystallization, we will get genistein monomer (5,7,4’-trihydroisoflavone through the physical and chemical properties and spectral identification and the HPLC purity test is above98%). As the research object to the hippocampus neurons of primary cultured rat, after using Aβ25-35to deal with hippocampus neurons and observing the antagonism of hippocampus neurons extracting form sophorae fructus to cure cytotoxic of Aβ25-35, it shows that genistein can effectively improve the morphology change of hippocampus neurons caused by inducing of Aβ25-35, reduce the damage of cell membrane, resist the activity reduction of hippocampus neurons caused by Aβ25-35, improve the survival rate of cells and reduce the apoptosis caused by oxidative injury. Comparing with the estradiol, both have the similar effect for resisting toxic effect of A(3. Genistein can restrain the enzyme activities of caspase-3, improve the impression of Inhibitory molecules apoptosis Bcl-2, and resist the change of ROS and MDA. The protection function of genistein may connect with its antioxidant effect, In addition, genistein can resist the downregulation of nAChR subunit a7mRNA and protein expression caused by Aβ, maintain the normal metabolization activity of hippocampus neurons, and thus exert neuroprotective effects.6. The Effect of Quercetin in Sophorae to restrain Bovine Insulin Protein Amyloid Fibrosis: The experiment applied bovine insulin as model protein to detect with ThT fluorescence, observe from transmission electron microscope, test the protein deposition rate, and conduct the hemolytic test and protein gel electrophoresis. Through vitro incubation, we studied the mechanism and effect of quercetin anti protein amyloid fibrils extracted from sophorae fructus. The experiment displayed that when insulin and quercetin (potency of25μg/mL or50μg/mL) were incubated together, the ThT fluorescence became weaker with the change of the dose and the S curve of insulin fibrils faded, which indicates the effect of quercetin to restrain the formation of insulin amyloid fibrils. However the quercetin of different potencies (10,25,50μg/mL) can damage the appearance of the insulin fibrils when processing it. Observed from the electric mirror, it became granulum and amorphous polymers without specific pattern. Bred by40μg/mL quercetin,13%insulin fibrils turned in to amorphous sediments within4hours. The deposition effect showed an obvious dependency on time and dosage. After the incubation, hemolysis caused by insulin fibrils of the same dosage was released with the dosage. According to the results of gel electrophoresis, insulin fibrils may lead to the gathering of spectrin proteins, while the quercetin weakened the formation of heavy polymer caused by insulin fibrils. The motivations of quercetin to develop its effect to resist amyloid fibrosis and destroy the fibrils formation might be hydrophobic interaction, aromatic amino acid overlap and hydrogen bonding.更多还原