基于谱效关系的黑骨藤药效物质辨识研究

发布时间：2018-09-06 17:52

【摘要】：黑骨藤为萝摩科(Asclepiasaceae)杠柳属(Periploca)植物黑龙骨(Periploca forrestii Schltr.)的根及根茎,大部分生长于阴暗潮湿之处,或者有水源的峭壁之上;主要分布于我国西部及南部地区如贵州、广西、四川、青海、西藏等地。黑骨藤全株入药,具有通经、活血、解毒、祛风的功效,民间广泛应用于治疗闭合性软组织损伤、风湿与类风湿等疾病。中药有效物质的辨识一直是中药现代化研究中的重点和难点。为了寻求一种黑骨藤药效物质辨识的方法,本论文在国家自然基金的资助下,于课题组前期对黑骨藤80%乙醇提取部位指纹图谱及谱效关系探索性研究的基础上,开展了相关药效物质辨识研究:通过对分离材料的筛选,优选出大孔树脂为分离材料,对黑骨藤80%乙醇提取部位组分群进行分离制备;并对分离得到的各个组分群进行体外抗过敏、体外抗氧化、乙酰胆碱酯酶抑制活性、细胞毒性等药效学优选研究,从而选出活性较强的组分;然后采用紫外吸收光谱和高效液相色谱法对其进行药效物质辨识研究,结果显示,组分中主要成分为黄酮类化合物;基于药效物质辨识的结果,以黑骨藤总黄酮提取率为指标进行超声预处理微波提取工艺研究,优化提取工艺条件,并进一步对其进行体外抗过敏活性验证。通过上述研究,为黑骨藤及其制剂的深度开发利用和质量控制提供了科学依据和理论基础。主要结果如下:1.黑骨藤有效成分群的分离与富集此部分研究对黑骨藤80%乙醇提取液进行了分离制备:通过预实验得到大孔树脂为最佳分离材料,并对溶剂洗脱系统进行了优化:采用5%、10%、15%、20%、25%、30%、35%至95%乙醇分段洗脱,通过高效液相色谱法检测洗脱液出峰情况,对其进行调整,合并收集相同组分,历时6个月,分离富集了6段黑骨藤组分群A(18 g)、B(8 g)、C(5 g)、D(10 g)、E(10 g)、F(10 g)。根据组分群的高效液相色谱图,与黑骨藤80%乙醇提取部位指纹图谱进行归属分析:指纹图谱上标记了8个特征峰,其归属关系为A(1、2)B(3)C(4、5)D(6)E(7、8)F(30-35 min成分群)。这为下一步组分群的药效研究奠定了基础。2.黑骨藤各组分群的药效学优选研究此部分研究中,以上述富集的黑骨藤80%乙醇提取部位组分群为基础,结合黑骨藤传统药效和现代化药理活性研究进展,选择抗氧化活性(ABTS·+自由基清除能力、DPPH·自由基清除能力)、乙酰胆碱酯酶抑制活性、抗过敏活性、细胞毒性对其进行药效学优选研究。结果显示:ABTS·+自由基清除能力大小:EDBCAFG;DPPH·自由基清除能力大小:EFDCABG;乙酰胆碱酯酶抑制活性大小:EDFBACG;体外抗过敏活性大小为EACFBDG;对MH7A类风湿关节炎细胞毒性大小为EABCDGF。课题组前期通过谱效关系研究推测的抗炎有效物质峰为2、6、8号,包含于A(1、2)、D(6)E(7、8)中,在此部分试验中,证实了2、8号峰位于的A、E段拥有较好的活性;在体外抗氧化与体外乙酰胆碱酯酶抑制活性中,E段的活性都为最强,因此,E段组分群可作为后续药效物质辨识研究的对象。3.黑骨藤E段组分群药效物质辨识研究此部分研究以活性最优的E段组分群为对象,采用紫外吸收光谱法和高效液相色谱法对其进行药效物质辨识研究。E段高效液相色谱图中主要有5个特征峰,通过样品与对照品紫外吸收波长和高效液相色谱图保留时间对比,指认了两个黄酮类化合物,分别是槲皮素-3-O-β-D-吡喃葡萄糖苷和槲皮素-3-O-α-L-吡喃阿拉伯糖苷。4.黑骨藤总黄酮提取工艺研究前述药效学优选出来活性最佳的E段组分群中含有黄酮类化合物,这为黑骨藤的进一步开发利用提供了科学参考。本项研究中,采用超声预处理微波提取技术,通过单因素实验筛选出超声预处理最佳时间25 min,运用中心组合(BBD)响应面法,以黑骨藤总黄酮提取率为指标优化提取工艺参数:微波功率(W)、乙醇浓度(%)、液料比(m L·g-1)及微波时间(s),通过实验得到最佳提取工艺参数为:乙醇浓度59.92%、液料比21.24 m L·g-1、微波时间为209.53 s、微波功率274.14 W。在此工艺参数下,黑骨藤总黄酮提取率为9.11±0.08%,与微波提取法相比增加了14.30%,与超声波提取法相比增加了19.86%;通过聚酰胺纯化后,超声预处理微波提取的黑骨藤总黄酮的抗过敏活性半抑制率为IC50=1.033 mg·m L-1,分别优于超声提取法(IC50=12.913 mg·m L-1)与微波提取法(IC50=1.762 mg·m L-1)。
[Abstract]:The roots and rhizomes of Periploca forrestii Schltr. grow mostly in shady and humid places or on cliffs with water sources. They are mainly distributed in Western and southern China, such as Guizhou, Guangxi, Sichuan, Qinghai and Tibet. In order to find a method for identifying the effective substances of the Chinese herbal medicine, this paper, with the support of the National Natural Fund of China, has been widely used in the treatment of closed soft tissue injury, rheumatism and rheumatoid diseases. On the basis of exploratory study on fingerprint and Spectrum-Effect relationship of 80% ethanol extract part of Celastrus vicina, the research group carried out identification of related pharmacodynamic substances. Through screening the separation materials, macroporous resin was selected as the separation material, and the fractions of 80% ethanol extract part of Celastrus vicina were separated and prepared. The constituents were selected by the pharmacodynamic optimization of anti-allergy, anti-oxidation, acetylcholinesterase inhibitory activity and cytotoxicity in vitro, and then identified by ultraviolet absorption spectroscopy and high performance liquid chromatography. According to the results of identification of the effective substances, the extraction rate of total flavonoids from Celastrus Vicia was used as the index to study the ultrasonic pretreatment microwave extraction process, optimize the extraction process conditions, and further verify its anti-allergic activity in vitro. The main results are as follows: 1. Separation and enrichment of the effective components of the vine were studied. The 80% ethanol extract of the vine was separated and prepared. The porous resin was obtained as the best separation material by preliminary experiment, and the solvent elution system was optimized: 5%, 10%, 15%, 20%, 25%, 30%, 35% to 95% ethanol was eluted by stages. The eluent peaking was detected by high performance liquid chromatography (HPLC), and the eluent was adjusted to collect the same components. After six months, the eluent components A (18 g), B (8 g), C (5 g), D (10 g), E (10 g), F (10 g) were separated and enriched. According to the high performance liquid chromatogram of the eluent group, the fingerprints of the eluent and 80% ethanol extract fraction of Celasted for six months. Eight characteristic peaks were labeled on the stripe map, and the attribution relationship was A(1,2)B(3)C(4,5)D(6)E(7,8)F(30-35 min constituent group). This laid a foundation for the further study on the pharmacodynamics of each constituent group. 2. The pharmacodynamics optimization of each constituent group was studied in this part. The antioxidant activity (ABTS (+) radical scavenging capacity, DPPH (+) radical scavenging capacity), acetylcholinesterase inhibitory activity, anti-allergic activity and cytotoxicity were selected to optimize the pharmacodynamics of Guteng. Scavenging capacity: EFDCABG; acetylcholinesterase inhibitory activity: EDFBACG; in vitro antiallergic activity: EACFBDG; cytotoxicity to MH7A rheumatoid arthritis: EABCDGF. The peak values of anti-inflammatory active substances predicted by the study group were 2,6,8 and included in A (1,2), D (6) E (7,8). The results showed that the A and E segments of the 2 and 8 peaks had good activity, and the E segment had the strongest activity in vitro and in vitro, so the E segment group could be used as the object of follow-up study on identification of effective substances. Two flavonoids, quercetin-3-O-beta, were identified by ultraviolet absorption spectroscopy and high performance liquid chromatography. Five characteristic peaks were identified in the E-segment high performance liquid chromatogram. Study on the extraction technology of total flavonoids from Celastrus nigra L. The flavonoids in E-segment group with the best pharmacodynamics were selected, which provided a scientific reference for the further development and utilization of Celastrus nigra L. In this study, ultrasonic pretreatment microwave extraction technology was used. The optimum time of ultrasonic pretreatment was 25 minutes by single factor experiment. The extraction parameters of total flavonoids were optimized by using central combination (BBD) response surface methodology with the extraction rate of total flavonoids as the index: microwave power (W), ethanol concentration (%), liquid-to-material ratio (m L.g-1) and microwave time (s). The optimum extraction parameters were obtained by experiment as follows: ethanol concentration 59. 92%, liquid-to-material ratio 21.24 m L g-1, microwave time 209.53 s, microwave power 274.14 W. Under these parameters, the total flavonoids extraction rate was 9.11 0.08%, 14.30% higher than that of microwave extraction method, and 19.86% higher than that of ultrasonic extraction method. The semi-inhibitory rate of allergic activity was IC50=1.033 mg/ml-1, which was superior to ultrasonic extraction (IC50=12.913 mg/ml-1) and microwave extraction (IC50=1.762 mg/ml-1).
【学位授予单位】：贵州师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657;R284.1

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