珠子参化学成分及生物活性研究

发布时间：2018-05-20 21:56

本文选题：珠子参 + 化学成分　；参考：《吉林大学》2017年博士论文

【摘要】：珠子参(Panacis majoris Rhizoma)为五加科(Araliaceae)人参属(Panax)植物珠子参(Panax japonicus C.A.Mey.var.major(Burk.)C.Y.Wu et K.M.Feng)或羽叶三七(Panax japonicus C.A.Mey.var.bipinnatifidus(Seem.)C.Y Wu et K.M.Feng)的干燥根茎,具有抗肿瘤、抗血栓、提高免疫力等药理作用。珠子参的化学成分主要为三萜皂苷,但与人参、西洋参、三七等同属植物相比,珠子参的研究报道较少,其化学成分及药理作用的研究尚存在许多空白。为完善珠子参的质量标准,研究其药效物质基础,进而开发其新的医药用途,本文对珠子参的化学成分与生物活性进行了研究。本文首先使用硅胶柱层析、ODS柱层析、制备色谱等分离方法并结合MS、1D、2D NMR等波谱学手段从珠子参乙醇提取物中分离并鉴定了42个单体化合物,其中包括6个新化合物和16个首次从珠子参中分离出的化合物。新化合物分别为(20S,24S,25R*)-6-O-[β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖基]-达玛-20,24-环氧-3β,6α,12β,25,26-五醇(1)、(20S,24R,25R)-6-O-[β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖基]-达玛-20,24-环氧-3β,6α,12β,25,26-五醇(2)、(20S)-6-O-[β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖基]-达玛-20,25-环氧-3β,6α,12β,24α-四醇(3)、6-O-[β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖基]-达玛-3β,6α,12β,20S,24R,25-六醇(4)、6-O-[β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖基]-达玛-25(26)-烯-3β,6α,12β,20S,24R-五醇(5)和3-O-[β-D-吡喃葡萄糖基-(1→2)-β-D-(6′-O-乙基)-吡喃葡萄糖醛基]-齐墩果酸-28-O-β-D-吡喃葡萄糖苷(6)。首次分离出的化合物分别为齐墩果酸-3-O-α-L-呋喃阿拉伯糖基-(1→4)-β-D-吡喃葡萄糖醛酸苷(12)、3-O-{β-D-吡喃葡萄糖基-(1→2)-[α-L-呋喃阿拉伯糖基-(1→4)]-β-D-吡喃葡萄糖醛基}-齐墩果酸-28-O-β-D-吡喃葡萄糖苷(18)、6″-O-乙酰基人参皂苷Rb1(20)、越南皂苷R4(21)、6″′-O-乙酰基人参皂苷Re(27)、拟人参皂苷RT2(32)、拟人参皂苷F11(33)、越南皂苷R8(34)、三七皂苷E(35)、越南皂苷R9(36)、越南皂苷R13(37)、ginsenoside I(38)、人参皂苷Re5(39)、ginsenjilinol(40)、西洋参皂苷L11(41)、yesanchinoside R2(42)。所分离出的化合物分别为竹节参皂苷V(7)、人参皂苷Ro甲酯(8)、齐墩果酸-3-O-β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖醛酸苷(9)、竹节参皂苷IV(10)、araloside A methyl ester(11)、pseudoginsenoside RT1(13)、pseudoginsenoside RT1 methyl ester(14)、竹节参皂苷IVa(15)、竹节参皂苷IVa甲酯(16)、齐墩果酸-28-O-β-D-吡喃葡萄糖苷(17)、人参皂苷Rb1(19)、人参皂苷Rf(22)、20-葡萄糖人参皂苷Rf(23)、三七皂苷R2(24)、三七皂苷R1(25)、人参皂苷Re(26)、人参皂苷Rd(28)、珠子参苷R1(29)、24(R)-珠子参苷R1(30)和珠子参苷R2(31)。上述分离出的皂苷类成分按苷元结构不同,分为齐墩果烷型皂苷、达玛烷型皂苷和奥克梯隆型皂苷。为明确上述不同类型的皂苷类成分在珠子参中的含量,本文分别建立了三种利用新的前处理手段的HPLC检测方法。(1)首次使用离子液体双水相结合HPLC法对珠子参中5种齐墩果烷型皂苷进行了提取与含量测定。通过实验条件的优化,最终建立了0.2 g珠子参样品,3.0 m L去离子水,0.5m L[Hmim]Br离子液体,2.5 g K2HPO4的样品前处理方法。该方法的检出限在0.31~0.88μg·m L-1之间,稳定性实验的RSD值在0.26%~2.69%之间,回收率在90.00%~106.00%之间,显示该方法灵敏度高、稳定性和回收率良好,适用于测定珠子参中齐墩果烷型皂苷的含量。在此条件下,测得5种齐墩果烷型皂苷的含量分别为47.10 mg·g~(-1)、24.00 mg·g~(-1)、0.35 mg·g~(-1)、0.17 mg·g~(-1)和0.44 mg·g~(-1);(2)首次使用离子液体提取-SPE富集结合HPLC法对珠子参中5种达玛烷型皂苷进行了提取与含量测定。通过实验条件的优化,最终建立了50.0 mg珠子参样品,0.8 m L去离子水,0.2 m L[Bmim]Br离子液体,3.0 m L 60%丙酮-水溶液洗脱的样品前处理方法。该方法的检出限在0.43~0.90μg·m L-1之间,稳定性实验的RSD值在0.63%~1.73%之间,回收率在92.00%~106.40%之间,显示该方法灵敏度高、稳定性和回收率良好,适用于测定珠子参中达玛烷型皂苷的含量。在此条件下,测得5种达玛烷型皂苷的含量分别为92.0μg·g~(-1)、106.0μg·g~(-1)、100.0μg·g~(-1)、33.0μg·g~(-1)和470.0μg·g~(-1);(3)首次使用基质固相分散提取结合HPLC-ELSD法对珠子参中5种奥克梯隆型皂苷进行了提取与含量测定。通过实验条件的优化,最终建立了以硅藻土为分散剂,甲醇为洗脱剂的样品前处理方法。该方法稳定性实验的RSD值在0.57%~1.96%之间,回收率在85.71%~103.00%之间,显示该方法灵敏度高、稳定性和回收率良好,适用于测定珠子参中奥克梯隆型皂苷的含量。在此条件下测得5种奥克梯隆型皂苷的含量分别为916.0μg·g~(-1)、35.7μg·g~(-1)、720.0μg·g~(-1)、19.2μg·g~(-1)和12.9μg·g~(-1)。上述三种类型的皂苷中,含量最高的是齐墩果烷型5种皂苷,在珠子参中总含量达到7.02%,远高于其他两种类型。与传统方法相比,上述三种样品前处理方法均具有便捷、快速、环保等优点,适于珠子参中有效成分的快速提取与准确分析。据文献报道,齐墩果烷型皂苷当C3位连糖C28位不连糖时其活性最强,据此,我们推测将C3,C28-双糖链齐墩果烷型皂苷降解成C3-单糖链皂苷后其药理活性有可能增强。为此本文对珠子参总皂苷进行了降解并对降解产物的化学成分进行了分离与结构鉴定,同时对降解前后的总皂苷的生物活性进行了对比研究。首先利用大孔吸附树脂法制备珠子参总皂苷(PJTS),经HPLC分析可知,PJTS中的主要成分为5种C3,C28-双糖链齐墩果烷型皂苷,它们分别为竹节参皂苷IVa(I)、竹节参皂苷IV(II)、pseudoginsenoside RT1(III)、竹节参皂苷V(IV)和3-O-{β-D-吡喃葡萄糖基-(1→2)-[α-L-呋喃阿拉伯糖基-(1→4)]-β-D-吡喃葡萄糖醛基}-齐墩果酸-28-O-β-D-吡喃葡萄糖苷(V),它们在PJTS中含量分别为168.1mg·g~(-1)、115.7 mg·g~(-1)、65.4 mg·g~(-1)、270.9 mg·g~(-1)和79.3 mg·g~(-1),总含量达到69.94%。接着采用碱降解的方法对上述珠子参总皂苷进行了降解,获得了珠子参总皂苷降解产物(DPJTS)。化学成分研究结果表明,上述5种C3,C28-双糖链齐墩果烷型皂苷(化合物I~V)完全降解为C3-单糖链齐墩果烷型皂苷,它们分别为齐墩果酸-3-O-β-D-吡喃葡萄糖醛酸苷(P-1)、齐墩果酸-3-O-α-L-呋喃阿拉伯糖基-(1→4)-β-D-吡喃葡萄糖醛酸苷(P-2)、齐墩果酸-3-O-β-D-吡喃木糖基-(1→2)-β-D-吡喃葡萄糖醛酸苷(P-3)、齐墩果酸-3-O-β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖醛酸苷(P-4)和齐墩果酸-3-O-β-D-吡喃葡萄糖基-(1→2)-[α-L-呋喃阿拉伯糖基-(1→4)]-β-D-吡喃葡萄糖醛酸苷(P-5),它们在DPJTS中的含量分别为128.1mg·g~(-1)、99.7 mg·g~(-1)、39.2 mg·g~(-1)、302.6 mg·g~(-1)和63.7 mg·g~(-1),总含量达到63.33%。接着利用大鼠心肌缺血再灌注损伤模型,对比考察了PJTS及DPJTS对大鼠心肌缺血再灌注损伤(MIRI)的保护作用,并初步探讨了其作用机制。实验结果显示,PJTS及DPJTS对大鼠实验性心肌缺血再灌注损伤均具有保护作用,其机制可能与抑制血小板黏附、聚集,抑制血液高黏滞状态及增强机体的抗氧化能力有关。DPJTS的上述作用略优于PJTS,但两者之间无明显差异,没有观察到文献所述的单糖链皂苷活性显著大于双糖链皂苷的现象,具体原因有待深入研究。另据文献报道,珠子参总皂苷具有抗血栓类疾病的作用,另外本文的研究结果也证明珠子参总皂苷具有抑制血小板黏附、聚集的作用。为探明珠子参中皂苷类单体成分与其抗血栓作用的关系,本实验利用体外抗血小板凝集实验模型,研究了珠子参中17种皂苷类单体成分对由二磷酸腺苷(ADP)和花生四烯酸(AA)作为诱导剂引起的血小板聚集的抑制作用,探讨了其构效关系。结果表明,上述皂苷对由二磷酸腺苷(ADP)和花生四烯酸(AA)引起的血小板聚集均有一定的抑制作用,在糖基数目相等的情况下,抑制活性随着羟基个数的增加而增强。综上所述,本文对珠子参的化学成分与生物活性进行了较为深入的研究,在化学成分的分离鉴定、含量测定、总皂苷降解产物的制备及生物活性研究等方面取得了创新性研究成果,为珠子参的开发利用提供了新的科学依据。
[Abstract]:Panacis majoris Rhizoma is the dry rhizome of the Panax plant (Panax japonicus C.A.Mey.var.major (Burk.) C.Y.Wu et K.M.Feng) or the pinnate leaf 37. It has antitumor, antithrombotic and immunity enhancement. The chemical composition of the ginseng is mainly three terpenoid saponins. But compared with ginseng, Panax quinquefolium and 37, there are few reports on the study of Pearl ginseng, and there are still many gaps in the study of its chemical composition and pharmacological action. The chemical composition and biological activity of the ginseng were studied. In this paper, 42 monomers were isolated and identified by silica gel column chromatography, ODS column chromatography, preparative chromatography and MS, 1D, 2D NMR and other spectroscopic methods, including 6 new compounds and 16 first from Pearl ginseng. The compounds are separated. The new compounds are (20S, 24S, 25R*) -6-O-[beta -D- Piran glucosamine - (1 - 2) - beta -D- piranosamine] - DMA -20,24- epoxy -3 beta, 6 alpha, 12 beta, 25,26- five alcohol (1), (20S, 24R, 25R) - beta glucosamine - (1 - 2) - beta glucosamine] - DMA beta epoxide beta, 6 A, 12 beta, five alcohol (2) -6-O-[beta -D- glucosyl group - (1 - 2) - beta -D- piranolyl] - DMA -20,25- epoxy -3 beta, 6 alpha, 12 beta, 24 - four alcohol (3), 6-O-[beta -D- glucosyl - (1 - 2) - beta -D- Piran glucosyl] - DMA -3 beta, 6, 12 beta, 20S, 24R, 25- six alcohol (4), beta glucosyl - glucosyl - beta glucosyl - beta glucosyl Alpha, 12 beta, 20S, 24R- five alcohol (5) and 3-O-[beta -D- glucolyl - (1 - 2) - beta -D- (6 '-O- ethyl) - glycosylaldehyde] - oleanolic acid -28-O- beta -D- piranoside (6). The first isolated compounds are oleanolic acid -3-O- alpha -L- furan - (1 - 4) - beta -D- glucuronidoside (12), 3-O-{beta glucan grape Glycosyl - (1 - 2) - [alpha -L- furan - Arabia sugar - (1 - 4)] - beta -D- piranolyl glucoside -28-O- beta -D- glucoside (18), 6 "-O- acetyl ginsenoside Rb1 (20), Vietnamese saponin R4 (21), 6 '-O- acetyl ginsenoside Re (27), pseudo ginseng saponins RT2 (32), saponins F11 (33), Viet Nam saponins R8 (34), 37 saponins 5) saponins R9 (36), Vietnamese saponin R13 (37), ginsenoside I (38), ginsenoside Re5 (39), ginsenjilinol (40), Panax ginseng L11 (41), yesanchinoside R2 (42). The separated compounds are V (7), ginseng soap glucoside Ro methyl ester (8), and oleanolic acid -3-O- beta glucoside (1 to 2) - beta glucuronide glucuronidoside. 9) Panax japonicus saponins IV (10), Araloside A methyl ester (11), pseudoginsenoside RT1 (13), pseudoginsenoside RT1 methyl ester (14), Panax japonicus saponins IVa (15), Rhizoma japonicus saponins (16), oleanolic acid, beta glucoside (17), ginsenoside 19, 22, 23, 37 saponins R2 (24), 37 saponins (25), ginsenoside Re (26), ginsenoside Rd (28), ginseng glucoside R1 (29), 24 (R) - Pearl glycoside R1 (30) and Pearl glycoside R2 (31). The above separated saponins are divided into oleanane saponins, damanane saponins and orktrolon saponins according to the structure of the glycosides. In this paper, three kinds of HPLC detection methods were established in this paper. (1) the extraction and content determination of 5 oleanane saponins from pearls with ionic liquid biaqueous phase combined with HPLC method was first used. By optimizing the experimental conditions, the 0.2 g Pearson samples were established, and 3 m L was removed. Sub water, 0.5m L[Hmim]Br ionic liquid, 2.5 g K2HPO4 sample pretreatment method. The detection limit of this method is between 0.31~0.88, G, m L-1. The RSD value of the stability test is between 0.26%~2.69%, and the recovery rate is between 90.00%~106.00%. It shows that the method is sensitive, stable and recovery is good. It is suitable for the determination of oleanane type in Pearl ginseng. Under this condition, the content of 5 kinds of oleanane saponins were 47.10 mg. G~ (-1), 24 mg. G~ (-1), 0.35 mg. G~ (-1), 0.17 mg. G~ (-1) and 0.44 respectively. (2) the extraction and determination of 5 damanane saponins in Pearl were first used. The test conditions are optimized. Finally, the samples of 50 mg Pearson ginseng, 0.8 m L deionized water, 0.2 m L[Bmim]Br ionic liquid, 3 m L 60% acetone water solution elution sample pretreatment method. The detection limit of this method is between 0.43~0.90 mu g m L-1, the RSD value of the stability experiment is 0.63% This method has high sensitivity, stability and recovery. It is suitable for the determination of the content of damanine saponins in Panax japonicus. Under this condition, the content of 5 kinds of damanine saponins is 92 G. G~ (-1), 106 mu g. G~ (-1), 100 mu g. G~ (-1), 33 mu g. G~ (-1) and 470 mu. The LC-ELSD method was used to extract and determine the content of 5 kinds of ootrolon saponins in the ginseng. Through the optimization of the experimental conditions, a sample pretreatment method was established with diatomite as a dispersant and methanol as a eluant. The RSD value of the stability test was between 0.57%~1.96% and 85.71%~103.00%, showing the spirit of the method. High sensitivity, good stability and recovery, it is suitable for the determination of the content of ootrolone saponins in Panax japonicus. Under this condition, the content of 5 kinds of oktrolone saponins is 916 G. G~ (-1), 35.7 mu g. G~ (-1), 720 mu g. G~ (-1), 19.2 mu g. G~ (-1) and 12.9 mu. The total content of 5 kinds of saponins in the Panax japonicus is 7.02%, far higher than the other two types. Compared with the traditional methods, the above three sample pretreatment methods are convenient, fast, and environmentally friendly. It is suitable for the rapid extraction and accurate analysis of the effective components in the Pearl ginseng. According to the literature, the oleanane saponins are C28 bit in C3 position sugar. The activity of C3, C28- double sugar chain oleanane saponins from C3- monosaccharide chain saponins could be enhanced. The degradation of total saponins from pearls and the identification of the chemical constituents of the degradation products were carried out in this paper, and the total saponins before and after degradation were also studied. First, the total saponins (PJTS) were prepared by macroporous adsorption resin. By HPLC analysis, the main components of PJTS were 5 kinds of C3, C28- double sugar chain oleanane saponins, they were I, IV (II), pseudoginsenoside RT1 (III), bamboo ginseng saponins and beta The glucosamine - (1 - 2) - [alpha -L- furan - Arabia sugar - (1 - 4)] - beta -D- piranolyl - glucuronic acid -28-O- beta -D- glucoside (V) in PJTS are 168.1mg. G~ (-1), 115.7 mg. G~ (-1), 65.4, 270.9, and 79.3. Methods the total saponins from the above pearl were degraded and the total saponins degradation product (DPJTS) was obtained. The results of chemical composition study showed that the above 5 kinds of C3, C28- chain oleanin saponins (compound I~V) were completely degraded to C3- monosaccharide chain oleanin saponins, which were oleanolic acid -3-O- beta -D- glucuronide glycoside respectively. P-1), oleanolic acid -3-O- alpha -L- furan - Arabia glycosyl - (1 - 4) - beta -D- glucuronidoside (P-2), oleanolic acid -3-O- beta -D- piranolyl - (1 - 2) - beta -D- piranoside (P-3), oleanolic acid -3-O- beta -D- Piran glucuronidyl - (1 - 2) - beta -D- Piran glucuronidoside and oleanolic acid - (1 - 2) - [alpha -L- furan - (1 - 4)] - beta -D- glucuronidoside (P-5), their contents in DPJTS were 128.1mg / g~ (-1), 99.7 mg. G~ (-1), 39.2 mg g~, 302.6 and 63.7. The protective effect of S on myocardial ischemia reperfusion injury (MIRI) in rats and its mechanism are preliminarily discussed. The experimental results show that PJTS and DPJTS have protective effects on experimental myocardial ischemia reperfusion injury in rats. The mechanism may be related to the inhibition of platelet adhesion, aggregation, inhibition of high viscosity of blood and the enhancement of the antioxidant capacity of the body. The above effect of.DPJTS is slightly better than that of PJTS, but there is no obvious difference between the two. There is no observation that the monosaccharide saponins in the monosaccharide chain are significantly greater than the double sugar chain saponins. The total saponins of Panax japonicus have the effect of inhibiting platelet adhesion and aggregation. In order to explore the relationship between saponins and the antithrombotic effect of saponins in the Panax japonicus, this experiment uses an in vitro antiplatelet aggregation experiment model to study 17 kinds of saponins in the ginseng, which are used as inducers of adenosine two phosphate (ADP) and peanut four enoic acid (AA). The inhibitory effect of platelet aggregation was discussed, and its structure-activity relationship was discussed. The results showed that the above saponins could inhibit the platelet aggregation caused by adenosine two (ADP) and arachidic acid (AA). In the case of the equal number of glycosyl groups, the inhibitory activity was enhanced with the increase of the number of hydroxyl groups. The chemical composition and biological activity of the sub ginseng have been deeply studied. The innovative research results have been obtained in the separation and identification of the chemical components, the determination of the content, the preparation of the total saponins degradation products and the study of biological activity, which provides a new scientific basis for the development and utilization of the Pearl ginseng.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O652;R284.1

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