朱砂七粗多糖的提取及其生物活性研究
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摘要
朱砂七为蓼科植物(Polygonum cillinerve(Nakai)Ohwi)的块根,产于太白山区以及中西部士也区,为“太白七药”之一,始载于《图经本草》。其味苦,性凉,具有滋肝养肾、抗肿瘤、抗衰老,抗炎、抗菌、抗病毒等作用。临床上主治扁桃体炎、肠炎、胃炎、肾炎、尿路感染及跌打损伤、风湿腰痛等症。关于朱砂七化学成分的研究已有报道,但尚未见到关于朱砂七多糖的研究报道。
本论文包括综述和研究报告两部分内容。
第一部分:绪论
从下面三方面进行了综述:
1.朱砂七研究概况
从朱砂七化学成分研究进展和朱砂七药理研究现状两方面对朱砂七的研究概况做了综述。
2.多糖研究概况
包括多糖的研究进展、多糖的来源、多糖的药理研究现状以及多糖的研究应用及发展方向四方面的内容。
3.抗氧化作用研究进展
抗氧化作用研究进展部分涉及自由基及其特点、氧自由基与疾病和衰老、体内抗氧化系统、评价体外抗氧化能力的自由基体系、中药的抗氧化作用与应用等几部分内容。
第二部分:研究报告
分为四章内容:朱砂七粗多糖的提取、分离及纯化;朱砂七多糖体外抗氧化活性研究;朱砂七粗多糖的体内抗氧化活性和朱砂七粗多糖的抗肿瘤作用。
一、朱砂七粗多糖的提取、分离及纯化
采用石油醚回流和无水乙醇脱脂,然后使用水提醇沉的方法提取朱砂七总多糖;用透析袋透析除去小分子化合物:采用苯酚.硫酸法测定PCCP中总糖的含量。然后经过DEAE-纤维素柱初步脱色纯化,得朱砂七粗多糖。结果表明,水提纯沉朱砂七效果良好,提取率达到5.1%;用苯酚.硫酸法测得PCCP总糖含量达到91.8%,表明PCCP中的主要成分为多糖类化合物;DEAE-纤维素柱起到初步纯化粗多糖的作用。
二、朱砂七多糖体外抗氧化活性研究
实验通过测定PCCP对·OH自由基、O_2~(?)自由基和DPPH咱由基的清除能力以及抗脂质过氧化能力,探讨其体外抗氧化活性。结果显示:PCCP的浓度在8.48 mg/mL时,对·OH、O_2~(?)和DPPH·的清除率分别达到了52.3%、54.7%和38.3%;PCCP的浓度在8.48 mg/mL时,对脂质过氧化的抑制率达到了62.0%;表明PCCP具有一定的体外抗氧化活性。
三、朱砂七粗多糖的体内抗氧化活性
实验利用环磷酰胺所致免疫功能低下小鼠模型,研究了朱砂七粗多糖的体内生物活性,考察了CY小鼠模型给药后的肝、脾和胸腺指数的变化,测定了心、肝和肾匀浆的总抗氧化能力(T-AOC)、丙二醛(MDA)含量、过氧化氢酶(CAT)、活性超氧化物歧化酶(SOD)活性以及谷胱甘肽过氧化物酶(GSH-Px)活力。结果表明,治疗组小鼠三种器官的指数以及心、肝和肾的T-AOC、CAT、SOD和GSH-Px活力均高于阴性对照组,与正常组相比也有所提高;MDA与阴性组相比有不同层度的下降,并呈现出量效关系,说明PCCP具有体内抗氧化、增强机体免疫能力的作用。
四、朱砂七粗多糖的体外抗肿瘤作用
选用人肝癌HepG-2细胞,经MTT染色法研究了朱砂七粗多糖的体外抗肿瘤活性,为朱砂七粗多糖开发为抗肿瘤药物积累资料。结果显示PCCP溶液浓度在92.6μg/mL、168.9μg/mL、277.78μg/mL时,对人肝癌HepG-2细胞的抑制率分别达到了37.5%、40.1%和42.6%,表明PCCP能抑制人肝癌HepG-2细胞的生长,具有一定的体外抗肿瘤活性。
本文从朱砂七中提取出多糖,经过初步分离、纯化得到粗多糖,重点研究了它的抗氧化活性,对体外抗肿瘤也进行了初步研究,这些研究为朱砂七多糖的进一步研究与开发奠定了基础。
Polygonum cillinerve(Nakai) Ohwi,one of the most valued traditional Chinese medicines,is a member of "Taibai Qiyao" and first year's growth liane,commonly found in hillside,forest,bosk, and lawn in the Taibai mountains and many northwestern provinces in China.As a drug,it can be traced back to tu jing ben cao.Whole plant may be taken for medicine.It has functions of nourishing the kidney,nourishing the liver,and anti-aging,immuno-modulating,anticancer,antibacterial, antivirus,and anti-microorganism activities.It is commonly used in China for over 2000 years for the treatment of quinsy,enteritis,gastritis diarrhea,nephritis dropsy,allantois infection and so on.In this paper,polysaccharide in Polygonum cillinerve(Nakai) Ohwi is studied,about which there was no reports as yet.
There are two parts in this thesis:
Part one:review
1.A survey of study on Polygonum cillinerve(Nakai) Ohwi
It evolved chemical component and pharmacological activity of P.cillinerve(Nakai) Ohwi.
2.A survey of study on polysaccharide
There are four faces in the research advancements of polysaccharide:research advance of polysaccharides;sources of polysaccharides;a review on the situation of the pharmacological activities of polysaccharides;and the application and the development direction of the studies on polysaccharides.
3.Research advance of antioxidation
Characteristics of free radical;relation between oxygen free radical and diseases,aging; antioxidation system in vivo;the system of evaluation of antioxidation in vitro;and antioxidant activity and applications of traditional chinese medicine.
Part two:research papers
In this part,four departments are involved:extraction,isolation and purification of the polysaccharides in Polygonum cillinerve(Nakai) Ohwi;Anti-oxidation of polysaccharides in vitro; Anti-oxidation of polysaccharides;anti-tumor activity in vitro of polysaccharides.
First,extraction,isolation and purification of the crude polysaccharides in Polygonum cillinerve (Nakai) Ohwi(PCCP).The sample was defatted with petroleum ether,remoned the essential oils with steam distillation.The water-soluble polysaccharide was extracted by boiling water from the sediment,and then was precipitated by 80%(v/v) of ethanol.The crude polysaccharides in Polygonum cillinerve(Nakai) Ohwi obtained is red.The compounds with small molecular weight were removed with dialyser and a part-pure product of polysaccharide was obtained,and the total contents of polysaccharides were determined by the method of sulphuric acid-phenol.PCCP is decolorized by DEAE-cellulose chromatography.The results showed extraction yield of polysaccharide by boiling water was about 5.1%;the total contents of polysaccharides in PCCP was about 91.8%.The DEAE-cellulose chromatography could purify polysaccharides preliminarily
Second,hydroxyl radical scavenging,super-oxide anion radical scavenging,DPPH.radical scavenging and lipid per-oxidation inhibiting were introduced to assay the antioxidant activity of PCCP in vitro.The results showed:hydroxyl radical,super-oxide anion radical,DPPH.radical was scavenged by 35.1%,40.7%and 38.3%when the concentration of PCCP comes to 8.48 mg/mL; Lipid per-oxidation inhibition rate of PCCP was 62.0%at 8.48 mg/mL.The results indicated that PCCP has antioxidant activities in vitro.
Third,PCCP were studied for their anti-oxidation and immune responses activity in mice.The in vivo investigation demonstrated that administration of PCCP by gavage is able to overcome cyclophosphamide-induced immunosuppression and significantly raised total antioxidant capacity, catalase activity,superoxidase dismutase activity,glutathione peroxidase level.It also raises liver, spleen and thymus indices,and decreases malondialdehyde in a dose-dependent manner in mice.The results indicated that the PCCP,possessing pronounced free radical scavenging and antioxidant activities,could play an important role in prevention of oxidative damage in immunological system.
Lastly,the anti-tumor activity of PCCP was evaluated by MTT colorimetric assay with HepG-2. HepG-2 cell is a cell line derived from HCC.This investigation showed that PCCP could inhibite HepG-2 cell proliferation in vitro.HepG-2 inhibition rate of PCCP was 42.6%when the concentration of PCCP comes to 277.78μg/mL,which indicated that PCCP has anti-tumor effects in vitro.
The polysaccharides of Polygonum Cillinerve(Nakai) Ohwi was firstly studied in this paper.It was preliminarily proved that PCCP has antioxidation and antitumor activities.The biological activities have made a foundation for the further study and application of the polysaccharides of Polygonum Cillinerve(Nakai) Ohwi.
本论文包括综述和研究报告两部分内容。
第一部分:绪论
从下面三方面进行了综述:
1.朱砂七研究概况
从朱砂七化学成分研究进展和朱砂七药理研究现状两方面对朱砂七的研究概况做了综述。
2.多糖研究概况
包括多糖的研究进展、多糖的来源、多糖的药理研究现状以及多糖的研究应用及发展方向四方面的内容。
3.抗氧化作用研究进展
抗氧化作用研究进展部分涉及自由基及其特点、氧自由基与疾病和衰老、体内抗氧化系统、评价体外抗氧化能力的自由基体系、中药的抗氧化作用与应用等几部分内容。
第二部分:研究报告
分为四章内容:朱砂七粗多糖的提取、分离及纯化;朱砂七多糖体外抗氧化活性研究;朱砂七粗多糖的体内抗氧化活性和朱砂七粗多糖的抗肿瘤作用。
一、朱砂七粗多糖的提取、分离及纯化
采用石油醚回流和无水乙醇脱脂,然后使用水提醇沉的方法提取朱砂七总多糖;用透析袋透析除去小分子化合物:采用苯酚.硫酸法测定PCCP中总糖的含量。然后经过DEAE-纤维素柱初步脱色纯化,得朱砂七粗多糖。结果表明,水提纯沉朱砂七效果良好,提取率达到5.1%;用苯酚.硫酸法测得PCCP总糖含量达到91.8%,表明PCCP中的主要成分为多糖类化合物;DEAE-纤维素柱起到初步纯化粗多糖的作用。
二、朱砂七多糖体外抗氧化活性研究
实验通过测定PCCP对·OH自由基、O_2~(?)自由基和DPPH咱由基的清除能力以及抗脂质过氧化能力,探讨其体外抗氧化活性。结果显示:PCCP的浓度在8.48 mg/mL时,对·OH、O_2~(?)和DPPH·的清除率分别达到了52.3%、54.7%和38.3%;PCCP的浓度在8.48 mg/mL时,对脂质过氧化的抑制率达到了62.0%;表明PCCP具有一定的体外抗氧化活性。
三、朱砂七粗多糖的体内抗氧化活性
实验利用环磷酰胺所致免疫功能低下小鼠模型,研究了朱砂七粗多糖的体内生物活性,考察了CY小鼠模型给药后的肝、脾和胸腺指数的变化,测定了心、肝和肾匀浆的总抗氧化能力(T-AOC)、丙二醛(MDA)含量、过氧化氢酶(CAT)、活性超氧化物歧化酶(SOD)活性以及谷胱甘肽过氧化物酶(GSH-Px)活力。结果表明,治疗组小鼠三种器官的指数以及心、肝和肾的T-AOC、CAT、SOD和GSH-Px活力均高于阴性对照组,与正常组相比也有所提高;MDA与阴性组相比有不同层度的下降,并呈现出量效关系,说明PCCP具有体内抗氧化、增强机体免疫能力的作用。
四、朱砂七粗多糖的体外抗肿瘤作用
选用人肝癌HepG-2细胞,经MTT染色法研究了朱砂七粗多糖的体外抗肿瘤活性,为朱砂七粗多糖开发为抗肿瘤药物积累资料。结果显示PCCP溶液浓度在92.6μg/mL、168.9μg/mL、277.78μg/mL时,对人肝癌HepG-2细胞的抑制率分别达到了37.5%、40.1%和42.6%,表明PCCP能抑制人肝癌HepG-2细胞的生长,具有一定的体外抗肿瘤活性。
本文从朱砂七中提取出多糖,经过初步分离、纯化得到粗多糖,重点研究了它的抗氧化活性,对体外抗肿瘤也进行了初步研究,这些研究为朱砂七多糖的进一步研究与开发奠定了基础。
Polygonum cillinerve(Nakai) Ohwi,one of the most valued traditional Chinese medicines,is a member of "Taibai Qiyao" and first year's growth liane,commonly found in hillside,forest,bosk, and lawn in the Taibai mountains and many northwestern provinces in China.As a drug,it can be traced back to tu jing ben cao.Whole plant may be taken for medicine.It has functions of nourishing the kidney,nourishing the liver,and anti-aging,immuno-modulating,anticancer,antibacterial, antivirus,and anti-microorganism activities.It is commonly used in China for over 2000 years for the treatment of quinsy,enteritis,gastritis diarrhea,nephritis dropsy,allantois infection and so on.In this paper,polysaccharide in Polygonum cillinerve(Nakai) Ohwi is studied,about which there was no reports as yet.
There are two parts in this thesis:
Part one:review
1.A survey of study on Polygonum cillinerve(Nakai) Ohwi
It evolved chemical component and pharmacological activity of P.cillinerve(Nakai) Ohwi.
2.A survey of study on polysaccharide
There are four faces in the research advancements of polysaccharide:research advance of polysaccharides;sources of polysaccharides;a review on the situation of the pharmacological activities of polysaccharides;and the application and the development direction of the studies on polysaccharides.
3.Research advance of antioxidation
Characteristics of free radical;relation between oxygen free radical and diseases,aging; antioxidation system in vivo;the system of evaluation of antioxidation in vitro;and antioxidant activity and applications of traditional chinese medicine.
Part two:research papers
In this part,four departments are involved:extraction,isolation and purification of the polysaccharides in Polygonum cillinerve(Nakai) Ohwi;Anti-oxidation of polysaccharides in vitro; Anti-oxidation of polysaccharides;anti-tumor activity in vitro of polysaccharides.
First,extraction,isolation and purification of the crude polysaccharides in Polygonum cillinerve (Nakai) Ohwi(PCCP).The sample was defatted with petroleum ether,remoned the essential oils with steam distillation.The water-soluble polysaccharide was extracted by boiling water from the sediment,and then was precipitated by 80%(v/v) of ethanol.The crude polysaccharides in Polygonum cillinerve(Nakai) Ohwi obtained is red.The compounds with small molecular weight were removed with dialyser and a part-pure product of polysaccharide was obtained,and the total contents of polysaccharides were determined by the method of sulphuric acid-phenol.PCCP is decolorized by DEAE-cellulose chromatography.The results showed extraction yield of polysaccharide by boiling water was about 5.1%;the total contents of polysaccharides in PCCP was about 91.8%.The DEAE-cellulose chromatography could purify polysaccharides preliminarily
Second,hydroxyl radical scavenging,super-oxide anion radical scavenging,DPPH.radical scavenging and lipid per-oxidation inhibiting were introduced to assay the antioxidant activity of PCCP in vitro.The results showed:hydroxyl radical,super-oxide anion radical,DPPH.radical was scavenged by 35.1%,40.7%and 38.3%when the concentration of PCCP comes to 8.48 mg/mL; Lipid per-oxidation inhibition rate of PCCP was 62.0%at 8.48 mg/mL.The results indicated that PCCP has antioxidant activities in vitro.
Third,PCCP were studied for their anti-oxidation and immune responses activity in mice.The in vivo investigation demonstrated that administration of PCCP by gavage is able to overcome cyclophosphamide-induced immunosuppression and significantly raised total antioxidant capacity, catalase activity,superoxidase dismutase activity,glutathione peroxidase level.It also raises liver, spleen and thymus indices,and decreases malondialdehyde in a dose-dependent manner in mice.The results indicated that the PCCP,possessing pronounced free radical scavenging and antioxidant activities,could play an important role in prevention of oxidative damage in immunological system.
Lastly,the anti-tumor activity of PCCP was evaluated by MTT colorimetric assay with HepG-2. HepG-2 cell is a cell line derived from HCC.This investigation showed that PCCP could inhibite HepG-2 cell proliferation in vitro.HepG-2 inhibition rate of PCCP was 42.6%when the concentration of PCCP comes to 277.78μg/mL,which indicated that PCCP has anti-tumor effects in vitro.
The polysaccharides of Polygonum Cillinerve(Nakai) Ohwi was firstly studied in this paper.It was preliminarily proved that PCCP has antioxidation and antitumor activities.The biological activities have made a foundation for the further study and application of the polysaccharides of Polygonum Cillinerve(Nakai) Ohwi.
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