西瓜藤化学成分研究及药效学筛选
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摘要
西瓜藤为葫芦科Cucurbitaceae植物西瓜Citrullus lanatus的藤茎,其作为一种农作物废弃物,资源非常丰富,但目前研究开发却非常少,往往被废弃或焚烧,造成严重的环境污染和资源浪费。开展其药用价值研究,对开发新的药用资源,实现中药资源的可持续发展,解决当前中药资源日益枯竭的局面具有重要的意义。本论文开展了西瓜藤不同部位提取物抗炎镇痛及其机理、降血糖、抑菌、抗肿瘤等几个方面的药效学筛选研究,并对具有活性的部位进行了化学成分研究,为西瓜藤的进一步开发研究提供了理论依据。
第一部分西瓜藤化学成分研究
实验一四种不同品种西瓜藤化学成分预实验
目的：采用系统预试法对四种不同品种西瓜藤可能的化学成分进行预实验,初步探索四种不同品种西瓜藤的化学成分。方法：采用试管反应法、层析法,对西瓜藤的水、95%乙醇、石油醚提取物进行研究,通过多种指示剂和显色剂的沉淀反应或颜色反应,对四种不同品种西瓜藤可能含有的化学成分进行初步研究。结果：四种不同品种西瓜藤中可能含有糖类、有机酸、皂苷、黄酮类、生物碱、甾体等化学成分。结论：初步确定四种不同品种西瓜藤含有多种有效成分,为西瓜藤的进一步开发利用提供了实验基础。
实验二西瓜藤石油醚部位化学成分研究
目的：研究西瓜藤石油醚部位的化学成分。方法：硅胶色谱、聚酰胺色谱分离纯化,根据理化性质和核磁共振结构数据鉴定化合物结构。结果：从西瓜藤乙醇提取物的石油醚萃取部分中共分离得到10个化合物,分别鉴定为p-谷甾醇(β-sitosterol,1)、胡萝卜苷(daucosterol,2)、豆甾醇(stigmasterol,3)、肉豆蔻酸甘油酯(monomyristin,4)、十六烷酸(palmitic acid,5)、棕榈酸甘油酯(monopalmitin,6)、二十二烷酸甘油酯(monobehenin,7)、熊果酸(ursolic acid,8)、二十一烷酸甘油酯(monoheneicosanoin,9)、硬脂酸(stearic acid,10)。结论：化合物(1-10)均为首次从西瓜藤中分离得到。
实验三西瓜藤乙酸乙酯部位化学成分研究
目的：研究西瓜藤乙酸乙酯部位的化学成分。方法：硅胶色谱、聚酰胺色谱分离纯化,根据理化性质和核磁共振结构数据鉴定化合物结构。结果：从西瓜藤乙醇提取物的乙酸乙酯萃取部分中共分离得到10个化合物,分别鉴定为十五烷酸(pentadecanoic acid,1),十五烷酸单甘油酯(monop entadecanoin,2),十九烷酸-1-甘油酯(2,3-dihydroxypropyl nonadecoate,3),二十四烷酸-α-单甘油酯(lignoceric acid-2,3-dihydroxy-propanenyl ester,4),2-[2’-羟基(2’R,13Z)十六酰胺]-3-羟基-(2S,3R,4Z)-十八碳烯-1-0-β-D-吡喃葡萄糖苷(lancerebroside5,5),2-羟基苯甲酸(salicylic acid,6)对羟基苯甲酸(4-hydroxybenzoic acid,7),对羟基苯酚(hydroquinone,8),琥珀酸(succinic acid,9),香草酸(vanillic acid,10)。结论：化合物(1-10)均为首次从西瓜藤中分离得到,其中化合物5为首次发现的新化合物。
第二部分西瓜藤总提取物抗炎镇痛作用实验研究
目的：研究西瓜藤总提取物(EWRL)的抗炎镇痛作用及急性毒性反应。方法：采用二甲苯致小鼠耳廓肿胀模型、大鼠足趾肿胀及棉球肉芽肿胀模型观察EWRL的抗炎作用。采用热板法、冰醋酸扭体法观察EWRL对小鼠的镇痛作用。采用Bliss法,观察EWRL灌胃给药的急性毒性反应。结果：EWRL对二甲苯致小鼠耳廓肿胀、棉球肉芽肿胀、角叉菜胶致足趾肿胀均具有显著的抑制作用,并能显著提高小鼠的热板痛阈值,减少小鼠的扭体次数。急性毒性实验中,连续观察14d,未见明显毒性反应症状,无动物死亡,计算得最大给药量为87.0g/kg。结论：EWRL具有显著的抗炎和镇痛活性,且经口服属无毒级。
第三部分西瓜藤不同部位提取物抗炎活性筛选及其机理研究
目的：研究西瓜藤不同部位提取物的抗炎作用,并初步阐明其作用机理。方法：采用LPS刺激RAW264.7细胞的炎症模型,以对NO释放水平影响为标志,对西瓜藤的石油醚、乙酸乙酯、正丁醇、50%乙醇、水部位进行抗炎活性筛选。确定活性部位后,采用ELISA法测定促炎介质TNF-α, IL-6, IL-1β, iNOS, COX-2, HO-1的浓度；免疫荧光法检测NF-κB的激活核转运；实时荧光定量PCR法测定IL-1β, IL-6, TNF-α、 COX-2、 iNOS和HO-1mRNA水平对其抗炎机理进行探讨。结果：西瓜藤各提取部位对LPS刺激RAW264.7细胞的炎症模型的NO释放均具有显著抑制作用,以石油醚部位最强。石油醚部位对炎症模型中TNF-α, IL-6, IL-1β、iNOS, COX-2的过度表达具有显著抑制作用；对HO-1水平无影响。同时还可以抑制NF-κB的激活核转运,降低IL-1β、IL-6、TNF-α、 COX-2、 iNOS和HO-1mRNA水平。结论：石油醚部位为西瓜藤的抗炎活性部位,其机理可能是通过抑制NF-κB的激活核转运来达到抗炎的作用。
第四部分西瓜藤不同部位提取物降血糖活性筛选研究
实验一西瓜藤不同提取物对四氧嘧啶致糖尿病小鼠的降血糖作用
目的：研究西瓜藤不同部位提取物对糖尿病小鼠的降血糖作用。方法：小鼠腹腔注射四氧嘧啶建立糖尿病小鼠模型,西瓜藤不同提取物喂养干预治疗。测定小鼠的空腹血糖、葡萄糖耐量、血清中胰岛素水平、TG、 TC、 NO和iNOS含量；肝组织匀浆中NO和iNOS水平；并观察小鼠胰腺组织病理学改变。结果：西瓜藤乙酸乙酯高剂量组和石油醚高剂量组TC显著性降低,TG值也有所降低,血糖水平及糖耐量均有改善,胰岛素含量显著升高,NO和iNOS含量显著降低。结论：西瓜藤乙酸乙酯部位、石油醚部位对糖尿病小鼠具有一定的降血糖作用。作用机理可能与其促进胰岛素释放及降低NO和iNOS含量有关。
实验二西瓜藤乙酸乙酯提取物对DN大鼠肾小管的保护作用
目的：探讨西瓜藤乙酸乙酯提取物对DN大鼠肾小管的保护作用。方法：大鼠腹腔注射链脲佐菌素(STZ)建立DN大鼠模型,西瓜藤乙酸乙酯提取物喂养干预治疗。检测血、尿中血糖、血肌酐、尿肌酐、尿清蛋白、血胰岛素等指标,计算内生肌酐清除率(Ccr),并观察大鼠肾脏组织病理学改变。结果：与DN组比较,西瓜藤乙酸乙酯高、中剂量组大鼠血糖、血胰岛素、24h尿清蛋白排泄量、肾重指数及肾小管损伤指数均明显下降(P<0.05),内生肌酐清除率升高(P<0.05)。结论：西瓜藤乙酸乙酯提取物具有保护DN大鼠肾小管的作用。
第五部分西瓜藤不同部位提取物抑菌活性筛选研究
目的：筛选西瓜藤不同部位提取物的抑菌活性部位。方法：采用琼脂稀释法对西瓜藤不同部位提取物进行体外抑菌试验；采用小鼠腹腔注射金黄色葡萄球菌法进行西瓜藤80%醇提取物体内抑菌实验。结果：西瓜藤提取物对金黄色葡萄球菌、大肠埃希氏菌、铜绿假单胞菌、伤寒沙门氏菌、枯草芽孢杆菌和肺炎克雷伯氏菌均有不同程度的抑制作用,但对链球菌作用不明显。其中80%醇提取物和乙酸乙酯萃取物抑制金黄色葡萄球菌活性最好,其最低抑菌浓度(MIC)分别为4.2mg·mL-1,8.4mg·mL-1。体内实验也表明,80%醇提取物具有较好的抑菌作用。结论：西瓜藤提取物具有较好的抑菌活性,在抑菌方面有一定的开发前景。
第六部分西瓜脑苷V体外抗肿瘤活性筛选研究
目的：研究西瓜藤中西瓜脑苷Ⅴ (lancerebroside Ⅴ)的体外抗肿瘤活性。方法：采用MTT法,测定西瓜藤中的lancerebroside Ⅴ体外分别对前列腺癌细胞株PC3,紫杉醇耐药株PC3-RTX,鼻咽癌细胞株SUNE1,HONE1,肺癌细胞株A549的增殖抑制率,并计算相应的IC50值。结果：得到lancerebroside Ⅴ对五种肿瘤细胞株IC5o值的范围为3.9～8.1μg·mL-1。结论：西瓜藤中的lancerebroside V对所选择的5种肿瘤细胞株均具有一定的抗肿瘤活性。
综上所述,西瓜藤总提取物具有较好的抗炎镇痛作用,且毒副作用很低；西瓜藤石油醚萃取物对LPS诱导的巨噬细胞炎症反应具显著抑制作用,其作用机理可能是通过选择性抑制COX-2的活性,抑制NF-κB核转运激活,从而抑制LPS诱导的促炎介质IL-1beta、IL-6、TNF-α释放以及iNOS的活性,减少NO的释放；西瓜藤乙酸乙酯萃取物和石油醚萃取物对四氧嘧啶致糖尿病小鼠具有一定的降血糖作用,西瓜藤乙酸乙酯萃取物对2型糖尿病大鼠肾小管具有一定的保护作用；西瓜藤提取物对8种临床常见细菌除了链球菌外,均有不同程度的抑制作用,其中,80%醇提物和乙酸乙酯萃取物作用效果较好；从西瓜藤中分离得到20个化合物,其中一个为新化合物：西瓜脑苷Ⅴ(lancerebroside Ⅴ),通过体外抗肿瘤实验发现,其对前列腺癌细胞株PC3,紫杉醇耐药株PC3-RTX,鼻咽癌细胞株SUNE1, HONE1,肺癌细胞株A549等五种肿瘤细胞均具有一定的抗肿瘤作用。
Part Ⅰ. Study on Chemical Constituents of the Citrullus Lanatus Vine Experiment1:Preliminary Tests of Chemical Components for the Four Different Species Citrullus Lanatus Vine
Objective:To identify the chemical components of the four different species citrullus lanatus vine using comprehensive preliminary tests. Methods:The mixture was extracted from the four different species citrullus lanatus vine with purified water95%ethanol, and petrol ether was studied using test tube and chromatography method. The chemical components of the four different species citrullus lanatus vine were preliminarily demonstrated using precipitation reaction and color reaction of multiple indicators. Results:The four different species citrullus lanatus vine might contain sugar, organic acid, sapaomin, flavonoids, alkaloids, steride. Conclusion: The research provides foundation for further study and application of citrullus lanatus vine.
Experiment2:Study on Chemical Constituents of Citrullus Lanatus Vine (I)
Objective:To study the chemical constituents of citrullus l anatus vine in extracting with petroleum ether. Methods:Compounds wer e isolated and purified by polyamide column chromatography. Their chemi cal structures were elucidated on the basis of physicochemical properties, and spectral data. Results:Ten compounds were isolated from citrullus la natus vine andelucidated as β-sitosterol,daucosterol, stigmasterol, monomyri stin, palmitic acid, monopalmitin, monobehenin, ursolic acid, monoheneicos anoin, stearic acid. Conclusion:Compounds of1-10were obtained from c itrullus lanatus vine for the first time.
Experiment3:Study on Chemical Constituents of Citrullus Lanatus Vine (Ⅱ)
Objective:To study the chemical constituents of citrullus lanatus vine in extracting with ethyl acetate. Methods:Compounds were isolated and purified by polyamide column chromatography. Their chemical structures were elucidated on the basis of physicochemical properties, and spectral data. Results:Ten compounds were isolated from citrullus lanatus vine and elucidated as pentadecanoic acid, monopentadecanoin,2,3-Dihydroxypropyl nonadecoate, lignocericacid-2,3-dihydroxy-propanenyl ester, lancerebroside5, salicylic acid,4-Hydroxybenzoic acid, hydroquinone, succinic acid, vanillic acid. Conclusion:Compounds of1-10were obtained from citrullus lanatus vine for the first time.
Part Ⅱ. Experimental Study on Anti-inflammation and Analgesia Effect and Acute Toxicity of Extract from Citrullus Lanatus Vine
Objective:To study the effect of extract from citrullus lanatus vine (EWRL) on anti-inflammation and analgesia and acute toxicity. Methods: The models of mice ear edema induced by xylene, carrageenin and cotton pellet granuloma in rats to observe the anti-inflammation effect of EWRL via oral administration. The effect of EWRL on analgesia was tested by measuring the latent period licking hind foot with the hot plate method and counting body twisting induced by acetic acid in mice. The acute toxicity of EWRL was measured by the method of Bliss. Results:In acute toxic reactions which were observed for14days, all mice were survived, and no toxic reaction was shown during the observed period. The maximum administration dosage was87.0g/kg of EWRL given to mice per kilogram. EWRL could significantly inhibit the ear edema caused by xylene in mice, granuloma hyperplasia caused by cotton in rats, paw oedema caused by carrageen in rats. It could significantly prolong the pain threshold on hot-plate in mice, reduce the writhing times in mice. Conclusion: EWRL has obvious effect on anti-inflammation and analgesia.
Part Ⅲ. Study on Screening and Mechanism of Anti-inflammatory Activity of Different Fractions from Citrullus Lanatus Vine
Objective:To screening the extract parts of citrullus lanatus vine on anti-inflammatory activity and study its mechanism. Methods: Inflammatory model of LPS-stimulated RAW264.7cells were used, the level of impact on the release of NO as a symbol, and the petroleum ether, ethyl acetate, n-butanol,50%ethanol and soluble parts of anti-inflammatory activity on citrullus lanatus vine wered screeninged. The concentration of proinflammatory mediators TNF-α, IL-6, IL-1β, iNOS and COX-2HO-1by ELISA were determined. The activation nuclear translocation of NF-κB by immunofluorescence assay were detected, Real-time PCR for the determination level of IL-1β, IL-6, TNF-α, COX-2, iNOS and HO-1mRNA, and explored its anti-inflammatory mechanism. Results:Extract parts of citrullus lanatus vine significantly inhibited NO release of the LPS-stimulated RAW264.7cells inflammatory model, and the petroleum ether part was the strongest. The petroleum ether fraction significantly inhibited the overexpression of TNF-a, IL-6, IL-1β, iNOS, COX-2, but have no effect on the levels of HO-1in inflammatory model. It could also inhibit the activation of NF-κB nuclear translocation, and reduce the level of IL-1β, IL-6, TNF-α, COX-2, iNOS and HO-1mRNA. Conclusion:Petroleum ether fraction of the watermelon vine was the anti-inflammatory active site, which may achieve the anti-inflammatory role by inhibiting the activation of NF-κB nuclear translocation.
Part IV. The Screening of Hypoglycemic Activities of Different Fractions from Citrullus Lanatus Vine
Experiment1:The Hypoglycemic Effects of Different Fractions from Citrullus Lanatus Vine on Diabetic Model Induced by Alloxan in Mice
Objective:To investigate the hypoglycemic effects of different fractions from citrullus lanatus vine on diabetic model induced by alloxan in mice. Methods:Healthy mices were used to establish diabetic models by peritoneal injection administration of alloxan. The different fractions from citrullus lanatus vine was applied as raw materials for potential marine drug. The levels of fasting blood glucose(FBG)、 fasting blood insulin (FBI) and glucose tolerance were detected by automatic blood glucose device. The concentrations of total cholesterol (TC)、 total glycerin (TG) nitric oxide (NO) and nitric oxide synthase (iNOS) in blood were respectively measured. The concentrations in liver homogenate of nitric oxide (NO) and nitric oxide synthase (iNOS) were determined. Pathology of pancreas was observed. Results:After treatment, ethyl acetate high dose group and Petroleum ether high dose group of citrullus lanatus vine all significantly decreased the levels of TC、 TG、 NO and iNOS. Meanwhile, blood glucose level and glucose tolerance were improved. Insulin levels was also improved. Conclusion:Ethyl acetate group and Petroleum ether group of citrullus lanatus vine shows significant hypoglycemic effect on diabetic model induced by alloxan in mice. The mechanism of its hypoglycemic effect may be promoted the insulin release and decreased the levels of NO and iNOS.
Experiment2:The Protective Renal Tubular Effects of Ethyl Acetate Extract from Citrullus Lanatus Vine in Rats with DN
Objective:To investigate the protective renal tubular effects of ethyl acetate extract from citrullus lanatus vine in rats with DN. Methods: Healthy rates were used to establish DN models by peritoneal injection administration of streptozotocin. The ethyl acetate extract from citrullus lanatus vine was applied as raw materials for potential marine drug. The levels of fasting blood glucose(FBG)、fasting blood insulin (FBI)、serum creatinine (Scr)、urine creatinine (Ucr) and urinary albumin(UAlb) were detected. Pathology of live was observed. Results:After treatment, ethyl acetate high dose group and medial dose group of citrullus lanatus vine all significantly decreased the levels of GLU、FBI、UAlb、KWI and TBI.Meanwhile, Ccr was also improved. Conclusion:The ethyl acetate extract from citrullus lanatus vine can protect renal tubular in rats with DN.
Part Ⅴ. Antimicrobial Activity of the Extracts from Citrullus Lanatus Vine
Objective:To study antimicrobial activity in vitro of citrullus lanatus vine extract. Methods:Agar dilution method was applied to detect antimicrobial effect of citrullus lanatus vine extract in vitro; the mice celiac injection of staphylococcus aureus method was applied to detect the antimicrobial effect of citrullus lanatus vine of ethyl acetate extraction in vivo. Results:Citrullus lanatus vine extract had different inhibition effects on staphylococcus aureus, escherichia coli, bacillus aeruginosus, salmonellatyph, bacillus subtilis and klebsiella pneumoniae, but it showed weak inhibition effect on chain coccus. Effects of80%alcohol extract and ethylacetate extract were better than others on staphylococcus aureus. The minimum inhibition concentration (MIC), respectively is4.2mg·mL-1,8.4mg·mL-1. Conclusion: Citrullus lanatus vine extract have antibacterial action, and provide a basis to exploit new antimicro-bic drug.
Part VI. Anti-tumor Active of Lancerebroside V from Citrullus Lanatus Vine in Vitro
Objective:To study the anti-tumor active of Lancerebroside Ⅴ extracted from citrullus lanatus vine in vitro. Methods:MTS method was used to test inhabitation of Lancerebroside Ⅴ from citrullus lanatus vine with five kinds of human tumor cells in vitro and IC50values were calculated respectively. Results:IC50values of these LancerebrosideV tested with five cancer cell lines were3.9～8.1μg·mL-1. Conclusion:To some extent, Lancerebroside V from citrullus lanatus vine have positive effect on inhabiting those human tumor lines in vitro.
第一部分西瓜藤化学成分研究
实验一四种不同品种西瓜藤化学成分预实验
目的：采用系统预试法对四种不同品种西瓜藤可能的化学成分进行预实验,初步探索四种不同品种西瓜藤的化学成分。方法：采用试管反应法、层析法,对西瓜藤的水、95%乙醇、石油醚提取物进行研究,通过多种指示剂和显色剂的沉淀反应或颜色反应,对四种不同品种西瓜藤可能含有的化学成分进行初步研究。结果：四种不同品种西瓜藤中可能含有糖类、有机酸、皂苷、黄酮类、生物碱、甾体等化学成分。结论：初步确定四种不同品种西瓜藤含有多种有效成分,为西瓜藤的进一步开发利用提供了实验基础。
实验二西瓜藤石油醚部位化学成分研究
目的：研究西瓜藤石油醚部位的化学成分。方法：硅胶色谱、聚酰胺色谱分离纯化,根据理化性质和核磁共振结构数据鉴定化合物结构。结果：从西瓜藤乙醇提取物的石油醚萃取部分中共分离得到10个化合物,分别鉴定为p-谷甾醇(β-sitosterol,1)、胡萝卜苷(daucosterol,2)、豆甾醇(stigmasterol,3)、肉豆蔻酸甘油酯(monomyristin,4)、十六烷酸(palmitic acid,5)、棕榈酸甘油酯(monopalmitin,6)、二十二烷酸甘油酯(monobehenin,7)、熊果酸(ursolic acid,8)、二十一烷酸甘油酯(monoheneicosanoin,9)、硬脂酸(stearic acid,10)。结论：化合物(1-10)均为首次从西瓜藤中分离得到。
实验三西瓜藤乙酸乙酯部位化学成分研究
目的：研究西瓜藤乙酸乙酯部位的化学成分。方法：硅胶色谱、聚酰胺色谱分离纯化,根据理化性质和核磁共振结构数据鉴定化合物结构。结果：从西瓜藤乙醇提取物的乙酸乙酯萃取部分中共分离得到10个化合物,分别鉴定为十五烷酸(pentadecanoic acid,1),十五烷酸单甘油酯(monop entadecanoin,2),十九烷酸-1-甘油酯(2,3-dihydroxypropyl nonadecoate,3),二十四烷酸-α-单甘油酯(lignoceric acid-2,3-dihydroxy-propanenyl ester,4),2-[2’-羟基(2’R,13Z)十六酰胺]-3-羟基-(2S,3R,4Z)-十八碳烯-1-0-β-D-吡喃葡萄糖苷(lancerebroside5,5),2-羟基苯甲酸(salicylic acid,6)对羟基苯甲酸(4-hydroxybenzoic acid,7),对羟基苯酚(hydroquinone,8),琥珀酸(succinic acid,9),香草酸(vanillic acid,10)。结论：化合物(1-10)均为首次从西瓜藤中分离得到,其中化合物5为首次发现的新化合物。
第二部分西瓜藤总提取物抗炎镇痛作用实验研究
目的：研究西瓜藤总提取物(EWRL)的抗炎镇痛作用及急性毒性反应。方法：采用二甲苯致小鼠耳廓肿胀模型、大鼠足趾肿胀及棉球肉芽肿胀模型观察EWRL的抗炎作用。采用热板法、冰醋酸扭体法观察EWRL对小鼠的镇痛作用。采用Bliss法,观察EWRL灌胃给药的急性毒性反应。结果：EWRL对二甲苯致小鼠耳廓肿胀、棉球肉芽肿胀、角叉菜胶致足趾肿胀均具有显著的抑制作用,并能显著提高小鼠的热板痛阈值,减少小鼠的扭体次数。急性毒性实验中,连续观察14d,未见明显毒性反应症状,无动物死亡,计算得最大给药量为87.0g/kg。结论：EWRL具有显著的抗炎和镇痛活性,且经口服属无毒级。
第三部分西瓜藤不同部位提取物抗炎活性筛选及其机理研究
目的：研究西瓜藤不同部位提取物的抗炎作用,并初步阐明其作用机理。方法：采用LPS刺激RAW264.7细胞的炎症模型,以对NO释放水平影响为标志,对西瓜藤的石油醚、乙酸乙酯、正丁醇、50%乙醇、水部位进行抗炎活性筛选。确定活性部位后,采用ELISA法测定促炎介质TNF-α, IL-6, IL-1β, iNOS, COX-2, HO-1的浓度；免疫荧光法检测NF-κB的激活核转运；实时荧光定量PCR法测定IL-1β, IL-6, TNF-α、 COX-2、 iNOS和HO-1mRNA水平对其抗炎机理进行探讨。结果：西瓜藤各提取部位对LPS刺激RAW264.7细胞的炎症模型的NO释放均具有显著抑制作用,以石油醚部位最强。石油醚部位对炎症模型中TNF-α, IL-6, IL-1β、iNOS, COX-2的过度表达具有显著抑制作用；对HO-1水平无影响。同时还可以抑制NF-κB的激活核转运,降低IL-1β、IL-6、TNF-α、 COX-2、 iNOS和HO-1mRNA水平。结论：石油醚部位为西瓜藤的抗炎活性部位,其机理可能是通过抑制NF-κB的激活核转运来达到抗炎的作用。
第四部分西瓜藤不同部位提取物降血糖活性筛选研究
实验一西瓜藤不同提取物对四氧嘧啶致糖尿病小鼠的降血糖作用
目的：研究西瓜藤不同部位提取物对糖尿病小鼠的降血糖作用。方法：小鼠腹腔注射四氧嘧啶建立糖尿病小鼠模型,西瓜藤不同提取物喂养干预治疗。测定小鼠的空腹血糖、葡萄糖耐量、血清中胰岛素水平、TG、 TC、 NO和iNOS含量；肝组织匀浆中NO和iNOS水平；并观察小鼠胰腺组织病理学改变。结果：西瓜藤乙酸乙酯高剂量组和石油醚高剂量组TC显著性降低,TG值也有所降低,血糖水平及糖耐量均有改善,胰岛素含量显著升高,NO和iNOS含量显著降低。结论：西瓜藤乙酸乙酯部位、石油醚部位对糖尿病小鼠具有一定的降血糖作用。作用机理可能与其促进胰岛素释放及降低NO和iNOS含量有关。
实验二西瓜藤乙酸乙酯提取物对DN大鼠肾小管的保护作用
目的：探讨西瓜藤乙酸乙酯提取物对DN大鼠肾小管的保护作用。方法：大鼠腹腔注射链脲佐菌素(STZ)建立DN大鼠模型,西瓜藤乙酸乙酯提取物喂养干预治疗。检测血、尿中血糖、血肌酐、尿肌酐、尿清蛋白、血胰岛素等指标,计算内生肌酐清除率(Ccr),并观察大鼠肾脏组织病理学改变。结果：与DN组比较,西瓜藤乙酸乙酯高、中剂量组大鼠血糖、血胰岛素、24h尿清蛋白排泄量、肾重指数及肾小管损伤指数均明显下降(P<0.05),内生肌酐清除率升高(P<0.05)。结论：西瓜藤乙酸乙酯提取物具有保护DN大鼠肾小管的作用。
第五部分西瓜藤不同部位提取物抑菌活性筛选研究
目的：筛选西瓜藤不同部位提取物的抑菌活性部位。方法：采用琼脂稀释法对西瓜藤不同部位提取物进行体外抑菌试验；采用小鼠腹腔注射金黄色葡萄球菌法进行西瓜藤80%醇提取物体内抑菌实验。结果：西瓜藤提取物对金黄色葡萄球菌、大肠埃希氏菌、铜绿假单胞菌、伤寒沙门氏菌、枯草芽孢杆菌和肺炎克雷伯氏菌均有不同程度的抑制作用,但对链球菌作用不明显。其中80%醇提取物和乙酸乙酯萃取物抑制金黄色葡萄球菌活性最好,其最低抑菌浓度(MIC)分别为4.2mg·mL-1,8.4mg·mL-1。体内实验也表明,80%醇提取物具有较好的抑菌作用。结论：西瓜藤提取物具有较好的抑菌活性,在抑菌方面有一定的开发前景。
第六部分西瓜脑苷V体外抗肿瘤活性筛选研究
目的：研究西瓜藤中西瓜脑苷Ⅴ (lancerebroside Ⅴ)的体外抗肿瘤活性。方法：采用MTT法,测定西瓜藤中的lancerebroside Ⅴ体外分别对前列腺癌细胞株PC3,紫杉醇耐药株PC3-RTX,鼻咽癌细胞株SUNE1,HONE1,肺癌细胞株A549的增殖抑制率,并计算相应的IC50值。结果：得到lancerebroside Ⅴ对五种肿瘤细胞株IC5o值的范围为3.9～8.1μg·mL-1。结论：西瓜藤中的lancerebroside V对所选择的5种肿瘤细胞株均具有一定的抗肿瘤活性。
综上所述,西瓜藤总提取物具有较好的抗炎镇痛作用,且毒副作用很低；西瓜藤石油醚萃取物对LPS诱导的巨噬细胞炎症反应具显著抑制作用,其作用机理可能是通过选择性抑制COX-2的活性,抑制NF-κB核转运激活,从而抑制LPS诱导的促炎介质IL-1beta、IL-6、TNF-α释放以及iNOS的活性,减少NO的释放；西瓜藤乙酸乙酯萃取物和石油醚萃取物对四氧嘧啶致糖尿病小鼠具有一定的降血糖作用,西瓜藤乙酸乙酯萃取物对2型糖尿病大鼠肾小管具有一定的保护作用；西瓜藤提取物对8种临床常见细菌除了链球菌外,均有不同程度的抑制作用,其中,80%醇提物和乙酸乙酯萃取物作用效果较好；从西瓜藤中分离得到20个化合物,其中一个为新化合物：西瓜脑苷Ⅴ(lancerebroside Ⅴ),通过体外抗肿瘤实验发现,其对前列腺癌细胞株PC3,紫杉醇耐药株PC3-RTX,鼻咽癌细胞株SUNE1, HONE1,肺癌细胞株A549等五种肿瘤细胞均具有一定的抗肿瘤作用。
Part Ⅰ. Study on Chemical Constituents of the Citrullus Lanatus Vine Experiment1:Preliminary Tests of Chemical Components for the Four Different Species Citrullus Lanatus Vine
Objective:To identify the chemical components of the four different species citrullus lanatus vine using comprehensive preliminary tests. Methods:The mixture was extracted from the four different species citrullus lanatus vine with purified water95%ethanol, and petrol ether was studied using test tube and chromatography method. The chemical components of the four different species citrullus lanatus vine were preliminarily demonstrated using precipitation reaction and color reaction of multiple indicators. Results:The four different species citrullus lanatus vine might contain sugar, organic acid, sapaomin, flavonoids, alkaloids, steride. Conclusion: The research provides foundation for further study and application of citrullus lanatus vine.
Experiment2:Study on Chemical Constituents of Citrullus Lanatus Vine (I)
Objective:To study the chemical constituents of citrullus l anatus vine in extracting with petroleum ether. Methods:Compounds wer e isolated and purified by polyamide column chromatography. Their chemi cal structures were elucidated on the basis of physicochemical properties, and spectral data. Results:Ten compounds were isolated from citrullus la natus vine andelucidated as β-sitosterol,daucosterol, stigmasterol, monomyri stin, palmitic acid, monopalmitin, monobehenin, ursolic acid, monoheneicos anoin, stearic acid. Conclusion:Compounds of1-10were obtained from c itrullus lanatus vine for the first time.
Experiment3:Study on Chemical Constituents of Citrullus Lanatus Vine (Ⅱ)
Objective:To study the chemical constituents of citrullus lanatus vine in extracting with ethyl acetate. Methods:Compounds were isolated and purified by polyamide column chromatography. Their chemical structures were elucidated on the basis of physicochemical properties, and spectral data. Results:Ten compounds were isolated from citrullus lanatus vine and elucidated as pentadecanoic acid, monopentadecanoin,2,3-Dihydroxypropyl nonadecoate, lignocericacid-2,3-dihydroxy-propanenyl ester, lancerebroside5, salicylic acid,4-Hydroxybenzoic acid, hydroquinone, succinic acid, vanillic acid. Conclusion:Compounds of1-10were obtained from citrullus lanatus vine for the first time.
Part Ⅱ. Experimental Study on Anti-inflammation and Analgesia Effect and Acute Toxicity of Extract from Citrullus Lanatus Vine
Objective:To study the effect of extract from citrullus lanatus vine (EWRL) on anti-inflammation and analgesia and acute toxicity. Methods: The models of mice ear edema induced by xylene, carrageenin and cotton pellet granuloma in rats to observe the anti-inflammation effect of EWRL via oral administration. The effect of EWRL on analgesia was tested by measuring the latent period licking hind foot with the hot plate method and counting body twisting induced by acetic acid in mice. The acute toxicity of EWRL was measured by the method of Bliss. Results:In acute toxic reactions which were observed for14days, all mice were survived, and no toxic reaction was shown during the observed period. The maximum administration dosage was87.0g/kg of EWRL given to mice per kilogram. EWRL could significantly inhibit the ear edema caused by xylene in mice, granuloma hyperplasia caused by cotton in rats, paw oedema caused by carrageen in rats. It could significantly prolong the pain threshold on hot-plate in mice, reduce the writhing times in mice. Conclusion: EWRL has obvious effect on anti-inflammation and analgesia.
Part Ⅲ. Study on Screening and Mechanism of Anti-inflammatory Activity of Different Fractions from Citrullus Lanatus Vine
Objective:To screening the extract parts of citrullus lanatus vine on anti-inflammatory activity and study its mechanism. Methods: Inflammatory model of LPS-stimulated RAW264.7cells were used, the level of impact on the release of NO as a symbol, and the petroleum ether, ethyl acetate, n-butanol,50%ethanol and soluble parts of anti-inflammatory activity on citrullus lanatus vine wered screeninged. The concentration of proinflammatory mediators TNF-α, IL-6, IL-1β, iNOS and COX-2HO-1by ELISA were determined. The activation nuclear translocation of NF-κB by immunofluorescence assay were detected, Real-time PCR for the determination level of IL-1β, IL-6, TNF-α, COX-2, iNOS and HO-1mRNA, and explored its anti-inflammatory mechanism. Results:Extract parts of citrullus lanatus vine significantly inhibited NO release of the LPS-stimulated RAW264.7cells inflammatory model, and the petroleum ether part was the strongest. The petroleum ether fraction significantly inhibited the overexpression of TNF-a, IL-6, IL-1β, iNOS, COX-2, but have no effect on the levels of HO-1in inflammatory model. It could also inhibit the activation of NF-κB nuclear translocation, and reduce the level of IL-1β, IL-6, TNF-α, COX-2, iNOS and HO-1mRNA. Conclusion:Petroleum ether fraction of the watermelon vine was the anti-inflammatory active site, which may achieve the anti-inflammatory role by inhibiting the activation of NF-κB nuclear translocation.
Part IV. The Screening of Hypoglycemic Activities of Different Fractions from Citrullus Lanatus Vine
Experiment1:The Hypoglycemic Effects of Different Fractions from Citrullus Lanatus Vine on Diabetic Model Induced by Alloxan in Mice
Objective:To investigate the hypoglycemic effects of different fractions from citrullus lanatus vine on diabetic model induced by alloxan in mice. Methods:Healthy mices were used to establish diabetic models by peritoneal injection administration of alloxan. The different fractions from citrullus lanatus vine was applied as raw materials for potential marine drug. The levels of fasting blood glucose(FBG)、 fasting blood insulin (FBI) and glucose tolerance were detected by automatic blood glucose device. The concentrations of total cholesterol (TC)、 total glycerin (TG) nitric oxide (NO) and nitric oxide synthase (iNOS) in blood were respectively measured. The concentrations in liver homogenate of nitric oxide (NO) and nitric oxide synthase (iNOS) were determined. Pathology of pancreas was observed. Results:After treatment, ethyl acetate high dose group and Petroleum ether high dose group of citrullus lanatus vine all significantly decreased the levels of TC、 TG、 NO and iNOS. Meanwhile, blood glucose level and glucose tolerance were improved. Insulin levels was also improved. Conclusion:Ethyl acetate group and Petroleum ether group of citrullus lanatus vine shows significant hypoglycemic effect on diabetic model induced by alloxan in mice. The mechanism of its hypoglycemic effect may be promoted the insulin release and decreased the levels of NO and iNOS.
Experiment2:The Protective Renal Tubular Effects of Ethyl Acetate Extract from Citrullus Lanatus Vine in Rats with DN
Objective:To investigate the protective renal tubular effects of ethyl acetate extract from citrullus lanatus vine in rats with DN. Methods: Healthy rates were used to establish DN models by peritoneal injection administration of streptozotocin. The ethyl acetate extract from citrullus lanatus vine was applied as raw materials for potential marine drug. The levels of fasting blood glucose(FBG)、fasting blood insulin (FBI)、serum creatinine (Scr)、urine creatinine (Ucr) and urinary albumin(UAlb) were detected. Pathology of live was observed. Results:After treatment, ethyl acetate high dose group and medial dose group of citrullus lanatus vine all significantly decreased the levels of GLU、FBI、UAlb、KWI and TBI.Meanwhile, Ccr was also improved. Conclusion:The ethyl acetate extract from citrullus lanatus vine can protect renal tubular in rats with DN.
Part Ⅴ. Antimicrobial Activity of the Extracts from Citrullus Lanatus Vine
Objective:To study antimicrobial activity in vitro of citrullus lanatus vine extract. Methods:Agar dilution method was applied to detect antimicrobial effect of citrullus lanatus vine extract in vitro; the mice celiac injection of staphylococcus aureus method was applied to detect the antimicrobial effect of citrullus lanatus vine of ethyl acetate extraction in vivo. Results:Citrullus lanatus vine extract had different inhibition effects on staphylococcus aureus, escherichia coli, bacillus aeruginosus, salmonellatyph, bacillus subtilis and klebsiella pneumoniae, but it showed weak inhibition effect on chain coccus. Effects of80%alcohol extract and ethylacetate extract were better than others on staphylococcus aureus. The minimum inhibition concentration (MIC), respectively is4.2mg·mL-1,8.4mg·mL-1. Conclusion: Citrullus lanatus vine extract have antibacterial action, and provide a basis to exploit new antimicro-bic drug.
Part VI. Anti-tumor Active of Lancerebroside V from Citrullus Lanatus Vine in Vitro
Objective:To study the anti-tumor active of Lancerebroside Ⅴ extracted from citrullus lanatus vine in vitro. Methods:MTS method was used to test inhabitation of Lancerebroside Ⅴ from citrullus lanatus vine with five kinds of human tumor cells in vitro and IC50values were calculated respectively. Results:IC50values of these LancerebrosideV tested with five cancer cell lines were3.9～8.1μg·mL-1. Conclusion:To some extent, Lancerebroside V from citrullus lanatus vine have positive effect on inhabiting those human tumor lines in vitro.
引文
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[1]邓家刚.农作物废弃物药用研究的战略意义与基本思路[J].广西中医药,2010,33(1)：1-3
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