高粱原花青素提取纯化及生物活性研究
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摘要
原花青素(procyanidins,简称PC)是一大类多酚化合物的总称。具有抗氧化、清除自由基、抗辐射、抗肿瘤、抗炎症、抗衰老和保护心血管活性、护肤美容、改善视觉等诸多生物功效。
高粱[Sorghum bicolor(Linn.)Moench]是禾本科高粱族高粱属一年生草本植物。它含有原花青素等很多化学成分,为了更好地利用高粱原花青素资源,本文对其做了初步研究。
1.采用乙醇提取高粱原花青素,正交试验得出最优提取工艺为:用70%乙醇,在70℃,提取2次,每次1.0h,原料和乙醇用量比为1:10;影响顺序是乙醇浓度＞提取时间＞料液比＞提取温度。
2.采用AB-8大孔树脂吸附,经过水洗脱,去掉了大部分水溶性杂质,再用30%乙醇水溶液洗脱,富集了绝大部分高粱原花青素,经比色测定原花青素含量为86.47%;经聚酰胺树脂纯化后,高粱原花青素含量为79.62%。
3.高速逆流色谱分离纯化原花青素,采用正丁醇-甲醇-水体系(20:1:15)分离得到3个物质峰。经过HPLC-TOFMS分析,推测包含原花青素单体儿茶素、一种原花青素二聚体和一种原花青素三聚体。
4.抗氧化试验。总抗氧化力,三个样品中以聚酰胺部位最高,AB-8树脂部位次之,70%乙醇粗提物部位最低;聚酰胺部位、AB-8树脂部位和70%乙醇粗提物部位均表现出清除自由基活性,且都具有明显的量效关系;与模型组比较,聚酰胺部位、AB-8树脂部位和70%乙醇粗提物部位的MDA水平都明显下降(P＜0.05);三个样品的小鼠血清中GSH-PX和SOD活力普遍提高,与模型组比较,差异显著(P＜0.05)。
5.抗Lewis肺癌试验。高粱原花青素高、中、低三个剂量组,给药量分别为每公斤体重1g、2g和4g,给药天数为20d。结果:①高剂量组抑制肿瘤率为17.59%(P＜0.05):②高、中、低剂量组肿瘤转移抑制率分别为80.56%(P＜0.01)、72.12%(P＜0.01)和61.1%(P＜0.01),抑制率与药物剂量呈明显量效关系。
6.VEGF免疫组化实验,结果表明高粱原花青素高、中、低剂量组可明显抑制肿瘤细胞VEGF的表达,从而抑制肿瘤转移,预示高粱原花青素具有比较好的肿瘤抑制前景。
7.建立高粱原花青素含量快速检测方法。利用近红外反射光谱技术,采用偏最小二乘法(PLS)建立高粱原花青素含量预测模型。模型的内部交叉验证决定系数(R~2)为0.8657,标准差(RMSECV)为0.277;外部验证决定系数(R~2)为0.8919,标准差(RMSEP)为0.312。高含量和低含量样品的预测值与化学值的基本吻合。结论:用PLS建立的回归方程能比较准确地预测高粱原花青素的含量。
8.高粱原花青素的资源研究表明:各省区高粱的原花青素含量范围为0.01%～4.1%。原花青素平均含量超过1.5%有黑龙江等9个省区,平均含量超过2.0%的有云贵川三省;平均含量低于1.0%的有新疆等5个省区,平均含量最低的为新疆,仅为0.025%。总体来说,南方产区比北方产区原花青素含量高。
Sorghum belongs to genus sorghum,family Oryza,which is a one-year herb plant.It contains many chemical components such as proanthocyanidins,and possesses widely nutritional and pharmacological actions.The paper aimed to study the proanthocyanidins of sorghum.The main conclusions are as follows:
1.Experiments were conducted to optimizing the extraction condition for extracting proanthocyanidins from sorghum grain.Results showed that ethanol concentration had the biggest effect to the extraction,followed by refluxing extraction time,material-liquid ratio and refluxing extraction temperature orderly.The optimized extraction conditeion is ethanol concentration of 70%,extraction temperature of 80℃,material-liquid ratio of 1:10 and extracting duration of 0.5 hour,two times.
2.The proanthocyanidins was purified and condensed by diluting through macroporous resin AB-8.Collecting 30%ethanol eluting fraction,the proanthocyanidins content was 86.47%.After the polyamide resin purification,.the proanthocyanidins content was 79.62%.
3.The extract derived before was purified by High-Speed Copunter-Current Chromatography(HSCCC).Three compounds were obtained and analyzed by HPLC-TOFMS;they were esteemed to be catechin,dimmer- catechin,and trimer- catechin.
4.Total antioxidant activity was measured using animal model.Ageing rats were oral administrated with polymide extract,AB-8 resin extracts and 70%ethanol extracts, respectively.The level of MDA of all groups decreased significantly(p<0.05).The activities of GSH-px and SOD enhanced significantly(p<0.05) of all groups too.
5.Lung cancer animal model was constructed by Lewis lung cancer cell intraperitoneal injection.And sorghum extracts were oral administrated with three levels,which were 1,2 and 4 g/kg body wt,respectively,for 20 days.The inhibition rate of high dose was 17.59%(p<0.05);and the trrans-inhibition effect of high,medium and low dose were 80.56%(P<0.01),72.12%(P<0.01) and 61.1%(P<0.01),respectively.It could conclude that the sorghum extracts could inhibited cancer cell proliferation in a dosage manner.
6.VEGF immune histochemistry terst showed that the extracts of sorghum could significantly inhibited the expression of tumour cell at high,medium and low concentration.
7.Near-infrared reflect spectroscopy(NIRS) technology was applied on the determination of the procyanidins content of sorghum.The main purpose is to investigate the feasibility to construct the mathematic model for procyanidins content of sorghum with partial least-squares(PLS) test.Regression model was evaluated in terms of root mean square error of cross-validation(RMSECV),the root mean square error of prediction(RMSEP) and inner/outer cross-validation test correlation coefficient(R~2).The inner corss-validation test correlation coefficient(R~2) is 0.8657 and RMSECV is 0.277,outer corss-validation test correlation coefficient(R~2) is 0.8919 and RMSECP is 0.312.The results showed that NIRS,a well-established and widely applied technique could be applied to determine procyanidins content in sorghum rapidly and nondestructively.
8.Materials about resource of sorghum proanthocyanidins showed that proanthocyanidins contents covered from 0.01%to 4.1%.Sorghums produced in Heilongjiang,Hebei,Shaanxi,Shandong,Anhui,Sichuan,Guizhou,Yunnan and Guangxi Province contain more than 1.5%proanthocyanidins.Yunnan,Guizhou and Sichuan sorghums contain more than 2.0%proanthocyanidins in average.Sorghums resources from five Provinces and zone contain proanthocyanidins less than 1.0%,they are Xinjiang,Jilin, Liaoning,Mongolia,Beijing and Henan,and sorghum produced in Xinjiang contains the lowest proanthocyanidins by 0.025%.To conclude,sorghums form northern place of production contain higher proanthocyanidins than southern zones.
高粱[Sorghum bicolor(Linn.)Moench]是禾本科高粱族高粱属一年生草本植物。它含有原花青素等很多化学成分,为了更好地利用高粱原花青素资源,本文对其做了初步研究。
1.采用乙醇提取高粱原花青素,正交试验得出最优提取工艺为:用70%乙醇,在70℃,提取2次,每次1.0h,原料和乙醇用量比为1:10;影响顺序是乙醇浓度＞提取时间＞料液比＞提取温度。
2.采用AB-8大孔树脂吸附,经过水洗脱,去掉了大部分水溶性杂质,再用30%乙醇水溶液洗脱,富集了绝大部分高粱原花青素,经比色测定原花青素含量为86.47%;经聚酰胺树脂纯化后,高粱原花青素含量为79.62%。
3.高速逆流色谱分离纯化原花青素,采用正丁醇-甲醇-水体系(20:1:15)分离得到3个物质峰。经过HPLC-TOFMS分析,推测包含原花青素单体儿茶素、一种原花青素二聚体和一种原花青素三聚体。
4.抗氧化试验。总抗氧化力,三个样品中以聚酰胺部位最高,AB-8树脂部位次之,70%乙醇粗提物部位最低;聚酰胺部位、AB-8树脂部位和70%乙醇粗提物部位均表现出清除自由基活性,且都具有明显的量效关系;与模型组比较,聚酰胺部位、AB-8树脂部位和70%乙醇粗提物部位的MDA水平都明显下降(P＜0.05);三个样品的小鼠血清中GSH-PX和SOD活力普遍提高,与模型组比较,差异显著(P＜0.05)。
5.抗Lewis肺癌试验。高粱原花青素高、中、低三个剂量组,给药量分别为每公斤体重1g、2g和4g,给药天数为20d。结果:①高剂量组抑制肿瘤率为17.59%(P＜0.05):②高、中、低剂量组肿瘤转移抑制率分别为80.56%(P＜0.01)、72.12%(P＜0.01)和61.1%(P＜0.01),抑制率与药物剂量呈明显量效关系。
6.VEGF免疫组化实验,结果表明高粱原花青素高、中、低剂量组可明显抑制肿瘤细胞VEGF的表达,从而抑制肿瘤转移,预示高粱原花青素具有比较好的肿瘤抑制前景。
7.建立高粱原花青素含量快速检测方法。利用近红外反射光谱技术,采用偏最小二乘法(PLS)建立高粱原花青素含量预测模型。模型的内部交叉验证决定系数(R~2)为0.8657,标准差(RMSECV)为0.277;外部验证决定系数(R~2)为0.8919,标准差(RMSEP)为0.312。高含量和低含量样品的预测值与化学值的基本吻合。结论:用PLS建立的回归方程能比较准确地预测高粱原花青素的含量。
8.高粱原花青素的资源研究表明:各省区高粱的原花青素含量范围为0.01%～4.1%。原花青素平均含量超过1.5%有黑龙江等9个省区,平均含量超过2.0%的有云贵川三省;平均含量低于1.0%的有新疆等5个省区,平均含量最低的为新疆,仅为0.025%。总体来说,南方产区比北方产区原花青素含量高。
Sorghum belongs to genus sorghum,family Oryza,which is a one-year herb plant.It contains many chemical components such as proanthocyanidins,and possesses widely nutritional and pharmacological actions.The paper aimed to study the proanthocyanidins of sorghum.The main conclusions are as follows:
1.Experiments were conducted to optimizing the extraction condition for extracting proanthocyanidins from sorghum grain.Results showed that ethanol concentration had the biggest effect to the extraction,followed by refluxing extraction time,material-liquid ratio and refluxing extraction temperature orderly.The optimized extraction conditeion is ethanol concentration of 70%,extraction temperature of 80℃,material-liquid ratio of 1:10 and extracting duration of 0.5 hour,two times.
2.The proanthocyanidins was purified and condensed by diluting through macroporous resin AB-8.Collecting 30%ethanol eluting fraction,the proanthocyanidins content was 86.47%.After the polyamide resin purification,.the proanthocyanidins content was 79.62%.
3.The extract derived before was purified by High-Speed Copunter-Current Chromatography(HSCCC).Three compounds were obtained and analyzed by HPLC-TOFMS;they were esteemed to be catechin,dimmer- catechin,and trimer- catechin.
4.Total antioxidant activity was measured using animal model.Ageing rats were oral administrated with polymide extract,AB-8 resin extracts and 70%ethanol extracts, respectively.The level of MDA of all groups decreased significantly(p<0.05).The activities of GSH-px and SOD enhanced significantly(p<0.05) of all groups too.
5.Lung cancer animal model was constructed by Lewis lung cancer cell intraperitoneal injection.And sorghum extracts were oral administrated with three levels,which were 1,2 and 4 g/kg body wt,respectively,for 20 days.The inhibition rate of high dose was 17.59%(p<0.05);and the trrans-inhibition effect of high,medium and low dose were 80.56%(P<0.01),72.12%(P<0.01) and 61.1%(P<0.01),respectively.It could conclude that the sorghum extracts could inhibited cancer cell proliferation in a dosage manner.
6.VEGF immune histochemistry terst showed that the extracts of sorghum could significantly inhibited the expression of tumour cell at high,medium and low concentration.
7.Near-infrared reflect spectroscopy(NIRS) technology was applied on the determination of the procyanidins content of sorghum.The main purpose is to investigate the feasibility to construct the mathematic model for procyanidins content of sorghum with partial least-squares(PLS) test.Regression model was evaluated in terms of root mean square error of cross-validation(RMSECV),the root mean square error of prediction(RMSEP) and inner/outer cross-validation test correlation coefficient(R~2).The inner corss-validation test correlation coefficient(R~2) is 0.8657 and RMSECV is 0.277,outer corss-validation test correlation coefficient(R~2) is 0.8919 and RMSECP is 0.312.The results showed that NIRS,a well-established and widely applied technique could be applied to determine procyanidins content in sorghum rapidly and nondestructively.
8.Materials about resource of sorghum proanthocyanidins showed that proanthocyanidins contents covered from 0.01%to 4.1%.Sorghums produced in Heilongjiang,Hebei,Shaanxi,Shandong,Anhui,Sichuan,Guizhou,Yunnan and Guangxi Province contain more than 1.5%proanthocyanidins.Yunnan,Guizhou and Sichuan sorghums contain more than 2.0%proanthocyanidins in average.Sorghums resources from five Provinces and zone contain proanthocyanidins less than 1.0%,they are Xinjiang,Jilin, Liaoning,Mongolia,Beijing and Henan,and sorghum produced in Xinjiang contains the lowest proanthocyanidins by 0.025%.To conclude,sorghums form northern place of production contain higher proanthocyanidins than southern zones.
引文
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