紫草抗氧化与抗癌活性成分的研究
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摘要
本文采用OSI方法，从7种我国常见中草药中选择具有较强抗氧化活性的中药。结果发现，紫草和虎杖对底物猪油具有明显的抗氧化作用。其中，紫草的抗氧化活性远强于合成抗氧化剂BHT和天然抗氧化剂α-tocopherol，且关于其抗氧化方面的报道还很有限，故本文对其进行了一系列研究。
对紫草不同浓度药材粉末和不同极性的提取物进行抗氧化活性测定，发现紫草粉末在0.02％-1.5％剂量下，可分别将猪油的氧化诱导期从4.5h延长至6.0h-87.4h，而且呈现良好的量效关系。其石油醚提取物、氯仿提取物也表现出很强的抗氧化作用。说明紫草中富含抗氧化活性成分。
以抗氧化活性跟踪为导向，通过柱色谱方法对具有抗氧化活性的石油醚提取物和氯仿提取物进行活性成分的分离纯化，结合理化性质和波谱分析，从紫草中分离并鉴定了7个化合物，它们是：去氧紫草素(deoxyshikonin，1)、β，β-二甲基丙烯酰紫草素(β，β-dimethylacrylshikonin，2)、异丁酰紫草素(isobutyl--shikonin，3)、紫草素(shikonin，4)、甲基紫草素[5，8-dihydroxy-2-(1-methoxy--4-methyl-3-pemenyl)-1，4-naphthalenedione，5]、β-谷甾醇(β-sitosterol，6)和咖啡酸脂肪醇酯(a mixture of two caffeic acid esters，7)。其中化合物5为首次从该植物中分离得到的己知化合物。同时选用Rancimat法、还原力法、DPPH自由基清除法和ABTS自由基清除法4种抗氧化活性测定方法对它们的抗氧化活性进行了比较和评价，并初步探讨了抗氧化机理。结果表明，除β-谷甾醇外，其余6个化合物均具有较强抗氧化作用。其中在Rancimat法和还原力法测定中，它们抗氧化活性的强弱顺序分别为：化合物7＞4＞BHT＞2＞3＞5＞1＞6和化合物7＞BHT＞4＞2～3～5＞1＞6；此外，化合物7对两种自由基的清除能力均较强，而5个萘醌类化合物则对ABTS自由基的清除能力强于DPPH。这些结果提示，从紫草中分离出的化合物可以抑制猪油的氧化，有效清除自由基，并且参与氧化还原反应，这为从紫草中寻求天然高效的抗氧化剂增添了部分实验依据。
本文首次在动物体内考察了紫草提取物的抗氧化和抗疲劳作用。实验以α-tocopherol为阳性对照，测定了紫草抗氧化活性部位对小鼠血清中SOD酶活力和MDA含量的影响，以及对负重小鼠游泳时间的影响。结果显示，用紫草石油醚、氯仿提取物连续喂饲小鼠3周后，与空白组比较，紫草提取物能提高小鼠血清中SOD活力和降低MDA含量(P＜0.05或P＜0.01)，并能显著延长负重小鼠的游泳时间在22％-56％之间。说明紫草具有生物抗氧化和抗疲劳作用。
本文研究了从紫草中分离得到的7个化合物在体外的抗癌作用。选取人胃癌细胞(MGC-803)和人肝癌细胞(BEL-7402)进行体外细胞培养，以顺氯氨铂(DDP)为阳性对照，通过MTT实验测定各化合物对2株癌细胞的生长抑制作用。结果发现，在细胞培养液中加入各测试样品24h后，7个化合物对两种癌细胞的抑制活性有所不同。以上化合物(1-7)以及DDP对胃癌细胞MGC-803的IC_(50)分别为：1.7、1.4、7.0、0.5、1.5、＞100、＞100和4.4μg／mL；对肝癌细胞BEL-7402的IC_(50)分别为：1.5、1.3、34.0、1.3、1.4、＞100、＞100和7.6μg／mL。证明从紫草分离得到的萘醌类化合物(1～5)对人胃癌细胞MGC-803和人肝癌细胞BEL-7402的生长有较强的抑制作用，呈明显的剂量-药效依赖关系，其中化合物1、2、4、5的抗肿瘤活性远强于阳性药物DDP。化合物6、7则没有抑制这两株癌细胞的作用。
本研究建立了同时测定紫草中活性成分紫草素、异丁酰紫草素和β，β-二甲基丙烯酰紫草素含量的反相高效液相色谱分析方法。紫草素、异丁酰紫草素和β，β-二甲基丙烯酰紫草素浓度分别在1.22～12.2μg·mL~(-1)(r=0.9999)，2.48～24.8μg·mL~(-1)(r=0.9998)和11.28～112.8μg·mL~(-1)(r=0.9998)范围内与峰面积呈良好线性关系(n=5)，方法回收率分别为100.3％，98.6％和98.4％，RSD分别为3.2％，3.0％和3.3％(n=9)。方法简便、准确，为紫草质量控制方法提供了定量依据。
本研究在自由基病源学说的理论和实验指导下，对抗氧化活性较强的中药紫草进行了系统分析，这对寻求新型高效的天然抗氧化剂和进一步开发利用药用紫草资源提供了部分实验依据。
Some familiar traditional Chinese medicines were selected and their antioxidant effects were evaluated using the Oxidative Stability Instrument (OSI) method. Among them, Lithospermum erythrorhizon and Polygonum cuspidatum were found to have significant antioxidant effects on retarding the peroxidation of lard. The antioxidant activity of the former one was even stronger than the synthetic antioxidant BHT and the natural antioxidantα-tocopherol. Few reports, however, have addressed the antioxidant activity of L. erythrorhizon. So it may be worth of studying as a potential antioxidant resource and a series of research on its antioxidant activity had been performed in this paper.
The antioxidant effects of crude powder and different polarity extracts of L. erythrorhizon at different concentrations were tested in lard. As a result, the 0.02%-1.5% of the powder obviously prolonged the induction period of lard oxidation from 4.5 h to 6.0 h-87.4 h respectively in a good dose-dependent manner. The petroleum ether extract and chloroform extract of L. erythrorhizon were also exerted strong antioxidant activity. These results suggested that L. erythrorhizon could be considered as a promising source of natural antioxidants.
On the basis of antioxidant-guiding, various chromatographic techniques were performed to isolate active constituents from the above two main fractions of L. erythrorhizon. Seven compounds were isolated and identified by UV, IR, NMR and MS spectra data and physical-chemical properties. They were identified as deoxyshikonin (1),β,β-dimethylacrylshikonin (2), isobutylshikonin (3), shikonin (4), 5,8-dihydroxy-2-(1-methoxy-4-methyl-3-pentenyl)-1,4-naphthalenedione (5),β-sitosterol (6) and a mixture of two caffeic acid esters (7). Among them, compound 5 was isolated from this plant species for the first time. The antioxidant activities of the seven compounds were compared and evaluated through Rancimat method, reducing power and radical scavenging activity. Their action mechanisms were also preliminarily explored. Results showed that, except compound 6, other six compounds all exhibited obvious antioxidant activities against four different methods. Compounds 4 and 7 exerted much more potent antioxidant effects on retarding the lard oxidation than that of BHT and both were found to exhibit strong reducing power. Their antioxidant activities, assessed by Rancimat method and reducing power, decreased in the following order respectively: compound 7 > 4 > BHT> 2 > 3 > 5 >1 >6 and compound 7 > BHT > 4 > 2～3～5 > 1 >6. In addition, compounds 1-5 all exerted very good radical scavenging activities toward ABTS~+ but showed moderate scavenging activities toward DPPH, while compound 7 presented as a powerful radical scavenger against both ABTS~+ and DPPH. Thus, this study furnishes additional dada to the literature about compounds with antioxidant activity from L. erythrorhizon.
In the experimental research, the antioxidant activities and anti-fatigue effects of L. erythrorhizon were performed in vivo for the first time andα-tocopherol was used for the comparison purpose. After mice were orally treated with extracts of L. erythrorhizon for 3 weeks, the serum level of SOD was significantly increased (P<0.05 or P<0.01) and the level of MDA was significantly decreased compared to the control group (P<0.05 or P<0.01). Further, they also could lessen fatigue by prolonging the swimming time of the burdened mice. These evidenced that L. erythrorhizon could exhibit bioactivity in vivo.
The study examined the in vitro antitumor activities of the 7 compounds isolated from L. erythrorhizon. MTT assays were used to explore their inhibiting effect on the proliferation of MGC-803 and BEL-7402 human cancer cell lines. Diamminedichloroplatinum (DDP) was used as comparison sample and the cytotoxicity effect was evaluated using growth inhibition ratio and IC_(50) value. The results showed that seven compounds exhibited different antitumor effects. The IC_(50) values of compounds 1-7 and DDP toward MGC-803 cell were 1.7、1.4、7.0、0.5、1.5、> 100、> 100 and 4.4μg/mL, respectively; and toward BEL-7402 cell were 1.5、1.3、34.0、1.3、1.4、> 100、>100 and 7.6μg/mL, respectively. It can be concluded that naphthoquinone compounds (1-5) from L. erythrorhizon possessed strong antitumor activities against MGC-803 and BEL-7402 in vitro and in a concentration dependent manner. In addition, among the isolated compounds, compounds 1, 2, 4 and 5 exhibited stronger antitumor activity than DDP while compounds 6 and 7 had no antitumor effect in this study.
RP-HPLC method for the simultaneous determination of shikonin, isobutylshikonin andβ,β-dimethylaerylshikonin in L. erythrorhizon was developed and validated. The linear ranges for shikonin, isobutylshikonin andβ,β-dimethylacrylshikonin were 1.22-12.2μg.mL~(-1) (r=0.9999), 2.48-24.8μg-mL-1 (r=0.9998) and 11.28- 112.8μg-mL~(-1) (r=0.9998), respectively. The average recoveries were 100.3% with RSD 3.2%, 98.6% with RSD 3.0%, and 98.4% with RSD 3.3%, respectively. The methods were simple and accurate and could be used as the quality control methods of L. erythrorhizon.
Under the direction of pathobiology theory of free radicals, this paper studied the antioxidant activity of L. erythrorhizon systematically, which may help the development and utilization of more effective and newly antioxidants from natural origins and endow the deep going research on L. erythrorhizon a great practical significance.
对紫草不同浓度药材粉末和不同极性的提取物进行抗氧化活性测定，发现紫草粉末在0.02％-1.5％剂量下，可分别将猪油的氧化诱导期从4.5h延长至6.0h-87.4h，而且呈现良好的量效关系。其石油醚提取物、氯仿提取物也表现出很强的抗氧化作用。说明紫草中富含抗氧化活性成分。
以抗氧化活性跟踪为导向，通过柱色谱方法对具有抗氧化活性的石油醚提取物和氯仿提取物进行活性成分的分离纯化，结合理化性质和波谱分析，从紫草中分离并鉴定了7个化合物，它们是：去氧紫草素(deoxyshikonin，1)、β，β-二甲基丙烯酰紫草素(β，β-dimethylacrylshikonin，2)、异丁酰紫草素(isobutyl--shikonin，3)、紫草素(shikonin，4)、甲基紫草素[5，8-dihydroxy-2-(1-methoxy--4-methyl-3-pemenyl)-1，4-naphthalenedione，5]、β-谷甾醇(β-sitosterol，6)和咖啡酸脂肪醇酯(a mixture of two caffeic acid esters，7)。其中化合物5为首次从该植物中分离得到的己知化合物。同时选用Rancimat法、还原力法、DPPH自由基清除法和ABTS自由基清除法4种抗氧化活性测定方法对它们的抗氧化活性进行了比较和评价，并初步探讨了抗氧化机理。结果表明，除β-谷甾醇外，其余6个化合物均具有较强抗氧化作用。其中在Rancimat法和还原力法测定中，它们抗氧化活性的强弱顺序分别为：化合物7＞4＞BHT＞2＞3＞5＞1＞6和化合物7＞BHT＞4＞2～3～5＞1＞6；此外，化合物7对两种自由基的清除能力均较强，而5个萘醌类化合物则对ABTS自由基的清除能力强于DPPH。这些结果提示，从紫草中分离出的化合物可以抑制猪油的氧化，有效清除自由基，并且参与氧化还原反应，这为从紫草中寻求天然高效的抗氧化剂增添了部分实验依据。
本文首次在动物体内考察了紫草提取物的抗氧化和抗疲劳作用。实验以α-tocopherol为阳性对照，测定了紫草抗氧化活性部位对小鼠血清中SOD酶活力和MDA含量的影响，以及对负重小鼠游泳时间的影响。结果显示，用紫草石油醚、氯仿提取物连续喂饲小鼠3周后，与空白组比较，紫草提取物能提高小鼠血清中SOD活力和降低MDA含量(P＜0.05或P＜0.01)，并能显著延长负重小鼠的游泳时间在22％-56％之间。说明紫草具有生物抗氧化和抗疲劳作用。
本文研究了从紫草中分离得到的7个化合物在体外的抗癌作用。选取人胃癌细胞(MGC-803)和人肝癌细胞(BEL-7402)进行体外细胞培养，以顺氯氨铂(DDP)为阳性对照，通过MTT实验测定各化合物对2株癌细胞的生长抑制作用。结果发现，在细胞培养液中加入各测试样品24h后，7个化合物对两种癌细胞的抑制活性有所不同。以上化合物(1-7)以及DDP对胃癌细胞MGC-803的IC_(50)分别为：1.7、1.4、7.0、0.5、1.5、＞100、＞100和4.4μg／mL；对肝癌细胞BEL-7402的IC_(50)分别为：1.5、1.3、34.0、1.3、1.4、＞100、＞100和7.6μg／mL。证明从紫草分离得到的萘醌类化合物(1～5)对人胃癌细胞MGC-803和人肝癌细胞BEL-7402的生长有较强的抑制作用，呈明显的剂量-药效依赖关系，其中化合物1、2、4、5的抗肿瘤活性远强于阳性药物DDP。化合物6、7则没有抑制这两株癌细胞的作用。
本研究建立了同时测定紫草中活性成分紫草素、异丁酰紫草素和β，β-二甲基丙烯酰紫草素含量的反相高效液相色谱分析方法。紫草素、异丁酰紫草素和β，β-二甲基丙烯酰紫草素浓度分别在1.22～12.2μg·mL~(-1)(r=0.9999)，2.48～24.8μg·mL~(-1)(r=0.9998)和11.28～112.8μg·mL~(-1)(r=0.9998)范围内与峰面积呈良好线性关系(n=5)，方法回收率分别为100.3％，98.6％和98.4％，RSD分别为3.2％，3.0％和3.3％(n=9)。方法简便、准确，为紫草质量控制方法提供了定量依据。
本研究在自由基病源学说的理论和实验指导下，对抗氧化活性较强的中药紫草进行了系统分析，这对寻求新型高效的天然抗氧化剂和进一步开发利用药用紫草资源提供了部分实验依据。
Some familiar traditional Chinese medicines were selected and their antioxidant effects were evaluated using the Oxidative Stability Instrument (OSI) method. Among them, Lithospermum erythrorhizon and Polygonum cuspidatum were found to have significant antioxidant effects on retarding the peroxidation of lard. The antioxidant activity of the former one was even stronger than the synthetic antioxidant BHT and the natural antioxidantα-tocopherol. Few reports, however, have addressed the antioxidant activity of L. erythrorhizon. So it may be worth of studying as a potential antioxidant resource and a series of research on its antioxidant activity had been performed in this paper.
The antioxidant effects of crude powder and different polarity extracts of L. erythrorhizon at different concentrations were tested in lard. As a result, the 0.02%-1.5% of the powder obviously prolonged the induction period of lard oxidation from 4.5 h to 6.0 h-87.4 h respectively in a good dose-dependent manner. The petroleum ether extract and chloroform extract of L. erythrorhizon were also exerted strong antioxidant activity. These results suggested that L. erythrorhizon could be considered as a promising source of natural antioxidants.
On the basis of antioxidant-guiding, various chromatographic techniques were performed to isolate active constituents from the above two main fractions of L. erythrorhizon. Seven compounds were isolated and identified by UV, IR, NMR and MS spectra data and physical-chemical properties. They were identified as deoxyshikonin (1),β,β-dimethylacrylshikonin (2), isobutylshikonin (3), shikonin (4), 5,8-dihydroxy-2-(1-methoxy-4-methyl-3-pentenyl)-1,4-naphthalenedione (5),β-sitosterol (6) and a mixture of two caffeic acid esters (7). Among them, compound 5 was isolated from this plant species for the first time. The antioxidant activities of the seven compounds were compared and evaluated through Rancimat method, reducing power and radical scavenging activity. Their action mechanisms were also preliminarily explored. Results showed that, except compound 6, other six compounds all exhibited obvious antioxidant activities against four different methods. Compounds 4 and 7 exerted much more potent antioxidant effects on retarding the lard oxidation than that of BHT and both were found to exhibit strong reducing power. Their antioxidant activities, assessed by Rancimat method and reducing power, decreased in the following order respectively: compound 7 > 4 > BHT> 2 > 3 > 5 >1 >6 and compound 7 > BHT > 4 > 2～3～5 > 1 >6. In addition, compounds 1-5 all exerted very good radical scavenging activities toward ABTS~+ but showed moderate scavenging activities toward DPPH, while compound 7 presented as a powerful radical scavenger against both ABTS~+ and DPPH. Thus, this study furnishes additional dada to the literature about compounds with antioxidant activity from L. erythrorhizon.
In the experimental research, the antioxidant activities and anti-fatigue effects of L. erythrorhizon were performed in vivo for the first time andα-tocopherol was used for the comparison purpose. After mice were orally treated with extracts of L. erythrorhizon for 3 weeks, the serum level of SOD was significantly increased (P<0.05 or P<0.01) and the level of MDA was significantly decreased compared to the control group (P<0.05 or P<0.01). Further, they also could lessen fatigue by prolonging the swimming time of the burdened mice. These evidenced that L. erythrorhizon could exhibit bioactivity in vivo.
The study examined the in vitro antitumor activities of the 7 compounds isolated from L. erythrorhizon. MTT assays were used to explore their inhibiting effect on the proliferation of MGC-803 and BEL-7402 human cancer cell lines. Diamminedichloroplatinum (DDP) was used as comparison sample and the cytotoxicity effect was evaluated using growth inhibition ratio and IC_(50) value. The results showed that seven compounds exhibited different antitumor effects. The IC_(50) values of compounds 1-7 and DDP toward MGC-803 cell were 1.7、1.4、7.0、0.5、1.5、> 100、> 100 and 4.4μg/mL, respectively; and toward BEL-7402 cell were 1.5、1.3、34.0、1.3、1.4、> 100、>100 and 7.6μg/mL, respectively. It can be concluded that naphthoquinone compounds (1-5) from L. erythrorhizon possessed strong antitumor activities against MGC-803 and BEL-7402 in vitro and in a concentration dependent manner. In addition, among the isolated compounds, compounds 1, 2, 4 and 5 exhibited stronger antitumor activity than DDP while compounds 6 and 7 had no antitumor effect in this study.
RP-HPLC method for the simultaneous determination of shikonin, isobutylshikonin andβ,β-dimethylaerylshikonin in L. erythrorhizon was developed and validated. The linear ranges for shikonin, isobutylshikonin andβ,β-dimethylacrylshikonin were 1.22-12.2μg.mL~(-1) (r=0.9999), 2.48-24.8μg-mL-1 (r=0.9998) and 11.28- 112.8μg-mL~(-1) (r=0.9998), respectively. The average recoveries were 100.3% with RSD 3.2%, 98.6% with RSD 3.0%, and 98.4% with RSD 3.3%, respectively. The methods were simple and accurate and could be used as the quality control methods of L. erythrorhizon.
Under the direction of pathobiology theory of free radicals, this paper studied the antioxidant activity of L. erythrorhizon systematically, which may help the development and utilization of more effective and newly antioxidants from natural origins and endow the deep going research on L. erythrorhizon a great practical significance.
引文
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