藏茵陈活性成分的结构修饰及生物活性研究
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摘要
藏茵陈是一种传统藏药,具有保肝、抗炎、抗氧化损伤等多种生物活性。该药主要含有环烯醚萜、口山酮和三萜类成分。近些年,其所含环烯醚萜类和口山酮类成分逐渐成为研究热点。但这类成分普遍存在活性不高或不易吸收等不足之处。本文旨在通过生物学方法对藏茵陈所含主要成分进行修饰,以期获得具有一定活性的化合物。主要工作如下：
以獐牙菜苦苷、当药醇苷的平均转移率为评价指标,通过正交试验对药材中含有的这两种化合物的提取工艺进行优化,并利用大孔树脂、硅胶等分离手段进行纯化,使其纯度达到80%以上。
考察黑曲霉菌对獐牙菜苦苷、当药醇苷的转化作用。采用獐牙菜苦苷与黑曲霉菌共同孵育,并获取了(5Z)-5-乙叉基-8-羟基-3,4,5,6,7,8-六氢-1H-吡喃[3,4-c]嘧啶-1-酮(EHPO),獐牙菜苷等产物。经考察,确定了适于EHPO生成的培养基为：葡萄糖5g／L,蛋白胨7.5g/L, KH2PO4 5g/L, MgSO4 1.2g/L, MnSO4 1.51 g/L, NaCl 0.59 g/L,初始pH值为6.0。
以转移率为主要指标确立了EHPO的制备工艺条件为：经石油醚(60-90℃)脱脂后,采用HPD-300型树脂纯化：依次采用水(3BV),10%乙醇(3BV)除杂,再用50%乙醇洗脱3BV。50%乙醇洗脱部分经Al2O3柱层析进一步纯化。依次采用乙酸乙酯-甲醇8：2溶液(9BV),乙酸乙酯-甲醇6：4溶液(4BV),乙酸乙酯-甲醇2：8溶液(12BV)洗脱。再经制备液相精制,可得到产物EHPO,并回收部分獐牙菜苦苷(纯度>95%)。
采用Caco-2细胞模型进行的体外吸收试验表明：给予不同剂量的EHPO后,在Caco-2细胞模型中的转运中对应时间点的表观渗透系数值PappAP-BL约等于PappBL-AP.在所测浓度范围内转化产物的Papp值基本保持恒定。在Caco-2细胞模型中,EHPO的渗透性较好,以被动扩散为主的方式跨膜转运,但pH值、维拉帕米对其跨膜转运仍有一定的影响。
通过大鼠肝匀浆脂质过氧化模型的研究结果表明：EHPO具有一定的体外抗氧化作用；在羟自由基致红细胞溶血模型中,0.8mg/mL的EHPO可显著性抑制羟自由基致氧化损伤。
四氯化碳致小鼠急性肝损伤试验结果表明：EHPO能显著性降低小鼠血清ALT、AST水平,减少MDA含量,增强SOD活力(P<0.05),对四氯化碳引起的肝细胞损伤有一定的保护作用。
总之,上述研究结果表明：藏茵陈所含主要化合物——獐牙菜苦苷、当药醇苷均可被黑曲霉菌降解转化。通过本文建立的分离、纯化工艺可有效的分离、制备前体化合物以及产物。初步研究结果表明,獐牙菜苦苷与黑曲霉菌共同孵育获得的化合物EHPO易于吸收,具有一定的抗氧化,保肝活性。
'Zang-Yin-Chen'(ZYC) is a traditional Tibetan medicine for the activity of hepatoprotective effect, antiinflammatory, antioxidative damage. It is suggested by phytochemistry study that iridoids、xanthones and triterpenoids are the mainly components in ZYC. In recent years, the compounds of xanthones and iridoids were studied widely. But the activity of these compouds were not good. For the purpose of new active substance discovery, the activity compounds of ZYC was modified by biology methods.The main research works are as follows:
With the average transfer rate of swertiamarin and swertianolin as index, the best extraction technology of these constituents in ZYC was optimized by orthogonal experimental design. And macroporous resin, silica gel column chromatography were used for the separation of swertiamarin、swertianolin. And the purity is more than 80%.
The utilization of swertiamarin、swertianolin by Aspergillus niger was investigated. And result showed these compouds could be transformed by it. For the purpose of compounds preparation, swertiamarin was coincubated with Aspergillus niger. The compouds including sweroside and (5Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c] pyridin-1-one were prepared. The medium was optimizated for the output of EHPO. The optimizated medium composition was glucose 5 g/L; Tryptone 7.5g/L; KH2PO45 g·/L; MnSO4 1.51 g/L; MgSO4 1.21 g/L, NaCl 0.59 g/L, pH of the medium was adjusted to 6.0.
With the transfer rate of EHPO as index, the separation technology of EHPO was established. The fermentation broth was concentrated under reduced pressure and partitioned with petroleum (60-90℃). The aqueous layer was isolated by macroporous resin column chromatography. And the column was eluted with H2O (3BV),10% ethanol (3BV) followed by 50% ethanol (3BV). The faction eluted by 50% ethanol was isolated by Al2O3 column chromatography. And the column was eluted with acetoacetate-methanol (8:2) solution (9BV), acetoacetate-methanol (6:4) solution (4BV), followed by acetoacetate-methanol (2:8) solution (12BV). Finaly, the compounds such as EHPO and swertiamarin were isolated by preparative HPLC. The purity of them is more than 95%.
In the Caco-2 cell model, the PappAP-BL of the EHPO was equal to the PappBL-AP, and Papp keep almost constant within the concentration investigated. The permeability of EHPO is good. In Caco-2 cell model, the transcellular transport mechanism of it mainly is a passive one. But the Papp still could be effected by pH, Verapamil.
The antioxidative property of EHPO was evaluated by malondialdehyde (MDA) formation in a hepatic homogenates model, and a model of H2O2-induced hemolysis. The result showed EHPO could inhibit hepatic homogenate peroxidation. In the model of H2O2-induced hemolysis model, it could inhibit hemolysis significantly(0.8mg/mL). (P<0.05).
The hepatoprotective effect was evaluated by the model of CCl4 induced acute hepatic injure in mice. EHPO could decrease the serum ALT, AST levels, the concentration of MDA and increase SOD activity in the mice treated with CCl4. It exhibited significant hepatoprotective effect.
In summary, the results showed that swertiamarin and swertianolin could be transformed by Aspergillus niger. The technology we set could separate the precurosor compounds and the product. The compound of EHPO yielded by co-incubated swertiamarin with Aspergillus niger could be absorbed soon. And it has the activity of antioxidative, hepatoprotective effect.
以獐牙菜苦苷、当药醇苷的平均转移率为评价指标,通过正交试验对药材中含有的这两种化合物的提取工艺进行优化,并利用大孔树脂、硅胶等分离手段进行纯化,使其纯度达到80%以上。
考察黑曲霉菌对獐牙菜苦苷、当药醇苷的转化作用。采用獐牙菜苦苷与黑曲霉菌共同孵育,并获取了(5Z)-5-乙叉基-8-羟基-3,4,5,6,7,8-六氢-1H-吡喃[3,4-c]嘧啶-1-酮(EHPO),獐牙菜苷等产物。经考察,确定了适于EHPO生成的培养基为：葡萄糖5g／L,蛋白胨7.5g/L, KH2PO4 5g/L, MgSO4 1.2g/L, MnSO4 1.51 g/L, NaCl 0.59 g/L,初始pH值为6.0。
以转移率为主要指标确立了EHPO的制备工艺条件为：经石油醚(60-90℃)脱脂后,采用HPD-300型树脂纯化：依次采用水(3BV),10%乙醇(3BV)除杂,再用50%乙醇洗脱3BV。50%乙醇洗脱部分经Al2O3柱层析进一步纯化。依次采用乙酸乙酯-甲醇8：2溶液(9BV),乙酸乙酯-甲醇6：4溶液(4BV),乙酸乙酯-甲醇2：8溶液(12BV)洗脱。再经制备液相精制,可得到产物EHPO,并回收部分獐牙菜苦苷(纯度>95%)。
采用Caco-2细胞模型进行的体外吸收试验表明：给予不同剂量的EHPO后,在Caco-2细胞模型中的转运中对应时间点的表观渗透系数值PappAP-BL约等于PappBL-AP.在所测浓度范围内转化产物的Papp值基本保持恒定。在Caco-2细胞模型中,EHPO的渗透性较好,以被动扩散为主的方式跨膜转运,但pH值、维拉帕米对其跨膜转运仍有一定的影响。
通过大鼠肝匀浆脂质过氧化模型的研究结果表明：EHPO具有一定的体外抗氧化作用；在羟自由基致红细胞溶血模型中,0.8mg/mL的EHPO可显著性抑制羟自由基致氧化损伤。
四氯化碳致小鼠急性肝损伤试验结果表明：EHPO能显著性降低小鼠血清ALT、AST水平,减少MDA含量,增强SOD活力(P<0.05),对四氯化碳引起的肝细胞损伤有一定的保护作用。
总之,上述研究结果表明：藏茵陈所含主要化合物——獐牙菜苦苷、当药醇苷均可被黑曲霉菌降解转化。通过本文建立的分离、纯化工艺可有效的分离、制备前体化合物以及产物。初步研究结果表明,獐牙菜苦苷与黑曲霉菌共同孵育获得的化合物EHPO易于吸收,具有一定的抗氧化,保肝活性。
'Zang-Yin-Chen'(ZYC) is a traditional Tibetan medicine for the activity of hepatoprotective effect, antiinflammatory, antioxidative damage. It is suggested by phytochemistry study that iridoids、xanthones and triterpenoids are the mainly components in ZYC. In recent years, the compounds of xanthones and iridoids were studied widely. But the activity of these compouds were not good. For the purpose of new active substance discovery, the activity compounds of ZYC was modified by biology methods.The main research works are as follows:
With the average transfer rate of swertiamarin and swertianolin as index, the best extraction technology of these constituents in ZYC was optimized by orthogonal experimental design. And macroporous resin, silica gel column chromatography were used for the separation of swertiamarin、swertianolin. And the purity is more than 80%.
The utilization of swertiamarin、swertianolin by Aspergillus niger was investigated. And result showed these compouds could be transformed by it. For the purpose of compounds preparation, swertiamarin was coincubated with Aspergillus niger. The compouds including sweroside and (5Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c] pyridin-1-one were prepared. The medium was optimizated for the output of EHPO. The optimizated medium composition was glucose 5 g/L; Tryptone 7.5g/L; KH2PO45 g·/L; MnSO4 1.51 g/L; MgSO4 1.21 g/L, NaCl 0.59 g/L, pH of the medium was adjusted to 6.0.
With the transfer rate of EHPO as index, the separation technology of EHPO was established. The fermentation broth was concentrated under reduced pressure and partitioned with petroleum (60-90℃). The aqueous layer was isolated by macroporous resin column chromatography. And the column was eluted with H2O (3BV),10% ethanol (3BV) followed by 50% ethanol (3BV). The faction eluted by 50% ethanol was isolated by Al2O3 column chromatography. And the column was eluted with acetoacetate-methanol (8:2) solution (9BV), acetoacetate-methanol (6:4) solution (4BV), followed by acetoacetate-methanol (2:8) solution (12BV). Finaly, the compounds such as EHPO and swertiamarin were isolated by preparative HPLC. The purity of them is more than 95%.
In the Caco-2 cell model, the PappAP-BL of the EHPO was equal to the PappBL-AP, and Papp keep almost constant within the concentration investigated. The permeability of EHPO is good. In Caco-2 cell model, the transcellular transport mechanism of it mainly is a passive one. But the Papp still could be effected by pH, Verapamil.
The antioxidative property of EHPO was evaluated by malondialdehyde (MDA) formation in a hepatic homogenates model, and a model of H2O2-induced hemolysis. The result showed EHPO could inhibit hepatic homogenate peroxidation. In the model of H2O2-induced hemolysis model, it could inhibit hemolysis significantly(0.8mg/mL). (P<0.05).
The hepatoprotective effect was evaluated by the model of CCl4 induced acute hepatic injure in mice. EHPO could decrease the serum ALT, AST levels, the concentration of MDA and increase SOD activity in the mice treated with CCl4. It exhibited significant hepatoprotective effect.
In summary, the results showed that swertiamarin and swertianolin could be transformed by Aspergillus niger. The technology we set could separate the precurosor compounds and the product. The compound of EHPO yielded by co-incubated swertiamarin with Aspergillus niger could be absorbed soon. And it has the activity of antioxidative, hepatoprotective effect.
引文
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