泄浊除痹方治疗高尿酸血症物质基础研究
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摘要
中药复方物质基础研究是中药现代化和国际化的前提和基础,是目前亟待解决的中药(复方)现代化关键问题之一。中药(复方)现代化的进程中,首先要揭示中药(复方)发挥作用的药效物质基础。中药复方物质基础是中药复方中发挥药理学治疗作用的化学成分的集合。目前中药复方物质基础研究方法包括中药化学与药效学结合的方法、血清药物化学和血清药理学结合的方法、细胞膜色谱法等。
本研究采用中药化学与药效学结合的方法对中药泄浊除痹方治疗高尿酸血症的物质基础进行了初步探讨：采用现代液相分析技术,对泄浊除痹方中活性成分进行了含量测定；采用色谱技术对泄浊除痹方含量较高的总黄酮部位进行了分离纯化研究,并系统研究了泄浊除痹方总黄酮部位体外对小鼠肾脏近曲小管上皮细胞(RTECs)增值、尿酸吸收及小鼠尿酸转运蛋白(RST)基因的负性调控,初步证实泄浊除痹方总黄酮为其治疗高尿酸血症的有效部位之一。
本研究主要包括以下研究内容：
1、泄浊除痹方中活性物质含量测定
本研究采用HPLC法测定泄浊除痹方中薯蓣皂苷元、蜕皮甾酮、落新妇苷的含量。Agilent HC-18柱(250 mm×4.6 mm,5μm)；以乙腈-甲醇(2：3)为流动相,流速为1mL-min"1,检测波长为206 nm,测定方中薯蓣皂苷元的含量,回收率为104.51%；AgilentTC-18柱(250 mm×4.6 mm,5μm),以乙腈-水(17：83)为流动相,流速为1mL-min-1,检测波长为243 nm,测定方中蜕皮甾酮的含量,回收率为93.94%；KromasilC-18柱(250 mm×4.6 mm,5μm),以乙腈-磷酸-水(20：0.025：80)为流动相,检测波长为290 nm,测定方中落新妇苷的含量,回收率为99.46%；以上含量测定方法经方法学验证,分析快速、准确、灵敏度高、重现性好。
2、泄浊除痹方总黄酮有效部位纯化分离研究
本研究以黄酮类成分为研究指标,完善补充了君药土茯苓的定性、定量方法,采用薄层色谱法进行了落新妇苷的鉴别,高效液相法(HPLC)建立了落新妇苷、黄杞苷的含量测定方法；采用高效液相法测定了提取溶液中落新妇苷含量,比色法测定了提取溶液中总黄酮含量,以正交试验进行了土茯苓总黄酮有效部位的提取工艺优化,考察了乙醇浓度、提取温度、固液比、提取次数等因素,确定提取工艺为以10倍量60%乙醇,沸水浴回流提取2次,经验证,总黄酮提取率可达95.99%。以土茯苓总黄酮、落新妇苷、黄杞苷含量为指标,通过考察静态、动态吸附试验,筛选了聚酰胺颗粒分离纯化土茯苓及泄浊除痹方总黄酮有效部位的科学合理的工艺条件,聚酰胺富集物中总黄酮含量为75.63%,转移率为68.24%,落新妇苷含量为12.77%,转移率为73.05%。
3、泄浊除痹方化学成分分离纯化及结构研究
本研究采用系统溶剂法、硅胶柱色谱法、聚酰胺柱色谱法等分离纯化技术分离泄浊除痹方化学成分；利用理化性质、红外吸收光谱、核磁共振碳谱与氢谱、质谱等鉴别化合物结构,并与相关文献对照,确认化合物。从泄浊除痹方中分离出7个化学成分,其中六个已鉴定结构,分别是p-谷甾醇(晶Ⅰ,β-Sitosterol)、齐墩果酸(晶Ⅱ,Oleanolic acid)、熊果酸(晶Ⅲ,Ursolic acid)、落新妇苷(晶Ⅳ, Astilbin)、黄杞苷(晶V, Engelitin)及p-蜕皮甾酮(晶Ⅵ,β-Ecdysterone)。
4、泄浊除痹方总黄酮降尿酸机理研究
进行了小鼠肾脏近曲小管上皮细胞原代培养条件优化及功能测试研究：经机械破碎的肾脏皮质组织在400U胶原酶Ⅰ处理20 min时,肾小管节段贴壁率达到最高,为54.5%；首次换液最佳时间为72 h；细胞在原代培养第4-7天生长达到对数期,在第10天状态开始下降。在1500μmol-L-1尿酸浓度的吸收培养基中,吸收实验进行30 min时,其对尿酸吸收速率达到最大。阳性药物丙磺舒和苯溴马隆在不同的浓度下,对尿酸吸收的抑制率不同,苯溴马隆的抑制率高于丙磺舒。
泄浊除痹方总黄酮对小鼠肾脏近曲小管上皮细胞增殖及尿酸吸收的影响：将原代培养的小鼠肾脏近曲小管上皮细胞用不同浓度泄浊除痹方总黄酮孵育48h,四甲基偶氮唑蓝(MTT)法检测细胞活性；进行尿酸吸收实验,采用尿酸检测试剂盒测定尿酸吸收值。结果显示：高浓度组总黄酮(10、7.5、5 g·L-1)对细胞生长有不同程度的抑制作用,低浓度组药物(2.5、0.5 g·L-1)对细胞生长活性无显著影响；在对细胞生长活性无显著影响的低浓度梯度范围内(2.5、2、1.5、1.0、0.5 g·L-1),药物对尿酸吸收表现出不同程度的抑制作用。该研究表明泄浊除痹方总黄酮对小鼠肾脏近曲小管上皮细胞尿酸吸收功能有一定的抑制作用。
泄浊除痹方总黄酮对RST基因的负性调控：利用分子生物学实验手段检测不同药物作用浓度下,细胞中RST蛋白的基因扩增。结果显示：泄浊除痹方总黄酮在1-2.5 g·L-1浓度范围内对尿酸吸收有显著抑制,且RST基因扩增明显下调。该研究表明泄浊除痹方有能够通过下调RST基因扩增,降低RTECs尿酸吸收作用。
Study on material basis for efficacy of Chinese herbal formula is the precondition and base of modernization and internationalization of Chinese materia medica, also is one of the problems demanded to be settled urgently of this course. In this course, it is the most important thing to open out material basis for efficacy of Chinese herbal formula. Material basis for efficacy of Chinese herbal formula is an aggregate of all effective components. At present, the methods to study on material basis for efficacy of Chinese herbal formula include combining Chinese medicinal chemistry and pharmacodynamics together, pharmaceutical chemistry and pharmacyology in serum, cell membrane chromatography, and also.
This research investigates the substance foundation of Xiezhuo Chubi Fang (XZCBF) for hyperuricemia using the first method:contents of active elements of XZCBF were determined by modern analyzing technology (HPLC); Total flavonoids in XZCBF were isolated and purified by chromatography, its effects on renal tubular epithelial cells' (RTECs) proliferation, uric uptake and RST gene expression were investigated systematically, these experimentation results show that Total flavonoids in XZCBF were one of the effective part to treat hyperuricemia.
The research include follow contents:
1. Contents determination of active elements of XZCBF by HPLC
Contents of diosgenin, ecdysterone and astilbin in XZCBF were determined by HPLC:For diosgenin, with a agilent HC-18 column (250 mm×4.6 mm,5μm), acetonitrile:methanol (3:2) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 206 nm, the recovery of diosgenin was 104.51%; For ecdysterone, with a agilent TC-18 (250 mm×4.6 mm,5μm) column, acetonitrile: water (17:83) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 243 nm, the recovery of ecdysterone was 93.94%; For astilbin, with a KromasilC-18 (250 mm×4.6 mm,5μm) column, acetonitrile-phosphoric acid-water (20:0.025:80) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 290 nm, the recovery of astilbin was 99.46%. These methods are sensitive, accurate, reproducible, specific and can be used for quality control of XZCBF efficiently.
2. Separation and purification of total flavonoids in XZCBF
Quality and quantity analysis methods of Smilax Glabra Roxb.(principal drug) were established, astilbin in it was identified by TLC, contents of astilbin and engelitin in Smilax Glabra Roxb. were determined by HPLC simultaneously; extraction procedure of total flavonoids was optimized by using orthogonal experiment, four factors-alcohol concentration, temperature, dosage and extraction times-were investigated, content of total flavonoid was assessed by colorimetry, and astilbin was assessed by HPLC. The best extraction procedure is the medical material should be extracted by 10 times (V/W) 60% alcohol twice in boiling water bath, the extraction efficiency is as high as 95.99%. With the contents of total flavonoids, astilbin and engelitin as indexes, the optimum process of separating and purifying flavonoid was screened by static and dynamic adsorption tests. The content of total flavonoids was 75.63% in the extractive, the transfer rate was 68.24%; the content of astilbin was 12.77% in the extractive, the transfer rate was 73.05%.
3. Isolations and structure identifications of chemical constituents of XZCBF
In this research, chemical constituents of XZCBF were isolated with systematic solvent, silica gel-column chromatogram and polyamide-column chromatogram; the structures of chemical constituents were identified with dates of IR,1HNMR,13CNMR, physical-chemical characters, compared with related documents reported.7 chemical compounds were isolated in this research; among them six have been identified, they wereβ-Sitosterol (I), Oleanolic acid (II), Ursolic acid (Ⅲ), Astilbin (IV), Engelitin (V) andβ-Ecdysterone (VI).
4. Study of action mechanism of total flavonoids in XZCBF in treating hyperuricemia
Firstly, conditions of RTECs primary culture were optimized and cultures underwent functional tests. The adherent rate of segments of renal proximal tubules reached 54.5% after the digestion using 400U collagenase type I for 20 minutes; the most suitable time for the first change of the culture medium was 72 hours; Log phase began from the fifth day and the cell's growth conditions became unsuitable in the tenth day. In the transport medium contained 1500μmol-L-1 uric acid, the 30-min uptake of uric acid was the highest and was taken as a measure of an initial rate. Different uric acid-uptake inhibitory effects appeared due to different concentrations of the indicate drugs probenecid and benzbromarone. The inhibition influence of benzbromarone was higher than probenecid. The mouse renal proximal tubular epithelial cells cultured by the modified method will yield rich and homogeneous harvest, and present well function of uric acid uptake.
Next, effects of total flavonoids on cell proliferation and uric acid-uptake were investigated. The RTECs were isolated from the renal of mouse by the utilized method and were cultured with total flavonoids from XZCBF at different concentrations. The proliferation of RTECs was assessed by MTT assay. The uric uptake medium was prepared and the uric acid-uptake values were determined by the kit. The interest cells were isolated and it has the function of uric acid-uptake. The proliferation of the RTECs was inhibited by high concentrations (10,7.5,5 g-L-1) of the drug, while no significant effects on the proliferation of the RTECs were observed at the low concentrations (2.5,0.5 g-L-1). Moreover, uric acid-uptake tests were undertaken at this range of concentrations, and different inhibitory degrees by the drug were also determined.
Effects of total flavonoids on gene RST expression were detected to reveal its molecular mechanism. The uric acid-uptake essay was carried out on the mouse renal tubular epithelia cells and the different effects of different levels of total flavonoids in XZCBF on the uric acid uptake were studied. The expression of mouse renal-specific transporter (RST) in the cells was detected by RT-PCR. Results indicated that total flavonoid in XZCBF at the concentration range from 0.5 g-L-1 to 2.5 g-L-1 was proved to inhibit the uric acid-uptake function of RTECs and the expression of RST was lower under treatment at these concentrations. The uric acid-uptake can be reduced by total flavonoids in XZCBF by inhibiting the expression of RST.
本研究采用中药化学与药效学结合的方法对中药泄浊除痹方治疗高尿酸血症的物质基础进行了初步探讨：采用现代液相分析技术,对泄浊除痹方中活性成分进行了含量测定；采用色谱技术对泄浊除痹方含量较高的总黄酮部位进行了分离纯化研究,并系统研究了泄浊除痹方总黄酮部位体外对小鼠肾脏近曲小管上皮细胞(RTECs)增值、尿酸吸收及小鼠尿酸转运蛋白(RST)基因的负性调控,初步证实泄浊除痹方总黄酮为其治疗高尿酸血症的有效部位之一。
本研究主要包括以下研究内容：
1、泄浊除痹方中活性物质含量测定
本研究采用HPLC法测定泄浊除痹方中薯蓣皂苷元、蜕皮甾酮、落新妇苷的含量。Agilent HC-18柱(250 mm×4.6 mm,5μm)；以乙腈-甲醇(2：3)为流动相,流速为1mL-min"1,检测波长为206 nm,测定方中薯蓣皂苷元的含量,回收率为104.51%；AgilentTC-18柱(250 mm×4.6 mm,5μm),以乙腈-水(17：83)为流动相,流速为1mL-min-1,检测波长为243 nm,测定方中蜕皮甾酮的含量,回收率为93.94%；KromasilC-18柱(250 mm×4.6 mm,5μm),以乙腈-磷酸-水(20：0.025：80)为流动相,检测波长为290 nm,测定方中落新妇苷的含量,回收率为99.46%；以上含量测定方法经方法学验证,分析快速、准确、灵敏度高、重现性好。
2、泄浊除痹方总黄酮有效部位纯化分离研究
本研究以黄酮类成分为研究指标,完善补充了君药土茯苓的定性、定量方法,采用薄层色谱法进行了落新妇苷的鉴别,高效液相法(HPLC)建立了落新妇苷、黄杞苷的含量测定方法；采用高效液相法测定了提取溶液中落新妇苷含量,比色法测定了提取溶液中总黄酮含量,以正交试验进行了土茯苓总黄酮有效部位的提取工艺优化,考察了乙醇浓度、提取温度、固液比、提取次数等因素,确定提取工艺为以10倍量60%乙醇,沸水浴回流提取2次,经验证,总黄酮提取率可达95.99%。以土茯苓总黄酮、落新妇苷、黄杞苷含量为指标,通过考察静态、动态吸附试验,筛选了聚酰胺颗粒分离纯化土茯苓及泄浊除痹方总黄酮有效部位的科学合理的工艺条件,聚酰胺富集物中总黄酮含量为75.63%,转移率为68.24%,落新妇苷含量为12.77%,转移率为73.05%。
3、泄浊除痹方化学成分分离纯化及结构研究
本研究采用系统溶剂法、硅胶柱色谱法、聚酰胺柱色谱法等分离纯化技术分离泄浊除痹方化学成分；利用理化性质、红外吸收光谱、核磁共振碳谱与氢谱、质谱等鉴别化合物结构,并与相关文献对照,确认化合物。从泄浊除痹方中分离出7个化学成分,其中六个已鉴定结构,分别是p-谷甾醇(晶Ⅰ,β-Sitosterol)、齐墩果酸(晶Ⅱ,Oleanolic acid)、熊果酸(晶Ⅲ,Ursolic acid)、落新妇苷(晶Ⅳ, Astilbin)、黄杞苷(晶V, Engelitin)及p-蜕皮甾酮(晶Ⅵ,β-Ecdysterone)。
4、泄浊除痹方总黄酮降尿酸机理研究
进行了小鼠肾脏近曲小管上皮细胞原代培养条件优化及功能测试研究：经机械破碎的肾脏皮质组织在400U胶原酶Ⅰ处理20 min时,肾小管节段贴壁率达到最高,为54.5%；首次换液最佳时间为72 h；细胞在原代培养第4-7天生长达到对数期,在第10天状态开始下降。在1500μmol-L-1尿酸浓度的吸收培养基中,吸收实验进行30 min时,其对尿酸吸收速率达到最大。阳性药物丙磺舒和苯溴马隆在不同的浓度下,对尿酸吸收的抑制率不同,苯溴马隆的抑制率高于丙磺舒。
泄浊除痹方总黄酮对小鼠肾脏近曲小管上皮细胞增殖及尿酸吸收的影响：将原代培养的小鼠肾脏近曲小管上皮细胞用不同浓度泄浊除痹方总黄酮孵育48h,四甲基偶氮唑蓝(MTT)法检测细胞活性；进行尿酸吸收实验,采用尿酸检测试剂盒测定尿酸吸收值。结果显示：高浓度组总黄酮(10、7.5、5 g·L-1)对细胞生长有不同程度的抑制作用,低浓度组药物(2.5、0.5 g·L-1)对细胞生长活性无显著影响；在对细胞生长活性无显著影响的低浓度梯度范围内(2.5、2、1.5、1.0、0.5 g·L-1),药物对尿酸吸收表现出不同程度的抑制作用。该研究表明泄浊除痹方总黄酮对小鼠肾脏近曲小管上皮细胞尿酸吸收功能有一定的抑制作用。
泄浊除痹方总黄酮对RST基因的负性调控：利用分子生物学实验手段检测不同药物作用浓度下,细胞中RST蛋白的基因扩增。结果显示：泄浊除痹方总黄酮在1-2.5 g·L-1浓度范围内对尿酸吸收有显著抑制,且RST基因扩增明显下调。该研究表明泄浊除痹方有能够通过下调RST基因扩增,降低RTECs尿酸吸收作用。
Study on material basis for efficacy of Chinese herbal formula is the precondition and base of modernization and internationalization of Chinese materia medica, also is one of the problems demanded to be settled urgently of this course. In this course, it is the most important thing to open out material basis for efficacy of Chinese herbal formula. Material basis for efficacy of Chinese herbal formula is an aggregate of all effective components. At present, the methods to study on material basis for efficacy of Chinese herbal formula include combining Chinese medicinal chemistry and pharmacodynamics together, pharmaceutical chemistry and pharmacyology in serum, cell membrane chromatography, and also.
This research investigates the substance foundation of Xiezhuo Chubi Fang (XZCBF) for hyperuricemia using the first method:contents of active elements of XZCBF were determined by modern analyzing technology (HPLC); Total flavonoids in XZCBF were isolated and purified by chromatography, its effects on renal tubular epithelial cells' (RTECs) proliferation, uric uptake and RST gene expression were investigated systematically, these experimentation results show that Total flavonoids in XZCBF were one of the effective part to treat hyperuricemia.
The research include follow contents:
1. Contents determination of active elements of XZCBF by HPLC
Contents of diosgenin, ecdysterone and astilbin in XZCBF were determined by HPLC:For diosgenin, with a agilent HC-18 column (250 mm×4.6 mm,5μm), acetonitrile:methanol (3:2) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 206 nm, the recovery of diosgenin was 104.51%; For ecdysterone, with a agilent TC-18 (250 mm×4.6 mm,5μm) column, acetonitrile: water (17:83) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 243 nm, the recovery of ecdysterone was 93.94%; For astilbin, with a KromasilC-18 (250 mm×4.6 mm,5μm) column, acetonitrile-phosphoric acid-water (20:0.025:80) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 290 nm, the recovery of astilbin was 99.46%. These methods are sensitive, accurate, reproducible, specific and can be used for quality control of XZCBF efficiently.
2. Separation and purification of total flavonoids in XZCBF
Quality and quantity analysis methods of Smilax Glabra Roxb.(principal drug) were established, astilbin in it was identified by TLC, contents of astilbin and engelitin in Smilax Glabra Roxb. were determined by HPLC simultaneously; extraction procedure of total flavonoids was optimized by using orthogonal experiment, four factors-alcohol concentration, temperature, dosage and extraction times-were investigated, content of total flavonoid was assessed by colorimetry, and astilbin was assessed by HPLC. The best extraction procedure is the medical material should be extracted by 10 times (V/W) 60% alcohol twice in boiling water bath, the extraction efficiency is as high as 95.99%. With the contents of total flavonoids, astilbin and engelitin as indexes, the optimum process of separating and purifying flavonoid was screened by static and dynamic adsorption tests. The content of total flavonoids was 75.63% in the extractive, the transfer rate was 68.24%; the content of astilbin was 12.77% in the extractive, the transfer rate was 73.05%.
3. Isolations and structure identifications of chemical constituents of XZCBF
In this research, chemical constituents of XZCBF were isolated with systematic solvent, silica gel-column chromatogram and polyamide-column chromatogram; the structures of chemical constituents were identified with dates of IR,1HNMR,13CNMR, physical-chemical characters, compared with related documents reported.7 chemical compounds were isolated in this research; among them six have been identified, they wereβ-Sitosterol (I), Oleanolic acid (II), Ursolic acid (Ⅲ), Astilbin (IV), Engelitin (V) andβ-Ecdysterone (VI).
4. Study of action mechanism of total flavonoids in XZCBF in treating hyperuricemia
Firstly, conditions of RTECs primary culture were optimized and cultures underwent functional tests. The adherent rate of segments of renal proximal tubules reached 54.5% after the digestion using 400U collagenase type I for 20 minutes; the most suitable time for the first change of the culture medium was 72 hours; Log phase began from the fifth day and the cell's growth conditions became unsuitable in the tenth day. In the transport medium contained 1500μmol-L-1 uric acid, the 30-min uptake of uric acid was the highest and was taken as a measure of an initial rate. Different uric acid-uptake inhibitory effects appeared due to different concentrations of the indicate drugs probenecid and benzbromarone. The inhibition influence of benzbromarone was higher than probenecid. The mouse renal proximal tubular epithelial cells cultured by the modified method will yield rich and homogeneous harvest, and present well function of uric acid uptake.
Next, effects of total flavonoids on cell proliferation and uric acid-uptake were investigated. The RTECs were isolated from the renal of mouse by the utilized method and were cultured with total flavonoids from XZCBF at different concentrations. The proliferation of RTECs was assessed by MTT assay. The uric uptake medium was prepared and the uric acid-uptake values were determined by the kit. The interest cells were isolated and it has the function of uric acid-uptake. The proliferation of the RTECs was inhibited by high concentrations (10,7.5,5 g-L-1) of the drug, while no significant effects on the proliferation of the RTECs were observed at the low concentrations (2.5,0.5 g-L-1). Moreover, uric acid-uptake tests were undertaken at this range of concentrations, and different inhibitory degrees by the drug were also determined.
Effects of total flavonoids on gene RST expression were detected to reveal its molecular mechanism. The uric acid-uptake essay was carried out on the mouse renal tubular epithelia cells and the different effects of different levels of total flavonoids in XZCBF on the uric acid uptake were studied. The expression of mouse renal-specific transporter (RST) in the cells was detected by RT-PCR. Results indicated that total flavonoid in XZCBF at the concentration range from 0.5 g-L-1 to 2.5 g-L-1 was proved to inhibit the uric acid-uptake function of RTECs and the expression of RST was lower under treatment at these concentrations. The uric acid-uptake can be reduced by total flavonoids in XZCBF by inhibiting the expression of RST.
引文
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