西青果治疗感染性创面的有效组分及作用机制研究
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摘要
目的
本文选择藏药西青果为主要研究对象，以传统中医药理论和现代医药理论为基础，采用现代分离分析技术，利用体外中药有效组分筛选方法，首次对西青果有效组分进行提取、分离和纯化，确定相关最佳工艺；首次结合体内外药效学试验，对筛选出的最佳有效组分进行抗菌、抗氧化和急性毒性实验，并首次对最佳有效组分的作用机理进行深入研究，从而为西青果的开发提供理论和实验依据。
研究方法：
1.采用现代生药鉴别技术，对实验所用的西青果的原植物形态、药材性状和显微特征进行了研究，从而保证后续实验用药的准确和一致性。
2.采用平皿挖沟灌药法，以西青果不同提取组分为研究对象，选择金黄色葡萄球菌和肺炎克雷伯杆菌，分别观察不同组分抑制细菌生长情况的影响；根据血清药理学方法检测西青果有效组分总鞣质含药血清对两种菌的抑制作用。
3．首次采用改良的干酪素法，考察西青果中主要抗菌有效组分总鞣质的含量；通过单因素考察和L49(3)正交试验，对西青果总鞣质的提取工艺进行优选研究，确定了其最佳提取条件。
4.参照中药新药指南和药典规定，首次采用UV法和HPLC法，分别考察总鞣质含量和没食子酸含量，通过静态实验筛选出大孔吸附树脂类型，再以动态实验确定大孔吸附树脂对总鞣质的最佳分离纯化条件。
5.采用柱色谱和半制备色谱等分离方法，从最佳有效组分中分离得到单体化合物，通过理化和波谱解析等方法确定了它们的化学结构。
6.首次采用HPLC法，对最佳有效组分中的没食子酸和没食子酸乙酯进行含量测定。
7．首次从抗菌、抗氧化、急毒和促进创面愈合4个方面对有效组分及没食子酸乙酯治疗感染性创面及相关机制进行研究。在抗菌方面，分别考察了有效组分对两种菌的MIC、MBC及杀菌曲线，采用扫描和透射电镜对不同时间段对菌的影响进行观察，从机制上探讨其抑菌作用；在抗氧化方面，采用ABTS法、FRAP法和DPPH法分别考察了有效组分的体外抗氧化作用；在急毒方面，观察了有效组分对小鼠的急毒及肝病理切片；在创面愈合实验中，分别对有效组分对大鼠创面的愈合情况，病理切片，VEGF和β-FGFmRNA表达及免疫组化实验进行了系统的研究。
结果
1.通过对西青果的原植物形态、药材性状和显微特征进行了研究，确保使用的西青果为使君子科植物诃子Terminalia chebula Retz.的干燥幼果。
2.采用平皿挖沟灌药法和血清药理学方法，发现西青果总鞣质提取物及含药血清对两种菌均有明显的抑制作用，同时确定总鞣质为其有效组分。
3．未提取纯化的西青果中主要抗菌有效组分总鞣质的含量仅为9.57%左右；对西青果总鞣质的提取工艺进行优选研究，其鞣质含量达到30%左右，并确定了其最佳提取条件为：15倍量水溶剂，浸泡20min，提取3次，每次1h。
4. UV法中对总鞣质的最佳分离纯化条件确定为：选用AB-8树脂，树脂柱径高比为1：1.5，洗脱剂为50%乙醇，洗脱速度为1ml/min为最佳洗脱条件；HPLC法中，最佳分离纯化条件确定为：洗脱剂为50%乙醇，选用HPD-400树脂，上样体积为40mL。
5.从最佳提取纯化工艺制备的有效组分中分离得到13个化合物，鉴定了11个化合物，其中9个酚酸类及衍生物，1个三萜类化合物。并确定了它们的化学结构，其中诃子次酸三正丁酯为一个未见报道的新化合物，其它均为西青果中首次分离得到。
6.利用HPLC法测定最佳有效组分中的没食子酸和没食子酸乙酯含量分别为0.2%和0.392%。
7．从抗菌、抗氧化、急毒和促进创面愈合4个方面对有效组分治疗感染性创面及相关机制进行研究。发现有效组分及没食子酸乙酯对两种菌具有明显的抑制作用，而且抗氧化作用明显，并且体内急毒实验证明其毒性小，安全性高；对感染性创面作用及相关机制研究中发现有效组分具有明显的促进创面愈合作用。
结论
1．西青果有效组分总鞣质的最佳提取纯化工艺稳定、可行，适合进一步的中试及放大实验。
2．首次对有效组分中的化学成分进行分离和结构鉴定，并对单一活性成分进行含量测定，为该药的开发提供相关的物质基础
3．体内外药效实验证明西青果有效组分具有明显的抗菌、抗氧化和促进创面愈合作用，并且毒性小，安全性高的特点。
4．首次利用电镜技术对其对抗菌机制进行研究，并通过免疫组化等生物技术方法对西青果有效组分促进创面愈合的作用机制进行深入探讨。
Objective
In this study, based on traditional Chinese and modern medicine theory,effective compositionof chebulae fructus immaturus was extracted、seperated and purified in order to select theoptimal processe for the first time by using modern separation&analysis technology andscreening of effective composition in vitro method; With the combination of vivo and vitropharmacology, the antibacterial test、 anti-oxidation test and acute toxicity test wereundertaken to the resulting effective composition whose mechanism was assessed for the fisrttime, which can be provided as theoretical and experimental basis for the future research ofChebula fructus immaturus.
Methods
1. Using modern pharmacognostical identification method, we check the plantmorphology、 macroscopic and microscopic characteristics of the chebulae fructusimmaturus used in the study to confirm the same source of the chebulae fructusimmaturus.
2. We collected extracts from chebulae fructus immaturus and selected staphylococcusaureus and klebsiella pneumonia, to observe the effects of different compositions onbacteria growth by plating-digging-perfusion method; the inhibition effect of serumcontainning effective composition (total tannin)on two bacteria growth was tested basedon serum pharmacology.
3. For the first time, we used improved Casein method to determine the total tannins ofantibacterial effective composition; we sort out the optimal extract process and conditionswith the application of single factor exploration and L9(34)orthogonal test.
4. For the fisrt time, we determined the total tannin and gallic acid content by UV andHPLC method according to Chinese new drug guideline and Pharmacopoeia, and usedstatic test to sort out the macro porous adsorption resin type, and dynamic test todetermine the optimal separation and purification conditions of the macro porousadsorption resin for total tannin.
5. We used column chromatography and half-prepared chromatography to separatemonocase compounds from effective composition which had been extracted by optimalprocess, and determine the chemical structure by physical and chemical method andspectral analysis etc.
6. We tested the gallic acid and ethyl gallate content of effective composition by HPLCmethod for the first time.
7．For the first time,we evaluated effective composition’s treatment for infectious wound and its working mechanism from4aspects: antibacterial, anti-oxidation, acute toxicity andwound healing. As for antibacterial, effective composition’s MIC, MBC and killing curveof two bacterias were surveyed. And also effective composition’s effects on bacteriagrowth at different times were explored by scan and transmission electron microscope toevaluate the antibacterial effect from mechanism point of view; As for anti-oxidationactivities, ABT, FRAP and DPPH were used to evaluate effective composition’santi-oxidation activities in vitro; As for acute toxicity, acute toxicity test and pathologicalsection were conducted to mice; during wound healing experiment, a systematic studywas conducted which included effective composition’s treatment for the mice woundhealing, mice pathological section, VEGF and β-FGFmRNA expression andimmunohistochemistry.
Results
1. Through the check of the plant morphology, macroscopic and microscopic characteristics,the chebulae fructus immaturus used in the study were identified to be dry immature fruitof Terminalia chebula Retz.
2. By plating-digging-perfusion method and serum pharmacology method, it was found thattatol tannin and serum both had antibacterial effect on two bacterias, and also proved thattotal tannin was the effective composition.
3. Before extraction, total tannin content of main antibacterial effective composition inchebulae fructus immaturus was only9.57%; however, produced from selected optimalextraction processes, the tannin content can reach up to30%, and the optimal extractionconditions are:15times amount aqueous solvent, soaking for20min,and extract in3times, each for1h.
4. For UV method which was used for the separation and purification of total tannin, thebest conditions were as followings: AB-8resin, resin balde diameter: length: ratio is1:1.5,eluent is50%ethonal, elute speed is1ml/min.For HPLC method, the best conditions areas followings: eluent is50%ethonal, use HPD resin, sample volume is40ml.
5.13compounds were extracted from effective composition by optimal separation andpurification process. And11of them were identified and chemical structures determined:9phenolic acids,1triterpenes compound. And one of them has never been reported before,and the rest were firstly obtained in the chebulae fructus immaturus separation.
6. The gallic acid and ethyl gallate content of effective composition produced from optimalprocess were determined by HPLC to be0.2%and0.392%.
7. Effective composition’s and ethyl gallate’s treatment for wound healing and relativemechanism were evaluated from4aspects: antibacterial, anti-oxidation, acute toxicityand wound healing. It was found that effective composition and ethyl gallate both hadinhibition effects on bacteria growth, and the anti-oxidation effects were obvious, and proved to be minor toxic and highly safe by acute toxicity test in vitro; And the effectivecomposition were proved to have wound healing effect as a result of the evaluationmentioned above.
Conclusions
1．The optimal extraction process of effective composition (total tannin) from Chebulaefructus immaturus is stable, feasible and suitable for pilot and scale-up experiment.
2．The chemical composition of the effective composition was fisrtly separated and itschemical structure determined. Single active composition was tested, which provides amaterial basis for the further research.
3．Pharmacology in vivo and vitro proved that the effective composition of chebulae fructusimmaturus had the characteristic of antibacterial, anti-oxidation, wound healing, minortoxic and high-safety.
4．For the first time, electron microscope was used to evaluate the antibaterial mechanism.With the application of immunohistochemistry, working mechanism of the effectivecomposition’s treatment for wound healing was insightly assessed.
本文选择藏药西青果为主要研究对象，以传统中医药理论和现代医药理论为基础，采用现代分离分析技术，利用体外中药有效组分筛选方法，首次对西青果有效组分进行提取、分离和纯化，确定相关最佳工艺；首次结合体内外药效学试验，对筛选出的最佳有效组分进行抗菌、抗氧化和急性毒性实验，并首次对最佳有效组分的作用机理进行深入研究，从而为西青果的开发提供理论和实验依据。
研究方法：
1.采用现代生药鉴别技术，对实验所用的西青果的原植物形态、药材性状和显微特征进行了研究，从而保证后续实验用药的准确和一致性。
2.采用平皿挖沟灌药法，以西青果不同提取组分为研究对象，选择金黄色葡萄球菌和肺炎克雷伯杆菌，分别观察不同组分抑制细菌生长情况的影响；根据血清药理学方法检测西青果有效组分总鞣质含药血清对两种菌的抑制作用。
3．首次采用改良的干酪素法，考察西青果中主要抗菌有效组分总鞣质的含量；通过单因素考察和L49(3)正交试验，对西青果总鞣质的提取工艺进行优选研究，确定了其最佳提取条件。
4.参照中药新药指南和药典规定，首次采用UV法和HPLC法，分别考察总鞣质含量和没食子酸含量，通过静态实验筛选出大孔吸附树脂类型，再以动态实验确定大孔吸附树脂对总鞣质的最佳分离纯化条件。
5.采用柱色谱和半制备色谱等分离方法，从最佳有效组分中分离得到单体化合物，通过理化和波谱解析等方法确定了它们的化学结构。
6.首次采用HPLC法，对最佳有效组分中的没食子酸和没食子酸乙酯进行含量测定。
7．首次从抗菌、抗氧化、急毒和促进创面愈合4个方面对有效组分及没食子酸乙酯治疗感染性创面及相关机制进行研究。在抗菌方面，分别考察了有效组分对两种菌的MIC、MBC及杀菌曲线，采用扫描和透射电镜对不同时间段对菌的影响进行观察，从机制上探讨其抑菌作用；在抗氧化方面，采用ABTS法、FRAP法和DPPH法分别考察了有效组分的体外抗氧化作用；在急毒方面，观察了有效组分对小鼠的急毒及肝病理切片；在创面愈合实验中，分别对有效组分对大鼠创面的愈合情况，病理切片，VEGF和β-FGFmRNA表达及免疫组化实验进行了系统的研究。
结果
1.通过对西青果的原植物形态、药材性状和显微特征进行了研究，确保使用的西青果为使君子科植物诃子Terminalia chebula Retz.的干燥幼果。
2.采用平皿挖沟灌药法和血清药理学方法，发现西青果总鞣质提取物及含药血清对两种菌均有明显的抑制作用，同时确定总鞣质为其有效组分。
3．未提取纯化的西青果中主要抗菌有效组分总鞣质的含量仅为9.57%左右；对西青果总鞣质的提取工艺进行优选研究，其鞣质含量达到30%左右，并确定了其最佳提取条件为：15倍量水溶剂，浸泡20min，提取3次，每次1h。
4. UV法中对总鞣质的最佳分离纯化条件确定为：选用AB-8树脂，树脂柱径高比为1：1.5，洗脱剂为50%乙醇，洗脱速度为1ml/min为最佳洗脱条件；HPLC法中，最佳分离纯化条件确定为：洗脱剂为50%乙醇，选用HPD-400树脂，上样体积为40mL。
5.从最佳提取纯化工艺制备的有效组分中分离得到13个化合物，鉴定了11个化合物，其中9个酚酸类及衍生物，1个三萜类化合物。并确定了它们的化学结构，其中诃子次酸三正丁酯为一个未见报道的新化合物，其它均为西青果中首次分离得到。
6.利用HPLC法测定最佳有效组分中的没食子酸和没食子酸乙酯含量分别为0.2%和0.392%。
7．从抗菌、抗氧化、急毒和促进创面愈合4个方面对有效组分治疗感染性创面及相关机制进行研究。发现有效组分及没食子酸乙酯对两种菌具有明显的抑制作用，而且抗氧化作用明显，并且体内急毒实验证明其毒性小，安全性高；对感染性创面作用及相关机制研究中发现有效组分具有明显的促进创面愈合作用。
结论
1．西青果有效组分总鞣质的最佳提取纯化工艺稳定、可行，适合进一步的中试及放大实验。
2．首次对有效组分中的化学成分进行分离和结构鉴定，并对单一活性成分进行含量测定，为该药的开发提供相关的物质基础
3．体内外药效实验证明西青果有效组分具有明显的抗菌、抗氧化和促进创面愈合作用，并且毒性小，安全性高的特点。
4．首次利用电镜技术对其对抗菌机制进行研究，并通过免疫组化等生物技术方法对西青果有效组分促进创面愈合的作用机制进行深入探讨。
Objective
In this study, based on traditional Chinese and modern medicine theory,effective compositionof chebulae fructus immaturus was extracted、seperated and purified in order to select theoptimal processe for the first time by using modern separation&analysis technology andscreening of effective composition in vitro method; With the combination of vivo and vitropharmacology, the antibacterial test、 anti-oxidation test and acute toxicity test wereundertaken to the resulting effective composition whose mechanism was assessed for the fisrttime, which can be provided as theoretical and experimental basis for the future research ofChebula fructus immaturus.
Methods
1. Using modern pharmacognostical identification method, we check the plantmorphology、 macroscopic and microscopic characteristics of the chebulae fructusimmaturus used in the study to confirm the same source of the chebulae fructusimmaturus.
2. We collected extracts from chebulae fructus immaturus and selected staphylococcusaureus and klebsiella pneumonia, to observe the effects of different compositions onbacteria growth by plating-digging-perfusion method; the inhibition effect of serumcontainning effective composition (total tannin)on two bacteria growth was tested basedon serum pharmacology.
3. For the first time, we used improved Casein method to determine the total tannins ofantibacterial effective composition; we sort out the optimal extract process and conditionswith the application of single factor exploration and L9(34)orthogonal test.
4. For the fisrt time, we determined the total tannin and gallic acid content by UV andHPLC method according to Chinese new drug guideline and Pharmacopoeia, and usedstatic test to sort out the macro porous adsorption resin type, and dynamic test todetermine the optimal separation and purification conditions of the macro porousadsorption resin for total tannin.
5. We used column chromatography and half-prepared chromatography to separatemonocase compounds from effective composition which had been extracted by optimalprocess, and determine the chemical structure by physical and chemical method andspectral analysis etc.
6. We tested the gallic acid and ethyl gallate content of effective composition by HPLCmethod for the first time.
7．For the first time,we evaluated effective composition’s treatment for infectious wound and its working mechanism from4aspects: antibacterial, anti-oxidation, acute toxicity andwound healing. As for antibacterial, effective composition’s MIC, MBC and killing curveof two bacterias were surveyed. And also effective composition’s effects on bacteriagrowth at different times were explored by scan and transmission electron microscope toevaluate the antibacterial effect from mechanism point of view; As for anti-oxidationactivities, ABT, FRAP and DPPH were used to evaluate effective composition’santi-oxidation activities in vitro; As for acute toxicity, acute toxicity test and pathologicalsection were conducted to mice; during wound healing experiment, a systematic studywas conducted which included effective composition’s treatment for the mice woundhealing, mice pathological section, VEGF and β-FGFmRNA expression andimmunohistochemistry.
Results
1. Through the check of the plant morphology, macroscopic and microscopic characteristics,the chebulae fructus immaturus used in the study were identified to be dry immature fruitof Terminalia chebula Retz.
2. By plating-digging-perfusion method and serum pharmacology method, it was found thattatol tannin and serum both had antibacterial effect on two bacterias, and also proved thattotal tannin was the effective composition.
3. Before extraction, total tannin content of main antibacterial effective composition inchebulae fructus immaturus was only9.57%; however, produced from selected optimalextraction processes, the tannin content can reach up to30%, and the optimal extractionconditions are:15times amount aqueous solvent, soaking for20min,and extract in3times, each for1h.
4. For UV method which was used for the separation and purification of total tannin, thebest conditions were as followings: AB-8resin, resin balde diameter: length: ratio is1:1.5,eluent is50%ethonal, elute speed is1ml/min.For HPLC method, the best conditions areas followings: eluent is50%ethonal, use HPD resin, sample volume is40ml.
5.13compounds were extracted from effective composition by optimal separation andpurification process. And11of them were identified and chemical structures determined:9phenolic acids,1triterpenes compound. And one of them has never been reported before,and the rest were firstly obtained in the chebulae fructus immaturus separation.
6. The gallic acid and ethyl gallate content of effective composition produced from optimalprocess were determined by HPLC to be0.2%and0.392%.
7. Effective composition’s and ethyl gallate’s treatment for wound healing and relativemechanism were evaluated from4aspects: antibacterial, anti-oxidation, acute toxicityand wound healing. It was found that effective composition and ethyl gallate both hadinhibition effects on bacteria growth, and the anti-oxidation effects were obvious, and proved to be minor toxic and highly safe by acute toxicity test in vitro; And the effectivecomposition were proved to have wound healing effect as a result of the evaluationmentioned above.
Conclusions
1．The optimal extraction process of effective composition (total tannin) from Chebulaefructus immaturus is stable, feasible and suitable for pilot and scale-up experiment.
2．The chemical composition of the effective composition was fisrtly separated and itschemical structure determined. Single active composition was tested, which provides amaterial basis for the further research.
3．Pharmacology in vivo and vitro proved that the effective composition of chebulae fructusimmaturus had the characteristic of antibacterial, anti-oxidation, wound healing, minortoxic and high-safety.
4．For the first time, electron microscope was used to evaluate the antibaterial mechanism.With the application of immunohistochemistry, working mechanism of the effectivecomposition’s treatment for wound healing was insightly assessed.
引文
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