桃叶珊瑚苷及其衍生物的分子结构与药理活性研究
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摘要
杜仲籽中主要的环烯醚萜苷类成分为桃叶珊瑚苷(AU),而AU的生物活性大多是通过其苷元(AUG)来实现的。本课题以杜仲籽中的AU为研究对象,从分子结构、结构修饰、抗氧化、抗菌、药物代谢、毒性和药效等方面展开系统研究,为开发利用、寻找新药奠定实验基础。
一、AU、AUG和异杜仲醇(ISE)的分子结构研究
本实验从杜仲籽中提取分离AU,通过p-葡萄糖苷酶37℃水解AU获得AUG,再用NaBH4还原AUG获得ISE,并用UV, IR,1HNMR,13CNMR,MS等波谱学方法鉴定结构,用单晶X射线衍射分析阐明这三种晶体的绝对构型和立体空间结构。晶体学研究发现AU属于正交晶系,P212121空间群；AUG属于单斜晶系,P21空间群；ISE属于三斜晶系,P1空间群。AU、AUG和ISE晶体均广泛存在分子间作用力,这为研究药物分子的结构-效应关系奠定了理论基础。
二、AU和AUG的抗氧化、抗菌活性研究
选用DPPH法、邻苯三酚自氧化法和Fenton反应法三种测定方法,用紫外分光光度计对AU和AUG清除DPPH自由基、超氧阴离子自由基和羟基自由基的能力进行初步探讨。选用大肠杆菌,铜绿假单胞菌,金黄色葡萄球菌,沙门氏菌四种标准菌株,用滤纸片扩散法和微量稀释法相结合来测定AU和AUG的抗菌活性。结果发现AU具有清除超氧阴离子和羟基自由基的活性,而AUG具有清除DPPH自由基和羟基自由基的活性。抗菌实验发现AU对四种标准菌株均无抑制作用,而AUG均显示出了一定的抗菌活性,尤其对金黄色葡萄球菌的抗菌活性最强,是一种潜在的植物抗生素。
三、AU的药代动力学研究
本实验建立了一种快速、灵敏的测定兔血中AU浓度的HPLC法,初步研究了AU在家兔体内的药代动力学行为。AU以500 mg·kg-1灌胃和100 mg·kg-1静脉注射两种给药途径对家兔进行药代动力学实验。结果发现,AU经灌胃和静脉给药后分布迅速,药物代谢过程符合二室模型,这为进一步研究AU的药理作用及体内代谢过程奠定了实验基础。
四、AU的急性毒性实验和长期毒性研究
AU急性毒性实验结果表明,小鼠灌胃AU的最大耐受量为40 g·kg-1。对大鼠进行6个月的AU长期毒性实验显示,大鼠的体重、毛发、活动、饮食等一般状况无明显异常；各组大鼠的血液学和血液生化学指标均在正常生理值范围内；大鼠的心、肝、脾、肺、肾、胃、肠等脏器和组织的病理学检查结果未显示有与给药有关的病理改变；在6个月给药期内未发现大鼠有死亡等其它毒性反应出现；在恢复期内大鼠的各项观察指标均无异常,无慢性毒性或迟发性毒性作用。
五、AU对正常大鼠的骨微结构和骨力学研究
AU长期毒性实验后,取大鼠两侧股骨,用Micro-CT分析股骨干骺端的骨小梁微结构,通过三点弯曲力学实验分析股骨生物力学性能。实验结果显示,三个给药组与对照组相比,均不同程度的提高了骨密度、相对骨体积、骨小梁数量、骨小梁厚度和连接密度指数,降低了骨小梁分离度和结构模型指数,从整体上优化了骨小梁的微体系结构。三点弯曲力学实验发现,各给药组均显著提高了骨折力,增加了骨强度。本实验首次发现AU能增加正常大鼠股骨的骨密度和骨强度,是一种潜在的治疗骨质疏松的新药。
Aucubin (AU), which is the main iridoid component from the seeds of Eucommia ulmoides Oliv., has been found to have various pharmacological activities such as high liver-protective activity, antiviral activity, antitumor, anti-inflammatory activity and so on. These actions require the presence of aβ-glucosidase to hydrolyse aucubin to its aglycone, aucubigenin (AUG).
In our studies, the chemistry and efficacy of AU and its derivatives were investigated, including molecular structure, chemical synthesis, antioxidant and antimicrobial activities, pharmacokinetics, acute toxicity and long-term toxicity. The detailed descriptions are listed below.
Part one:The crystallographic study on AU, AUG and isoeucommiol (ISE).
We obtained AU from the seeds of Eucommia ulmoides Oliv. by a procedure of extraction with ethanol. We also have improved the procedure for the extraction of AUG from theβ-glucosidase hydrolysis of AU at 37℃. ISE was obtained through NaBH4 reduction of AUG. The crystal structures of the three compounds were determined by a single-crystal X-ray diffraction analysis. It has been found that crystals of AU are orthorhombic, crystallize in the P212121 space group, and have four molecules in the crystal unit cell. While the crystals of AUG are monoclinic, space group P21, and have two molecules in the unit cell. The crystals of ISE adopts an envelope conformation, presenting a triclinic system, space group P1 with one molecule in the unit cell. Moreover, intensive strength O-H…O hydrogen bonds in all crystal lattices can be observed.
Part two:The bioactivities study on AU and AUG.
AU and its aglycone AUG have been investigated for their in vitro free radical scavenging and antibacterial activities. Various methods, such as the scavenging activities towards DPPH radical, superoxide anion radical, and hydroxyl radical, were established. The antibacterial activities of AU and AUG were also determined by the disc diffusion method and micro-well dilution assay with spectrophotometric method. AU and AUG were individually tested against Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Salmonella enterica (ATCC 14028), respectively. The results showed for the first time that the two compounds exhibited significant differences in free radical scavenging activities. AU showed little DPPH radical scavenging activity. However, a dose response relationship is found in DPPH scavenging activity of AUG. Both AU and AUG showed a moderate scavenging activity against superoxide anion radical. AUG appeared to have little scavenging activity on hydroxyl radical scavenging effects but AU exhibited moderate scavenging activity in a dose dependent manner. The results also indicated that AUG exhibited considerable antibacterial activity against all tested strains, and was particularly more effective against Staphylococcus aureus. As expected, AU had no inhibitory activity against the up growth of all tested strains.
Part three:The pharmacokinetic study on AU.
A simple and accurate high performance liquid chromatographic method was developed and validated for the determination of AU in rabbit plasma. For the experimental group, a dose of 500 mg·kg-1 oral administration of AU and 100 mg·kg-1 intravenous injection of AU were finished. The result showed that both drug concentration-time data were fitted to a two-compartment model with first order absorption. This method could determine the AU level in plasma and are suitable for its pharmacological study.
Part four:The toxicity study on AU.
The acute toxicology experiment study on AU showed that MTD (maximal tolerance dose) administration to mice was 40 g·kg-1. Results of 6 months' long-term toxicity studies indicate that AU has no effect on weight, hair, activity, diet and general condition of rats. Hematologic parameters and blood biochemical indices were within normal physiological range. Heart, liver, spleen, lung, kidney, stomach, intestines and other organs and tissues do not contain any visible pathological changes.Every organ has no change of histopathology. In the recovery period, the rats showed no significant chronic toxicity or delayed toxicity.
Part five:Effect of AU on bone microarchitecture and biomechanics in rats.
To our knowledge, the direct effect of AU on bone structure in vivo has not been investigated yet. Therefore we became interested to study the effect of AU on bone structural and biomechanical analysis in rats after 6 months'long-term toxicity studies. Then the rats were killed and the femurs were dissected for measurement of trabecular microarchitecture and biomechanical analysis. Micro-CT analysis of the distal femur showed that AU significantly increased in bone volume/tissue volume, connect density, trabecular number and trabecular thickness, and decrease in trabecular separation and structure model index in normal rats. In the three-point bending test, the maximum load (ultimate strength) was determined until the specimen was broken. Daily oral administration of AU was found to be able to significantly increase biomechanical quality of femur. The mechanical changes were associated with the bone mineral density increase or even with some improvements in microarchitecture. We demonstrated for the first time that AU taken orally increased bone density and bone strength of rats at the femur. It might be a potential alternative medicine to be used for the treatment of bone deficient diseases such as osteoporosis.
一、AU、AUG和异杜仲醇(ISE)的分子结构研究
本实验从杜仲籽中提取分离AU,通过p-葡萄糖苷酶37℃水解AU获得AUG,再用NaBH4还原AUG获得ISE,并用UV, IR,1HNMR,13CNMR,MS等波谱学方法鉴定结构,用单晶X射线衍射分析阐明这三种晶体的绝对构型和立体空间结构。晶体学研究发现AU属于正交晶系,P212121空间群；AUG属于单斜晶系,P21空间群；ISE属于三斜晶系,P1空间群。AU、AUG和ISE晶体均广泛存在分子间作用力,这为研究药物分子的结构-效应关系奠定了理论基础。
二、AU和AUG的抗氧化、抗菌活性研究
选用DPPH法、邻苯三酚自氧化法和Fenton反应法三种测定方法,用紫外分光光度计对AU和AUG清除DPPH自由基、超氧阴离子自由基和羟基自由基的能力进行初步探讨。选用大肠杆菌,铜绿假单胞菌,金黄色葡萄球菌,沙门氏菌四种标准菌株,用滤纸片扩散法和微量稀释法相结合来测定AU和AUG的抗菌活性。结果发现AU具有清除超氧阴离子和羟基自由基的活性,而AUG具有清除DPPH自由基和羟基自由基的活性。抗菌实验发现AU对四种标准菌株均无抑制作用,而AUG均显示出了一定的抗菌活性,尤其对金黄色葡萄球菌的抗菌活性最强,是一种潜在的植物抗生素。
三、AU的药代动力学研究
本实验建立了一种快速、灵敏的测定兔血中AU浓度的HPLC法,初步研究了AU在家兔体内的药代动力学行为。AU以500 mg·kg-1灌胃和100 mg·kg-1静脉注射两种给药途径对家兔进行药代动力学实验。结果发现,AU经灌胃和静脉给药后分布迅速,药物代谢过程符合二室模型,这为进一步研究AU的药理作用及体内代谢过程奠定了实验基础。
四、AU的急性毒性实验和长期毒性研究
AU急性毒性实验结果表明,小鼠灌胃AU的最大耐受量为40 g·kg-1。对大鼠进行6个月的AU长期毒性实验显示,大鼠的体重、毛发、活动、饮食等一般状况无明显异常；各组大鼠的血液学和血液生化学指标均在正常生理值范围内；大鼠的心、肝、脾、肺、肾、胃、肠等脏器和组织的病理学检查结果未显示有与给药有关的病理改变；在6个月给药期内未发现大鼠有死亡等其它毒性反应出现；在恢复期内大鼠的各项观察指标均无异常,无慢性毒性或迟发性毒性作用。
五、AU对正常大鼠的骨微结构和骨力学研究
AU长期毒性实验后,取大鼠两侧股骨,用Micro-CT分析股骨干骺端的骨小梁微结构,通过三点弯曲力学实验分析股骨生物力学性能。实验结果显示,三个给药组与对照组相比,均不同程度的提高了骨密度、相对骨体积、骨小梁数量、骨小梁厚度和连接密度指数,降低了骨小梁分离度和结构模型指数,从整体上优化了骨小梁的微体系结构。三点弯曲力学实验发现,各给药组均显著提高了骨折力,增加了骨强度。本实验首次发现AU能增加正常大鼠股骨的骨密度和骨强度,是一种潜在的治疗骨质疏松的新药。
Aucubin (AU), which is the main iridoid component from the seeds of Eucommia ulmoides Oliv., has been found to have various pharmacological activities such as high liver-protective activity, antiviral activity, antitumor, anti-inflammatory activity and so on. These actions require the presence of aβ-glucosidase to hydrolyse aucubin to its aglycone, aucubigenin (AUG).
In our studies, the chemistry and efficacy of AU and its derivatives were investigated, including molecular structure, chemical synthesis, antioxidant and antimicrobial activities, pharmacokinetics, acute toxicity and long-term toxicity. The detailed descriptions are listed below.
Part one:The crystallographic study on AU, AUG and isoeucommiol (ISE).
We obtained AU from the seeds of Eucommia ulmoides Oliv. by a procedure of extraction with ethanol. We also have improved the procedure for the extraction of AUG from theβ-glucosidase hydrolysis of AU at 37℃. ISE was obtained through NaBH4 reduction of AUG. The crystal structures of the three compounds were determined by a single-crystal X-ray diffraction analysis. It has been found that crystals of AU are orthorhombic, crystallize in the P212121 space group, and have four molecules in the crystal unit cell. While the crystals of AUG are monoclinic, space group P21, and have two molecules in the unit cell. The crystals of ISE adopts an envelope conformation, presenting a triclinic system, space group P1 with one molecule in the unit cell. Moreover, intensive strength O-H…O hydrogen bonds in all crystal lattices can be observed.
Part two:The bioactivities study on AU and AUG.
AU and its aglycone AUG have been investigated for their in vitro free radical scavenging and antibacterial activities. Various methods, such as the scavenging activities towards DPPH radical, superoxide anion radical, and hydroxyl radical, were established. The antibacterial activities of AU and AUG were also determined by the disc diffusion method and micro-well dilution assay with spectrophotometric method. AU and AUG were individually tested against Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Salmonella enterica (ATCC 14028), respectively. The results showed for the first time that the two compounds exhibited significant differences in free radical scavenging activities. AU showed little DPPH radical scavenging activity. However, a dose response relationship is found in DPPH scavenging activity of AUG. Both AU and AUG showed a moderate scavenging activity against superoxide anion radical. AUG appeared to have little scavenging activity on hydroxyl radical scavenging effects but AU exhibited moderate scavenging activity in a dose dependent manner. The results also indicated that AUG exhibited considerable antibacterial activity against all tested strains, and was particularly more effective against Staphylococcus aureus. As expected, AU had no inhibitory activity against the up growth of all tested strains.
Part three:The pharmacokinetic study on AU.
A simple and accurate high performance liquid chromatographic method was developed and validated for the determination of AU in rabbit plasma. For the experimental group, a dose of 500 mg·kg-1 oral administration of AU and 100 mg·kg-1 intravenous injection of AU were finished. The result showed that both drug concentration-time data were fitted to a two-compartment model with first order absorption. This method could determine the AU level in plasma and are suitable for its pharmacological study.
Part four:The toxicity study on AU.
The acute toxicology experiment study on AU showed that MTD (maximal tolerance dose) administration to mice was 40 g·kg-1. Results of 6 months' long-term toxicity studies indicate that AU has no effect on weight, hair, activity, diet and general condition of rats. Hematologic parameters and blood biochemical indices were within normal physiological range. Heart, liver, spleen, lung, kidney, stomach, intestines and other organs and tissues do not contain any visible pathological changes.Every organ has no change of histopathology. In the recovery period, the rats showed no significant chronic toxicity or delayed toxicity.
Part five:Effect of AU on bone microarchitecture and biomechanics in rats.
To our knowledge, the direct effect of AU on bone structure in vivo has not been investigated yet. Therefore we became interested to study the effect of AU on bone structural and biomechanical analysis in rats after 6 months'long-term toxicity studies. Then the rats were killed and the femurs were dissected for measurement of trabecular microarchitecture and biomechanical analysis. Micro-CT analysis of the distal femur showed that AU significantly increased in bone volume/tissue volume, connect density, trabecular number and trabecular thickness, and decrease in trabecular separation and structure model index in normal rats. In the three-point bending test, the maximum load (ultimate strength) was determined until the specimen was broken. Daily oral administration of AU was found to be able to significantly increase biomechanical quality of femur. The mechanical changes were associated with the bone mineral density increase or even with some improvements in microarchitecture. We demonstrated for the first time that AU taken orally increased bone density and bone strength of rats at the femur. It might be a potential alternative medicine to be used for the treatment of bone deficient diseases such as osteoporosis.
引文
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