绵萆薢中抗骨质疏松活性成分的研究
摘要
骨质疏松症是当今及今后的老龄化社会面临的一个严重疾患。为寻找具有抗骨质疏松活性的化学成分，我们以成骨样细胞株UMR106，破骨样细胞及PTH诱导的小鼠颅骨骨吸收亢进器官等细胞、组织培养模型为筛选手段，动物体内实验为验证方法，在天然药物提取物中筛选有活性的化学成分并探讨其作用机理。
在对历代本草记载有治疗“骨痹、骨痿”之功的29种中药的水、甲醇提取物进行的促成骨样细胞增殖活性实验中，7种中药水提取物和4种醇提取物表现出明显的促成骨样细胞增殖活性作用，在进一步对它们进行的抑制破骨样细胞形成实验中，4个提取物在高浓度时完全抑制了破骨细胞的形成，其中绵萆薢水提取物既显示了非常强的促成骨样细胞增殖活性(浓度为400微克／毫升时，细胞增殖率为66.9％)，也明显地抑制了破骨样多核细胞的形成(浓度为200微克／毫升时，抑制率为100％，并呈明显的量效关系)。随后进行的体内实验表明，绵萆薢水提取物对双侧卵巢摘出(OVX)所致雌性骨质疏松大鼠有较显著的治疗作用，可改善骨质疏松发生时引起的椎骨骨密度(BMD)降低及胫骨近端松质骨骨矿量、松质骨骨密度及皮质骨的骨矿含量的下降。
对绵萆薢水提取物经Diaion 101柱层析分离后的4个部位：水，30％乙醇，60％乙醇，90％乙醇部位．进行了促成骨样细胞增殖活性，抑制破骨样多核细胞形成活性及抑制PTH诱导的小鼠颅骨骨吸收亢进等一系列体外活性筛选实验，从中发现90％乙醇部位具有较强的促成骨样细胞增殖，抑制破骨细胞形成的活性，及较明显的抑制PTH诱导的小鼠颅骨骨吸收亢进的活性。进一步的体内实验表明，90％乙醇部位可显著提高OVX大鼠胫骨近端全骨、松质骨的骨矿含量和骨密度、皮质骨的骨矿含量和厚度，改善骨强度。其中尤为重要的是90％乙醇部位高剂量组可显著提高胫骨的抗扭力强度，这是许多抗骨质疏松药物所达不到的。所以我们认为90％乙醇部位具有良好医疗价值。
对抗骨质疏松活性部位90％乙醇部位进行的化学成分研究过程中，运用了Diaion HP-20，Silica gel，ODS等柱层析及正反相制备薄层层析方法，共分离得到了28个化合物，运用化学及光谱学手段鉴定了它们的化学结构及立体构型。通过文献检索确证，其中9个是新化合物，7个是首次从本属中分得。对它们的抗骨质疏松活性的研究系首次报道。
Spongiosin A，B，C是3个首次从薯蓣科分得的新diarylheptanoid类化合物，piperitol(4)，sesaminone(5) and syringaresinol(6)，是3个首次从薯蓣科分得的木脂素类化合物，在低浓度下spongiosin B，C及3个木脂素类化合物就显示出了很强的抑制破骨细胞形成作用，并且经过显微观察及细胞毒性实验，发现这种抑制作用并非源于细胞毒性，而是可能通过抑制了破骨细胞前体而实现的。在随后的抑制PTH诱导的小鼠颅骨骨吸收亢进试验中，spongiosin B、C、piperitol(4)，sesaminone(5)也显示了非常强的抑制活性。Spongiosin A与B虽然是同分
沈阳药科大学博士论文 绵草蕉中抗骨质疏松括性成分的研究
异构体，但由于立体结构不同，在抑制破骨细胞形成及抑制骨吸收作用上却截然
不同，这可能与破骨细胞受体的结构有关系。
在90％乙醇活性部位中，还分离到了6个新的笛体皂贰类化合物，其中
s卯n轩oside Eu人连同己知皂贰类化合物7，n及葡萄糖贰类化合物27，显
示了较强的抑制PTH诱导的小鼠颅骨骨吸收亢进活性。尤其是化合物ZI，作为90％
乙醇部位中的主要成分，在此试验中显示了比阳性对照药elcitonin还要强的抑
制活性，因而以双侧卵巢摘出所致雌性骨质疏松小鼠为模型进行了化合物ZI的动
物体内活性试验，在提高OVX小鼠腔骨近端全骨、皮质骨的骨矿含量和骨密度、
皮质骨的厚度及松质骨的骨矿含量和改善骨强度方面；在提高OVX小鼠股骨远端
全骨、皮质骨的骨矿含量和骨密度、松质骨的骨矿含量等方面都显示了显著性活
性，能抑制OVX小鼠腔骨近端骨强度的下降，但对股骨远端骨强度无作用。
通过90％乙醇部位及化合物21都与阳性对照药雌H醇的纵向比较后，在大鼠
OVX模型中，90％乙醇部位高剂量组在提高全骨的骨矿含量和骨密度、松质骨的骨
矿含量和骨密度及骨的抗扭力方面，作用强于阳性对照组雌激素类化合物，化合
物 21在小鼠 OVX模型中，抑制了全骨的骨矿含量、骨密度和全骨断面面积的降低，
松质骨骨密度的降低，皮质骨的外膜周长的增长，皮质骨骨矿含量量、皮质骨厚
度和皮质骨密度的下降及抗扭力的减弱。具有较强的抗骨质疏松作用。但除抑制体
重的增加外，在其他方面的作用弱于阳性对照组。所以化合物21抗骨质疏松作用
可能弱于与90％乙醇部位，但在对皮质骨及骨强度方面要强于总皂贰组。因而，gCh
乙醇部位被认为更具有新药开发价值。
Osteoporosis, which has been defined as a state of low bone mass, is one of the most serious disorder associated with aging. According to the theory of Chinese traditional medicine, the formulations used to tonify the kidney and strengthen the bone are always useful in the treatment of osteoporosis syndrome. So 29 kinds of crude drugs from this type of formulation were selected and the aqueous ext. and MeOH ext. of them were screened for their antiosteoporotic activity on stimulation of proliferation of osteoblast-like cell lines UMR106, among all the ext., the water ext. of rhizomes of Dioscorea spongiosa showed the strongest stimulatory activity.
As we know, the imbalance between the bone formation related to the osteoblast closely and bone resorption related to the osteoclast closely, make the osteoporosis occur. So a good agent for anti-osteoporosis should be effective both to osteoblast and to osteoclast. Therefore, the water ext. of D. spongiosa, together with another 10 ext showed the stronger stimulatory activity on osteoblast, were screened further on its inhibitory activity on formation of osteoclast multinuclear cell. It also showed the stronger activity in this in vitro experiment. Then, the result of in vivo experiment revealed that it prevented the decrease of BMD of the spinal vertebra, trabecular mineral content, trabecular density, cortical mineral content of proximal tibia in OVX rat significantly.
The water ext. of D. spongiosa was subjected to the chromatographic separation on Dion 101 column to get H20,30%, 60%, 90%EtOH fractions successively, among them, the 90%EtOH fraction showed the strongest anti-osteoporotic activity, both on stimulation of osteoblast proliferation and inhibition of osteoclast formation, and also showed the inhibitory activity on bone resorption induced by PTH. In vivo experiment, it can prevent the decrease in total mineral content, total density, trabecular mineral content, trabecular density, cortical mineral content and the anti-twist strength of the proximal tibia in OVX rat significantly. So it was further separated by a series of chromatography to afford 28 compounds, in which, 3 new diarylheptanoids and 6 new steroid saponins.
Diarylheptanoids and lignans were evaluated their anti-osteoporotic activity both on the inhibition of the osteoclast formation and on bone resorption induced by PTH, while the glycosides were perfermed only on bone resorption induced by PTH due to small amount of some of them. All the diarylheptanoid and lignan compounds can inhibit the formation of osteoclast significantly but did not show any cytotoxic to cells, while they also showed the inhibitory activity on bone resorption significantly. Interestingly, 2 showed strong inhinition in both experiment, while 1 showed only weak
activity on the osteoclast formation, but no activity on bone resorption, though they are diasteromers. It seems that they acted on the receptors of the osteoclast, where the stereochemistry is crucial for the activity. Among steroid saponin compounds, some compounds behaved the inhibitory activity on osteoclast formation but they also showed the cytotoxic also. 3 saponins and 1 glycoside possessed the inhibition on bone resorption and compound 21, the major compound of this fraction, showed the strongest activity among the glycosides. So it was supposed to do in vivo experiment in OVX mice. From the result data, compound 21, showed the inhibitory activity on the decrease of total mineral content, total mineral density, trabecular mineral density, cortical mineral content, cortical mineral density, cortical thickness and the anti-twist strength of the proximal tibia, and total mineral content, total mineral density, trabecular mineral density, cortical mineral content, cortical mineral density of the distal femora in OVX mice.
Comparison the in vivo activity of 90%EtOH fraction and compound 21 with estradiol, indicated the 90% EtOH fraction behaved the stronger activity on total mineral content, total density, trabecular mineral content, trabecular d
在对历代本草记载有治疗“骨痹、骨痿”之功的29种中药的水、甲醇提取物进行的促成骨样细胞增殖活性实验中，7种中药水提取物和4种醇提取物表现出明显的促成骨样细胞增殖活性作用，在进一步对它们进行的抑制破骨样细胞形成实验中，4个提取物在高浓度时完全抑制了破骨细胞的形成，其中绵萆薢水提取物既显示了非常强的促成骨样细胞增殖活性(浓度为400微克／毫升时，细胞增殖率为66.9％)，也明显地抑制了破骨样多核细胞的形成(浓度为200微克／毫升时，抑制率为100％，并呈明显的量效关系)。随后进行的体内实验表明，绵萆薢水提取物对双侧卵巢摘出(OVX)所致雌性骨质疏松大鼠有较显著的治疗作用，可改善骨质疏松发生时引起的椎骨骨密度(BMD)降低及胫骨近端松质骨骨矿量、松质骨骨密度及皮质骨的骨矿含量的下降。
对绵萆薢水提取物经Diaion 101柱层析分离后的4个部位：水，30％乙醇，60％乙醇，90％乙醇部位．进行了促成骨样细胞增殖活性，抑制破骨样多核细胞形成活性及抑制PTH诱导的小鼠颅骨骨吸收亢进等一系列体外活性筛选实验，从中发现90％乙醇部位具有较强的促成骨样细胞增殖，抑制破骨细胞形成的活性，及较明显的抑制PTH诱导的小鼠颅骨骨吸收亢进的活性。进一步的体内实验表明，90％乙醇部位可显著提高OVX大鼠胫骨近端全骨、松质骨的骨矿含量和骨密度、皮质骨的骨矿含量和厚度，改善骨强度。其中尤为重要的是90％乙醇部位高剂量组可显著提高胫骨的抗扭力强度，这是许多抗骨质疏松药物所达不到的。所以我们认为90％乙醇部位具有良好医疗价值。
对抗骨质疏松活性部位90％乙醇部位进行的化学成分研究过程中，运用了Diaion HP-20，Silica gel，ODS等柱层析及正反相制备薄层层析方法，共分离得到了28个化合物，运用化学及光谱学手段鉴定了它们的化学结构及立体构型。通过文献检索确证，其中9个是新化合物，7个是首次从本属中分得。对它们的抗骨质疏松活性的研究系首次报道。
Spongiosin A，B，C是3个首次从薯蓣科分得的新diarylheptanoid类化合物，piperitol(4)，sesaminone(5) and syringaresinol(6)，是3个首次从薯蓣科分得的木脂素类化合物，在低浓度下spongiosin B，C及3个木脂素类化合物就显示出了很强的抑制破骨细胞形成作用，并且经过显微观察及细胞毒性实验，发现这种抑制作用并非源于细胞毒性，而是可能通过抑制了破骨细胞前体而实现的。在随后的抑制PTH诱导的小鼠颅骨骨吸收亢进试验中，spongiosin B、C、piperitol(4)，sesaminone(5)也显示了非常强的抑制活性。Spongiosin A与B虽然是同分
沈阳药科大学博士论文 绵草蕉中抗骨质疏松括性成分的研究
异构体，但由于立体结构不同，在抑制破骨细胞形成及抑制骨吸收作用上却截然
不同，这可能与破骨细胞受体的结构有关系。
在90％乙醇活性部位中，还分离到了6个新的笛体皂贰类化合物，其中
s卯n轩oside Eu人连同己知皂贰类化合物7，n及葡萄糖贰类化合物27，显
示了较强的抑制PTH诱导的小鼠颅骨骨吸收亢进活性。尤其是化合物ZI，作为90％
乙醇部位中的主要成分，在此试验中显示了比阳性对照药elcitonin还要强的抑
制活性，因而以双侧卵巢摘出所致雌性骨质疏松小鼠为模型进行了化合物ZI的动
物体内活性试验，在提高OVX小鼠腔骨近端全骨、皮质骨的骨矿含量和骨密度、
皮质骨的厚度及松质骨的骨矿含量和改善骨强度方面；在提高OVX小鼠股骨远端
全骨、皮质骨的骨矿含量和骨密度、松质骨的骨矿含量等方面都显示了显著性活
性，能抑制OVX小鼠腔骨近端骨强度的下降，但对股骨远端骨强度无作用。
通过90％乙醇部位及化合物21都与阳性对照药雌H醇的纵向比较后，在大鼠
OVX模型中，90％乙醇部位高剂量组在提高全骨的骨矿含量和骨密度、松质骨的骨
矿含量和骨密度及骨的抗扭力方面，作用强于阳性对照组雌激素类化合物，化合
物 21在小鼠 OVX模型中，抑制了全骨的骨矿含量、骨密度和全骨断面面积的降低，
松质骨骨密度的降低，皮质骨的外膜周长的增长，皮质骨骨矿含量量、皮质骨厚
度和皮质骨密度的下降及抗扭力的减弱。具有较强的抗骨质疏松作用。但除抑制体
重的增加外，在其他方面的作用弱于阳性对照组。所以化合物21抗骨质疏松作用
可能弱于与90％乙醇部位，但在对皮质骨及骨强度方面要强于总皂贰组。因而，gCh
乙醇部位被认为更具有新药开发价值。
Osteoporosis, which has been defined as a state of low bone mass, is one of the most serious disorder associated with aging. According to the theory of Chinese traditional medicine, the formulations used to tonify the kidney and strengthen the bone are always useful in the treatment of osteoporosis syndrome. So 29 kinds of crude drugs from this type of formulation were selected and the aqueous ext. and MeOH ext. of them were screened for their antiosteoporotic activity on stimulation of proliferation of osteoblast-like cell lines UMR106, among all the ext., the water ext. of rhizomes of Dioscorea spongiosa showed the strongest stimulatory activity.
As we know, the imbalance between the bone formation related to the osteoblast closely and bone resorption related to the osteoclast closely, make the osteoporosis occur. So a good agent for anti-osteoporosis should be effective both to osteoblast and to osteoclast. Therefore, the water ext. of D. spongiosa, together with another 10 ext showed the stronger stimulatory activity on osteoblast, were screened further on its inhibitory activity on formation of osteoclast multinuclear cell. It also showed the stronger activity in this in vitro experiment. Then, the result of in vivo experiment revealed that it prevented the decrease of BMD of the spinal vertebra, trabecular mineral content, trabecular density, cortical mineral content of proximal tibia in OVX rat significantly.
The water ext. of D. spongiosa was subjected to the chromatographic separation on Dion 101 column to get H20,30%, 60%, 90%EtOH fractions successively, among them, the 90%EtOH fraction showed the strongest anti-osteoporotic activity, both on stimulation of osteoblast proliferation and inhibition of osteoclast formation, and also showed the inhibitory activity on bone resorption induced by PTH. In vivo experiment, it can prevent the decrease in total mineral content, total density, trabecular mineral content, trabecular density, cortical mineral content and the anti-twist strength of the proximal tibia in OVX rat significantly. So it was further separated by a series of chromatography to afford 28 compounds, in which, 3 new diarylheptanoids and 6 new steroid saponins.
Diarylheptanoids and lignans were evaluated their anti-osteoporotic activity both on the inhibition of the osteoclast formation and on bone resorption induced by PTH, while the glycosides were perfermed only on bone resorption induced by PTH due to small amount of some of them. All the diarylheptanoid and lignan compounds can inhibit the formation of osteoclast significantly but did not show any cytotoxic to cells, while they also showed the inhibitory activity on bone resorption significantly. Interestingly, 2 showed strong inhinition in both experiment, while 1 showed only weak
activity on the osteoclast formation, but no activity on bone resorption, though they are diasteromers. It seems that they acted on the receptors of the osteoclast, where the stereochemistry is crucial for the activity. Among steroid saponin compounds, some compounds behaved the inhibitory activity on osteoclast formation but they also showed the cytotoxic also. 3 saponins and 1 glycoside possessed the inhibition on bone resorption and compound 21, the major compound of this fraction, showed the strongest activity among the glycosides. So it was supposed to do in vivo experiment in OVX mice. From the result data, compound 21, showed the inhibitory activity on the decrease of total mineral content, total mineral density, trabecular mineral density, cortical mineral content, cortical mineral density, cortical thickness and the anti-twist strength of the proximal tibia, and total mineral content, total mineral density, trabecular mineral density, cortical mineral content, cortical mineral density of the distal femora in OVX mice.
Comparison the in vivo activity of 90%EtOH fraction and compound 21 with estradiol, indicated the 90% EtOH fraction behaved the stronger activity on total mineral content, total density, trabecular mineral content, trabecular d
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52. Pike J. W., Ann. Rev. Nutr., 1991, 11: 189-216
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54．殷军，王大为等，沈阳药科大学学报，2002，18：279-282
55. Li, H.; Li, J.; Prasain, J. K. et al Bio. Pharm. Bull., 1998, 21: 594-598.
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59. Nonaka K. Clinical Calcium, 2000, 10(6): 51-57
60. Abe, F.; Yahara, S.; Kubo, K.; Nonaka, G.; Okabe, H.; Nishioka, I. Chem. Pharm. Bull. 1974, 22, 2650-2655.
61. Chiung, Y. -M.; Hayashi, H.; Matsumoto, H.; Otani, T.; Yoshida, K.; Huang, M. -Y.; Chen, R. -X.; Liu, J. -R.; Nakayama, M. J. Antibiot. 1994, 47, 487-491.
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63. Hu, K.; Yao, X. et al J. Nat. Prod., 1999, 62: 299-301.
64. Kawasaki, T.; Yamauchi, T.; Yamauchi, R. Chem. Pharm. Bull., 1962, 10: 698-702.
65 Liu, C. -L.; Chen, Y. -Y. Acta Bot. Sin. 1985, 27, 635-639.
66. Abbas, F. A. Sci. Pharm. 2001, 69, 219-234.
67. Dong, M.; Feng, X.; Wu, L. et al Planta Med., 2001, 67: 853-857. Zhang, K. Chinese Herbs, 1990, 21, 25-27.
68. Watanabe, Y.; Sanada, S.; Ida, Y.; Shoji, J. Chem. Pharm. Bull., 1983, 31: 3486-3495.
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71. Okamura, N.; Yagi, A.; Nishioka, I. Chem. Pharm. Bull. 1981, 29, 3507-3514.
72. Mimaki, Y.; Sashida, Y.; Nakamura, O.; Nikaido, T.; Ohmoto, T. Phytochemistry 1993, 33, 675-682.
73. Ahmad, V. U.; Khaliq-uz-Zaman, S. M.; Shameel, S.; Perveen, S.; Ali, Z. Phytochemistry 1998, 50, 481-484.
74. Craveiro A.A., Prado A.C., Gottlieb O. R., Welerson P. C., Phytochemistry, 1970, 9, 1860—1862.
75. Abe, F.; Yamauchi, T. Phytochemistry 1988, 27, 575-577.
76. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
77. Rychnovsky, S. D.; Skalitzky, D. J. Tetrahedron Lett. 1990, 31, 945-948.
78. Rychnovsky, S. D.; Richardson, T. I.; Rogers, B. N. J. Org. Chem. 1997, 62, 2925-2934.
79. Harada, N.; Saito, A.; Ono, H.; Gawronski, J.; Gawronska, K.; Sugioka, T.; Uda, H.; Kuriki, T. J. Am. Chem. Soc. 1991, 113, 3842-3850.
80. Kasai, R.; Okihara, M.; Asakawa, J.; Mizutani, K., Tanaka, O. Tetrahedron 1979, 35, 1427-1432.
81. Kawashima, K.; Mimaki, Y.; Sashida, Y. Chem. Pharm. Bull. 1991, 39,
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82. Nes, W. D.; Koike, K.; Jia, Z.; Sakamoto, Y.; Satou, T.; Nikaido, T.; Griffin, J. F. J. Am. Chem. Soc. 1998, 120, 5970-5980.
83. Kuroda, M.; Mimaki, Y.; Sashida, Y. Phytochemistry 1999, 52, 445-452.