促进成骨细胞骨形成的中药及其活性成分的研究
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摘要
成骨细胞是骨形成的主要功能细胞，因此刺激成骨细胞骨形成的药物将具
有独特的抗骨质疏松的活性。为了筛选和开发新型、高效的防治骨质疏松的药
物，本研究首次建立了成骨样UMR106细胞体外培养为抗骨质疏松药物的活性
筛选模型。以成骨样细胞的增殖和分化为活性检测指标，成骨样细胞的增殖以
MTT法测定，氟化钠为阳性对照药。成骨样细胞的分化采用测定细胞内碱性磷
酸酶活性的方法，地塞米松为阳性对照药，同时考察了溶剂乙醇和二甲基亚砜
对细胞增殖和分化的影响。
依据传统中医“肾主骨”的理论，选择补肾中药为主要研究对象，考察它
们对成骨样细胞骨形成的活性。将中药和一些未作药用的植物的提取物与成骨
样细胞体外共同培养，在所研究的15种补肾中药中对成骨样细胞增殖有促进作
用的有9种，在所研究的15种其它中药中对成骨样细胞增殖有促进作用的有4
种，而对53种还未作药用的植物的活性测试发现只有2种有显著活性；12种
中药中有6种能促进成骨样细胞分化。其中有4种补肾中药既能促进成骨样细
胞增殖，又能促进成骨样细胞分化，它们是：补骨脂、槲寄生、淫羊藿、川牛
膝。
对补骨脂不同极性萃取物进一步进行成骨样细胞增殖和分化活性测试，发
现补骨脂乙酸乙酯萃取物为补骨脂中的主要活性部分。又选用雌性大鼠双侧去
卵巢所致的骨质疏松模型，依替膦酸二钠盐为阳性药，考察补骨脂乙酸乙酯萃
取物对骨质疏松模型大鼠的作用。结果表明，给予补骨脂乙酸乙酯萃取物的大
鼠与模型组大鼠相比，骨密度、骨钙含量显著增加。
为了追踪分离补骨脂乙酸乙酯萃取物中的活性成分，本实验采用多种色谱
分离技术，以活性测试为导向，结合波谱技术及理化数据，从补骨脂乙酸乙酯
萃取物中分离并鉴定了4个活性化合物的化学结构，它们是具有促进细胞增殖
和分化作用的补骨脂异黄酮（corylin）和补骨脂二氢黄酮（bavachin），对细胞
分化有一定促进作用的异补骨脂素（isopsoralen）和补骨脂素（psoralen）。
由于补骨脂中黄酮类化合物的分析测定未见报道，因此，本研究首次建立
了同时测定补骨脂中补骨脂异黄酮和补骨脂二氢黄酮的HI，LC分析方法。色谱
靴为：ODS柱，流动相：甲醇20 nnnoM醋酸铰缓冲液，pH4刀的733，州；
流速：0．SInljlnin；检测波长：240nln。本实验采用普通回流6小时后用乙酸乙
酪苹取三次的样品制备方法，以补骨脂二氢黄酮和补骨脂异黄酮计RSD分别为
3＊％和3．6％，回收率分别为94．9％和96．2％。并对8个不同产地的补骨脂药材
中补骨脂二氢黄酮和补骨脂异黄酮进行了含量测定。
此外，本研究考察了一些黄酮类化合物对成骨样细胞的作用。结果发现：
中药淫羊蕾中的主要成分淫羊蕾昔及其大鼠体内主要代谢物淫羊霍次苦H具有
类似的作用，可显著促进成骨样细胞的增殖：中药懈寄生总黄酮对成骨样细胞
的增殖有一定的促进作用，并可显著提高细胞内ALP活性；大豆昔元及其衍生
物对U’MR106细胞的增殖作用不明显。对于中药补骨脂中分离的两个促成骨样
细胞增殖的活性成分补骨脂H氢黄酮和补骨脂异黄酮，采用 Western blotting的
方法考察不同时间对 U’MR106细胞周期蛋白 CyClin E的表达，结果它们对 CyClin
E的表达没有显著影响。
本实验所建立的细胞培养方法具有快速、所需样品量少和作用直接的优点，
尤其适用于中药中微量成分的药效及活性成分的追踪研究，而且动物体内实验
结果证实了本方法的可靠性，为从天然药物中寻找和开发新型有效的防治骨质
疏松的药物提供了一种便捷的手段。大量中药及植物促进成骨样细胞骨形成活
性筛选结果表明，补肾中药的“壮骨”作用可能与其对成骨细胞活性的促进作
用有关，中药补骨脂中的黄酮成分可能具有防治骨质疏松的活性。
Osteoblasts are the important cells in bone formation and, therefore, agents stimulating osteoblastic bone formation would have potential activity of antiosteoporosis. In order to screen and exploit novel and effective medicines to prevent and cure osteoporosis, osteoblast-like UMR1O6 cells were employed as a screening model of anti-osteoporosis medicines for the first time. The cell proliferation and differentiation were measured as index of stimulating osteoblastic bone formation. Cell proliferation was assayed by M~F method with fluoride sodium as positive control. And cell differentiation was assayed by measuring the cellular alkaline phosphatase activity with dexaniethasone as positive control. The influence of ethanol and dimethyl sulfoxide on cell proliferation and differentiation assay was also investigated.
Some 搆idney-tonifying?traditional Chinese medicines were selected on the basis of the theory of traditional Chinese medicines and examined for the activity of stimulating osteoblastic bone formation in vitro. After co-culturing the crude extracts of 15 搆idney-tonifying?traditional Chinese medicines with UMIR1 06 cells, 9 were found to have significant promoting effect on osteoblast proliferation. Fifteen traditional Chinese medicines other than 搆idney-tonifying?and 53 plants not yet for medicinal use were also studied, 4 from the former and 2 from the later were found to stimulate the cell proliferation significantly. And 6 from 12 traditional Chinese medicines provided promoting effect on the cell differentiation. Four traditional Chinese medicines showed activities of stimulating both osteoblastic proliferation and differentiation, they are Psoralea coryl~folia L., Viscum coloratum (Kom.) Nakai, Epimedium sagittatum Maxim and Cyathula officinalis Kuan.
The effect of various polar fractions from Psoralea corylzfolia L. ethanol extract on cell proliferation and differentiation were further examined and its ethyl acetate fraction were found to be active fraction. Also the effect of its ethyl acetate fraction on ovariectimized rats, as a pharmacological model of osteoporosis, was studied and biphosphate served as positive control. Compared with OVX, the ethyl acetate fraction from Psoralea coryl~folia L. significantly increased bone mineral density and bone calcium content of rats.
In order to isolate the active compounds in ethyl acetate fraction of Psoralea coryl~folia L., activity-guided isolation was performed along with chromatographic
3
techniques. Four active compounds were isolated from ethyl acetate fraction and identified by their NMR spectral data and physical-chemical properties. They are corylin and bavachin, which showed stimulating effect on osteoblastic proliferation and differentiation, isopsoralen and psoralen exhibited activity of promoting cell differentiation in some degree.
There was no report, up to dale, on the determination of flavonoids in Psoralea cory4folia L.. In this study, an HPLC method for simultaneous determination of corylin and bavachin was set up. HIPLC analysis was carried out on an ODS column
(25mmX 4.6mm). The mobile phase was methanol and 2OmmoIIl ammonium acetate buffer, pH 4.0 (67: 33). The flow rate was 0.8 mllmin and the wavelength of UV detector was set at 240 nra. The sample was prepared by first refluxing raw materials for 6 h and then extract 3 times with ethyl acetate. And the RSD of bavachin and corylin was 3.1% and 3.6%, respectively; the recovery was 94.9% and 96.2%. The contents of bavachin and corylin in Psoralea coiyljfolia L. from 8 regions were determined.
In addition, the effect of some flavonoid compounds on osteoblast-like cells was also studied. Icariin, the main constituent of Epimedium sagittatum Maxim, and its metabolite icariside II showed similar activity in cell proliferation. The total flavonoids from J?scum coloratum (Kom.) Nakai could stimulate cell proliferation and significantly promote cellular ALP activity. Daidzein and its derivatives exhibited no significant activity in cell
有独特的抗骨质疏松的活性。为了筛选和开发新型、高效的防治骨质疏松的药
物，本研究首次建立了成骨样UMR106细胞体外培养为抗骨质疏松药物的活性
筛选模型。以成骨样细胞的增殖和分化为活性检测指标，成骨样细胞的增殖以
MTT法测定，氟化钠为阳性对照药。成骨样细胞的分化采用测定细胞内碱性磷
酸酶活性的方法，地塞米松为阳性对照药，同时考察了溶剂乙醇和二甲基亚砜
对细胞增殖和分化的影响。
依据传统中医“肾主骨”的理论，选择补肾中药为主要研究对象，考察它
们对成骨样细胞骨形成的活性。将中药和一些未作药用的植物的提取物与成骨
样细胞体外共同培养，在所研究的15种补肾中药中对成骨样细胞增殖有促进作
用的有9种，在所研究的15种其它中药中对成骨样细胞增殖有促进作用的有4
种，而对53种还未作药用的植物的活性测试发现只有2种有显著活性；12种
中药中有6种能促进成骨样细胞分化。其中有4种补肾中药既能促进成骨样细
胞增殖，又能促进成骨样细胞分化，它们是：补骨脂、槲寄生、淫羊藿、川牛
膝。
对补骨脂不同极性萃取物进一步进行成骨样细胞增殖和分化活性测试，发
现补骨脂乙酸乙酯萃取物为补骨脂中的主要活性部分。又选用雌性大鼠双侧去
卵巢所致的骨质疏松模型，依替膦酸二钠盐为阳性药，考察补骨脂乙酸乙酯萃
取物对骨质疏松模型大鼠的作用。结果表明，给予补骨脂乙酸乙酯萃取物的大
鼠与模型组大鼠相比，骨密度、骨钙含量显著增加。
为了追踪分离补骨脂乙酸乙酯萃取物中的活性成分，本实验采用多种色谱
分离技术，以活性测试为导向，结合波谱技术及理化数据，从补骨脂乙酸乙酯
萃取物中分离并鉴定了4个活性化合物的化学结构，它们是具有促进细胞增殖
和分化作用的补骨脂异黄酮（corylin）和补骨脂二氢黄酮（bavachin），对细胞
分化有一定促进作用的异补骨脂素（isopsoralen）和补骨脂素（psoralen）。
由于补骨脂中黄酮类化合物的分析测定未见报道，因此，本研究首次建立
了同时测定补骨脂中补骨脂异黄酮和补骨脂二氢黄酮的HI，LC分析方法。色谱
靴为：ODS柱，流动相：甲醇20 nnnoM醋酸铰缓冲液，pH4刀的733，州；
流速：0．SInljlnin；检测波长：240nln。本实验采用普通回流6小时后用乙酸乙
酪苹取三次的样品制备方法，以补骨脂二氢黄酮和补骨脂异黄酮计RSD分别为
3＊％和3．6％，回收率分别为94．9％和96．2％。并对8个不同产地的补骨脂药材
中补骨脂二氢黄酮和补骨脂异黄酮进行了含量测定。
此外，本研究考察了一些黄酮类化合物对成骨样细胞的作用。结果发现：
中药淫羊蕾中的主要成分淫羊蕾昔及其大鼠体内主要代谢物淫羊霍次苦H具有
类似的作用，可显著促进成骨样细胞的增殖：中药懈寄生总黄酮对成骨样细胞
的增殖有一定的促进作用，并可显著提高细胞内ALP活性；大豆昔元及其衍生
物对U’MR106细胞的增殖作用不明显。对于中药补骨脂中分离的两个促成骨样
细胞增殖的活性成分补骨脂H氢黄酮和补骨脂异黄酮，采用 Western blotting的
方法考察不同时间对 U’MR106细胞周期蛋白 CyClin E的表达，结果它们对 CyClin
E的表达没有显著影响。
本实验所建立的细胞培养方法具有快速、所需样品量少和作用直接的优点，
尤其适用于中药中微量成分的药效及活性成分的追踪研究，而且动物体内实验
结果证实了本方法的可靠性，为从天然药物中寻找和开发新型有效的防治骨质
疏松的药物提供了一种便捷的手段。大量中药及植物促进成骨样细胞骨形成活
性筛选结果表明，补肾中药的“壮骨”作用可能与其对成骨细胞活性的促进作
用有关，中药补骨脂中的黄酮成分可能具有防治骨质疏松的活性。
Osteoblasts are the important cells in bone formation and, therefore, agents stimulating osteoblastic bone formation would have potential activity of antiosteoporosis. In order to screen and exploit novel and effective medicines to prevent and cure osteoporosis, osteoblast-like UMR1O6 cells were employed as a screening model of anti-osteoporosis medicines for the first time. The cell proliferation and differentiation were measured as index of stimulating osteoblastic bone formation. Cell proliferation was assayed by M~F method with fluoride sodium as positive control. And cell differentiation was assayed by measuring the cellular alkaline phosphatase activity with dexaniethasone as positive control. The influence of ethanol and dimethyl sulfoxide on cell proliferation and differentiation assay was also investigated.
Some 搆idney-tonifying?traditional Chinese medicines were selected on the basis of the theory of traditional Chinese medicines and examined for the activity of stimulating osteoblastic bone formation in vitro. After co-culturing the crude extracts of 15 搆idney-tonifying?traditional Chinese medicines with UMIR1 06 cells, 9 were found to have significant promoting effect on osteoblast proliferation. Fifteen traditional Chinese medicines other than 搆idney-tonifying?and 53 plants not yet for medicinal use were also studied, 4 from the former and 2 from the later were found to stimulate the cell proliferation significantly. And 6 from 12 traditional Chinese medicines provided promoting effect on the cell differentiation. Four traditional Chinese medicines showed activities of stimulating both osteoblastic proliferation and differentiation, they are Psoralea coryl~folia L., Viscum coloratum (Kom.) Nakai, Epimedium sagittatum Maxim and Cyathula officinalis Kuan.
The effect of various polar fractions from Psoralea corylzfolia L. ethanol extract on cell proliferation and differentiation were further examined and its ethyl acetate fraction were found to be active fraction. Also the effect of its ethyl acetate fraction on ovariectimized rats, as a pharmacological model of osteoporosis, was studied and biphosphate served as positive control. Compared with OVX, the ethyl acetate fraction from Psoralea coryl~folia L. significantly increased bone mineral density and bone calcium content of rats.
In order to isolate the active compounds in ethyl acetate fraction of Psoralea coryl~folia L., activity-guided isolation was performed along with chromatographic
3
techniques. Four active compounds were isolated from ethyl acetate fraction and identified by their NMR spectral data and physical-chemical properties. They are corylin and bavachin, which showed stimulating effect on osteoblastic proliferation and differentiation, isopsoralen and psoralen exhibited activity of promoting cell differentiation in some degree.
There was no report, up to dale, on the determination of flavonoids in Psoralea cory4folia L.. In this study, an HPLC method for simultaneous determination of corylin and bavachin was set up. HIPLC analysis was carried out on an ODS column
(25mmX 4.6mm). The mobile phase was methanol and 2OmmoIIl ammonium acetate buffer, pH 4.0 (67: 33). The flow rate was 0.8 mllmin and the wavelength of UV detector was set at 240 nra. The sample was prepared by first refluxing raw materials for 6 h and then extract 3 times with ethyl acetate. And the RSD of bavachin and corylin was 3.1% and 3.6%, respectively; the recovery was 94.9% and 96.2%. The contents of bavachin and corylin in Psoralea coiyljfolia L. from 8 regions were determined.
In addition, the effect of some flavonoid compounds on osteoblast-like cells was also studied. Icariin, the main constituent of Epimedium sagittatum Maxim, and its metabolite icariside II showed similar activity in cell proliferation. The total flavonoids from J?scum coloratum (Kom.) Nakai could stimulate cell proliferation and significantly promote cellular ALP activity. Daidzein and its derivatives exhibited no significant activity in cell
引文
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