Part I ： Action Mechanisms of Traditional Traumatological Chinese Herbal Medicine on Newborn Rat Bone Cells

第一部份：傳統傷科中藥對新生鼠骨細胞作用機制的探討 第二部份：嘧啶類及其類似物對活性氧之清除作用

 第一部份：傳統傷科中藥對新生鼠骨細胞作用機制的探討

傳統傷科中草藥骨碎補（ Drynaria fortunei ）、續斷（ Dipsacus asper ）、淫羊藿（ Epimedium brevicornum ）、杜仲（ Eucommia ulmoide s ）、蛇床子（ Cnidium monnieri ）、蟅蟲（ Eupolyphaga sinensis ）、牛膝（ Achyranthes bidentata ）、自然銅（ Pyrite ）、沒藥（ Commip hora myrrha ）、紅花（ Carthamus tinctorius ）、丹參（ Salvia miltiorrhiza ）、三七 (Panax psedoginseng var. notoginseng) 、熟地黃（ Rehma nnia glutinosa ）、白花菜（ Cleome gynandra ），與複方六味地黃丸、續骨活血湯、接骨散、龜鹿膠、龜鹿二仙膠用於補腎、促進血液循環 與治療骨折，然而其作用機轉未經證實，故本實驗利用骨母細胞與蝕骨細胞體外培養模式進行各項生化反應分析。

中草藥單味以 70%acetone ，複方以水煮方式萃取，骨母細胞或蝕骨細胞取自 3-5 天大的 Wistar 鼠頭蓋骨，以不同時間、不同中藥濃度處理 後，測定細胞存活率、鈣化、鹼性磷酸酵素（ ALP ）、酸性磷酸酵素（ ACP ）、乳酸脫氫酵素（ LDH ）等酵素活性、總蛋白質含量與 m RNA 表現。我們發現骨碎補與自然銅可增加骨細胞存活率與酸性磷酸酵素（ ACP ）、鹼性磷酸酵素（ ALP ）活性；杜仲、牛膝、熟地黃

、白花菜、續骨活血湯、龜鹿二仙膠可促進骨母細胞增生；淫羊藿、蟅蟲、蛇床子、紅花、丹參、六味地黃丸、龜鹿膠可促進骨母細胞增 生與鹼性磷酸酵素（ ALP ）增加；續斷可增加鹼性磷酸酵素（ ALP ）含量（ p<0.05 ）。乳酸脫氫酵素（ LDH ）用於測定細胞毒性，在本 實驗中的中藥對細胞呈現幾乎近於無毒性的反應，由此可知中藥在骨細胞上之作用機轉各異。希望藉由加入中藥後的骨母細胞與蝕骨細胞 之生化表現，推測其作用機制，以期理論與臨床應用可相互佐證，並在骨折癒合與骨質疏鬆症的治療上有所貢獻。

第二部份：嘧啶類及其類似物對活性氧之清除作用

毒性氧自由基、過氧化氫、超氧陰離子與羥自由基已廣泛被證實和老化、許多疾病和症狀如細胞損傷、缺血後再灌流組織損害、腦瘤和發 炎等有重要關聯。這些自由基會引起生物體內核酸、蛋白質、氨基酸、脂質、脂蛋白、醣類和組織連結分子可逆或不可逆性的破壞。黃嘌 呤氧化酵素能連續催化兩個氧化反應：將次黃嘌呤轉變成黃嘌呤再轉變成尿酸，並產生氧化產物過氧化氫、超氧陰離子與羥自由基。於本 研究中，我們使用電子自旋共振法測試黃嘌呤氧化酵素 ; 抑制劑 2-mercaptopyrimidine 、 2-mercapto-4(3H)-quinazolinone 和 sulfasalazine 清除 超氧陰離子與羥自由基之活性。

黃嘌呤氧化酵素的活性是以黃嘌呤為受質分析 295nm 下的光譜，超氧陰離子從次黃嘌呤 - 黃嘌呤氧化酵素產生，羥自由基從 Fenton 反應系 統產生，經 5,5-dimethyl-l-pyrroline N-oxide （ DMPO ）捕捉。

Sulfasalazine （ IC50 = 25.11 μM ， Ki = 50.88μM ）與 2-mercapto- 4(3H)-quinazolinone （ IC50 = 98.71 μM ， Ki = 158.38μM ）對黃嘌呤氧化 酵素之抑制型態屬混合型中的不競爭型 — 非競爭型作用， 2-mercaptopyrimidine （ IC50 =136.14μM, Ki = 62.46μM ）之抑制型態為非競爭 型作用。在自由基清除活性上， 200 μM 的 2-mercaptopyrimidine 、 2-mercapto-4(3H)-quinazolinone 與 sulfasalazine 抑制超氧陰離子形成百分 比各為 46.90% 、 14.94% 與 5.24% ；而 200 μM 的 2-mercapto-4(3H)-quinazolinone 、 sulfasalazine 和 2-mercaptopyrimidine 對羥自由基抑制 百分比各為 33.34% 、 23.85% 與 21.23% 。

在本研究中， 2-mercaptopyrimidine 有最有佳的超氧陰離子清除作用，且抑制效果於 100-500 μM 濃度下隨劑量增加，但並非強效的黃嘌呤 氧化酶抑制劑。 2-mercapto-4(3H)-quinazolinone 有較顯著的羥自由基（ hydroxyl radical ）清除效果， sulfasalazine 對黃嘌呤氧化酶具有強的 抑制效果，但非有效的超氧陰離子驅除劑。

Part I ： Action Mechanisms of Traditional Traumatological Chinese He rbal Medicine on Newborn Rat Bone Cells Part II ： Scavenging Effect

of Reactive Oxygen by



Part I ： Action Mechanisms of Traditional Traumatological Chinese Herbal Medicine on Newborn Rat Bone Cells

The traditional traumatological Chinese herbal medicine （ Drynaria fortunei, Dipsacus asper, Epimedium brevicornum, Eucommia ulmoides, Cnidium monnieri, Eup olyphaga sinensis, Achyranthes bidentata, Pyrite, Commiphora myrrha, Carthamus tinctorius, Salvia miltiorrhiza, Panax psedoginseng var. notoginseng, Rehmannia gl utinosa and Cleome gynandra ） and some compound prescribes （ Liuwei Dihuang Wan, Xugu Hwoshiueh Tang, Jiegu San, Gueiluh Jiau and Gueiluh Ellshain Jia u ） have been claimed to warming the kidney, promoting blood circulation or therapeutic effects on bone healing; however, clinical mechanisms for their effect have not been identified. In this study, we investigated their biological effects via in vitro osteoblast and osteoclast cell culture.

The Chinese herbal medicine was extracted by 70% acetone and compound prescribes with boiling water. Osteoblasts or osteoclasts were prepared from newborn Wist ar rat (3-5 days old) calvarias and treated with different concentration of the Chinese medicine herbs for various time intervals in this study. Cell viability (%), calcifica tion, alkaline phosphatase (ALP), acid phosphatase (ACP), lactate dehydrogenase (LDH) activity, total protein and mRNA expression were determined. We found that the ACP activities, cell viabilities and ALP activities were significantly increased by Drynaria fortunei and Pyrite. Eucommia ulmoides, Achyranthes bidentata, Rehma nnia glutinosa, Cleome gynandra, Xugu Hwoshiueh Tang, and Gueiluh Ellshain Jiau significantly increased the cell viabilities. Both of cell viabilities and ALP activiti es were significantly increased by Epimedium brevicornum, Eupolyphaga sinensis, Cnidium monnieri, Carthamus tinctorius, Salvia miltiorrhiza, Liuwei Dihuang Wan and Gueiluh Jiau. Dipsacus asper increased the ALP activity of osteoblast cells (p<0.05). Assays for LDH have been used for detecting cellular cytotoxicity and the res ult showed a little or not changed during the experimental. This means different preparations of the Chinese medicine had different effects on the bone cell activities.

We try to find the action mechanism of each sample on osteoblast and osteoclast cells by its biochemical expression. Base on these theories and clinical practice, it may contribute to the treatment of bone healing and osteoporosis.

Part II ： Scavenging Effect of Reactive Oxygen by Pyrimidines and It’s Analogues

Toxic oxygen free radicals, hydrogen peroxide, superoxide anion and hydroxyl radical have been implicated as important pathologic mediators in many clinical disord ers and diseases such as cellular injury, hypoxia-reoxygenation injury, brain tumors and inflammation. These radicals are capable of reversibly or irreversibly damagin g compounds of all biochemical classes, including nucleic acids, protein and free amino acids, lipids and lipoproteins, carbohydrates, and connective tissue macromole cules. Xanthine oxidase catalyses two consecutive oxidation reactions: from hypoxanthine to xanthine and from xanthine to uric acid, and generates the oxidants hydro gen peroxide, superoxide anion and hydroxyl radical. In this study, we tested the xanthine oxidase inhibitiors 2-mercaptopyrimidine, 2-mercapto- 4(3H)-quinazolinone and sulfasalazine by measuring their scavenging activity to superoxide anion (O2-˙) and hydroxyl radical (OH˙) using electron spin resonance technique.

The activity of xanthine oxidase towards xanthine as a substrate was assayed spectrophotometrically at 295 nm. The superoxide anion generated from a hypoxanthine - xanthine oxidase reaction system and the hydroxyl radical (OH˙) generated by Fenton reaction system, were trapped by 5,5-dimethyl-l-pyrroline N-oxide （ DMPO ） . Sulfasalazine (IC50 = 25.11μM, Ki = 50.88μM) and 2-mercapto-4(3H)- quinazolinone (IC50=98.71μM, Ki=158.38μM) induced a mixed type (non-competitive-unco mpetitive) inhibition, and 2-mercaptopyrimidine （ IC50 =136.14μM, Ki = 62.46μM ） induced uncompetitive type inhibitions on xanthine oxidase, respectively. T he free radical scavenging activities were showed that the percentage inhibition of superoxide anion formation of 2-mercaptopyrimidine, 2-mercapto-4(3H)-quinazolin one and sulfasalazine were 46.90, 14.94 and 5.24% at 200μM, but the percentage inhibition of hydroxyl radical formation were 33.34, 23.85 and 21.23% for 2-mercapt o-4(3H)-quinazolinone, sulfasalazine, 2-mercaptopyrimidine at 200 μM, respectively.

In this study, 2-mercaptopyrimidine had a potent superoxide anion scavenging effect, and showed a dose dependent manner from 100 to 500μM, but not a powerful xa

nthine oxidase inhibitor. It was found that 2-mercapto-4(3H)- quinazolinone showed the significant effect on hydroxyl radical scavenger activity. Sulfasalazine showed

a potent inhibitory effect on xanthine / xanthine oxidase system and induced mixed type inhibition, but not a efficient superoxide anion scavenger.