地乌三萜皂苷W1对破骨细胞分化和骨吸收功能的影响

目的探讨地乌三萜皂苷类成分W1对体外破骨细胞分化及骨吸收功能的影响。方法3个不同浓度（0．0625,0．125,0．25μg／ml）的W1与核因子-κB受体活化因子配体（receptor activator of nuclear factor kB ligand,RANKL）诱导的小鼠单核／巨噬细胞RAW264．7共孵育7天,抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色观察TRAP阳性多核细胞的形成,甲苯胺蓝染色及扫描电镜观察骨吸收陷窝的形成,图像分析计算骨吸收陷窝面积百分比;同3个浓度的W1与RANKL诱导的RAW264．7细胞共孵育24小时后收集细胞上清液,放射免疫法检测肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）的含量;甲基噻唑基四唑（methyl thiazolyl tetrazolium,MTT）法检测W1对RAW264．7细胞毒性影响。结果RANKL组诱导RAW264．7细胞形成多量TRAP阳性多核细胞,形成明显的骨吸收陷窝,并诱导TNF-α在RAW264．7细胞上清中异常高表达;与RANKL组相比,3个浓度的W1均能显著减少TRAP阳性细胞数（P〈0．01）,显著减少骨吸收陷窝面积（P〈0．01,P〈0．001,P〈0．001）,明显降低TNF-α的表达量（P〈0．05,P〈0．01,P〈0．01）,且未观察到对RAW264．7的细胞毒性。结论W1对由RANKL诱导的体外破骨细胞分化和骨吸收功能有一定的负调节作用。     

Inhibition of Osteoclast Differentiation by Ginsenoside Rg3 in RAW264.7 Cells via RANKL,JNK and p38 MAPK Pathways Through a Modulation of Cathepsin K: An In Silico and In Vitro Study

Various studies have demonstrated that overexpression of cathepsin K (Cat‐K) causes excessive bone loss, which ultimately leads to a variety of bone diseases including osteoporosis. Therefore, inhibition of Cat‐K signifies a potential therapeutic target in osteoporosis treatment. Ginsenoside Rg3 is one of the most promising compound of Panax ginseng Meyer (P. ginseng) with numerous biological activities. Thus, in recent study the inhibitory effect of Rg3 isolated from P. ginseng was investigated in order to impede the osteoclast activity by an in silico approach followed by in vitro study validation using RAW264.7 cells through the investigation of different biological activity prediction such as absorption distribution metabolism and excretion (ADMET) properties against Cat‐K protein. The docking results of our study showed that Rg3 is a non‐toxic compound and may act as a drug‐like molecule. Additionally, the molecular interaction of Rg3 with the active residues of Cat‐K markedly describes its inhibitory effects on osteoclastogenesis. Findings of the present study exhibited that Rg3 significantly reduced receptor activator of nuclear factor kappa B ligand (RANKL)‐induced tartrate‐resistant acid phosphatase (TRAP) activity, pit formation (actin rings), and TRAP‐positive multinucleated cells development in RAW264.7 cells. Furthermore, Rg3 dose‐dependently reduced the mRNA expression levels of osteoclast‐specific markers such as RANK, TRAP, and Cat‐K induced by RANKL through the down regulation of p38, extracellular signal‐regulated kinase, and c‐Jun N‐terminal kinase (JNK) pathways. In conclusion, in silico docking study and in vitro validation together suggested that Rg3 inhibits osteoclastogenesis and reduces bone resorption through the inhibition of Cat‐K. Therefore, Rg3 might be a useful therapeutic agent for the treatment of osteoporosis and proper bone formation. Copyright © 2015 John Wiley & Sons, Ltd.     

右归饮对体外培养破骨细胞分化与功能的影响

目的：探讨右归饮对体外培养破骨细胞的分化、凋亡及骨吸收活性的影响。方法：分离培养大鼠破骨细胞,分别以空白对照血清、地塞米松＋对照血清、地塞米松＋右归饮血清的培养液来培养破骨细胞,通过抗酒石酸酸性磷酸酶（TRAP）染色对TRAP阳性多核破骨细胞计数;采用吖啶橙荧光染色法计数破骨细胞的凋亡率;用RT-PCR测定ATPase a3基因相对表达变化分析破骨细胞骨吸收活性。结果：与空白对照血清组和对照血清＋地塞米松组相比,地塞米松＋右归饮血清组TRAP染色阳性的破骨细胞数量显著减少（分别为P〈0.01,P〈0.05）,破骨细胞的凋亡率明显增加（P〈0.01）,ATPase a3基因表达明显减少（P〈0.01）。结论：右归饮可抑制破骨细胞的形成和分化及骨吸收活性,促进破骨细胞的凋亡,从而发挥其抗激素性股骨头坏死的作用。     

Stimulative effects of Ulmus davidiana Planch (Ulmaceae) on osteoblastic MC3T3-E1 cells

Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions. To treat rheumatoid arthritis (RA), a herbal medicine, Ulmus davidiana Planch (Ulmaceae) extract (UD) is being used in traditional oriental medicine. The effect of UD on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. UD dose-dependently increased DNA synthesis (significant at 5-20 microg/ml). UD increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (5-20 microg/ml). Antiestrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1, which was induced by UD. UD at concentrations ranged from 30 to 100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that UD directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and UD is effective for bone anti-resorptive action in bone cells.     

黑骨藤抑制破骨细胞分化及骨吸收能力的活性部位研究

目的:探究黑骨藤对破骨细胞分化的影响并确定其活性部位。方法:采用维生素D3(VD3)诱导兔骨髓细胞法获得破骨细胞,以形态学观察、苏木素和伊红(HE)染色、抗酒石酸酸性磷酸酶(TRAP)染色鉴定破骨细胞,然后通过测定黑骨藤各极性部位低、中、高剂量组(1,10,100 mg·L-1)对破骨细胞TRAP酶活力,TRAP+细胞计数及骨吸收陷窝面积的影响来确定活性部位。结果:黑骨藤水饱和正丁醇层的高剂量组及乙酸乙酯层的中、高剂量组均对骨髓细胞向破骨细胞分化具有显著的抑制作用(P<0.01),并具有剂量依赖性。在抑制破骨细胞骨吸收能力实验中,黑骨藤乙酸乙酯层各剂量组均有显著抑制骨吸收陷窝面积的作用(P<0.01),而正丁醇层中、高剂量组具有显著抑制作用(P<0.01),均呈剂量依赖性。结论:黑骨藤抑制骨髓细胞向破骨细胞分化及骨吸收能力的主要活性部位是乙酸乙酯部位。     

6,7,4′‐Trihydroxyflavone inhibits osteoclast formation and bone resorption in vitro and in vivo

The balance between the osteoblasts and the osteoclasts is important for the maintenance of the skeleton of the human body. The osteoclasts absorb bone after differentiated into polymorphonuclear cells by the fusion of monocytes/macrophages. We have found that 6,7,4′‐Trihydroxyflavone (THF), a compound from the heartwood of Dalbergia Odorifera inhibits receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast differentiation, actin ring formation, and bone resorption in RAW 264.7 cells and bone marrow macrophage. THF significantly inhibited the c‐Jun‐N‐terminal kinase signaling pathway without affecting extracellular signal‐regulated kinase, p38, and AKT signaling. Moreover, THF inhibited the expression of c‐Fos, nuclear factor‐activated T cells cytoplasm 1, cathepsin K, and c‐src by RANKL. We used a lipopolysaccharide (LPS)‐induced bone loss model in mice. Consequently, bone volume per tissue volume, trabecular number's reduction was recovered in THF‐treated mice, and trabecular separation's augmentation was also attenuated by THF administration. In summary, THF inhibits RANKL‐induced osteoclast differentiation by MAPK signaling pathway and inhibits bone resorption by destroying the actin ring in mature osteoclasts. THF also prevented LPS‐induced bone loss in a mice model. Thus, THF may be useful in the treatment of bone diseases associated with excessive osteoclast differentiation and bone resorption.     

Suppressive effects of a water extract of Ulmus davidiana Planch (Ulmaceae) on collagen-induced arthritis in mice

Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone resorption. Therefore, this study was undertaken to address (1) whether the water extract of the bark of Ulmus davidiana Planch (Ulmaceae) (UD) can modulate the expression of inducible inflammatory cytokines in mice; (2) in order to assess the therapeutic effects of UD in collagen-induced arthritis (CIA) in mice. DBA/1 mice were immunized with bovine type II collagen. After a second collagen immunization, mice were treated with UD orally at 100mg/kg once a day for 3 weeks. Paws were evaluated macroscopically for redness, swelling and deformities. The levels of TNF-alpha and IL-1beta in the ankle were examined. The severity of arthritis within the knee joints was evaluated by histological assessment of cartilage destruction and pannus formation. Administration of UD significantly suppressed the progression of CIA and inhibited the production of TNF-alpha and IL-1beta in the paws. The erosion of cartilage was dramatically reduced in mouse knees after treatment with UD. In the serum of UD-treated mice, the levels of IL-4 and IL-10, anti-inflammatory cytokines, were increased. From the results, it was concluded that administration of UD has therapeutic effects on CIA including protection of cartilage and RA for a potential therapy.     

刺老苞根皮黄酮类化合物对破骨细胞分化的影响

目的：研究刺老苞根皮黄酮类化合物对体外破骨细胞分化的影响。方法：建立由骨保护素配体（RANKL）和巨噬细胞集落刺激因子（M-CSF）共同细胞因子的小鼠破骨细胞骨髓诱导体系,将不同浓度的刺老苞根皮黄酮类化合物作用于破骨细胞。受试细胞分为对照组、刺老苞根皮黄酮类化合物低剂量组（10-8mol·L^-1）、刺老苞根皮黄酮类化合物中剂量组（10^-7mol·L^-1）和刺老苞根皮黄酮类化合物高剂量组（10^-6mol·L^-1）,并设立空白对照组。7天后取细胞玻片进行抗酒石酸酸性磷酸酶（TRAP）染色,观察破骨细胞并计数;测量抗酒石酸酸性磷酸酶（TRAP）活性以及破骨细胞表面NF—kB活化受体（RAVK）mRNA表达量。结果：诱导培养的破骨细胞形态特征明显;刺老苞根皮黄酮类化合物中、高剂量组在细胞数量、TRAP活性上与对照组相比有明显统计学差异（P〈0．05）;刺老苞根皮黄酮类化合物各剂量组在（RANK）mRNA表达量上与对照组相比有明显统计学差异（P〈0．05）,且呈量效关系。结论：刺老苞根皮黄酮类化合物可以抑制体外培养的破骨细胞分化。     

骨稳态中成骨细胞与破骨细胞的阴阳属性

从成骨细胞和破骨细胞来源的同一性,其间存在的生、克、制、化关系,阳性、阴性调控因子,阴阳属性的相对性等方面,阐释骨稳态中成骨细胞、破骨细胞的阴阳属性。认为成骨细胞和破骨细胞在骨稳态内是典型的阴阳关系,成骨细胞及其主导的骨生成属于"阴",破骨细胞及其主导的骨吸收属于"阳",二者动态平衡发挥维持骨稳态的作用。     

Herbal formulation, Yukmi-jihang-tang-Jahage, regulates bone resorption by inhibition of phosphorylation mediated by tyrosine kinase Src and cyclooxygenase expression

Anti-bone resorption properties of the Korean herbal medicine, Yukmi-jihang-tang (YJ), which is comprised of seven herbs such as Rehmannia glutinosa Libosch, Dioscorea japonica THUNB, Cornus officinalis SIEB et. ZUCC, Smilax glabra ROXB, Paeonia suffruticosa ANDR, Alisma platago-aquatica var. orientale SAMUELS and Hominis placenta, were investigated. Cyclooxygenase-2 (COX-2) and tyrosine kinase involve on prostaglandin E2 (PGE2) production in mouse calvarial osteoblasts stimulated by cytokine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-6 (IL-6). IL-1beta and IL-6 and to a lesser extent TNF-alpha, enhanced COX-2 mRNA levels in calvarial osteoblasts. TGF-beta, YJ (100microg/ml) and their combinations of YJ+TGF-beta reduced the COX-2 mRNA level, PGE2 biosynthesis and bone resorption induced by IL-1beta, TNF-alpha, IL-6 or their combination. Finally, YJ inhibits in vitro and in vivo bone resorption by inhibition of phosphorylation of peptide substrates. The parathyroid hormone-induced bone resorption in mouse fetal long bone cultures was inhibited with an IC(50) of 16microg/ml. YJ dose-dependently reduced the hypercalcemia induced in mice by IL-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. These results indicate that the synergy between IL-beta, TNF-alpha, IL-6 on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in mouse calvarial osteoblasts. Thus, YJ as a possible Src family kinase inhibitor may be useful for the treatment of diseases associated with elevated bone loss. This result also suggested that the YJ extracts is effective for bone resorptive action in bone cells.     

结合状态伊班膦酸盐对破骨细胞骨吸收作用的影响

 目的 观察伊班膦酸盐（ibandronate,Iba）与羟基磷灰石（hydroxyapatite,HA）结合后对破骨细胞骨吸收作用的影响。方法 将Iba与HA复合成伊班膦酸盐-羟基磷灰石（Iba-HA）作为实验组,未结合Iba的HA作为对照组。分离、培养、纯化大鼠破骨细胞,Giemsa染色和抗酒石酸酸性磷酸酶(TRAP)染色显示破骨细胞及其细胞核的形态和数目。用两组材料与破骨细胞复合培养,透射电镜观察细胞内结构变化,SNAFL染色测定破骨细胞内氢离子浓度,骨吸收陷窝面积测定并辅以上清液内胶原C端肽片段（CTx）的浓度测定观察破骨细胞骨吸收功能。结果 Gimsa染色标本可见多核的破骨细胞、细胞核染成蓝紫色,TRAP染色标本可见TRAP酶活性的部位被染成红色,分布于破骨细胞胞浆内。透射电镜观察发现,破骨细胞与Iba-HA复合培养后,较之与HA复合培养的破骨细胞生物活性下降。SNAFL染色显示Iba-HA组破骨细胞荧光强度小于HA组,统计学有差异（P<0.05）;骨吸收检测时面积显示,Iba-HA组小于HA组,统计学有差异（P<0.05）,上清液CTx浓度,Iba-HA组小于HA组,统计学有差异（P<0.05）。结论 与ＨＡ处于结合状态的Iba对破骨细胞的骨吸收作用仍有显著影响。     

Protective action of aqueous black tea (Camellia sinensis) extract (BTE) against ovariectomy‐induced oxidative stress of mononuclear cells and its associated progression of bone loss

The protective action of aqueous black tea extract (BTE) against ovariectomy‐induced oxidative stress of mononuclear cells and its associated progression of bone loss was demonstrated in this study. Eighteen female adult 6‐month‐old Wistar albino rats were divided into three groups: sham‐control (A), bilaterally ovariectomized (B) and bilaterally ovariectomized + BTE supplemented (C). Studies included the measurement of oxidative (nitric oxide, lipid peroxidation) and antioxidative (superoxide dismutase, catalase) markers, inflammatory cytokines (IL‐6, TNF‐α), osteoclast differentiation factor (RANKL) and bone resorption markers (tartrate‐resistant acid phosphatase and hydroxyproline). Also quantitative histomorphometry and histological studies were undertaken. The bone breaking force was measured. The results indicate that BTE was effective in preserving and restoring skeletal health by reducing the number of active osteoclasts. Such changes with BTE supplementation were steadily linked with the reduced oxidative stress of mononuclear cells, serum levels of bone resorbing cytokines, osteoclast differentiation factor and resorption markers. The results of the bone breaking force, histological and histomorphometric analyses further supported the hypothesis. This study suggests that BTE has both protective and restorative actions against ovariectomy‐induced mononuclear cell oxidative stress and associated bone loss. Copyright © 2009 John Wiley & Sons, Ltd.     

大黄素对兔主动脉平滑肌细胞CatK,CatDmRNA表达的影响

目的:探讨大黄素对兔主动脉平滑肌细胞(CCC-SMC-1)组织中蛋白酶K(Cat K),组织蛋白酶D(Cat D)mRNA表达的影响。方法:以2×104/m L密度接种细胞,实验分5个实验组,大黄素0.01,0.05,0.1,0.15,0.2,0.25 mmol·L-1组,对照组为培养基空白组,培养24 h,每组设5个复孔。MTT法观察大黄素对兔主动脉平滑肌细胞增殖的抑制作用,PCR法检测大黄素对兔主动脉平滑肌细胞Cat K,Cat D mRNA的表达。结果:MTT法显示大黄素在0.05~0.25 mmol·L-1浓度对CCCSMC-1细胞增殖有抑制作用,作用24 h后的半数抑制浓度(IC50)为0.10 mmol·L-1,且IC50随作用时间的延长显著前移。PCR法显示,不同浓度的大黄素作用于兔主动脉平滑肌细胞24 h后,与正常组细胞相比,Cat K,Cat D mRNA表达上调(P0.05)。结论:大黄素能抑制CCC-SMC-1细胞的增殖,其机制可能与上调Cat K,Cat D mRNA表达有关。     

Inhibitory effect of a Korean traditional medicine, Honghwain-Jahage (water extracts of Carthamus tinctorius L. seed and Hominis placenta) on interleukin-1-mediated bone resorption.

A Korean herbal formulation, Honghwain-Jahage (HJ), which is comprised of a herb of Carthamus tinctorius L. seed and Hominis Placenta, was investigated for inhibiting effects on IL-1 beta-stimulated bone resorption in the fetal mouse bone culture system. Results of in vitro cytotoxicities showed that HJ extracts have no any cytotoxicities in concentrations of 1-200 microg/ml on the cultured osteoblast cells derived from mouse calvarial bone explants. Cell viability was not significantly affected by treatment with the indicated concentration of the extracts. The HJ extracts were shown to have inhibitory effects against the synthesis of PGE(2). We also examined the effect of the pretreatment with various concentrations of the HJ extracts then treated by the PGE(2)-induction agents. Pretreatment of the HJ extracts for 1 h, which by itself had little effect on cell survival, reduced the synthesis of PGE(2). Furthermore, the HJ extracts were shown to have protective effects against plasminogen dependent fibrinolysis induced by IL-1 beta. Pretreatment of the HJ extracts for 1 h did not enhance the plasminogen dependent fibrinolysis. Finally, co-treatment of HJ with calcitonin showed significant inhibitory activity on the IL-1 beta-stimulated bone resorption. From these results, it was found that HJ extracts inhibited IL-1 beta-induced bone resorption.     

补肾壮骨中药对大鼠去卵巢后早期白细胞介素6介导的骨髓源性破骨细胞形成的影响

目的 观察补肾壮骨中药对大鼠去卵巢后早期骨髓源性破骨细胞形成能力和骨髓细胞表达白细胞介素6(IL-6)、IL-6受体(IL-6R)、gp130基因的影响。方法 健康3月龄雌性SD大鼠72只,其中24只为假手术对照组;48只去卵巢,随机分为去卵巢组和中药组,每组24只。分别于去卵巢后2、4、6、12周每组各取6只大鼠骨髓细胞作细胞培养和提取RNA,培养第6天分别作瑞氏-吉姆萨染色和抗酒石酸酸性磷酸酶(TRAP)染色,骨髓细胞TRIZOL提取总RNA。结果 去卵巢后2周,去卵巢组破骨细胞形成数即多于对照组(P<0．05),骨髓细胞IL-6和IL-6R基因表达均显著升高(P<0．05,P<0．01),第4～6周。上述改变达高峰,并持续至第12周;从4～12周,上述各指标中药组均明显低于去卵巢组(P<0．05,P<0．01)。以上各组全程未见gp130基因表达水平有明显变化。结论 本方所用补肾壮骨中药可以抑制大鼠去卵巢后骨髓源性破骨细胞的生成,这一效应至少部分与其抑制骨髓细胞在去卵巢后过度表达IL-6、IL-6R有关。     

湖北海棠总黄酮对成骨细胞增殖分化及破骨细胞活性的影响

目的：探讨湖北海棠总黄酮及其主要成分根皮苷对大鼠成骨细胞增殖分化及破骨细胞活性的影响。方法：取新生大鼠颅骨,用改良组织块消化法分离成骨细胞。MTT法检测药物对成骨细胞增殖的影响,对硝基苯二钠基质动力学法测定细胞内碱性磷酸酶（AKP）的活性。分离培养大鼠破骨细胞。加药共培后检测抗酒石酸酸性磷酸酶（TRACP）活性,检查骨吸收陷窝变化。结果：湖北海棠总黄酮能明显促进成骨细胞增殖;提高成骨细胞内碱性磷酸酶活性。湖北海棠主要成分根皮苷能提高成骨细胞内碱性磷酸酶活性;降低抗酒石酸酸性磷酸酶活性,减少骨吸收陷窝数目,与对照组比较差异有统计学意义。结论：湖北海棠总黄酮能促进成骨细胞的增殖和分化,其主要成分根皮苷能促进成骨细胞的分化,抑制破骨细胞的活性和嗜骨活性。     

Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation

Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant‐derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin‐treated murine osteoblastic MC3T3‐E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone‐resorption activity through slightly decreased the phosphorylation of extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, and inhibitor of nuclear factor kappa Bα; however, the phosphorylation of Akt and p38 slightly increased in bone marrow‐derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T‐cell cytoplasmic‐1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases. Copyright © 2015 John Wiley & Sons, Ltd.     

Inhibitory effect of Carthamus tinctorius L. seed extracts on bone resorption mediated by tyrosine kinase,COX-2 (cyclooxygenase) and PG (prostaglandin) E2

Anti-bone resorption properties of the Korean herbal formulation, Honghwain (HHI; Carthamus tinctorius L. seed) was biochemically investigated. On processing bone metabolism, PGE2 accelerated production of IL-1beta in fetal mouse osteoblast and stimulated physiological activation substance, IL-1beta. The novel class of Src tyrosine kinase inhibitors, Herbimycin A (HERB) and HHI reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta, TNF-alpha and IL-6. HHI inhibited in vitro and in vivo bone resorption by inhibition of phosphorylation of peptide substrates. HHI dose-dependently reduced the hypercalcemia induced in mice by IL-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. These results indicate that the synergy between IL-beta, TNF-alpha, IL-6 on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase (s) are involved in the signal transduction of COX-2 in mouse calvarial osteoblasts. Thus, HHI as a possible Src family kinase inhibitor may be useful for the treatment of diseases associated with elevated bone loss.     

Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula

AIM OF THE STUDY: Er Xian Decoction (EXD), a traditional Chinese medicine formula, has long been used for the treatment of osteoporosis and menopausal syndrome in China. The present study was designed to investigate the antiosteoporotic constituents of EXD, and evaluate their antiosteoporotic effects in ovariectomized rats. MATERIALS AND METHODS: Osteoblasts in neonatal calvaria cultures and osteoclasts derived from rat marrow cells were used to bioactivity-guided screen the active constituents. The proliferation of osteoblast was assayed by MTT methods. The activity of ALP and TRAP was measured by p- nitrophenyl sodium phosphate assay. The antiosteoporotic effects of icariin (1), anemarsaponin B II (8) and berberine (6) were verified by using OVX rats model. The bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry using the small animal scan mode. The undecalcified longitudinal proximal tibial metaphysical (PTM) sections were cut and stained for the bone histomorphometric analysis. RESULTS: Bioactivity-guided fractionation has led to the successful isolation of antiosteoporotic constituents, i.e., icariin (1), icariside I (2), baohuoside I (3), mangiferin (4), neomangiferin (5), berberine (6), anemarsaponin B (7), anemarsaponin BII (8), anemarsaponin C (9), anemarrhenasaponin I (10), rubiadin-1-methyl ether (11) and obaculactone (12) from EXD. Further study showed that icariin (1), anemarsaponin BII (8) and berberine (6) increased the BMD in ovariectomized rats, and icariin (1) not only increased the bone formation, but also inhibited bone resorption; anemarsaponin BII (8) mainly increased bone formation and berberine (6) only inhibited the bone resorption in ovariectomized rats. CONCLUSION: Our findings demonstrate that multiple ingredients are responsible for antiosteoporotic activity in traditional Chinese medicine formula Er-Xian decoction.