菱叶山蚂蝗抗骨质疏松物质基础及作用机制研究
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摘要
菱叶山蚂蝗是豆科Leguminosae长柄山蚂蝗属Podocarpium植物长柄山蚂蝗Podocarpium podocarpum (DC.) Yang et Huang的根、叶或全草，具有发表散寒、止血功能，主治风寒感冒、急性黄疸型肝炎、风湿痹痛、跌打损伤、刀伤等。药理活性筛选方面，课题组前期研究表明菱叶山蚂蝗醇提物具有镇痛、解热和抗炎作用；化学成分研究方面，有文献报道从其变种尖叶长柄山蚂蝗Podocarpiumpodocarpum (DC.) Yang et Huang var. oxyphyllum (DC.) Yang et Huang中分离得到11个化合物（包括6个黄酮、2个三萜和1个甾体），还没有关于菱叶山蚂蝗化学成分分离及成分活性筛选方面的文献报道。
骨质疏松是以骨量减少、骨的微观结构退化为特征，致使骨的脆性增加以及易于发生骨折的一种全身性骨骼疾病。化学药治疗一般采用联合用药，其毒副作用大，疗程长，从天然药用植物中寻找副作用小治疗骨质疏松疾病的有效提取物、有效部位及单体化合物已成为研究热点。目前抗骨质疏松作用的研究多从补肾类中药和富含黄酮的豆科植物着手，天然产物中具有抗骨质疏松活性的化合物结构类型主要包括黄酮类、甾体类、苯丙素类、生物碱类等，其中黄酮类化合物抗骨质疏松报道较多。菱叶山蚂蝗是豆科蝶形花亚科植物，富含黄酮类化合物，并且菱叶山蚂蝗传统可用于治疗风湿痹痛、跌打损伤和刀伤，提示菱叶山蚂蝗可能具有抗骨质疏松的作用。课题组前期针对菱叶山蚂蝗进行了体外抗骨质疏松作用初步筛选，结果显示粗提物具有良好的体外促成骨细胞增殖作用。
基于以上原因，本论文对菱叶山蚂蝗全草提取物进行了体内抗骨质疏松作用研究，并对全草进行了系统的的化学成分分离鉴定，对分离得到的化合物进行体外和体内抗骨质疏松作用筛选，阐明菱叶山蚂蝗抗骨质疏松的物质基础。采用分子对接技术筛选化合物抗骨质疏松的作用靶点，围绕靶点蛋白进行菱叶山蚂蝗抗骨质疏松作用机制探讨。
一、菱叶山蚂蝗体内抗骨质疏松作用筛选
本论文采用大鼠去卵巢致骨质疏松模型(OVX)对菱叶山蚂蝗乙醇提物（100mg/kg～300mg/kg）进行预防骨质疏松症药效学研究。取大鼠右侧股骨测定总骨密度(t-BMD)，采用显微CT观察股骨的骨组织尤其是骨小梁形态并比较相关计量学参数，以确定有效性；取尿液测定钙(Ca)、磷(P)和肌酐(Cr)含量观察骨Ca、P吸收和流失情况；取血清测定骨形成相关指标ALP和骨钙素(BGP)含量、骨吸收相关指标组织蛋白酶K(Cathepsin K)、TRAP和脱氧胶原吡啶交联(DPD)含量，探索药物与骨形成和骨吸收的联系；取血清测定氧化应激相关酶(谷胱甘肽过氧化物酶GSH-Px、超氧化物歧化酶SOD、一氧化氮合酶NOS、丙二醛MDA)的活力，探讨药物可能的作用机制。结果显示，醇提物能显著提高去卵巢大鼠t-BMD，micro-CT直观反映出醇提物对去卵巢造成的大鼠股骨骨小梁厚度和数量减少及连接间隙增大有显著的改善；血清生化指标测定的结果表明醇提物对骨吸收指标有显著抑制作用，对骨形成指标无明显影响，提示醇提物很大程度上是通过抑制骨吸收起到抗骨质疏松作用的；另外，醇提物能够提高血清中GSH-Px、NOS和SOD酶活力，提示其抗骨质疏松作用可能与氧化应激机制有关。
二、菱叶山蚂蝗化学成分研究
本论文应用萃取、硅胶、凝胶、反相柱层析和制备HPLC等多种分离方法和分离材料对菱叶山蚂蝗全草乙醇提取物进行了系统的化学成分分离，共计分离鉴定73个化合物，其中2个新化合物(1个双黄酮和1个苯丙素苷酯类化合物)。化合物类型包括：黄酮类(DP1～25)(11个异黄酮、9个异戊烯基黄酮)，苯丙素类(DP26～33)，三萜皂苷和单萜(DP34～40)，甾体(DP41～42)，生物碱类(DP43～46)，酚酸类(DP47～56)，以及其它类化合物(DP57-73)。从正丁醇部位分得了约10g的山萘苷单体化合物，提示山萘苷是菱叶山蚂蝗主要的化学成分，其大量存在可能是菱叶山蚂蝗抗骨质疏松的主要药效物质。因此，建立了菱叶山蚂蝗中山萘苷HPLC含量测定方法，并对菱叶山蚂蝗全草、根、茎、叶及其变种尖叶长柄山蚂蝗全草中山萘苷的含量进行了测定。结果表明，菱叶山蚂蝗中山萘苷主要分布在叶中，其含量可达1.16±0.09%；根和茎中不含山萘苷。变种尖叶长柄山蚂蝗全草中不含山萘苷。提示菱叶山蚂蝗植物变种与原种在化学成分及其含量方面差异较大。
三、菱叶山蚂蝗化学成分体外抗骨质疏松作用研究
本论文对分离得到的18个黄酮类(DP1～18)化合物进行了体外成骨细胞和破骨细胞活性的筛选。采用新生大鼠颅盖骨成骨细胞筛选化合物对成骨细胞增殖、碱性磷酸酶(ALP)活性和骨结节诱导能力的影响；采用成骨细胞和骨髓单核细胞经1,25-(OH)2-VD3和地塞米松共同诱导得到的成熟破骨细胞筛选化合物对破骨细胞酒石酸酸性磷酸酶（TRAP）活性的抑制作用。结果显示，12个黄酮类化合物同时具有体外促成骨细胞增殖、提高成骨细胞ALP活性、促进骨结节诱导能力以及抑制破骨细胞TRAP酶的活性。其中2个化合物（DP3和DP16）均不具有以上活性。
四、菱叶山蚂蝗中山萘苷体内抗骨质疏松作用筛选
鉴于菱叶山蚂蝗全草具有抗骨质疏作用，全草中山萘苷含量高于1%，山萘苷体外又具有成骨和破骨细胞活性，因此采用大鼠去卵巢致骨质疏松模型(OVX)对山萘苷(8～32mg/kg)单体化合物进行预防骨质疏松症药效学研究。结果显示，山萘苷高、中剂量均能显著提高去卵巢大鼠t-BMD，micro-CT显示山萘苷对去卵巢造成的大鼠股骨骨小梁厚度和数量减少及连接间隙增大有显著的改善；血清生化指标测定的结果表明山萘苷对骨形成指标ALP有提高作用，对骨吸收指标均有显著抑制作用，提示山萘苷兼有促进骨形成和抑制骨吸收的作用，但后者占主导地位；另外，山萘苷高剂量组能够提高血清中NOS和SOD酶活力，提示其抗骨质疏松作用可能与氧化应激机制有关。
五、山萘苷抗骨质疏松作用机制研究
采用分子对接法寻找山萘苷抗骨质疏松的作用靶点。通过SYBYL软件构建山萘苷3D结构并进行结构分子力学优化，将优化后的结构文件上传至药物靶标数据库搜寻潜在的药物靶点，将与骨质疏松有关的靶点按照打分高低筛选出来后，再应用DOCK软件进行正向对接验证，最终确定药靶。分子对接结果显示，山萘苷作用靶点是与骨吸收直接相关的由破骨细胞分泌的关键酶－Cathepsin K。紧接着，采用体外破骨细胞验证山萘苷对Cathepsin K的抑制作用。结果显示，山萘苷对破骨细胞Cathepsin K有显著的抑制作用，抑制率达50%。大鼠去卵巢动物试验也显示山萘苷对去卵巢大鼠Cathepsin K的升高有明显抑制作用。以上初步证实山萘苷抗骨质疏松的作用靶点可能是Cathepsin K。检索文献，目前Cathepsin K参与骨质疏松的信号通路有NF-κB通路、MAPK通路（包括ERK1/2、P38和JNK）、PI3K/Akt和CN/NFATC通路。采用Western-blot技术对p-ERK、p-JNK、p-P38和p-IκB等关键蛋白酶进行表达，结果显示山萘苷对p-JNK和p-IκB有明显的下调作用，说明山萘苷主要通过NF-κB和MAPK/JNK这两条信号通路发挥作用，抑制破骨前体细胞的分化成熟，从而抑制骨吸收起到抗骨质疏松作用。
综上所述，菱叶山蚂蝗具有抗骨质疏松作用，其物质基础是黄酮类化合物群，主要药效物质是山萘苷，作用靶点可能是组织蛋白酶K，其抗骨质疏松作用与NF-κB和MAPK/JNK这两条信号通路相关。
Podocarpium podocarpum (DC.) Yang et Huang belongs to genus Podocarpium(Leguminosae). The leaves and roots have been taken to acute icteric hepatitis,rheumatism, bone bruises and knife trauma in Traditional Chinese Medicine. Its ethanolextract has been proved possess significant analgesic, antipyretic and anti-inflammatoryactivities in our previous study. So far, few phytochemical investigations have beencarried out on this species, only a total of6flavonoids,2triterpenoids,1steroidoid and2other compounds have been isolated and identified from Podocarpium podocarpum(DC.) Yang et Huang var. oxyphyllum (DC.) Yang et Huang, a variety of Podocarpiumpodocarpum.
Osteoporosis is a chronic, progressive disease of the skeleton characterized by bonefragility due to a reduction in bone mass and possibly alteration in bone architecturewhich leads to a propensity to fracture with minimum trauma. At present, the chemicaldrugs, which are clinically used as effective medications, are associated with longtreated time and numerous side effects. Therefore, the natural plant extracts, effectivefrations and compounds, with few side effects, attracted our attention. The plants blongsto the family Leguminosae or had the effect of strengthening the sexual behavior wereproved effective in anti-osteoporosis. The natural products, including flavonoids,steroidal, phenylpropanoids and alkaloids, have been proved possess anti-osteoporosisactivity, especially flavonoids. As Podocarpium podocarpum is a species of familyPapilionoideae (Leguminosae), a big sub-family rich in flavonoids, and it has been usedin treatment of rheumatism, bone bruises and knife trauma in traditional medical uses,both of the two implied that this plant probably possess anti-osteoporosis activity. Inaddition, our previous in vitro experiments also proved that the ethanol extract ofPodocarpium podocarpum showed potent stimulated effects on osteoblasticproliferation.
This dissertation is therefore conducted to investigate the anti-osteoporosis activityof Podocarpium podocarpum in vivo, followed by the isolation of the chemicalconstituents from its extract. In order to determine the chemical responsible for theanti-osteoporosis activity of this species, both in vivo and in vitro experiments wereused to test the anti-osteoporosis activity of the isolated compounds. In addition, themolecular docking technique was applied to discover the anti-osteoporotic targets ofkaempferitrin, so as to discuss the molecular mechanism of this action.
1In vivo antiosteoporotic evaluation of Podocarpium podocarpum extracts
The anti-osteoporotic effect of the ethanol extract of Podocarpium podocarpum(100～300mg/kg) in ovariectomized (OVX) rats model of osteoporosis wasinvestigated. The right femur of rats was used to detect the total bone mineral density(BMD) and bone tissue morphology, especially the tissue of trabecular and relatedparameters, which were determined by micro-CT; the urine was taken to evaluate thecontent of Ca, P and Cr; the serum was employed to determined the bone formationindicators including bone gla-protein (BGP) and alkaline phosphatase (ALP), the boneresorption indicators including cathepsin K, tartrate-resistant acid phosphatase (TRAP)and deoxypyridinoline (DPD); in addition, markers of oxidative stress, glutathioneperoxidase (GSH-Px), super oxidase dimutase (SOD), nitric oxide synthase (NOS) andmalondialdehyde (MDA) were also test to explore the possible mechanism of action.The results showed that the ethanol extract of Podocarpium podocarpum cansignificantly promote the BMD in OVX rats; the bone tissue of trabecular, includingtrabecular thickness (Tb. Th), trabecular number (Tb. N), connectivity density (CD) andtrabecular separation (Tb. Sp) were also improved; the findings assessed on the basis ofbiochemical, bone formation and resorption indicators strongly suggested that theethanol extract of Podocarpium podocarpum had a definite antiosteoporotic effectthrough inhibition of bone resorption. Furthermore, the activity of GSH-Px, SOD andNOS were also stimulate, which revealed that the anti-osteoporotic activity of ethanolextract of Podocarpium podocarpum probably related with oxidative stress.
2Chemical constituents of Podocarpium podocarpum
A total of73chemical compounds have been isolated from the ethanol extracts ofwhole plant of Podocarpium podocarpum whole plant by using silica gel columnchromatography, Sephadex LH-20and reversed-phase HPLC. The structures of thesecompounds, including flavonoids (DP1~25), phenylpropanoids (DP26~33),triterpenoids (DP34~40), steroidals (DP41~42), alkaloids (DP43~46), phenolic acids(DP47~56), and others (DP57~73), were elucidated on the basis of chemical andspectroscopic analyses. A total of about10g kaempferitrin was isolated from then-butanol fraction, implied that this compound was the main constituents ofPodocarpium podocarpum, and probably the main chemical responsible for theanti-osteoporotic activity of the plant. Therefore, a HPLC method for the determination of kaempferitrin was established. Its content in different part of Podocarpiumpodocarpum, including leaf, root and whole plant, as well as its variety, Podocarpiumpodocarpum var. oxyphyllum, have been detected and compared. The results showedthat the content of kaempferitrin in leaves was1.16±0.09%; wheras the root and stemas well as its variety were not detected. It is implied that the plant is differs markedlyfrom its variety in chemical constituents and their content.
3In vitro antiosteoporotic evaluation of the compounds isolated from Podocarpiumpodocarpum
Atotal of18flavonoids (DP1～18) were evaluated for their proliferative effects onosteoblasts derived from neonatal rat calvaria and inhibitory effects on multinucleatedosteoclasts from rat marrow cells. The stimulated effects on osteoblastic proliferation,ALP activity and calcium deposition, as well as the inhibited effects on osteoclasticTRAP activity were evaluated. The osteoclast was induced by1,25-(OH)2-VD3anddexamethasone by using both osteoblast and bone marrow cells. The results showed that12flavonoids exhibited significant effects both on osteoblasts and osteoclasts, whereasthe two compounds, DP3and DP16, exhibited no activity. In addition, nocorresponding dose-dependent phenomenon was observed.
4In vivo antiosteoporotic evaluation of kaempferitrin
As the whole plant of Podocarpium podocarpum possessed anti-osteoporoticactivity, and the content of kaempferitrin in the leaves of this species was high then1%,as well as the kaempferitrin showed proliferative effects on osteoblasts and inhibitoryeffects on multinucleated osteoclasts in vitro, the anti-osteoporotic activity ofkaempferitrin (8～32mg/kg) in ovariectomized (OVX) rats model of osteoporosis wasinvestigated. The results showed that the high and moderate dosage of kaempferitrin cansignificantly promote the BMD in OVX rats; the bone tissue of trabecular, includingtrabecular thickness (Tb. Th), trabecular number (Tb. N), connectivity density (CD) andtrabecular separation (Tb. Sp) were also significantly improved; the findings assessedon the basis of biochemical, bone formation and resorption indicators stronglysuggested that the kaempferitrin had a definite antiosteoporotic effect both throughstimulation of bone formation and inhibition of bone resorption, but the latter was thedominant mechanism. Furthermore, the activity of SOD and NOS were also stimulate,which revealed that the anti-osteoporotic activity of and kaempferitrin probably related with oxidative stress.
5Molecular mechanism of anti-osteoporotic activity
A molecular docking technique was used to discover the anti-osteoporotic targetsof kaempferitrin. The3D structure of kaempferitrin was constructed with SYBYLsoftware, followed by the energy optimization; then searched for the potential targetsfrom drug target database by upload the structure to internet, recorded the high scoredrug targets which related to osteoporosis disease, using forward docking to validatethose aimed drug targets, then the anti-osteoporotic targets of kaempferitrin wasconfirmed. The cathepsin K, a key enzyme responsible for osteoclast-mediated boneresorption, was identified as the antiosteoporotic targets of this herb by moleculardocking technology. The inhibitory effect on cathepsin K of multinucleated osteoclastsin vitro was investigated. Results showed that the kaempferitrin exhibited significantinhibitory effect on cathepsin K of osteoclasts, the inhibition rate was almost50%. Theprevious in vivo experiment in ovariectomized (OVX) rats model of osteoporosis hadalso proved the inhibition effect of kaempferitrin on cathepsin K in the serum ofrats.Therefore, both in vitro and in vivo valuated the result of molecular docking. So far,the anti-osteoporosis signal pathways involved with cathepsin K were NF-κB, MAPK(ERK1/2, P38and JNK), PI3K/Akt and CN/NFAT. The western-blot was used toexpress the key proteins p-ERK, p-JNK, p-P38and p-IκB. The results showed that theexpression of p-JNK and p-IκB were obviously inhibited by kaempferitrin, whichimplied that the anti-osteoporotic activivity of kaempferitrin is related to the NF-κB andJNK signaling pathways.
Summarily, the whole plant of Podocarpium podocarpum exhibited a definiteantiosteoporotic activity, the chemical responsible for this effect are flavonoidscompounds, and the kaempferitrin is the main constituents. The cathepsin K isidentified as the antiosteoporotic targets of this herb, and the anti-osteoporotic activivityis related to the NF-κB and JNK signaling pathways.
骨质疏松是以骨量减少、骨的微观结构退化为特征，致使骨的脆性增加以及易于发生骨折的一种全身性骨骼疾病。化学药治疗一般采用联合用药，其毒副作用大，疗程长，从天然药用植物中寻找副作用小治疗骨质疏松疾病的有效提取物、有效部位及单体化合物已成为研究热点。目前抗骨质疏松作用的研究多从补肾类中药和富含黄酮的豆科植物着手，天然产物中具有抗骨质疏松活性的化合物结构类型主要包括黄酮类、甾体类、苯丙素类、生物碱类等，其中黄酮类化合物抗骨质疏松报道较多。菱叶山蚂蝗是豆科蝶形花亚科植物，富含黄酮类化合物，并且菱叶山蚂蝗传统可用于治疗风湿痹痛、跌打损伤和刀伤，提示菱叶山蚂蝗可能具有抗骨质疏松的作用。课题组前期针对菱叶山蚂蝗进行了体外抗骨质疏松作用初步筛选，结果显示粗提物具有良好的体外促成骨细胞增殖作用。
基于以上原因，本论文对菱叶山蚂蝗全草提取物进行了体内抗骨质疏松作用研究，并对全草进行了系统的的化学成分分离鉴定，对分离得到的化合物进行体外和体内抗骨质疏松作用筛选，阐明菱叶山蚂蝗抗骨质疏松的物质基础。采用分子对接技术筛选化合物抗骨质疏松的作用靶点，围绕靶点蛋白进行菱叶山蚂蝗抗骨质疏松作用机制探讨。
一、菱叶山蚂蝗体内抗骨质疏松作用筛选
本论文采用大鼠去卵巢致骨质疏松模型(OVX)对菱叶山蚂蝗乙醇提物（100mg/kg～300mg/kg）进行预防骨质疏松症药效学研究。取大鼠右侧股骨测定总骨密度(t-BMD)，采用显微CT观察股骨的骨组织尤其是骨小梁形态并比较相关计量学参数，以确定有效性；取尿液测定钙(Ca)、磷(P)和肌酐(Cr)含量观察骨Ca、P吸收和流失情况；取血清测定骨形成相关指标ALP和骨钙素(BGP)含量、骨吸收相关指标组织蛋白酶K(Cathepsin K)、TRAP和脱氧胶原吡啶交联(DPD)含量，探索药物与骨形成和骨吸收的联系；取血清测定氧化应激相关酶(谷胱甘肽过氧化物酶GSH-Px、超氧化物歧化酶SOD、一氧化氮合酶NOS、丙二醛MDA)的活力，探讨药物可能的作用机制。结果显示，醇提物能显著提高去卵巢大鼠t-BMD，micro-CT直观反映出醇提物对去卵巢造成的大鼠股骨骨小梁厚度和数量减少及连接间隙增大有显著的改善；血清生化指标测定的结果表明醇提物对骨吸收指标有显著抑制作用，对骨形成指标无明显影响，提示醇提物很大程度上是通过抑制骨吸收起到抗骨质疏松作用的；另外，醇提物能够提高血清中GSH-Px、NOS和SOD酶活力，提示其抗骨质疏松作用可能与氧化应激机制有关。
二、菱叶山蚂蝗化学成分研究
本论文应用萃取、硅胶、凝胶、反相柱层析和制备HPLC等多种分离方法和分离材料对菱叶山蚂蝗全草乙醇提取物进行了系统的化学成分分离，共计分离鉴定73个化合物，其中2个新化合物(1个双黄酮和1个苯丙素苷酯类化合物)。化合物类型包括：黄酮类(DP1～25)(11个异黄酮、9个异戊烯基黄酮)，苯丙素类(DP26～33)，三萜皂苷和单萜(DP34～40)，甾体(DP41～42)，生物碱类(DP43～46)，酚酸类(DP47～56)，以及其它类化合物(DP57-73)。从正丁醇部位分得了约10g的山萘苷单体化合物，提示山萘苷是菱叶山蚂蝗主要的化学成分，其大量存在可能是菱叶山蚂蝗抗骨质疏松的主要药效物质。因此，建立了菱叶山蚂蝗中山萘苷HPLC含量测定方法，并对菱叶山蚂蝗全草、根、茎、叶及其变种尖叶长柄山蚂蝗全草中山萘苷的含量进行了测定。结果表明，菱叶山蚂蝗中山萘苷主要分布在叶中，其含量可达1.16±0.09%；根和茎中不含山萘苷。变种尖叶长柄山蚂蝗全草中不含山萘苷。提示菱叶山蚂蝗植物变种与原种在化学成分及其含量方面差异较大。
三、菱叶山蚂蝗化学成分体外抗骨质疏松作用研究
本论文对分离得到的18个黄酮类(DP1～18)化合物进行了体外成骨细胞和破骨细胞活性的筛选。采用新生大鼠颅盖骨成骨细胞筛选化合物对成骨细胞增殖、碱性磷酸酶(ALP)活性和骨结节诱导能力的影响；采用成骨细胞和骨髓单核细胞经1,25-(OH)2-VD3和地塞米松共同诱导得到的成熟破骨细胞筛选化合物对破骨细胞酒石酸酸性磷酸酶（TRAP）活性的抑制作用。结果显示，12个黄酮类化合物同时具有体外促成骨细胞增殖、提高成骨细胞ALP活性、促进骨结节诱导能力以及抑制破骨细胞TRAP酶的活性。其中2个化合物（DP3和DP16）均不具有以上活性。
四、菱叶山蚂蝗中山萘苷体内抗骨质疏松作用筛选
鉴于菱叶山蚂蝗全草具有抗骨质疏作用，全草中山萘苷含量高于1%，山萘苷体外又具有成骨和破骨细胞活性，因此采用大鼠去卵巢致骨质疏松模型(OVX)对山萘苷(8～32mg/kg)单体化合物进行预防骨质疏松症药效学研究。结果显示，山萘苷高、中剂量均能显著提高去卵巢大鼠t-BMD，micro-CT显示山萘苷对去卵巢造成的大鼠股骨骨小梁厚度和数量减少及连接间隙增大有显著的改善；血清生化指标测定的结果表明山萘苷对骨形成指标ALP有提高作用，对骨吸收指标均有显著抑制作用，提示山萘苷兼有促进骨形成和抑制骨吸收的作用，但后者占主导地位；另外，山萘苷高剂量组能够提高血清中NOS和SOD酶活力，提示其抗骨质疏松作用可能与氧化应激机制有关。
五、山萘苷抗骨质疏松作用机制研究
采用分子对接法寻找山萘苷抗骨质疏松的作用靶点。通过SYBYL软件构建山萘苷3D结构并进行结构分子力学优化，将优化后的结构文件上传至药物靶标数据库搜寻潜在的药物靶点，将与骨质疏松有关的靶点按照打分高低筛选出来后，再应用DOCK软件进行正向对接验证，最终确定药靶。分子对接结果显示，山萘苷作用靶点是与骨吸收直接相关的由破骨细胞分泌的关键酶－Cathepsin K。紧接着，采用体外破骨细胞验证山萘苷对Cathepsin K的抑制作用。结果显示，山萘苷对破骨细胞Cathepsin K有显著的抑制作用，抑制率达50%。大鼠去卵巢动物试验也显示山萘苷对去卵巢大鼠Cathepsin K的升高有明显抑制作用。以上初步证实山萘苷抗骨质疏松的作用靶点可能是Cathepsin K。检索文献，目前Cathepsin K参与骨质疏松的信号通路有NF-κB通路、MAPK通路（包括ERK1/2、P38和JNK）、PI3K/Akt和CN/NFATC通路。采用Western-blot技术对p-ERK、p-JNK、p-P38和p-IκB等关键蛋白酶进行表达，结果显示山萘苷对p-JNK和p-IκB有明显的下调作用，说明山萘苷主要通过NF-κB和MAPK/JNK这两条信号通路发挥作用，抑制破骨前体细胞的分化成熟，从而抑制骨吸收起到抗骨质疏松作用。
综上所述，菱叶山蚂蝗具有抗骨质疏松作用，其物质基础是黄酮类化合物群，主要药效物质是山萘苷，作用靶点可能是组织蛋白酶K，其抗骨质疏松作用与NF-κB和MAPK/JNK这两条信号通路相关。
Podocarpium podocarpum (DC.) Yang et Huang belongs to genus Podocarpium(Leguminosae). The leaves and roots have been taken to acute icteric hepatitis,rheumatism, bone bruises and knife trauma in Traditional Chinese Medicine. Its ethanolextract has been proved possess significant analgesic, antipyretic and anti-inflammatoryactivities in our previous study. So far, few phytochemical investigations have beencarried out on this species, only a total of6flavonoids,2triterpenoids,1steroidoid and2other compounds have been isolated and identified from Podocarpium podocarpum(DC.) Yang et Huang var. oxyphyllum (DC.) Yang et Huang, a variety of Podocarpiumpodocarpum.
Osteoporosis is a chronic, progressive disease of the skeleton characterized by bonefragility due to a reduction in bone mass and possibly alteration in bone architecturewhich leads to a propensity to fracture with minimum trauma. At present, the chemicaldrugs, which are clinically used as effective medications, are associated with longtreated time and numerous side effects. Therefore, the natural plant extracts, effectivefrations and compounds, with few side effects, attracted our attention. The plants blongsto the family Leguminosae or had the effect of strengthening the sexual behavior wereproved effective in anti-osteoporosis. The natural products, including flavonoids,steroidal, phenylpropanoids and alkaloids, have been proved possess anti-osteoporosisactivity, especially flavonoids. As Podocarpium podocarpum is a species of familyPapilionoideae (Leguminosae), a big sub-family rich in flavonoids, and it has been usedin treatment of rheumatism, bone bruises and knife trauma in traditional medical uses,both of the two implied that this plant probably possess anti-osteoporosis activity. Inaddition, our previous in vitro experiments also proved that the ethanol extract ofPodocarpium podocarpum showed potent stimulated effects on osteoblasticproliferation.
This dissertation is therefore conducted to investigate the anti-osteoporosis activityof Podocarpium podocarpum in vivo, followed by the isolation of the chemicalconstituents from its extract. In order to determine the chemical responsible for theanti-osteoporosis activity of this species, both in vivo and in vitro experiments wereused to test the anti-osteoporosis activity of the isolated compounds. In addition, themolecular docking technique was applied to discover the anti-osteoporotic targets ofkaempferitrin, so as to discuss the molecular mechanism of this action.
1In vivo antiosteoporotic evaluation of Podocarpium podocarpum extracts
The anti-osteoporotic effect of the ethanol extract of Podocarpium podocarpum(100～300mg/kg) in ovariectomized (OVX) rats model of osteoporosis wasinvestigated. The right femur of rats was used to detect the total bone mineral density(BMD) and bone tissue morphology, especially the tissue of trabecular and relatedparameters, which were determined by micro-CT; the urine was taken to evaluate thecontent of Ca, P and Cr; the serum was employed to determined the bone formationindicators including bone gla-protein (BGP) and alkaline phosphatase (ALP), the boneresorption indicators including cathepsin K, tartrate-resistant acid phosphatase (TRAP)and deoxypyridinoline (DPD); in addition, markers of oxidative stress, glutathioneperoxidase (GSH-Px), super oxidase dimutase (SOD), nitric oxide synthase (NOS) andmalondialdehyde (MDA) were also test to explore the possible mechanism of action.The results showed that the ethanol extract of Podocarpium podocarpum cansignificantly promote the BMD in OVX rats; the bone tissue of trabecular, includingtrabecular thickness (Tb. Th), trabecular number (Tb. N), connectivity density (CD) andtrabecular separation (Tb. Sp) were also improved; the findings assessed on the basis ofbiochemical, bone formation and resorption indicators strongly suggested that theethanol extract of Podocarpium podocarpum had a definite antiosteoporotic effectthrough inhibition of bone resorption. Furthermore, the activity of GSH-Px, SOD andNOS were also stimulate, which revealed that the anti-osteoporotic activity of ethanolextract of Podocarpium podocarpum probably related with oxidative stress.
2Chemical constituents of Podocarpium podocarpum
A total of73chemical compounds have been isolated from the ethanol extracts ofwhole plant of Podocarpium podocarpum whole plant by using silica gel columnchromatography, Sephadex LH-20and reversed-phase HPLC. The structures of thesecompounds, including flavonoids (DP1~25), phenylpropanoids (DP26~33),triterpenoids (DP34~40), steroidals (DP41~42), alkaloids (DP43~46), phenolic acids(DP47~56), and others (DP57~73), were elucidated on the basis of chemical andspectroscopic analyses. A total of about10g kaempferitrin was isolated from then-butanol fraction, implied that this compound was the main constituents ofPodocarpium podocarpum, and probably the main chemical responsible for theanti-osteoporotic activity of the plant. Therefore, a HPLC method for the determination of kaempferitrin was established. Its content in different part of Podocarpiumpodocarpum, including leaf, root and whole plant, as well as its variety, Podocarpiumpodocarpum var. oxyphyllum, have been detected and compared. The results showedthat the content of kaempferitrin in leaves was1.16±0.09%; wheras the root and stemas well as its variety were not detected. It is implied that the plant is differs markedlyfrom its variety in chemical constituents and their content.
3In vitro antiosteoporotic evaluation of the compounds isolated from Podocarpiumpodocarpum
Atotal of18flavonoids (DP1～18) were evaluated for their proliferative effects onosteoblasts derived from neonatal rat calvaria and inhibitory effects on multinucleatedosteoclasts from rat marrow cells. The stimulated effects on osteoblastic proliferation,ALP activity and calcium deposition, as well as the inhibited effects on osteoclasticTRAP activity were evaluated. The osteoclast was induced by1,25-(OH)2-VD3anddexamethasone by using both osteoblast and bone marrow cells. The results showed that12flavonoids exhibited significant effects both on osteoblasts and osteoclasts, whereasthe two compounds, DP3and DP16, exhibited no activity. In addition, nocorresponding dose-dependent phenomenon was observed.
4In vivo antiosteoporotic evaluation of kaempferitrin
As the whole plant of Podocarpium podocarpum possessed anti-osteoporoticactivity, and the content of kaempferitrin in the leaves of this species was high then1%,as well as the kaempferitrin showed proliferative effects on osteoblasts and inhibitoryeffects on multinucleated osteoclasts in vitro, the anti-osteoporotic activity ofkaempferitrin (8～32mg/kg) in ovariectomized (OVX) rats model of osteoporosis wasinvestigated. The results showed that the high and moderate dosage of kaempferitrin cansignificantly promote the BMD in OVX rats; the bone tissue of trabecular, includingtrabecular thickness (Tb. Th), trabecular number (Tb. N), connectivity density (CD) andtrabecular separation (Tb. Sp) were also significantly improved; the findings assessedon the basis of biochemical, bone formation and resorption indicators stronglysuggested that the kaempferitrin had a definite antiosteoporotic effect both throughstimulation of bone formation and inhibition of bone resorption, but the latter was thedominant mechanism. Furthermore, the activity of SOD and NOS were also stimulate,which revealed that the anti-osteoporotic activity of and kaempferitrin probably related with oxidative stress.
5Molecular mechanism of anti-osteoporotic activity
A molecular docking technique was used to discover the anti-osteoporotic targetsof kaempferitrin. The3D structure of kaempferitrin was constructed with SYBYLsoftware, followed by the energy optimization; then searched for the potential targetsfrom drug target database by upload the structure to internet, recorded the high scoredrug targets which related to osteoporosis disease, using forward docking to validatethose aimed drug targets, then the anti-osteoporotic targets of kaempferitrin wasconfirmed. The cathepsin K, a key enzyme responsible for osteoclast-mediated boneresorption, was identified as the antiosteoporotic targets of this herb by moleculardocking technology. The inhibitory effect on cathepsin K of multinucleated osteoclastsin vitro was investigated. Results showed that the kaempferitrin exhibited significantinhibitory effect on cathepsin K of osteoclasts, the inhibition rate was almost50%. Theprevious in vivo experiment in ovariectomized (OVX) rats model of osteoporosis hadalso proved the inhibition effect of kaempferitrin on cathepsin K in the serum ofrats.Therefore, both in vitro and in vivo valuated the result of molecular docking. So far,the anti-osteoporosis signal pathways involved with cathepsin K were NF-κB, MAPK(ERK1/2, P38and JNK), PI3K/Akt and CN/NFAT. The western-blot was used toexpress the key proteins p-ERK, p-JNK, p-P38and p-IκB. The results showed that theexpression of p-JNK and p-IκB were obviously inhibited by kaempferitrin, whichimplied that the anti-osteoporotic activivity of kaempferitrin is related to the NF-κB andJNK signaling pathways.
Summarily, the whole plant of Podocarpium podocarpum exhibited a definiteantiosteoporotic activity, the chemical responsible for this effect are flavonoidscompounds, and the kaempferitrin is the main constituents. The cathepsin K isidentified as the antiosteoporotic targets of this herb, and the anti-osteoporotic activivityis related to the NF-κB and JNK signaling pathways.
引文
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