| 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 extractsThe 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 podocarpumA 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 PodocarpiumpodocarpumAtotal 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 kaempferitrinAs 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 activityA 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. |
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