Isolation And Identification Of The Saponins From The Root Bark Of The Tujia Medicine Acanthopanax Japonicus And Its Inhibitory Effect On Primary Osteoclasts

Experimental object:The aim of this thesis is to systematically purify and identify the saponin chemical constituents from the root bark of the Aralia echinocaulis Hand.-Mazz.and obtain the saponin monomer compound.Then,the isolated monomer compounds were screened for activity,and the inhibitory effects of the chemical components on the primary osteoclasts were explored.Thus,we can some extent reveal the material basis of the treatment of osteoporosis.Experimental content:1.Study on the chemical composition of the root bark of the Aralia echinocaulis Hand.-Mazz.Based on the conventional method of extraction and separation of saponins,modern methods of separation and analysis were used to purify and identify the saponin chemical components of the root bark of the Aralia echinocaulis Hand.-Mazz.Monomer compounds were obtained.2.Study on the inhibitory effects of chemical components on the primaryosteoclasts.The primary osteoclasts were cultured in vitro and identified.The cultured primary osteoclasts were used as carriers to screen the isolated monomer compounds for their activity on primary osteoclasts.Experimental method:1.Study on the chemical composition of the root bark of the Aralia echinocaulis Hand.-Mazz.By heating and refluxing,we used 70%ethanol to extract the root bark of the Aralia echinocaulis Hand.Mazz,and concentrated the extract to obtain extracts.We used macroporous adsorption resin for preliminary separation,separated the extracts by ethanol gradient elution,and combined eluents by thin layer chromatography.The fractions eluted from 30%to 70%ethanol were separated on a silica gel column using methylene chloride-methanol-water as the mobile phase.The acetonitrile-water(0.01%formic acid)mobile phase was used for the liquid phase preparation to obtain monomer compounds.The molecular formulas and structures were identified by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.2.Study on the inhibitory effects of chemical components on the primary osteoclasts.Primary osteoclasts were cultured in vitro and identified using tartrate-resistant acid phosphatase positive staining and bone resorption toluidine blue staining.CCK-8 cytotoxicity assay was used to detect the effect of monomer on the activity of osteoclast precursor cells,excluding the false positive result of high concentration of inhibitory cells,and screening the inhibitory concentration range of osteoclasts.Then the number of TRAP positive cells was used to test the inhibitory effect of monomer on the osteoclasts.Experimental results:1.Study on the chemical composition of the root bark of the Aralia echinocaulis Hand.-Mazz.Eighteen compounds were isolated and eleven compounds were identified,nine of which are oleanolic acid saponins,including:Tarasaponin Ⅳ(compound Ⅰ),Stipuleanoside R2(compound Ⅱ),Araloside C(compound Ⅲ),Decaisneanaside(compound Ⅵ),Chikuselsusaponin V(compound Ⅶ),Araloside A(compoundⅧ),Chikuselsusaponin IVa(compound Ⅺ),Narcissiflorine(compoundⅫ),Ealenduloside E(compound ⅩⅢ).Two novel compounds compound V and compound IV were obtained.The aglycone of this class of compounds is the new framework aglycone.2.Study on the inhibitory effects of chemical components on the primary osteoclasts.In the range of 0～50 μg/mL,the false positive result of inhibition of osteoclasts by monomeric compounds can be excluded.The IC50 of Stipuleanoside R2,Araloside C,Araloside A,and Chikuselsusaponin IVa were 81.217±1.910、478.012±2.679.71.207± 1.853.5.286±0.723 μg/mL,respectively.Low,medium,and high concentrations of Stipuleanoside R2,Araloside C,Araloside A,andChikuselsusaponin Ⅳa(0.5,5,and 50 μg/mL)showed a decrease in the number of osteoclasts compared with the control group,indicating a certain inhibitory effect.There was statistically significant difference(P<0.05 or P<0.01).Among them,the number of osteoclasts at each concentration of Stipuleanoside R2 decreased,and the inhibitory effect was more obvious.The number of osteoclasts at Araloside C and Araloside A decreased as the concentration increased.The number of osteoclasts at each concentration of Chikuselsusaponin IVa decreased.However,the change with increasing concentration is not obvious.Comparing the results of Araloside C,Araloside A and Chikuselsusaponin IVa experiments,when the amount of sugar is different,the inhibitory effect of the compound on primary osteoclasts is different,but the difference is not obvious;through the comparison of the experimental results of Stipuleanoside R2 and Araloside C,when the amount of sugar and the connection position are the same,the type of sugar is different,and the inhibitory effect of the compound on the primary osteoclast is also different,and the difference is obvious.Based on the above,it is presumed that when the aglycones of the compounds are the same,the effect of the sugar species on the side chain on the proliferation of the primary osteoclast is greater than the quantity of sugar.Experimental conclusion:1.The saponins in the bark of the Aralia echinocaulis Hand.-Mazz.weremainly oleanolic acid triterpene saponins.They also contained the novelsaponins such as compound V and compound Ⅳ.The aglycone of this compound is a new skeleton.2.The oleanolic acid saponins Stipuleanoside R2,Araloside C,Araloside A,and Chikuselsusaponin Ⅳa extracted from the root bark inhibited the proliferation of primary osteoclasts in a concentration range of 0～50 μg/mL.When the aglycone of the compound is the same,the effect of the sugar species on the side chain on the proliferation of the primary osteoclast is greater than the quantity of sugar.