Ethyl acetate extract of Smilax glabra Roxb roots and its major active compound astilbin promote osteoblastogenesis in vitro by upregulating bone cell differentiation-associated genes

Objective:To investigate the osteoblastogenic activity of the ethyl acetate(EtOAc)extract of Smilax glabra Roxb roots and its major active compound astilbin.Methods:Astilbin was isolated from EtOAc extract using silica gel chromatography combined with fraction crystallization.Chemical structure of astilbin was determined by analysis of the spectroscopic data in comparison with the literature.MTT method was used to detect the toxicity.Alkaline phosphatase(ALP)activity was determined by the spectrophotometric method at 405 nm using p-nitrophenyl phosphate as a substrate.Calcium deposition was stained with alizarin red-S,distained with cetylpyridium chloride,and quantified at 562 nm.In silico model for astilbin-ALP interaction was analyzed using AutoDock 4.2.6.The changes in expression of osteoblast differentiation related genes were determined using quantitative real-time PCR.Results:Both the EtOAc extract and astilbin had no toxicity toward osteoblast MC3T3-E1 cells at 5.0,10,25,and 50μg/mL.At 25μg/mL,they enhanced ALP activity and mineralization of osteoblasts up to 30%and 55%for the EtOAc extract and 22%and 41%for astilbin,respectively.Molecular docking analysis of astilbin-ALP interaction revealed Arg167,Asp320,His324,and His437 were key residues participating in hydrophobic interaction;meanwhile,His434 and Thr436 residues were involved in hydrogen bond formation in the active site of human tissue-nonspecific ALP.Moreover,the expression level of genes opn,col1,osx,and runx2 were up-regulated in astilbin treated samples with the fold changes as 2.2;3.7;4.1;2.3,respectively at 10μg/mL(P<0.05).Conclusions:The EtOAc extract and its major compound astilbin exhibit osteoblastogenic activity by up-regulating important markers for bone cell differentiation.It could be a new and promising osteogenic agent with dual actions for therapeutic applications.

Effects of Ethyl Acetate Extract of Phyllanthus reticulatus Leaves on Autophagy-related Proteins Beclin-1,ATG5 and LC3 in Nude Mice Xenograft Model of HCC

[Objectives]To explore the effects of ethyl acetate extract of Phyllanthus reticulatus leaves on autophagy-related proteins Beclin-1,ATG5 and LC3 by immunohistochemistry,and to preliminarily explore their effects on autophagy.[Methods]BEL-7404 Hepatocellular Carcinoma(HCC)nude mice model was established,and blank group(same volume of pure water),positive control group(20 mg/kg fluorouracil),high dose drug group(600 mg/kg),and medium dose drug group(300 mg/kg),and low dose drug group(150 mg/kg)were set up.After 2 weeks of intragastric administration,the nude mice were sacrificed,and the tumor tissues were taken out,processed by immunohistochemistry,and then made into paraffin sections.Photos were taken under an optical microscope(10×40),and evaluation and analysis were performed with the aid of the Image-Pro Plus 6.0 image analysis software.Differences were calculated using SPSS 20.0 software.The effects of drugs on autophagy-related proteins LC3,Beclin-1 and ATG5 were observed.[Results]Compared with the blank group,the medium and high dose groups of ethyl acetate extract of P.reticulatus leaves had the effect of promoting the increase of autophagy-related proteins LC3,Beclin-1 and ATG5(P<0.05).However,there was no significant difference between the low dose group of ethyl acetate extract of P.reticulatus leaves and the blank group(P>0.05).[Conclusions]The ethyl acetate extract of P.reticulatus leaves has a promoting effect on autophagy-related proteins LC3,Beclin-1,and ATG5.

Preventive Effect and Mechanism of Ethyl Acetate Extract of Sceptridium ternatum in Monocrotaline-Induced Pulmonary Arterial Hypertension

Objective:To observe the effect and molecular mechanism of ethyl acetate extract of Sceptridium ternatum(STE)on the monocrotaline(MCT)-induced pulmonary arterial hypertension(PAH).Methods:The main chemical components of Sceptridium ternatum were determined,and the effects in PAH rats were observed.A total of 140 Sprague Dawley rats were randomly and equally divided into the normal group,the model group,the Bosentan group,and the STE groups(2.5,5,10 g/kg)by the random number table method.The characteristic indicators of PAH were measured,and immunohistochemistry was used to observe the lung tissue of rats.Morphological changes of the lung tissue were observed under the light microscope.Results:Compared with the normal group,rats in the model group showed a significant increase in right ventricular free wall thickness(RVFWT),mean pulmonary arterial pressure(mPAP),mean right ventricular pressure(mRVP),max right ventricular pressure(max RVP),weight of right ventricle(RV),and lung index(LI),while a significant decrease in pulmonary artery acceleration time(PAAT,P<0.01).Compared with the model group,rats treated with STE had a significant decrease of RVFWT,mPAP,mRVP,max RVP,and RV,while a significant increase of PAAT(P<0.01).After injection of MCT,nuclear factor-κB(NF-κB)p65 andα-smooth muscle actin(α-SMA)expression levels were up-regulated,and on the contrary,the treatment groups showed a significant downregulation without dose-dependent trend.Conclusions:STE can relieve the PAH in rats.STE may relieve pulmonary vascular disease and pulmonary injury by down-regulating the expression of NF-κB p65 andα-SMA.

Quantitative variation of bioactive phyto compounds in ethyl acetate and methanol extracts of Pergularia daemia(Forsk.)Chiov.

Dear Editor： Pergularia daemia Forsk （Asclepiadaceae） is a perennial twining herb commonly known as veliparuthi in Tamil. The plant has anthelmintic, laxative, antidiabetic, hepatoprotective and anti-inflammatory activities. The pharmacological properties of this plant come from bioactive phytochemicals such as alkaloids, triterpenes, saponins and flavonoids. Phytochemically, the plant has been investigated for the presence of cardenolides, alkaloids, saponins and steroidal compoundst. In the present study, we developed a rapid method for identification and quantitative determination of putative phyto compounds in the crude extracts of ethyl acetate and methanol from whole plant of Pergularia daemia.

Antibacterial activity of the ethyl acetate part of Abrus cantoniensis against Staphylococcus aureus

Objective:The aim of this work was to measure the antibacterial activity(against Escherichia coli and Staphylococcus aureus[S.aureus])of the ethyl acetate part of Abrus cantoniensis and assess their potential as medicines.Methods:The experiment was divided into four groups:negative control group[with Mueller-Hinton broth(MHB)],positive control group(with 75%ethanol),blank group(with MHB)and test group(with the ethyl acetate part of Abrus cantoniensis).The antibacterial activities of the extracts were evaluated by the Oxford cup assay and minimum inhibitory concentration(MIC).Time-kill curve experiments,scanning electron microscopy,the content of DNA,RNA and protein were used to study the antibacterial mechanism of the ethyl acetate extract part on the growth and viability of S.aureus.The study procedures were approved by the Animal Care and Use Committee of Xi’an Jiaotong University(approval No.XJTULAC2016-412)on January 22,2016.Results:The ethyl acetate part of Abrus cantoniensis extract exhibited the highest inhibitory activity against the growth of S.aureus with an inhibition zone diameter of 16.4mm and MIC value of 0.5mg/mL.The general activity range of the ethyl acetate part,determined using a time-killing curve,was found to be 0.5mg/mL to 40mg/mL(MIC to 80
MIC).Changes in the scanning electron microscopy images and of DNA,RNA and proteins of S.aureus indicated possible mechanisms of the inhibitory activity of the ethyl acetate part.Conclusion:The ethyl acetate part of Abrus cantoniensis damaged bacterial cell structures,which results in protoplasm leakage,and eventually bacterial cell death.