Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy / 中国中药杂志

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1β and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

Triterpene saponins in Panax japonicus and their ~(13)C-NMR spectroscopic characteristics / 中国中药杂志

Panax japonicus( PJ) is a valuable medicinal plant belonging to the genus Panax of Araliaceae,the recumbent rhizome of which is widely used in clinic therapy,healthcare products and as cosmetic additives with functions of dissipating stasis,reducing swelling,stanching bleeding,and reinforcing deficiency,etc. PJ contains abundant levels of oleanane-and dammarane-type triterpene saponins,which are considered as the material basis for exerting pharmacodynamic action. Based on the previous researches,more than110 triterpene saponins have been reported from PJ. These triterpene saponins were summarized in this review,and could be classified into dammarenediol Ⅱ,protopanaxadiol,protopanaxatiol,ocotillol,oleanolic acid,ursolic acid and miscellaneous subtypes,according to their molecular skeletons in biosynthesis processes. Further more,the structural features of these triterpene saponins in the seven different subtypes,together with their~(13)C-NMR spectroscopic characteristics were described,hoping to provide available information for chemical diversity research of PJ.

Improved effects of saponins from Panax japonicus on decline of cognitive function in natural aging rats via NLRP3 inflammasome pathway / 中国中药杂志

The aim of this paper was to investigate the effect of total saponins from Panax japonicus( SPJ) on cognitive decline of natural aging rats and its mechanism. Thirty male SD rats of eighteen month old were randomly divided into three groups: aged group,10 mg·kg~(-1) SPJ-treated group and 30 mg·kg~(-1) SPJ-treated group. The SPJ-treated groups were given SPJ at the dosages of 10 mg·kg~(-1) and 30 mg·kg~(-1),respectively,from the age of 18 to 24 months. Aged group were lavaged the same amount of saline,10 six-month-old rats were used as control group,with 10 rats in each group. The open field test,novel object recognition and Morris water maze were performed to detect the changes of cognitive function in each group. The changes of synaptic transmission of long-term potentiation( LTP) in hippocampal CA1 region were detected by field potential recording. Western blot was used to detect the protein levels of NLRP3,ASC,caspase-1 and the changes of Glu A1,Glu A2,CAMKⅡ,CREB and phosphorylation of CAMKⅡ,CREB in each group.The results showed that SPJ could improve the decline of cognitive function in aging rats,reduce the damage of LTP in the hippocampal CA1 region of aged rats,and decrease the expression of NLRP3,ASC,caspase-1 in aging rats. At the same time,SPJ could enhance the membrane expression of AMPA receptor( Glu A1 and Glu A2),and increase the expression of p-CAMKⅡand p-CREB in aging rats.SPJ could improve cognitive decline of natural aging rats,and its mechanism may be related to regulating NLRP3 inflammasome,thus regulating the membrane expression of AMPA receptor,and enhancing the expression phosphorylation of CAMKⅡ and CREB.

Effect of saponins extracted from Panax japonicas on inhibiting myocardial fibrosis by TGF-β1/Smad3 signaling pathway in aging rats / 中国中药杂志

To investigate the amelioration effect of saponins extracted from Panax japonicas (SPJ) on myocardial fibrosis in natural aging rats and its mechanisms, male SD rats aged 18 months were randomly divided into 3 groups (aging model group, low-dose SPJ group and high-dose SPJ group), with 10 rats in each group. SPJ groups were given SPJ at different doses (10, 60 mg·kg⁻¹·d⁻¹) consecutively for 6 months, meanwhile, aging model group was treated with the equal volume of saline for 6 months until 24 months old. Another 10 rats aged 6 month were used as young control group. The changes of myocardial morphological were observed by haematoxylin-eosin (HE) staining. Masson staining was used to observe the changes of collagen deposition in rat hearts. RT-PCR was used to detect the mRNA expression levels of myofibroblast marker α-SMA, collagen-related protein COL1α2, COL3α1 and matrix metalloproteinase MMP2, MMP9. Western blot was used to test the changes of the protein expressions of TGF-β1, p-Smad3, IL-1β and TNF-α in heart tissues. SPJ can effectively improve the arrangement of myocardial fibers, decrease inflammatory infiltration and reduce collagen deposition in aging rats. SPJ can effectively down-regulate the mRNA expression levels of COL1α2, COL3α1, α-SMA, MMP9, MMP2 and inhibit the protein expressions of TGF-β1, p-Smad3, TNF-α, IL-1β in the natural aging heart tissues. SPJ can effectively alleviate myocardial fibrosis in natural aging rats, and its mechanisms was related to the inhibition of the protein expressions of TGF-β1, p-Smad3 and the reduction of myocardial inflammation in rat hearts.

Protective effects of total saponins of Panax japonicas on HepG2 cell apoptosis induced with palmitic acid / 中国中药杂志

This study aimed to investigate the molecular mechanism and protective effect of total saponins of Panax japonicas (TSPJ) on HepG2 cells apoptosis induced by palmitic acid (PA).The HepG2 cells were cultured , and divided into five groups: the control group, the model group, the high-dose group (50 mg·L⁻¹), the middle-dose group (25 mg·L⁻¹) and the low-dose group (12.5 mg·L⁻¹).The cells of the five groups were cultured continuously for 24 hours. The cell viability was measured with MTT. HepG2 cells apoptosis was detected by Hoechest staining and Annexin V-FITC/PI staining. The protein expressions of BCL-2, CHOP and TLR4 were measured with western blotting and flow cytometry analysis. The mRNA expressions of TNF-α, IL-1β, BCL-2, CHOP and GAPDH were measured with RT-PCR. The results suggested that compared with the control group, the number of HepG2 cells of the model group were reduced significantly (<0.01), while the number of apoptotic HepG2 cells were increased. Compared with the model group, the number of HepG2 cells of the high-dose group and the middle-dose group were increased significantly (<0.01), whereas the number of apoptotic HepG2 cells were reduced. Compared with the control group, TNF-α, IL-1β and CHOP mRNA expressions and CHOP and TLR4 protein expressions in the model group were significantly up-regulated (<0.01), while BCL-2 protein and mRNA expressions in the model group were significantly decreased (<0.01). Compared with the model group, TNF-α, IL-1β and CHOP mRNA expressions and CHOP and TLR4 protein expressions in the high-dose group were significantly decreased (<0.01), while BCL-2 protein and mRNA expressions in the high-dose group were significantly up-regulated (<0.01).In conclusion, TSPJ can reduce inflammation and apoptosis induced by palmitic acid, with a certain protective effect on liver cells.

Protective effect of Wuzi Yanzong recipe on testicular germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress / 中国中药杂志

To study the protective effects of Wuzi Yanzong recipe on testis germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress (ERS), 16-month-old male SPF grade SD rats were randomly divided into three groups: ageing model group, and low and high-dose Wuzi Yanzong recipe groups (WZ, 1 and 4 g·kg⁻¹), with 10 rats in each group. In addition, 2-month-old SD male rats were used as adult control group. The ageing model group and the adult control group were fed with normal diet for 4 months. WZ groups were given the medicated feed for 4 months. After fasting for 12 hours, the rats were put to death. Then, the testes were immediately collected. The change of testicular tissue morphology was observed by HE staining. The expression levels of ER stress-related proteins GRP78, p-PERK, p-eif2, ATF4, p-IRE1, XBP1, ATF6 and apoptosis-related proteins CHOP, caspase12 and p-JNK in testes were detected by Western blot. Compared with the ageing model group, Wuzi Yanzong recipe alleviated the morphological changes of testicular tissue. Western blot results showed that Wuzi Yanzong recipe significantly increased the expression levels of endoplasmic reticulum stress-related proteins GRP78, p-PERK, p-eif2, ATF4, p-IRE1, XBP1, ATF6 and significantly decreased the expression levels of endoplasmic reticulum-induced apoptosis-related proteins CHOP, caspase 12 and p-JNK. In conclusion, Wuzi Yanzong recipe can alleviate the ageing-related apoptosis of testicular germ cells in natural ageing rats by regulating endoplasmic reticulum stress.

Study on protective effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 cell inflammation through NF-kappaB pathway / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages.</p><p><b>METHOD</b>The effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot.</p><p><b>RESULT</b>The safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65.</p><p><b>CONCLUSION</b>This study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.</p>

Studies on preparative technology and quantitative determination for extracts of total saponin in roof of Panax japonicus / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the optimum extraction and purification condition of the total saponins in the root of Panax japonicus (RPJ), and establish its quality control methods.</p><p><b>METHOD</b>Designed L16 (4(5)) orthogonal test with the extraction rate of total saponins as index, to determine the rational extraction process, and the techniques of water-saturated n-butanol extraction and acetone precipitation were applied to purify the alcohol extract of RPJ. Total saponins were detected by spectrophotometry and its triterpenoidal sapogenin oleanolic acid detected by HPLC.</p><p><b>RESULT</b>The optimum conditions of total saponins from RPJ was as follows: the material was pulverized, dipped in 60% ethanol aqueous solution as extract solvent at 10 times of volume, and refluxed 3 times for 3 h each time. Extractant of water-saturated n-butanol with extraction times of 3 and precipitant of acetone with precipitation amount of 4-5 times were included in the purification process, which would obtain the quality products. The content of total saponins could reach to 83.48%, and oleanolic acid to 38.30%.</p><p><b>CONCLUSION</b>The optimized preparative technology is stable, convenient and practical. The extract rate of RPJ was high and steady with this technology, which provided new evidence for industrializing production of the plant and developing new drug.</p>