Mechanism of Pachymic Acid in Inhibiting Invasion and Metastasis of Renal Carcinoma Cells via Regulating MMP/TIMP Balance by Smads / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of pachymic acid （PA） in Poria on the invasion and metastasis of renal carcinoma cells. MethodThe effect of PA （0， 20， 40， 80， 160 μmol·L-1） on cell viability was detected by cell counting kit-8（CCK-8）， and the dose of PA was selected for subsequent experiments. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell proliferation was evaluated by colony formation assay. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell adhesion ability was observed by cell adhesion assay. The effect of PA （0， 20， 40， and 80 μmol·L-1） on cell invasion and metastasis was investigated by Wound healing assay and Transwell invasion assay. The inhibitory effect of PA （0， 20， 40， 80 μmol·L-1） on cell motility was further observed and verified by high-content imaging technology. The effects of PA （0， 20， 40， 80 μmol·L-1） on the expression of matrix metalloproteinase （MMP）/tissue inhibitor of metalloproteinasas （TIMP） related to invasion and metastasis and Smads were detected by Western blot. ResultCCK-8 results showed that compared with the blank group， the PA groups showed decreased cell viability（P<0.01）， with the half-maximal inhibitory concentration （IC50） of ACHN cells of 70.42 μmol·L-1 at 24 h. Colony formation assay showed that the number of cell clonal groups in the PA groups was reduced compared with that in the blank group（P<0.01）. Cell adhesion assay showed that compared with the blank group， the PA groups displayed reduced cell adhesion（P<0.01）. Wound healing assay showed that the wound healing rate of cells in the PA groups was lower than that in the blank group （P<0.05，P<0.01）. Transwell invasion assay showed that compared with the blank group， the number of transmembrane cells in PA groups was reduced（P<0.01）. High-content imaging showed that the cumulative migration distance of cells in the PA groups was shorter than that in the blank group（P<0.01）. The results of Western blot showed that the protein expression of MMP-2 and MMP-9 in the PA groups decreased （P<0.01）， and TIMP-1 protein expression increased （P<0.01） compared with those in the blank group. In addition， compared with the blank group， the PA groups showed decreased protein expression of Smad2 and Smad3 （P<0.01）. ConclusionPA can inhibit the invasion and metastasis of renal carcinoma cells presumably through regulating the homeostasis of MMP/TIMP by Smad2/3.

Pachymic acid protects against Crohn's disease-like intestinal barrier injury and colitis in miceby suppressingintestinal epithelial cell apoptosis via inhibiting PI3K/AKT signaling / 南方医科大学学报

OBJECTIVE@#To investigate the effect of pachymic acid (PA) against TNBS-induced Crohn's disease (CD)-like colitis in mice and explore the possible mechanism.@*METHODS@#Twenty-four C57BL/6J mice were randomized equally into control group, TNBS-induced colitis model group and PA treatment group. PA treatment was administered via intraperitoneal injection at the daily dose of 5 mg/kg for 7 days, and the mice in the control and model groups were treated with saline. After the treatments, the mice were euthanized for examination of the disease activity index (DAI) of colitis, body weight changes, colon length, intestinal inflammation, intestinal barrier function and apoptosis of intestinal epithelial cells, and the expressions of TNF-α, IL-6 and IL-1β in the colonic mucosa were detected using ELISA. The possible treatment targets of PA in CD were predicted by network pharmacology. String platform and Cytoscape 3.7.2 software were used to construct the protein-protein interaction (PPI) network. David database was used to analyze the GO function and KEGG pathway; The phosphorylation of PI3K/AKT in the colonic mucosal was detected with Western blotting.@*RESULTS@#PA significantly alleviated colitis in TNBS-treated mice as shown by improvements in the DAI, body weight loss, colon length, and histological inflammation score and lowered levels of TNF-α, IL-6 and IL-1β. PA treatment also significantly improved FITC-dextran permeability, serum I-FABP level and colonic transepithelial electrical resistance, and inhibited apoptosis of the intestinal epithelial cells in TNBS-treated mice. A total of 248 intersection targets were identified between PA and CD, and the core targets included EGFR, HRAS, SRC, MMP9, STAT3, AKT1, CASP3, ALB, HSP90AA1 and HIF1A. GO and KEGG analysis showed that PA negatively regulated apoptosis in close relation with PI3K/AKT signaling. Molecular docking showed that PA had a strong binding ability with AKT1, ALB, EGFR, HSP90AA1, SRC and STAT3. In TNBS-treated mice, PA significantly decreased p-PI3K and p-AKT expressions in the colonic mucosa.@*CONCLUSION@#PA ameliorates TNBS-induced intestinal barrier injury in mice by antagonizing apoptosis of intestinal epithelial cells possibly by inhibiting PI3K/AKT signaling.

Simultaneous quantitative analysis of four triterpenes in Poria Cocos from different habitats by QAMS / 中草药

Objective To establish a new method and validate its feasibilities by the simultaneous quantitative assay of four triterpenes in Poria cocos. Methods A new quality evaluation method of multi-components by single marker (QAMS) was established and validated for P. cocos. Pachymic acid (PA), dehydropachymic acid (DPA), dehydrotumulosic acid (DTA), and dehydrotrametenolic acid (DMA) were selected as analytes while pachymic acid was chosen as internal reference substance to evaluate the quality. The relative correction factor (RCF) of pachymic acid to the other three triterpenes were calculated. The method was evaluated by the comparison of quantity between external standard method and QAMS method. Results The contents of four triterpenes in 17 batches of P. cocos from QAMS method were not significantly different from those from external standard method. Conclusion The method with a single marker is accurate and feasible to determine PA, DPA, DTA, and DMA when some authentic standard substance are unavailable.

Simultaneous Determination of 4 Active Components in Compound Xiaosuanzao Chewable Tablets by HPLC-ELSD / 中国药房

OBJECTIVE:To establish the method for the content determination of 4 active components in Compound xiaosuanzao chewable tablets.METHODS:HPLC-ELSD method was adopted.The determination was performed on a Grace Brava C18-BDS column with mobile phase consisted of acetonitrile-water (gradient elution) at the flow rate of 1.0 mL/min.The column temperature was 25 ℃,and sample size was 20 μL.The drift tube temperature is 100 ℃,and the carrier gas flow rate is 2.9 L/min.RESULTS:The linear ranges of betulic acid,betulinol,pachymic acid and glycyrrhizic acid were 44.50-890.0 μg/mL (r=0.999 3),20.28-405.6 μg/mL (r=0.999 7),20.50-656.0 μg/mL(r=0.999 7) and 10.50-336.0 μg/mL(r=0.999 6),respectively.RSDs of precision,stability and reproducibility were all lower than 3.0％.The recoveries were 99.44％-101.12％ (RSD=0.57％,n=6),99.41％-100.39％ (RSD=0.34％,n=6),99.31％-100.46％ (RSD=0.51％,n=6),98.96％-101.19％ (RSD=0.84％,n=6),respectively.CONCLUSIONS:The method is simple,precise,stable and reproducible,and can be used for simultaneous determination of 4 active components in Compound xiaosuanzao chewable tablets.

Induction of pachymic acid on apoptosis of human breast cancer MDA-MB-231 cells by activating Poly ADP ribose polymerase / 中草药

Objective: To study the potential apoptotic effect of pachymic acid isolated from Poria cocos, a traditional Chinese medicinal fungi, on human breast cancer cells. Methods: Pachymic acid was isolated and purified from the crude ethanol extract of P. cocos. Human breast cancer cell line MDA-MB-231 cells were treated with pachymic acid. Relative cell viabilities were determined by the Cell Counting Kit-8 assay; Apoptosis was analyzed by Annexin V/PI dual staining with flow cytometry; The protein expression levels of poly ADP ribose polymerase (PARP) and other apoptotic related proteins were detected by Western blotting. Results: Pachymic acid reduced the viability of MDA-MB-231 cells dose-and time-dependently. Moreover, pachymic acid induced the apoptosis in MDA-MB-231 cells. The data of Western blotting showed that pachymic acid up-regulated the expression level of PARP, in a dose-dependent manner. In addition, apoptosis-related proteins like Bax, Bcl-2, and casapases were all affected by pachymic acid. Conclusion: Pachymic acid could induce the apoptosis in human breast cancer cells, which provides a novel information for clinical application of pachymic acid and its preparations for the breast cancer patients.

Comparison on six active constituents between formula granule and traditional decoction of Rehmanniae Decoction of six ingredients / 中草药

Objective: To compare the content of chemical components in each drug in formula granule and traditional decoction of Rehmanniae Decoction with six ingredients. Methods: By using high performance liquid chromatography analysis and various chromatographic conditions, the contents were determined respectively, which were listed as follows: acteoside, loganin, paeonol, allantoin, pachymic acid and alisol B 23-acetate. Results: The contents of acteoside, loganin, paeonol, allantoin, pachymic acid and alisol B 23-acetate in traditional decoction were 304.5, 2 473.6, 3 135.1, 708.8, 5.9, and 104.4 μg/g, and they were 289.6, 3 685.7, 706.5, 714.2, 17.4, and 217.8 μg/g correspondingly in formula granule. The contents of acteoside and allantoin were basically the same between them; The contents of loganin, pachymic acid, and alisol B 23-acetate in formula granule were significantly higher than those in the traditional decoction; The content of paeonol in formula granule was significantly lower than that in the traditional decoction. Conclusion: The content difference of the chemical components is related to its chemical character between formula granule and traditional decoction.

Poria cocos ethanol extract and its active constituent, pachymic acid, modulate sleep architectures via activation of GABA(A)-ergic transmission in rats / 동물의과학연구지

Poria cocos is a well-known traditional Chinese traditional medicine (TCM) that grows around roots of pine trees in China, Korea, Japan, and North America. Poria cocos has been used in Asian countries to treat insomnia as either a single herb or part of an herbal formula. In a previous experiment, pachymic acid (PA), an active constituent of Poria cocos ethanol extract (PCE), increased pentobarbital-induced sleeping behaviors. The aim of this experiment was to evaluate whether or not PCE and PA modulate sleep architectures in rats as well as whether or not their effects are mediated through GABA(A)-ergic transmission. PCE and PA were orally administered to individual rats 7 days after surgical implantation of a transmitter, and sleep architectures were recorded by Telemetric Cortical encephalogram (EEG) upon oral administration of test drugs. PCE and PA increased total sleep time and non-rapid eye movement (NREM) sleep as well as reduced numbers of sleep/wake cycles recorded by EEG. Furthermore, PCE increased intracellular chloride levels, GAD65/67 protein levels, and alpha-, beta-, and gamma-subunits of GABA(A) receptors in primary cultured hypothalamic neuronal cells. These data suggest that PCE modulates sleep architectures via activation of GABA(A)-ergic systems. Further, as PA is an active component of PCE, they may have the same pharmacological effects.

Pachymic Acid Enhances Pentobarbital-Induced Sleeping Behaviors via GABA(A)-ergic Systems in Mice

This study was investigated to know whether pachymic acid (PA), one of the predominant triterpenoids in Poria cocos (Hoelen) has the sedative-hypnotic effects, and underlying mechanisms are mediated via gamma-aminobutyric acid (GABA)-ergic systems. Oral administration of PA markedly suppressed locomotion activity in mice. This compound also prolonged sleeping time, and reduced sleep latency showing synergic effects with muscimol (0.2 mg/kg) in shortening sleep onset and enhancing sleep time induced by pentobarbital, both at the hypnotic (40 mg/kg) and sub-hypnotic (28 mg/kg) doses. Additionally, PA elevated intracellular chloride levels in hypothalamic primary cultured neuronal cells of rats. Moreover, Western blotting quantitative results showed that PA increased the amount of protein level expression of GAD65/67 over a broader range of doses. PA increased alpha- and beta-subunits protein levels, but decreased gamma-subunit protein levels in GABA(A) receptors. The present experiment provides evidence for the hypnotic effects as PA enhanced pentobarbital-induced sleeping behaviors via GABA(A)-ergic mechanisms in rodents. Taken together, it is proposed that PA may be useful for the treatment of sleep disturbed subjects with insomnia.

UPLC determination of determine dehydrotumulosic acid etc. six active components in poria / 中国药学杂志

OBJECTIVE: To establish an analytical method to determine dehydrotumulosic acid, tumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid and pachymic acid in poria quickly and accurately. METHODS: An UPLC method was established on an HSS T3 Column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of water (containing 0.05% phosphoric acid) and acetonitrile in gradient elution mode, and the detection wavelength was set at 210 nm. RESULTS: The standard curves of dehydrotumulosic acid, tumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid, and pachymic acid showed good linearity in 5.400-108.0, 2.040-40.80, 5.020-100.4, 2.120-42.40, 5.060-101.2 and 5.100-102.0 μg · mL-1, respectively and the average recoveries were 98.0% with RSD of 2. 9% for dehydrotumulosic acid, 99. 0% with RSD of 2.8% for tumulosic acid, 101.5% with RSD of 2.5% for polyporenic acid C, 97.1% with RSD of 2.7% for 3-epi-dehydrotumulosic acid, 101.5% with RSD of 2.1% for dehydropachymic acid and 99.6% with RSD of 1.1% for pachymic acid, respectively. CONCLUSION: The method is quick, accurate, and can be used to determine multiple triterpenoid acids in Poria simultaneously.

Study of Poria in different parts and different localities / 中成药

AIM: The contents of pachymic acid in Poria in different parts and in different localities were measured and compared. METHODS: 9 samples were measured with HPLC. RESULTS: The content of pachymic acid in Fushen is the highest in different parts and the content of pachymic acid in Guangxi province is the highest in five localities. CONCLUSION: The content of pachymic acid was obviously variable in different parts and this result made the basis of the application of with the different parts of Poria. The content of pachymic acid wasn't obviously variable in different localities.