Punicalagin inhibits hepatic lipid deposition in obese mice via AMPK/ACC pathway / 中国中药杂志

Hepatic lipid deposition is one of the basic manifestations of obesity, and nowadays pharmacological treatment is the most important tool. Punicalagin(PU), a polyphenol derived from pomegranate peel, is a potential anti-obesity substance. In this study, 60 C57BL/6J mice were randomly divided into a normal group and a model group. After establishing a model of simple obesity with a high-fat diet for 12 weeks, the successfully established rat models of obesity were then regrouped into a model group, an orlistat group, a PU low-dose group, a PU medium-dose group, and a PU high-dose group. The normal group was kept on routine diet and other groups continued to feed the high-fat diet. The body weight and food intake were measured and recorded weekly. After 8 weeks, the levels of the four lipids in the serum of each group of mice were determined by an automatic biochemical instrument. Oral glucose tole-rance and intraperitoneal insulin sensitivity were tested. Hemoxylin-eosin(HE) staining was applied to observe the hepatic and adipose tissues. The mRNA expression levels of peroxisome proliferators-activated receptor γ(PPARγ) and C/EBPα were determined by real-time quantitative polymerase chain reaction(Q-PCR), and the mRNA and protein expression levels of adenosine 5'-monophosphate-activated protein kinase(AMPK), anterior cingulate cortex(ACC), and carnitine palmitoyltransferase 1A(CPT1A) were determined by Western blot. Finally, the body mass, Lee's index, serum total glyceride(TG), serum total cholesterol(TC), and low-density lipoprotein cholesterol(LDL-C) levels were significantly higher and high-density lipoprotein cholesterol(HDL-C) levels were significantly lower in the model group as compared with the normal group. The fat deposition in the liver was significantly increased. The mRNA expression levels of hepatic PPARγ and C/EBPα and the protein expression level of ACC were increased, while the mRNA and protein expression levels of CPT-1α(CPT1A) and AMPK were decreased. After PU treatment, the above indexes of obese mice were reversed. In conclusion, PU can decrease the body weight of obese mice and control their food intake. It also plays a role in the regulation of lipid metabolism and glycometabolism metabolism, which can significantly improve hepatic fat deposition. Mechanistically, PU may regulate liver lipid deposition in obese mice by down-regulating lipid synthesis and up-regulating lipolysis through activation of the AMPK/ACC pathway.

Network pharmacological study and experimental verification of punicalagin in treatment of depression / 中国药理学通报

Aim To study and verify the effeet and po¬tential mechanism of punicalagin ( Pun) in the treat-ment of depression by preliminary experiments based on network pharmacology.Methods The intersection genes of Pun and depression were obtained through the database, and protein interaction ( PPI ), GO and KEGG were enriched and analyzed.Molecular docking technology was used to preliminarily verify the binding ability of Pun active components to core therapeutic targets.The depression model of CUMS mice was es¬tablished by chronic stress, and Pun was administered by gavage.Open field experiments were conducted to investigate behavior changes.The content of neuro¬transmitters in hippocampus was detected by liquid chromatography-mass spectrometry ( LC-MS / MS ).Results The results of network pharmacology showed that Pun had 76 targets involved in the occurrence of depression, and PPI network showed that the intersec¬tion genes were closely related.Proteoglycans, lipids and atherosclerosis enriched in cancer.The results of molecular docking showed that there was a good bind¬ing between the compound and the target protein.The results of animal experiments showed that Pun could in¬crease the exploration desire of open field experimental mice.The levels of DA and 5-HT in hippocampus in-creased ( P < 0.05, P < 0.01 ).Conclusions Pun can significantly reduce the depressive state of mice, and its mechanism may act on ALB and AKT1 targets, mediate proteoglycans, lipids and atherosclerotic path¬ways in cancer, so as to improve the secretion of neu¬rotransmitters.

Effects of knocking down TSPO on proliferation and autophagy of BV2 cells in OGD/R models / 中国药理学通报

Aim To investigate the effeets of prolifera¬tion and autophagy of BV2 eells in OGD/R models when the 18 ku transloeator protein( TSPO) was inhibi¬ted.Methods BV2 microglia were eultured in vitro and the model established by oxygen-glueose depriva- tion/reperfusion( OGD/R) , the eells were divided into eontrol group and OGD/R group, OGD/R + small hair¬pin RNA negative eontrol group ( OGD/R + NCshR- NA) , OGD/R + TSPO small hairpin RNA group (OGD/R + TSPOshRNA ).The expression of TSPO mRNA and TSPO protein were deteeted by qRT-PCR and Western blot, respectively.In order to study the effeet of TSPO on BV2 microglial eells in OGD/R inju¬ry and autophagy, the cell viability was tested by CCK- 8 assey, the cytotoxicity was deteeted by reactive oxy¬gen speeies ( ROS) , autophagy-related mRNA ( p62 mRNA, LC3B mRNA, Beolin-1 mRNA) expressions were detected by qRT-PCR, and the expression levels of autophagy -related proteins ( p62 , LC3 II /LC3 1 , Beclin-1 ) were detected by Western blot in each group.Result The expression of TSPO mRNA and protein increased significantly in OGD/R group while compared to control group, the cell death and cytotox¬icity increased significantly, the expression levels of LC3B mRNA and Beclin-1 mRNA increased, while the p62 mRNA decreased significantly, the levels of LC3 II/LC3 1 and Beclin-1 protein increased, the expres¬sion of p62 protein decreased significantly in OGD/R group, and the autophagy was activated; compared with OGD/R group, the different levels of cell viabili¬ty, cytotoxicity and autophagy in OGD/R + NCshRNA group were not statistically significant.But the survival rate of cells in OGD/R + TSPOshRNA group signifi¬cantly increased, the levels of cytotoxicity and autoph¬agy were significantly reduced.Conclusions The in¬hibition of TSPO has a significant protective effect on OGD/R injury model in BV2 microglial cells, which may be related to the inhibition of autophagy.

Supplementing punicalagin reduces oxidative stress markers and restores angiogenic balance in a rat model of pregnancy-induced hypertension

Pre-eclampsia (PE) is a pregnancy disorder that is characterised by severe hypertension and increased risks of foetal and maternal mortality. The aetiology of PE not completely understood; however, maternal nutrition and oxidative stress play important roles in the development of hypertension. The treatment options for PE are currently limited to anti-hypertensive drugs. Punicalagin, a polyphenol present in pomegranate juice, has a range of bioactive properties. The effects of supplementation with punicalagin on angiogenesis and oxidative stress in pregnant rats with induced hypertension were investigated. The pregnant rats were randomly divided into five experimental groups (n=12 per group). Hypertension was induced using an oral dose of NG-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day) on days 14–19 of pregnancy. Punicalagin (25, 50 or 100 mg/kg) was given orally on days 14–21 of pregnancy. Punicalagin treatment at the tested doses significantly reduced diastolic, systolic, and mean arterial blood pressure in L-NAME treated rats from day 14. Punicalagin also restored angiogenic balance by increasing the expression of vascular endothelial growth factor and downregulating vascular endothelial growth factor receptor-1/fms-like tyrosine kinase-1. Punicalagin, significantly increased the placental nitric oxide levels as compared to PE group. The increased levels of oxidative stress in rats with PE were markedly decreased by treatment with punicalagin. Punicalagin at the tested doses markedly (p < 0.05) enhanced the placental antioxidant capacity in L-NAME-treated rats. The raised catalase activity observed following L-NAME induction was significantly (p < 0.05) and restored to normal activity levels in punicalagin treatment. Further, 100 mg dose of punicalagin exhibited higher protective effects as compared to lower doses of 25 and 50 mg. This study shows that supplementation with punicalagin decreased blood pressure and oxidative stress and restored angiogenic balance in pregnant rats with induced PE.

Study on the Transport Mechanism of Punicalagin in MDCK Cells Monolayer Model / 中国药师

Objective:To investigate the transport mechanism of punicalagin in MDCK monolayer model .Methods:The safe con-centration of punicalagin in MDCK cells was determined by CCK8 assay.Millicell -ERS was used to measure cell monolayer TEER value to determine the integrity of the cell monolayer .The effects of direction , drug concentration , time, P-gp inhibitor and EDTA-Na2 on the absorption and transport of punicalagin were studied systematically .And then the drug concentration was analyzed by HPLC to calculate the apparent permeability coefficient (Papp) and efflux ratio(ER).Results: Punicalagin transport in MDCK cells was time and concentration dependent .Punicalagin showed poor absorption in MDCK cells .Papp from apical to basolateral side ( AP-BL) within the concentration range of 100-300μg· ml-1 was (6.13 ±0.12) ×10 -7 cm· s-1 , (6.96 ±0.26) ×10 -7 cm· s-1 and (5.94 ±0.10) ×10 -7 cm· s-1 , respectively .P-gp inhibitor and EDTA-Na2 could significantly increase the transport of punicalagin in AP-BL direc-tion, while the transport decreased at 4℃.Conclusion:The transport mechanism of punicalagin might be passive diffusion as the dom-inating process involving active transportation .Punicalagin is one of P-gp substrates with exocytosis and absorbed via the paracellular route.

Optimization of Purification and Isolation of Punicalagin from Pomegranate Peel / 中国药师

Objective: To establish the methods for extraction and isolation of punicalagin from pomegranate peel, and further study the purification and quantification of punicalagin. Methods: Using an ultrasonic-assisted extraction method, punicalagin in pomegranate peel was extracted at room temperature by 50% ethanol with 20-fold volume of raw material. The content of punicalagin in the crude extract was determined by HPLC. To optimize the purification process of punicalagin, static adsorption and desorption experi-ments were employed to study five kinds of macroporous adsorbent resins (D101, A8-8, NKA-9, HPD-100 and HPD-500) for the one with the highest purification efficacy of punicalagin. In addition, the technical parameters of the macroporous adsorbent resin were opti-mized to obtain punicalagin with higher purity. Punicalagin was further separated and purified by using a reverse phase MCI GEL CHP20P column. Results:HPD500 resin showed the best ability to absorb and separate punicalagin in among five kinds of macro-porous adsorbent resins. The best technical parameters were as follows:the mass concentration of sample solution was 15 mg·ml-1 , the loading amount was 2BV, the pH was 2 and the eluting solvent was 8BV of 30% ethanol. With the best process as described a-bove, the content of punicalagin extracted from pomegranate peel increased from 10. 3% to 30. 7%. The obtained punicalagin could be further purified to 61. 3% from 30% in ethanol eluate by the reverse phase MCI GEL CHP20P column. Conclusion:HPD500 resin is the most effective in the purification of punicalagin from pomegranate peel, and the content of punicalagin can be dramatically increased after the purification by a reverse phase MCI GEL CHP20P column. The optimized process shows good reproducibility and stability.

Potential role of punicalagin against oxidative stress induced testicular damage / 亚洲男科学杂志(英文版)

Punicalagin is isolated from pomegranate and widely used for the treatment of different diseases in Chinese traditional medicine. This study aimed to evaluate the effect of Punicalagin (purity ≥98%) on oxidative stress induced testicular damage and its effect on fertility. We detected the antioxidant potential of punicalagin in lipopolysaccharide (LPS) induced oxidative stress damage in testes, also tried to uncover the boosting fertility effect of Punicalagin (PU) against oxidative stress-induced infertility. Results demonstrated that 9 mg kg-1 for 7 days treatment significantly decreases LPS induced oxidative damage in testes and nitric oxide production. The administration of oxidative stress resulted in a significant reduction in testes antioxidants GSH, T-SOD, and CAT raised LPO, but treatment with punicalagin for 7 days increased antioxidant defense GSH, T-SOD, and CAT by the end of the experiment and reduced LPO level as well. PU also significantly activates Nrf2, which is involved in regulation of antioxidant defense systems. Hence, the present research categorically elucidates the protective effect of punicalagin against LPS induced oxidative stress induced perturbation in the process of spermatogenesis and significantly increased sperm health and number. Moreover, fertility success significantly decreased in LPS-injected mice compared to controls. Mice injected with LPS had fertility indices of 12.5%, while others treated with a combination of PU + LPS exhibited 75% indices. By promoting fertility and eliminating oxidative stress and inflammation, PU may be a useful nutrient for the treatment of infertility.

Protective effects of pomegranate glycosides on LPS-induced acute lung injury to mice / 中国免疫学杂志

Objective:To investigate the protective effect of punicalagin in LPS induced ALI.Methods: ALI mice model was induced by intraperitoneal injection of LSP(20 mg/kg bodyweight) to induce acute lung injury in mice.A total of 60 mice were divided into 6 groups,10 rats in each group:normal control group,LPS group,LPS+punicalagin group (10,20,40 mg/kg bodyweight),LPS+dexamethasone group.2 h before LPS injection,punicalagin and dexamethasone were intraperitoneal injection in mice,and then by intra-peritoneal injection of a lethal dose of LPS, normal control group mice were given intraperitoneal injection of an equal volume of PBS.HE staining,observed the lung tissue of mice in the different treatment conditions of the pathological changes and the degree of lung injury,weighted the wet lung and dry lung of the mice respectively, observed degree of pulmonary edema in mice, inspect MPO activity of lung homogenate, analyzed the activation and infiltration of polymorphonuclear, counted the total number of cells and the number of inflammatory cells in alveolar lavage fluid, detected the concentration of inflammatory cytokines in bronchoalveolar lavage fluid with ELISA,and analyzed the impact of punicalagin on inflammation of lung tissue.Results: HE staining of lung tissue in mice and found that the LPS-induced lung inflammatory cell infiltration, interstitial edema and pulmonary parenchymal damage were significantly reduced.Different dose of punicalagin could significantly reduce the lung wet/dry lung weight ratio of ALI in mice ( P<0.05).Punicalagin ALI could reduce lung tissue caused exudation,edema.The different doses of punicalagin could significantly reduce the inflammatory cells in bronchoalveolar lavage fluid,the sum of the number of neutrophils and macrophages (P<0.05).Punicalagin could significantly reduce the concentration in the bronchoalveolar lavage cytokines IL-6,IL-12,TNF-αand IL-1β(P<0.05).The MPO activity was detected that punicalagin reduce neutrophil infiltration induced by LPS in the lung tissue.Conclusion: Punicalagin has protective effects on LPS induced acute lung injury.

Uptake Property Study on Punicalagin in Caco-2 Cells / 中国中医药信息杂志

Objective To study the uptake property of punicalagin in Caco-2 cells.Methods Caco-2 cell monolayer model was used to research the effects of punicalagin on cell uptake in different times, concentrations, pH values, temperatures and P-gp inhibitors. HPLC was used to determine the content of punicalagin in cells.Results The linearity of punicalagin concentration curve was found in the range of 0.024 8-0.992μg (r=0.997 9). Uptake determination of punicalagin in Caco-2 cells absorbed a certain amount of time, concentration and pH dependence and P-gp inhibitors could significantly increase the punicalagin uptake.Conclusion This method is simple and sensitive to determine the punicalagin concentration in Caco-2 cells, which is mainly passively transported. P-gp is involved in punicalagin transport process.

Preventive effects of punicalagin against ultraviolet B-induced damage to human HaCaT keratinocytes / 中华皮肤科杂志

Objective To investigate the mechanisms underlying the protection by punicalagin against ultraviolet B (UVB)-induced damage to keratinocytes.Methods Cultured human HaCaT keratinocytes were divided into several groups:blank control group receiving no treatment,punicalagin groups treated with various concentrations of punicalagin,UVB group irradiated with UVB at 30 mJ/cm2,combination groups pretreated with different concentrations of punicalagin followed by UVB radiation at 30 mJ/cm2.The concentrations of punicalagin were 5,10,20,40 and 80 μmol/L in the cell proliferation assay,10,20 and 40 μmol/L in the other assays.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of HaCaT cells,Hoechst and propidium iodide (PI) staining as well as flow cytometry to detect the apoptosis in cells,reverse transcription-PCR to quantify the mRNA expressions of matrix metalloproteinase-1 (MMP1) and tissue inhibitor of metalloproteinase-1 (TIMP1) in HaCaT cells,Western blot to determine the phosphorylation levels of the mitogen-activated protein kinase (MAPK) pathway-related proteins including P38,JNK and ERK.Statistical analysis was carried out by t test,one-way analysis of variance,and Dunnett's t-test.Results As the MTT assay showed,punicalagin at 10-40 μmol/L showed stronger pre-protective effects against UVB-induced damage to HaCaT cells compared with punicalagin at the other concentrations.The number of cells highly positive for both Hoechst and PI staining was larger in the UVB group than that in the blank control group,but smaller in the combination groups than in the UVB group.The percentage of apoptotic cells increased significantly in the UVB group compared with the blank control group (9.82％ ± 0.11％ vs.1.24％ ± 0.91％,P ＜ 0.01),but decreased significantly in the three combination groups (punicalagin (10,20 and 40 μmol/L) + UVB) compared with the UVB group (6.38％ ± 0.14％,5.24％ ± 0.17％ and 3.77％ ± 0.11％ vs.9.82％ ± 0.11％,all P＜ 0.01).Theexpression of MMP1 mRNA was significantly higher,but that of TIMP1 mRNA was significantly lower in the UVB group than in the blank control group (both P ＜ 0.01),whereas no statistically significant difference was observed in the expression of MMP1 or TIMP1 mRNA between the punicalagin groups and blank control group (all P ＞ 0.05).The pretreatment with punicalagin significantly reduced the expression level of MMP1 mRNA (P ＜ 0.01),but elevated that of TIMP1 mRNA (P ＜ 0.01) in the combination groups compared with the UVB group.As Western blot showed,the phosphorylation levels of P38,JNK and ERK were markedly increased in the UVB group (all P ＜0.01),but experienced no significant changes in the punicalagin groups (all P ＞ 0.05) compared with the blank control group,and decreased to different degrees in the combination groups compared with the UVB group (all P ＜0.01).Conclusion Punicalagin has a pre-protective effect on UVB-induced damage to HaCaT cells.

Antifungal activity of the ethanolic extracts of Punica granatum L. and evaluation of the morphological and structural modifications of its compounds upon the cells of Candida spp.

Ethanolic crude extracts prepared from the arils and seeds, pericarp, peels and from the whole fruit of Punica granatum, known as pomegranate, had their antifungal activity tested against Candida spp. The ethanolic crude extracts were analyzed by Mass Spectrometry and yielded many compounds such as punicalagin and galladydilacton. The extracts from the pericarp and peel showed activity against Candida spp., with MICs of 125 µg/mL. The effect of pericarp and peel extracts upon the morphological and structure of C. albicans and C. krusei were examined by scanning and transmission electron microscopy, with the visualization of an irregular membrane and hyphae, formation of vacuoles and thickening of the cell wall. The data obtained revealed potential antimicrobial activity against yeasts cells of the Candida genus, and the bioactive compounds could be responsible for changes in cell morphology and structure. The data obtained open new perspectives for future research in continuation to this study, where information such as determination of the site of action of the compounds could contribute to an alternative therapy against these organisms.