Simultaneous determination of five active ingredients including gallic acid in different dosage forms of Sana preparation by reverse-phase high-performance liquid chromatography / 中国基层医药

Objective:To establish reverse-phase high-performance liquid chromatography and simultaneously determine gallic acid, methyl gallate, corilagin, Sennoside B, and Sennoside A levels in Sana preparations.Methods:From January to December 2021, Phenomenex Hydro-RP 80A column (4.60 mm × 250 mm, 4 μm) was used. Elution was conducted using mobile phases methanol (A)-0.2% formic acid (B). The following gradients were applied: 1%-3%A for 0-18 minutes, 3%-15%A for 18-19 minutes, 15%-17%A for 19-40 minutes, 17%-25%A for 40-45 minutes, 25%-35%A for 45-65 minutes, 35%-60%A for 65-95 minutes, 60%-90%A for 95-96 minutes, 90%-1%A for 96-97 minutes. The flow rate was 1.0 mL/minute. The column temperature was 35 ℃. The detection wavelength was 270 nm.Results:The linear ranges of gallic acid, methyl gallate, corilagin, Sennoside B and Sennoside A were 0.182-1.099 μg ( r = 0.999 9), 0.046-0.278 μg ( r = 0.999 2), 0.266-1.598 μg ( r = 0.999 4), 0.172-1.036 μg ( r = 0.999 2), and 0.176-1.056 μg ( r = 0.999 9). The average dosing recovery rates were 100.02%, 99.14%, 99.38%, 101.77%, and 100.92%, respectively. Conclusion:Reverse-phase high-performance liquid chromatography can be used for quality control of Sana preparations because of high accuracy, sensitivity, reliability, and reproduction.

Shabyar Ameliorates High Glucose Induced Retinal Pigment Epithelium Injury Through Suppressing Aldose Reductase and AMPK/mTOR/ULK1 Autophagy Pathway.

Shabyar (SBA) is a traditional medicine formula for relieving vision loss caused by factors including diabetic retinopathy (DR) in clinics. However, the mechanism of it on retina protective effect still unclear. The present study aimed to investigate whether its protective effect was related to aldose reductase (AR) inhibition and retinal pigment epithelial cell injury mediated by autophagy or not. Human retinal pigment epithelial cells (ARPE-19) induced by high glucose was used as a model in vitro, with Epalrestat (EPL, AR inhibitor) and Difrarel (DFR, DR therapeutic drug) as positive controls. Western blotting and Polyol pathway products assay showed that SBA reduced the expression of AR protein and the content of ROS, and sorbitol, increased the level of Na+-K+-ATPase and alleviated cell edema. Western blotting and DCFH-DA probe assay showed that SBA decreased pAMPK/AMPK and pULK1/ULK1 which associated with autophagy initiation, down-regulated Beclin-1, Atg3, Atg5, Atg7, LC3 II and Bax/Bcl2 ratio, and up-regulated pmTOR/mTOR, SQSTM1/p62 and mitochondrial membrane potential (MMP), reduces intracellular autophagosomes. Real-Time PCR assay showed that SBA had no significant effect on mRNA expression of AR and mTOR. These data demonstrated that SBA treatment inhibits the autophagy of ARPE-19 through the AMPK/mTOR/ULK1 signaling pathway, and reduced early-stage apoptosis occurred by high glucose. These findings reveal the protective role and mechanism of SBA on retinal pigment epithelium, and provide experimental basis for the clinical application of SBA in the treatment of DR.

Chemical Components and Hepatoprotective Mechanism of Xwak Granule in Mice Treated with Acute Alcohol.

OBJECTIVE: To evaluate the hepatoprotective mechanism of Xwak granule (Xwak) in treatment of mice with alcoholic liver injury via activating ERK/NF-κB and Nrf/HO-1 signaling pathways. METHODS: The chemical composition of Xwak was tested by liquid chromatography coupled with mass spectrometry (LC-MS). Herein, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical tests were performed in vitro. The hepatoprotective effect of Xwak was assessed at different concentrations (1.5, 3, and 6 g/kg) in a mouse model of alcoholic liver injury. RESULTS: Totally, 48 compounds, including 16 flavonoids, 8 tannins, 9 chlorogenic acids, and 15 other compounds, were identified from Xwak. Xwak showed to have a satisfactory antioxidant activity in vitro. In a group of Xwak-treated mice, the serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were decreased compared with a group of the mouse model of alcoholic liver injury. In addition, the levels of antioxidant enzymes, such as glutathione peroxidase (GSH-PX), total superoxide dismutase (T-SOD), and catalase (CAT), were noticeably increased and the levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), and interleukin-6 (IL-6) were markedly reduced in the liver of mice. The state of oxidative stress in the mouse model of alcoholic liver injury was improved after treatment with Xwak. The improvement of inflammation-mediated disruption may conducive to the Xwak activity in the control of liver injury. The signals of p-ERK1/2, p-NF-κB, COX-2, iNOS, CYP2E1, Nrf, and HO-1 were significantly induced in the liver of mice after treatment with Xwak. CONCLUSIONS: The abovementioned findings indicated that the hepatoprotective mechanism of Xwak could be achieved by activating ERK/NF-κB and Nrf/HO-1 signaling pathways to alleviate oxidative stress and inflammatory.

Optimization of Extraction Technology of Majun Mupakhi Ela and its Effect on Hydrocortisone-induced Kidney Yang Deficiency in Mice.

We used Box-Behnken design-based (BBD) response surface methodology (RSM) in this research to optimize the extraction process of Traditional medicine Majun Mupakhi Ela (MME) and evaluate its effect on hydrocortisone-induced kidney yang deficiency. Three independent parameters were applied to evaluate the maximum phosphodiesterase type 5 (PDE5) inhibition activity of MME extracts in vitro. The optimal processing conditions (extraction time 2 h, solid-liquid ratio 1:16, extraction once) gave a maximum PDE5 inhibition rate of 84.10%, flavonoid content of 0.49 mg/ml, icariin content of 0.028 mg/ml and targeted extraction yield of 26.50%. In animal experiments, MME extracts significantly increased the adrenal mass index, semen weight index, preputial gland weight index, and penis weight index in mice; in the middle and high dose group, the level of serum testosterone increased by 7664.29% and 14207.14% respectively, compared with the model group, and the level of PDE5 decreased by 67.22% and 74.69% respectively compared with the control group. These results indicate that MME has a significant positive effect on the hypothalamus-pituitary-gonadal axis, improve mating ability and not only has inhibits PDE5 activity but also significantly inhibits the expression of PDE5 in penile tissues, potential to become erectile dysfunction (ED) therapies for the clinical management of patients with kidney yang deficiency.

Anti-Inflammatory Effect of Rosa rugosa Flower Extract in Lipopolysaccharide-Stimulated RAW264.7 Macrophages.

Rosa rugosa Thunb, a deciduous shrub of the genus Rosa, has been widely used to treat stomach aches, diarrhoea, pain, and chronic inflammatorydisease in eastern Asia. In recent years, our research team has extensively studied the Rosa rugosa flowerextract, and specificallyundertook pharmacological experiments which have optimized the extraction process. Our methods have yielded a standard extract enriched in phenolic compounds, named PRE. Herein, we expand our efforts and evaluated the antiinflammatoryactivity of PRE on lipopolysaccharide (LPS)-induced inflammationin RAW 264.7 macrophages. PRE significantlyinhibited production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and interleukin 1ß (IL-1ß), as well as expression of their synthesizing enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX-2). Furthermore, PRE inhibited activity of mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappa B (NF-κB) signaling pathway. Our findingsare the firstto explain the anti-inflammatorymechanism by PRE in LPS-stimulated macrophages. Given these results, we propose that PRE has therapeutic potential in the prevention of inflammatory disorders.

Simultaneous Determination of Gallic Acid and Ellagic Acid in Xihe Majun Ointment by HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of gallic acid and ellagic acid in Xihe majun ointment. METHODS:HPLC method was performed on the column of X-Bridge C18 with mobile phase of methanol-acetoni-trile-0.2% Phosphoric acid solution (gradient elution) at a flow rate of 1.0 ml/min,detection wavelength was 254 nm,detection temperature was 30 ℃,and injection volume was 10 μl. RESULTS:The linear range was 0.132-0.792 μg for gallic acid (r=0.999 9) and 0.108 0-0.648 2 μg for ellagic acid (r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 3%;recoveries were 95.15%-99.69%(RSD=1.64%,n=9) and 97.24%-100.43%(RSD=1.24%,n=9). CONCLUSIONS:The method is simple,accurate and sensitive,and can be used for the content determination of gallic acid and ellagic acid in Xihe majun ointment.

Anti-Arthritic and Antiinflammatory Effects of the Traditional Uighur Formula Kursi Caper In Vivo.

Kursi Caper (KC) is a Uighur medicine based on caper which is widely used to treat arthritis and rheumatism, and preliminary studies in our laboratory showed that this traditional formula may possess potent antiinflammatory effects. This study confirms the antiinflammatory effect of KC in the adjuvant induced arthritis (AIA) model, the carrageenan and cotton-pellet induced granuloma rat models, and further investigates in vivo the mechanism of action by measuring relevant indicators of anti-arthritic activity. KC showed significant and dose-dependent anti-arthritic and antiinflammatory effects, demonstrated by reduced paw edema and arthritic scores in all animal models. Histopathological examination showed that KC reduced levels of synovial inflammatory factors in AIA rats. The overproduction of TNF-α and IL-1ß was attenuated, and CAT, MDA and SOD levels were restored to normal in KC-treated rats. KC also significantly reduced LPS-induced proliferation of B lymphocytes and ConA induced proliferation of T lymphocytes in a dose-dependent manner. Flow cytometry showed that the high dose KC-treated group had a significantly decreased frequency of Th17 cells. This study indicates that KC can significantly attenuate arthritis and inflammation in rats by decreasing the levels of inflammatory cytokines, regulating oxidative stress, reducing lymphocyte proliferation and decreasing Th17. This supports the traditional use of KC as a potential modern therapeutic agent for the treatment of arthritis and related conditions.

[Study on mechanism of abnormal savda munziq flavonoids in induction of apoptosis of Hep2 cells].

OBJECTIVE: To investigate the effect of Abnormal Savda Munziq (ASMq) flavonoids on proliferation, apoptosis and apoptosis-related gene expression in human hepatoma (HepG2) cells in vitro and to probe the mechanism. METHOD: The effects of ASMq flavonoids on proliferation, apoptosis and apoptosis-related gene expression of HepG2 cells were investigated respectively by MTT assay, gel electrophoresis, flow cytometry and RT-PCR. RESULT: ASMq flavonoids significantly inhibited growth of HepG2 cells in vitro, arrested HepG2 in the sub-G, phase, induced cell apoptosis and significantly down-regulated expression level of Bcl-2 mRNA, and up-regulated expression of p53, p21, Bax gene mRNA expressions. CONCLUSION: ASMq flavonoids has significantly regulative action on growth, apoptosis and apoptosis-related gene expression of cancer cells in vitro, which possibly are the important way to excert anticancer effect, and flavonoids are possibly a main active component of ASMq for exerting the anticancer effect.