Overexpressed SIRT6 ameliorates doxorubicin-induced cardiotoxicity and potentiates the therapeutic efficacy through metabolic remodeling

Since the utilization of anthracyclines in cancer therapy, severe cardiotoxicity has become a major obstacle. The major challenge in treating cancer patients with anthracyclines is minimizing cardiotoxicity without compromising antitumor efficacy. Herein, histone deacetylase SIRT6 expression was reduced in plasma of patients treated with anthracyclines-based chemotherapy regimens. Furthermore, overexpression of SIRT6 alleviated doxorubicin-induced cytotoxicity in cardiomyocytes, and potentiated cytotoxicity of doxorubicin in multiple cancer cell lines. Moreover, SIRT6 overexpression ameliorated doxorubicin-induced cardiotoxicity and potentiated antitumor efficacy of doxorubicin in mice, suggesting that SIRT6 overexpression could be an adjunctive therapeutic strategy during doxorubicin treatment. Mechanistically, doxorubicin-impaired mitochondria led to decreased mitochondrial respiration and ATP production. And SIRT6 enhanced mitochondrial biogenesis and mitophagy by deacetylating and inhibiting Sgk1. Thus, SIRT6 overexpression coordinated metabolic remodeling from glycolysis to mitochondrial respiration during doxorubicin treatment, which was more conducive to cardiomyocyte metabolism, thus protecting cardiomyocytes but not cancer cells against doxorubicin-induced energy deficiency. In addition, ellagic acid, a natural compound that activates SIRT6, alleviated doxorubicin-induced cardiotoxicity and enhanced doxorubicin-mediated tumor regression in tumor-bearing mice. These findings provide a preclinical rationale for preventing cardiotoxicity by activating SIRT6 in cancer patients undergoing chemotherapy, but also advancing the understanding of the crucial role of SIRT6 in mitochondrial homeostasis.

Optimization of the ellagic acid synthesis process at the bioreactor level using non-conventional yeasts

Abstract Ellagic acid (EA) is a phenolic biomolecule. For its biosynthesis, a source of ellagitannins is required, such as strawberries and yeasts, as precursors of the tannase and ß-glucosidase enzymes responsible for hydrolysis of ellagitannins. Two experimental mixture designs were applied., varying the yeast concentration and the number of ellagitannins in the culture medium, evaluating the enzymatic activity and ellagic acid biosynthesis. Aiming to find the optimal compositions of the non-conventional yeasts assessed in the research to biosynthesize ellagic acid feasibly and efficiently using a response surface performing the statistical analysis in the StatGraphics® program for obtaining a higher yield and optimizing the ellagic acid synthesis process, the results indicate that the strains Candida parapsilosis ITM LB33 and Debaryomyces hansenii ISA 1510 have a positive effect on the synthesis of ellagic acid, since as its concentration increases in the mixture the concentration of ellagic acid in the medium also increases; on the other hand, the addition of Candida utilis ITM LB02 causes a negative effect, resulting in the compositions of 0.516876, 0.483124 and 2.58687E-9 respectively, for a treatment under the same conditions, an optimal value of ellagic acid production would be obtained. With an approximate value of 7.33036 mg/mL

Antitumor activity of irinotecan with ellagic acid in C6 glioma cells

SUMMARY OBJECTIVE: Irinotecan-based combination chemotherapies in malignant gliomas need to be examined. The aim of this study was to investigate the synergetic effect of ellagic acid, a natural polyphenolic antioxidant compound, with irinotecan, an inhibitor of topoisomerase I enzyme, on the growth, cadherin switch, and angiogenic processes of a glioma cell line. METHODS: A combination of 100 μM ellagic acid and 100 μM irinotecan was applied to rat C6 glioma cells for 24th, 48th, and 72nd h. The cell proliferation was evaluated by 5-bromo-2′-deoxyuridine immunocytochemistry. The expression levels of vascular endothelial growth factor, E-cadherin, and N-cadherin were measured using real-time polymerase chain reaction and their immunoreactivities using immunocytochemistry. RESULTS: The treatment of irinotecan with combining ellagic acid enhanced antitumor activity and the synergistic effect of these reduced the cell proliferation of C6 glioma by inhibiting the cadherin switch and promoting the antiangiogenic processes. CONCLUSIONS: Further research is required to prove a negative relationship between C6 glial cell proliferation and irinotecan with ellagic acid application. Our preliminary data suggest that even with the extremely short-term application, irinotecan with ellagic acid may affect glioma cells at the level of gene and protein expression.

Ellagic acid on the quality of the adhesive interface of class I composite resin restorations after aging

To evaluate the effect of ellagic acid on the inhibition of matrix metalloproteinase by analyzing the quality of the adhesive interface with bond strength measures in periods of 24 hours and six months of storage. Method: 40 healthy human third molars were prepared with class I cavities (5x4x3mm). The teeth were divided into four experimental groups: Group 1- without application of ellagic acid and storage time of 24 hours; Group 2- with ellagic acid/24 hours; G3- without ellagic acid/six months; Group 4- with ellagic acid/six months. Then, the cavities were restored with Single Bond Universal adhesive and Z350 composite resin, with and without the previous application of ellagic acid. Subsequently, hourglass-shaped specimens were obtained and subjected to the bond strength (BS) test (n = 10) in a universal testing machine. The bond test was performed after 24 hours and six months of storage. For the standard evaluation (n = 3) the samples were infiltrated with silver nitrate and placed in a developing solution for analysis in a Scanning Electron Microscope (SEM). The data obtained were analyzed with the Kruskal-Wallis non-parametric test, showing a statistically significant difference. Results: The highest bond strength values were found for the 24-hour groups followed by the groups with six months of storage. For nano-infiltration, groups G1 and G2 showed lower infiltration than groups G3 and G4. Conclusion: The previous application of ellagic acid did not affect the BS of the adhesive interface of the adhesive system analyzed, regardless of storage time.

Anti-obesity Effects of Extracts from Irvingia gabonensis on High Fat Diet-induced Obese Mice / 日本補完代替医療学会誌

Irvingia gabonensis seed extract is known to have an anti-obesity effect. In this study, we confirmed this effect in high-fat diet-fed mice using the commercially available “Africamangonoki” food product with functional claims. As a result, significant reductions in body weight and visceral fat (excluding paratesticular fat) were observed in the high-fat food intake group containing the commercially available “Africamangonoki” product, compared with the high-fat food only intake group. Therefore, it was considered that the seed extract of Irvingia gabonensis has an excellent anti-obesity effect. Conversely, no anti-obesity effect was observed with ellagic acid, which is a component involved, suggesting that components other than ellagic acid may also be involved in the anti-obesity effect observed in relation to Irvingia gabonensis.

Bioactivities of Alchemilla mollis, Alchemilla persica and Their Active Constituents

Abstract The aim of this study was to determine antimicrobial activities of Alchemilla mollis, Alchemilla persica as well as ellagic acid and miquelianin against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans by using microbroth dilution method and anti-inflammatory activity by using human red blood cell (HRBC) membrane stabilization method. Microbroth dilution method was used to determine the antimicrobial activities. Extracts possessed activity having MIC values of 2.5-5-10mg/ mL, compounds possessed activity having MIC values of 1.25-2.5-4-5mg/mL. A.mollis aerial parts displayed the highest anti-inflammatory activity (IC50=1.22±0.07mg/mL). Ellagic acid and miquelianin were also determined as anti-inflammatory agents with 0.57±0.01mg/mL and 1.23±0.02mg/mL IC50 values, respectively. Total phenolic content and tannin content of the A.mollis and A.persica were determined as 357.00±75.80mg, 282.50±28.70mg PGE/g plant material and 18.02%, 18.63% respectively according to the method described by European Pharmacopoeia. Ellagic acid, miquelianin and catechin were analyzed by HPLC. The highest catechin content was detected in A.persica roots (6.69±0.05g/100g plant material). A.mollis aerial parts contain higher miquelianin (0.39±0.02g/plant material) and ellagic acid (1.56±0.01g/ plant material) than A.persica.

Inhibition of α-glucosidase, pancreatic lipase, and antioxidant property of Myrcia hatschbachii D. Legrand containing gallic and ellagic acids / Inhibición de la α-glucosidasa, la lipasa pancreática y la propiedad antioxidante de Myrcia hatschbachii D. Legrand que contiene ácidos gálico y elágico

Several species of the Myrcia genus have been used in folk medicine to treat diabetes. Therefore, the aim of this work was to investigate the inhibitory activity of α-glucosidase and pancreatic lipase in the crude extract (EBF) and in the ethyl acetate fraction (FFA) of Myrcia hatschbachii, as well as to identify isolated phenolic compounds and to evaluate the antioxidant property and preliminary in vitro toxicity against Artemia salina. EBF (IC50: 3.21 µg/mL) and FFA (IC50: 1.14 µg/mL) showed inhibitory activity superior to acarbose (IC50: 193.65 µg/mL). In addition, they showed inhibitory effects of pancreatic lipase (IC50: 556.58 µg/mL for EBF and 532.68 µg/mL for FFA), antioxidant potential, absence of preliminary toxicity and presence of gallic andellagic acids in FFA. The relevant results in the inhibition of α-glucosidase and pancreatic lipase motivate new studies for the development of herbal medicines that assist in the treatment of diabetic patients.

Varias especies del género Myrcia se han utilizado en la medicina popular para tratar la diabetes. Por lo tanto, el objetivo de este trabajo fue investigar la actividad inhibitoria de la α-glucosidasa y la lipasa pancreática en el extracto crudo (EBF) y en la fracción de acetato de etilo (FFA) de Myrcia hatschbachii, así como identificar compuestos fenólicos aislados y evaluar la propiedad antioxidante y toxicidad in vitro preliminar contra Artemia salina. EBF (IC50: 3.21 µg/mL) y FFA (IC50: 1.14 µg/mL) mostraron una actividad inhibitoria superior a la acarbosa (IC50: 193.65 µg/mL). Además, mostraron efectos inhibitorios de la lipasa pancreática (IC50: 556.58 µg/mL para EBF y 532.68 µg/mL para FFA), potencial antioxidante, ausencia de toxicidad preliminar y presencia de ácidos gálico y elágico en FFA. Los resultados relevantes en la inhibición de la α-glucosidasa y la lipasa pancreática motivan nuevos estudios para el desarrollo de medicamentos a base de hierbas que ayudan en el tratamiento de pacientes diabéticos.

Biological and Oxidative Effect of Ellagic Acid on Saccharomyces cerevisiae: A New Way for Culture Developing

Abstract In this study, the effects of Ellagic acid (EA) on protein expression in yeasts and cellular development were investigated. Four groups were formed. Groups: 1) Control group; yeast only cultivated group; 2) Ellagic Acid (EA) group: EA (10%) given group; 3) Hydrogen peroxide (H2O2) Group: The group given H2O2 (15 mM); 4) EA + H2O2 group: EA (10%) + H2O2 (15 mM) group. After sterilization, EA (10%) and H2O2 (15 mM) were added to the Saccharomyces cerevisiae (S. cerevisiae) cultures and the cultures were grown at 30 °C for 1 hour, 3 hours, 5 hours and 24 hours (overnight). S. cerevisiae cell growth, lipid peroxidation MDA (malondialdehyde) analysis and GSH (glutathione) level were analyzed by spectrophotometer. Total protein changes were determined by SDS-PAGE electrophoresis and measured by the Bradford method. According to the obtained results, compared with the H2O2 group, cell development (1, 3, 5 and 24 hours), GSH level and total protein synthesis (24 hours) were increased with EA, while MDA level (24 hours) decreased. These results show that EA reduces oxidative damage, increases cell growth and it has a protective effect to promote protein synthesis in S. cerevisiae culture.

Establishmend of Content Determination of 5 Components and Their Content Difference Analysis in the Branch and Root of Juglans man dshurica / 中国药房

OBJECTIVE：To esta blish a method for si multaneous determination of 5 components in the branch and root of Juglans mandshurica as gallic acid ，ellagic acid ，1，6-di-O-galloyl-β-D-glucose，1，2，6-tri-O-galloyl-β-D-glucose and 1，2，3， 6-tetra-O-galloyl-β-D-glucose，and to analyze the content difference of above 5 components between the branch and root samples. METHODS：HPLC method was adopted. The determination was performed on Agilent Poroshell 120 SB-C18 column with mobile phase consisted of water （containing 0.2％ formic acid ）-acetonitrile （containing 0.2％ formic acid ）. A gradient elution was performed at a flow rate of 0.3 mL/min. The column temperature was 30 ℃ and the detection wavelength was 270 nm. The sample size was 5 μL. Independent samples t-test and partial least squares-discriminant analysis （PLS-DA）were applied for statistical analysis of 5 components. RESULTS ：The linear range of gallic acid ，ellagic acid ，1，6-di-O-galloyl-β-D-glucose，1，2， 6-tri-O-galloyl-β-D-glucose and 1，2，3，6-tetra-O-galloyl-β-D-glucose were 0.989-63.3，1.58-101，1.01-64.7，3.31-212，3.34-214 μg/mL （r≥0.997 3），respectively. RSDs of precision ，reproducibility and stability tests （12 h）were all lower than 3.2％. The average recoveries of the 5 components were 103.2％（RSD＝4.85％），99.1％（RSD＝2.80％），101.5％（RSD＝1.31％），102.9％（RSD＝ 2.73％）and 104.7％（RSD＝1.28％），respectively. The average contents of the above components in the branch of J. mandshurica were 0.296 5，0.621 1，0.562 5，3.111 7 and 3.451 3 mg/g，respectively. The average contents of above components in the root were 0.673 4，2.755 5，0.964 0，2.946 6 and 4.836 4 mg/g，respectively. The total contents of the 5 components in the branch and roo t of J. mandshurica were 8.043 2 and 12.175 9 mg/g，respectively. The contents of gallic acid ，ellagic acid and 1，6-di-O-galloyl- β-D-glucose in roots were significantly higher than those in branches （P＜0.05 or P＜0.01）. There were no significant differences in the contents of the other 2 components and the total contents of the 5 components in branches and roots （P＞0.05）. The cumulative interpretability （R 2X，R 2Y） and cumulative predictability （Q 2） of the model established by PLS-DA were 0.943，0.745，and 0.710 respectively. The model load diagram showed that the distance between the ellagic acid and the origin was the farthest ，and only variable projection importance of the content of the ellagic acid was greater than 1. CONCLUSIONS：The established method can be used for the content determination of 5 components in the branch and root of J. mandshurica . Except for 1，2，6-tri-O-galloyl-β-D-glucose，the contents of other 4 components and total contents of the 5 components in the root of J. mandshurica are higher than those of the branch. Ellagic acid is selected as the potential marker for discriminating the branch and root samples.

The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats

Abstract Objective This study aims to evaluate the effect of ellagic acid (EA) by measuring the levels of alveolar bone resorption and inflammatory and oxidative stress markers in the periodontal tissues and serum on the periodontal repair process related to experimental periodontitis in rats. Methodology Forty Wistar rats were divided into four study groups as follows: Group 1=healthy control (n=10); Group 2=EA control (15 mg/kg)(n=10); Group 3=periodontitis (n=10); Group 4=periodontitis+EA (15 mg/kg) (n=10). The periodontitis model was established by ligating bilateral mandibular first molars for 14 days. Then, rats were given normal saline or EA for another 14 days by gavage administration. Serum and gingiva myeloperoxidase (MPO) activity, 8-hydroxydeoxyguanosine(8-OHdG), and glutathione (GSH) levels were analyzed by ELISA. İmmunohistochemical analysis was used to detect Interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α) immunoreactivities in the periodontal tissues. Alveolar bone loss (ABL) and attachment loss (AL) was evaluated by histomorphometry analysis. Results ABL and AL were statistically higher in group 3 than in groups 1, 2 and 4 and in group 4 than in groups 1 and 2 (p<0.05). MPO activities in gingival tissue and serum were significantly increased in group 3 compared to groups 1 and 2 (p<0.05). Significantly higher serum GSH levels, lower gingiva, and serum 8-OHdG levels, and MPO activity were observed in group 4 compared to group 3 (p<0.05). Rats with periodontitis (group 3) expressed significantly higher immunoreactivities of IL-6 and TNF-α and lower IL-10 immunoreactivity compared to those other groups (p<0.05). IL-6 and TNF-α immunoreactivities significantly decreased and IL-10 immunoreactivity increased in group 4 after the use of EA compared to group 3 (p<0.001). Conclusions Our findings showed that EA provides significant improvements on gingival oxidative stress and inflammatory markers and alveolar bone resorption in the repair process associated with experimental periodontitis. Therefore, EA may have a therapeutic potential on periodontitis.

WHO prescribed shelf life assessment of Syzygium cumini extract through chromatographic and biological activity analyses

Background: Regulatory guidelines recommend shelf life of herbal products to be established throughsystematic stability studies.Objective: The study was designed to establish shelf life of Syzygium cumini extract through acceleratedand long-term stability testing as per WHO guidelines.Material and methods: The extract was stored under accelerated (40_x005F_x005F_x0001_C/75 %RH) and long-term (25_x005F_x005F_x0001_C/60%RH) stability conditions for 6 and 30 months, respectively. Samples were withdrawn at periodic intervals and analysed through two validated HPLC-UV methods (I and II) for fingerprint and quantitativeanalysis of markers. Antidiabetic activity of control and stability samples was evaluated by a-glucosidaseinhibitory model.Results: Method I generated a well resolved fingerprint of the control sample that was found to containgallic acid (GA, 1.45 % w/w) and ellagic acid (EA, 3.97 % w/w). The content of GA did not change underboth the stability conditions, but that of EA varied insignificantly (3.97e4.77 % w/w) under long-termconditions up to 24 months and subsequently decrease to 3.15 % w/w after 30 months. There was novisible change in LC-UV fingerprint of any stability sample with respect to control. a-Glucosidaseinhibitory activity of all stability samples also remained unaltered as compared to control sample (IC501.48 mg/mL). GA and EA did not elicit any activity at the concentrations present in the extract.Conclusion: Phytochemical composition and antidiabetic efficacy of S. cumini extract remain unchangedduring its storage under both accelerated and long-term stability conditions, which suggest its shelf lifeto be 30 months. Also, GA and EA are not appropriate anti-diabetic markers

Ellagic acid production using polyphenols from orange peel waste by submerged fermentation

Background: Biotechnological processes are part of modern industry as well as stricter environmental requirements. The need to reduce production costs and pollution demands for alternatives that involve the integral use of agro-industrial waste to produce bioactive compounds. The citrus industry generates large amounts of wastes due to the destruction of the fruits by microorganisms and insects together with the large amounts of orange waste generated during the production of juice and for sale fresh. The aim of this study was used orange wastes rich in polyphenolic compounds can be used as source carbon of Aspergillus fumigatus MUM 1603 to generate high added value compounds, for example, ellagic acid and other molecules of polyphenolic origin through submerged fermentation system. Results: The orange peel waste had a high concentration of polyphenols, 28% being condensed, 27% ellagitannins, 25% flavonoids and 20% gallotannins. The major polyphenolic compounds were catechin, EA and quercetin. The conditions, using an experimental design of central compounds, that allow the production of the maximum concentration of EA (18.68 mg/g) were found to be: temperature 30°C, inoculum 2 × 107 (spores/g) and orange peel polyphenols 6.2 (g/L). Conclusion: The submerged fermentation process is an effective methodology for the biotransformation of molecules present in orange waste to obtain high value-added as ellagic acid that can be used as powerful antioxidants, antibacterial and other applications.

Simultaneous determination of six components in Mongolia medicine Gurigumu-13 Pills by QAMS / 中草药

Objective: To establish a quantitative analysis of multi-components by single marker (QAMS) method for the simultaneous determination of six components of gallic acid, hydroxysafflor yellow A, geniposide, ellagic acid, costunolide and dehydrocostus lactone in Gurigumu-13 Pill, which is proved to be a scientific and feasible method in the quality analysis in Gurigumu-13 Pill. Methods: The relative factor (fs/i) of gallic acid, ellagic acid, hydroxysafflor yellow A, costunolide and dehydrocostus lactone were established by HPLC method with geniposide as internal standard, which were used to calculate the content of five constituents in the samples of Gurigumu-13 Pill. Meanwhile, external standard method (ESM) was used to calculate the content of six constituents. The difference between QAMS and ESM was analyzed to evaluate the accuracy of QAMS. Results: The fs/i of gallic acid, hydroxysafflor yellow A, ellagic acid, costunolide and dehydrocostus lactone were 0.481 0, 0.906 4, 0.170 9, 0.971 2 and 1.261 5, respectively. The content determination results of six batches of Gurigumu-13 Pill were calculated by the method of QAMS and ESM, with no significant difference in RSD < 2.0%. Conclusion: The fs/i established in the QAMS method with geniposide as the internal reference substance is accurate and simple. The QAMS method can be used for the multi-index quality evaluation of Gurigumu-13 Pill.

Chemical constituents and hypoglycemic activity of Phlomis tuberosa / 中草药

Objective: To study the chemical constituents and hypoglycemic activity of Phlomis tuberosa. Methods: The db/db diabetic mice was used to screen the hypoglycemic active site of P. tuberosa. The chemical constituents were isolated and purified by various separation and analysis techniques. The structures of these compounds were identified by spectroscopic analysis (1H-, 13C-NMR and MS). The hypoglycemic activities of these compounds were verified by the DPP-4 inhibitory activity in vitro. Results: Ethyl acetate extract of P. tuberosa showed significant hypoglycemic effect. Twenty-five compounds were isolated from the active site, containing β-stiosterol (1), stigmasterol (2), daucosterol (3), clerosterol-stigmast-4-ene-3,6-dione (4), 22-dehydro- stigmast-4-ene-3,6-dione (5), ellagic acid (6), ethyl gallate (7), gllagic acid (8), 4-hydroxybenzoic acid (9), 3,4-diohydroxybenzoic acid (10), cinnamic acid (11), p-hydroxy-cinnamic acid (12), caffeic acid (13), 5-hydromethylfuraldehyde (14), quinic acid (15), chlorogenic acid (16), ferulic (17), 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-2,6,10,14,18-eicosapentaen-1- yl)-2,5-cyclohexadiene-1,4-dione (18), 1-O-caffeoyl- quinic acid (19), 3,5-dimethoxy-4-hydroxy-benzene carbonic-1-O-β-D-glucoside (20), 2-O-butyl-α-D-fructofuranoside (21), n-octadecanoic acid (22), stearic acid (23), methyl-5-(hydroxymethyl) furan-2-carboxylate (24) and 4-hydroxy-3-methoxy-benzaldehyde(25). Nine compounds were obtained from the genus Phlomis for the first time, in which ellagic acid (6), quinic acid (15), and 1-O- caffeoylquinic acid (19) showed strong DPP-4 inhibitory activity with IC50 of 72.3, 89.2, and 103.4 μmol/L, respectively. The IC50 of the positive drug diprotin A was 50 μmol/L. Conclusion: Compounds (3-7 and 18-21) are obtained from the genus Phlomis for the first time. Compound 6, 15, and 19 show DPP-4 inhibitory activities.

Analysis of Chemical Components in Supernatant and Precipitate of Sanajon Oral Liquid Based on UPLC-Q-TOF-MSE Technology / 中国实验方剂学杂志

Objective:To analyze the main chemical components in the supernatant and precipitate of Sanajon oral liquid, so as to provide basis for establishing its quality standard and precipitation control technology. Method:UPLC-Q-TOF-MSE was used to analyze the chemical components in the supernatant and precipitate of this oral liquid. The analysis was performed on Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 μm) with the mobile phase of 0.1% formic acid solution (A) and acetonitrile (B) for gradient elution (0-1 min, 2%B; 1-2 min, 2%-5%B; 2-4 min, 5%-7%B; 4-6 min, 7%-24%B; 6-10 min, 24%-42%B; 10-12 min, 42%-54%B; 12-15 min, 54%-76%B; 15-18 min, 76%-100%B), the flow rate was set to 0.3 mL·min-1, the column temperature was 30 ℃, the injection volume was 2 µL. The mass spectrographic analysis was used with electrospray ionization (ESI), sample MS data was acquired by time-dependent MSE in negative ion mode, the collection range was m/z 50-1 200 (supernatant) and m/z 50-3 000 (precipitate). Then the chemical constituents were identified by the information of retention time, accurate relative molecular mass and secondary mass spectrum fragment. Result:Totally 61 compounds were identified in the supernatant, including tannins, phenolic acids, flavonoids, amino acids, organic acids, fatty acids, etc. Totally 15 compounds were identified in the precipitate, including tannins, phenolic acids, flavonoids, fatty acids, etc. Conclusion:The hydrolyzed tannin of Sanajon oral liquid may be the potential material basis of its precipitate, and its precipitate is likely to be a complex precipitate mainly composed of ellagic acid and tanned red. The established UPLC-Q-TOF-MSE can quickly and comprehensively analyze the chemical composition of Sanajon oral liquid, which can provide a scientific basis for the researches of its material basis, precipitation mechanism and quality control.

Study on in vitro Anti-bacterial Activity and Mechanism of Ellagic Acid on Streptococcus mutans / 中国药房

OBJECTIVE：To study the in vitro anti-bacterial activity and potential mechanism of ellagic acid on Streptococcus mutans，and to provide evidence for its prevention and treatment of dental caries. METHODS ：Using Compound chlorhexidine gargle as positive control ，5％DMSO as negative control ，bacteriostasis experiment was conducted by the method of drilling hole ， and bacteriostatic effects of 50，25，12.5，6.25，3.125，1.562 5 mg/mL ellagic acid on S. mutans was preliminarily determined by measuring the diameter of bacteriostatic ring. The minimal inhibitory concentration （MIC）and minimal bactericidal concentration （MBC）of ellagic acid on S. mutans were determined by microdilution method. Using 5％ DMSO as negative control ，the effects of 1/8 MIC，1/4 MIC，1/2 MIC and MIC ellagic acid on the formation of S. mutans biomembrane was determined by crystal violet staining. The changes of the biomembrane structure under the action of 1/2 MIC ellagic acid were observed by microscopy after fluorescence staining. Phenol sulfuric acid method and reducing coenzyme Ⅰoxidation method were used to determine inhibitory effects of 1/8MIC，1/4MIC，1/2MIC，MIC ellagic acid on S. mutans on extracellular polysaccharide （EPS）as well as effect on the activity of lactate dehydrogenase （LDH）in extracellular matrix. RESULTS ：Ellagic acid with concentration of 12.5～50 mg/mL produced an inhibitory ring on S. mutans with diameter greater than 15 mm. Under the action of 50 mg/mL ellagic acid ，the diameter of bacteriostatic ring was the same as that of Compound chlorhexidine gargle . MIC and MBC of ellagic acid to S. mutans were 12.5 mg/mL and 25 mg/mL. The survival rate of bacterial biomembrane after 1/8MIC-MIC ellagic acid treatment was significantly lower than that of the negative control （P＜0.01），and had a certain dose-response trend. After MIC ellagic acid treatment，the survival rate of bacterial biomembrane was （16.41±1.346）％. After fluorescence staining ，the structure of bacterial biomembrane was destroyed by 1/2 MIC ellagic acid. After treated with 1/8MIC-MIC ellagic acid ，its inhibitory rates on water-soluble EPS and water-insoluble EPS were increased significantly ，compared with negative control （P＜0.01）. After treated with 1/4MIC-MIC ellagic acid ，the activity of LDH in the extracellular matrix of bacteria increased significantly ，compared with negative control （P＜0.01），in dose-effect dependent trend. CONCLUSIONS ：Ellagic acid can inhibit the growth of S. mutans ，the mechanism of which may be associated with inhibiting EPS production ，reducing bacterial adhesion ，destroying bacterial cell membrane.

Hptlc Estimation Of Gallic Acid And Ellagic Acid In Amrtottara Kvatha Prepared In Two Ratios

Amrtottara kvatha is a decoction which is used primarily in the management of hyperpyrexia (jwara). Fresh stem of Tinospora cordifolia (Willd.) Miers (Guduchi), dried fruit rind of Terminalia chebula Retz (Haritaki), and dried rhizome of Zingiber offficinale Roscoe (Shunti) in the ratio 3:2:1 are its ingredients. From the point of view of drug design, it has the peculiarity that, its ingredients are specified to be added in a particular ratio, rather than in equal amounts as is the case in most of the compound formulations in Ayurveda. But, the rationale behind the drug design are not detailed enough to give much information regarding the effect of alteration of the drug ratio etc. Thus, in the present study an exploration was made with respect to its phytochemistry, by comparing with the decoction prepared in the general ratio of 1:1:1. The compounds Gallic acid and Ellagic acid were used for quantitative evaluation and comparison using HPTLC method. Estimation of Gallic acid in the samples showed that the amount of Gallic acid in Amrtottara kvatha prepared in classical ratio of 3:2:1 is significantly higher than that prepared in the altered ratio of 1:1:1 with a p value <0.05, though the amount by weight of Haritaki (source of Gallic acid) was unaltered in both, thus indicating the possibility of some complex phytochemical interactions among the constituents. With respect to Ellagic acid, there was no statistically significant difference in its quantity in the two decoctions compared. The method developed for HPTLC analysis in this study can be used as a technique for standardization of Amrtottara kvatha.

Antileishmanial activity and chemical composition from Brazilian geopropolis produced by stingless bee Melipona fasciculata

ABSTRACT Geopropolis is produced by some stingless bee species such as Melipona fasciculata and consists of a mixture of plant resins, salivary secretions of the bee, wax, and soil. This study evaluated the antileishmanial activity in vitro, cytotoxicity and chemical composition of geopropolis produced by M. fasciculata in the savannah region of Maranhão, Brazil. The geopropolis extract was obtained through maceration using in 70% ethanol. The hydroalcoholic extract of geopropolis after liquid-liquid partition yielded the hexane, chloroformic, ethyl acetate and hydroalcoholic fractions. Antileishmanial activity was evaluated against promastigote and intracellular amastigote of Leishmania amazonensis. Cytotoxic was realized in BALB/c mice peritoneal macrophages. Chemical analysis was performed by gas chromatography-mass spectrometry and high performance liquid chromatography coupled to an ultraviolet-visible detector. The geopropolis inhibited the L. amazonensis promastigotes growth and was effective in reducing the infection of murine macrophages since the number of internalized amastigotes were smaller in cells treated with the geopropolis extract in relation to the untreated group. The ethyl acetate fraction was the most active and showed the highest index of selectivity as antileishmanial product. The geopropolis from M. fasciculata had an antileishmanial effect, mainly after the obtention of the ethyl acetate fraction that improved the activity without increasing the cytotoxicity against murine macrophages. Analysis for gas chromatography-mass spectrometer identified as main compounds the gallic and ellagic phenolic acids, either in the extract or in the active fraction. The results obtained by high performance liquid chromatography it was possible to confirm the presence and quantify the concentration gallic and ellagic acid either in the extract or in the active fraction. These results suggest that the antileishmanial activity of geopropolis is related to the presence of derivatives of these phenolic acids, mainly gallic and ellagic acids.

Pharmaceutical mechanism and control strategy for storage of ellagic acid stably in traditional Chinese medicine oral liquid / 药学学报

Ellagic acid is ubiquitous in plants and is considered as a potential candidate for antioxidant and antineoplastic drugs. However, ellagic acid has poor solubility and precipitates easily even after initial solubilization. Improvement of its bioavailability has been a concern of pharmaceutical industry. It was found that storage in Sanlejiang oral liquid at low temperature keeps its stability. Ellagic acid is anomalous in a way that is easily soluble at low temperatures but precipitates at high temperatures. In order to reveal the mechanism of this phenomenon and develop precipitation prevention and control strategies, ellagic acid in Sanlejiang oral liquid was stored at high, medium and low temperatures for three months. The changes of composition and phase state of the whole system during storage were systematically tracked and studied by means of precipitation amount or morphology, HPLC chemical profile of supernatant versus precipitates, and comprehensive characterization of physical phase state. The results show that the amount of precipitation at low temperature is only 1/3 of that at normal room temperature. As the temperature rises, the sedimentation increases sharply. HPLC analyses of supernatant versus precipitates revealed that ellagic acid precipitation originated from two ways: chemical degradation and physical deposition. The chemical sedimentation is related to the hydrolysis of tannins under acidic condition, forming chebulagic acid and corilagin. Physical sedimentation is related to the decrease of the association degree and viscosity of polyphenol colloids when temperature rises. This study elucidated the stability mechanism of ellagic acid in liquid preparations of TCM, and provided the mechanistic basis for efficient utilization and solution prepartion of ellagic acid.

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy / 中国病理生理杂志

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy.METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group.The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 m L/kg, suspended in corn oil, ig).After 24 h of operation, the rats from each group were sacrificed and their brains were collected.TTC staining and HE staining were used to define the infarct areas and brain structure.The autophagy-related proteins beclin-1, P62, LC3-II/-I and Atg5 in the cortex in each group were compared by Western blot.In vitro, PC12 cells were divided into 3 groups:control group, Coand CoEA pretreatment group.Co800μmol/L was added to the PC12 cells to induce an anoxic environment.The PC12 cells were pretreated with EA at 8μmol/L and the cell viability was measured by CCK-8 assay.The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining.MDC staining and TM-RE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively.The autophagy-related proteins in PC12 cells were also investigated.RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining.HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage.EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group.Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-II/-I in the cortex were increased (P<0.01) , and P62 protein expression was decreased (P<0.01) in HIE group.Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-II/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01).In vitro, compared with CoPC12 cells in CoEA pretreatment group showed a lower ROS level.Moreover, the cells in CoEA pretreatment group exhibited higher mitochondrial membrane potential than that in CoMDC staining in Coshowed high value of fluorescence and increased number of autophagosomes.EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12cells.Furthermore, the protein levels of Atg5, beclin-1 and LC3-II/-I in Cowere higher (P<0.01) , and the protein expression of P62 was lower (P<0.01) than those in control group.In CoEA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-II/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with Co (P<0.01).CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.