Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation,chemical profiling and biochemical assay / 药物分析学报

Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood.Recently,we assessed the inhibitory potentials of several HMs against human pancreatic lipase(hPL,a key therapeutic target for human obesity),among which the root-extract of Rhodiola crenulata(ERC)showed the most potent anti-hPL activity.In this study,we adopted an integrated strategy,involving bioactivity-guided fractionation techniques,chemical profiling,and biochemical assays,to identify the key anti-hPL constituents in ERC.Nine ERC fractions(retention time=12.5-35 min),obtained using reverse-phase liquid chromatography,showed strong anti-hPL activity,while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS/MS).Among the identified ERC constituents,1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(PGG)and catechin gallate(CG)showed the most potent anti-hPL activity,with pIC50 values of 7.59±0.03 and 7.68±0.23,respectively.Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner,with inhibition constant(Ki)values of 0.012 and 0.082 μM,respectively.Collectively,our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC,as well as to elucidate their anti-hPL mechanisms.These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

Chemical constituents of tannin from roots of Rheum palmatum / 中草药

Objective: To study the chemical constituents of tannin from the roots of Rheum palmatum. Methods: Chemical constituents of tannins were isolated by silica gel column chromatography, medium pressure column chromatography, and semi-preparative HPLC from the roots of R. palmatum. The structures were elucidated on the basis of spectral data analysis. Results: Four tannins, 3-O-cinnamoyl-1-O-galloyl-β-D-glucopyranoside (1), 2-O-cinnamoyl-1-O-galloyl-β-D-glucopyranoside (2), 2-O-cinnamoyl-1,6-di-O-galloyl-β-D-glucopyranoside (3), and 6-O-cinnamoyl-1-O-galloyl-β-D-glucopyranoside (4) were isolated from the roots of R. palmatum. Conclusion: Compound 1 is a new compound named palmatoside.

Correlation between bone protection of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose and Nrf2/HO-1 signaling pathway / 药学学报

To study the osteoprotective effect of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose (PGG) its anti-osteoblast apoptosis related mechanism was investigated. A model of zebrafish osteoporosis induced by prednisolone (Pred, 25 μmol·L-1) was established in vivo, and calcein staining was used to detect the effect of PGG on the bone area of ​​zebrafish. Bone marrow mesenchymal stem cells were cultured in vitro, and the number of calcified nodules was observed by alizarin red staining, and the relevant indexes of osteoblast differentiation runt-related transcription factor 2 (Runx 2), osteocalcin (OCN) mRNA level were detected by qRT-PCR. The osteoblast cell line MC3T3-E1 cells was cultured in vitro, and 400 μmol·L-1 hydrogen peroxide (H2O2) was used to intervene the injury to detect the effect of PGG on osteoblasts under oxidative stress. The effect of PGG on osteoblast activity was detected by MTT assay. The effect of PGG on apoptosis was observed by Hoechst 33342 staining. Western blot was used to detect the expression of Bcl-2, Bax, nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). DCFH-DA fluorescence staining for detection of reactive oxygen species (ROS) levels. JC-1 staining was used to detect mitochondrial membrane potential levels. The results showed that PGG could significantly increase the vertebral area of ​​the zebrafish model when compared with the model group. On the 14 th day of osteoblast differentiation, the number of calcified nodules in the PGG group was significantly increased when compared with the control group and the mRNA levels of Runx 2 and OCN were also significantly increased. In addition, under oxidative stress, PGG could increase osteoblast viability, significantly reduce the number of apoptotic cells, and increase the ratio of Bcl-2/Bax. Fluorescence staining results show that PGG decreased intracellular ROS fluorescence density and increased mitochondrial membrane potential. Western blot data showed that PGG could promote the expression of Nrf2 in the nuclear and enhance the expression of downstream protein HO-1. In conclusion, PGG could improve osteoporosis in zebrafish, and this effect may be related to the regulation of Nrf2/HO-1 signaling pathway to improve mitochondrial dysfunction, anti-oxidative stress in osteoblast apoptosis and promote bone formation. This study provides new ideas and clues for the discovery of anti-osteoporosis drugs.

Simultaneous determination of thirteen components of Mortan Cortex in different regions by HPLC / 中草药

Objective: To establish an HPLC method for the analysis of thirteen main chemical constituents (gallicacid, 5-hydroxymethylfurfural, methylgallate, oxypaeoniflora, catechins, paeoniflorin, 1,2,3,6-tetra- O-galloyl-D-glucose, 1,2,4,6-tetra-O- galloyl-D-glucose, benzoicacid, 1,2,3,4,6-penta-O-galloyl-D-glucose, mudanpioside C, benzoyloxypaeoniflorin, and paeonal) in Moutan Cortex from different origins. Methods: The methanol (50%) reflux extraction was adopted to Moutan Cortex. Separation was carried out on C18 (250 mm × 4.6 mm, 5 μm), the mobile phase was eluted with acetonitrile-0.5% phosphoric acid. The flow rate was 1.0 mL/min, the detection wavelength was 254 nm, and the column temperature was 30 ℃. Results :The linear relation of thirteen main active components measured in the range of mass concentration was good with perfect precision, repeatability, and stability, the value of which was all more than 0.999 5. Conclusion: The HPLC method established for simultaneous determination of thirteen main chemical compositions in Mortan Cortex is effective, accurate, and reproducible, which can be used for the quality control of Mortan Cortex.

Inhibitory effects of tannins from leaves of Hippophae rhamnoids and their anti-inflammatory and anti-obesity effects / 中草药

Objective: To study the anti-inflammatory and anti-obesity constituents from the leaves of Hippophae rhamnoids. Methods: Several open-column chromatographic techniques and semi-preparative HPLC were used to separate and purify the compounds in H. rhamnoids. The structures of isolated compounds were elucidated by the spectroscopic analysis. Their inhibitory effects on nitric oxide production in RAW264.7 cells, and triglyceride accumulation in 3T3-L1 cells were examined. Results: Eighteen tannins and other compounds were isolated and identified as 1,2,6-tri-O-galloyl-β-D-glucopyranose (1), 1,3,6-tri-O-galloyl-β-D- glucopyranose (2), 1,4,6-tri-O-galloyl-β-D-glucopyranose (3), 1,3,4,6-tetra-O-galloyl-β-D-glucopyranose (4), 1,2,3,6-tetra-O-galloyl- β-D-glucopyranose (5), 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6), 1-O-galloyl-4,6-(S)-HHDP-β-D-glucopyranose (7), 1-O- galloyl-2,3-(S)-HHDP-β-D-glucopyranose (8), 1,3-di-O-galloyl-4,6-(S)-HHDP-β-D-glucopyranose (9), 1,6-di-O-galloyl-2,3-(S)- HHDP-β-D-glucopyranose (10), casuarictin (11), 1,2,3-tri-O-galloyl-4,6-(S)-HHDP-β-D-glucopyranose (12), 1,4,6-tri-O-galloyl-2,3-(S)- HHDP-β-D-glucopyranose (13), hippophaenin B (14), pedunculagin (15), casuarinin (16), ellagic acid (17), and pinitol (18). Conclusion: Tannins from the leaves of H. rhamnoides showed anti-inflammatory and anti-obesity activities. Compounds 2, 3, 5, 6, 8, 10, 12, and 13 were isolated from this genus for the first time.

Virtual screening of Chinese herbal monomer against EV71 3C protease / 中草药

Objective: To search for Chinese herbal monomer with the antiviral activity of enterovirus 71 (EV71) 3C protease by virtual screening. Methods: A total of 1998 monomers from antiviral Chinese herbs downloaded from TCM Database@Taiwan database were chosen as screening ligands, and EV71 3C protease was selected as a target. The binding mode of Chinese herbal monomer and EV71 3C protease was simulated through the molecular docking tools of AutoDock Vina. The monomers were sorted according to the binding free energy. The binding poses of the monomers with docking scores less than -37.673 kJ/mol were visualized by Discovery Studio 2018 Visualizer program. Results: Two monomers, namely, procyanidin B5 and 1,2,3,6-tetra-O-galloyl- β-D-glucose, were obtained with the lowest binding energies. Conclusion: The promising candidate monomers with anti-EV71 3C protease activity were screened out from traditional Chinese medicine, providing an alternative method for further development of therapeutically useful anti-EV71 agents.

Simultaneous Determination of 8 Non-anthraquinone Constituents in Rheum palmatum by HPLC / 中国药房

OBJECTIVE： To establish a method for simultaneous determination of 8 non-anthraquinone constituents in Rheum palmatum. METHODS： HPLC method was adopted. The determination was performed on Symmetry C18 column with mobile phase consisted of methanol-0.1％ phosphoric acid （gradient elution） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃ and detection wavelength was 280 nm. Sample size was 30 μL. RESULTS： The linear range of gallic acid， catechin， epicatechin， resveratrol 4′-O-glucopyranoside， epicatechin gallate， resveratrol 4′-O-β-D-（6″-O-galloyl）-glucopyranoside， sennoside A， 4′-hydroxyphenyl-2-butanone-4′-O-β-D-（2″-O-galloyl-6″-O-p-hydroxy cinnamyl）-glucopyranoside were 6.16-2 464 ng（r＝0.999 9）， 37.4-14 960 ng（r＝0.999 9）， 7.635-3 054 ng（r＝0.999 7）， 7.63-3 052 ng（r＝0.999 9）， 8.32-3 328 ng（r＝0.999 9）， 11.5-4 600 ng（r＝0.999 9）， 16.08-6 432 ng（r＝0.999 9）， 29.3-11 720 ng（r＝0.999 9）， respectively. The limits of quantitation were 3.48， 4.30， 6.40， 4.40， 3.39， 2.87， 8.40 and 4.95 ng， respectively. The limits of detection were 2.32， 2.58， 2.40， 2.64， 2.26， 1.23， 4.20， 2.97 ng， respectively. RSDs of precision， stability and reproducibility tests were all lower than 5％. Recoveries were 94.32％- 100.54％（RSD＝2.78％，n＝6）， 91.15％-99.36％（RSD＝3.72％，n＝6）， 92.16％-98.04％（RSD＝2.39％，n＝6）， 93.41％-100.73％（RSD＝3.17％，n＝6）， 93.89％-98.40％（RSD＝1.99％，n＝6）， 92.61％-101.74％（RSD＝3.71％，n＝6）， 92.66％-103.40％（RSD＝3.76％，n＝6）， 95.45％-102.70％（RSD＝3.06％，n＝6）， respectively. CONCLUSIONS： The established method is simple， accurate and specific， and can be used for the simultaneous determination of 8 non-anthraquinone constituents in R. palmatum.

Intestinal absorption of phenolic acids in Rhus chinensis extracts by in situ single-pass perfusion model in rats / 中国中药杂志

The intestinal absorption properties of four main effective components(gallic acid, ocinolglucoside, ethyl gallate and penta-O-galloyl-β-D-glucose) in Rhus chinensis extracts were investigated by in situ single-pass intestinal perfusion model in rats. The liquid accumulation of perfusion was corrected by gravimetry. The HPLC method was established to determine the concentration of the four effective components in the intestinal perfusion. It showed significant differences(Pethyl gallate>gallic acid>ocinolglucoside, with significant differences between them(P<0.05). In conclusion, gallic acid, orpheolglucoside, ethyl gallate and pentacyl-glucose could be absorbed in the whole intestine. Their absorption rate and permeation ability were related to the intestinal section and the perfusate concentration. These results indicated potential active transport or facilitated diffusion in the intestinal transport process of the four effective components.

Metabolite characterization of Penta-O-galloyl-β-D-glucose in rat biofluids by HPLC-QTOF-MS / 中草药·英文版

Objective: To understand the in vivo metabolic fate of 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) naturally existed in many medicinal herbs and food plants such as Rhus chinensis, Paeonia suffruticosa, Paeonia lactiflora and Mango. Methods: The metabolites of PGG in rat biofluids were characterized using high performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS). Results: Ten metabolites in urine, five metabolites in feces and two metabolites in plasma, were observed when the rats were administrated with a single intravenous injection of PGG (20 mg/kg). Conclusion: PGG is firstly metabolized to gallic acid, then gallic acid undergoes sulfation, glucuronidation and methylation by rat liver. The determination of metabolites and the proposed metabolic pathway of PGG in vivo will be benefit to gain deeper insights into its pharmacological activities.

Schinus terebinthifolius: phenolic constituents and in vitro antioxidant, antiproliferative and in vivo anti-inflammatory activities

ABSTRACT Schinus terebinthifolius Raddi, Anacardiaceae, native to Brazil, is referred to as "pimento-rosa" and is used to treat inflammatory disease in folk medicine. Studies have reported important pharmacological properties, but these effects have still not been fully exploited. This study reports that the crude extract and isolated compounds of S. terebinthifolius (leaves) have in vitro antioxidant, antiproliferative, and in vivo anti-inflammatory activities. The samples were evaluated for antioxidant activity using 2, 2-diphenyl-1-picrylhydrazyl, β-carotene/linoleic acid and 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulphonic acid reagents. The anti-inflammatory effects were assayed against a carrageenan-induced paw oedema model in mice to test doses of 10, 100 and 300 mg/kg at different time points in addition to myeloperoxidase activity analysis. The antiproliferative activity was evaluated using ten human tumour cell lines. Two derivatives of gallic acid and four flavonoids were isolated and exhibited considerable antioxidant activity. The extract and its compounds showed selectivity towards ovarian cancer cells, with growth inhibitory activity values ranging from 1.9 to 6.5 µg/ml. Sample extracts and methyl gallate significantly inhibited carrageenan-induced oedema in the mice paw oedema experimental model. The calculated topological polar surface area for methyl gallate (86.98 Å2) showed good intestinal absorption. The effects reported herein are be related to the presence of flavonoids and the galloyl phenolic derivative content.

Comparison on chemical constituent changes of gallnut fermented Chinese gall leaven by HPLC fingerprint / 中草药

Objective To study and establish the HPLC fingerprint standard for the quality analysis and compare effects on the chemical composition of gallnut by ferment of Chinese gall leaven. Methods The fingerprint of Chinese gall leaven was built by Waters Symmmetry ShieldTM RP18 (250 mm × 4.6 mm, 5 μm) C18 column, and acetonitrile-0.1% trifluoroacetic acid aqueous in gradient as mobile phase, the flow rate was 0.8 mL/min, and the detecting wavelength was set at 280 nm. The chemical fingerprint similarity of 10 batches of Chinese gall leaven was calculated with the Chromap Chromafinger 2005 beta 0.1 standard substance comparison and HPLC-MS were adopted to identify the common peaks. Results The fingerprint chromatography for the 10 batches of Chinese gall leaven included 10 common peaks, with a good separation at each peak. The relative retention time for common peaks of each batch was less than 1.0%, and the similarities among 10 samples were greater than 0.90. Gallic acid (peak 1), (-)-epigallocatechin (EGC, peak 2), methyl gallate (peak 3), ethyl gallate (peak 5), epigallocatechin gallate (EGCG, peak 6), 2,4,6-tri-O-galloyl-β-D-glucose (peak 7), and 2,4,6-tri-O-galloyl-α-D-glucose (peak 9) were identified. The gallnut fermented made the content of gallic acid and 2,4,6-tri-O-galloyl-α-D-glucose increased and the contents of methyl gallate, ethyl gallate, and (-)-epigallocatechin gallate decreased. It was found that (-)-epigallocatechin and 2,4,6-tri-O-galloyl-β-D-glucose had formed in the process for the first time. Conclusion The processing mechanism of Chinese gall leaven is related to gallnut fermentation process change and create new chemical composition and fingerprint can be used to monitor the quality of fermentation processing of Chinese gall leaven.

Difference analysis of chemical compositions in Moutan Cortex from different origins by UPLC-Q-TOF-MS / 中草药

Objective: To identify and analyze the components of Moutan Cortex from different origins by UPLC-Q-TOF-MS technology. Methods: UPLC-Q-TOF-MS technology combined with chemical composition database was used to identify and analyze the components of Moutan Cortex. The results were analyzed and verified by multivariate statistical analysis and the biomarkers were identified. Results: The 37 components were identified in Moutan Cortex, the major components were galloyl glucoses, benzoic acids, paeonols, peoniflorins, and flavonoids. There were certain differences in the composition and content of Moutan Cortex from different origins. The medicinal materials from different origins were clustered into different groups respectively by principal component analysis (PCA). Five biomarkers were collected by partial least squares-discriminant analysis (PLS-DA) model and orthogonal partial least squares-discriminant analysis (OPLS-DA). Conclusion: The experimental results provide a theoretical basis for understanding chemical material basis and quality evaluation of Moutan Cortex, also provide the basis for the development of Chinese medicinal materials specifications and grades.

A Study on the Statistical Method for the Target Component of a Glucose-lowering Functional Material in Extracts of Evening Primerose Seeds

BACKGROUND: The use of the extracts from evening primrose seeds as a health functional food has been gradually increased. Therefore, the monitoring and screening process has been considerably required for its quality control. OBJECTIVE: This study aimed to estimate the measurement uncertainty associated with determination of penta-o-galloyl β-D-glucose (PGG) in extracts from evening primrose seeds by high-performance liquid chromatography. METHODS: The sources of measurement uncertainty was expressed in accordance with mathematical/statistical theories of GUM & EURACHEM Guide. The expanded uncertainty was calculated by using the relative standard uncertainty between analytical result and sources of uncertainty in measurement (sample weight, final volume, extraction volume, standard solution, matrix and instrument etc). RESULTS: In the results of 95% confidence interval, the uncertainty in measurement was 10,253.34 ± 1,844.50 µg/kg (k = 2.0). CONCLUSION: In this study, it showed that the value of uncertainty in measurement for determination of PGG in extracts from evening primrose seeds by HPLC has about 18.0% influence on PGG contents of the analytical results. The results would be very useful for the monitoring and screening of evening primrose seeds marketed in Korea for its quality control as dietary supplement.

The Longevity Properties of 1,2,3,4,6-Penta-O-Galloyl-beta-D-Glucose from Curcuma longa in Caenorhabditis elegans

Here in this study, we isolated 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) from Curcuma longa L. and elucidated the lifespanextending effect of PGG using Caenorhabditis elegans model system. In the present study, PGG demonstrated potent lifespan extension of worms under normal culture condition. Then, we determined the protective effects of PGG on the stress conditions such as thermal and oxidative stress. In the case of heat stress, PGG-treated worms exhibited enhanced survival rate, compared to control worms. In addition, PGG-fed worms lived longer than control worms under oxidative stress induced by paraquat. To verify the possible mechanism of PGG-mediated increased lifespan and stress resistance of worms, we investigated whether PGG might alter superoxide dismutase (SOD) activities and intracellular ROS levels. Our results showed that PGG was able to elevate SOD activities of worms and reduce intracellular ROS accumulation in a dose-dependent manner.

Effects of 1,2,4,6-tetra-O-galloyl-β-D-glucose from P. emblica on HBsAg and HBeAg Secretion in HepG2.2.15 Cell Culture / 中国病毒学·英文版

A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.

A SEDOHEPTULOSE GALLATE FROM THE FRUITS OF CORNUS OFFICINALIS / 药学学报

AIM: To investigate the chemical constituents of the plant drug Cornus officinalis Sieb et Zucc. which is widely used in Chinese traditional medicine, having actions of invigorating the liver and kidney, strengthening the body, and of an astringent, etc. METHODS: A sedoheptulose gallate was isolated from the water extracts of fruits of C.officinalis using various chromatographies. The structure determination was based on spectral (UV, IR, 1H & 13CNMR and MS) and chemical evidence. RESULTS: Its structure was characterized to be 7-O-galloyl-D-sedoheptulose (I). The proportion of its three major conformation forms of β-F, α-F and α-P in aqueous solution was estimated to be 57:24:19 according to their C-1 peak heights in the 13CNMR spectrum. CONCLUSION: I was found in natural world for the first time and its β-D-heptufuranosyl form was the predominant existing tautomer in its equilibrated aqueous solution according to NMR determination.