Biotransformation of Ginsenosides (Rb1 , Rb2 , Rb3 , Rc) in Human Intestinal Bacteria and Its Effect on Intestinal Flora.

Ginsenosides, including Rb1 , Rb2 , Rb3 and Rc, belong to protopanaxadiol-type saponins in Panax ginseng C. A. Mey. Their contents are high in P. ginseng. They could inhibit oxidant stress, enhance immunity, lower blood sugar, resist tumor cells and facilitate other physiological activities. This study aimed to explore the interaction between ginsenosides Rb1 , Rb2 , Rb3 and Rc and the intestinal flora of healthy people. It also sought to analyse the biotransformation products and pathways of these ginsenosides in in-vitro human intestinal bacteria and their effects on the diversity of human intestinal flora. Human intestinal bacteria were incubated with ginsenosides Rb1 , Rb2 , Rb3 and Rc at 37 °C under anaerobic conditions. Samples were taken at different timepoints. The transformed products were identified by rapid high-resolution liquid chromatography-quadrupole time-of-flight mass spectrometry. After 48 h of transformation, the transformed product of ginsenosides Rb1 , Rb2 , Rb3 and Rc was ginsenoside compound K. The transformation rates were 83.5 %, 88.7 %, 85.6 %, and 84.2 %. 16S rRNA sequencing technology was applied to the bioinformatic analysis of faecal samples incubated for 48 h. Relative to the blank control, the relative abundance of Firmicutes and Proteobacteria significantly increased at the phylum level. Moreover, the relative abundance of Bacteroidetes significantly decreased in ginsenosides Rb1 , Rb2 , Rb3 and Rc. At the genus level, the relative abundance of Escherichia significantly increased, whereas that of Dorea, Prevotella and Megasphaera significantly decreased in all groups. These results showed that Rb1 , Rb2 , Rb3 and Rc could improve the structure and diversity of human intestinal flora and balance the metabolic process.

Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway.

OBJECTIVE: To investigate whether ginsenoside-Rb1 (Gs-Rb1) improves the CoCl-induced autophagy of cardiomyocytes via upregulation of adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway. METHODS: Ventricles from 1- to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group (normal level oxygen), hypoxia group (500 µmol/L CoCl2), Gs-Rb1 group (200 µmol/L Gs-Rb1 + 500 µmol/L CoCl2), Ara A group (500 µmol/L Ara A + 500 µmol/L CoCl2), Ara A+ Gs-Rb1 group (500 µmol/L Ara A + 200 µmol/L Gs-Rb1 + 500 µmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + 500 µmol/L CoCl2], and AICAR+Gs-Rb1 group (1 mmol/L AICAR + 200 µmol/L Gs-Rb1 + 500 µmol/L CoCl2). Cells were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cardiac troponin I (cTnI) levels were detected by enzyme-linked immunosorbent assay (ELISA). AMPK activity was assessed by 2',7'-dichlorofluorescein diacetate (DCFH-DA) ELISA assay. The protein expressions of Atg4B, Atg5, Atg6, Atg7, microtubule-associated protein 1A/1B-light chain 3 (LC3), P62, and active-cathepsin B were measured by Western blot. RESULTS: Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes (P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A (P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A (P<0.01) and was not affected by AICAR =0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B II, the LC3B II/I ratio and cathepsin B activity of hypoxia cardiomyocytes (P<0.05), each of these protein levels was significantly enhanced by Ara A (all P<0.01), but was not affected by AICAR (all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes (P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A (P<0.05) and were not affected by AICAR (P=0.871). CONCLUSION: Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.

Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate whether ginsenoside-Rb1 (Gs-Rb1) improves the CoCl-induced autophagy of cardiomyocytes via upregulation of adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway.@*METHODS@#Ventricles from 1- to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group (normal level oxygen), hypoxia group (500 μmol/L CoCl), Gs-Rb1 group (200 μmol/L Gs-Rb1 + 500 μmol/L CoCl), Ara A group (500 μmol/L Ara A + 500 μmol/L CoCl), Ara A+ Gs-Rb1 group (500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + 500 μmol/L CoCl], and AICAR+Gs-Rb1 group (1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl). Cells were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cardiac troponin I (cTnI) levels were detected by enzyme-linked immunosorbent assay (ELISA). AMPK activity was assessed by 2',7'-dichlorofluorescein diacetate (DCFH-DA) ELISA assay. The protein expressions of Atg4B, Atg5, Atg6, Atg7, microtubule-associated protein 1A/1B-light chain 3 (LC3), P62, and active-cathepsin B were measured by Western blot.@*RESULTS@#Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes (P0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes (P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A (P<0.05) and were not affected by AICAR (P=0.871).@*CONCLUSION@#Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.

Conversion of Ginsenoside Rb1 into Six Types of Highly Bioactive Ginsenoside Rg3 and Its Derivatives by FeCl3 Catalysis.

Ginsenoside Rb1 is an important saponin of ginseng(s); however, Rb1, with 3-O- and 20-O-sugar moieties, has low bioavailability. Here, we report the derivatization of ginsenoside Rb1 to completely generate six types of highly bioactive minor ginsenoside Rg3 and its derivatives by FeCl3 catalysis, the reaction conditions are similar to enzymatic reaction conditions. In FeCl3 catalysis, the only 20-O-sugar-moiety of ginsenoside Rb1 was decomposed into the minor ginsenosides Rk1 and Rg5 with newly produced C-20 ethylene bands; but also hydrolyzed into 20(S)-Rg3 and 20(R)-Rg3; subsequently the C-24(25) ethylene bands of 20(S)-Rg3 and 20(R)-Rg3 were hydrated to 20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3. After separation of reaction mixture from 34 g ginsenoside-Rb1 by silica-gel-column, the 3.3 g sample I of TLC top-band consisting of Rg5 and Rk1, 8.7 g sample II of TLC middle-band consisting of 20(S)-Rg3 and 20(R)-Rg3, 3.5 g sample III of TLC bottom-band consisting of unknown product-I and -II including 20(S)-25-OH-Rg3, were obtained. The sample III consisting of unknown product-I and -II was purified by crystallization, and identified to 20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3 by HPLC-Evaporative Light Scattering Detector (ELSD) and NMR. Therefore, six types of minor-ginsenosides Rk1, Rg5, 20(S)-Rg3, 20(R)-Rg3, 20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3 were successfully prepared from ginsenoside Rb1 by FeCl3 catalysis. FeCl3 has low toxicity and is inexpensive, and the reaction conditions are similar to enzymatic reaction conditions; thus, this method is applicable to the development of ginseng-based drugs.

Study on Quality Standard for Fushiming Capsule / 中国药房

OBJECTIVE:To establish quality standard of Fushiming capsule. METHODS:TLC was used to qualitatively identify the ligustilide,aurantio obtusin,chrysophanol,fruit of Chinese wolfberry and Whitmania pigra,respectively. HPLC method was used to determine the contents of puerarin and ginsenosides Rb1. The determination was performed on Intersil C18 column with mobile phase consisted of acetonitrile-0.3% phosphoric acid(gradient elution)at flow rate of 1.0 mL/min;the detection wavlength was set at 203 nm,and column temperature was 25 ℃;the sample size was 10 μL. RESULTS:TLC spots of ligustilide,aurantio obtusin,chrysophanol,the fruit of C. wolfberry and W. pigra were clear and well separated without negative interference. The linear range of puerarin and ginsenoside Rb1were 10.56-337.92 μg/mL(r=0.999 7)and 17.80-569.70 μg/mL(r=0.999 6). The limits of quantitation were 2.20,1.86 μg/mL,and the limits of detection were 0.12,0.13 μg/mL,respectively. RSDs of precision,stability and reproducibility tests were lower than 2.0%. The recoveries were 95.65%-99.66%(RSD=1.45%,n=6) and 96.95%-98.52%(RSD=0.77%,n=6),respectively. CONCLUSIONS:Established quality standard can be used for the quality control of Fushiming capsule.

Optimization of extraction technique for Shenxi Oral Liquid by orthogonal design based on AHP-CRITIC analysis / 中草药

Objective The extraction process of Shenxi Oral Liquid by exploring the optional extraction process parameters based on determining the index weight. Methods Using the contents of cyasterone, ginsenosides Rg1, Re, Rb1, panaxadiol, ratio of dry extraction, total polysaccharides, and total peak area as comprehensive evaluation indexes, the weighting coefficient in eight synthetic evaluation indexes was determined by analytic hierarchy process (AHP), criteria importance through intercriteria correlation (CRITIC) method, and mixed weighted AHP-CRITIC. Compared mixed weighting method and single weighting method, the optimal parameters of compound extraction process were optimized by orthogonal test. Results Mixed weighted AHP-CRITIC was more scientific, reasonable, and comprehensive than the single weighting method. According to the weighting coefficient determined by comprehensive evaluation, the optimal process parameters were as follow: compound herbs plus nine times of water, extracting three times, each for 80 min. The mean of three batches of comprehensive evaluation was 99.06 and RSD value was 0.18%. Conclusion AHP combined with CRITIC could be used to establish weighting coefficient of the compound formula extraction process and the optimized process of extraction had been verified to be reasonable, stable, and reproducible, which could be used for industrial production.

Effect of Bletilla striata polysaccharides on pharmacokinetics of 10 components in Panax notoginseng saponins / 中草药

Objective: To establish a UPLC-MS/MS method for simultaneous determination of 10 components in Panax notoginseng saponins (PNS), and the study the effect of Bletilla striata polysaccharides (BSP) on the pharmacokinetic parameters of PNS. Methods: Rats were divided into PNS group (P group) and PNS-BSP compatibility group (BP group) by ig administration. The plasma concentration of 10 saponins was determined by UPLC-MS/MS. The pharmacokinetic parameters were calculated by DAS software. Results: The determination method corresponded to the biological sample measurement requirements. Compared with P group, the AUC of Rb1 significantly reduced in BP group (P<0.01) and the AUCs of notoginsenoside R1, Rg1, Rf, Rd, CK, Rc, Rh1 were lower but without significant difference; The total AUC of 10 components significantly reduced in BP group (P<0.01); The plasma concentration of Rg1 in BP group was lower compared with that in P group (P<0.05); The tmax of Rg1, Rb1, and Rf significantly delayed (P<0.01); The tmax of notoginsenoside R1 and Rg2 delayed compared with P group (P<0.05). Conclusion: The established UPLC-MS/MS analysis method is suitable for the simultaneous determination of 10 components in PNS in rat plasma; PNS-BSP compatibility can reduce the plasma concentration of PNS and AUC exposure and prolong the tmax. This illustrates that BSP may increase the exposure levels of PNS in the gastrointestinal tract so that increase the effect in the gastrointestinal tract.

Study on the quality standard of Fufang-Shiwu-Zhixue powder / 国际中医中药杂志

Objective To establish the quality standard of Fufang-Shiwu-Zhixue powder. Methods The microscopical identification was adopted to analyze charred typha pollen and cuttlebone. TLC was used to identity rhubarb and radix notoginseng. UV-HPLC was used to determine the contents of notoginsenosides R1, ginsenosides Rg 1, ginsenosides Rb1. Results Microscopic identifications shower the characteristics of harred typha pollen and cuttlebone. The identified characteristics of rhubarb and radix notoginseng by TLC were distinct and the spots were clear. Notoginsenosides R1, ginsenosides Rg 1, ginsenosides Rb1 showed good linearity in the range of 0.16-1.58 μg (r=0.9998), 0.58-5.81 μg (r=0.9999), 0.33-3.29 μg (r=1.0000), respervtively.The average recoveries were 98.51% (RSD=1.81%), 97.80% (RSD=2.04%), 98.22%(RSD=1.45%). Conclusions The method is accurate, simple and repeatable, which could be used for the quality control of Fufang-Shiwu-Zhixue powder.

The effect of ginsenosides Rb1 on cerebral blood flow of cerebral ischemia/reperfusion injury in rats / 中国中西医结合急救杂志

ObjectiveTo observe the effect of ginsenosides Rb1 on cerebral blood flow of rat models with cerebral ischemia/reperfusion (I/R) injury, which could provide a new theory of cerebral protective mechanism about ginsenosides Rb1.Methods Twenty-four rats were randomly divided into sham-operation group, model group, normal saline control group and ginsenosides Rb1 group, 6 rats in each group. The middle cerebral artery occlusion (MCAO) model was established by thread embolism method. At the end of I/R, in the rat of ginsenosides Rb1 group, ginsenosides Rb1 40 mg/kg was immediately intraperitoneally injected, while in the rat of normal saline control group, an equal volume of normal saline was injected intraperitoneally. After I/R for 24 hours, the cerebral local amount of blood flow was measured, the rats' behavior score was observed, and the volume of cerebral infarction was monitored by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining.Results The percentage of volume of cerebral infarction [(64.23±8.12)% vs. 0%] and behavior score [3.0 (2.0-4.0) vs. 0 (0-0),P< 0.05] in model group were significantly higher than those in sham-operation group, while the cerebral local amount of blood flow in model group was obviously lower than that in sham-operation group (mL/min: 125.75±57.65 vs. 225.01±78.25,P< 0.05); Compared with the model group and normal saline control group, the percentage of volume of cerebral infarction [(23.62±8.74)% vs. (64.23±8.12)%, 56.72±8.92] and behavior score [0.5 (0.0-2.0) vs. 3.0 (2.0-4.0), 3.5 (1.0-4.0)] in the ginsenosides Rb1 group were significantly lower, the cerebral local amount of blood flow was markedly increased in the ginsenosides Rb1 group (177.25±75.36 vs. 125.75±57.65, 132.65±58.65,P< 0.05).Conclusion Ginsenosides Rb1 can increase the cerebral blood flow in rats with cerebral I/R injury, which maybe one of the mechanisms of cerebral protection of Ginsenosides Rb1.

Simultaneous determination of the contents of astragaloside IV, danshensu, protocatechualdehyde, ginsenosides Rg1 and Rb1 in Qishenyiqi dropping pills by LC-MS / 中国药学杂志

OBJECTIVE: To establish a LC-MS method for simultaneous determination of 5 compounds (astragaloside IV, danshensu, protocatechualdehyde, ginsenosides Rg1 and Rb1) in Qishenyiqi Dropping Pills. METHODS: The chromatographic separation was carried out at 30°C on a Wonda Sil C18 column (4.6 mm × 150 mm, 5 μm) eluted in a gradient program. The mobile phase consisted of water containing 10 mmol · L-1 ammonium formate and 0.2% formic acid-acrtonitrile containing 0.2% formic acid. The mass spectra were obtained by Agilent 6410 triple quad mass spectrometer with electrospray ionization source in negative ion mode. RESULTS: The linear ranges for astragaloside IV, danshensu, protocatechualdehyde, ginsenosides Rg1 and Rb1 were 10.72-536.0, 89.10-4 455, 1.337-66.87, 22.60-1 130 and 23.55-1 177 ng · mL-1, respectively. The average recoveries ranged between 98%-102%. CONCLUSION: The established method is convenient, sensitive and accurate. It can be used for the determination of the contents of astragaloside IV, danshensu, protocatechualdehyde, ginsenosides Rg1 and Rb1 in Qishenyiqi Dropping Pills for quality control.