ABSTRACT
Although the tumor suppressor P53 is known to regulate a broad network of signaling pathways, it is still unclear how certain drugs influence these P53 signaling networks. Here, we used a comprehensive single-cell multiomics view of the effects of ginsenosides on cancer cells. Transcriptome and proteome profiling revealed that the antitumor activity of ginsenosides is closely associated with P53 protein. A miRNA-proteome interaction network revealed that P53 controlled the transcription of at least 38 proteins, and proteome-metabolome profiling analysis revealed that P53 regulated proteins involved in nucleotide metabolism, amino acid metabolism and "Warburg effect". The results of integrative multiomics analysis revealed P53 protein as a potential key target that influences the anti-tumor activity of ginsenosides. Furthermore, by applying affinity mass spectrometry (MS) screening and surface plasmon resonance fragment library screening, we confirmed that 20()-protopanaxatriol directly targeted adjacent regions of the P53 DNA-binding pocket and promoted the stability of P53-DNA interactions, which further induced a series of omics changes.
ABSTRACT
Panax notoginseng saponins (PNS) are the major components of Panax notoginseng, with multiple pharmacological activities but poor oral bioavailability. PNS could be metabolized by gut microbiota in vitro, while the exact role of gut microbiota of PNS metabolism in vivo remains poorly understood. In this study, pseudo germ-free rat models were constructed by using broad-spectrum antibiotics to validate the gut microbiota-mediated transformation of PNS in vivo. Moreover, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was developed for quantitative analysis of four metabolites of PNS, including ginsenoside F1 (GF1), ginsenoside Rh2 (GRh2), ginsenoside compound K (GCK) and protopanaxatriol (PPT). The results showed that the four metabolites could be detected in the control rat plasma, while they could not be determined in pseudo germ-free rat plasma. The results implied that PNS could not be biotransformed effectively when gut microbiota was disrupted. In conclusion, gut microbiota plays an important role in biotransformation of PNS into metabolites in vivo.
Subject(s)
Animals , Male , Anti-Bacterial Agents , Pharmacology , Biotransformation , Chromatography, High Pressure Liquid , Feces , Microbiology , Gastrointestinal Microbiome , Physiology , Ginsenosides , Blood , Panax notoginseng , Chemistry , Rats, Sprague-Dawley , Sapogenins , Blood , Saponins , Metabolism , Tandem Mass SpectrometryABSTRACT
Objective: To study the minor triterpenoid saponins from the roots of Panax notoginseng, which provided basis for the systematic research, quality control and safety evaluation of P. notoginseng. Methods The compounds were isolated and purified by MCI resin, ODS, along with Preparative-HPLC, and the structures were identified by spectroscopic analysis, and comparing with the pubished literature values. Results: Twelve monomeric compounds isolated from the roots of P. notoginseng, were identified as notoginsenoside P1 (1), notoginsenoside T5 (2), ginsenoside Rk3 (3), ginsenoside Rh4 (4), notoginsenoside T3 (5), 20(S)-protopanaxatriol (6), dammar 20 (21),24-diene-3β,6α,12β-triol (7), ginsenoside Rg3 (8), gypenoside XIII (9), ginsenoside Rk1 (10), ginsenoside Rg5 (11), and 20 (S)-ginsenoside Rh2 (12). Conclusion: Compound 1 is a new dammarane-type triterpenoid saponin
ABSTRACT
UDP-glycosyltransferase PgUGT74AE2 from Panax ginseng can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxadiol (PPD) to produce ginsenoside Rh2. However, no report demonstrates that PgUGT74AE2 can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxatriol (PPT) to produce a PPT-type ginsenoside. In this study, the expression plasmid pET-32a-PgUGT74AE2 was constructed for expression of the recombinant protein and transferred into Escherichia coli Transetta (DE3) to generate the recombinant strain Transetta-PgUGT74AE2. The recombinant enzyme PgUGT74AE2 was expressed by induction of isopropyl-β-D-thiogalactoside (IPTG). An in vitro enzymatic reaction system was established with the recombinant enzyme PgUGT74AE2 and the substrate PPT. PgUGT74AE2 catalyzed the glycosylation of the free C-3 hydroxyl of PPT to produce 3-O-β-D-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol, a new PPT-type ginsenoside. This study provides an efficient approach for the biosynthesis of a new PPT-type ginsenoside through in vitro enzymatic reaction, which may pave a way to produce promising lead in drug discovery.
ABSTRACT
Ginseng is a traditional medicine in Asian countries. Ginsenoside has the main active ingredient, exhibit cardiovascular, tumor, and central nervous system activities. In particular, protopanaxatriol-type ginsenosides Rh1, exhibits anti-inflammatory, anti-allergic, and memory improvement activities. Ginsenoside Rh1 is only found in trace amounts in Panax ginseng, Panax pseudoginseng var. notoginseng, and Panax quinquefolius. Biotransformation of rare ginsenosides has become an effective way. In this paper, the research progress of transformation of ginsenoside saponins by biotransformation to produce rare ginsenoside Rh1 is reviewed, which provides a useful reference for the further development and preparation of ginsenoside Rh1.
ABSTRACT
Objective: For making full use of our country's rich ginseng resources to look for 25-OH-PPT\ and provide the scientific basis for the innovation and development of ginsenoside hypoglycemic activity. Methods: Using chromatographic column Agilent C18 (250 mm × 4.6 mm, 5 μm); mobile phase was CH3OH-H2O (70:30); Column temperature was 35 ℃; Flow rate was 1.0 mL/min; the boiling chamber temperature of ELSD was 87 ℃; Flow rate of carrier gas was 2.20 L/min; injection volume was 10 μL. Using acetone as the solvent and ultrasonic extraction to extract the medicinal materials, then using the HPLC-ELSD method to determine the contents of 20(R)-25-OH-PPT in different parts of plants in Panax ginseng. Results: 20(R)-25-OH-PPT had a good linear relationship when the concentration was 0.001-0.05 mg/mL. The 20(R)-25-OH-PPT contents in gingseng cauline leaf, gingseng root, gingseng fruit, gingseng flower, cauline leaf of American ginseng, root of American ginseng, and the basal part of stem of P. notoginseng were 3.74%, 2.59%, 0.40%, 2.37%, 0.68%, 0.22%, and 0.39%. The recovery rates were 98.54%, 97.54%, 100.35%, 101.46%, 97.46%, 99.14%, and 99.38%. Conclusion :25(R)-OH-PPT exists in different parts of P. ginseng, and it can be an important basis for the use and development of ginsenoside with hypoglycemic activity.
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Objective To explore the excretion of the 20 (S)-protopanaxatriol (PPT) and its metabolites ocotillol type epimers (M1 and M2) in urine, feces samples and the excretion of Ml and M2 in bile samples. Methods The concentration of PPT, Ml and M2 in urine, feces samples and the concentration of Ml and M2 in bile samples were determined by the LC-MS/MS methods with or without the hydrolization by β-glucuronidase. Results After intragastric(ig) administration of PPT, the cumulative excretion rate for 72 h of PPT, Ml and M2 in feces were 14.88%, 1.34% and 0.084%, respectively. With the hydrolization by β-glucuronidase, the cumulative excretion rate for 72 h of PPT, Ml and M2 in feces were 14.77%, 1.36% and 0.085%, respectively. However, the epimers and PPT were hardly detected in urine. After ig administration of M1 or M2, the accumulation excretion rate were 4.41% for M1 and 47.2% for M2 in feces, while both epimers were hardly detected in urine. After ig administration of M1 or M2, the 36 h cumulative biliary excretion rate was 3.01% for M1, and only 0.068% for M2. The 36 h cumulative biliary excretion rate of M1 was 8.80% after intravenous administration, while only 1.24% for M2. Conclusion After ig administration of PPT, a small amount of PPT and its metabolites (Ml, M2) are excreted by the feces but little via urine, and there are stereoselectivity differences in biliary excretion between M1 and M2.
ABSTRACT
Objective To explore the excretion of the 20(S)-protopanaxatriol(PPT)and its metabolites ocotillol type epi?mers(M1 and M2)in urine,feces samples and the excretion of M1 and M2 in bile samples. Methods The concentration of PPT,M1 and M2 in urine,feces samples and the concentration of M1 and M2 in bile samples were determined by the LC-MS/MS methods with or without the hydrolization byβ-glucuronidase. Results After intragastric(ig)administration of PPT,the cumulative excretion rate for 72 h of PPT,M1 and M2 in feces were 14.88%,1.34%and 0.084%,respectively. With the hydrolization byβ-glucuronidase,the cumulative excretion rate for 72 h of PPT,M1 and M2 in feces were 14.77%,1.36%and 0.085%,respectively. However,the epimers and PPT were hardly detected in urine. After ig administration of M1 or M2,the accumulation excretion rate were 4.41%for M1 and 47.2%for M2 in feces,while both epimers were hardly detected in urine. After ig administration of M1 or M2,the 36 h cumulative bili?ary excretion rate was 3.01%for M1,and only 0.068%for M2. The 36 h cumulative biliary excretion rate of M1 was 8.80%after intra?venous administration ,while only 1.24%for M2. Conclusion After ig administration of PPT,a small amount of PPT and its metabo?lites(M1,M2)are excreted by the feces but little via urine ,and there are stereoselectivity differences in biliary excretion between M1 and M2.
ABSTRACT
Ginsenoside Rg1 (Rg1), the major effective component of ginseng, has been shown to have multiple bioactivities, but low oral bioavailability. The aim of this study was to develop a simple, sensitive and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which could be used to validate and quantify the concentrations of Rg1 and its metabolites in Sprague-Dawley rat bile, urine, and feces after oral administration (25 mg/kg). Calibration curves offered satisfactory linearity (>0.995) within the determined ranges. Both intra-day and inter-day variances were less than 15%, and the accuracy was within 80-120%. The excretion recoveries of Rg1, ginsenoside Rh1 (Rh1), and protopanaxatriol (Ppt) in bile, urine, and feces combined were all greater than 70%. The fecal excretion recoveries of Rg1, Rh1, and Ppt were 40.11%, 22.19%, and 22.88%, respectively, whereas 6.88% of Rg1 and 0.09% of Rh1 were excreted in bile. Urinary excretion accounted for only 0.04% of Rg1. In conclusion, the observed excretion profiles for Rg1 and its metabolites after oral administration are helpful for understanding the poor oral bioavailability of Rg1 and will aid further investigations of Rg1 as a pharmacologically active component.
ABSTRACT
Objective: In order to evaluate the quality of Panax ginseng and its preparation, a simple and accurate HPLC method for determining the contents of 16 ginsenosides from P. ginseng was established. Methods: The chromatographic separation was achieved on a C18 column (150 mm × 4.6 mm, 5 μm) using a mobile phase made up of acetonitrie and water at a flow rate of 1.0 mL/min. The detection wavelength and column temperature were set as 203 nm and 35°C, respectively. Results: Sixteen ginsenosides (Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, Rb3, F1, Rd, F2, Rg3, protopanaxatriol, compound K, Rh2, and protopanaxadiol) were separated at baseline with good linearity (r ≥ 0.9990). The recovery rates were 95%-102% (RSD < 2%). Conclusion: The method is simple, fast, accurate, and could be applied to the quality control of P. ginseng and its preparation.
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Objective To investigate the derivatives of ginsenoside sapogenins and their activities against HL-60 cells.Methods The total ginsenoside extract was treated by Smith degradation.All products were isolated by silica gel chromatography and purified by preparative HPLC.On the basis of 1D and 2D NMR data,structures were elucidated.The activity against HL-60 cells was measured by MTT method.Results Three compounds were isolated and identified as 20(S)-protopanaxadiol(PD),20(S)-protopanaxatriol(PT),and 24,25-en-3?,6?-dihydroxy-12,20-(1′-hydroxy) ethanedioxy-dammarane(1).Conclusion Compound 1,named 1′-hydroxy ethanedioxy PT,is a novel derivate of sapogenin with higher inhibitory activity against HL-60 cells than PD and PT.