Network Pharmacology and Transcriptomics to Explore the Pharmacological Mechanisms of 20(S)-Protopanaxatriol in the Treatment of Depression.

Depression is one of the most common psychological disorders nowadays. Studies have shown that 20(S)-protopanaxatriol (PPT) can effectively improve depressive symptoms in mice. However, its mechanism needs to be further explored. In this study, we used an integrated approach combining network pharmacology and transcriptomics to explore the potential mechanisms of PPT for depression. First, the potential targets and pathways of PPT treatment of depression were screened through network pharmacology. Secondly, the BMKCloud platform was used to obtain brain tissue transcription data of chronic unpredictable mild stress (CUMS) model mice and screen PPT-altered differential expression genes (DEGs). Gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using network pharmacology and transcriptomics. Finally, the above results were verified by molecular docking, Western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we demonstrated that PPT improved depression-like behavior and brain histopathological changes in CUMS mice, downregulated nitric oxide (NO) and interleukin-6 (IL-6) levels, and elevated serum levels of 5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) after PPT treatment compared to the CUMS group. Eighty-seven potential targets and 350 DEGs were identified by network pharmacology and transcriptomics. Comprehensive analysis showed that transthyretin (TTR), klotho (KL), FOS, and the phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling pathway were closely associated with the therapeutic effects of PPT. Molecular docking results showed that PPT had a high affinity for PI3K, AKT, TTR, KL, and FOS targets. Gene and protein level results showed that PPT could increase the expression of PI3K, phosphorylation of PI3K (p-PI3K), AKT, phosphorylation of AKT (p-AKT), TTR, and KL and inhibit the expression level of FOS in the brain tissue of depressed mice. Our data suggest that PPT may achieve the treatment of depression by inhibiting the expression of FOS, enhancing the expression of TTR and KL, and modulating the PI3K-AKT signaling pathway.

20(S)-protopanaxatriol inhibited D-galactose-induced brain aging in mice via promoting mitochondrial autophagy flow.

Previous reports have confirmed that saponins (ginsenosides) derived from Panax ginseng. C. A. Meyer exerted obvious memory-enhancing and antiaging effects, and the simpler the structure of ginsenosides, the better the biological activity. In this work, we aimed to explore the therapeutic effect and underlying molecular mechanism of 20(S)-protopanaxatriol (PPT), the aglycone of panaxatriol-type ginsenosides, by establishing D-galactose (D-gal)-induced subacute brain aging model in mice. The results showed that PPT treatment (10 and 20 mg/kg) for 4 weeks could significantly restore the D-gal (800 mg/kg for 8 weeks)-induced impaired memory function, choline dysfunction, and redox system imbalance in mice. Meanwhile, PPT also significantly reduced the histopathological changes caused by D-gal exposure. Moreover, PPT could increase TFEB/LAMP2 protein expression to promote mitochondrial autophagic flow. Importantly, the results from molecular docking showed that PPT had good binding ability with LAMP2 and TFEB, suggesting that TFEB/LAMP2 might play an important role in PPT to alleviate D-gal-caused brain aging.

Neuroprotective Effects and Metabolomics Study of Protopanaxatriol (PPT) on Cerebral Ischemia/Reperfusion Injury In Vitro and In Vivo.

Stroke, one of the leading causes of disability and death worldwide, is a severe neurological disease that threatens human life. Protopanaxatriol (PPT), panaxatriol-type saponin aglycone, is a rare saponin that exists in Panax ginseng and Panax Noto-ginseng. In this study, we established an oxygen-glucose deprivation (OGD)-PC12 cell model and middle cerebral artery occlusion/reperfusion (MCAO/R) model to evaluate the neuroprotective effects of PPT in vitro and in vivo. In addition, metabolomics analysis was performed on rat plasma and brain tissue samples to find relevant biomarkers and metabolic pathways. The results showed that PPT could significantly regulate the levels of LDH, MDA, SOD, TNF-α and IL-6 factors in OGD-PC12 cells in vitro. PPT can reduce the neurological deficit score and infarct volume of brain tissue in rats, restore the integrity of the blood-brain barrier, reduce pathological damage, and regulate TNF-α, IL-1ß, IL-6, MDA, and SOD factors. In addition, the results of metabolomics found that PPT can regulate 19 biomarkers involving five metabolic pathways, including amino acid metabolism, arachidonic acid metabolism, sphingolipid metabolism, and glycerophospholipid metabolism. Thus, it could be inferred that PPT might serve as a novel natural agent for MCAO/R treatment.

Inclusion complex of 20(S)-protopanaxatriol with modified ß-cyclodextrin: Characterization, solubility, and interaction with bovine serum albumin.

20(S)-protopanaxatriol (PPT) is one of the ginsenosides isolated from Panax ginseng which have many pharmaceutical activities. However, the poor water solubility of PPT restrict its applications. Herein, a novel bridged-bis-[6-(3,3'-(ethylenedioxy) bis (propylamine))-6-deoxy-ß-cyclodextrin] (EDBA-bis-ß-CD) was designed and synthesized, and the inclusion complex (IC) of EDBA-bis-ß-CD with PPT was successfully prepared in the solid state, and characterized by UV, 1H NMR, 2D ROESY, FT-IR, XRD and SEM and molecular modelling methods. The continuous variation method analysis indicated that the stoichiometry of the IC was 1:1. UV-vis spectral analysis demonstrated the binding constant Ks was 995.94 M-1, and the solubility study showed that the solubility of PPT improved 290 times. The interaction of the IC with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy. The results indicated that fluorescence quenching of BSA by IC was static quenching. Thermodynamic studies showed that van der Waals forces and hydrogen bonding play significant roles in interaction. The esterase-like activity of BSA in the presence of IC showed that it reduce the esterase activity of BSA in a competitive manner. Furthermore, molecular docking and molecular dynamics simulations for EDBA-bis-ß-CD/PPT and BSA/IC systems were generated to provide information on the stability and the forces in the binding.

Integrated Metabolomics and Network Pharmacology to Decipher the Latent Mechanisms of Protopanaxatriol against Acetic Acid-Induced Gastric Ulcer.

Gastric ulcer (GU) is a peptic disease with high morbidity and mortality rates affecting approximately 4% of the population throughout the world. Current therapies for GU are limited by the high relapse incidence and side effects. Therefore, novel effective antiulcer drugs are urgently needed. Ginsenosides have shown good anti-GU effects, and the major intestinal bacterial metabolite of ginsenosides, protopanaxatriol (PPT), is believed to be the active component. In this study, we evaluated the anti-GU effect of PPT in rats in an acetic acid-induced GU model. High (H-PPT) and medium (M-PPT) doses of PPT (20.0 and 10.0 mg/mg/day) significantly reduced the ulcer area and the ET-1, IL-6, EGF, SOD, MDA and TNF-α levels in serum were regulated by PPT in a dose-dependent manner. We also investigated the mechanisms of anti-GU activity of PPT based on metabolomics coupled with network pharmacology strategy. The result was that 16 biomarkers, 3 targets and 3 metabolomic pathways were identified as playing a vital role in the treatment of GU with PPT and were further validated by molecular docking. In this study, we have demonstrated that the integrated analysis of metabolomics and network pharmacology is an effective strategy for deciphering the complicated mechanisms of natural compounds.

Protective Effects of Protopanaxatriol Saponins on Ulcerative Colitis in Mouse Based on UPLC-Q/TOF-MS Serum and Colon Metabolomics.

Ulcerative colitis (UC) is a chronic, nonspecific inflammation of the bowel that mainly affects the mucosa and submucosa of the rectum and colon. Ginsenosides are the main active ingredients in ginseng and show many therapeutic effects in anti-inflammatory diseases, cancer, and nervous system regulation. Protopanaxatriol saponin (PTS) is an important part of saponins, and there is no research on its pharmacological effects on colitis. In this study, a model of ulcerative colitis in mice was induced by having mice freely drink 3.5% dextran sodium sulfate (DSS) solution, and UPLC-Q-TOF-MS-based metabolomics methods were applied to explore the therapeutic effect and protective mechanism of PTS for treating UC. The results showed that PTS could significantly prevent colon shortening and pathological damage and alleviate abnormal changes in UC mouse physiological and biochemical parameters. Moreover, PTS intervention regulated proinflammatory cytokines such as TNF-α, IL-6, and IL-1 in serum, and MPO and NO in colon. Interestingly, PTS could significantly inhibit UC mouse metabolic dysfunction by reversing abnormal changes in 29 metabolites and regulating eleven metabolic pathways. PTS has potential application in the treatment of UC and could alleviate UC in mice by affecting riboflavin metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, retinol metabolism, and steroid hormone biosynthesis and by regulating pentose and glucuronate conversion, linoleic acid metabolism, phenylalanine metabolism, ether lipid metabolism, sphingolipid metabolism, and tyrosine metabolism, which points at a direction for further research and for the development of PTS as a novel natural agent.

Ginsenoside (20S)-protopanaxatriol induces non-protective autophagy and apoptosis by inhibiting Akt/mTOR signaling pathway in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) lacks a recognized therapeutic molecular target and has an unfavorable prognosis. (20S)-Protopanaxatriol (g-PPT, PPT) is an active metabolite extracted from ginseng. Accumulating evidence suggests that it has good anti-cancer activity in vivo and in vitro. In this study, we aimed to elucidate the anti-tumor effects of PPT in TNBC cells and tumor-bearing mice, as well as the relevant molecular mechanisms of autophagy and apoptosis. In vitro, we have found that PPT is capable of inducing non-protective autophagy and apoptosis, thus exerting some anti-proliferative and anti-migration activity in TNBC cells. And in vivo, the therapeutic effects of PPT were evaluated by xenograft mouse models. The potential binding mode of PPT and Akt was predicted by molecular docking. Our findings indicated that PPT treatment induced non-protective autophagy in TNBC cells by inhibiting the Akt/mTOR signaling pathway. Therefore, PPT may be a potential treatment for TNBC in the future.

High-level sustainable production of the characteristic protopanaxatriol-type saponins from Panax species in engineered Saccharomyces cerevisiae.

The Chinese medicinal plant Panax notoginseng has been traditionally used to activate blood flow and circulation, and to prevent blood stasis. P. notoginseng contains protopanaxatriol (PPT)-type saponins as its main active compounds, thus distinguishing it from the other two famous Panax species, P. ginseng and P. quinquefolius. Ginsenoside Rg1 (Rg1), notoginsenoside R1 (NgR1), and notoginsenoside R2 (NgR2) are three major PPT-type saponins in P. notoginseng and possess potential cardiovascular protection activities. However, their use in medical applications has long been hampered by the lack of sustainable and low-cost industrial-scale preparation methods. In this study, a PPT-producing yeast chassis strain was designed and constructed based on a previously constructed and optimized protopanaxadiol (PPD)-producing Saccharomyces cerevisiae strain, and further optimized by systemically engineering and optimizing the expression level of its key P450 biopart. Rg1-producing yeast strains were constructed by introducing PgUGT71A53 and PgUGT71A54 into the PPT chassis strain. The fermentation titer of Rg1 reached 1.95 g/L. A group of UDP-glycosyltransferases (UGT) from P. notoginseng and P. ginseng were characterized, and were found to generate NgR1 and NgR2 by catalyzing the C6-O-Glc xylosylation of Rg1 and Rh1, respectively. Using one of these UGTs, PgUGT94Q13, and the previously identified PgUGT71A53 and PgUGT71A54, the biosynthetic pathway to produce saponins NgR1 and NgR2 from PPT could be available. The NgR1 cell factory was further developed by introducing PgUGT94Q13 and a heterologous UDP-xylose biosynthetic pathway from Arabidopsis thaliana into the highest Rg1-producing cell factory. The NgR2-producing cell factory was constructed by introducing PgUGT71A54, PgUGT94Q13, and the UDP-xylose biosynthetic pathway into the PPT chassis. De novo production of NgR1 and NgR2 reached 1.62 g/L and 1.25 g/L, respectively. Beyond the realization of artificial production of the three valuable saponins Rg1, NgR1, and NgR2 from glucose, our work provides a green and sustainable platform for the efficient production of other PPT-type saponins in engineered yeast strains, and promotes the industrial application of PPT-type saponins as medicine and functional foods.

In vitro modulatory effects of ginsenoside compound K, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol on uridine 5'-diphospho-glucuronosyltransferase activity and expression.

We explored the inhibitory effect of ginsenoside compound K (CK), 20(S)-protopanaxadiol (PPD), and 20(S)-protopanaxatriol (PPT) on six uridine 5'-diphospho-glucuronosyltransferase (UGT) enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) activities in human liver microsomes (HLMs) and 10 UGT enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17) activities in recombinant UGT isoforms.PPD was a potent inhibitor of UGT1A3 activity with half-maximal inhibitory concentration values of 5.62 and 3.38 µM in HLMs and recombinant UGT1A3, respectively. UGT1A3 inhibition by CK and PPD was competitive with inhibitory constant (Ki) values of 17.4 and 1.21 µM, respectively, and inhibition by PPT was non-competitive with a Ki value of 8.07 µM in HLMs. PPD exhibited more than 3.4-fold selectivity for UGT1A3 inhibition compared with other UGT isoforms inhibition, while CK and PPT showed more than 2.16- and 2.21-fold selectivity, respectively.PPD did not significantly increase the mRNA expression of UGT1A1, 1A3, 1A4, 1A9, and 2B7 in hepatocytes.Given the low plasma concentrations of PPD in healthy human subjects and the absence of induction potential on UGT isoforms, we conclude that PPD cause no pharmacokinetic interactions with other co-administered drugs metabolised by UGT1A3.

20(S)-Protopanaxadiol Saponins Mainly Contribute to the Anti-Atherogenic Effects of Panax notoginseng in ApoE Deficient Mice.

Atherosclerosis mainly contributes to cardiovascular disease, a leading cause of global morbidity and mortality. Panax notoginseng saponins (PNS) are proved to therapeutically attenuate the formation of atherosclerotic lesions. According to different sapogenin, PNS are generally classified into 20(S)-protopanaxadiol saponins (PDS) and 20(S)-protopanaxatriol saponins (PTS). It was reported that PDS and PTS might exert diverse or even antagonistic bioactivities. In this study, the probable effects of PTS and PDS on atherosclerotic development were investigated and compared in ApoE-deficient mice (ApoE-/-). Male mice were gavaged daily by PNS (200 mg/kg/d), PTS (100 mg/kg/d), or PDS (100 mg/kg/d), respectively for eight weeks. The treatments of PNS and PDS, but not PTS, showed decreased atherosclerotic lesions in the entire aorta by 45.6% and 41.3%, respectively, as evaluated by an en-face method. Both PNS and PDS can improve the plaque vulnerability, as evidenced by the increased collagen fiber, increased expression of α- smooth muscle actin (α-SMA), and decreased Cluster of differentiation 14 (CD14). Additionally, PDS also inhibit the nuclear factor kappa B (NF-κB)-mediated vascular inflammation in the aorta. In conclusion, PDS, but not PTS, might mainly contribute to the anti-atherosclerosis of P. notoginseng.

Neuroprotective effects of 20(S)-protopanaxatriol (PPT) on scopolamine-induced cognitive deficits in mice.

20(S)-protopanaxatriol (PPT), one of the ginsenosides from Panax ginseng, has been reported to have neuroprotective effects and to improve memory. The present study was designed to investigate the protective effect of PPT on scopolamine-induced cognitive deficits in mice. Male Institute of Cancer Research mice were pretreated with 2 different doses of PPT (20 and 40 µmol/kg) for 27 days by intraperitoneal injection, and scopolamine (0.75 mg/kg) was injected intraperitoneally for 9 days to induce memory impairment. Thirty minutes after the last pretreatment, the locomotor activity was firstly examined to evaluate the motor function of mice. Then, memory-related behaviors were evaluated, and the related mechanism was further researched. It was founded that PPT treatment significantly reversed scopolamine-induced cognitive impairment in the object location recognition experiment, the Morris water maze test, and the passive avoidance task, showing memory-improving effects. PPT also significantly improved cholinergic system reactivity and suppressed oxidative stress, indicated by inhibition of acetylcholinesterase activity, elevation of acetylcholine levels, increasing superoxide dismutase activity and lowering levels of malondialdehyde in the hippocampus. In addition, the expression levels of Egr-1, c-Jun, and cAMP responsive element binding in the hippocampus were significantly elevated by PPT administration. These results suggest that PPT may be a potential drug candidate for the treatment of cognitive deficit in Alzheimer's disease.

Potential Dissociative Glucocorticoid Receptor Activity for Protopanaxadiol and Protopanaxatriol.

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist.

Integrated Chemical and Transcriptomic Analysis Reveals the Distribution of Protopanaxadiol- and Protopanaxatriol-Type Saponins in Panax notoginseng.

Panax notoginseng is famous for its important therapeutic effects and commonly used worldwide. The active ingredients saponins have distinct contents in different tissues of P. notoginseng, and they may be related to the expression of key genes in the synthesis pathway. In our study, high-performance liquid chromatography results indicated that the contents of protopanaxadiol-(Rb1, Rc, Rb2, and Rd) and protopanaxatriol-type (R1, Rg1, and Re) saponins in below ground tissues were higher than those in above ground tissues. Clustering dendrogram and PCA analysis suggested that the below and above ground tissues were clustered into two separate groups. A total of 482 and 882 unigenes were shared in the below and above ground tissues, respectively. A total of 75 distinct expressions of CYPs transcripts (RPKM ≥ 10) were detected. Of these transcripts, 38 and 37 were highly expressed in the below ground and above ground tissues, respectively. RT-qPCR analysis showed that CYP716A47 gene was abundantly expressed in the above ground tissues, especially in the flower, whose expression was 31.5-fold higher than that in the root. CYP716A53v2 gene was predominantly expressed in the below ground tissues, especially in the rhizome, whose expression was 20.1-fold higher than that in the flower. Pearson's analysis revealed that the CYP716A47 expression was significantly correlated with the contents of ginsenoside Rc and Rb2. The CYP716A53v2 expression was associated with the saponin contents of protopanaxadiol-type (Rb1 and Rd) and protopanaxatriol-type (R1, Rg1, and Re). Results indicated that the expression patterns of CYP716A47 and CYP716A53v2 were correlated with the distribution of protopanaxadiol-type and protopanaxatriol-type saponins in P. notoginseng. This study identified the pivotal genes regulating saponin distribution and provided valuable information for further research on the mechanisms of saponin synthesis, transportation, and accumulation.

A validated LC-MS/MS method for the simultaneous determination of 20-(S)-protopanaxatriol and its two active metabolites in rat plasma: Application to a pharmacokinetics study.

We present a validated liquid chromatography with tandem mass spectrometry method for simultaneous determination of 20-(S)-protopanaxatriol and its two oxidative stereoisomeric metabolites (20S,24S)-epoxy-dammarane-3,6,12,25-tetraol (M1) and (20S,24R)-epoxy-dammarane-3,6,12,25-tetraol (M2) in rat plasma. 20-(S)-Protopanaxatriol, M1, and M2 were extracted with methanol and separated on an ACQUITY HSS T3 column. The mass spectrometry detection was accomplished in selected reaction monitoring mode with precursor-to-product ion transitions of m/z 493.4→143.1 for M1 and M2, m/z 475.4→391.3 for 20-(S)-protopanaxatriol, and m/z 459.4→375.3 for 20-(S)-protopanaxadiol (internal standard). The method showed good linearity over the concentration ranges of 1-1000 ng/mL for 20-(S)-protopanaxatriol and 0.5-200 ng/mL for M1 and M2, with correlation coefficients of more than 0.995. The lower limits of quantification for 20-(S)-protopanaxatriol, M1, and M2 were 1, 0.5, 0.5 ng/mL, respectively. The intra- and interday precisions (RSD, %) were less than 10.41% while the accuracy (relative error, %) ranged from -3.14 to 8.73%. Under the current conditions, M1 and M2 were completely separated within 3 min. The validated assay was successfully applied to evaluating pharmacokinetic profiles of 20-(S)-protopanaxatriol, M1, and M2 in rat.

Potential accumulation of protopanaxadiol-type ginsenosides in six-months toxicokinetic study of SHENMAI injection in dogs.

SHENMAI injection (SMI), derived from famous Shen Mai San, is a herbal injection widely used in China. Ginsenosides are the major components of SMI. To monitor the exposure level of SMI during long-term treatment, a 6-month toxicokinetic experiment was performed. Twenty-four beagle dogs were dived into four groups (n = 6 in each group): a control group (0.9% NaCl solution) and three SMI groups (2, 6 or 3 mg/kg). The dogs were i.v. infused with vehicle or SMI daily for 180 d. Blood samples for analysis were collected at specific time points as follows: pre-dose (0 h); at 10, 30, and 60 min during infusion; and at 10, 30, 60, 90, 120, 240, and 300 min post-administration. Concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 in the plasma were determined simultaneously by liquid chromatography-tandem mass spectrometry. Non-compartmental parameters were further calculated and analyzed. Significant differences were found between the kinetic behavior of 20(S)-protopanaxadiol-type (PPD-type) and 20(S)-protopanaxatriol-type (PPT-type) ginsenosides. Increasing in the exposure level of PPD-type ginsenosides was observed in dogs during the experiment. Therefore, PPD-type ginsenosides are closely related to the immunity modulation effect of SMI. Increased PPD-type ginsenoside exposure level may present potential toxicity and induce drug-drug interaction risks during SMI administration. As such, PPD-type ginsenoside accumulation must be carefully monitored in future SMI research.

20(S)-protopanaxatriol inhibits liver X receptor α-mediated expression of lipogenic genes in hepatocytes.

20(S)-protopanaxatriol (PPT) is an aglycone of ginsenosides isolated from Panax ginseng and has several interesting activities, including anti-inflammatory and anti-oxidative stress effects. Herein, PPT was identified as an inhibitor against the ligand-dependent transactivation of liver X receptor α (LXRα) using a Gal4-TK-luciferase reporter system. LXRα is a transcription factor of nuclear hormone receptor family and stimulates the transcription of many metabolic genes, such as lipogenesis- or reverse cholesterol transport (RCT)-related genes. Quantitative RT-PCR analysis showed that PPT inhibited the LXRα-dependent transcription of lipogenic genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase, and stearoyl CoA desaturase 1. These inhibitory effects of PPT are, at least in part, a consequence of the reduced recruitment of RNA polymerase II to the LXR response element (LXRE) of the SREBP-1c promoter. Furthermore, LXRα-dependent triglyceride accumulation in primary mouse hepatocytes was significantly reduced by PPT. Interestingly, PPT did not inhibit the LXRα-dependent transcription of ABCA1, a crucial LXRα target gene involved in RCT. Chromatin immunoprecipitation assays revealed that PPT repressed recruitment of the lipogenic coactivator TRAP80 to the SREBP-1c LXRE, but not the ABCA1 LXRE. Overall, these data suggest that PPT has selective inhibitory activity against LXRα-mediated lipogenesis, but not LXRα-stimulated RCT.

Stereoselective oxidation metabolism of 20(S)-protopanaxatriol in human liver microsomes and in rats.

1. In this study, the oxidative metabolites of 20(S)-protopanaxatriol (PPT) were identified in human liver microsomes (HLMs) and in rats using liquid chromatography-electrospray ionization tandem mass spectrometry. 2. Twelve oxidative metabolites were found in HLM, eight of which have not been previously reported. Twenty-four oxidative metabolites were found in rat feces after oral administration and 20 of these, including six found in HLM, were first reported. The results indicated PPT was more extensively metabolized in rats than in HLM. C20-24 epoxides, a pair of epimers (namely, M1-1 and M1-2) were the major metabolites, and other metabolites were derived from their further metabolism. 3. Enzyme kinetics experiments showed that the apparent formation Vmax of M1-1 was 10.4 folds and 2.4 folds higher than that of M1-2 in HLM and in rat liver microsomes (RLMs), respectively. The depletion rate of M1-2 was 11.0 folds faster than M1-1 in HLM, and was similar in RLM. Hence, the remarkable species differences of PPT metabolism mainly resulted from the stereoselective formation and further metabolic elimination of M1-1 and M1-2. 4. Chemical inhibition study and recombinant human P450 isoforms analysis showed that CYP3A4 was the predominant isoform involved in the oxidative metabolism of M1-1 and M1-2.

Biotransformation of 20(S)-protopanaxatriol by Mucor racemosus and the anti-cancer activities of some products.

OBJECTIVE: To produce new derivatives of 20(S)-protopanaxatriol by fungal biotransformation. RESULT: Biotransformation of 20(S)-protopanaxatriol (1) by Mucor racemosus AS 3.205 afforded six products. Their structures were elucidated on the basis of extensive spectroscopic analyses. M. racemosus could selectively catalyze dehydrogenation at C-12 and further hydroxylation at C-7, C-11, and C-15, as well as rearrangement of double bond at C-26. Two of these new compounds exhibited potent inhibitory activity against SH-SY5Y and HepG2 cell lines. CONCLUSION: Biotransformation by M. racemosus AS 3.205 was an effective approach to produce new derivatives of 20(S)-protopanaxatriol.

Cloning and heterologous expression of UDP-glycosyltransferase genes from Bacillus subtilis and its application in the glycosylation of ginsenoside Rh1.

UNLABELLED: Bacillus subtilis CCTCC AB 2012913 can transform ginsenoside Rh1 to 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol. Based on its genome sequence, strain B. subtilis 168 contains three UDP-glycosyltransferase genes. Here, we cloned the three UDP-glycosyltransferase genes (ydhE1, yojK1 and yjiC1) from B. subtilis CCTCC AB 2012913 and expressed in Escherichia coli BL21 (DE3) with His-tag. The crude enzyme extracts were assayed, respectively, for their activities to transform ginsenoside Rh1. Extracts containing enzymes YojK1 and YjiC1 could use ginsenoside Rh1 as a substrate to produce 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol, which had an additional glucopyranosyl linked with C-3 over the substrate. Enzyme YjiC1 was purified by affinity chromatography on Ni-NTA His Binding resin. The molecular mass of purified YjiC1 was c. 47 kDa as determined by SDS-PAGE. This is the first report of an in vitro biotransformation of ginsenoside Rh1 to 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol using the recombinant UDP-glycosyltransferase. SIGNIFICANCE AND IMPACT OF THE STUDY: The Chinese traditional medicinal plant Panax is reported to have multiple health benefits. Its main active ingredient is saponin, and different saponins have different activity spectrum. In the study, three UDP-glycosyltransferase genes, ydhE1, yojK1 and yjiC1, were cloned from Bacillus subtilis CCTCC AB2012913 and the three genes were expressed in Escherichia coli BL21 (DE3). The enzyme YjiC1 was purified and converted ginsenoside Rh1 to 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol in vitro. The compound is the first saponin possessing ß-glucopyranosyl at both C-3 and C-6 sites. We showed that the in vitro biotransformation was effective, and the reaction condition was easy to control. Our research suggests that a diversity of saponins could be generated through efficient and directed enzymatic biotransformation.

Conformational studies of dammarane-type triterpenoids using computational and NMR spectroscopic methods.

Natural triterpenoids are of great interest to researchers of various fields as they possess diverse physicochemical and biological properties. In medicinal chemistry, detailed information about the chemical structures of bioactive triterpenoids often helps find new lead compounds. Herein, the low-energy structures of (20S)-protopanaxadiol and (20S)-protopanaxatriol, the aglycones of various triterpenoid saponins found in Panax ginseng, and their (20R)-epimers have been predicted by the geometry optimization of the conformers extracted from molecular dynamics simulations with the self-consistent-charge density functional tight-binding method. By performing quantum mechanical calculations on the low-energy conformers, we have estimated the NMR chemical shifts of the compounds, which display good agreement with the most recently reported experimental values within an expected range of errors. Our results indicate that theoretical estimation of the NMR parameters of a relatively large molecule with a molecular mass of 500 is feasible.