Pseudoginsenoside

Novel glycosidase from Paenibacillus lactis 154 hydrolyzing the 28-O-beta-D-glucopyranosyl ester bond of oleanane-type saponins.[Pubmed:38573330]

Appl Microbiol Biotechnol. 2024 Apr 4;108(1):282.

Oleanane-type ginsenosides are a class of compounds with remarkable pharmacological activities. However, the lack of effective preparation methods for specific rare ginsenosides has hindered the exploration of their pharmacological properties. In this study, a novel glycoside hydrolase PlGH3 was cloned from Paenibacillus lactis 154 and heterologous expressed in Escherichia coli. Sequence analysis revealed that PlGH3 consists of 749 amino acids with a molecular weight of 89.5 kDa, exhibiting the characteristic features of the glycoside hydrolase 3 family. The enzymatic characterization results of PlGH3 showed that the optimal reaction pH and temperature was 8 and 50 degrees C by using p-nitrophenyl-beta-D-glucopyranoside as a substrate, respectively. The K(m) and k(cat) values towards ginsenoside Ro were 79.59 +/- 3.42 microM and 18.52 s(-1), respectively. PlGH3 exhibits a highly specific activity on hydrolyzing the 28-O-beta-D-glucopyranosyl ester bond of oleanane-type saponins. The mechanism of hydrolysis specificity was then presumably elucidated through molecular docking. Eventually, four kinds of rare oleanane-type ginsenosides (calenduloside E, Pseudoginsenoside RP1, zingibroside R1, and tarasaponin VI) were successfully prepared by biotransforming total saponins extracted from Panax japonicus. This study contributes to understanding the mechanism of enzymatic hydrolysis of the GH3 family and provides a practical route for the preparation of rare oleanane-type ginsenosides through biotransformation. KEY POINTS: * The glucose at C-28 in oleanane-type saponins can be directionally hydrolyzed. * Mechanisms to interpret PlGH3 substrate specificity by molecular docking. * Case of preparation of low-sugar alternative saponins by directed hydrolysis.

Pseudoginsenoside GQ mitigates chronic intermittent hypoxia-induced cognitive damage by modulating microglia polarization.[Pubmed:37977071]

Int Immunopharmacol. 2024 Jan 5;126:111234.

Obstructive sleep apnea (OSA), a state of sleep disruption, is characterized by recurrent apnea, chronic intermittent hypoxia (CIH) and hypercapnia. Previous studies have showed that CIH-induced neuroinflammatory plays a crucial role in cognitive deficits. Pseudoginsenoside GQ (PGQ) is a new oxytetracycline-type saponin formed by the oxidation and cyclization of the 20(S) Rg3 side chain. Rg3 has been found to afford anti-inflammatory effects, while whether PGQ plays a role of anti-neuroinflammatory remains unclear. The purpose of this study was to investigate whether PGQ attenuates CIH-induced neuroinflammatory and cognitive impairment and the possible mechanism it involves. We found that PGQ significantly ameliorated CIH-induced spatial learning deficits, and inhibited microglial activation, pro-inflammatory cytokine release, and neuronal apoptosis in the hippocampus of CIH mice. In addition, PGQ pretreatment promoted microglial M1 to M2 phenotypic transition in IH-induced BV-2 microglial, as well as indirectly inhibited IH-induced neuronal injury via modulation of microglia polarization. Furthermore, we noted that activation of HMGB1/TLR4/NF-kappaB signaling pathway induced by IH was inhibited by PGQ. Molecular docking results revealed that PGQ could bind to the active sites of HMGB1 and TLR4. Taken together, this work supports that PGQ inhibits M1 microglial polarization via the HMGB1/TLR4/NF-kappaB signaling pathway, and indirectly exerts neuroprotective effects, suggesting that PGQ may be a potential therapeutic strategy for cognitive impairment accompanied OSA.

Diversity and Isolation of Endophytic Fungi in Panax japonicus and Biotransformation Activity on Saponins.[Pubmed:37767797]

Curr Pharm Biotechnol. 2023 Sep 25.

OBJECTIVE: This study reports the diversity and community structure differences of the endophytic fungi of Panax japonicus of different ages to obtain novel endophytic fungi with glycoside hydrolytic activity for rare saponins production. METHODS: This study used the high-throughput sequencing method to analyze the diversity and community structure of endophytic fungi of P. japonicus. The endophytic fungi were processed by traditional isolation, culture, conservation, and ITS rDNA sequence analyses. Then the total saponins of P. japonicus were used as the substrate to evaluate the glycoside hydrolytic activity. RESULTS: The composition analysis of the community structure showed that the abundance, evenness, and diversity of endophytic fungi of nine-year-old P. japonicus were the best among all samples. A total of 210 endophytic fungi were isolated from P. japonicus samples and further annotated by sequencing the internal transcribed spacer. Then the biotransformation activity of obtained strains was further examined on total saponins of P. japonicus (TSPJ), with a strain identified as Fusarium equiseti (No.30) from 7-year-old P. japonicus showing significant glycoside hydrolytic activity on TSPJ, including ginsenoside Ro-->zinglbroside R1, Pseudoginsenoside RT1-->Pseudoginsenoside RP1, chikusetsusaponin IV-->tarasaponin VI and chikusetsusaponin IVa -->calenduloside E. CONCLUSION: These results reveal the diversity and community structure differences of the endophytic fungi of P. japonicus with different ages and establish a resource library of endophytic fungi of P. japonicus. More importantly, we identified a valuable endophytic fungus with glycoside hydrolytic activity and provided a promising convenient microbial transformation approach to produce minor deglycosylated ginsenosides.

A new ocotillol-type ginsenoside from American ginseng berry.[Pubmed:37667570]

Nat Prod Res. 2023 Sep 4:1-6.

A new ocotillol-type ginsenoside, named Pseudoginsenoside F(12) (1), was isolated from American ginseng berry, whose structure was elucidated as 6-O-[alpha-L-2,3-epoxy-rhamnopyranosyl-(1-2)-beta-D-glucopyranosyl]-dammar-20S,24R-epoxy-3beta, 6alpha,12beta,25-tetraol. In addition, the known alkaloids beta-carboline-1-carboxylic acid (2) and anoectochine (3) were isolated for the first time from the Araliaceae family. The new compound 1 was evaluated for cytotoxicity against MDA-MB-231 breast cancer cell line.

Therapeutic Candidates for Alzheimer's Disease: Saponins.[Pubmed:37445682]

Int J Mol Sci. 2023 Jun 22;24(13):10505.

Drug development for Alzheimer's disease, the leading cause of dementia, has been a long-standing challenge. Saponins, which are steroid or triterpenoid glycosides with various pharmacological activities, have displayed therapeutic potential in treating Alzheimer's disease. In a comprehensive review of the literature from May 2007 to May 2023, we identified 63 references involving 40 different types of saponins that have been studied for their effects on Alzheimer's disease. These studies suggest that saponins have the potential to ameliorate Alzheimer's disease by reducing amyloid beta peptide deposition, inhibiting tau phosphorylation, modulating oxidative stress, reducing inflammation, and antiapoptosis. Most intriguingly, ginsenoside Rg1 and Pseudoginsenoside-F11 possess these important pharmacological properties and show the best promise for the treatment of Alzheimer's disease. This review provides a summary and classification of common saponins that have been studied for their therapeutic potential in Alzheimer's disease, showcasing their underlying mechanisms. This highlights the promising potential of saponins for the treatment of Alzheimer's disease.

Identification of two key UDP-glycosyltransferases responsible for the ocotillol-type ginsenoside majonside-R2 biosynthesis in Panax vietnamensis var. fuscidiscus.[Pubmed:37178342]

Planta. 2023 May 13;257(6):119.

Two UDP-glycosyltransferases from Panax vienamensis var. fuscidiscus involved in ocotillol-type ginsenoside MR2 (majonside-R2) biosynthesis were identified. PvfUGT1 and PvfUGT2 sequentially catalyzes 20S,24S-Protopanxatriol Oxide II and 20S,24R-Protopanxatriol Oxide I to Pseudoginsenoside RT4/RT5 and RT4/RT5 to 20S, 24S-MR2/20S, 24S-MR2. Ocotilol type saponin MR2 (majonside-R2) is the main active component of Panax vietnamensis var. fuscidiscus (commonly known as 'jinping ginseng') and is well known for its diverse pharmacological activities. The use of MR2 in the pharmaceutical industry currently depends on its extraction from Panax species. Metabolic engineering provides an opportunity to produce high-value MR2 by expressing it in heterologous hosts. However, the metabolic pathways of MR2 remain enigmatic, and the two-step glycosylation involved in MR2 biosynthesis has not been reported. In this study, we used quantitative real-time PCR to investigate the regulation of the entire ginsenoside pathway by MeJA (methyl jasmonate), which facilitated our pathway elucidation. We found six candidate glycosyltransferases by comparing transcriptome analysis and network co-expression analysis. In addition, we identified two UGTs (PvfUGT1 and PvfUGT2) through in vitro enzymatic reactions involved in the biosynthesis of MR2 which were not reported in previous studies. Our results show that PvfUGT1 can transfer UDP-glucose to the C6-OH of 20S, 24S-protopanaxatriol oxide II and 20S, 24R-protopanaxatriol oxide I to form Pseudoginsenoside RT4 and Pseudoginsenoside RT5, respectively. PvfUGT2 can transfer UDP-xylose to Pseudoginsenoside RT4 and Pseudoginsenoside RT5 to form 20S, 24S-MR2 and 20S, 24S-MR2. Our study paves the way for elucidating the biosynthesis of MR2 and producing MR2 by synthetic biological methods.

LC-MS Analysis of Ginsenosides in Different Parts of Panax quinquefolius and Their Potential for Coronary Disease Improvement.[Pubmed:36940929]

Planta Med. 2023 Jun;89(7):764-772.

Seven main ginsenosides, including ginsenoside Re, ginsenoside Rb(1), Pseudoginsenoside F(11), ginsenoside Rb(2), ginsenoside Rb(3), ginsenoside Rd, and ginsenoside F(2), were identified by LC-QTOF MS/MS from root, leaf and flower extracts of Panax quinquefolius. These extracts promoted intersegmental vessel growth in a zebrafish model, indicating their potential cardiovascular health benefits. Network pharmacology analysis was then conducted to reveal the potential mechanisms of ginsenoside activity in the treatment of coronary artery disease. GO and KEGG enrichment analyses elucidated that G protein-coupled receptors played a critical role in VEGF-mediated signal transduction and that the molecular pathways associated with ginsenoside activity are involved in neuroactive ligand-receptor interaction, cholesterol metabolism, the cGMP-PKG signaling pathway, etc. Moreover, VEGF, FGF2, and STAT3 were confirmed as the major targets inducing proliferation of endothelial cells and driving the pro-angiogenic process. Overall, ginsenosides could be potent nutraceutical agents that act to reduce the risks of cardiovascular disease. Our findings will provide a basis to utilize the whole P. quinquefolius plant in drugs and functional foods.

Comparison of Phytochemical Profiles of Wild and Cultivated American Ginseng Using Metabolomics by Ultra-High Performance Liquid Chromatography-High-Resolution Mass Spectrometry.[Pubmed:36615206]

Molecules. 2022 Dec 20;28(1):9.

American ginseng (Panax quinquefolius L.) has been recognized as a valuable herb medicine, and ginsenosides are the most important components responsible for the health-beneficial effects. This study investigated the secondary metabolites responsible for the differentiation of wild and cultivated American ginsengs with ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based metabolomic approach. An in-house ginsenoside library was developed to facilitate data processing and metabolite identification. Data visualization methods, such as heatmaps and volcano plots, were utilized to extract discriminated ion features. The results suggested that the ginsenoside profiles of wild and cultivated ginsengs were significantly different. The octillol (OT)-type ginsenosides were present in greater abundance and diversity in wild American ginsengs; however, a wider distribution of the protopanaxadiol (PPD)-and oleanolic acid (OA)-type ginsenosides were found in cultivated American ginseng. Based on the tentative identification and semi-quantification, the amounts of five ginsenosides (i.e., notoginsenoside H, glucoginsenoside Rf, notoginsenoside R1, Pseudoginsenoside RT2, and ginsenoside Rc) were 2.3-54.5 fold greater in wild ginseng in comparison to those in their cultivated counterparts, and the content of six ginsenosides (chicusetsusaponin IVa, malonylginsenoside Rd, Pseudoginsenoside Rc1, malonylfloralginsenoside Rd6, Ginsenoside Rd, and malonylginsenoside Rb1) was 2.6-14.4 fold greater in cultivated ginseng compared to wild ginseng. The results suggested that the in-house metabolite library can significantly reduce the complexity of the data processing for ginseng samples, and UHPLC-HRMS is effective and robust for identifying characteristic components (marker compounds) for distinguishing wild and cultivated American ginseng.

Identification of key pharmacodynamic markers of American ginseng against heart failure based on metabolomics and zebrafish model.[Pubmed:36313322]

Front Pharmacol. 2022 Oct 14;13:909084.

Background: American ginseng (Panax quinquefolium L., AG) is a traditional Chinese medicine with multiple cardiovascular protective properties. Many bioactive components have been discovered in AG over these years. However, the understanding of these key pharmacodynamic components of activity against heart failure is insufficient. Methods: A heart failure model was established using AB line wild-type zebrafish (Danio rerio) to evaluate the anti-heart failure activity of AG. Untargeted metabolomics analysis based on ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap-mass spectrometry technology (UHPLC-QE-Orbitrap-MS) was performed to screen differential components from AG samples. The potential active components were verified using the zebrafish model. Simultaneously, network pharmacology and molecular docking techniques were used to predict the possible mechanism. Finally, the key targets of six key pharmacodynamic components were verified in zebrafish using quantitative real-time-polymerase chain reaction (Q-PCR) techniques. Results: The heart failure model was successfully established in 48 h of post-fertilization (hpf) zebrafish larvae by treating with verapamil hydrochloride. The zebrafish assay showed that the anti-heart failure effects of AG varied with producing regions. The result of the herbal metabolomic analysis based on UHPLC-QE-Orbitrap-MS indicated that ginsenoside Rg3, ginsenoside Rg5, ginsenoside Rg6, malic acid, quinic acid, L-argininosuccinic acid, 3-methyl-3-butenyl-apinosyl (1-->6) glucoside, Pseudoginsenoside F11, and annonaine were differential components, which might be responsible for variation in efficacy. Further analysis using zebrafish models, network pharmacology, and Q-PCR techniques showed that ginsenoside Rg3, ginsenoside Rg5, ginsenoside Rg6, malic acid, quinic acid, and Pseudoginsenoside F11 were the pharmacodynamic markers (P-markers) responsible for anti-heart failure. Conclusion: We have rapidly identified the P-markers against heart failure in AG using the zebrafish model and metabolomics technology. These P-markers may provide new reference standards for quality control and new drug development of AG.

Integrative SMRT sequencing and ginsenoside profiling analysis provide insights into the biosynthesis of ginsenoside in Panax quinquefolium.[Pubmed:36031233]

Chin J Nat Med. 2022 Aug;20(8):614-626.

Panax quinquefolium is one of the most common medicinal plants worldwide. Ginsenosides are the major pharmaceutical components in P. quinquefolium. The biosynthesis of ginsenosides in different tissues of P. quinquefolium remained largely unknown. In the current study, an integrative method of transcriptome and metabolome analysis was used to elucidate the ginsenosides biosynthesis pathways in different tissues of P. quinquefolium. Herein, 22 ginsenosides in roots, leaves, and flower buds showed uneven distribution patterns. A comprehensive P. quinquefolium transcriptome was generated through single molecular real-time (SMRT) and second-generation sequencing (NGS) technologies, which revealed the ginsenoside pathway genes and UDP-glycosyltransferases (UGT) family genes explicitly expressed in roots, leaves, and flower buds. The weighted gene co-expression network analysis (WGCNA) of ginsenoside biosynthesis genes, UGT genes and ginsenoside contents indicated that three UGT genes were positively correlated to Pseudoginsenoside F11, notoginsenoside R1, notoginsenoside R2 and Pseudoginsenoside RT5. These results provide insights into ginsenoside biosynthesis in different tissues ofP. quinquefolium.

Comprehensive investigation on metabolites of Panax quinquefolium L. in two main producing areas of China based on ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.[Pubmed:34905806]

J Mass Spectrom. 2021 Nov 5;56(12):e4791.

Jilin Province and Shandong Province are two main American ginseng (AG) producing areas in China. The geographical difference existed in these two provinces. Aiming at evaluating the similarities and differences of the secondary metabolites, the comprehensive metabolite profiling of AG from Jilin Province (AG(J) ) and Shandong Province (AG(S) ) was performed based on UPLC-QTOF-MS for the first time. In screening analysis, a total of 111 shared compounds, with ginsenosides being major components, were identified or tentatively characterized, which indicated that AG(J) and AG(S) were all rich in phytochemicals and contained similar structural types. Untargeted metabolomics analysis indicated that there were significant differences in the contents of certain constituents in AG(J) and AG(S) . Nineteen (12 for AG(J) , 7 for AG(S) ) potential producing area-dependent chemical markers were discovered. Based on the contents and MS responses, ginsenoside Rg(1) , Re, and Pseudoginsenoside F(11) could be considered as the characteristical markers of AG(J) , whereas ginsenoside Rg(3) and Rh(2) of AG(S) . This comprehensive phytochemical profile study could provide valuable chemical evidence for evaluating the characteristics qualities of AG from various producing areas.

Neuroprotective effect of pseudoginsenoside-F11 on permanent cerebral ischemia in rats by regulating calpain activity and NR2A submit-mediated AKT-CREB signaling pathways.[Pubmed:34836744]

Phytomedicine. 2022 Feb;96:153847.

BACKGROUND: N-methyl-d-aspartate receptors (NMDARs) have been demonstrated to play central roles in stroke pathology and recovery, including dual roles in promoting either neuronal survival or death with their different subtypes and locations. PURPOSE: We have previously demonstrated that Pseudoginsenoside-F11 (PF11) can provide long-term neuroprotective effects on transient and permanent ischemic stroke-induced neuronal damage. However, it is still needed to clarify whether NMDAR-2A (NR2A)-mediated pro-survival signaling pathway is involved in the beneficial effect of PF11 on permanent ischemic stroke. MATERIAL AND METHODS: PF11 was administrated in permanent middle cerebral artery occlusion (pMCAO)-operated rats. The effect of PF11 on oxygen-glucose deprivation (OGD)-exposed primary cultured neurons were further evaluated. The regulatory effect of PF11 on NR2A expression and the activation of its downstream AKT-CREB pathway were detected by Western blotting and immunofluorescence in the presence or absence of a specific NR2A antagonist NVP-AAM077 (NVP) both in vivo and in vitro. RESULTS: PF11 dose- and time-dependently decreased calpain1 (CAPN1) activity and its specific breakdown product alpha-Fodrin expression, while the expression of Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKII-alpha) was significantly upregulated in the cortex and striatum of rats at 24 h after the onset of pMCAO operation. Moreover, PF11 prevented the downregulation of NR2A, p-AKT/AKT, and p-CREB/CREB in both in vivo and in vitro stroke models. Finally, the results indicated treatment with NVP can abolish the effects of PF11 on alleviating the ischemic injury and activating NR2A-mediated AKT-CREB signaling pathway. CONCLUSIONS: Our results demonstrate that PF11 can exert neuroprotective effects on ischemic stroke by inhibiting the activation of CAPN1 and subsequently enhancing the NR2A-medicated activation of AKT-CREB pathway, which provides a mechanistic link between the neuroprotective effect of PF11 against cerebral ischemia and NR2A-associated pro-survival signaling pathway.

Evaluation of the Saponin Content in Panax vietnamensis Acclimatized to Lam Dong Province by HPLC-UV/CAD.[Pubmed:34500805]

Molecules. 2021 Sep 3;26(17):5373.

Panax vietnamensis, or Vietnamese ginseng (VG), an endemic Panax species in Vietnam, possesses a unique saponin profile and interesting biological activities. This plant is presently in danger of extinction due to over-exploitation, resulting in many preservation efforts towards the geographical acclimatization of VG. Yet, no information on the saponin content of the acclimatized VG, an important quality indicator, is available. Here, we analyzed the saponin content in the underground parts of two- to five-year-old VG plants acclimatized to Lam Dong province. Nine characteristic saponins, including notoginsenoside-R1, ginsenoside-Rg1, -Rb1, -Rd, majonoside-R1, -R2 vina-ginsenoside-R2, -R11, and Pseudoginsenoside-RT4, were simultaneously determined by HPLC coupled with UV and with a charged aerosol detector (CAD). Analyzing the results illustrated that the detection of characteristic ocotillol-type saponins in VG by CAD presented a superior capacity compared with that of UV, thus implying a preferential choice of CAD for the analysis of VG. The quantitative results indicating the saponin content in the underground parts of VG showed an increasing tendency from two to five years old, with the root and the rhizome exhibiting different saponin accumulation patterns. This is the first study that reveals the preliminary success of VG acclimatization and thereby encourages the continuing efforts to develop this valuable saponin-rich plant.

Simultaneous quantitative assays of 15 ginsenosides from 119 batches of ginseng samples representing 12 traditional Chinese medicines by ultra-high performance liquid chromatography coupled with charged aerosol detector.[Pubmed:34487881]

J Chromatogr A. 2021 Oct 11;1655:462504.

Despite the extensive consumption of ginseng, precise quality control of different ginseng products is highly challenging due to the containing of ginsenosides in common for different Panax species or different parts (e.g. root, leaf, and flower) of a same species. Herein we performed a comparative investigation of diverse ginseng products by simultaneously assaying 15 saponins (notoginsenoside R1, ginsenosides Rg1, -Re, -Rf, -Ra2, -Rb1, -Rc, -Ro, -Rb2, -Rb3, -Rd, 20(R)-ginsenoside Rg3, 24(R)-Pseudoginsenoside F11, chikusetsusaponins IV, and -IVa) using an ultra-high-performance liquid chromatography/charged aerosol detector (UHPLC-CAD) approach. Twelve Panax-derived ginseng products (involving P. ginseng root, P. quinquefolius root, P. notoginseng root, Red ginseng, P. ginseng leaf, P. quinquefolius leaf, P. notoginseng leaf, P. ginseng flower, P. quinquefolius flower, P. notoginseng flower, P. japonicus root, and P. japonicus var. major root) were considered. Benefiting from the condition optimization, the baseline resolution of 15 ginsenosides was achieved on a CORTECS UPLC Shield RP18 column. This method was validated as specific, precise (0.81-1.94% intra-day variation; 0.86-2.35% inter-day variation), and accurate (recovery: 90.73-107.5%), with good linearity (R(2) > 0.999), high sensitivity (limit of detection: 0.02-0.21 mug; limit of quantitation: 0.04-0.42 mug) and sample stability (1.49-4.74%). Its application to 119 batches of ginseng samples unveiled vital information enabling the authentication of these different ginseng products. Detection of ginsenosides by CAD exhibited superiority over UV in sensitivity and the ability to monitor chromophore-free structures. Large-scale comparative studies by quantifying multiple markers provide methodological reference to the precise quality control of herbal medicine.