Induction and Suspension Culture of Panax japonicus Callus Tissue for the Production of Secondary Metabolic Active Substances.

Using Panax japonicus as research material, callus induction and culture were carried out, and high-yielding cell lines were screened to establish a suspension culture system that promotes callus growth and the accumulation of the "total saponins" (total content of triterpenoid glycosides or ginsenosides). Using the root as an explant, the medium for callus induction and proliferation was optimized by adjusting culture conditions (initial inoculation amount, carbon source, shaking speed, hormone concentration, culture time) and a high-yielding cell line with efficient proliferation and high total saponins content was screened out. The conditions of suspension culture were refined to find out the most suitable conditions for the suspension culture of callus, and finally, the suspension culture system was established. We found that the lowest (5%) contamination rate was achieved by disinfecting the fresh roots with 75% alcohol for 60 s, followed by soaking in 10% NaClO for 15 min. The highest induction rate (88.17%) of callus was obtained using the medium MS + 16.11 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 sucrose + 7.5 g·L-1 agar. The callus was loose when the callus subcultured on the proliferation medium (MS + 5.37 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 sucrose + 3.8 g·L-1 gellan gum) for 21 days. The callus growth was cultured in a liquid growth medium (MS + 5.37 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 sucrose) with an initial inoculation amount of 40 g·L-1, a shaking speed of 110 r/min and darkness. Cell growth was fastest with a culture period of 21 days. We replaced the growth medium with the production medium (MS + 5.37 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 glucose) for maximum accumulation of total saponins. [Conclusion] A callus induction and suspension culture system for the root of P. japonicus was established. In this way, we can promote the accumulation of total saponins in callus cells and provide a basis for large-scale cell culture and industrial production of medicinal total saponins.

Integrated metabolome and transcriptome analysis provide insight into the biosynthesis of flavonoids in Panax japonicus.

Panax japonicus is an important medicinal plant, and flavonoids are one of its main secondary metabolites. In this study, the main roots, fibrous roots, stems, leaves and flowers of P. japonicus were analyzed using transcriptomics and widely targeted metabolomics. Through correlation analysis of transcription and metabolism, the flavonoid biosynthesis pathway in P. japonicus was analyzed, and the accumulation of flavonoid metabolites and the expression of related genes were investigated. Metabolomics revealed a total of 209 flavonoid metabolites in P. japonicus, among which flavonoids, flavonols, flavanones and flavanonols significantly accumulated in the flowers and leaves. Transcriptome sequencing revealed that key genes in the flavonoid pathway exhibited increased expression in the flowers and leaves. The expression patterns of key genes involved in flavonoid biosynthesis, including PjC4H, Pj4CL, PjCHS, PjCHI, PjF3H, PjF3'H, PjCYP, and PjPAL, are consistent with their upstream and downstream metabolites, demonstrating a significant positive correlation among them. In addition, the PjUGT gene is highly expressed in five tissues of P. japonicus, indicating that PjUGT is one of the key factors for the diversity of flavonoid glycosides. The WGCNA results showed that WRKY transcription factors exist widely in the candidate modules, and it was possible that PjWRKY transcription factors are involved in regulating the expression of key genes involved in flavonoid biosynthesis and the biosynthesis of flavonoid metabolites. This study reveals spatial differences in the accumulation patterns of flavonoid metabolites in different tissues and provides important clues for further understanding the regulatory mechanisms of flavonoid metabolism in P. japonicus, thus contributing to the optimization of germplasm resources of P. japonicus and the promotion of genetic diversity analysis.

Transcriptomic Analysis Under Drought and Salt Stress Provides Insight into Genes Putatively Involved in Ginsenoside Biosynthesis in Panax japonicus Meyer.

Panax japonicus Meyer, a perennial herb of the dicotyledonaceae family Araliaceae, is a rare folk traditional Chinese medicine, known as "the king of herbal medicine" in China. To understand the genes involved in secondary pathways under drought and salt stress, the transcriptomic analysis of P. japonicus is of vital importance. The transcriptome of underground rhizomes, stems, and leaves under drought and salt stress in P. japonicus were performed using the Illumina HiSeq platform. After de novo assembly of transcripts, expression profiling and identified differentially expressed genes (DEGs) were performed. Furthermore, putative functions of identified DEGs correlated with ginsenoside in P. japonicus were explored using Gene Ontology terms and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis. A total of 221,804 unigenes were obtained from the transcriptome of P. japonicus. The further analysis revealed that 10,839 unigenes were mapped to 91 KEGG pathways. Furthermore, a total of two metabolic pathways of P. japonicus in response to drought and salt stress related to triterpene saponin synthesis were screened. The sesquiterpene and triterpene metabolic pathways were annotated and finally putatively involved in ginsenoside content and correlation analysis of the expression of these genes were analyzed to identify four genes, ß-amyrin synthase, isoprene synthase, squalene epoxidase, and 1-deoxy-D-ketose-5-phosphate synthase, respectively. Our results paves the way for screening highly expressed genes and mining genes related to triterpenoid saponin synthesis. It also provides valuable references for the study of genes involved in ginsenoside biosynthesis and signal pathway of P. japonicus.

Total saponins from Panax japonicus regulated the intestinal microbiota to alleviate lipid metabolism disorders in aging mice.

Total saponins from Panax japonicus (TSPJ) have many beneficial physiological activities, particularly in alleviating the damages of aging and abnormal lipid metabolism. This work used mice models to investigate if TSPJ reduced obesity and regulated metabolic functions via the intestinal microbiota, the disturbance of which has been shown to cause aging-related diseases. The results showed that TSPJ significantly reduced the weight and blood lipid level of aging mice. Further analyses showed that TSPJ significantly inhibited adipogenesis, changed the composition of the intestinal flora, and protected the integrity of the intestinal barrier. It was inferred from the accumulated experimental data that TSPJ helped to combat obesity in aging mice by regulating the intestinal microbiota and promoting microbial metabolism.

The antiangiogenic effect of total saponins of Panax japonicus C.A. Meyer in rheumatoid arthritis is mediated by targeting the HIF-1α/VEGF/ANG-1 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal medicine Panax japonicus C.A. Meyer has a long history in clinical treatment of rheumatoid arthritis (RA). Total saponins of Panax japonicus C.A. Meyer (TSPJs) were extracted from the root of Panax japonicus C.A. Meyer, and its anti-rheumatism mechanism is still unclear. AIM OF THE STUDY: To investigate whether TSPJs attenuated synovial angiogenesis in RA and explore the potential mechanisms. MATERIALS AND METHODS: Potential TSPJs targets involving gene function were predicted by network pharmacology related databases. Bioinformatics analysis and molecular docking technology were used to predict the mechanism of TSPJs in the treatment of RA. The predicted results were validated by cell experiments and a collagen-induced arthritis (CIA) mouse model. RESULTS: Bioinformatics analysis results showed that TSPJs may inhibit RA-related angiogenesis through the hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) pathways. In vitro, different doses of TSPJs showed a good inhibitory effect on the tube formation of EA.hy926 cells. The results of the cellular thermal shift assay indicated that TSPJs can bind to the HIF-1α, VEGFA, and angiopoietin-1 (ANG-1) proteins. In vivo, the administration of TSPJs alleviated the symptoms of CIA mice, including the arthritis index, hind paw thickness, and swollen joint count. The histological results demonstrated that TSPJs inhibited inflammation, angiogenesis, bone damage, and cartilage destruction. Furthermore, TSPJs decreased the number of vessels and the expression level of CD31. The mechanistic results revealed that TSPJs decreased the expression of HIF-1α, VEGFA, and ANG-1 in the serum or synovial tissues of CIA mice. CONCLUSION: These results suggest that TSPJs effectively inhibit angiogenesis in RA, and the mechanism may be related to inhibiting the HIF-1α/VEGF/ANG-1 axis.

[Mechanism of total saponins of Panax japonicus against liver injury induced by acetaminophen in mice based on ERK/NF-κB/COX-2 signaling pathway].

This study investigated the effects and mechanisms of total saponins of Panax japonicus(TSPJ) against liver injury induced by acetaminophen(APAP). Male Kunming mice were randomly divided into a blank control group, TSPJ group(200 mg·kg~(-1), ig), model group, APAP+ TSPJ low-dose group(50 mg·kg~(-1), ig), APAP+ TSPJ medium-dose group(100 mg·kg~(-1), ig), APAP+ TSPJ high-dose group(200 mg·kg~(-1), ig), and APAP+ N-acetyl-L-cysteine group(200 mg·kg~(-1), ip). The administration group received the corresponding medications via ig or ip once a day for 14 consecutive days. After the last administration for one hour, except for the blank control group and TSPJ group, all groups of mice were given 500 mg·kg~(-1) APAP by gavage. After 24 hours, mouse serum and liver tissue were collected for serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), reactive oxygen species(ROS), tumor necrosis factor alpha(TNF-α), interleukin-1 beta(IL-1ß), cyclooxygenase-2(COX-2), IL-6, IL-4, IL-10, as well as lactate dehydrogenase(LDH), glutathione(GSH), superoxide dismutase(SOD), catalase(CAT), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and myeloperoxidase(MPO) liver tissue. Hematoxylin-eosin staining was used to observe the morphological changes of liver tissue. The mRNA expression levels of lymphocyte antigen 6G(Ly6G), galectin 3(Mac-2), TNF-α, IL-1ß, COX-2, IL-6, IL-4, and IL-10 in liver tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expression levels of Ly6G, Mac-2, extracellular regulated protein kinases(ERK), phosphorylated extracellular regulated protein kinases(p-ERK), COX-2, inhibitor of nuclear factor κB protein α(IκBα), phosphorylated inhibitor of nuclear factor κB protein α(p-IκBα), and nuclear factor-κB subunit p65(NF-κB p65) in cytosol and nucleus in liver tissue. The results manifested that TSPJ dramatically reduced liver coefficient, serum ALT, AST, ROS, TNF-α, IL-1ß, IL-6, and COX-2 levels, LDH, MPO, and MDA contents in liver tissue, and mRNA expressions of TNF-α, IL-1ß, and IL-6 in APAP-induced liver injury mice. It prominently elevated serum IL-4 and IL-10 levels, GSH, CAT, SOD, and T-AOC contents, and mRNA expressions of IL-4 and IL-10 in liver tissue, improved the degree of liver pathological damage, and suppressed neutrophil infiltration and macrophage recruitment in liver tissue. In addition, TSPJ lessened the mRNA and protein expressions of neutrophil marker Ly6G, macrophage marker Mac-2, and COX-2 in liver tissue, protein expressions of p-ERK, p-IκBα, and NF-κB p65 in nuclear, and p-ERK/ERK and p-IκBα/p-IκBα ratios and hoisted protein expression of NF-κB p65 in cytosol. These results suggest that TSPJ has a significant protective effect on APAP-induced liver injury in mice, and it can alleviate APAP-induced oxidative damage and inflammatory response. Its mechanism may be related to suppressing ERK/NF-κB/COX-2 signaling pathway activation, thus inhibiting inflammatory cell infiltration, cytokine production, and liver cell damage.

Comprehensive Characterization of Triterpene Saponins in Rhizoma Panacis Japonici by Offline Two-Dimensional Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry.

Rhizoma Panacis Japonici (RPJ) is an ancient herbal medicine from China that has long been employed for its medicinal benefits in relieving arthritis physical debility and diverse afflictions. The primary bioactive constituents found in RPJ are triterpene saponins, which exhibit numerous pharmacological actions, including anti-inflammatory, antioxidant, and immunomodulating effects. The present study established a straightforward and effective approach for characterizing triterpene saponins in RPJ. An offline HILIC × RP LC/QTOF-MS method was developed, along with a self-constructed in-house database containing 612 saponins reported in the Panax genus and 228 predicted metabolites. The approach achieved good chromatographic performance in isolating triterpene saponins of RPJ, with the HILIC column as the first dimension (1D) and the BEH C18 column as the second dimension (2D). The developed two-dimensional liquid chromatography system exhibited an orthogonality of 0.61 and a peak capacity of 1249. Detection was performed using a QTOF mass spectrometer in a data-independent manner (MSE) in a negative ion mode. Using the in-house database, the collected MS data were processed by an automatic workflow on UNIFI 1.8.2 software, which included data correction, matching of precursor and product ions, and peak annotation. In this study, 307 saponins were characterized from RPJ and 76 saponins were identified for the first time in Panax japonicus. This research not only enhances our understanding of the chemical characteristics of RPJ but also offers a simple and efficient method for analyzing the complex composition of herbal medicine.

[Total saponins of Panax japonicus alleviates CCl4-induced acute liver injury in rats by regulating the PI3K/AktNF-κB signaling pathway].

OBJECTIVE: To investigate the protective effect of total saponins of Panax japonicus (TSPJ) against CCl4-induced acute liver injury (ALI) in rats and explore the underlying pharmacological mechanisms. METHODS: Male SD rat models of CCl4-induced ALI were given intraperitoneal injections of distilled water, 100 mg/kg biphenyl bisabololol, or 50, 100, and 200 mg/kg TSPJ during modeling (n=8). Liver functions (AST, ALT, TBil and ALP) of the rats were assessed and liver pathologies were observed with HE staining. Immunohistochemistry was used to detect the expressions of PI3K/Akt/NF-κB signaling pathway molecules in liver tissue; ELISA was used to determine the levels of T-SOD, GSH-Px, and MDA. Western blotting was performed to detect the expression levels of PI3K-Akt and SIRT6-NF-κB pathways in the liver tissue. RESULTS: Network pharmacological analysis indicated that the key pathways including PI3K/Akt mediated the therapeutic effect of TSPJ on ALI. In the rat models of ALI, treatments with biphenyl bisabololol and TSPJ significantly ameliorated CCl4-induced increase of serum levels AST, ALT, ALP, TBil and MDA and decrease of T-SOD and GSH-Px levels (all P < 0.01). The rat models of ALI showed significantly increased expression of p-NF-κB (P < 0.01), decreased expressions of PI3K, p-Akt and SIRT6 proteins, and elevated expression levels of p-NF-κB, TNF-α and IL-6 proteins in the liver, which were all significantly improved in the treatment groups (P < 0.05 or 0.01). CONCLUSION: TSPJ can effectively alleviate CCl4-induced ALI in rats by suppressing inflammatory responses and oxidative stress in the liver via regulating the PI3K/Akt and SIRT6/NF-κB pathways.

Polysaccharide from Panax japonicus C.A. Mey prevents non-alcoholic fatty liver disease development based on regulating liver metabolism and gut microbiota in mice.

In this study, a new polysaccharide (PSPJ) with specific molecular weight and monosaccharide compositions was isolated and purified from the water extract of Panacis Japonici Rhizoma (PJR). 16S rRNA analysis and untargeted metabolomic analysis were used to assess PSPJ's efficacy in averting non-alcoholic fatty liver disease (NAFLD). This study indicated that PSPJ significantly reduced liver fat accumulation, the increase in blood lipids and ALT caused by HFD, indicating that PSPJ can prevent NAFLD. We demonstrated through cell experiments that PSPJ does not directly affect liver cells. The gut microbiota disorder and alterations in short-chain fatty acids (SCFAs) induced by the high-fat diet (HFD) were ameliorated by PSPJ, as evidenced by the analysis of 16S rRNA. In particular, supplementing PSPJ reduced the abundance of Turicibacter, Dubosiella, and Staphylococcus, and increased the abundance of Bacteroides, Blautia, and Lactobacillus. Untargeted metabolomic analysis shows that PSPJ improves liver metabolic disorders by regulating arachidonic acid metabolism, carbohydrate digestion and absorption, fatty acid biosynthesis, fatty acid metabolism and retinol metabolism. The findings of our investigation indicate that PSPJ has the potential to modulate liver metabolism through alterations in the composition of intestinal bacteria, hence preventing NAFLD.

Panax japonicus C.A. Meyer: a comprehensive review on botany, phytochemistry, pharmacology, pharmacokinetics and authentication.

BACKGROUND: Panax japonicus C.A. Meyer (Zhujieshen) is widely used in traditional medicine as a tonic hemostatic and anti-inflammatory agent in China, Japan, and Korea. Furthermore, it is used as an important substitute for ginseng roots by minority ethnic groups in China. The purpose of this review is to summarize the latest research on Zhujieshen in recent years, aiming at providing a systematic overview of the current knowledge, and perspectives for future research and exploitation. MAIN BODY: This review examines the research advances in botanical profile, phytochemicals, pharmacology, pharmacokinetics, and authentication of Zhujieshen. Various compounds have been reported as active components, mainly including saponins, volatile oils, and polysaccharides. Pharmacological investigations have demonstrated that Zhujieshen is an important herb with significant bioactivities, such as anti-inflammatory, hepato-protective, cardio-protective, neuro-protective, anti-tumor, anti-oxidant, anti-thrombotic and immunomodulatory activities. CONCLUSION: Currently, research on Zhujieshen is in the preliminary stages, and further research is required to understand the active compounds present and mechanisms of action. We hope that this comprehensive review of Zhujieshen will serve as a background for future research and exploitation.

Saponins of Panax japonicus ameliorates cardiac aging phenotype in aging rats by enhancing basal autophagy through AMPK/mTOR/ULK1 pathway.

Heart disease is a significant health concern for elderly individuals, with heart aging being the primary cause. Recent studies have shown that autophagy can play a protective role in preventing cardiac aging. Our previous research confirmed that Chikusetsu saponin IVa, a fundamental component of Saponins of Panax japonics (SPJ), can enhance basic autophagy levels in cardiomyocyte of isoproterenol induced cardiac fibrosis mice. However, it remains unclear whether SPJ possesses a protective effect on cardiac dysfunction during the natural aging process. Rats were randomly divided into four groups: adult control group (6 months old), aging group (24 months old), aging group treated with 10 mg/kg SPJ, and aging group treated with 30 mg/kg SPJ. The heart function, blood pressure, and heart mass index (HMI) were measured. Hematoxylin and eosin staining (H&E) and Wheat Germ Agglutinin (WGA) staining were used to observe the changes in morphology, while Masson staining was used to examine collagen deposition in the rat hearts and CD45 immunohistochemistry was conducted to examine the macrophage infiltration in heart tissues. TUNEL kit was used to detect apoptosis level of cardiomyocyte, and western blot was used to evaluate autophagy-related proteins as well as AMPK/mTOR/ULK1 pathway-related markers. SPJ treatment improved the cardiac function, reduced HMI, attenuated myocardial fiber disorder, inhibited inflammatory cell infiltration, and decreased collagen deposition and cardiomyocyte apoptosis in aging rats. Additionally, SPJ treatment decreased the expression of aging-related proteins and restored the expression of autophagy-related markers. SPJ activated autophagy through the activation of AMPK, which in turn increased the phosphorylation of ULK1(Ser555), while inhibited the phosphorylation of mTOR and ULK1(Ser757). Our study demonstrates that SPJ improves the cardiac function of aging rats by enhancing basal autophagy through the AMPK/mTOR/ULK1 pathway. These results offer a theoretical foundation and empirical evidence to support the clinical advancement of SPJ in enhancing age-related cardiac dysfunction.

Characterization of ginsenosides from Panax japonicus var. major (Zhu-Zi-Shen) based on ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry and desorption electrospray ionization-mass spectrometry imaging.

BACKGROUND: Panax japonicus var. major (PJM) belongs to the well-known ginseng species used in west China for hundreds of years, which has the effects of lung tonifying and yin nourishing, and exerts the analgesic, antitussive, and hemostatic activities. Compared with the other Panax species, the chemical composition and the spatial tissue distribution of the bioactive ginsenosides in PJM have seldom been investigated. METHODS: Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) and desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) were integrated for the systematic characterization and spatial tissue distribution studies of ginsenosides in the rhizome of PJM. Considering the great difficulty in exposing the minor saponins, apart from the conventional Auto MS/MS (M1), two different precursor ions list-including data-dependent acquisition (PIL-DDA) approaches, involving the direct input of an in-house library containing 579 known ginsenosides (M2) and the inclusion of the target precursors screened from the MS1 data by mass defect filtering (M3), were developed. The in situ spatial distribution of various ginsenosides in PJM was profiled based on DESI-MSI with a mass range of m/z 100-1500 in the negative ion mode, with the imaging data processed by the High Definition Imaging (HDI) software. RESULTS: Under the optimized condition, 272 ginsenosides were identified or tentatively characterized, and 138 thereof were possibly not ever reported from the Panax genus. They were composed by 75 oleanolic acid type, 22 protopanaxadiol type, 52 protopanaxatriol type, 16 octillol type, 19 malonylated, 35 C-17 side-chain varied, and 53 others. In addition, the DESI-MSI experiment unveiled the differentiated distribution of saponins, but the main location in the cork layer and phloem of the rhizome. The abundance of the oleanolic acid ginsenosides was high in the rhizome slice of PJM, which was consistent with the results obtained by UHPLC/QTOF-MS. CONCLUSION: Comprehensive characterization of the ginsenosides in the rhizome of PJM was achieved, with a large amount of unknown structures unveiled primarily. We, for the first time, reported the spatial tissue distribution of different subtypes of ginsenosides in the rhizome slice of PJM. These results can benefit the quality control and further development of PJM and the other ginseng species.

[Protective effects of total saponins from Panax japonicus against high-fat diet-induced testicular Sertoli cell junction damage in mice].

OBJECTIVE: To investigate the protective effects of total saponins from Panax japonicus (TSPJ) against high-fat dietinduced testicular Sertoli cell junction damage in mice. METHODS: Forty male C57BL/6J mice were randomized into normal diet group, high-fat diet group, and low-dose (25 mg/kg) and high-dose (75 mg/kg) TSPJ treatment groups (n=10). The mice in the normal diet group were fed a normal diet, while the mice in the other groups were fed a high-fat diet. After TSPJ treatment via intragastric administration for 5 months, the testes and epididymis of the mice were collected for measurement of weight, testicular and epididymal indices and sperm parameters. HE staining was used for histological evaluation of the testicular tissues and measurement of seminiferous tubule diameter and seminiferous epithelium height. The expression levels of ZO-1, occludin, claudin11, N-cadherin, E-cadherin and ß-catenin in Sertoli cells were detected with Western blot, and the localization and expression levels of ZO-1 and ß-catenin in the testicular tissues were detected with immunofluorescence assay. The protein expressions of LC3B, p-AKT and p-mTOR in testicular Sertoli cells were detected using double immunofluorescence assay. RESULTS: Treatment with TSPJ significantly improved high-fat diet-induced testicular dysfunction by reducing body weight (P < 0.001), increasing testicular and epididymal indices (P < 0.05), and improving sperm concentration and sperm viability (P < 0.05). TSPJ ameliorated testicular pathologies and increased seminiferous epithelium height of the mice with high-fat diet feeding (P < 0.05) without affecting the seminiferous tubule diameter. TSPJ significantly increased the expression levels of ZO-1, occludin, N-cadherin, E-cadherin and ß-catenin (P < 0.05) but did not affect claudin11 expression in the testicular tissues. Immunofluorescence assay showed that TSPJ significantly increased ZO-1 and ß-catenin expression in the testicular tissues (P < 0.001), downregulated LC3B expression and upregulated p-AKT and p-mTOR expressions in testicular Sertoli cells. CONCLUSION: TSPJ alleviates high-fat diet-induced damages of testicular Sertoli cell junctions and spermatogenesis possibly by activating the AKT/mTOR signaling pathway and inhibiting autophagy of testicular Sertoli cells.

Total saponins from Panax japonicus attenuate acute alcoholic liver oxidative stress and hepatosteatosis by p62-related Nrf2 pathway and AMPK-ACC/PPARα axis in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax japonicus (T. Nees) C.A. Mey. (PJ) has been used as a tonic traditional Chinese medicine (TCM) for years. Based on its meridian tropism in liver, spleen, and lung, PJ was popularly used to enhance the function of these organs. It is originally recorded with detoxicant effect on binge drink in Ben Cao Gang Mu Shi Yi, a persuasive Chinese materia medica. And binge dink has a close relationship with alcoholic liver disease (ALD). Hence, it's meaningful to investigate whether PJ exerts liver protection against binge drink toxicity. AIM OF THE STUDY: This investigation was carried out not only to emphasize the right recognition of total saponins from PJ (SPJ), but also to study on its sober-up effectiveness and defensive mechanism against acute alcoholic liver injury in vivo and in vitro. MATERIALS AND METHODS: SPJ constituents were verified by HPLC-UV analysis. In vivo, acute alcoholic liver oxidative stress and hepatosteatosis were established by continuous ethanol gavage to C57BL/6 mice for 3 days. SPJ was pre-administered for 7 days to investigate its protective efficacy. Loss of righting reflex (LORR) assay was employed to assess anti-inebriation effect of SPJ. Transaminases levels and hematoxylin and eosin (H&E) staining were measured to indicate the alcoholic liver injury. Antioxidant enzymes were measured to evaluate the oxidative stress degree in liver. Measurement of hepatic lipid accumulation was based on Oil Red O staining. Levels of inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA). In vitro, HepG2 cells were treated with ethanol for 24 h, and SPJ was pre-administered for 2 h. 2,7-dichlorofluorescein diacetate (DCFH-DA) was used as a probe to indicate reactive oxygen species (ROS) generation. Nrf2 activation was verified by the favor of specific inhibitor, ML385. The nuclear translocation of Nrf2 was indicated with immunofluorescence analysis. Proteins expressions of related pathways were determined by Western blotting. RESULTS: Oleanane-type saponins are the most abundant constituents of SPJ. In this acute model, SPJ released inebriation of mice in a dose dependent manner. It decreased levels of serum ALT and AST, and hepatic TG. Besides, SPJ inhibited CYP2E1 expression and reduced MDA level in liver, with upregulations of antioxidant enzymes GSH, SOD and CAT. p62-related Nrf2 pathway was activated by SPJ with downstream upregulations of GCLC and NQO1 in liver. AMPK-ACC/PPARα axis was upregulated by SPJ to alleviate hepatic lipidosis. Hepatic IL-6 and TNF-α levels were downregulated by SPJ, which indicated a regressive lipid peroxidation in liver. In HepG2 cells, SPJ reduced ethanol-exposed ROS generation. Activated p62-related Nrf2 pathway was verified to contribute to the alleviation of alcohol-induced oxidative stress in hepatic cells. CONCLUSION: This attenuation of hepatic oxidative stress and steatosis suggested the therapeutic value of SPJ for ALD.

Therapeutic effect of the total saponin from Panax Japonicus on experimental autoimmune encephalomyelitis by attenuating inflammation and regulating gut microbiota in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizomes of Panax japonicus (RPJ), a traditional herbal medicine, was used for treating arthritis and physical weakness in China from the Ming dynasty. Triterpene saponins are the main bioactive components of RPJ. In this work, for the first time, we evaluate the therapeutic effect of the total saponin from RPJ (TSPJ) on experimental autoimmune encephalomyelitis (EAE) mice induced by myelin oligodendrocyte glycoprotein (MOG) 35-55, a commonly used animal model of Multiple sclerosis (MS). AIM OF THE STUDY: To evaluate the therapeutic effect of TSPJ on EAE and explored its possible underlying mechanisms. MATERIALS AND METHODS: EAE was induced by MOG 35-55. Mice were administrated with TSPJ (36.5 mg/kg, 73 mg/kg) and prednisone acetate (positive control) orally once daily up to 28 days postimmunization, and their neurological deficit was scored. Hematoxylin and Eosin (HE), Luxol Fast Blue (LFB), and transmission electron microscopy (TEM) were carried out to evaluate the EAE-induced pathological changes in the brain and spinal cord. IL-17a and Foxp3 levels in central nervous system (CNS)were evaluated by immunohistochemical staining. The changes in IL-1ß, IL-6, and TNF-α levels in serum and CNS were measured with ELISA. Quantitative reverse transcription PCR (qRT-PCR) was used to access mRNA expression in CNS of the above indices. The percentages of Th1, Th2, Th17and Treg cells in spleen were determined by Flow Cytometry (FCM). Furthermore, 16S rDNA sequencing was used to detect the intestinal flora of mice in each group. In vitro studies, lipopolysaccharides (LPS)-induced BV2 microglia cells were used and the expression of TLR4, MyD88, p65, and p-p65 in cells was detected by Western blot. RESULTS: TSPJ treatment significantly alleviated neurological impairment caused by EAE. Histological examination confirmed the protective effects of TSPJ on myelin sheath and the reduction of inflammatory cell infiltration in the brain and spinal cord of EAE mice. TSPJ notably downregulated the ratio of IL-17a/Foxp3 at protein and mRNA levels in CNS, as well as Th17/Treg and Th1/Th2 cell ratios in the spleen of EAE mice. The levels of TNF-α, IL-6, and IL-1ß in CNS and peripheral serum also decreased post-TSPJ treatment. In vitro, TSPJ suppressed LPS-induced production of inflammatory factors in BV2 cells via TLR4-MyD88-NF-κB signaling pathway. More importantly, TSPJ interventions altered the composition of gut microbiota and restored the ratio of Firmicutes to Bacteroidetes in EAE mice. Furthermore, Spearman's correlation analysis revealed that a relationship existed between statistically significantly altered genera and CNS inflammatory indices. CONCLUSION: Our results demonstrated TSPJ had therapeutic effects on EAE. Its anti-neuroinflammation property in EAE was related to modulating gut microbiota and inhibiting TLR4-MyD88-NF-κB signaling pathway. Our study indicated that TSPJ may be a potential candidate for the treatment of MS.

Total saponins from Panax japonicus reduce inflammation in adipocytes through the miR155/SOCS1/NFκB signaling pathway.

BACKGROUND: The rising incidence of metabolic diseases due to chronic inflammation in the adipose tissue has been attributed to factors such as high fat diet (HFD). Previous studies have demonstrated that the total saponins from Panax japonicus (TSPJ) can reduce HFD-induced adipocyte inflammation, but the underlying mechanism remains unclear. In this work, we explored the molecular mechanism by which TSPJ reduces inflammation response in adipocytes. METHODS: We first established C57BL/6 mouse and 3T3-L1 adipocyte models. Lentiviruses packaged with the plasmids were injected into mice through the tail vein or into adipocytes to generate the in vivo and in vitro models with miR155 knockdown and overexpression. The mice were fed with HFD to trigger inflammation and administered TSPJ (25 mg/kg∙d and 75 mg/kg∙d) by gavage. The adipocytes were treated with palmitic acid (PA) to trigger inflammation response, then treated with TSPJ (25 µg/ml and 50 µg/ml). Finally, the expression of miR155, inflammatory factors, SOCS1, and NFκB pathway-related proteins was explored. RESULTS: TSPJ significantly inhibited the expression of inflammation-related genes and the miR155 expression in adipocytes both in vitro and in vivo. The dual luciferase reporter gene assay revealed that miR155 mediated the downregulation of SOCS1. TSPJ significantly inhibited and upregulated the phosphorylation of the NFκB protein and the SOCS1 proteins, respectively. CONCLUSION: TSPJ inhibits miR155 to upregulate the SOCS1 expression, which subsequently inhibits the NFκB signaling pathway, thereby mitigating the inflammatory response in the adipocytes of HFD mice.

The Hypoglycemic and Hypocholesterolemic Activity of Dioscorea deltoidea, Tribulus terrestris and Panax japonicus Cell Culture Biomass in Rats with High-Fat Diet-Induced Obesity.

Obesity, and its consequences for human health, is a huge and complicated problem that has no simple solution. The constant search for natural and safe compounds with systemic action that can be used for obesity prophylactics and treatment is hampered by the limited availability and variable quality of biomass of wild medicinal plants. Plant cell biotechnology is an alternative approach for the sustainable production of vegetative biomass or individual phytochemicals with high therapeutic potential. In this study, the suspension cell biomass of the medicinal plants, Dioscorea deltoidea Wall., Tribulus terrestris L., and Panax japonicus (T. Nees) C.A. Mey, produced in 20 L and 630 L bioreactors, were tested for therapeutic effects in rat models with alimentary-induced obesity. Three-month intake of water infusions of dry cell biomass (100 mg/g body weight) against the background of a hypercaloric diet reduced weight gain and the proportion of fat mass in the obese animals. In addition, cell biomass preparation reduced the intracellular dehydration and balanced the amounts of intra- and extracellular fluids in the body as determined by bioimpedance spectroscopy. A significant decrease in the glucose and cholesterol levels in the blood was also observed as a result of cell biomass administration for all species. Hypocholesterolemic activity reduced in the line P. japonicus > D. deltoidea > T. terrestris/liraglutide > intact group > control group. By the sum of parameters tested, the cell culture of D. deltoidea was considered the most effective in mitigating diet-induced obesity, with positive effects sometimes exceeding those of the reference drug liraglutide. A safety assessment of D. deltoidea cell phytopreparation showed no toxic effect on the reproductive function of the animals and their offspring. These results support the potential application of the biotechnologically produced cell biomass of medicinal plant species as safe and effective natural remedies for the treatment of obesity and related complications, particularly for the long-term treatment and during pregnancy and lactation periods when conventional treatment is often contraindicated.

Integrating metabolomics and network pharmacology to investigate Panax japonicus prevents kidney injury in HFD/STZ-induced diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax japonicus C. A. Meye (PJ) has unique effects on diseases by "qi" stagnation and blood stasis in ancient. Modern studies have shown that PJ can treat diabetic kidney disease (DKD) caused by deficiency and blood stasis. AIM OF THE STUDY: This study evaluated the potential effects of PJ on DKD, a microvascular complication, and investigated its possible mechanisms. MATERIALS AND METHODS: In this study, the chemical constituents of PJ were analyzed by HPLC. In vivo studies, we constructed a diabetic mice model by HDF combined with STZ, then administered PJ to diabetic mice for 6 weeks. Blood lipid, BUN, 24h urine protein, and renal tissue HE staining were detected to comprehensively evaluate the protective effect of PJ on DKD. Metabolomics investigated the metabolic pathways influenced by PJ in the treatment of DKD. Moreover, the potential targets and signal pathways were investigated using network pharmacology. Finally, molecular docking predicts affinity of active compounds and core targets, and western blotting was used to detect core target expression levels. RESULTS: In vivo study, PJ can reduce hyperlipidemia, serum BUN, and 24-h urinary protein in diabetic mice, and protect the pathological changes in renal tissue. Metabolomics results showed that PJ had significant regulatory effect on unsaturated fatty acids, glycerophospholipid metabolism, and purine metabolism. Network pharmacology showed that MAPK1, MAPK8, Bcl-2, and Caspase 3 were the core targets in PJ against DKD. Molecular docking revealed that Bcl-2 and Caspase 3 have a strong affinity for Chikusetsusaponin Iva, Ginsenoside Rb1, and Ginsenoside Rg1. Moreover, when compared to the model group, the PJ group had higher levels of anti-apoptosis protein Bcl-2 and lower levels of pro-apoptosis protein Caspase 3. CONCLUSION: PJ can reduce blood lipids, regulate the biosynthesis of unsaturated fatty acids and purine metabolism, thereby alleviating the renal injury of diabetic mice. Moreover, it can regulate the Bcl-2/caspase 3 apoptosis signaling pathway to prevent the apoptosis of renal cells and protect the renal function of diabetic mice.

Identification of the Origin, Authenticity and Quality of Panax Japonicus Based on a Multistrategy Platform.

BACKGROUND: Panax Japonicus (PJ) is a widely used Chinese herbal medicine, functional food and tonic. However, its origin has a great influence on the quality of PJ, and with the increasing demand for PJ, fake and inferior products, such as Panax Stipuleanatus (PS), often appear. The identification of the origin and authenticity of PJ is critical for ensuring the quality, safety and effectiveness of drugs. OBJECTIVE: Proposing a strategy to identify the origin, authenticity, and quality of PJ using HPLC fingerprints, chemometrics, and network pharmacology. METHODS: The chromatographic fingerprint method was established to analyze the origin and authenticity of PJ. Multiple chemometric methods were performed to analyze the fingerprints, including a Hierarchical Cluster Analysis (HCA), Principal Component Analysis (PCA), and Counter Propagation Artificial Neural Network (CP-ANN). Finally, the network pharmacology method was used to construct the "active ingredient-target" network, predict and assist in analyzing potential Qmarkers in PJ. RESULTS: Ward's method was used for the HCA. The results showed that PJ samples from different origins had significant regional differences and could be accurately distinguished from PS. The PCA classification results are consistent with the HCA classification results, further illustrating the model's accuracy. The CP-ANN model can analyze and predict PJ and PS and accurately obtain PJ and PS chemical markers to identify PJ and PS correctly. The network pharmacology of PJ was constructed, and three PJ Q-markers, namely, ginsenoside Ro, ginsenoside Rb1, and chikusetsu saponin Ⅳa, were identified, which lays a foundation for the establishment of PJ quality standards. CONCLUSION: This research provides a feasible platform for the quality evaluation of PJ in the future.

Saponins from Panax japonicus alleviate adipose tissue fibrosis and metabolic dysfunction in high-fat-diet-induced obese mice.

INTRODUCTION: Adipose tissue fibrosis is a typical feature of adipose tissue dysfunction in obese individuals, which is closely related to metabolic diseases. OBJECTIVE: To explore the effect and mechanism of Saponins from Panax japonicus (SPJ) on adipose tissue fibrosis in obese mice induced by high fat diet (HFD). MATERIALS AND METHODS: We established a HFD induced obese mice model. Then the obese mice were treated with 90 mg/kg SPJ by oral gavage for 10 weeks. The levels of adipose tissue fibrosis and molecules related to signalling pathways were measured. Then the effects of SPJ on TGFß1-induced fibrosis in 3T3-L1 differentiated adipocytes were evaluated. RESULTS: SPJ reduced body weight, fat accumulation, and improved glucose and lipid metabolism in obese mice. SPJ decreased collagen deposition and expressions of fibrotic genes in epididymal white adipose tissue (eWAT) of obese mice. SPJ decreased the levels of TGFß1 protein and pSmad2, and increased the expression of PPARγ and PGC1α, thus alleviating oxidative stress in eWAT. Consistently, SPJ inhibited TGFß1-induced fibrosis in 3T3-L1 differentiated adipocytes. CONCLUSIONS: SPJ may alleviate adipose tissue fibrosis and improve obesity by inhibiting TGFß1/Smad2 and activating PPARγ/PGC1α pathway. SPJ is expected to become an efficient medicine for treatment of obesity.