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.

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.

Study on purification technic of total saponins in Panacis Japonici Rhizoma with macroporous resin / 国际中医中药杂志

Objective:Select a suitable macroporous resin for the purification technic of total saponins from Panacis Japonici Rhizoma and determine the parameter of purification technic. Methods:Made the content of total saponins as the index, used static adsorption test and combined the adsorption kinetic parameters to select the type of macroporous resin. By using dynamic adsorption experiment to investigate the technical parameters of the purified macroporous resin extracted from Panacis Japonici Rhizoma. Then the preparation technic of the total saponins of Panacis Japonici Rhizoma was determined. Results:The D101 macroporous resin could absorpt and desorpt total saponins of Panacis Japonici Rhizoma effectively. The optimal purification parameters were as follow: the loading mass concentration was 0.1 g/ml (based on crude drug), and the loading volume was 100 ml (which means the loading volume of resin per ml was equivalent to 3.3 grams of crude drug). During the elution process, distilled water (3 BV) and 20% ethanol (3 BV) were used to remove impurity, and then 70% ethanol elution (6 BV) was used to enrich the total saponins. The flow rate of loading and elution was 0.5 ml/min. The transfer rate of total saponins could reache 85.6%. Conclusion:The D101 macroporous resin can effectively enrich and purify the total saponins of Panacis Japonici Rhizoma, which provides the scientific basis for the development and utilization of Panacis Japonici Rhizoma.