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Objective: The isotopic ratios of light elements (C/N/H/O) in Panax japonicus from six producing areas were determined with isotope ratio mass spectrometry.Methods: Three methods, including linear discrimination (LD), gaussian kernel support vector machine (SVM), and the back-propagation learning algorithm of pattern recognition based on neural network toolbox (BPN) were employed to establish a model for P. japonicus geographical origin discrimination. Results: The results showed that stable isotope carbon (δ 13C) had obvious regional characteristics, which will be used to effectively distinguish the origin of P. japonicus. The methods of LD and BPN could classify the geographical origin of P. japonicus from six producing areas, both of which showed that the accuracy rates were 100% using training dataset. Conclusion: Therefore, the stable isotope technique combined with LD and BPN method can effectively trace the origin of P. japonicus.
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Objective: To provide a scientific basis for the modern identification of Panax japonicus and ensure the clinical efficacy and medication accuracy, the molecular identification of P. japonicus and its related species or adulterants was carried out. Methods: ITS2 sequences of P. japonicus were amplified and sequenced directionally. ITS2 sequences of related species and adulterants were downloaded from GenBank. Cutting with ITS2 database, the final dataset consisted of 102 sequences from 24 species. DAMBE program was also implemented to detect substitution saturation of ITS2 sequences. MEGA 6.06 software was utilized for sequence alignment, calculating GC content, analyzing variation sites, estimating intra-specific and inter-specific genetic distances, and finally building NJ tree. Moreover, the secondary structure of ITS2 was predicted by using the ITS2 database. Results: The length of ITS2 sequences from P. japonicas was 230 bp and GC content was 63.7%. The average genetic distance analysis, NJ tree, and secondary structure characteristics of the ITS2 sequences showed that there were great differences between P japonicus and its non-identical adulterants or partial related species (P. stipuleanatus, P. pseudoginseng, P. trifolius, P. vietnamensis var. fuscidiscus, and P. vietnamensis). Howerer, the application of such method for the identification of P. japonicus and partial closely related species (P. quinquefolius, P. ginseng, P. notoginseng, P. japonicus var. major, P. japonicus var. bipinnatifidus, P. assamicus, P. variabilis, and P. japonicus var. angustifolius) had certain limitation. Conclusion: The ITS2 sequence can be used as one of the DNA barcodings for the identification of P. japonicus and its non-identical adulterants at high identification rate, however, its versatility of identification between P japonicus and related species needs to be further verified. Our study provides the basis for the identification of inter-specific genetic and affinity relationship of P. japonicas and its related species, the distinguishment between P. japonicas and non-identical adulterants, and the clinical safety of P japonicas.
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Objective: To establish a simple and effective extraction method for the preparation of total saponins of Panax japonicas (TSPJ). Methods: Combination of macroporous adsorption and ion exchange resin chromatography was adopted in the present study. For quality evaluation, chikusetsusaponin IVa was used as reference, and vanillin-perchloric acid was applied as chromogenic reagent to determine total saponin content at 545 nm. Results: X-5 macrophous resin offered better adsorption and desorption capacities for TSPJ than other macrophous resins. The optimum purification process was confirmed as follows: The sample solution concentration was 0.2 mg/L; The sample volume was 10 g/g, and eluting with 5 mL of 70% aqueous ethanol solutions on 1 g wet macrophous resin column. Followed this step, decoloring of TSPJ was studied and the decoloring capacity of two different types of ion exchange resins was evaluated. The result showed that 732-type cation exchange resin was the better resin for decolorization of the TSPJ. The total saponin products with higher purity and quality were obtained, with the mass fraction more than 85.0%, and the transfer rate of TSPJ was more than 70.0%. Conclusion: The results show that the total saponins can be separated and purified effectively from P. japonicus. The preparation method is simple, effective, and efficient for large-scale preparation of TSPJ.
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Objective To observe the effects of saponins from Panax japonicus on neuronal apoptosis of natural aging rats and its mechanisms based on NLRP1 and NLRP3 inflammasome pathway. Methods Male SD rats in a SPF grade were randomly divided into five groups: control group (9-month-old rats), model group (24-month-old rats), and SPJ treatment group (10, 30, and 60 mg/kg). From the beginning of 18 months, animals were treated with SPJ (or normal saline) by ig until 24 months, and stopped 2 d each week for six months of continuous administration. The neural apoptosis situation of cortex and hippocampus in aging rats were observed by TUNEL method. The protein expression of IL-1β, ASC, NLRP3, NLRP1, Caspase-1, and IL-18 of the cerebral cortex and hippocampal were detected by Western blotting. Results TUNEL results showed that there were a very small number of apoptotic cells in the cortex and hippocampus in control group. Compared with control group, the model group significantly increased the number of apoptotic cells. Compared with model group, the number of apoptotic cells was significantly decreased in rat cortex and hippocampus (CA1, CA3, and DG) after treated with SPJ (10, 30, and 60 mg/kg). Western blotting results showed a significant age-related increase in the expression of IL-1β, ASC, NLRP3, NLRP1, Caspase-1, and IL-18, while SPJ concentration-dependently decreased the levels of IL-1β, ASC, NLRP3, NLRP1, Caspase-1, and IL-18 after six-month treatment. Conclusion In conclusion, saponins from P. japonicus has protective effects on the brain (cortex and hippocampus) of aging rats. The mechanism is likely to be that saponins from P. japonicus can reduce nerve inflammation by regulating NLRP1 and NLRP3 inflammasome pathway.
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Objective: To analyze the genetic diversity of Panax japonicus var. major by inter simple sequence repeat (ISSR) molecular makers. Methods: Genetic diversity of 19 samples from main production areas was investigated by ISSR and analyzed by principal coordinate analysis and UPGMA cluster analysis. Results: A total of 181 bands were generated by 13 ISSR primers, among which 166 bands (91.71%) were polymorphic bands (PPB). The coefficient of genetic similarity ranged from 0.60 to 0.83. The results of UPGMA cluster analysis and principal coordinate analysis showed that the genetic diversity of P. japonicus var. major from the same region presented a geographical distribution regularity. Conclusion: The results of ISSR analysis reveals that P. japonicus var. major has a high genetic diversity level and the genetic relationship closely contacts with the geographical location.