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Ten compounds were isolated and purified from ethanol extracts of dried roots bark of Polygala tenuifolia Willd. by various chromatography techniques such as silica gel and Sephadex LH-20. Their structures were identified by analysis of physicochemical properties and spectral data, and determined as β-sitosterol (1), tenuifolin (2), 6-methoxy coumarin (3), 7-phenyl-1-hydroxy-2,3,6-trimethoxyxanthone (4), 1,8-dihydroxy-3,4,7-trimethoxyanthone (5), mangiferin (6), quercetin-3-O-β-D-glucoside (7), rutin (8), syringaldehyde (9), salicylicacid (10). Among them, compounds 3, 4 and 5 were isolated from the genus of Ploygala for the first time and compound 4 was a new xanthone. The acetylcholinesterase inhibitory activities of compounds 3, 4 and 5 were evaluated by Ellman colorimetric method, compounds 3 and 5 exhibited moderate inhibitory activity, compound 4 exhibited weak inhibitory activity.
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Objective: The contents of five main components in Polygala tenuifolia from different habitats were determined, which provided certain data support and theoretical basis for the quality evaluation system of P. tenuifolia. Methods: The contents of polygalaxanthone III, 3,6’-disinapoyl sucrose, polygalacic acid, senegenin and tenuifolin of roots from different wild samples were determined by HPLC. Then SPSS 20.0 and SIMCA 11.5 were used for difference analysis, correlation analysis, hierarchical cluster analysis (HCA), and principal component analysis (PCA). Results: The content of the five main components in wild P. tenuifolia samples from different habitats was significantly different. The 20 samples were placed into two clusters (I, II) by HCA and PCA. Cluster I comprised three samples with higher content of 3,6’-disinapoyl sucrose, polygalaxanthone III, senegenin and tenuifolin from Weinan, Xianyang in Shannxi Province, and Xinjiang in Shanxi Province, whereas cluster II contained the other 17 samples. Conclusion: The results showed that the main components of P. enuifolia from Weinan, Xianyang in Shannxi Province and Xinjiang in Shanxi Province were significantly higher than other origins, and which provided a reference for the quality control, selection of excellent germplasm and cultivation bases of P. tenuifolia.
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Objective: To obtain the transcriptome sequence database induced by methyl jasmonate (MeJA) and identify the genes related to the biosynthesis of triterpenoid saponin in Polygala tenuifolia. Methods: The seedlings grown for 30 d were respectively treated with sterile water, 50 μmol/L MeJA and 100 μmol/L MeJA for 24 h. The transcriptome data of seedlings of P. tenuifolia were obtained by Illunima HiseqTM 2000 150PE sequencing and de novo splicing of Unigene was realized by Trinity software. The GO classification, KOG functional annotation, metabolism of KEGG metabolic pathway, protein function annotation analysis, differential gene analysis and screening were completed based on BLAST. Results: A total of 52.19 Gb clean data were obtained after the transcriptome of P. tenuifolia being assembled by Trinity software, and 54 426 Unigenes were assembled with an average length of 1 604 bp. All Unigenes were annotated in the public databases NR, NT, KEGG, Swissprot, GO, and Pfam. Through differential analysis of genes responding to MeJA, a total of 3 390 differentially expressed genes (DEGs) were found, of which 1 287 were up-regulated and 2 103 were down-regulated. The response of DEGs showed that the total number and up-regulated number of P. tenuifolia seedlings treated by 100 μmol/L MeJA was the highest. KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in metabolic pathways including phenylpropanoid biosynthesis, cysteine and methionine metabolism, starch and sucrose metabolism, carbon fixation in photosynthetic organisms and terpenoid backbone biosynthesis. Furthermore, a total of 59 Unigenes involved in anthraquinones biosynthesis were found according to the assignment of KEGG pathway. Expression analysis showed that AACT, HMGS, HMGR, MK, PMK, MPD, DXS, IDI, FPPS, SQS, SE and β-AS were up-regulated after being induced by MeJA. Conclusion: In this study, the transcriptome of P. tenuifolia seedlings treated with methyl jasmonate was analyzed, and candidate genes related to triterpenoid skeleton biosynthesis of P. tenuifolia were obtained. MeJA can induce the expression of genes related to triterpenoid skeleton synthesis, which provided a wealth of data resources for the molecular biology research and also laid the foundation for the analysis of the secondary metabolic pathways of triterpenoid saponins in P. tenuifolia.
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Objective: To study the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on growth, activity of related enzymes and chemical components in Polygala callus. Methods: The callus of Polygala was used as material. After 30 d of dark culture at different concentrations of SA (0, 4, 8, 12, 16, 20, 24, 28, 32 mg/L) and MeJA (0, 100, 200, 400, 600, 800, 1 000 μmol/L), the growth of callus, anti-oxidant enzyme activity, the content of MDA, total phenolic, total flavonoid, polygalaxanthone III, and 3,6’-disinapoyl sucrose were determined. Results: MeJA inhibited the growth of Polygala callus, and 12 mg/L SA promoted the growth of Polygala callus. SA and MeJA promoted the activity of SOD, CAT, POD, and MDA in the callus of Polygala. With the increase of SA and MeJA concentration, the activities of SOD, CAT and POD increased first and then decreased, and the content of MDA continued to rise. When the concentration of SA was 20 mg/L, the activities of CAT and SOD reached the maximum, which were 248.45 U/mg and 4451.06 U/mg, respectively. When the concentration of SA was 16 mg/L, the activity of POD reached the maximum, which was 7.22 U/mg. When the concentration of SA was 32 mg/L, the MDA content reached the maximum value of 25.09 nmol/mg. When the concentration of MeJA was 600 μmol/L, the activities of CAT, SOD, and POD reached the maximum, which were 273.30, 1451.06 and 15.27 U/mg, respectively. When the concentration of MeJA was 1000 μmol/L, the MDA content reached the maximum value of 27.10 nmol/mg. SA inhibited the accumulation of total flavonoids in Polygala callus, and had no significant effect on total phenols. MeJA promoted the accumulation of total phenols and total flavonoids in Polygala callus. When MeJA concentration was 600 μmol/L, the content of total flavonoids was the highest. When the concentration of MeJA was 400 μmol/L, the total phenolic content was the highest. Both SA and MeJA promoted the accumulation of polygalaxanthone III and 3,6’-disinapoyl sucrose in Polygala callus. When the concentration of SA was 32 mg/L, the concentration of polygalaxanthone III, 3,6’-disinapoyl sucrose was the highest. When the concentration of MeJA was 1 000 μmol/L, the content of polygalaxanthone III, 3,6’-disinapoyl sucrose was the highest. Conclusion: MeJA had an inhibitory effect on the growth of Polygala callus, and 12 mg/L SA can promote this effect. SA and MeJA promoted the activity of SOD, POD, CAT, the content of MDA and the accumulation of polygalaxanthone III and 3,6’-disinapoyl sucrose in Polygala callus. SA inhibited the accumulation of total flavonoids in Polygala callus, and had no significant effect on total phenolics. MeJA promoted the accumulation of total phenolics and total flavonoids in Polygala callus.
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Objective: To provide a suitable expression analysis of Polygala tenuifolia in different growth years and tissues, so as to select the stable internal control genes. The spatial and temporal expression of four cytochrome P450 monooxygenase genes in the above-mentioned samples was analyzed. Methods: The soluble curve and Ct value of eight candidate internal reference genes including Tubulin 1, Tubulin 2, Elongation 1, Elongation 2, Actin 1, Actin 2 and cdc-42 in different growth years (1-3 years old) and tissues (root,stem, leaf, and flower) of P. tenuifolia were obtained by real-time quantitative PCR (qRT-PCR). The expression stability of candidate reference genes was assessed by geNorm and NormFinder. Then, the qRT-PCR technique was also used to identify the temporal and spatial expression of four P450 genes including CYP709B2, CYP71AP39, CYP88A85 and CYP714E38 in P. tenuifolia. Results: The geNorm results showed that the stable internal reference genes expressed in P. tenuifolia were Tubulin 2 and Elongation 1. The NormFinder results showed that the most stable and suitable internal reference gene for expression analysis was Elongation 1. The mRNA expression levels of CYP709B2 and CYP71AP39 genes in stems and leaves (1-3 years old) were higher than that in roots and flowers. The CYP88A85 and CYP714E38 genes had got a higher mRNA expression level in roots (1-3 years old). Conclusion: Elongation 1 is suitable as an ideal internal control gene for qRT-PCR analysis of P. tenuifolia. The expression trend of P450s in roots, stems, leaves and flowers of cultivated P. tenuifolia is inconsistent.
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Objective: To establish a method of fingerprint analysis and determination of index compounds on commercial Polygala tenuifolia by ultra performance liquid chromatography, and to evaluate the quality of P. tenuifolia with different commodity specification grades. Methods: UPLC was used to establish different commodity specification grade of P. tenuifolia and determine the contents of ten indicative compounds including sibiricose A5, sibiricose A6, lancerin, sibiricoxanthone B, glomeratose A, 7-O-methyl mangiferin, polygalaxanthone III, 3,6′-disinapoyl sucrose, tenuifoliside A, and tenuifoliside C, and establish comprehensive quality evaluation function at the same time. Results: The methods for fingerprint analysis and compound determination were in agreement with methodological requirements. A total of 37 peaks were selected as the common peaks, of which 22 peaks were identified, and the similarities were between 0.927 and 0.971. There are differences at different degrees in the contents of ten indicative compounds. The comprehensive quality evaluation function showed that the overall quality of Datong was better than that of Zhongtong; The overall quality of wild P. tenuifolia was better than that of cultivated. Conclusion: The validated UPLC fingerprint analysis and compound determination methods were successfully used in the quality control of P. tenuifolia.
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AIM:To screen and verify the effective ingredient of traditional Chines medicine (TCM) Q0409 in improving learning and memory ability of mice.METHODS:The mouse learning and memory impairment model was induced by intraperitoneal injection of scopolamine.The mice in each group were given the corresponding drug by gavage at the same time for 14 d in succession.Morris water maze test was used to measure the learning and memory ability of the mice, and then the hippocampal tissue homogenate was taken to determine the activity of acetylcholinesterase (AChE).The animals were divided into 8 groups according to L8(27) orthogonal table.The variance analysis and factorial analysis were used to analyze the pharmacological effects of seven kinds of single herbal in TCM Q0409 and determine the screening ingredients.The animals were divided into 6 groups according to the results of the preliminary screening results, and further testing and validation of TCM Q0409 screening ingredients were performed to get the final simplified ingredients.RESULTS:Three medicinal herbs of Polygalae, Panax ginseng and Acori graminei rhizome were screened by the orthogonal results of Morris water maze test and the activity of AChE in mouse hippocampal tissues.The simplified ingredients of TCM Q0409 were obtained through the variance results of Morris water maze test and the activity of AChE in mouse hippocampal tissues.CONCLUSION:Polygala and ginseng were eventually determined as simplified ingredients of TCM Q0409 and it was verified that they improve the learning and memory ability of the mice with learning and memory impairment.
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Objective To establish the main component analysis method of commodity grade Polygala tenuifolia, and to explore the correlation between the grade character of P. tenuifolia and the main chemistry. Methods P. tenuifolia of four markets were determined by high performance liquid chromatography (HPLC) to analyze the different level of medicine and explore the correlation between commodity grade and composition for P. tenuifolia. Results The main components determination of P. tenuifolia in different market grades were not completely consistent with the grade of commodity. Conclusion There are certain limitations of the commodity grading standards for P. tenuifolia in the medicinal materials market. It is necessary to establish a new grade quality standard for accurately evaluating the quality of P. tenuifolia. This study also provides some reference for the comprehensive quantitative evaluation of P. tenuifolia.
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Objective: To study the molecular phylogeography of natural populations of Polygala tenuifolia (Polygalaceae) in China, reveal the forming reasons of geographic distribution pattern of extent populations, and further determine the potential glacial refuges and colonization/migration routines of P. tenuifolia during the postglacial or interglacial periods. Methods: We investigated the distribution patterns of genetic variation of 308 individuals from 39 natural populations of P. tenuifolia in China based on one chloroplast DNA fragment (cpDNA trnL intron). Results: A total of 26 polymorphic sites were identified and 12 cpDNA haplotypes were recovered. Phylogenetic analysis of cpDNA haplotypes suggested that the natural populations of P. tenuifolia could be divided into two different groups: northern part (including populations of northeast, center, and northwest of China) and southern part. There were no shared cpDNA haplotypes between the two groups. And the population genetic analysis suggested that the high level of genetic differentiation (Gst = 0.783, P < 0.001) between the two groups, the high level of the genetic diversity were found (Ht = 0.755) at the species level. No significant phylogeographic structure was detected in the North group. Conclusion: The present studies revealed that the multiple glacial refuges of P. tenuifolia are identified in the northern and southern parts of China during the Quaternary periods. And northern populations have occurred the expansion events during the post-glacial or interglacial periods. The genetic divergence between the north and south groups of natural populations of P. tenuifolia may be caused by the long term geographic isolation.
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Objective: To discuss the genetic diversity of Polygala tenuifolia in its main production area. Methods: The genetic diversity of ten populations of P. tenuifolia from its main production area Shaanxi, Shanxi, and Hebei Provinces, was analyzed using ISSR markers. Results: Twelve selected ISSR primers generated a total of 276 fragments, 271 of which were polymorphic fragments. The cluster analysis showed that wild and cultivated populations from the same geographical area were clustered first. Genetic variation of both wild and cultivated populations occurred mainly among populations, and the genetic diversity of cultivated populations was lower than wild ones, but both of them were not consistent with the isolation-by-distance model. Conclusion: The results of cluster analysis agree with the fact that cultivated populations are derived directly from wild ones nearby. It was its own reproductive biological features and the current extensive cultivation that resulted in the genetic pattern of P. tenuifolia. Such analyses can offer necessary molecular biological reasons for the utilization and seed breeding of P. tenuifolia.
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OBJECTIVE:To study the pharmacological effects of saponin H,a new saponin isolated from Polygala tenuifolia Willd,on isolated smooth muscle and herat.METHODS:Isolated rabbit ileum,aorta strip,Langendorff heart,guinea pig trachea,and rat uterus were prepared.Their changes in tension 3 min after administration of H were recorded.RESULTS:19 and 76 μmol.L-1of H showed contracting on above-mentioned smooth muscles,with increasing their tensions by 64% and 167%,30% and 118%,20% and 50%,100% and 118%,respectively.19,38 and 76μmol.L-1 of H showed dose-dependent inhibition on heart,decreased contractile force by 20%,23% and 34%,and reduced the heart rate by 17%,20% and 35%,respectively,but failed to affect the coronary flow.The contracting action of H on uterus was not influnced by diphenhydramine,but partly attenuated by indomethacin.CONCLUSION:H excited the isolated smooth muscles but inhibited the heart.Its action on uterus was partly related to the activation of prostaglandin synthetase.
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Object To study the chemical constituents of the root of traditional Chinese medicine "Yuanzhi" (Polygala tenuifolia Willd ) Methods Seperation and purification were performed on silica gel, Sephadex LH 20 and ODS CC Their structures were established on the basis of physicochemical and spectral analysis Results Five compounds were isolated and identified as tenuifoliside B (Ⅰ), methyl 3, 4, 5 trimethoxycinnamate (Ⅱ), polygalaxanthone Ⅲ (Ⅲ), 7 O methylmangiferin (Ⅳ) and lancerin (Ⅴ), respectively Conclusion Compounds Ⅱ and Ⅳ were isolated from the plant of Polygala L for the first time and compound V was isolated from P tenuifolia for the first time
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Objective To investigate the chemical constituents of Polygala tenuifolia. Methods Silica gel column and Sephadex LH-20 column chromatography were used to separate and purify the chemical constituents. The structures were elucidated by spectral data. Results Six compounds were isolated and identified as: 2-hydroxy-4, 6-dimethoxybenzophone (hydrocotoin, Ⅰ), 1, 7-dihydroxy-3-methoxyxanthone (Ⅱ), 1, 7-dihydroxy-2, 3-dimethoxyxanthone (Ⅲ), ?-spinasterol (Ⅳ), 1, 5-anhydro-D-sorbitol (polygitol Ⅴ) and benzoic acid (Ⅵ). Conclusion Compound Ⅳ is isolated from the plant and compound Ⅰ is isolated from the plants of Polygala L. both for the first time.
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98 %).Conclusions Saponins are the major active ingredients in Radix Polygalae.Tenuifolin as an alkaline hydrolysis product of total saponins can be used as a chemical reference substance for the quality control of Radix Polygalae.
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Eighty-five endophytic fungal strains were isolated from the roots、stems and leaves of Polygala tenuifolia Willd, among which, fifty-two from natural plants and thirty-three from cultivated ones. Sev-enty-six strains were classified as twenty-three fungal genera. The inhibitory activity screening to fourteen microbe were conducted research. The results showed that some endophytic fungi had remarkble inhibitory activities to Bacillus subtilis, Shigella sonnei, Escherichia coli, Candida albicans, Fusarium kyrushuense and they were all belonged to Fusarium, Alternaria, Aphanocladium respectively. All of the endophytic fungi isolated from Polygala tenuifolia showed no inhibitory activities to Staphyloccocus aureus, Salmonel-lae enteritis, Bibrio parahemolyticus.