| Pseudostellariae Radix, the dried root of Pseudostellaria heterophylla(Miq.) Pax, a traditional Chinese herb had long been used as a traditional Chinese medicine and widely used in the vast majority of Chinense medicine prescription for replenishing qi to invigorate the splee, moistening lung. However, due to differences in environment and genetic germplasm, accumulation of active ingredients in Pseudostellariae Raidx had a great variation and varying quality. How to ensure quality and uniformity of Pseudostellariae Radix was become a key issue in medicine production process. In the formation process of the quality of Pseudostellariae Radix, many factors could influence the synthesis and accumulation of secondary metabolites in Pseudostellariae Radix. Effect and mechanism of secondary metabolites accumulation and synthesis in Pseudostellariae Radix by Ecological environment and the provenance had not been clarified, thus improving the quality of the Pseudostellariae Raidx medicinal materials engineering. Plant metabolomics technology was a biological method which studied whole plant metabolites and its variation, and used detection methods such as color-ms and nuclear magnetic resonance(1H-NMR) to study the difference between the metabolic products in plant samples. With the help of research ideas and methods of plant metabolomics, the topic studied on research of metabonomics in Pseudostellariae Radix by UPLC-Triple TOF-MS/MS, GC-MS and 1H-NMR. It Compared that the chemical composition of different regions, strains, growing periods, processing methods, samples between cultivated and wild species, and screened, identificatied the differences of chemical compositions. Correlation analysis was carried out on the differences in chemical compositions and ecological factors, and discussed the ecological environment of the influence of accumulation of chemical compositions in Pseudostellariae Radix. This paper provided basic data and scientific basis to explore the formation process of medicinal materials quality, suitable production and breeding, cultivation, proper harvest stage and reasonable processing.Research mainly included the following aspects:1. Literature Research A literature review study system Radix relevant literature, a literature review of studies conducted heterophylla.2. Studies on the metabolomics of Pseudostellariae Radix based on UPLC-Triple TOF MS/MS Studied on the differences in chemical constituents on the alcohol extract and water extract of Pseudostellariae Radix from different regions, germplasm, growing stages, processing methods, wild and cultivated by UPLC-Triple TOF-MS/MS. Peak matching, peak alignment and noise filtering were used in analyzing mass spectrometry data. Accurated m/z value analysis of MS data based on software of database search and MS/MS fragment analysis were applied to constituents identification. Data processing used PCA and PLS-DA to find the difference of chemical composition and change rule in Radix Pseudostellariae from different regions, germplasm, growing stages, processing methods, wild and cultivated species. The results showed that:(1) Alcohol extract of Pseudostellariae Radix ① The chemical compositions of Pseudostellariae Radix from different regions had obvious differences. 21 chemical compositions obtained by PLS-DA analysis. There were differences in the 10 components, the relative content of Pseudostellarin B, F, D, Heterophyllin A and Sphingosine in Fujian Zherong were higher; the relative amounts of Maltose, Sucrose and Thyronine in Guizhou Shibing were higher; relative content of Inosine triphosphate and Pseudostellarin A in Jiangsu Jurong were higher. ②The analysis of PCA concluded that the difference was significant among Pseudostellariae Radix of different germplasm. In 11 common components, the relative content of sucrose, Pseudostellarin A, Pseudostellarin G, Heterophyllin A, B and Cyclic AMP in germplasm of Jurong were higher; the relative content of Rutin, Inosine triphosphate, Pseudostellarin B, D, F were higher in germplasm of Fujian Zherong. ③ In different harvest periods of Pseudostellariae Radix from Fujian, samples complied with the local optinal rules. Relative content of rutin, Pseudostellarin B, Heterophyllin A etc. 7 kinds of chemical components in the June harvest samples of Pseudostellariae Radix had high-level; the relative content of 11 components such as Pseudostellariae A were higher in samples of harvested in July and Auguset. ④ In different harvest times of Pseudostellariae Radix from Jiangsu, the chemical compositions of traditional harvest time and other harvest times were different. There were differences in the chemical composition showed different variation, the relative amounts of Sucrose, Inosine triphosphate, Pseudostellarin A, F etc. 12 chemical compositions have peaked in the traditional harvest time of samples; relative content of Rutin, Heterophyllin B, Pseudostellarin E and Thyronine presented a tendency of decreasing as the delay of harvest time. The relative content of Heterophyllin A had high-level after the traditional harvest time. ⑤ It was obvious differences among samples with different processing methods, including Pseudostellariae Radix of drying and dryed in the sun. The relative content of α-aspartic acid in samples of dryed in the sun had high-level; the relative amounts of 7 chemical components including Canthaxanthin, Rutin, Heterophyllin B was higher in Pseudostellariae Radix rubbed. Samples of Pseudostellariae Radix drying at 40℃ had higher unknown compound(C23H42N8O5) and β-Sitosterol. The relative content of Sucrose higher in samples at 50℃, and samples at 60℃ had higher Heterophyllin A. ⑥ Chemical compositions of wild and cultivated Pseudostellariae Radix had distinguished significantly, and screened 22 differences in chemical compositions. There were 10 chemical compounds including Sucrose, Pseudostellarin A, B in cultivated Pseudostellariae Radix were higher relative amounts, and relative amounts of 12 chemical components including Heterophyllin A and B were greater in wild speiecs.(2) Water extract of Pseudostellariae Radix ① Water extract of Pseudostellariae Radix contained mainly amino acids, nucleosides, sugars, organic acids and other 17 kinds of chemical compositions, and its main component was arginine. Chemical compositions in water extract of Pseudostellariae Radix from four different regions were significant. Water extract of Pseudostellariae Radix from Fujian, Zherong had higher Uridine-5’-monophosphate, Isobutyryl glycine, Guanine, Adenosine and Inosine; and the extract from Guizhou, Shibing had higher Arginine, Phenylalanine and Isomaltose. The relative contents of Dodecanoic acid, 5-Methylcytidine, Glutamic acid and Histidine were higher in the water extract of samples from Anhui, Xuancheng, while samples from Jiangsu, Jurong contained more Melatonin. ② Samples of Pseudostellariae Radix in different harvest times from Fujian obtained a distinguish, and the common chemical compositions of differences showed different variation. In addition to the relative content of Diphenhydramine was higher in June harvest time than the rest of 13 common chemical components which the relative amounts were higher in July and August.This was accoreded with local tradition of Fujian. ③ Chemical composition of samples used by drying on different temperatures relatively closed to each other, and had large differences with the other two kinds of processing methods. Relative content of Isoacetylglycine, Adenine and Inosine etc. 5 chemical compounds in the extract from dried samples were higher; and relative content of Dodecanoic acid, Isomaltose and Guanosine triphosphate were higher in samples of dry on the sun. Water extract of Pseudostellariae Radix at 40℃ had higher Arginine, 5-Methylcytidine and Melatonin, while the relative contents of Uridine-5’-monophosphate were higher in samples dried at 60℃. ④ Differences in chemical components had exist in water extract of Wild and cultivated Pseudostellariae Radix. It screened and identified 15 differences in the chemical compositions. The results showed that Dodecanoic acid and Melatonin were higher in the extract of cultivated samples, and the remaining 13 compounds such as Adenine, Histidine which were the relative contents have higher in the extract from the wild speices.3. Studies on the metabolomics of Pseudostellariae Radix based on GC-MS GC-MS combined with ultivariate statistical analysis were used to compare the chemical components of volatile oils from Pseudostellariae Radix in different development stages, cultivated areas, wild and cultivated species. Peak alignment, incision and noise filtering were used in pretreating the data using principal component analysis(PCA) and partial least-squares discriminant analysis(PLS-DA). The results showed that: ① Samples of Pseudostellariae Radix from different cultivated areas influenced by the local ecological environment, resulting in changes in the chemical composition, in which a total of 10 kinds of differences in the chemical composition and showed a different variation. ② Samples of Pseudostellariae Radix from Fujian have been a clear distinction in June, early July, mid-July to August. The result was fitted with the traditional local harvest period(late July). 6 common volatile oils including hexanal and furfural had higher relative comtents in harvest of July to mid-August. ③ Samples of two harvest periods after mid-July had gathered, and distinguished with the other 3 harvest times of samples clearly. This result was the same with the local traditional harvest Guizhou(after mid-July). There are 9 differences in chemical compositions, which 4 common compounds such as 4-Butyl-3-methoxyl-2-cyclohexene-1-ketone and 2-Butyl-2-octenal had higher relative content in Pseudostellariae Radix harvested after mid-July. ④ Chemical compositions of Pseudostellariae Radix before and after the sulfur fumigation were significantly different. There were 10 differences in compositions effected in differentiation. The relative contents of 7 common components including hexanal, furfural and 2,5-Dimethylfuran were higher in samples before sulfur fumigation, and 4-Butyl-3-methoxyl-2,4-cyclohexadiene-1-ketone, 2-Heptanone, and 2-Octene,(E)- were higher in Pseudostellariae Radix after sulfur fumigation.4. Studies on the metabolomics of Pseudostellariae Radix based on 1H-NMR Extracts of different samples from Pseudostellariae Radix were analyzed by 1H-NMR, and the resulting spectra were identified. 1H-NMR spectra for all samples used TSP scaling(δ0.00). Nuclear magnetic resonance spectra would be integrated using MestReNova8.0 software, and the data which integraled used principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis(OPLS-DA). It can analyzed the difference between metabolites of samples and found the differences according to the results. The paper showed that the samples of Pseudostellariae Radix from different cultivation areas had significant differences, but the difference was not significant between samples from different garmglasm. It proved that ecological entironment may be the main factors contributed to the difference among samples. It showed that significant differentiation was obvious in cultivated and wild Pseudostellariae Radix according to compare the chemical compositions between these two species. It provided basis data to announce change rules of accumulation of metabolites in cultivated and wild Pseudostellariae Radix and explore influence of genetic variation on Pseudostellariae Radix.5. Correlation analysis between differences in chemical components from Pseudostellariae Radix and meteoprological factors 10 chemical compounds in alcohol extracts of Pseudostellariae Radix from different regions and climatic factors were analyzed by correlation analysis. The results showed that accumulated temperature in July, sunshine hours in July, minimum temperature in August, precipitation in 2011 four ecological attributes were the main meteorological factors which impacted the relative contents of differences in chemical compositions. Accumulated temperature and sunshine hours in July had a major impact on the relative content of cyclic peptides. It revealed the influence of environment on accumulation of secondary metabolites in Pseudostellariae Radix. It had a demonstration and guidance to study of medicinal plants ecological environment adaptation mechanisms, variety and quality changes. |
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