Assay of 18 amino acids in Chuanmingshen violaceum roots by pre-column derivative HPLC / 中国中药杂志

The roots of Chuanmingshen violaceum is a commonly used Chinese herb and food, which contains rich amino acids. However, the kinds and amounts of amino acids are variety in this herb among the geographical location and ecological environment. Therefore, this study firstly developed a new pre-column derived HPLC method to quantify the levels of 18 amino acids in Ch. violaceum roots. Then 24 Ch. violaceum samples were harvested from its main cultivating areas in Sichuan, China. These samples were divided into 4 producing areas based on their geographical sites. The 18 kinds of amino acids were quantified in these sample by the developed method. The differences of these amino acids were further analyzed among these herbal samples and the 4 producing areas by t-test and principal component analysis(PCA). The result indicated the peaks of the 18 kinds of amino acids were separated well in 70 min.The correlation coefficients between peak areas and concentration of these amino acids were more than 0.999 1(n=6). All of their recoveries were in the range of 97.38%-101.3%(n=6).Their detection limit was in the range of 0.003-0.379 μg·mL~(-1).It demonstrates that the developed HPLC method can accurately quantify the amounts of multi-amino acids in this herb. The results of t-test analysis showed the contents of histidine, cystine, leucine, valine, tryptophan, phenylalanine and threonine were significantly different(P<0.05) among the 4 producing areas. But the differences of other amino acids were not significant.The first five factors were extracted by PCA to calculate the comprehensive score. The order of comprehensive score for the 4 producing areas was B(0.603, n=10), C(0.206, n=3), A(-0.283, n=7) and D(-1.167, n=4). The total content of amino acids in Ch. violaceum collected in B producing area was largest(12.5 mg·g~(-1)). It is concluded the Ch. violaceum contains multi-kinds of amino acids. On the basis of amino acid amount, Langzhong city and Cangxi county in Sichuan province(producing area B) is the suitable areas for cultivating Ch. violaceum.

Comprehensive evaluation of yield and quality of Chuanmingshen violaceum based on principal component and cluster analysis / 中草药

Objective: To comprehensively compare and evaluate the yield and quality of Chuanmingshen violaceum and provide a basis for breeding and high-yield cultivation. Methods: The principal component analysis of eight main agronomic characters and two quality characters from 25 cultivated populations of C. violaceum came from different origins in Sichuan Province were analyzed, and the comprehensive evaluation and cluster analysis were carried out. Results: The results showed that fresh and dry weight of stem and leaf, fresh and dry weight of root and polysaccharide content had greater coefficients of variation among species, while plant height, length, and diameter of taproot had smaller coefficients of variation. Principal component analysis showed that the 10 main traits might be represented by four principal components, and the cumulative contribution rate was 87.304%, and induced that high yield factor, quality factor, plant type factor, and plant height factor respectively. The comprehensive score of sample 1 was the highest, which was the low-height, high-yield, and good-quality material, and the comprehensive score of sample 13 was the lowest, which was the high-height and high-quality and low-yield material. The test materials could be divided into five types by cluster analysis, namely, long-term and high-yield and high-quality material, heavy-root and poor-quality material, low-yield and poor-quality material, high-height and low-yield and good-quality material, and dwarf and high-yield and good-quality material. Conclusion: The comprehensive evaluation method is reliable by the principal component analysis and cluster analysis. The resource of C. violaceum in Sichuan is rich. We can choose some high quality resources among them to provide the basis for the selection of new varieties of Sichuan Province.

Effects of combined application of N, P, and K on yield and quality of Chuanmingshen violaceum / 中草药

Objective The best combination of nitrogen, phosphorus and potassium and application amount of fertilizer were studied to provide technical support and theoretical basis for rational fertilization in the production of Chuanmingshen violaceum. Methods The effects of N, P and K application on yield and main effective components of C. violaceum were studied by L9(34) orthogonal experiment design. Results Reasonable N, P, and K application could significantly increase the yield of C. violaceum, and was beneficial to the accumulation of total polysaccharides and the content of imperatorin. Among them, the yield of 75.22 kg/hm2 under the combination of N2P1K2 was the highest, the increase rate was 60.67%, the total polysaccharide content was 25.46%, and the content of imperatorin was 0.489 mg/g. The yield of 44.82 kg/hm under the combination of N3P2K2 was the lowest, the total polysaccharide content was 2.40%, and the content of imperatorin was 0.379 mg/g. The effects sequence of N, P, and K fertilizers on the yield of C. violaceum were N > P > K. The nitrogen fertilizer had the most obvious effect on the yield of C. violaceum, followed by phosphate fertilizer, and potassium fertilizer had the worst effect. Conclusion Under the conditions of this experiment, the best combination of fertilization was N2P1K2 or N2P3K1. The optimum amount of fertilization of N, P2O5, and K2O were 1.08, 0.73, and 0.83 kg/hm2, respectively; or 1.08, 1.10, and 0.50 kg/hm2, respectively.

Antifungal effects of three medicinal crops on Phytophthora nicotianae / 中国中药杂志

Tobacco black shank is one of the most harmful soil-borne diseases infected by Phytophthora parasitica. In order to probe the control method to this disease, in this study, the mycelial growth rate method was employed to investigate the antifungal effects of extracts from stem-leaf and root, root exudates, and their combination of Scrophularia ningpoensis, Chuanmingshen violaceum and Pinellia ternata The results showed that: ①Stem-leaf and root extracts of S. ningpoensis, C. violaceum and P. ternata exhibited different antifungal activities, and the inhibition increased with the increase of extract concentration. The antifungal effect of S. ningpoensis extracts at 0.5 g•mL⁻¹ was the strongest than other medicinal plants, the inhibition rate of steam-leaf and root extracts reached 74.88%, 69.27%, respectively. The inhibitory effect of C. violaceum and P. ternata was relatively lower, however, there is a significant gain effect after combination of steam-leaf and root extracts of C. violaceum. ②The root exudates of S. ningpoensis, C. violaceum and P. ternata showed fungistasis to Phytophthora nicotianae, and fungistasis was enhanced with the increase of root exudate concentration. The antifungal effect in the order of C. violaceum > S. ningpoensis > P. ternata. ③The antifungal activity of combination of extract and root exudate from S. ningpoensis was similar with the effect of C. violaceum, they were both stronger than P. ternata, and the antifungal activity for three combination were located between the antifungal activity of their extracts and root exudates. S. ningpoensis and C. violaceum can be potentially applied to prevent and control the tobacco black shank.

Determination of heavy metal elements in Chuanmingshen violaceum by ICP-MS / 中草药

Objective: To quantify the contents of heavy metal elements in the roots of Chuanmingshen violaceum (Chuanmingshinis Violacei Radix, and Chuanmingshen) and to evaluate its safety. Methods: An inductively coupled plasma mass spectrometric (ICP-MS) method was developed to quantify six heavy metal elements, i.e. lead (Pb), cadmium (Cd), mercury (Hg), copper (Cu), arsenic (As), and chromium (Cr) in Chuanmingshinis Violacei Radix. A total of 33 Chuanmingshinis Violacei Radix samples were collected from eight counties/districts in Sichuan province of China, processed and then quantified for the contents of these elements. Moreover, the eight counties/districts were divided into four cultivation areas of Chuanmingshinis Violacei Radix based on the geographical location and ecological environment. The differences of heavy metals were compared by statistical analysis. Results: The total content of each element in the 33 samples of Chuanmingshinis Violacei Radix were Pb ≤ 0.256 μg/g, Cd ≤ 0.235 μg/g, Hg ≤ 0.123 μg/g, Cu ≤ 3.963 μg/g, and Cr ≤ 2.145 μg/g; As below the detection limit, the findings were in line with the "Green Trade Standards of Importing and Exporting Medicinal Plants and Preparations" in China. Comparing the amount of heavy metal elements in the samples of Chuanmingshinis Violacei Radix, it was found that the contents of the six heavy metal elements were varied among the four cultivation areas. Except for the difference of Pb content which was significant between the two cultivation areas (P < 0.05), other heavy metal elements in these samples among the four cultivation areas were not significantly different. Conclusion: The developed ICP-MS method is suitable to accurately quantify the content of heavy metal elements in the samplesof Chuanmingshinis Violacei Radix. On the basis of the level of heavy metal elements, Chuanmingshinis Violacei Radix is considered safe and the current cultivation areas are suitable for the cultivation of C. violaceum.

Genetic diversity of Chuanmingshen violaceum by SRAP markers / 中草药

Objective: In order to correctly identify the different germplasm resources of Chuanmingshen violaceum and gain the excellent germplasm resources, SRAP molecular marker was used to analyze the genetic diversity of C. violaceum. Methods: C. violaceum was collected from seven different areas, which included 24 samples, the DNA fingerprint of C. violaceum was constructed with SRAP molecular marker, and the genetic diversity was analyzed. Results: Totally 374 bands were amplified by 37 primer pairs, of which 283 bands were polymorphic, and the polymorphic percentage was 75.67%. The SRAP-based genetic similarity coefficient of all samples ranged from 0.7267 to 0.9239, with a mean of 0.8150. The analysis of molecular variance showed higher percentages of genetic variation within population. All the accessions could be distinguished by SRAP markers. The cluster analysis results showed that 63 accessions were classified into seven groups, which were correlated with the geographical distribution of the accessions to some degree. The accessions from Langzhong and Jintang had higher diversity. Conclusion: There actually exists plentiful genetic diversity among the genetic resources of C. violaceum. SRAP marker is a useful method for analyzing the genetic diversity among C. violaceum accessions.

Effect of Chuanmingshen violaceum polysaccharides and its sulfated derivatives on immunosuppression induced by cyclophosphamide in mice / 中国免疫学杂志

Objective:In oder to investigate the effect of Chuanmingshen violaceum polysaccharides ( CVP) and Solfated Chua-nmingshen violaceum polysaccharides ( SCVP) on immunosuppression induced by cyclophosphamide ( CY) in mice.Methods: CY were used to induce immunosuppression in mice;Spleen and thymus indexes were used to evaluate the immune organs indexes;the [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide,MTT] method was used to detect the proliferation of spleen lymphocytes of each group;the concentrations of IFN-γand IL-2 were assayed by ELISA kit.Results: SCVP and CVP could resist immunosuppression by promoting lymphocyte proliferation, increasing the contents of IFN-γ and IL-2, promoting immune organs development in immunosuppressive mice induced by CY.Conclusion:SCVP and CVP exhibited the potential to used as immunopotentiator.