Inhibitory Effect of Cinobufotalin on Macrophage Inflammatory Factor Storm and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism.@*METHODS@#THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot.@*RESULTS@#1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway.@*CONCLUSION@#Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.

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.

Determination of common dyes in dyed safflower by near infrared spectroscopy / 中国中药杂志

Because the red and bright color of corolla is the main indicator for the quality assessment of good safflower,the dyed safflower is sometimes found at the herbal market,what is influence on this herb quality and efficacy. A total of 127 safflower samples was therefore collected from different cultivating areas and herbal markets in China to develop a rapid method to identify the dyed safflower. Near-infrared spectroscopy(NIRS) combined with characteristic identification,high performance liquid chromatography(HPLC),principal component analysis(PCA) and partial least squares regression analysis(PLS) were employed to differentiate safflower from dyed safflower samples,and further quantify the levels of the 6 dyes,i.e. tartrazine,carmine,sunset yellow,azorubine,acid red 73 and orange Ⅱ in the dyed safflower. The results indicated that the 50 safflower samples and 77 dyed safflower samples were located at different regions in PCA cluster diagram by NIR spectra. Tartrazine,carmineand and sunset yellow were found in the 77 dyed safflower samples with the amounts of 0. 60-3. 66,0. 11-1. 37,0. 10-0. 71 mg·g-1,respectively. It indicated that the three dyes were the common and main dyes in the dyed safflower. However,azorubine,acid red 73 and orange Ⅱ were not detected in all herb samples. A total of 62 dyed safflower samples were chosen as calibration samples to develop the model for estimating the amount of dyes in dyed safflower. The estimating accuracy was verified by another 15 dyed safflower samples. The values of tartrazine,carmine and sunset yellow in dyed safflower samples were compared between the NIRS and HPLC methods. Each value of mean absolute difference(MAD) was less than 5%. The correlation coefficients of tartrazine,carmineand and sunset yellow were 0. 970,0. 975,0. 971,respectively. It indicated the data quantified by NIRS and HPLC were consistence. It is concluded that NIRS can not only differentiate safflower from dyed safflower,but also quantify the amount of the dyes. NIRS is suitable for rapidly identify the quality of safflower.