Quality control of eight substances in Changwei San based on quantitative analysis of multi-components by single-marker / 国际药学研究杂志

Objective: To establish a quantitative analysis of multi-components by single marker(QAMS)method for the simultaneous determination of jaceosidin, eupatilin, limoni, evodiamine, rutaecarpine, cinnamyl alcohol, cinnamic acid and cinnamal-dehyde in Changwei San. Methods: The Waters Symmetry C18 column(250 mm×4.6 mm, 5 μm)was used for the separation, and the mobile phase was the acetonitrile(A)and 0.1% phosphoric acid(B)solution in a gradient elution at a flow rate of 1.0 ml/min. The detection wavelengths were set at 345 nm for jaceosidin and eupatilin, 215 nm for limoni, evodiamine and rutaecarpine, and 275 nm for cinnamyl alcohol, cinnamic acid and cinnamaldehyde. With evodiamine as an internal reference standard, the relative correction factors for the other 7 components were established and their contents were calculated with the relative correction factors to achieve the QAMS, and then the differences between the calculated values by QAMS and measured values by the external standard method(ESM) were compared to validate the accuracy and feasibility of the QAMS method. Results: Jaceosidin, eupatilin, limoni, evodiamine, rutaecarpine, cinnamyl alcohol, cinnamic acid and cinnamaldehyde showed good linear relationships within the ranges of 0.98-19.60, 2.67-53.40, 4.06-81.20, 1.98-39.60, 2.69-53.80, 0.56-11.20, 1.49-29.80, and 8.77-175.40 μg/ml(r≥0.9992), whose average recoveries(RSD) were 98.77%(0.96%), 99.38%(1.01%), 100.02%(0.83%), 97.80%(1.40%), 98.91%(1.18%), 96.99% (1.13%), 98.09%(1. 24%)and 99.10%(0.67%), respectively. No significant difference was observed between the calculated values by QAMS and the measured values by ESM. Conclusion: The established QAMS method is simple and accurate, which might be used to evaluate the quality of Changwei San.

Prediction of Cinnamomum cassia grade based on binary Logistic regression analysis / 中草药

Objective: In this study, a two-classification model based on the idea of “ingredient-efficacy” was established for the quality classification of Cinnamomum cassia with considerations to quality control components and biological activities. Methods: A method to determine quality control components was proposed by UPLC. The in vitro anti-oxidant activity of C. cassia was reflected by DPPH and hydroxyl radical scavenging experiment. The quality control index and anti-oxidant index were correlated by a Logistic algorithm. Finally, a binary logistic regression model for classification of C. cassia was established. Results: UPLC fingerprints of 20 samples of C. cassia were established, and their anti-oxidant activities were determined. Four quality control components (coumarin, cinnamyl alcohol, cinnamic acid, and cinnamaldehyde) were screened out by principal component analysis, and their methodological validation was carried out. According to the regression equation, 20 batches of C. cassia were divided into four grades: excellent, good, medium, and poor. Conclusion: The binary logistic regression model can describe the mapping relationship between the grade of C. cassia. It can better express the classification standard for the prepared C. cassia. This study provides a new idea for quality evaluation of C. cassia.

Protective effects of three phenylallyl compounds from Guizhi decoction ox-LDL-induced oxidative stress injury of human brain microvascular endothelial cells / 中国中药杂志

The main objective of this research is to observe protective effects of three phenylallyl compounds(cinnamyl alcohol,cinnamaldehyde and cinnamic acid)from Guizhi decoction against ox-LDL-induced oxidative stress injury on human brain microvascular endothelial cells(HBMEC).In this study,the toxicity and optimal protective concentration of three phenylallyl compounds from Guizhi decoction were determined by MTT assay.The HBMEC were divided into control group(DMSO),model group(ox-LDL),tert-butylhydroquinone (t-BHQ) group,cinnamyl alcohol group, cinnamaldehyde group and cinnamic acid group.The model group were treated with ox-LDL (50 mg•L⁻¹)for 24 h,other groups were separately treated with t-BHQ, cinnamyl alcohol, cinnamaldehyde and cinnamic acid of 20 μmol•L⁻¹, and exposed to ox-LDL (50 mg•L⁻¹) for 24 h at the same time.The survival rate of HBMEC was detected by MTT assay,reactive oxygen species(ROS) production of injured cells were detected using laser scanning confocal microscope (LSCM),the content of SOD, MDA, eNOS and NO in HBMEC was determined by ELISA, and the expressions of Nrf2 mRNA were detected by quantitative Real-time PCR(qRT-PCR).The results shows that oxidative stress injury of HBMEC could be induced by ox-LDL, the three phenylallyl compounds from Guizhi decoction did not affect morphology and viability of normal HBMEC.Compared with model group, the three phenylallyl compounds from Guizhi decoction could improve the above oxidative stress status and up-regulate Nrf2 mRNA expressions in injured HBMEC(P<0.05, P<0.01) .These findings suggested that the three phenylallyl compounds from Guizhi decoction have certain protective effects against ox-LDL-induced oxidative stress injury on HBMEC(cinnamaldehyde> t-BHQ> cinnamic acid>cinnamyl alcohol),the protective mechanism maybe related to regulation of antioxidant enzymes gene expression in HBMEC by Nrf2.

Screening target genes for bimolecular marking methods of Magnolia quality / 中国中药杂志

The study used use bimolecular marking methods to evaluate the lignans of Magnolia officinalis and M. officinalis var. biloba. First, we compare the chemical constituents between M. officinalis and M. officinalis var.biloba. There were significant differences in concentration of magnolignan I between leaves of these two varieties. Then we further select the p-hydroxyphenyl lignin to mining the key enzyme genes of biosynthesis from Magnolia transcriptome, and screened an encoding cinnamyl alcohol dehydrogease gene as the candidate marker of bimolecular marking methods of Magnolia quality by comparing of the expression level and structure variation in homologous gene between M. officinalis and M. officinalis var.biloba. The established method provides the technical support for bimolecular marking methods of Magnolia quality evaluation.

Phenylpropanoid enzymes, phenolic polymers and metabolites as chemical defenses to infection of Pratylenchus coffeae in roots of resistant and susceptible bananas (Musa spp.).

Activity differences of the first (phenylalanine ammonia lyase, PAL) and the last (cinnamyl alcohol dehydrogenase, CAD) enzymes of phenylpropanoid pathway in the roots of resistant (Yangambi Km5 and Anaikomban) and susceptible (Nendran and Robusta) banana cultivars caused by root lesion nematode, Pratylenchus coffeae, were investigated. Also, the accumulation of phenolics and deposition of lignin polymers in cell walls in relation to resistance of the banana cultivars to the nematode were analyzed. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher PAL activity and total soluble and cell wall-bound phenolics than in susceptible cultivars. The resistant cultivars responded strongly to the infection of the nematode by induction of several-time higher PAL and CAD enzymes activities, soluble and wall-bound phenolics and enrichment of lignin polymers in cell wall and these biochemical parameters reached maximum at 7th day postinoculation. In addition, profiles of phenolic acid metabolites in roots of Yangambi Km5 and Nendran were analyzed by HPLC to ascertain the underlying biochemical mechanism of bananas resistance to the nematode. Identification and quantification of soluble and cell wall-bound phenolic acids showed six metabolites and only quantitative, no qualitative, differences occurred between the resistant and susceptible cvs. and between constitutive and induced contents. A very prominent increase of p-coumaric, ferulic and sinapic acids, which are precursors of monolignols of lignin, in resistant cv. was found. These constitutive and induced biochemical alterations are definitely the chemical defenses of resistant cvs. to the nematode infection.

Chemical constituents of Empetrum nigrum var. japonicum / 中草药

Objective: To study the chemical constituents from the herbs of Empetrum nigrum var. japonicum. Methods: The compounds were isolated and purified by silica gel, MCI gel, Sephadex LH-20 column chromatographies, semi-preparative HPLC, and their structures were mainly elucidated on the basis of physicochemical characteristics and spectral analysis. Results: Twelve compounds were isolated from 95% EtOH extract of E. nigrum var. japonicum, and were identified as 3β-hydroxy-nigrum-21-en (1), 4'-hydroxy-α, β-dihydrochalcone-2'-O-β-D-glucopyranoside (2), 5-(2-phenylethyl)-3-hydroxyphenol-1-O-β-D-glucopyranoside (3), 2', 4'-dihydroxy-chalcone (4), 2'-methoxy-4'-hydroxy-α, β-dihydrochalcone (5), phenylpropionic acid (6), cinnamyl alcohol (7), quercetin (8), (-)-epicatechin (9), quercetin-3-O-α-L-arabinoside (10), hyperoside (11), and 8-methoxyquercetin-3-glucoside (12), respectively. Conclusion: Compound 1 is a new triterpenoid and compound 2 is a new dihydrochalcone.

Cinnamyl alcohol attenuates vasoconstriction by activation of K+ channels via NO-cGMP-protein kinase G pathway and inhibition of Rho-kinase

Cinnamyl alcohol (CAL) is known as an antipyretic, and a recent study showed its vasodilatory activity without explaining the mechanism. Here we demonstrate the vasodilatory effect and the mechanism of action of CAL in rat thoracic aorta. The change of tension in aortic strips treated with CAL was measured in an organ bath system. In addition, vascular strips or human umbilical vein endothelial cells (HUVECs) were used for biochemical experiments such as Western blot and nitrite and cyclic guanosine monophosphate (cGMP) measurements. CAL attenuated the vasoconstriction of phenylephrine (PE, 1 microM)-precontracted aortic strips in an endothelium-dependent manner. CAL-induced vasorelaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), methylene blue (MB; 10(-5) M) and 1 H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10one, (ODQ; 10(-6) or 10(-7) M) in the endothelium-intact aortic strips. Atrial natriuretic peptide (ANP; 10(-8) or 10(-9) M) did not affect the vasodilatory effect of CAL. The phosphorylation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) were stimulated by CAL treatment in HUVECs and inhibited by treatment with L-NAME. In addition, cGMP and PKG1 activation in aortic strips treated with CAL were also significantly inhibited by L-NAME. Furthermore, CAL relaxed Rho-kinase activator calpeptin-precontracted aortic strips, and the vasodilatory effect of CAL was inhibited by the ATP-sensitive K+ channel inhibitor glibenclamide (Gli; 10(-5) M) and the voltage-dependent K+ channel inhibitor 4-aminopyridine (4-AP; 2 x 10(-4) M). These results suggest that CAL induces vasorelaxation by activating K+ channels via the NO-cGMP-PKG pathway and the inhibition of Rho-kinase.