Quality research of Gastrodiae Rhizoma with UPLC fingerprint and content determination of multicomponent / 中草药

Objective To establish the analysis method of UPLC fingerprint and simultaneous determination of five active components (gastrodin, 4-hydroxybenzyl alcohol, parishin B, parishin C, and parishin A). Methods The analysis was performed on a Waters Acquity with a Waters ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column. The gradient mobile phase was acetonitrile and 0.1% formic acid-water solution, flow rate was set at 0.3 mL/min, the injection volume was 0.2 μL and the temperature of column was 35 ℃. The detection wavelengths were set at 270 nm (fingerprint), 220 nm (content determination). The 28 batches of samples fingerprint were analyzed by similarity evaluation, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). The identification and content determination of the main common peaks were carried out by comparison with reference. Results We set up the common mode of the fingerprint with 20 common peaks, and six of them were identified, which were gastrodin (1), 4-hydroxybenzyl alcohol (3), p-hydroxybenzaldehyde (7), parishin B (14), parishin C (15), and parishin A (18). The similarities among 28 samples were all above 0.90. PCA was used to divide the samples into five groups, and six different substances were found. The content of the same component and the fluctuation range among different components were all different. The content of the five components of each origin was Guizhou 2.52% > Yunnan 2.49% > Shaanxi 2.33% > Hubei 2.10% > Zhejiang 1.90% > Anhui 1.65%.Conclusion The establishment of UPLC fingerprint and simultaneous determination of five active components provide a reference for quality control of Gastrodia Rhizoma.

Protective effect and mechanism of p-hydroxybenzaldehyde on blood-brain barrier / 中国中药杂志

The disruption of blood-brain barrier(BBB) induced by oxidative stress is an important pathological reaction which results in secondary brain injury during the cerebral ischemia-reperfusion. This study was designed to investigate the protective effect and mechanism of p-hydroxybenzaldehyde (p-HBA) from Gastrodia elata on BBB. The BBB is mainly consisted of vascular endothelial cells and astrocytes, so brain microvascular endothelial cell line (bEnd.3) and astrocytes (Ast) in mice were used in this study to establish BBB model. H₂O₂-induced oxidative stress was employed to induct the BBB damage. The bEnd.3 cells or astrocytes were exposed to different concentrations of H₂O₂ (0.125, 0.25, 0.5, 0.75 mmol·L⁻¹) for 4 h, then exposed to 0.5 mmol·L⁻¹ H₂O₂ for different duration (1, 2, 4, 6 h) to detect the reasonable condition of oxidative injury. After intervention by different concentrations of p-HBA(12.5, 25, and 50 mg·L⁻¹), LDH leakage rate was detected for bEnd.3 and Ast cells; the expression levels of tight junction protein claudin-5 and occludin in bEnd.3 cells were determined by Western blot and immunofluorescence. Nrf2, HO-1 and NQO1 in normal bEnd.3 cells and astrocytes as well as H₂O₂-induced damaged in astrocytes were detected by western blot after treatment with p-HBA. The results showed that the optimal condition of H₂O₂ induced damage in bEnd.3 cells and astrocytes was set up as exposure the cells to 0.5 mmol·L⁻¹ H₂O₂ for 4 h. Different concentrations of p-HBA could decrease LDH leakage rate after bEnd.3 and Ast injury was induced by H₂O₂; increase the protein expression levels of claudin-5, occludin, Nrf2, HO-1 and NQO1; and increase the expression levels of Nrf2, HO-1 and NQO1 in normal and H₂O₂-induced damaged astrocytes. These findings indicate that the p-HBA has protective effect on the BBB, and the related mechanism seems to involve up-regulating tight junction protein of the bEnd.3 cells and enhancing endogenous antioxidant capacity by activating the Nrf2/ARE pathway in both of bEnd.3 cells and astrocytes.

Chemical Constituents from the Ethyl Acetate Parts of the Leaves of Cyclocarya paliurus / 中国药学杂志

OBJECTIVE: To investigate the chemical constituents from the leaves of Cyclocarya paliurus. METHODS: Chemical constituents were isolated and purified by column chromatography with silicagel, Sephadex LH - 20, and semi-preparative HPLC. RESULTS: Eleven compounds were isolated from the ethyl acetate extracts of the leaves of Cyclocarya paliurus. Their structures were elucidated as hirsutrin (1), contigoside B (2), kaempferol (3), sterubin (4), syringic acid (5), vanillic acid (6), 2-hydroxy-5-ethoxybenzoic acid (7), 2-methoxy-4-hydroxycinnamic acid (8), cis-p-hydroxycinnamic acid (9), p-hydroxybenzonic acid (10), and phydroxybenzaldehyde (11). CONCLUSION: All the compounds except 3 and 6 are obtained from Cyclocarya paliurus for the first time.

Biological activity and hydroxy safflor yellow A content of intermediates in preparation of Safflower Injection / 中国中药杂志

In order to investigate the effect of various production processes on the quality of Safflower Injection, the biological activities of the intermediates were evaluated by measuring activated partial thromboplastin time (APTT) and adenosine diphosphate (ADP) induced platelet aggregation in vitro. Intermediates were produced by key processes, such as extraction, concentration, twice alcohol precipitation, water sedimentation and two sterilizations during the production of Safflower Injection. The content of main chemical components in intermediates was determined by HPLC. The results showed that with the advance of the preparation process of Safflower Injection, the inhibition of ADP-induced platelet aggregation rate of each intermediate decreased gradually, and the trend of extending APTT activity decreased first and then increased. Meanwhile, the content of hydroxy safflor yellow A (HSYA) was gradually lowered, the content of p-hydroxy-cinnamic acid was increased, and new chemical component p-hydroxybenzaldehyde was produced. In conclusion, sterilization played a key role in the biological activity and HSYA content of Safflower injection.

Establishment of screening method for γ-aminobutyric acid transaminase inhibitor and its application in screening constituents in Gastrodiae Rhizoma / 中草药

Objective: To establish a screening model in vitro with γ-aminobutyric acid transaminase (GABA-T) as a target for screening the inhibitors of anti-epilepsy drugs for the treatment of nervous diseases and finish the structure-activity relationship analysis of p-hydroxybenzaldehyde as effective components in Gastrodiae Rhizoma and its analogs. Methods: Catalyst performed temperature, reaction time, the concentration of NAD+, α-ketoglutarate, and GABA were investigated to optimize the model. The test of p-hydroxybenzaldehyde (HBA) and its 11 analogs were selected as verification of this model. Results: The screening model in vitro based on GABA-T was established. The testing result of p-HBA and its 11 analogs was consistent with the practical activity recorded in the literature. Hydroxyl groups of -OH and -CHO in p-aldehyde on the benzene ring were pharmacophores. Conclusion: The model established in this paper can be used for high throughput screening the inhibitors of anti-epilepsy drugs, which provides the reference for the study on the inhibitors of anti-epilepsy drugs for nervous diseases and their mechanisam.

Simultaneous determination of eight active components in Gastrodiae Rhizoma by HPLC-MS / 中草药

Objective: To establish an HPLC method for the simultaneous determination of eight active components (gastrodin, 4-hydroxybenzyl alcohol, vanillyl alcohol, vanillin, p-hydroxybenzaldehyde, parishin B, parishin C, and parishin A). Methods: The analysis was performed on an Agilent 1220 system with a Hypersil C18 column (250 mm × 4.6 mm, 5 μm). The eight components were separated in 75 min with gradient mobile phase consisting of 0.5% CH3COOH-H2O (A) and 0.5% CH3COOH-CH3OH (B): 0-10 min, 98% A; 10-60 min, 98%-60% A; 60-75 min, 60% A. The temperature of column was 30 ℃, and the injection volume was 20 μL. The multiple-reaction monitoring (MRM) scanning was employed for the quantification with switching electrospray ion source polarity in negative mode. The ion spray voltage was set at -4500 V and the turbo spray temperature was maintained at 550 ℃. Results: The eight components had the good linearity (r ≥ 0.999 2) within the linear ranges. The average recovery rate was 94.51%-102.70% with RSD < 3.50%. The contents of the eight components of Gastrodiae Rhizoma varied according to the different habits. The contents of parishins A, B, and C were high while the contents of vanillyl alcohol and vanillin were low. Conclusion: The developed HPLC-MS method is simple, sensitive, and accurate, and has the good repeatability in separation, which is available for the quality control of Gastrodiae Rhizoma.

Chemical constituents of Opuntia milpa (I) / 中草药

Objective: To investigate the chemical constituents of Opuntia milpa. Methods: The compounds were isolated by repeated silica gel chromatography and were elucidated by chemical and spectral analyses. Results: Ten compounds were isolated from the ethyl acelate extraction of O. milpa and identified as β-sitosterol (1), 1-heptadecanol (2), 5-hyadroxymemyl-2-furancarboxaldehyde (3), syringaldehyde (4), vanillin (5), p-hydroxybenzaldehyde (6), coniferyl aldehyde (7), p-hydroxyl-cinnamaldehyde (8), quercetin-3-methyl ether (9), and 2,6-dimethoxyphenol (10). Conclusion Compounds 3-10 are isolated from O. milpa for the first time.