ABSTRACT
Objective: To establish an HPLC fingerprint of Cnidii Fructus formula granule analysis method for simultaneous determination of six main coumarin components, including osthol, xanthotoxin, xanthotol, bergapten, imperatorin and isopimpinellin, in order to provide reference for the study of the material basis of Cnidii Fructus formula granule. Methods: The method was performed by high performance liquid chromatography with a Waters XBridge C18 (250 mm × 4.6 mm, 5 μm) column and methanol (A)-0.1% acetic acid (B) as the mobile phase for gradient elution. The flow rate was 0.5 mL/min, the injection volume was 10 μL and the column temperature was 40 ℃. The detection wavelength was set at 320 nm. The chromatographic fingerprint evaluation system published by the State Pharmacopoeia Commission (2012 Edition) was used to establish the fingerprint of Cnidii Fructus formula granule, and the content of six main coumarin components was simultaneously determined. Results: The research on the 18 batches of Cnidii Fructus formula granule showed that the fingerprint similarity was greater than 0.992 and 19 common peaks were calibrated with satisfied peak resolution. The content determination results showed that the content of both xanthotoxin and osthol were the main coumarin components in Cnidii Fructus formula granule. According to the methodological investigation, the precision RSD values were all less than 1.6%. The sample was stable within 48 h and this method had good repeatability. The average recovery rates of xanthotol, xanthotoxin, imperatorin, isopimpinellin, bergapten and osthol were 100.69%, 101.03%, 99.48%, 100.88%, 101.27% and 100.35%, respectively. All of these coumarin components’ RSD were less than 2.5%. The six components showed a good linear relationship within a certain concentration range. The results of the content determination of xanthotol, xanthotoxin, isopimpinellin, bergapten, imperatorin and osthol respectively were 8.01-8.29, 2.37-2.63, 4.30-4.61, 4.04-4.40, 3.45-3.90 and 6.02-6.80 mg/g among the 18 batches of the Cnidii Fructus formula granule. Conclusion: The fingerprint method and the determination method of six main coumarin components in the Cnidii Fructus formula granule established in this study are simple, stable, accurate and reliable. This method can be used for the quality control of the Cnidii Fructus formula granule.
ABSTRACT
Objective:To observe the effect of isopimpinellin on primary hippocampal neuron cells γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and receptor genes expressions, in order to explore its hypnotic mechanism. Method:The primary hippocampal neurons of neonatal Sprague-Dawley rats were cultured in vitro. And subsequent experiments were conducted in the optimal state of cell growth, and the purity was identified by immunohistochemistry of neuron-specific enolase. Hippocampal neurons were randomly divided into five groups, namely blank control group, diazepam group (25 mg·L-1), and low-dose (5 mg·L-1), moderate-dose (10 mg·L-1) and high-dose (20 mg·L-1) isopimpinellin groups. Early apoptosis of hippocampus neuron cells were detected using flow cytometry technique after 24 h administration, and the changes in the levels of GABA and 5-HT were detected using enzyme-linked immunosorbent. The changes in mRNA expressions of receptor genes relating to gamma-aminobutyric acid type A receptor(GABAA) genes GABRA1,GABRA5,GABBR1, gamma-aminobutyric acid type B receptor genes (GABAB) GABRB2, 5-hydroxytryptamine 1A receptor (5-HT1A)5-HT1A(A),5-HT1A(B),5-HT1A(C) were detected by real-time quantitative PCR(Real-time PCR). Result:On the 7th day, the hippocampal neurons grew in a good condition, and the purity was above 90%. Apoptosis rates of hippocampal neurons in the low-dose and moderate-dose groups were significantly lower than that in the blank control group (PP1,GABRA5,5-HT1A(A),5-HT1A(C) in the moderate-dose and high-dose isopimpinellin groups were significantly higher than those in the blank control group (PP1,5-HT1A(B) in the low-dose, moderate-dose and high-dose isopimpinellin groups were significantly higher than those in the blank control group (PPConclusion:The hypnotic mechanism of isopimpinellin may be related to the inhibition of hippocampal neuron apoptosis, the increase of the content of inhibitory neurotransmitter GABA, and the up-regulation of GABA and 5-HT-related receptor genes.
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Objective To study the chemical constituents from the fresh fruits of Physalis pubescens. Methods Compounds were isolated and purified by repeated column chromatography on silica gel column, Sephadex LH-20 gel column, and preparative HPLC methods. Physicochemical properties and spectroscopic methods were employed for the structure elucidation. Results Twenty compounds were obtained and identified as osthol (1), xanthotoxin (2), isopimpinellin (3), imperatorin (4), (-)-meranzin hydrate (5), auraptenol (6), 1-(4-hydroxy-3,5-dimethoxyphenyl) ethanone (7), 2,5-dimethoxybenzoquinone (8), 2-(4-hydroxyphenyl) acetic acid (9), (S)-methyl 2-hydroxy-2-(4-hydroxyphenyl) acetate (10), pyrocatechol 1-O-β-D-glucopyranoside (11), benzyl β-D-glucopyranosyl (1→6)-β-D-glucopyranoside (12), 2-phenylethyl-O-β-D-glucopyranoside (13), p-hydroxybenzene propanoic acid (14), 3,4-dihydroxybenzene propionic acid (15), (1-O-p-coumaroyl)-(6-O-β-glucosyl)-β-glucoside (16), 1-O-trans-cinnamoyl-β-D- glucopyranosyl-(1→6)-β-D-glucopyranoside (17), N-trans-feruloyltyramine (18), kaempferol 3-O-β-D-glucopyranoside (19), and bergapten (20), respectively. Conclusion Compounds 1-16 are obtained from Physalis genus for the first time.
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Objective: The chemical constituents were isolated and identified in order to find the bioactive natural products from Gelsemium elegans. Methods: Silica gel, sephadex LH-20 and HPLC column chromatographic techniques were used for separation and purification of the compounds and extensive spectral analysis spectrum were employed for structural elucidation. Results: Fifteen compounds were separated from G. elegans, identified by physicochemical properties, spectral analysis and other means. They are koumine (1), gelsemine (2), gelsevirine (3), gelsemicine (4), xanthotoxin (5), bergapten (6), isopimpinellin (7), imperatorin (8), osthol (9), p-hydroxybenzoic acid (10), vanilla acid (11), β-sitosterol (12), β-daucosterol (13), gelsemamide (14), and 16-epivoacarpine (15). Conclusion: Compounds 5-9 are first isolated from the plants in genus Gelsemium Juss.
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Objective: To study the chemical constituents from the root bark of Changium smyrnioides. Methods: Compounds were isolated by various kinds of column chromatographies on silica gel, Sephadex LH-20, and recycling preparative HPLC from the ethanol extract in the root bark of C. smyrnioides, and their structures were elucidated by the physicochemical characteristics and spectral analyses. Results: Fifteen chemical constituents were obtained and identified as imperatorin (1), phellopterin (2), xanthotoxol (3), 5-hydroxy-8-methoxy-psoralen (4), vanillic acid (5), alloimperatorin (6), psoralen (7), bergapten (8), 8-O-β-D-glucopyranosyl- 5-methoxylpsoralen (9), isopimpinellin (10), caffeic acid (11), aurantiamide acetate (12), vaginatin (13), β-sitosterol (14), and succinic acid (15). Conclusion: Compounds 6-13 are isolated from the plants in Changium Wolff for the first time.
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Objective: To investigate the chemical constituents from Peucedanum decursivum. Methods: P. decursivum was extracted with 95% ethanol, the compounds were isolated via various chromatographic methods, and their structures were identified by detailed spectroscopic analyses. Results: Twelve compounds were isolated from the acetic ether fraction in the 95% extract of P. decursivum and were identified as isobergapten (1), bergapten (2), pimpinellin (3), isopimpinellin (4), columbianetin acetate (5), sphondin (6), qianhucoumarin E (7), xanthotoxin (8), osthole (9), ferulic acid (10), β-sitosterol (11), and psoralen (12). Conclusion: Compounds 1, 4, and 5 are isolated from the plants in Peucedanum L. for the first time, and compounds 2, 3, 6-10, and 12 are firstly isolated from P. decursivum.