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This study aims to establish a method for the determination of the concentration of five main components of phthalide target areas of Chaxiong(CPTA) and its inclusion of β-CD in the plasma of rats, and determine the pharmacokinetic parameters, absolute bioavailability and relative bioavailability of CPTA/β-CD inclusion compound in vivo. The plasma concentrations of senkyunolide A, N-butylphthalide, new osthol lactone, Z-ligustilide and butenyl phthalide were determined with UPLC-MS/MS. The content determination was conducted at the chromatographic conditions as follows: Shim-pack GIST C_(18)-AQ HP column(2.1 mm×100 mm, 3 μm), mobile phase of 0.1% formic acid solution(A)-acetonitrile(B), gradient elution, flow rate of 0.3 mL·min~(-1), column temperature of 35 ℃ and injection volume of 2 μL. The mass spectra were obtained with electrospray ion source(ESI), positive ion mode and multi reaction monitoring. CPTA/β-CD inclusion compound was prepared by grinding method, DAS 2.0 software was used to model the data, and the absolute bioavailability of CPTA and relative bioavailability of inclusion compound were calculated. Finally, the methods for the determination of five components of senkyunolide A, N-butylphthalide, new osthol lactone, Z-ligustilide and butenyl phthalide in CPTA, were successfully established. The linear relationship among the five components was good within their respective ranges, r>0.99. The absolute bioavailability of the five components in rats was 22.30%, 16.32%, 21.90%, 10.16% and 12.43%, respectively. After CPTA/β-CD inclusion was prepared, the relative bioavailability of the five components was 138.69%, 198.39%, 218.01%, 224.54% and 363.55%, respectively, significantly improved. This method is rapid, accurate and sensitive, so it is suitable for the pharmacokinetic study of extracts in traditional Chinese medicine and their preparations.
Subject(s)
Animals , Rats , Benzofurans , Chromatography, High Pressure Liquid , Chromatography, Liquid , Rats, Sprague-Dawley , Reproducibility of Results , Tandem Mass SpectrometryABSTRACT
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.
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OBJECTIVE： To prepare insoluble anti-tumor drug-loading polymer micelles， and to increase inhibitive effect of insoluble anti-tumor drug. METHODS： Chitosan （CSO） and stearic acid （SA） were used to prepare blank micelles （CSO-SA）， then modified with mPEG and folic acid （FA） to prepare PEG-CSO-SA and FA-PEG-CSO-SA. Characteristic functional groups of CSO-SA， PEG-CSO-SA and FA-PEG-CSO-SA were detected by infrared spectroscopy. The morphology of micelles was observed by transmission electron microscopy. The particle size and Zeta potential of micelles were measured by laser particle size analyzer. Osthole （OST） was used as the model drug and drug-loading micelles （FA-PEG-CSO-SA/OST） were prepared by dialysis. MTT assay was used to detect the inhibitory rate of FA-PEG-CSO-SA， OST solution and FA-PEG-CSO-SA/OST to human liver cancer cell HepG2. Half inhibitory concentration （IC50） was calculated. RESULTS： FA-PEG-CSO-SA was successfully prepared. CSO-SA， PEG-CSO-SA， FA-PEG-CSO-SA were oval in shape； particle sizes were （96.01±5.99）， （112.93±1.06）， （216.01±4.76） nm （n＝3） and Zeta potentials were （39.30±1.75）， （38.03±2.91）， （15.17±2.10） mV （n＝3）， respectively. Encapsulation efficiency and drug-loading amount of OST in FA-PEG-CSO-SA were （84.47±2.07）％ and （16.01±0.90）％ （n＝3）， respectively. The inhibition rates of FA-PEG-CSO-SA to HepG2 cells were＜20％. IC50 of OST solution and FA-PEG-CSO-SA/OST to HepG2 cells were （62.08±5.21）， （27.49±0.50） μg/mL （n＝3）， respectively. CONCLUSIONS： Prepared FA-PEG-CSO-SA can significantly increase inhibitive effect of insoluble drug OST to HepG2 cells， and it is expected to become a new anti-tumor drug carrier.
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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 main factors affecting the preparation of osthol ( Ost ) loaded solid lipid nanoparticies ( SLN ) . Methods The SLN were prepared by melt-homogenization method. The optimum formulation and process were selected by orthogonai design. The shape, particle size, loading capacity were investigated. Results The obtained Ost-loaded SLN were sphere or oval at a range of 100-200 nm, and were well distributed without adhesion, the loading capacity was 59. 78%. Conclusion The melt-homogenization method is available for the preparation of Ost loaded SLN.
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Objective To study the process of hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion techniques for osthol. Methods The inclusion complex of osthol and HP-β-CD was prepared by unsaturated water solution and freeze-drying technique. Inclusion techniques were selected by screening on quadratic orthogonal rotation combination design method, and the entrapment efficiency was identified by HPLC. Results The optimal technical conditions were as follows:the ratio of HP-β-CD and drug was 4.5∶1;temperature for electric mixer was 35 ℃;the stirring time for thermostatic waterbath was 210 min. Conclusion This method is reasonable and it may have a prosperous future of development and application.
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Objective To establish the determination method for osthol in Compound Shechuang Tincture. Methods Hypersil ODS column was used and methanol-water (70∶30) was used as a mobile phase. The flow rate was at 1.0 mL/min and the detection wave-length was at 322 nm. Results Osthol had a good linear relationship between 0.018～0.126 ?g (r =0.999 9). The average recovery was 98.55% and RSD was 1.45%. Conclusion This method is simple, rapid and accurate. It can be applied to determining osthol in Compound Shechuang Tincture.
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Aim To investigate the effects of osthol on bone marrow stromal stem cells in vitro under the con-ditions of the ability to differentiate into osteoblasts and the case of proliferation. Methods The rat bone marrow sample was obtained,and the all bone marrow cell culture methods were used to separate and collect the stratum of mononuclear cells. The cells were cultured in DMEM containing 10% fetal bovine serum. Three days later the culture medium was changed for the first time. Nine days later,serial subcultivation proceeded. The final concentration of osthol was 1 ? 10 -4,1 ? 10 -5,1 ? 10 -6,1 ? 10 -7 mol?L -1 respectively. MTT method was adopted in proliferation analysis. Under the induced condition,the Alkaline phosphatase activity, calcium salt sediment yield and osteocalcin were meas-ured on 4th,8th,12th,16th day. On the fifteenth day,his-tochemistry dyeing proceeded for calcified tubercle. Total RNA was isolated and the gene expression of bFGF, IGF-1,Osterix and Runx-2 was investigated by RT-Real Time PCR. Results The BMSCs proliferation was refrained by osthol dose-dependently. But it evidently led to osteogenesis. The ALP activity,calcium salt sediment yield and osteocalcin were raised,and calcified tubercle amount was increased. Besides,it also could enhance the mRNA level of bFGF,IGF-1,Osterix and Runx-2. Conclusion The osthol with final concentra-tion of 1 ? 10 -5 mol?L -1 can markedly promote BM-SCs differentiation to osteogenesis. which proves osthol is an active constituent of the traditional Chinese medi-cine Common Cnidium Fruit.
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OBJECTIVE:To evaluate the efficiency and safety of Hydroalcoholic gel of osthol for subacute eczema. METHODS:By a randomized,parallel controlled clinical trial,a total of 243 patients with subacute eczema were assigned to either group A(n=116) or group B(n=127).The patients in group A were applied locally with Hydroalcoholic gel of osthol in the morning and evening for 2 weeks,and those in group B with Hydrocortisone butyrate cream in the morning and evening for two weeks.The total scores for the target sites and adverse reactions were evaluated after the completion of the 2- week treatment.RESULTS:There were no significant differences between the two groups in effective rate(84.48%for group A vs. 81.10%for group B),however,the itching- relieving efficacy in group A was significantly better than in group B(P
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Object To estabish a method for the quantitative analysis of osthol in LOTIO FRUCTUS CNIDII COMPOSITA by RP HPLC Methods Megestrol acetate was used as the internal standard Results Chromatographic separation of osthol and megestrol acetate was accomplished within 12 min on Hypersil ODS 2(200 mm? 4 0 mm, 5 ?m) column and acetonitrile 0 01 mol/L sodium dihydrogen phosphate (48∶52) as mobile phase The flow rate was 1 2 mL/min and the detection wavelength was 320 nm A good linearity was obtained in the range of 0 123 8～2 970 ?g/mL (r=0 999 6) for osthol and the average method recovery was (98 4?0 90) % with RSD=0 91% Conclusion The method was simple, rapid, accurate, reliable, and suitable for the quality control of LOTIO FRUCTUS CNIDII COMPOSITA
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Objective To obtain the optimal solid dispersion prescription of the extract of Fructus Cnidii.Methods The release rate t63.2 was used as index and uniform design was used to investigate the effect of PEG,PVP,and Poloxamer on the release of the solid dispersion of the extract of Fructus Cnidii.Results PVP,PEG can delay the release of osthole from the solid dispersion,and tween-80 can promote its release.Conclusion The optimal solid dispersion prescription consists of tween-80(10.2 %),Poloxamer(79.8 %) and the extract(10 %).
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AIM: To establish the quality standard for Duhuo Jisheng Mixture (Radix Angelicae Pubescentis, Herba Taxilli, Cortex Fraxini, etc.) METHODS: Herbs in Duhuo Jisheng Mixture were identified by TLC, and osthol content was determined by TLCS. RESULTS: Rhizoma Chuanxiong; Radix Angelicae Sinensis; Radix Saposhnikoviae; Radix Gentianae Macrophyllae; Cortex Eucommiae; Herba Asari; Radix Paeoniae Alba could be determined. Osthol showed a good linear relationship at a range of 0.466 - 3.600 ?g,r= 0.999 2 . The average recovery was 95.4% . CONCLUSION:The methods are available with a reproducibility and can control the quality of this mixture.