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Objective: To compare the differences in pharmacokinetic behavior of six ingredients in Qikui Sustained-release Tablets in rabbit plasma. Qikui Granules was taken as reference. Methods: Diazepam was used as internal standard. LC-MS/MS detection methods of astragaloside, hyperin, isoquercitrin, rutin, morroniside, and loganin in rabbit plasma were established, and pharmacokinetic parameters of six components were calculated. Results: Six active ingredients’ equation of linear regressions were: astragaloside Y = 1.0 × 10-4 X - 0.009 9 (r = 0.999 7), morroniside Y = 1.0 × 10-4 X + 0.038 7 (r = 0.999 4), loganin Y = 3.0 × 10-5 X + 0.008 7 (r = 0.999 3), hyperin Y = 1.0 × 10-3 X - 0.016 1 (r = 0.999 0), rutin Y = 5.0 × 10-4 X - 0.011 5 (r = 0.999 4), isoquercitrin Y = 1.7 × 10-3X - 0.307 5(r = 0.999 2). Intra-day and inter-day precision and accuracy and recovery rate were up to the mustard. After Qikui Sustained-release Tablets and Qikui Granules being given by gavege, the maximal concentration (Cmax) of morroniside, loganin, astragaloside, rutin, hyperin, and isoquerctirin in Qikui Granules were (1.333 ± 0.051), (1.238 ± 0.164), (0.83 ± 0.079), (0.127 ± 0.017),(0.444 ± 0.048), and (0.223 ± 0.048) mg/L, t1/2 were (3.848 ± 0.311), (3.822 ± 0.757), (4.982 ± 1.14), (3.73 ± 0.298), (4.732 ± 0.642), and (5.132 ± 0.901) h, respectively, AUC(0-t) were (3.069 ± 0.307), (2.891 ± 0.943), (2.079 ± 0.306), (0.313 ± 0.068), (1.087 ± 0.177), (0.496 ± 0.129) mg∙h/L, respectively, Cmax of morroniside, loganin, astragaloside, rutin, hyperin, and isoquerctirin in Qikui Sustained-release Tablets were (0.985 ± 0.13), (0.961 ± 0.175), (0.693 ± 0.101), (0.094 ± 0.012), (0.354 ± 0.045), (0.201 ± 0.037) mg/L, t1/2 were (4.691 ± 0.337), (5.62 ± 1.64), (6.408 ± 0.707), (4.103 ± 0.341), (6.048 ± 0.882), (5.803 ± 0.59) h, AUC(0-t) were (5.191 ± 1.046), (6.168 ± 1.25), (4.293 ± 0.823), (0.485 ± 0.103), (1.84 ± 0.432), (0.924 ± 0.19) mg∙h/L. Contrast with Qikui Granules, relative bioavailability of morroniside, loganin, astragaloside, rutin, hyperin, and isoquerctirin in Qikui Sustained-release Tablets were 169.1%, 213.3%, 206.5%, 156.0%, 169.3%, and 186.3%, respectively. Conclusion: Qikui Sustained-release Tablets can significantly improve the bioavailability of each active ingredient in rabbit.
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Objective: To establish multiwavelength HPLC fingerprint of Aurea helianthus from different batches, and combine quantitative analysis, similarity evaluation, cluster analysis, and principal component analysis to evaluate the quality of A. helianthus. Methods: The chromatographic column was Phenomenex Kinetex C18 (250 mm × 4.6 mm, 5 μm). The mobile phase was composed of 0.08% phosphoric acid water (A) and acetonitrile (B) in gradient elution at a flow rate of 1.0 mL/min, the detection wavelength was set at 260 nm for protocatechuic acid during 0-8 min, 324 nm for caffeic acid during 8-15 min, 360 nm for rutin, hyperin, isoquercitrin, gossypetin-8-O-β-D-glucuronide, myricetin, quercetin-3’-O-glucoside, and quercetin during 15-60 min. The column temperature was set at 30 oC. And the HPLC fingerprint of A. helianthus was established by the similarity evaluation system for chromatographic fingerprint of TCM (Version 2004A) and SPSS19.0, which was used for similarity evaluation, cluster analysis, and principal component analysis. Results: A total of 25 common peaks were confirmed of A. helianthus HPLC fingerprint, and nine peaks were identified which were determined. The similarity of 16 batches of samples was between 0.879 and 0.983; The results of cluster analysis showed that A. helianthus was clustered into two groups, indicating that there were differences in the similarity; Ranked the quality of A. helianthus based on the main component composite score. Conclusion: The method is simple and accurate, which can be used for the comprehensive quality evaluation research of the medicinal materials of A. helianthus.
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OBJECTIVE： To establish the quality standard for Bushen quyu granules. METHODS： TLC was used for qualitative identification of Rosa laevigata， Cuscuta chinensis， processed Fallopia multiflora and Lithospermum erythrorhizon in Bushen quyu granules. And then， the content of total polysaccharides in Bushen quyu granules was determined by UV spectrophotometry. HPLC method was used for the content determination of rutin， quercetin and hyperin in Bushen quyu granules. The determination was performed on BDS C18 column with mobile phase consisted of acetonitrile-0.08％ phosphoric acid solution （gradient elution） at the flow rate of 1 mL/min. The column temperature was 30 ℃， and detection wavelength was set at 370 nm. The sample size was 10 μL. RESULTS： TLC test sample chromatogram of 4 medicinal materials showed the same spot or fluorescence at the corresponding position with the reference substance and control medicinal materials. The linear range of glucose， rutin， quercetin and hyperin were 0.003-0.018 mg/mL， 0.225-7.20 μg/mL， 0.07-2.24 μg/mL and 1.25-39.88 μg/mL（r＝0.999 5 or 0.999 9， n＝6）. RSDs of precision， stability and reproducibility tests were all less than 3％ （n＝6）. Average recoveries were 102.2％， 101.2％， 100.9％， 101.0％ （RSD＝1.28％， 2.93％， 2.41％， 1.59％， n＝6）. Average contents were 0.46 g/g， 5.48 μg/g， 8.18 μg/g and 102.88 μg/g（n＝3）. CONCLUSIONS： Established quality standard of Bushen quyu granules is accurate and reliable， and can provide scientific reference for quality control of Bushen quyu granules.
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Objective To analyze the accumulation patterns of active constituents in Farfarae Flos with different flower bud colors (yellow, purple, and deep purple) at different growth stages, and provide theoretical guidance for the production and quality control of Farfarae Flos. Methods The medicinal materials of Farfarae Flos of different growth stages with different colors were determinated by HPLC method. The Similarity Evaluation System for Chromatographic Fingerprint of TCM (2012 A edition) was used to evaluate the similarity of the samples. The differences among samples were identified by chemical pattern recognition methods including hierarchical principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA). Results The HPLC fingerprint of different flower bud colors of Farfarae Flos at different growth stages was obtained, 27 common peaks were found in the chromatography, and 11 of them were identified. Similarities of samples of all batches with reference fingerprint were among 0.901-0.995. There were differences in the accumulation characteristics of Farfarae Flos at different growth stages according to the peak area, and showing significant differences among different flower bud colors. PCA and PLS-DA results demonstrated obvious distinction among different flower bud colors. Twelve constituents, such as gallic acid, chlorogenic acid, rutin, hyperin, isochlorogenic acid B and quercetin, were screened as biomarkers, representing major differences among colors. The quality evaluation demonstrated that deep purple buds was the best, followed by purple buds and yellow buds for the worst. Conclusion The HPLC fingerprint can reflect the accumulation characteristics of the active constituents of Farfarae Flos in different growth stages and the differences among different flower bud colors. Combining chemical pattern recognition can provide reference for the production and quality evaluation of Farfarae Flos.
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Objective To study the preparation method and analytical technique of hyperoside from Flos Abelmoschus manihot.Methods Hyperoside was isolated and purified by solvent extract and chromatography, whose structure was determined by 1H-NMR and 13C-NMR. The purity was analyzed by TLC and HPLC.Results The TLC showed that the hyperoside had no impurity spot. The HPLC indicated that the purity reached more than 98.5%.Conclusions The mothod of isolation and purification for hyperoside reported in this paper was simple and economical.
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Objective To establish a quantitative analysis of multi-component with a single-marker (QAMS) method for the quality control of Wuzi Yanzong Pills (WYP). Methods Six main effective components (schisandrin, hyperin, quercitrin, kaempferol 3-O-rutinoside, deoxyschizandrin, and γ-schizandrin) of WYP were simultaneously separated on a reversed-phase column (Ultimate LP-C18) with high-resolution of each chromatographic peak by high performance liquid chromatography (HPLC). Schisandrin was selected as the internal reference, and the relative correlation factors (RCFs) of other five components were calculated to achieve QAMS. The ruggedness of RCFs was tested on different instruments and columns. Moreover, results of the QAMS were compared with the external standard method. Results Within a certain linear range, the RCFs of hyperin, quercitrin, kaempferol 3-rutinoside, deoxyschizandrin, and γ-schizandrin were 0.36, 4.86, 0.88, 7.34, and 6.35, respectively. The repeatability was good under different experimental conditions. There were no significant differences between the calculated value and estimated value on QAMS and external standard method. Conclusion The QAMS method can be used to assay the content of six components of WYP simultaneously and control the quality of WYP simplely, reliably, and accurately.
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Objective:To develop a method of quantitative analysis of multi-components by single marker ( QAMS) for five index components in Yinzhihuang oral liquid. Methods:Using baicalin as the reference,an HPLC method with a Venusil MP C18 (2) column (250 mm × 4. 6 mm,5 μm) was applied,the mobile phase was acetonitrile-0. 1% phosphoric acid with gradient elution,the flow rate was 1. 0 ml·min-1 ,the detection wavelength was 327 nm, the column temperature was 35℃ and the injection volume was 10μl. The relative correction factors among the five index components were detected by QAMS. The five index contents were determined respec-tively by an external standard method and QAMS, and the results obtained from the two different methods were compared to verify the practicability and stability of QAMS. Results: The established QAMS method was used to determine the five index components in 6 batches of Yinzhihuang oral liquid. There were no significant differences in the calculated values and the determined ones (P>0. 05). Conclusion:The QAMS method is simple,effective and accurate in determining the contents of five index components in Yinzhihuang oral liquid,which can be used for the quality control of Yinzhihuang oral liquid and provide reference for the further studies.
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Objective:To develop a method of quantitative analysis of multi-components by single marker ( QAMS) for five index components in Yinzhihuang oral liquid. Methods:Using baicalin as the reference,an HPLC method with a Venusil MP C18 (2) column (250 mm × 4. 6 mm,5 μm) was applied,the mobile phase was acetonitrile-0. 1% phosphoric acid with gradient elution,the flow rate was 1. 0 ml·min-1 ,the detection wavelength was 327 nm, the column temperature was 35℃ and the injection volume was 10μl. The relative correction factors among the five index components were detected by QAMS. The five index contents were determined respec-tively by an external standard method and QAMS, and the results obtained from the two different methods were compared to verify the practicability and stability of QAMS. Results: The established QAMS method was used to determine the five index components in 6 batches of Yinzhihuang oral liquid. There were no significant differences in the calculated values and the determined ones (P>0. 05). Conclusion:The QAMS method is simple,effective and accurate in determining the contents of five index components in Yinzhihuang oral liquid,which can be used for the quality control of Yinzhihuang oral liquid and provide reference for the further studies.
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Objective To establish an HPLC method for determination of 2,3,5,4’-tetrahydroxystibene-2-O-β-D-glucoside(SBGC)and hyperin in the effective fraction of Jiangzhining. Methods HPLC analysis was performed on a C18 column(4.6 mm×250 mm,5 μm),with acetonitrile-0.1% methanoic acid water solution(15.5: 84.5)serving as the mobile phase. The flow rate was 1.0 mL·min-1 ,the wavelength was 340 nm,and the injection volume was 20 μL. Results The calibration curve was linear for SBGC in the range of 24-120 μg·mL-1(r = 0.999 8)and for hyperin in the range of 2.4-12.0 μg·mL-1(r= 0.999 6),respectively.Their average recoveries were 100.07% and 100.14%,respectively.The contents of SBGC and hyperin were 2.26% and 0.23%,respectively Conclusion The method is convenient,precise and reliable for determination of the content of SBGC and hyperin in the effective fraction of Jiangzhining.
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Objective To establish a Goto-Kakizaki ( GK) rat model of duodenal-jejunal bypass( DJB) and ob-serve the changes in insulin-resistance after surgery, and to explore the mechanism of DJB surgery in treatment of type 2 di-abetes mellitus.Methods Male Goto-kakizaki diabetic rats(GK,n=36)were used as experiment group and 18 healthy male Wistar rats as blank group.GK rats were randomly divided into two groups: diabetic control group and DJB surgery group ( n=18) .Euglycemic-hyperinsulinemic clamp technique was performed in 6 rats randomly taken from each group at third week, sixth week and ninth week after surgery, respectively.The expression levels of Gck, G6P, PEPCK mRNA in the liver and GLUT4 content on skeletal muscle cell plasma membrance were detected one week after the clamp test. Results In the DJB surgery group at the end of third week and sixth week after surgery, the levels of glucose infusion rates and the expression levels of Gck, G6P, PEPCK mRNA in the liver showed no statistically significant difference as com-pared with the diabetic control group (P>0.05).In the DJB surgery group at the end of 9th week after surgery, the glu-cose infusion rate and expression level of Gck mRNA in the liver were significantly higher, and the expression of G6P and PEPCK mRNA was significantly lower than those in the diabetic control group ( P0.05 for all) .Conclusions Our results indicate that the mechanism of DJB surgery improving blood glucose level may be closely related to the amel-ioration of insulin-resistance in the liver, thereby augmenting glucose uptake and inhibiting gluconeogenesis in liver through the regulation of glucose metabolism-related enzymes.There is no significant improvement in insulin-resistance in the skele-tal muscles during the experiment period.This result implies that the effect of DJB surgery on type 2 diabetes is related to the duration of therapy.
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Objective: To establish an HPLC method for the simultaneous determination of the main flavonoids in the leaves of Apocynum venetum and Poacynum hendersonii, and to reveal the differences of flavonoids in Apocyni Veneti Herba located between Xinjiang and other areas in China. Methods: The leaves of the two kinds of Apocyni Veneti Herba were extracted with 60% ethanol by reflux extraction. The separation was carried out on an Agilent TC-C18 column (150 mm × 4.6 mm, 5 μm) eluted with the mobile phases of acetonitrile-0.1% phosphoric acid (16:84). The column temperature was 40°C, and the flow rate was 1.0 mL/min, the detection wavelength was 360 nm, and the column temperature was 40°C. Results: The linear ranges of rutin, hyperin, and isoquercitrin were 4.96-49.60 (r = 0.9992), 0.96-9.60 (r = 0.9990) and 10.24-102.40 μg/mL (r = 0.9970), respectively; and the average recoveries (n = 5) were 98.2% (RSD = 1.5%), 92.3% (RSD = 3.2%), and 100.1% (RSD = 2.7%), respectively. Conclusion: This method is simple, rapid, accurate, and reproducible, and the results show that the flavonoids in Apocyni Veneti Herba located in Xinjiang are remarkably different from those in the other areas in China. In the leaves of A. venetum located in Xinjiang, the lutin content is high, about 0.55%, close to the isoquercetin content, but the hypercetin content is very low, only about 0.07%, and in the leaves of P. hendersonii, the contents of both lutin and hypercetin are very small. Such study will offer the credible method for the quality control of A. venetum and P. hendersonii.
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Antihyperglycemic potential of hyperin at 25 and 50 mg/kg doses for 30 days to streptozotocin induced diabetic rats has been reported. In oral glucose tolerance test, hyperin treated rats showed a significant reduction in blood glucose level after 120 min. It was found that hyperin exhibited dose dependent and significant antihyperglycemic activity in streptozotocin induced diabetic rats which were nearly similar with standard drug glybenclamide. Activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, glycogen phosphorylase, glycosylated haemoglobin and level of serum urea and creatinine were significantly decreased in hyperin supplemented diabetic rats, dose dependently. Activities of hexokinase and glycogen synthase were increased with augmentation in liver glycogen, insulin and haemoglobin content in hyperin treated diabetic rats. General hematological parameters did not show any significant change in hyperin treated diabetic rats hence it is safe at these doses. Histopathological studies showed significant morphological changes in pancreatic β-cells of streptozotocin induced diabetic rats. A decreased number of secretory granules of β- cells were observed in diabetic rats and these pathological abnormalities were normalized after treatment with hyperin and standard drug glybenclamide. Further, hyperin decreases significant in serum total cholesterol, triglyceride, low density lipoprotein, very low density lipoprotein levels coupled with elevation of high density lipoprotein in diabetic rats. These results suggest that hyperin has a pivotal role in blood glucose level in streptozotocin induced hyperglycemia by improving the function of pancreatic islets and increasing glycolysis and decreasing gluconeogenesis.
Subject(s)
Animals , Diabetes Mellitus, Experimental/drug therapy , Glucose Tolerance Test , Glyburide/pharmacology , Glycogen/metabolism , Hexokinase/metabolism , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Lipids/chemistry , Liver/metabolism , Male , Models, Chemical , Quercetin/analogs & derivatives , Quercetin/chemistry , Quercetin/metabolism , Quercetin/pharmacology , Rats , Rats, Wistar , Rhododendron/metabolismABSTRACT
Objective: To establish an HPLC method for the simultaneous determination of cafferic acid, forsythoside A, forsythoside B, rutin, hyperoside, forsythin, and arctigenin in Forsythia suspensa. Methods: The analysis was carried out on an Inertsil ODS-3 C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase was composed of acetonitrile and 0.2% phosphorie acid aqueous with gradient elution. The detection wavelength was set at 275 nm. The flow rate was 1.0 mL/min at column temperature of 30 °C. Results: Cafferic acid, forsythoside A, forsythoside B, rutin, hyperoside, forsythin, and arctigenin were well separated by this method, and showed a good linearity in the ranges of 18.24-91.20, 5.88-29.40, 132.60-663.00, 8.34-41.70, 1.96-9.80, 7.60-38.00, and 11.34-56.70 μg/mL, respectively. The average recoveries of the seven components were 97.7%, 96.7%, 102.6%, 101.3%, 93.2%, 91.8%, and 96.7% and the RSD values were 2.3%, 1.4%, 2.4%, 2.2%, 1.0%, 1.0%, and 1.3%, respectively. Conclusion: The established method is accurate, reliable, and could be used for the simultaneous determination of the seven components in F. suspense, which provides a scientific basis for the quality evaluation of F. suspense.
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Objective: To establish HPLC fingerprint for the analysis on Ziziphora tenuior and Z. clinopodioides and the determination of hyperin, rutine, and pulegone in eight batches of Z. tenuior and 22 batches of Z. clinopodioides synchronously. Methods: The HPLC method was used with Kromasil C18 column (250 mm × 4.6 mm, 5 μm), and a mixture of methanol-0.08% HCOOH was used as mobile phase with gradient elution. The HPLC fingerprint for eight batches of Z. tenuior and 22 batches of Z. clinopodioides was established. Results: The HPLC fingerprints of the two species were obviously different, so two modes were established for two species, respectively. The eight batches of Z. tenuior and 22 batches of Z. clinopodioides were classified into two groups based on the result of principal component, hierarchical cluster, and similarity analyses. Hyperin and pulegone were the common compositions; quercetin (161.06-213.22 μg/g) was founded in Z. tenuior, but not in Z. clinopodioides; the contents of hyperin (174.15-802.24 μg/g), pulegone (77.43-353.45 μg/g) of Z. clinopodioides were higher than those of Z. tenuior (49.72-204.33 and 36.19-93.29 μg/g). Conclusion: Z. tenuior and Z. clinopodioides have the different fingerprint, which could provide the evidence for their quality control, species identification, and classification.
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Objective: To establish a method for simultaneous determination of the contents of six main active constituents (neochlorogenic acid, chlorogenic acid, rutin, hyperin, isoquercitrin, and quercitrin) in Houttuyniae Herba by quantitative analysis of multi-components by single marker (QAMS). Methods: The chromatographic separation was achieved on a Kromasil C18 column (250 mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile-0.1% phosphoric acid aqueous with gradient elution and changeable wavelength detection. Chlorogenic acid was used as the reference, the calibration factor of other five constituents to that of chlorogenic acid was calculated respectively to get each relative calibration factor. And the contents of the active constituents were calculated by the relative calibration factor to realize QAMS. At the same time, the external standard method was used to detect the contents of the six constituents, comparing the two methods to validate the accuracy and feasibility of the new method. Results: No significant differences were found in the quantitative results of the six active constituents in Houttuyniae Herba determined by measured value and calculated value. The amounts of the six active constituents in the different parts of Houttuyniae Herba were found to be varied, chlorogenic acids and flavonoids mainly distributed in the leaves. Conclusion: The active constituents of Houttuyniae Herba mainly distribute in the leaves. The QAMS is feasible and accurate to evaluate the contents of the six active constituents in Houttuyniae Herba.
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Objective To study the protective effect of hyperin( Hyp) on the acute liver injury in rats induced by CCl4 . Methods The acute liver injury model was induced by CCl4 . The effect of Hyp on acute liver injury of rat liver histopathology was observed;and the impact of changes of homogenates total superoxide dismutase ( T-SOD) ,glutathione ( GSH) activity and malondialdehyde ( MDA) in liver were de-tected. Results The acute liver injury of tissue induced by CCl4,apparent pathological inflammation and fibrous tissue degeneration and necrosis were observed by HE staining;At the high doses of 60 mg/kg and medium doses of 30 mg/kg treated by Hyp,liver pathology changes was significantly obvious. The T-SOD,GSH activity of liver tissue was significantly increased in the groups of Hyp treatment,MDA content was significantly decreased,which related to the dosages. Conclusion In the rat model of acute liver injury induced by CCl4 . A better therapeu-tic effect of Hyp was shown,the mechanism may be related to its antioxidant activity.
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Aim To study the action mechanism of hyperin(Hyp) on neonatal rat's neuron with anoxia/reoxygenation(A/R).Methods The dissociated neonatal rat brain cells were subjected to 30 min of anoxia or followed 40 min of reoxygenation.Lactate dehydrogenase(LDH),malondialdehyde(MDA)and nitric oxide(NO)in the supernatant were measured.The intracellular free calcium concentration([Ca~(2+)]_i)in brain cells was assayed with Fura 2-AM method.Results Anoxia induced a significant increase of LDH in the supernatant from (62.0±13.0) U·L~(-1)(Sham group)to (116.0±16.6) U·L~(-1)(Control group,P<0.01),and reoxygenation markedly increased LDH and MDA in the supernatant from (45.6±9.2) U·L~(-1) and (9.1±0.9) μmol·L~(-1)(Sham group)to (106.0±17.4) U·L~(-1) and (16.4±2.7) μmol·L~(-1)(Control group,P<0.01),respectively.In the range of 1.0 ～ 16.0 μmol·L~(-1),Hyp markedly and concentration-dependently inhibited anoxia-or reoxygenation-evoked increases of LDH and MDA.1.0～16.0 μmol·L~(-1) Hyp not only inhibited anoxia-induced increase of NO in the supernatant and rise of [Ca~(2+)]_i in brain cells(P<0.05 or P<0.01),but also attenuated reoxygenation-evoked increases of NO and[Ca~(2+)]_i(P<0.05 or P<0.01),Hyp 16.0 μmol·L~(-1) significantly reduced NO and[Ca~(2+)]_i from (34.4±6.3) μmol·L~(-1) and (640±94) nmol·L~(-1) to (25.0±5.1) μmol·L~(-1) and (331±56) nmol·L~(-1),respectively.Conclusion The protective effect of Hyp on A/R-injured neurons may be related to the inhibition of overload of[Ca~(2+)]_i,NO release and lipid peroxidation.
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Object To develop a method for the determination of flavonoids in Hypericum perforatum L processed by different drying methods to provide a basis for the processing of the natural herbs Methods The chromatographic conditions were Discovery C 18 column (5 ?m, 4.6 mm ? 25 cm). detection wavelength 365 nm; mobile phrase : water : acetonitrile : phosphoric acid (825∶175∶1); flow rate 1.0 mL/min Results The contents of rutin and hyperin were at their maximum when dried at 60 ℃ for 4 h Rutin had a good linearity in the range of 0.107～2 675 ?g, average recovery rate 99 32%, RSD 1 007%, and hyperin in the range of 0 107～2 675 ?g, average recovery rate 99 54%, RSD 3 591% Conclusion Temperature is the main factor influencing the content of flavonoids The HPLC determination was shown to be rapid, reliable and simple, and may be used for the quality control of H perforatum
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Objective To establish a method for preparing the reference substances of hyperin and isorhamnetin- 3- O- galactoside. Methods Methanol- extract of Fructus Evodia was isolated and purified by combining solvent extraction with column chromatography and recrystallization. Hyperin and isorhamnetin- 3- O- galactoside were identified by mass spectrometry (MS), 1H nuclear magnetic resonance (1HNMR ) and 13C nuclear magnetic resonance (13CNMR) and their content was determined by HPLC. Results The purity was 99.6 % and 98.0 % for hyperin and isorhamnetin- 3- O- galactoside respectively. Conclusion This method is simple cancl effective to yield high- purity products. And these high- purity products can be used as reference substances for the research of herbal medicine.
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After icv administration, the analgesic action of Hyp was found on the tail-flick and hot-plate tests. The analgesic action potency of icv Hyp was approximately equal to 1 ~20 of morphine and was about 100 times as potent as sc administration. No effect was seen on the analgesic action of Hyp after ip naloxone, the analgesic action of Hyp was no concern of L-enkephalin contents in discrete regions of rat brain either. But icv EGTA markedly enhanced while icv CaCl2 antagonized the analgesic action of Hyp. It was also found that calcium ion content in mice brain markedly reduced when the analgesic action of Hyp appeared. These results suggest that Hyp have significant central analgesic action and the central analgesic action of Hyp may relate to reducing of calcium ion in brain.