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Objective:To investigate the effect of Asarinin on the survival time of transplanted heart after allogeneic heterotopic heart transplantation and to further verify the anti-immune rejection effect of Asarinin in spleen and peripheral blood.Methods:Using 64 Wistar rats as donors, 64 SD rats as recipients to establish the allogeneic heterotopic heart transplantation model in rats.After successful transplantation, 64 rats were use simple randomization divided into control group, cyclosporine A(CsA) group, Asarinin group and half CsA + half Asarinin group with 16 rats in each group.CsA group was given 5 mg/kg by gavage; Asarinin group was given 25 mg/kg; half dose group was given CsA 2.5 mg/kg+ Asarinin 12.5 mg/kg and the control group was given the same volume of normal saline by gavage.After administration for 1 week, half of them were used to observe the survival time.The other half of the rats were fully anesthetized with chloral hydrate, spleen and peripheral blood were taken.Half of the spleen was taken to observe the slices under the microscope.The other half of spleen was used RT-PCR to detect the relative expression of IFN-γ and IL-4.The expression of co-stimulatory molecules CD80, CD86 and CD40 in peripheral blood were detected by flow cytometry.Results:Survival time of transplanted heart was control group (8.4±0.9), CsA group (30.5±8.3), Asarinin group (16.5±4.3) and half-dose group (26.1±5.2) days.Compared with control group, survival time of heart transplantation became prolonged in all groups and the difference was statistically significant ( P<0.05). HE staining of splenic tissue showed that, as compared with control group, the injury of each group was alleviated.The relative expression of IFN-γ in spleen was control group (1.055±0.083), CsA group (0.396±0.038), Asarinin group (0.833±0.094) and half-dose group (0.862±0.104). The last three groups were lower than control group and the difference was statistically significant ( P<0.05). The relative expression of IL-4 in spleen was control group (1.429±0.234), CsA group (3.808±0.729), Asarinin group (2.209±0.306) and half-dose group (2.323±0.321). The last three groups all spiked as compared with control group and the difference was statistically significant ( P<0.05). The expressions of CD80, CD86 and CD40 in peripheral blood were control group (98.21±0.54), (85.78±0.89) and (96.36±0.66), CsA group (89.26±0.36), (56.86±2.32) and (88.11±1.61), Asarinin group (94.19±0.47), (79.01±1.12) and (87.86±1.67) and half-dose group (94.87±0.74), (80.81±0.98) and (89.71±0.97) respectively.The last three groups were lower than control group and the difference was statistically significant ( P<0.05). Conclusions:Asarinin can prolong the survival time of transplanted heart after allogeneic heterotopic heart transplantation in rats, inhibit the immune injury of spleen after allogeneic heterotopic heart transplantation in rats, decrease IFN-γ in spleen, increase IL-4 in spleen and inhibit the expression of peripheral blood costimulatory molecules CD80, CD86 and CD40.
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Objective: Active ingredients of 47 Asarum samples from different habitats with different phenotypes were analyzed in this paper in order to reveal the relationship between the formation of active ingredients and genetics and geography, and provide some scientific basis for the breeding and untilizing of Asarum. Methods: Ethanol extract and volatile oil were extracted according to the methods of Chinese Pharmacopoeia. Asarinin was detected by HPLC. Volatile oil compositon was determined by GC-MS. The between-groups linkage clustering method was carried out. Results: Volatile oil content of 47 samples was 0.81%-3.32%, and five samples’ volatile oil content exceeded 3.0 %. Ethanol extract content of samples was 9.87%-29.40%, and ethanol extract content of 30 resources accounting for 63.8 percent of all samples exceeded 20%. Asarinin content of 26 resources was more than 0.25%. Totally 48-77 compounds from the volatile oil could be determined and relative content of each compound was different. Forty-seven samples from different habitats with different phenotypes were divided to ten groups by between-groups linkage clustering method according to the active ingredients. All the accessions could be classified clearly. The cluster of accessions was associated with resource region. Asarum heterotropoides var. mandshuricum and Asarum sieboldii var. seoulense from the same origin were grouped together. Conclusion: There were much more differences in active ingredients content among Asarum resources which were in different regions cultivated with same way and environment with different phenotypes. We could select the perfect fundamental material according to the phenotypes and introduce germplasm from different regions for breeding and utilizing of Asarum. Most of samples clustered together associated with their origins instead of phenotype or specie.
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OBJECTIVEP: To investigate ultrasonic-assisted estraction(UAE) and response surface methodology(RSM) for the extraction of asarinin from Asari Radix et Rhizoma(ARR). METHODS: The RSM was based on a three-level, four-variable Box-Behnken design (BBD). The independent variables were ultrasonic time, liquid to solid ratio, ultrasonic temperature, and ultrasonic power, the dependent variable was extraction rate of asarinin, which was used to estimate the relationship between independent and dependent variables. Box-Behnken design and RSM were used to optimize the process of extraction. The prediction was carried out through comparing the observed and predicted values. Antioxidant activity of the extract of ARR was determined by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH) and 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate(ABTS) radical scavenging assays in vitro, and good correlation between extraction rate of asarinin and antioxidant activity was observed. RESULTS: The results indicated that ultrasonic time, liquid to solid ratio, ultrasonic temperature, and ultrasonic power had a significant effect on extraction rate of asarinin. Overall process intensification was achieved with ultrasonic time of 56 min, liquid to solid ratio of 17:1 mL•g-1, ultrasonic temperature of 52℃, and ultrasonic power of 180 W by UAE method. Under optimal conditions, the yield of asarinin was (1.55±0.32) mg•g-1 (n=3), which was in accordance with the predicted yield of 1.58 mg•g-1. The IC50 values of the extract of ARR sample were 29.701 and 64.643 mg•mL-1, respectively. The antioxidant results indicate that the extract of ARR has excellent ability to scavenge free radicals and antioxidant capacity and is expected to be used as a natural antioxidant in industrial applications. CONCLUSION: The extraction technology is simple, reliable and highly predictive.The UAE method is effective for extraction of asarinin from ARR.
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Objective: To analyze the change rule of main chemical components in Asarum heterotropoidesvar.mandshuricum seedling during the growing process.Method:Whole seedling samples (one week and two weeks) and the mature plant (three months) of A.heterotropoidesvar.mandshuricum were collected and every sample was divided to aerial part (stems and leaves) and underground part (roots).The secondary metabolites were qualitatively identified by HPLC-TOF-MS and the quantitative identification was carried out at the same time with asarinin as index component.Result: A total of 6 known compounds were identified from the underground part of A.heterotropoidesvar.mandshuricum as α-asarone (1),N-isobutyl-2,4,8,10-dodecatetraenamide (2),9-methoxyaristolactam Ⅳ(3),asarinin (4),caulesnarinside (6) and chalcononaringenin 2',4'-di-O-β-D-glucopyranoside (7),respectively,the peak area values showed that the contents of these compounds increased gradually with the growth time.A total of 4 known compounds were identified from the aerial part of this herb as N-isobutyl-2,4,8,10-dodecatetraenamide (2),caulesauroneside (5),caulesnarinside (6) or chalcononaringenin 2',4'-di-O-β-D-glucopyranoside (7) and peonidin 3-caffeoylgentiobioside (8).Asarinin was identified only in the underground part of mature plant,its content was 155.4 μg·g-1.Conclusion: The species and contents of secondary metabolites are quite different in the aerial and underground parts of A.heterotropoidesvar.mandshuricum.At different growth stages of A.heterotropoides var.mandshuricum seedling,the types and contents of secondary metabolites in the same site are also different,while the contents of main components show an increasing trend with the growth time.
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Objective:To study the transdermal absorption in vitro of sinapine thiocyanate,tetrahydropalmatine and asarinin in Xiaochuan ointment so as to offer reference for its clinical application.Methods:A Franz diffusion cells method with isolated rat skins was carried out to study the percutaneous rate of sinapine thiocyanate,tetrahydropalmatine and asarinin determined by LC-MS/MS.Results:With the increase of administration dosage,the cumulative penetration of sinapine thiocyanate,tetrahydropalmatine and asarinin showed few changes.The results showed that the transdermal behavior of sinapine thiocyanate fit to a Higuchi equation,and that of tetrahydropalmatine and asarinin fit zero-order process.The penetration rate of tetrahydropalmatine and asarinin respectively was 0.362×10-1 and 0.330×10-2 μg·cm-2·h-1.Conclusion:Xiaochuan ointment exhibits transdermal penetration and absorption properties,which provides evidence for its transdermal administration.
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Objective: To establish an HPLC method for the simultaneous determination of catalpol, liquiritin, ammonium glycyrrhizinate, asarinin, verbascoside, atractylodin, ferulic acid, imperatorin, notopterol, isoimperatorin, baicalin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside in Jiuwei Qianghuo Oral Liquid (JQOL). Methods: The analysis was performed on Zorbax Eclipse XDB-C18 column (250 mm×4.6 mm, 5 μm) by gradient elution of acetonitrile-0.005 mol/L KH2PO4 (adjusting to pH 3.0 with phosphoric acid) (15:85). The flow rate was 1.0 mL/min. Results: The linear ranges of catalpol, liquiritin, ammonium glycyrrhizinate, asarinin, verbascoside, atractylodin, ferulic acid, imperatorin, notopterol, isoimperatorin, baicalin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside were 2.08-31.22 μg/mL (r=0.9995), 4.01-60.15 μg/mL (r=0.9992), 10.09-151.31 μg/mL (r=0.9992), 4.98-74.63 μg/mL (r=0.9994), 2.05-30.74 μg/mL (r=0.9996), 4.10-61.46 μg/mL (r=0.9996), 2.93-43.98 μg/mL (r=0.9993), 2.04-30.66 μg/mL (r=0.9995), 12.54-181.55 μg/mL (r=0.9997), 53.95-89.23 μg/mL (r=0.9995), 12.05-180.68 μg/mL (r=0.9995), 5.97-89.51 μg/mL (r=0.9994), and 7.99-119.82 μg/mL (r=0.9996). The average recoveries (n=6) were 98.8% (RSD=1.9%), 98.6% (RSD=1.8%), 101.2% (RSD=1.5%), 99.4% (RSD=0.8%), 100.1% (RSD=0.6%), 99.7% (RSD=0.9%), 98.9% (RSD=1.2%), 99.4% (RSD=2.0%), 100.5% (RSD=1.6%), 98.7% (RSD=0.8%), 101.2% (RSD=1.4%), 98.3% (RSD=1.5%), and 99.1% (RSD=1.7%), respectively. The contents of nine batches of the catalpol, liquiritin, ammonium glycyrrhizinate, asarinin, verbascoside, atractylodin, ferulic acid, imperatorin, notopterol, isoimperatorin, baicalin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside were 0.229-0.259 mg/L, 1.231-1.260 mg/L, 0.849-0.877 mg/L, 0.357-0.371 mg/L, 0.149-0.169 mg/L, 0.941-0.967 mg/L, 0.529-0.547 mg/L, 0.269-0.294 mg/L, 1.039-1.067 mg/L, 0.043-0.064 mg/L, 3.631-3.649 mg/L, 0.157-0.183 mg/L, and 0.068-0.084 mg/L. Conclusion: This method is simple and rapid, and can be used for the quality control of JQOL with satisfactory separation and repeatability.
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OBJECTIVE:To determinate the release rate and in vitro transdermal rate of asarinin in Cancer pain cataplasm. METHODS:Using homemade devices and modified France diffusion,isolated skin of rats as barrier,normal saline as solvent,the content of asarinin was determined by HPLC. Release rate of Cancer pain cataplasm within 20,50,80 and 120 min and transder-mal amount within 2,4,8,12,24 h were investigated,and accumulative release rate and accumulative transdermal rate were cal-culated. RESULTS:Accumulative release rate by 120 min of asarinin in Cancer pain cataplasm was 73.01%;24 h in vitro transder-mal rate was 26.01%,and transdermal kinetics equation of asarinin was Q=5.717 7t1/2-0.385 4(r=0.979). CONCLUSIONS:Cancer pain cataplasm has good release and transdermal performance. Its transdermal kinetics is in line with Higuchi equation.
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Objective:To simultaneously determine the contents of asarinin, prim-O-glucosylcimifugin and 5-O-methylvisammio-side in Xinqin granules. Methods:An HPLC method was used. The determination was performed on a ZORBAX Eclipse XDB-C18 col-umn(150 mm ×4.6mm,5 μm) with the mobile phase consisting of menthol (A)-water (B) with gradient elution. The flow rate was 1. 0 ml·min-1 . The column temperature was 30℃. The detection wavelength was set at 254 nm from 0 to 30 min and 287nm from 30 to 55 min. The injection volume was 10μl. Results:The linear range of prim-O-glucosylcimifugin, asarinin and 5-O-methylvisammio-side was 10.210-163.400 μg·ml-1(r=0.999 7),10.160-162.600 μg·ml-1(r=0.999 8) and 5.015-80.240 μg·ml-1(r=0. 999 8), respectively. The average recovery was 100. 30%(RSD=1. 6%, n=6),101. 53%(RSD=1. 1%,n=6) and 101. 12%(RSD=1. 2%, n=6), respectively. Conclusion: The method is simple and accurate, which can be used in the quality control of Xinqin granules.