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Objective: To improve the quality control level of ethanol extraction process of Corydalis Rhizoma (CR) by using quality by design (QbD), so as to meet the extraction requirements of Kedaling Tablets. Methods: The critical process parameters were solvent multiple, extraction time and soaking time, and the critical quality attributes were dry extract rate, content and transfer rate of dehydrocorydaline. The response surface methodology was used to establish the mathematical model between the critical process parameters and the critical quality attributes. The overlapping design space of multiple index design spaces was established to select the optimal operation space, where the process verification was carried out. Results: The operating space parameters of ethanol extraction of CR was 14-24 h of the immersion time, 3.0-4.0 times of the first time solvent multiple, 1.5-2.0 times of the second and third time solvent multiple, and 1.5-2.5 h of the extraction time. Under this operating space, the extraction yield of CR was ranged from 6%-8%, the content of dehydrocorydaline was more than 2.8%, and the transfer rate was not less than 85%. Conclusion: The concept of QbD is helpful to improve the ethanol extraction process of CR, and to obtain a reliable and suitable extraction operation space for the production of Kedaling Tablets.
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Objective: Based on the color space technology of CIE-LAB,the color of vinegar-processed Corydalis Rhizoma decoction pieces was digitized,combining with the contents of 10 major alkaloids in the decoction pieces,to discuss the correlation between the color and contents of main ingredients of vinegar-processed Corydalis Rhizoma decoction pieces,and investigate the intrinsic quality difference in the decoction pieces with different color. Method: The precision colorimeter was used to determine the color parameters of vinegar-processed Corydalis Rhizoma decoction pieces;HPLC was employed to determine contents of main chemical components in the decoction pieces,which was performed on Agilent ZORBAX SB-C18 column(4.6 mm×250 mm,5 μm) with mobile phase of acetonitrile(A)-0.1% potassium dihydrogen phosphate aqueous solution(B) for gradient elution(0-10 min,5%-22%A;10-30 min,22%-25%A;30-50 min,25%-60%A;50-70 min,60%-95%A),detection wavelength of 280 nm,column temperature at 30℃ and flow rate of 1.0 mL·min-1. Result: The quality of vinegar-processed Corydalis Rhizoma decoction pieces with different color was in line with the requirement of the 2015 edition of Chinese Pharmacopoeia,but there were differences in the intrinsic quality between the decoction pieces.The total content of chemical components in the samples showed a positive correlation with the a*(green-red axis) and total chromatic aberration value(ΔE) in the CIE-LAB color space, and it was significantly negative correlated with L*(lightness) and b*(blue-yellow axis).In the 10 tested components,except for D-tetrahydrojatrorrhizine and tetrahydrocoptisine,contents of protopine and other 6 components were positively correlated with color,and only the content of corydaline was negatively correlated with color. Conclusion: Color analysis technology can objectively quantify the color of the decoction pieces,and can achieve a quick evaluation of quality of the decoction pieces by analyzing correlation between the color and the contents of main active ingredients.
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Objective To determine the technological conditions for the purification of the total alkaloid from the Corydalis Rhizoma (CR) by macroporous adsorption resin. Methods Total alkaloids of CR were determined by acid dye colorimetry, palmatine hydrochloride, dehydrocorydaline, tetrahydropalmatine, and corydaline were determined by HPLC. Six macroporous adsorption resins were investigated with the absorption rates, elution rates, and the content of the total alkaloid and four alkaloids of CR by static and dynamic adsorbing experiments. The purification process conditions of the total alkaloid of CR were optimized by the loading amount and volume flow of sample, the type and volume of the impurity removal of solvent and elution solvent and so on. The stability of the purification process was investigated by 5, 10 times enlargement. Results D141 type macroporous adsorption resin was the best choice for the purification of the total alkaloid from CR, the optimized parameters were as follows: Drug concentration was 0.6 g/mL of medicinal material and was added to the D141 macroporous resin column that the ratio of diameter to height range from 1:5 to 1:9 at a flow rate of 2 BV/h to 2 BV, 1.3 BV of purified water was used to remove impurities, and then 6 BV 95% ethanol was used as eluent at a flow rate of 2 BV/h. The purity of the total alkaloid of CR was up to 68.19% after purification, and the content of palmatine hydrochloride, dehydrocorydaline, tetrahydropalmatine, and corydaline was 1.95%, 11.74%, 4.93%, and 6.36%, respectively. The purity of the total alkaloid of the CR can reach more than 65% by 5 times and 10 times enlargement. Conclusion The purity of the total alkaloids can reach more than 65% after verification test, and the transfer rate of total alkaloids and four alkaloid monomers of CR can reach more than 85%, indicating that D141 macroporous adsorption resin can effectively purify total alkaloids from CR, and can be applied to industrial production.
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Objective To investigate the effects of dehydrocorydaline(DHC) on complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia in mice.Methods 40 mice were divided randomly into 4 groups (CFA test:experiment group =8,control group =8;locomotor activity and organ coefficient test:experiment group=12,control group =12).Subcutaneously injected CFA in the plantar of mice to establish pain model.The experimental group mice were injected with 10 mg/kg DHC while the control group mice received 10％ DMSO.The paw withdrawal mechanical threshold(PWMT) of mice was tested before and after administration of DHC.The effects of DHC on spontaneous activity and organ coefficient were observed in mice.Results The basic values of PWMT showed there were no statistically significant differences between experimental group and control group ((10.27± 1.34)g vs (10.28 ±0.35)g,P＞0.05).Compared with the control group,the values of PWMT in experimental group at 0.5 h,1 h,2 h,3 h after administration of DHC were significantly increased(0.5 h:(8.18±0.87) g vs (4.85±0.65) g;1 h:(7.85±0.59) g vs (4.84±0.54) g;2 h:(7.36±0.49) g vs (4.90±0.59) g;3 h:(6.66±0.45) g vs (5.00±0.36) g;all P＜0.01).Compared with the control group,no significant effect was observed on the number mice crossed grids and lifted forelimb and stood in 2 min in the experimental group (P＞ 0.05).And no significant effect was observed on the liver,kidney,spleen,heart,lung and brain organ coefficient in the experiment group (P＞0.05).Conclusion DHC can alleviate CFA-induced mechanical hyperalgesia in mice.
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Objective: To establish an HPLC method for simultaneous assay of seven main active constituents (protopine, coptisine, palmatine, dehydrocorydaline, D-tetrahydro jatrorrhizine, tetrahydropalmatine, and corydaline) in Corydalis Rhizoma, and on this basis, establishing a methodology of quantitative analysis on multi-components by single marker (QAMS) to validate the feasibility of method and technical adaptability of quality control applications for Corydalis Rhizoma. Methods: Taking seven components in Corydalis Rhizoma as indicators, two correction methods were used to establish the relative correction factor (fk/s) between each component and tetrahydropalmatine. Then the correction factor was used to calculate the amount of each component in Corydalis Rhizoma and finally to achieve this method. In the meantime, the external standard method was used to measure the above seven components to compare the difference between the calculated and measured value of the two fk/s, and to validate the correctness and adaptability of QAMS. Results: The methodology of QAMS which was used to evaluate the seven kinds of alkaloids in Corydalis Rhizoma was established; There was no significant difference between the data calculated by QAMS in different columns and instruments and the values measured by the external standard method. Conclusion: The QAMS method for measuring the components of protopine, coptisine, palmatine, dehydrocorydaline, D-tetrahydro jatrorrhizine, tetrahydropalmatine, and corydaline in Corydalis Rhizoma is reliable and accurate, it could be used to control the quality of crude drugs and herbal pieces of Corydalis Rhizoma.
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Objective: To establish HPLC method for the simultaneous determination of protopine, palmatine hydrochloride, berberine hydrochloride, dehydrocorydaline, tetrahydropalmatine, tetrahydroberberine, and corydaline in Cuyanhusuo Granule. Methods: The analysis was performed on Ultimate AQ-C18 column (250 mm×4.6 mm, 5 μm) by gradient elution of acetonitrile-0.1% phosphoric acid (adjust to pH 6.0 with triethylamine) (10:90). The UV detection wavelength was set at 280 nm and the flow rate was 1.0 mL/min. Results: The linear ranges of protopine, palmatine hydrochloride, berberine hydrochloride, dehydrocorydaline, tetrahydropalmatine, tetrahydroberberine, and corydaline were 6.8-119.0 (r=0.9999), 24.38-426.65 (r=0.9999), 8.88-155.40 (r=0.9999), 77.66-1359.05 (r=0.9999), 41.4-724.5 (r=0.9999), 6.70-117.25 (r=0.9999), and 25.50-446.25 ng (r=0.9999). The average recoveries (n=6) were 98.2% (RSD=2.0%), 99.6% (RSD=2.8%), 100.2% (RSD=1.3%), 99.0% (RSD=2.2%), 100.8% (RSD=2.6%), 98.7% (RSD=2.5%), and 97.7% (RSD=2.2%), respectively. Conclusion: This method is simple and rapid, and can be used for the quality control of Cuyanhusuo Granule with satisfactory separation and repeatability.
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Objective: To isolate dehydrocorydaline and glaucine by high-speed counter-current chromatography (HSCCC) from the extraction of Corydalis Rhizoma (CR). Methods: A mixture of chloroform-n-butanol-methanol-water (4:1:2:5) was used as the two phase solvent system both in forward and reversal direction, with a flow rate of 10.0 mL/min and a rotary speed of 800 r/min eluting for 30 min. The detection wavelength was 282 nm and injection volume was 50 mg. The purity of the target product was analyzed by HPLC-UV and the structure was identified by ultra performance liquid chromatography-tandem quadrupole time-of- flight mass spectrometry (UPLC-Q-TOF-MS/MS). Results: Under optimized conditions, 7.1 mg and 3.4 mg of two compounds were obtained and their yields were 81.43% and 91.11% respectively. Their purities were 98.9% and 94.3% detected by HPLC. dehydrocorydaline and glaucine were identifiled through HPLC, ultraviolet absorbance, and UPLC-Q-TOF-MS/MS. Conclusion: The result indicate that HSCCC is a powerful technique for the purification of dehydrocorydaline and glaucine from CR.
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Objective: To establish a method of HPLC for simultaneous determination of six ingredients in Yuanhu Zhitong Dripping Pills. Methods: Separation was performed on a Diamonsil C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase was acetonitrile (A)-0.04% ammonium acetate (acetic acid tone pH 4.0) (B), gradient elution: 0-10 min, 20% A, 10-25 min, 20%-25% A, 25-70 min, 25%-75% A; flow rate was 1 mL/min; column temperature was 35 ℃; detection wavelength was 280 nm; and injection volume was 10 μL. The contents of dl-tetrahydropalmatine, palmatine, dehydrocorydaline, d-corydaline, imperatorin, and isoimperation were determined. Results: The linear ranges were 14.94-149.40 μg/mL for dl-tetrahydropalmatine, 1.97-19.72 μg/mL for palmatine, 15.94-159.36 μg/mL for dehydrocorydaline, 9.72-97.20 μg/mL for d-corydaline, 2.82-28.17 μg/mL for imperatorin, and 0.61-6.06 μg/mL for isoimperation, respectively, with the correlation r > 0.999 0. The extraction recoveries varied from 95% to 105% (RSD varied from 1.82% to 2.99%). The contents of dl-tetrahydropalmatine, palmatine, dehydrocorydaline, d-corydaline, imperatorin, and isoimperation was 0.368-0.483, 0.058-0.082, 0.532-0.766, 0.315-0.459, 0.145-0.212, and 0.030-0.053 mg/g, respectively. Conclusion: The method is accurate and reliable, which can be used for the quality control of Yuanhu Zhitong Dripping Pills.
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Objective To observe the effects of dehydrocorydaline (DHC) on proliferation and collagen secretion of cardiac fibroblasts (CFs) cultured by high glucose;To provide experimental evidence for clinical application of Rhizoma Corydalis. Methods CFs cultured in vitro with high glucose were made into models. Collagenase and trypsin were used for the combine digestion of CFs from ratsbornin 24 h. 2-4 generation CFs were cultured by high glucose (25 mmol/L), and then 100, 50, 25 mg/L dethydrocorydaline was added for intervention. Cellular morphology of CFs was observed after 24, 48 h. CFs proliferation was detected by MTT method. Cell cycle was assessed via flow cytometry. The levels of collagen Ⅰ and collagen Ⅲ were determined by ELISA. Results CFs began to grow adherence 3 hours after planting, and CFs cultured by high glucose significantly proliferated 24, 48 h later (P<0.05, P<0.01). The percentage of S+G2+M phase CFs increased significantly after 48 h (P<0.01). The secretion of collagen Ⅰ and collagen Ⅲ also increased significantly (P<0.01). After the intervention of DHC, CFs proliferation was significantly inhibited (P<0.01);the percentage of S+G2+M phase CFs decreased (P<0.01);the secretion of collagen Ⅰ and collagen Ⅲ was reduced (P<0.05, P<0.01). Conclusion DHC can reduce CFs proliferation, decrease collagen secretion of CFs cultured by high glucose, and has potential effects of anti-myocardial fibrosis.
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Objective To explore the effect of different processes on the contents of active ingredients in Rhizoma Corydalis. Methods Processes of vinegar-boiling, vinegar-frying and alcohol-frying were used to fresh corydalis, boiled corydalis and purchased corydalis. Then the method of extraction by ultrasound with water and by reflux with methanol were used to extract the sample. The contents of Protopine, Tetrahydropalmatine and Dehydrocorydaline in corydalis was determined by RP-HPLC. Agilent TC-C18 (25 mm?4.6 mm, 5 ?m) column was used at 35 ℃, with acetonitrile-0.1% phosphonic acid (pH 5.3 with triethylamine) (28∶72) as mobile phase. The flow rate was 1 mL/min and detection wavelength was set at 280 nm. Results In the sample extracted by water, the contents of active ingredients in processed products were higher than in corydalis. And in the sample extracted by methanol, it also has the tendency that the contents of active ingredients in processed products were higher than in corydalis. The content of Dehydrocorydaline in fresh corydalis which was processed by vinegar-boiling, vinegar-frying and alcohol- frying was high. The content of Tetrahydropalmatine in purchased corydalis which was processed by water-boiling at production place was high. Conclusion The method of determining active ingredients in Rhizoma Corydalis was established. It was convenient and the result was accurate. The process technology that fresh corydalis was boiled by water and then processed or fresh corydalis was processed in production place can be used.