Warm Kidney and Regulate Menstruation Granules is mainly composed of Angelica sinensis, Cortex Eucommiae, Cuscuta sinensis, Cistanchis sinensis, Cynomorium cuspidatum, and other medicines, and its prescription is from the clinical prescription, which has the efficacy of warming the kidneys and nourishing the blood, facilitating warming and resolving turbidity and eliminating stagnation and regulating menstruation, and it is used for the treatment of late menstruation, scanty menstruation, and even menstruation closure and impassability due to the insufficiency of kidney-yang, deficiency of the Chong Ren and the blood and the stagnation of the dampness and turbid stagnation with obvious effects. It is the only Chinese medicinal preparation in the field of polycystic ovary syndrome that has entered the clinical stage of research in China. In this study, we conducted qualitative and quantitative research on its quality standards, developed the qualitative identification of Cistanchis, Cuscuta and Cynanchum by thin-layer chromatography, and established a high performance liquid chromatographic method for the determination of ferulic acid in the preparation.
1. Instruments and reagents
Shimadzu SCL-10AVP liquid chromatograph***Japan***, SPD-10AVP detector, Class-VP workstation, KQ3200DE medical ultrasonic cleaner***Kunshan Ultrasonic Instrument Co. UP-Ⅱ-10T***Chengdu Ultrapure Technology Co.,Ltd***;1/100,000 and 1/100,000 Electronic Balance***Beijing Sartorius Instrument System Co. Warm Kidney and Regulate Menstruation Granules ***Homemade***;Mauricea Glycoside Control*** China Institute for Identification of Pharmaceutical and Biological Products, Batch No. 1530-200202***;Cuscuta Control*** China Institute for Identification of Pharmaceutical and Biological Products, Batch No. 21232-200401***;Cynanchum Control*** China Institute for Identification of Pharmaceutical and Biological Products, 121049-0301*** ; Ferulic acid control *** China Institute for Identification of Pharmaceutical and Biological Products, 100834-200701***; Silica G*** for thin-layer chromatography, Qingdao Ocean Chemical Factory***; methanol is chromatographically pure, water is homemade secondary redistilled water, and all other reagents are analytically pure.
2. Methods and results
2.1 Qualitative identification
2.1.1 Thin-layer chromatography identification of Cistanthus cistanches take 10g of this product, add 50ml of methanol, ultrasonic treatment for 30min, filtration, filtrate evaporation, residue with Anhydrous ethanol 1~2ml to dissolve, as a test solution. Take ergosterol control product, add ethanol to make a solution containing 2.5mg per 1ml. Take the sample without Cistanchis and make the negative control solution by the same method. According to the thin layer chromatography method *** "Chinese Pharmacopoeia" [1] 2010 version of a Appendix VI B *** test, absorb the above three solutions of 10μl, respectively, point in the same sodium carboxymethylcellulose as a binder on the silica gel G thin layer plate, ethyl acetate - methanol - 9% acetic acid *** 20:3:2 *** as the unfolding agent, unfolding, take out, drying, sprayed with 5% FeCl3 ethanol solution, and then heated at 105 ℃ until the spots show color. Heated at 105 ℃ until the spots show color clearly, the test chromatogram, in the corresponding position with the control chromatogram, show the same color spots, the negative control solution does not interfere.
2.1.2 Cuscuta thin layer chromatography identification take 5g of this product, add methanol 60ml, ultrasonic treatment for 30min, filtration, filtrate evaporation, residue with anhydrous ethanol to dissolve, filtration, filtrate concentrated to 1 ~ 2ml, as a test solution. Another take 1g of Cuscuta control herb, crushed, add methanol 20ml, ultrasonic treatment for 30min, filtration, filtrate evaporation, residue with anhydrous ethanol to dissolve, filtration, filtrate concentrated to 1~2ml, as the control herb solution. Then take the negative samples of Cuscuta chinensis to form the negative control solution by the same method. According to the thin-layer chromatography method *** "Chinese Pharmacopoeia" 2010 version of a Appendix VI B *** test, absorb the above three solutions of 10μl, respectively, point in the same sodium carboxymethylcellulose as a binder silica G thin-layer plate, with toluene - ethyl acetate - methanol ***5: 5:3 *** as the unfolding agent, unfolding, take out, air dry, and placed in the ultraviolet lamp *** 254nm*** under the examination, the test chromatogram in the negative samples of Cuscuta chinensis, in the negative samples in the same method into a negative control solution. In the chromatogram of the test material, in the position corresponding to the chromatogram of the control material, the fluorescent spots of the same color are shown, and the negative control solution is not interfered.
2.1.3 Thin-layer identification of cynomolgus take 10g of this product, add methanol 50 ml, ultrasonic treatment for 30min, filtration, filtrate evaporation, residue with chloroform to dissolve, filtration, filtrate concentrated to 1 ~ 2 ml, as a test solution. Take 1g of control herb of Cynanchum vulgare, crushed, add 10ml of methanol, ultrasonic treatment for 30min, the same method into a control herb solution. Then take the negative sample of Cynanchum vulgare to form a negative control solution in the same method. According to the thin-layer chromatography method *** "Chinese Pharmacopoeia" 2010 version of a Appendix VI B *** test, absorb the above three solutions of 10μl, respectively, point in the same sodium carboxymethylcellulose as a binder on the silicone G thin-layer plate, with petroleum ether *** 60 ~ 90 ℃ *** - acetone *** 4:1 *** for the unfolding agent, unfolding, take out, dry, and put in the ultraviolet lamp *** 254 nm ** under the examination, the test material for the test ** ***, and the test results are shown in the following table. ** under the ultraviolet lamp ***254nm **, the test chromatogram, in the corresponding position with the control chromatogram, the fluorescent spots of the same color, the negative control solution is not interfered with.
2.2 Determination of ferulic acid content
2.2.1 Chromatographic conditions Kromasil C18 column***250mm×4.6mm, 5μm***; mobile phase methanol-5% acetic acid***20:80***; flow rate of 1.0 ml/min; detection wavelength 323nm The column temperature was 35℃.
2.2.2 Preparation of control solution: Take appropriate amount of ferulic acid control, weigh it precisely, put it in a brown measuring flask, add methanol:5% acetic acid***1:4*** to make a solution containing 10 μg of ferulic acid per 1 ml, which is obtained.
2.2.3 Preparation of the test solution take the appropriate amount of the product, research fine, take 1.4g, weighing, placed in a stoppered conical flask, add methanol: 5% acetic acid ****1:4*** mixture of 25ml, weighing, ultrasonication **** power of 160W, frequency of 50kHz **** 40min, cooled, and then weigh, and the above solvents to supplement the loss of weight. Filtration, take the renewed filtrate through the microporous filter membrane ***0.45μm***, that is obtained.
2.2.4 Negative control solution preparation according to the prescription ratio weighing of herbs other than Angelica and Rhizoma Ligustici, according to the preparation process to make the lack of Angelica and Rhizoma Ligustici negative samples, according to the above method of preparation of the test solution made of negative control solution.
2.2.5 Exclusivity test: 20 μl of each of the control solution, test solution and negative control solution were sucked up precisely and injected into the liquid chromatograph, and the results showed that there was no interfering peaks in the relative retention times of the control and test solutions in the negative control solution, and the method was feasible for this study. Chromatographic peaks are shown in Figure 1.
2.2.6 Linearity range of the investigation of precision weighing of the appropriate amount of ferulic acid control, add methanol: 5% acetic acid ****1:4*** mixed solvent to make each 1ml containing 50μg of control solution, respectively, take up a precise amount of 0.5, 1.0, 1.5, 2.0, 4.0 ml of the solution to the 10 ml flask, dilute to the scale with the above solvent, shaking, and then inject 20μl into the liquid chromatography, respectively, and then injected into the liquid chromatograph. Then inject 20μl into the liquid chromatograph and measure according to the above chromatographic conditions. Calculate and regress the peak area against concentration, and get the regression equation Y=118234X+3026.98 r=0.9999, which showed that: ferulic acid showed good linear relationship in the concentration range of 2.50-20.00μg/ml.
2.2.7Precision test: The relative standard deviation (RSD) of peak area was calculated as 1.4%, which indicated that the precision of the method was good.
2.2.8 Repeatability test: 5 copies of the test solution were prepared and injected into the sample, 20μl each time, and the average mass fraction of ferulic acid was 0.1205mg/g, RSD 0.56%, which indicated that the method was reproducible.
2.2.9 Stability test on the same test solution every 3h into the sample 1 time, each time 20μl, ***4 times, according to the law, the results of ferulic acid peak area RSD 0.41%, indicating that the test solution is stable in 9h. A control; B negative control; C. test material
2.2.10 spiking recovery test take the known content of the sample powder 0.7g, precision weighing, precision addition of a certain amount of ferulic acid control, determined in accordance with the proposed method, calculate the recovery, the results are shown in Table 1. table 1 ferulic acid spiking recovery test***n=5***
2.2.11 sample assay The samples of different batches were taken 1.4g, weighed precisely, and the test solution was prepared according to the method of test solution preparation, 20μl of sample was injected, and the test content was calculated, and the results of ferulic acid content of 3 batches of warm kidney and regulate menstruation granules were 0.1205, 0.1221, and 0.1235 mg/g, respectively.
3. p>Cistanchis, Cuscuta sinensis and Cynanchum sativum are the main three herbs in this product. In this study, we referred to the literature[2],and established a TLC identification method for the above three herbs, and the results showed that the established method unfolded a systematic separation with good effect, clear spots, strong exclusivity, and negative and non-interfering. This product is a traditional Chinese medicine compound preparation, the composition is relatively complex, the prescription of Angelica sinensis is the monarch drug, so the active ingredient ferulic acid was selected as the quantitative detection index, and the HPLC content determination method was established, and the methodological study showed that the method has the characteristics of simplicity, sensitivity and good reproducibility, and can be used for the quality control method of this product.