In this study, the prescription of soybean isoflavone tablets was screened by orthogonal test, and the contents of daidzein and genistein, the main active components, were determined by HPLC.
1 Instruments and materials
1. 1 instrument Dionex high performance liquid system, Chlomelon chromatographic data workstation; YP- 1.5 tabletop electric continuous single punch tablet press produced by Nantong Huayu Pharmaceutical Machinery Co., Ltd.; ZRS-8G intelligent dissolution instrument produced by Tianjin Tianda Tianfa Technology Co., Ltd. ..
1.2 material daidzein and genistein reference substance, purchased from sigma company, the content is more than 99%; Soybean isoflavone extract, isoflavone purity >; 90%, purchased from Beijing Huaqing Meiheng Company; Sodium dodecyl sulfate (produced by Sinopharm Chemical Reagent Co., Ltd.); Lactose (produced by Huzhou Food and Chemical Company); Magnesium stearate (produced by Shanghai Pharmaceutical Accessories Factory).
2 methods and results
2. Prescription screening of1soybean isoflavone tablets
2. 1. 1 orthogonal test screening formula Based on the pre-test, lactose was used as filler, 10% starch slurry was used as binder, magnesium stearate was used as lubricant, and sodium dodecyl sulfate was used as disintegrant. Select 1000 tablets, lactose dosage, 10% starch slurry dosage, magnesium stearate dosage and sodium dodecyl sulfate dosage, and take three levels for each factor, and use orthogonal table L9(34) to optimize the prescription. Table 1 factor grade table
2. 1.2 preparation process: raw materials and auxiliary materials are respectively sieved with 100 mesh for later use. Weigh a specified amount of soybean isoflavone, lactose and sodium dodecyl sulfate, and mix them evenly. The soft material is made of an appropriate amount of adhesive, passes through a 16 mesh nylon screen, the wet particles are dried at 60℃ for 60 min, the dry particles pass through a 14 mesh nylon screen, the particles are sorted and mixed evenly, and then magnesium stearate is added. The hardness of tablets with a diameter of 8 mm and a tablet weight of 500 mg was controlled at (3.5 0.5) kg/mm2.
2. 1.3 See Table 2 for the experimental results and analytical results. Table 2 Orthogonal test and prescription screening results
The best formula is A3B3C2D3, that is, per kilogram of soybean isoflavone tablets, the dosage of lactose is 200 g, the dosage of 10% starch slurry is 20 ml, the dosage of sodium dodecyl sulfate is 12 g, and the dosage of magnesium stearate is10 g. Among the four factors, the dosage of lactose is factor A, followed by sodium dodecyl sulfate and stearic acid. C>D> B.
2.2 Content determination
2.2. 1 chromatographic column: Diamondsil TM C18 column (4.6 mm×200 mm, 5 microns); Mobile phase: the ratio of methanol to 0. 1% phosphoric acid is 42 ∶ 58; Column temperature: 30℃; Flow rate: 1.0 ml/min; Detection wavelength: 254 nm.
2.2.2 Preparation of reference solution Accurately weigh 65438±05mg daidzein and 20 mg genistein respectively, put them in the same volumetric flask, and adjust the volume to 50 ml with anhydrous ethanol to obtain the reference solution.
2.2.3 Preparation of test solution Take 3 pieces of soybean isoflavone tablets, accurately weigh about 0.6 g in a 100 ml volumetric flask after crushing, add 80 ml methanol for ultrasonic treatment for 30 min, after cooling, adjust the volume to scale with methanol, shake well, filter with a 0.45 micron microporous membrane, and take the continuous filtrate as the test solution.
2.2.4 investigation of linear relationship: inject 2, 4, 6, 8, 10, 12, 14, 16 μl daidzein and genistein into high performance liquid chromatograph, and take the sample volume (c) as the abscissa, the chromatographic peak area. At the same time, the regression equation Y=55.378X- 10.789, r=0.9996. The results show that the concentration of genistein is in the range of 8.0 ~ 64.0 μ g/ml, and the peak area has a good linear relationship with the concentration.
Figure 1 HPLC diagram of soybean isoflavone tablets
(a) chromatogram of reference substance, and (b) chromatogram of soybean isoflavone tablet; 1 is daidzein and 2 is genistein)
2.2.5 Precision test Take 30.0 μg/ml daidzein control solution and 40.0 μg/ml genistein control solution 10 μl, and inject samples for 6 times continuously. The relative standard deviations of daidzein and genistein peak areas are 0.86% and 0.78%, respectively.
2.2.6 In the stability test, the same concentration of test solution 10 μl was injected at 0, 2, 4, 8, 12 and 24 h respectively, and the RSD of daidzein and genistein peak areas were 1.64 1% and 0.836% respectively.
2.2.7 Repeatability test Take the same batch of samples, prepare 6 test solutions according to the method of 2.2.3, accurately measure 10 μl, and inject samples respectively. The RSD of daidzein and genistein are 1.03% and1.6% respectively.
2.2.8 recovery rate test: take 6 samples with known content and add 1.0 1 mg/ml daidzein reference solution 1 ml respectively, and take 6 samples with known content and add 1.85 mg/ml genistein reference solution/kloc-0 respectively. After injecting 10 μl in turn, the recovery rates of daidzein and genistein were calculated to be 98.3% and 97. 1% respectively, and the RSD was 1.45% and 1. 19% respectively.
2.2.9 Determination of finished product content: Weigh different batches of soybean isoflavone tablets about 0. 1 g, accurately weigh them, prepare the test solution according to the method of 2.3.3, and determine them according to the above chromatographic conditions. The results are shown in Table 3. Table 3 Determination Results of Finished Product Content
3 discussion
The optimum formula of soybean isoflavone tablets is as follows: per kilogram of soybean isoflavone tablets, the dosage of lactose is 200 g, the dosage of 10% starch slurry is 20 ml, the dosage of sodium dodecyl sulfate is 12 g, and the dosage of magnesium stearate is10 g. After large-scale production, the conditions are controllable, and the extruded tablets have smooth appearance, uniform color, rapid disintegration and taste.
The HPLC method established in this paper is simple, reproducible and can effectively control the quality of soybean isoflavone tablets.
Daidzein and genistein are the main active ingredients in soybean isoflavone tablets, which have obvious preventive and therapeutic effects on osteoporosis. Therefore, it is reasonable and feasible to take the content of daidzein and genistein as the quality control index of soybean isoflavone tablets.