Intervention Research Of Phenylethyl Alcohol GlycosidesinCistanche On Perimenopausal Syndrome Animal Model Ⅱ
Apr 16, 2024
Experiment 1 Preparation of Cistanche phenylethanoid glycoside 1 Source of medicinal materials
Cistanche tubulosa, Chengdu Wecistanche biotech Ltd.d, batch number: 20130501.
2 reagents
95% ethanol (medical grade); AB-8 macroporous adsorption resin, Tianjin Guangfu Fine Chemical Research Institute, batch number: 20130618.
Echinacoside reference substance, provided by China Institute for the Control of Pharmaceutical and Biological Products, batch number: 111670-200503.3
HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK?
Preparation method
Reference was made to the literature method [19] and was conducted under the guidance of Professor Feng Suxiang from the Department of Drug Analysis of Henan College of Traditional Chinese Medicine. Grind Cistanche into a coarse powder, add 10 times the amount of 70% ethanol to reflux, and extract twice, 1.5 hours each time, combine the two alcohol extracts, concentrate under reduced pressure until there is no alcohol smell, and disperse with distilled water (the concentration is 0.5g medicinal material/ ml) as the sample solution. Pass through AB-8 resin (column drug: resin is 1:10), load the sample in a flow, and let it stand for 5 hours until it is fully adsorbed. First, wash with 10 times the column volume of distilled water, discard the water, and then use 10 times the column volume. Use 10% ethanol to remove impurities, and finally elute with 7 times the column volume of 60% ethanol. Collect the eluate and evaporate to dryness to obtain a powder. 4 Measurement wavelength selection
Precisely measure 0.5 mL of the reference solution, add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 minutes; add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes; add 10% sodium hydroxide solution 10 mL, dilute to 25 mL with water, shake well, and let stand for 18 min to serve as the reference solution. 0.5ml of Cistanche phenylethanol glycoside solution was prepared in the same way as the test solution. Use the parallel sample without adding reference substance and test the solution as blank, and prepare the blank solution in the same way. On the UV spectrophotometer, perform a full wavelength scan on the above three solutions in the wavelength range of 200-800nm. Both the reference substance and the test solution have maximum absorption peaks at 507nm, so 507nm is determined as the absorption wavelength.
After color development of Cistanche phenylethanol glycoside sample + color development of Echinacea tangerine reference substance (superimposed comparison), see Figure 1.
Figure 1 After color development of Cistanche phenylethanoid glycoside sample + after color development of Echinacea mandarin orange reference substance Figure 5 Content detection method
Precisely measure 1 mL of the solution to be tested, add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min; add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min; add 10% sodium hydroxide The solution is 10 mL, dilute to 25 mL with water, shake well, and let stand for 18 minutes. Use the parallel sample without adding sample solution as a blank, and prepare a blank solution in the same way. Measured using UV spectrophotometer at 507nm. 6 Methodological review
6.1 Preparation of reference solution and sample solution
Preparation of the reference solution: Accurately weigh 3.06 mg of the echinacoside reference standard that has been dried to constant weight, place it in a 25mL volumetric flask, add 70% solution to dissolve and dilute to the mark, shake well, and set aside. The standard concentration is 0.1224 mg/ml.
Preparation of sample solution: Precisely weigh 5 mg of Cistanche phenylethanoid glycoside extract into a 10 ml volumetric flask, dissolve it with 70% ethanol and dilute to volume.
6.2 Preparation of standard curve
Accurately draw 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 mL of reference solution, develop color and dilute to volume according to the method in 4.2, and measure the absorbance value at 507 nm. Using concentration versus absorbance to make a regression curve, the regression equation is: Y=20.296X-0.0015 (r=0.9994), and the linear range is 4.90-29.4μg/ml.
6.3 Precision test
Take the sample solution and measure it six times in parallel according to the "Content Determination Method" item. Calculate the relative standard deviation of the absorbance. The absorbances are 0.299, 0.299, 0.298, 0.297, 0.297, 0.297 respectively. The average absorbance = 0.298, RSD = 0.330% (n =6).
6.4 Stability test
Take an appropriate amount of the sample solution, and according to the "Content Determination Method" item, measure the absorbance values at 0, 15, 30, 50, and 60 minutes after the solution is completely colored, and calculate the relative standard deviation. The absorbances are 0.304, 0.303, 0.300, and 0.296 respectively. , 0.295, 0.295, average absorbance = 0.299, RSD = 1.362% (n = 5).
6.5 Repeatability test
Take a sample, prepare 6 sample solutions according to the "Preparation of Sample Solution" item, and measure the absorbance value according to the "Content Determination Method" item, and calculate the content. The contents are 65.2%, 65.6%, 66.7%, 67.2%, and 66.5% respectively. , 65.8%, average content 66.2%, RSD=1.142% (n=6).
6.6 Sample recovery test
Precisely draw 0.5 mL of the sample solution with known content, a total of 9 parts, and add 200, 250, and 300 μl of the standard solution respectively. Measure and calculate the recovery rate and relative standard deviation of Cistanche phenylethanol glycoside. The recovery rates are 101.45% and 99.26% respectively. , 98.94%, 101.83%, 99.05%, 99.47%, 98.72%, 99.17%, 100.64%, the average recovery rate is 99.84%, RSD=1.164% (n=9).
7 Sample content determination
Table 1 Determination results of phenylethanoid glycoside content in Cistanche
