Abstract: Rutin is a vitamin medicine, which can reduce the permeability and fragility of capillaries, and maintain and restore the normal elasticity of capillaries. It is used to prevent hypertensive cerebral hemorrhage; diabetic retinal hemorrhage and hemorrhagic purpura, etc. It is also used as a food antioxidant and pigment.
Objective To quantify the content of rutin in thistle by high performance liquid chromatography. Methods A LichrospherC18 chromatographic column (4.6mm × 200mm, 5μm), column temperature 30 ℃, methanol-0.5% acetic acid (volume ratio 35:65) was used as mobile phase, flow rate was 1.0mL / min, and detection wavelength was 254nm. Results The content of rutin in thistle was 0.5808mg / g. Conclusion After systematic methodological investigation, this method is simple, exclusive, stable and repeatable, and can be used for the determination of rutin in thistle.
[Keywords] thistle; rutin; content determination; high performance liquid chromatography
The small thistle is the dry ground part or root of the Compositae plant thistle [Cirsiumsetosum (Willd.) MB.]. It has the functions of cooling blood to stop bleeding, removing blood stasis and reducing swelling. , Trauma bleeding, carbuncle swollen sores and so on. The organic acids contained in thistle include rutin and caffeic acid. Rutin and caffeic acid are documented as hemostatic active ingredients. In this experiment, the content of rutin in thistle was determined by high performance liquid chromatography. The method is stable, rapid and reproducible.
1 Experimental materials
1.1 Instrument
LC-10AT high performance liquid chromatograph, SPD-10A detector, CLASS-LC10 chromatography workstation; sartoriusBP211D electronic balance; SHIMADZUAY220 electronic balance; KQ-500 type ultrasonic cleaner.
1.2 Reagents and drugs
Methanol is chromatographically pure, water is double distilled water, and other reagents are analytically pure. Rutin was purchased from China National Institute for the Control of Pharmaceutical and Biological Products (Lot No. 0080-9705). The small thistle medicinal materials were purchased from Jiangsu (Nanjing Pharmaceutical Co., Ltd., batch number 020313), Anhui (Bozhou Yonggang Pieces Co., Ltd., batch number 031205), and Shanghai (Shanghai Huayu Pharmaceutical Co., Ltd., batch number 040802). Professor Chen Jianwei of the Department of Medicinal Appraisal was identified as a genuine thistle [Cirsiumsetosum (Willd.) MB.].
1.3 Chromatographic conditions and system suitability test
1.3.1 Selection of chromatographic conditions
Chromatography column: LichrospherC18 (4.6mm × 200mm, 5μm), column temperature 30 ℃, mobile phase: methanol-0.5% acetic acid (volume ratio 35:65), flow rate 1.0mL / min; detection wavelength: 254nm. The high-performance liquid chromatogram of the reference substance and sample is shown in Figure 1.
1.3.2 Investigation of extraction solvent
Take 2 parts of this product, 1.0g each, weigh accurately, place in two 50mL conical flasks with stoppers, add 50mL methanol and 50mL40% methanol respectively, weigh, immerse for 1h, ultrasonic treatment (power 500W, frequency 50kHz) 40min, make up the weight, filter; microporous filter membrane (0.45μm) filter, you get. Pipette 10μL each of the above two solutions into high performance liquid chromatograph and calculate the peak area according to the external standard method. The results show that it is better to use 40% methanol as the extraction solvent.
2 Methods and results
2.1 Solution preparation
2.1.1 Preparation of reference solution
Precisely weigh 0.98mg of rutin reference substance, put it in a 25mL volumetric flask, add 40% methanol to dissolve and dilute to the mark, shake well, and you get it.
2.1.2 Preparation of test solution
Take 3 medicinal powder of origin (through No. 2 sieve) 1.0g each, weigh accurately, place in a conical flask with stopper, add 50mL40% methanol respectively, weigh, immerse for 1h, ultrasonic treatment (power 500w, frequency 50kHz) 40min, make up the weight, filter, filter with microporous membrane (0.45μm), then you get it.
2.2 Investigation of linear relationship
Accurately draw the reference solution 1, 2, 3, 4, 5mL and dilute 10mL, and then accurately draw 10μL of the diluted reference solution into the liquid chromatograph and measure the peak area value. The concentration of rutin (g / L) is On the abscissa, the integral value of the peak area is on the ordinate, and linear regression is performed. The regression equation is: Y = 25845255X-28716, r = 0.9996. The results show that rutin has a good linear relationship in the range of 0.00392 ～ 0.0196g / L.
2.3 Precision test
Draw 10μL of No. 3 reference solution, repeat the injection 5 times, and determine the integral value of the peak area, RSD = 1.503%. This shows that the precision of this method is better.
2.4 Stability test
Accurately draw 20μL of the same sample solution at 0, 1, 2, 4, 6, 12, and 24h to measure the peak area integral value, and calculate RSD = 1.972% within 6h. The results show that the test solution is basically stable at room temperature for 6 hours.
2.5 Repeatability test
Take a total of 5 samples of the same batch of small thistle, 1.0g each, weigh accurately, and prepare 5 copies in parallel according to the preparation method of the test sample solution, respectively inject 10μL, each measured twice, the average content is 0.7035mg / g , RSD = 1.129%.
2.6 Recovery rate test
Accurately weigh 6 small thistle samples, 1.0g each, add the appropriate amount of rutin reference substance accurately, and prepare according to the preparation method of the test sample solution, each injection is 10μL, each measurement is 2 times, the average value is calculated, and the addition is calculated. Sample recovery rate, the average sample recovery rate was 98.70%, RSD = 2.88%. See Table 1. Table 1 Rutin sample recovery rate determination results (omitted)
2.7 Sample determination
Take small thistle samples from 3 places of origin and prepare according to the preparation method of the test solution. Accurately draw the reference solution and the sample solution, inject into the liquid chromatograph, measure the peak area, and calculate according to the external standard method 0.5808mg / g. See Table 2. Table 2 Determination results of rutin content in thistle (omitted)
3 summary
For the determination of rutin content in thistle, some people have used the polyamide thin layer scanning method [1]. This method is not stable due to the surface smoothness, uniformity, thickness and other factors of the film surface. Determination of rutin content in tartary buckwheat. According to the literature, no one has ever used HPLC to determine the content of rutin in thistle. In this experiment, the rutin content was determined by HPLC method, which has the characteristics of simple and rapid, accurate and reliable results, and can be used as a quality control method for thistle medicinal materials and decoction pieces.
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