Abstract: Rutin is a vitamin medicine, which can reduce the permeability and fragility of capillaries, and maintain and restore the normal elasticity of capillaries. For the prevention and treatment of hypertensive cerebral hemorrhage; diabetic retinal hemorrhage and hemorrhagic purpura.
Objective To establish a method for identification and content determination of rutin in trigonal supercritical CO2 extract. Methods Spotting on the polyamide membrane, using 80% ethanol as the developing agent to identify rutin, using SymmetryC18 column, using acetonitrile-tetrahydrofuran-0.2% citric acid (14: 1: 85) as mobile phase, column temperature 25 ℃ At a flow rate of 1.0 ml · min-1 and a detection wavelength of 360 nm, the rutin content was measured. Results The rutin was contained in the supercritical extract of the three ribs. The established liquid chromatography method has a good linear relationship in the range of rutin concentration of 0.00340 ～ 0.02432mg · ml-1, the sample is stable within 8h, and the average sample is added. The recovery rate was 98.56%, and the peak area was used for precision experiment. The RSD was 1.38%. Conclusion The supercritical extract of Trigonal contains rutin. The established high performance liquid chromatography method has a good linear relationship, high precision and high recovery rate, which is suitable for the determination of rutin in Trigonal.
Trigonal Sparganiumstoloniferum, originally published in "Compendium of Materia Medica", is a commonly used Chinese medicine. It is a dry tuber of the black trigonal plant of the Trigonaceae family. It mainly contains volatile oils and flavonoids [1 ~ 3]. It has anticoagulant and antithrombotic and analgesic properties. Role [4], clinically used to treat stroke, coronary heart disease, ectopic pregnancy, abscess around the appendix and chronic hepatitis, chronic pelvic inflammatory disease, etc. In addition, it is used with Curcuma to inhibit tumors [5], and plays a very important role in the formulation of anti-cancer Chinese medicine. At present, there are not many studies on the extraction of trigonometry in the literature, especially the basic research on its chemical composition and so on. The flavonoids reported in literature [6] are kaempferol and 5,7,3,5-tetrahydroxydihydrogen Flavonol-3-O-β-D-glucoside has not been reported in the literature with rutin in the three sides. Therefore, in this experiment, thin layer chromatography was used to identify the rutin in the three-sided supercritical carbon dioxide extract, and the content of rutin was determined by high performance liquid chromatography.
1 Instruments and materials
1.1 Instruments and reagents
Jiangsu Nantong Huaan Company's Ha120-50-01 supercritical extraction device; Shimadzu LC-10ATvp high-performance liquid chromatograph, Shimadzu ShimadzuCLASS-VPV6.12SP4 workstation; KQ-250B ultrasonic cleaner.
1.2 Reference and test products
Trigonal (Beijing Tongrentang Group); Rutin reference substance (China Institute of Biological Products); methanol (analytical purity); acetonitrile (chromatographically pure); tetrahydrofuran (chromatographically pure); citric acid (chromatographically pure); carbon dioxide (99.95%) ); The water used in this experiment is ultrapure water.
2 Methods and results
2.1 Thin layer identification of rutin in trigonal supercritical extract
2.1.1 Preparation of test solution
The trigonal crude drug was crushed, passed through a 40-mesh sieve, and dried at 60 ° C for 6h. Accurately weigh 1000g of sieve triangulated powder, add 180ml of entrainer, mix thoroughly overnight, and then place in an extraction kettle. The extraction kettle, analysis kettle I and analysis kettle II are heated separately, when the temperature reaches the set temperature respectively, the extraction kettle and the two analysis kettles are pressurized by a high-pressure pump, and when the pressure reaches the set pressure respectively, the static After the extraction, the cyclic extraction is started, and the temperature and pressure are kept constant, and the CO2 flow rate is 40L / h. After extraction for 2h, a yellow oily liquid was discharged from the outlets of the analytical kettles I and II. The extract was weighed, measured by volume, filtered, and the filtrate was transferred to a 100ml measuring flask, which was made up to volume with methanol and shaken to obtain the test solution.
2.1.2 Preparation of Rutin Reference Solution I
Precisely weigh 1.0mg of rutin reference substance, place it in a 50ml measuring flask, dissolve with methanol and dilute to the mark, shake well to make a test solution of 0.02mg · ml-1.
2.1.3 Thin layer identification
Pipette the above reference solution I and the test solution 20μl each with a quantitative capillary, spot on the polyamide membrane respectively, use 80% ethanol as the developing agent, unfold, dry, and use 1% aluminum trichloride ethanol solution Color development, inspection under the UV lamp, the test solution shows spots of the same color on the corresponding position of the chromatogram of the reference substance, indicating that the sample contains rutin.
2.2 Determination of rutin content in trigonal supercritical extract
The content of rutin in the samples was determined by high performance liquid chromatography.
2.2.1 Chromatographic conditions Chromatographic column: SymmetryC18 (250m × 4.6nm, 5μm); mobile phase: acetonitrile-tetrahydrofuran-0.2% citric acid (14: 1: 85), column temperature 25 ℃; detection wavelength: 360nm, flow rate is 1.0ml · min-1, 20μl injection (quantitative loop). Under this condition, the peak appears around 30 minutes, and the rutin and other substances in the sample are well separated.
2.2.2 The suitability experiment of the chromatographic system is calculated according to the rutin peak. The theoretical plate number of the chromatographic system is 57059; the resolution of rutin and its adjacent chromatographic peaks is 2.3 and 1.5, respectively, and that of rutin and the adjacent chromatographic peak Can be completely separated. The tailing factor of Rutin Peak is 1.07, and the peak shape symmetry is good.
2.2.3 Preparation of reference substance solution II Weigh 15.0mg of rutin reference substance precisely, place it in a 50ml measuring flask, dissolve with methanol and dilute to the mark, shake well to make a reference substance with a concentration of 0.3mg · ml-1 Solution II.
2.2.4 Linear relationship Precisely draw the reference solution II 0.1, 0.2, 0.4, 0.6, 0.8ml into a 10ml volumetric flask, dilute to the mark with methanol, and shake well. According to "2.2.1" chromatographic conditions. A standard curve is drawn with the peak area as the abscissa and the injection concentration as the ordinate, and the linear regression equation is obtained:
Y = -5.7380 × 10-5 6.2708 × 10-8X, r = 0.9999
Results: Rutin has a good linear relationship in the concentration range of 0.00304 to 0.02432 mg · ml-1.
2.2.5 Sample stability experiment The sample solution was placed at room temperature, and the sample was injected every 2 hours to determine the content of rutin, which was measured 5 times. The RSD was 1.87%, indicating that the sample solution was basically stable within 8 hours.
2.2.6 Precision experiment Take the sample solution of the same concentration and inject 6 times continuously to determine the content of rutin. The RSD is 1.38%.
2.2.7 Sample recovery rate experiment: Accurately draw 5 ml of test solution with known content of 5ml, put them in 10ml measuring flask, respectively, add 5ml of rutin control solution with concentration of 0.30mg · ml-1 accurately, shake Homogenize, filter, take the subsequent filtrate and inject separately, operate according to the selected chromatographic conditions, determine its concentration, and calculate the recovery rate.
2.2.8 Determination of samples Prepare the test solution according to the above "2.1.2" method, and measure according to the "2.2.1" chromatographic conditions, using standard linear equation to calculate the content of rutin in each test solution, and then Calculate the rutin content per gram of raw medicinal materials.
2.3 Determination of three-sided supercritical extraction samples The experiments were arranged according to the experimental conditions in Table 2. For the experimental method, see "2.1.1". Each experimental condition was repeated 3 times. The average result was taken and the content of rutin in the extract.
3 Discussion
TLC showed that rutin was contained in the three-sided supercritical extract.
Use UV spectrophotometry to scan the reference solution in the 200-500nm wavelength range, the reference substance has the maximum absorption at 360nm, and determine 360nm as the detection wavelength.
In the selection of HPLC mobile phase, the organic phases of methanol and acetonitrile were selected as mobile phases, respectively, and the two systems of ethanol-water-phosphoric acid and acetonitrile-tetrahydrofuran-0.2% citric acid were investigated at different ratios and different flow rates. The influence of the conditions on the determination of the sample, and finally determined that the mobile phase was acetonitrile-tetrahydrofuran-0.2% citric acid (14: 1: 85).
A SymmetryC18 (250mm × 4.6mm, 5μm) column was used, with acetonitrile-tetrahydrofuran-0.2% citric acid (14: 1: 85) as the mobile phase, at a flow rate of 1.0ml · min-1 and a detection wavelength of 360nm. The determination of rutin in trigonal supercritical extract was systematically studied. The established method has a good linear relationship in the range of rutin concentration of 0.00340 ～ 0.02432mg · ml-1, the sample is stable within 8h, and the average sample recovery rate is 98.56%. The method has high precision and is suitable for detection of three The content of rutin in the edge.
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