Abstract: Orthogonal design was used to optimize the technique of extracting Linarin from wild chrysanthemum by matrix solid-phase extraction method. The operation process was adding silica gel-alumina-pollen into the PTFE core chromatography column firstly, then polarity eluting with solution combination of hexane-ethyl acetate-acetone, and finally determining the linarin content by High Performance Liquid Chromatography. The results showed that the optimum condition of extracting linarin from chrysanthemum inflorescence was extracting 1.5 h at 30℃ with an elution solution ratio of 1∶2∶3.

Key words: Inflorescences of wild chrysanthemum; Linarin; Orthogonal design; HPLC

Wild chrysanthemum (Flos Chrysanthemi Indici) has anti-inflammatory and antioxidant effects, such as resistance to pathogenic microorganisms, treatment of cardiovascular diseases, antihypertensive activity, reducing platelet aggregation etc. In addition, it is also used to treat hypertension and hyperlipidemia, and has certain preventive effect on cold and epidemic meningitis.

Linarin is one of the most active flavonoid compounds, which consists of robinin and an aglycone. Linarin is also known as robinoside, acaciin, wild chrysanthemum flower flavone glycosides, robinoside 7-O-β-D-rutinoside and so on, in the form of two isomers of compounds α and β. Linarin and its derivatives are widely distributed in the plant kingdom and exist in the flowers, leaves and fruits of many plants, mostly in the form of glycosides. Among them, the content of wild chrysanthemum inflorescence is the most, which is an important pharmacodynamic component in wild chrysanthemum. In recent years, studies at home and abroad have shown that Linarin has a very wide range of physiological and pharmacological activities, it has a significant effect on the prevention and improvement of diabetes, and it also has the effects of inhibiting phosphodiesterase, aldose reductase, Staphylococcus aureus, beta hemolytic streptococcus, and antibacterial, which can protect liver, and has a certain effect on chronic bronchitis.

1. Materials and methods

1.1 Materials

1.1.1 The dried inflorescences of wild chrysanthemum used in the experiment were purchased from the TCM pharmacy of Zhejiang TCM Hospital. Put it into a mortar and grind it into powder, then put it into a beaker and seal it with plastic wrap, and set it aside.

1.1.2 Reagents Linarin, n-hexane, ethyl acetate, acetone, methanol, glacial acetic acid, alumina, silica gel, etc.

1.1.3 Instrument YP202N electronic balance (Shanghai Precision Scientific Instrument Co., Ltd); PTFE core chromatography column (Shanghai Liyatai Glass Instrument Manufacturing Co.,Ltd. Hangzhou Branch); Waters AcQuity High Performance Liquid Chromatography (Waters Corporation of the United States); UV 2501 PC ultraviolet -Visible spectrophotometer (Shimadzu); beaker, mortar, funnel, glass rod, pipette, volumetric flask, desiccator, etc.

1.2 Method

1.2.1 Extraction of Linarin. Precisely weigh 1.2g of wild chrysanthemum inflorescence powder, 2.4g of silica gel and 2.4g of alumina, add 20mL of deionized water, stir well, and put it into the PTFE core chromatography column fixed on the iron stand. Add n-hexane, ethyl acetate and acetone at intervals of 15minutes to polarity elute from small to large. Leach the eluate to obtain a yellow filtrate, and filter it again with filter paper to obtain a clear yellow filtrate, that is the Linarin extract solution, store it in a dark place for later use. 

1.2.2 Single factor test. Precisely weigh 1.2g of wild chrysanthemum inflorescence powder, and carry out single factor test under different extraction time, extraction temperature and eluent volume ratio (V n-hexane: V ethyl acetate: V acetone). Then leach the eluate to obtain a yellow filtrate, and filter it again with filter paper to obtain the Linarin extract solution, fill the volume to 50mL with deionized water. Measure the absorbance of Linarin at 326nm, and calculate the corresponding content of Linarin.  

1.2.3 Orthogonal optimization test. Accurately weigh 1.2g of dry wild chrysanthemum inflorescence powder and use different extraction time, extraction temperature, eluent volume ratio as the investigation factors, according to the results of the single factor test, and then select

1.2.4 Detection of high performance liquid chromatography [9] Chromatographic column: Symmetry-C18 column (3.9mm×150.0mm, 5μm); Mobile Phase: methanol-water-glacial acetic acid (52:46:2); Flow Rate: 1.0mL/min; Detection Wavelength: 326nm; Column Temperature: 30°C; Injection Mode: manual injection; Injection Volume: 5μL.

Dissolve 1.2mg of the standard Linarin in methanol, and dilute to volume in a 50mL volumetric flask to obtain Linarin standard solution. Take 2mL from it and transfer it to a small beaker for the liquid phase as standard 1. Again take 2mL each into 4 test tubes and dilute to 2, 3, 4 and 5 times respectively, and transfer to a small beaker to be marked as standard 2, 3, 4 and 5. Take 2mL of each of the constant volume extracts into 50mL volumetric flask, dilute to the mark with 30% ethanol. After 15min, use a blank standard solution as a control, determine the absorbance at 326nm, and calculate the content of Linarin in the extract solution.

2. Results and Analysi

2.1 Detection by High Performance Liquid Chromatography

2.1.1 Selection of Detection Wavelength. Take the standard sample of Linarin, and make a solution with methanol to scan the spectrum at 200-400nm. The spectrogram shows that it has large absorption peaks near 207, 267, and 326nm. The peak area at 326nm is the largest. In order to reduce the interference of other components on the determination of Linarin, 326nm was selected as the detection wavelength.

2.1.2 Investigation of the linear relationship. Take 5μL samples from standard 1, standard 2 and standard 3 respectively, and determine them according to the conditions described in 1.2.4. The results showed that Linarin had a good linear relationship with the peak area in the range of 0.048~2.130μL. Taking the peak area (y) as the ordinate, and the concentration of Linarin (x) as the abscissa, draw a standard curve, and the regression equation of the curve = 1 980x-7.560, r=0.9999. 

2.1.3 Precision test. Precisely draw 5μL of the reference solution and inject 6 times continuously, and the CV of the peak area of Linarin is 0.78%. 

2.1.4 Repeatability test. Take about 2.4mg of sample powder, prepare 6 test solutions according to the preparation method and testing conditions of the above-mentioned test solution, and the CV of the test result of the content of Linarin is 1.30%. 

2.1.5 Stability test. Prepare the reference solution and the test solution respectively, place them at room temperature, inject at 0, 2, 4, 6, 8, and 12 hours for measurement, record the peak area of the chromatographic peak area of Linarin, and calculate the variation coefficient, the results show that the CV of the chromatographic peak area of the reference solution is 0.48%, and the CV of the chromatographic peak area of the test solution is 0.81%, it indicates that the reference solution and the test solution are stable within 12h.

2.1.6 The recovery test is carried out by the added sample recovery. Take 0.12g of wild chrysanthemum powder with known content, total 6 parts, and accurately weigh them. According to the ratio of the added amount of reference substance and the content of the test substance as 1:1, add the reference substance of Linarin to prepare the test solution, measure respectively, and calculate the recovery rate. The results show that the average recovery rate of Linarin is 97.4%, and the CV is 2.42%.

2.1.7 Chromatographic analysis. According to the spectrum of the reference substance, it can be seen that the retention time is about 1.89 min, and it can be concluded that this is Linarin, that is, the third absorption peak is Linarin’s absorption peak.
 

2.2 Single factor test

2.2.1 The effect of extraction time on the extraction amount of Linarin. Extract Linarin at room temperature with different extraction times, and determine the content of Linarin in the extraction solution, the results showed that the content of Linarin of wild chrysanthemum increased with the extension of extraction time, but after extraction of 1.5h, the content of Linarin began to decrease, so the extraction effect was the best when the extraction time was 1.5h. 

2.2.2 The effect of extraction temperature on the extraction amount of Linarin. At different temperatures, after extracting for 1.5h with the eluent volume ratio of 1:1:1, determine the content of Linarin in the extract solution, and it can be seen that the content of Linarin in the wild chrysanthemum is the highest when the extraction temperature is 25℃, so the appropriate extraction temperature is 25℃.

2.2.3 The effect of eluent volume ratio on the extraction amount of Linarin. Extract for 1.5h with different volume ratio of elution reagent at 25°C, and determine the content of Linarin in the extract solution. The results showed that under the same other conditions, from an economic point of view, the extraction under eluent volume ratio of 1:2:3 is appropriate.

3. Conclusion

Using a new type of matrix solid-phase dispersion extraction technology to extract the natural active ingredient Linarin in the dried inflorescence of wild chrysanthemum, by comparing different factors and levels of extraction conditions of the samples, and using orthogonal experimental design for multi-factor comprehensive analysis, the optimal experimental scheme of extraction time, extraction temperature and eluent volume ratio was optimized, and a practical, simple, convenient, and easy-to-operate technology for the matrix solid-phase dispersion extraction of Linarin was established. The optimal conditions of extraction technology are: extraction time 1.5h, extraction temperature 30℃, solution ratio 1:2:3.