Abstract: Gentian contains the bitter taste components of cymene iridoid glycosides: gentiopicroside, medicinal bitter glycosides, medicinal glycosides, bitter gentioside glycosides, trace bitter medicinal ester glycosides; the total bitter glycoside content can be as high as 7.33 %, And the content of gentiopicroside can reach 6.34%.
Objective To establish a reversed-phase high performance liquid chromatography method for the simultaneous determination of the content of ascorbic acid, swertiamarin, gentiopicrin, swertiaside in gentian flowers. Methods ZORBAXSB-CM18 (250mm × 4.6mm, 5μm) column was used; the mobile phase methanol and water (containing 0.04% phosphoric acid) were eluted at a ratio of 15:85; the detection wavelength was 238nm; the flow rate was 1ml · min-1; the column temperature 30 ℃. Results The four components reached baseline separation. The linear ranges of ascorbic acid, swertiamarin, gentiopicroside, swertiamarin were 0.10 ～ 6.25μg (r = 0.9999), 0.076 ～ 3.5μg (r = 0.9998), 0.04 ～ 5.65μg (r = 0.9999) and 1.27 ～ 7.62μg (r = 0.9998); the average recovery rate is 99.2% (RSD = 2.6%, n = 5), 103.0% (RSD = 1.5%, n = 5), 101.0% (RSD = 1.1%, n = 5), 99.7% (RSD = 3.6%, n = 5). Conclusion The method is rapid, accurate and reliable.
The Tibetan medicine gentian flower is a flower of the Gentianaveitchiorum Hemsl, a plant of the gentian family Gentianaveitchiorum Hemsl. The Tibetan name is "Bangjian". Sore throat, pulmonary fever, cough, gastritis, meningitis, urethritis, tracheitis, pruritus, scrotal eczema, smallpox, etc. [1].
The gentian flower mainly contains active components of the iridoid glycosides, such as loganicacid, swertiamarin, gentiopicroside, sweroside, etc. [ 2]. Gentian flower is often used as a monarch or prince of Tibetan medicine, such as Longdanhuawan, Shiweiweilongdanwan, Sanweilongdanwan, etc. The only active ingredient in the determination of gentian flower and preparation is gentiopicroside [ 3], and the method of simultaneous determination of gentiopicroside of gentian flower and the above three active ingredients has not been reported. In this paper, high-performance liquid chromatography (HPLC) method was used to simultaneously determine the contents of the above four active ingredients in gentian flower, which provides a more scientific basis for comprehensive evaluation of the quality of gentian flower and Tibetan patent medicine.
1 Instruments and reagents
Agilent1100 high performance liquid chromatograph (configuration: manual sampler, online degasser, high-pressure binary gradient pump, constant temperature column oven, DAD detector, Agilent1100 chromatography workstation), ZORBAXSB-C18 (250mm × 4.6mm, 5μm) Chromatography column; KQ3200 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).
Swertiamarin and gentiopicroside reference products were purchased from the China National Institute for the Control of Pharmaceutical and Biological Products; lyophyllic acid and swertiamarin reference products were isolated by our laboratory, and the purity was greater than 98% as determined by the normalization method; Methanol is chromatographically pure, phosphoric acid is analytically pure, and water is homemade triple distilled water.
Gentian flower was collected in Maqin County, Guoluo Prefecture, Qinghai Province in 2007-08. It was identified by Sun Hongfa, a researcher of the Institute of Tibetan Medicine of Qinghai Province. After it was dried, it was crushed through an 80-mesh sieve and stored under refrigeration.
2 Methods and results
2.1 Chromatographic conditions
The column is ZORBAXSB-C18 (250mm × 4.6mm, 5μm); the mobile phase is methanol: water (containing 0.04% phosphoric acid) 15:85; the flow rate is 1ml · min-1; the detection wavelength is 238nm; the column temperature is 30 ℃. Under the chromatographic conditions, the four components of dry acid, swertiamarin, gentiopicroside and swertiamarin were all eluted and reached baseline separation. The chromatogram is shown in Figure 1.
2.2 Preparation of reference solution
Accurately weigh the appropriate amount of aspartic acid, swertiamarin, gentiopicroside and swertiarin, and dissolve in methanol, and set the volume in a 10ml volumetric flask. Shake well as a reference solution. Dilute to the desired concentration when using.
2.3 Preparation of the test product solution Weigh accurately 0.5g of the sample crushed to 80 mesh, add 15ml of methanol, extract with ultrasonic for 40min, filter with suction, wash the filter residue twice with methanol, 1ml / time. Repeat the above operation, the obtained filtrate is combined with the previous filtrate, after concentration, the volume is fixed in a 50ml volumetric flask with methanol. Pass the 0.45μm filter membrane and measure the content of active ingredients under the selected chromatographic conditions.
2.4 Investigation of linear relationship
Accurately measure the appropriate amount of 4 kinds of reference stock solutions, place them in a 2ml volumetric flask, dilute to the mark with methanol, and obtain the mixed reference standard solution series. Take 10μl of the mixed reference standard solution series and measure according to the chromatographic conditions under “2.1â€. The linear equations and correlation coefficients of each component injection volume (μg) and peak area A are shown in Table 1. Table 14 Linear equations and linear ranges of the active ingredients (omitted)
2.5 detection limit
Under the selected chromatographic conditions, when the signal-to-noise ratio S / N = 3, the minimum detection limit of each component was determined. The results showed that the acid, swertiamarin, gentiopicrin and swertia The minimum detection limits of glycosides were 0.56, 0.25, 0.45, and 0.84 ng.
2.6 Precision experiment According to the chromatographic conditions under "2.1", 10μl of mixed reference solution was injected to determine the peak area of ​​the chromatographic peak. The RSDs of the four active ingredients were calculated as 0.93%, 0.81%, 0.56% and 1.2 respectively % (n = 5), indicating that the precision of the instrument is good.
2.7 Stability experiment Weigh accurately 0.5g of the sample, according to the chromatographic conditions under the item "2.1", analyze the sample on the same day, the next day and the third day to determine the dry acid, swertiamarin, gentiopicroside The peak areas of swertiamarin were calculated to have RSDs of 1.2%, 1.6%, 1.3% and 2.1%, respectively, indicating that the test solution was stable within 3 days.
2.8 Repeatability experiment
Accurately weigh 6 parts of gentian flower coarse powder (80 mesh), and prepare the test solution according to the method under "2.3". According to the chromatographic conditions under "2.1", sample analysis, record the peak area, and calculate the component content according to the regression equation, the RSDs of ascorbic acid, swertiamarin, gentiopicroside and swertiamarin are respectively 0.87%, 1.11%, 1.87% and 1.75%.
2.9 Recovery rate experiment Weigh accurately 5 parts of gentian flower coarse powder of known content, add a certain amount of mixed reference solution, prepare the test solution according to the method under "2.3", and press the chromatography under "2.1" The conditions were measured, and the average recovery rates of ascorbic acid, swertiamarin, gentiopicroside, swertiamarin were 99.2% (RSD = 2.6%), 103.0% (RSD = 1.5%), 101.0% ( RSD = 1.1%) and 99.7% (RSD = 3.6%).
2.10 Determination of sample content
Take the sample solution prepared under "2.3" and analyze it according to the chromatographic conditions under "2.1". After the DAD detector detects the peak purity of the above four test components and its UV spectrum is consistent with the reference, press The standard method is calculated based on the integral value of the peak area, and the content of the four active ingredients (repeatedly injected 5 times for each sample, and the average value) is 1.16%, 0.15%, 5.47%, and 3.86%, respectively.
3 Discussion
3.1 Determination of detection wavelength
The solutions of ascorbic acid, swertiamarin, gentiopicroside, and swertiarin were scanned in the range of 200-400nm, and the results were absorbed in the vicinity of 254nm and 238nm. At 238nm, there is less impurity interference, so 238nm is selected as the detection wavelength.
3.2 Determination of extraction method
The experiment investigated the extraction rate of methanol reflux extraction method and ultrasonic extraction. The results showed that the active components of the iridoid glycosides were partially destroyed during reflux extraction, so ultrasonic extraction was used. During the ultrasonic extraction, the pulverization degree of the medicinal materials and the extraction rate under different solvents at different times were investigated. The results suggested that the methanol was extracted twice through an 80-mesh sieve, 40 minutes each time, and the extraction was complete.
3.3 Selection of mobile phases In the process of screening the chromatographic conditions for the determination of the active constituents of cycloheteroid glycosides, I have tried using different proportions of methanol and water as mobile phases and found that methanol-water (0.4% phosphoric acid) is 15 : 85, all four components were eluted and reached baseline separation.
The proposed method has the characteristics of extraction, simple and rapid analysis method, accurate and reliable results, etc. It has an important role in the comprehensive evaluation of the quality of gentian flower and its stored proprietary medicines.
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