Abstract: The high content of ursolic acid is glossy prismatic (anhydrous ethanol) or fine hair-like needle crystals (thin ethanol), the low content is brownish yellow or yellow-green powder, with a special smell, it is present in natural plants Is a pentacyclic triterpenoid.
Objective To determine the content of ursolic acid in Shanxiangyuan by high performance liquid chromatography (HPLC). Methods Chromatographic conditions: HypersiODSC18 chromatographic column (4.6mm × 250mm; 5μm); mobile phase was acetonitrile: methanol (volume ratio 68:32), detection wavelength was 210nm, mobile phase flow rate was 1.0ml / min, column temperature was 25 ℃. Results The linear range of ursolic acid detection was 0.50 ～ 10.05mg (R2 = 0.9987), the average recovery rate was 99.28%, and the relative standard deviation (RSD) was 1.96% (n = 5). Conclusion The method is accurate, rapid, sensitive and reproducible, and can be used as an effective method for the quality control of Shanxiangyuan and related preparations.
ã€Key words】 High performance liquid chromatography determination of arbutin
TurpiniaargutaSeen is a plant belonging to the genus Proaceae in the province. It is also known as Thousand Beats, Seven-inch Nail, Moth Medicine, Hedyotis diffusa, Hedyotis diffusa, etc. It is mainly produced in Jiangxi, Fujian, Guangdong, Guangxi and other provinces. , With the effect of clearing away heat and detoxification, diuresis and swelling [1]. The main active ingredients in the mountain leaf are ursolic acid, oleanolic acid, p-coumaric acid, myristic acid, etc. [2-5]. Ursolic acid (UA), also known as ursolic acid and ursolic acid, is a triterpenoid compound found in natural plants (Figure 1), with a relative molecular mass of 456 and a wide range of biological effects. It has antibacterial activity against G and G-bacteria and yeast in vitro; it has anti-diabetic, anti-ulcer and blood lipid lowering effects. At the same time, it has anti-carcinogenic and anti-cancer effects, induces differentiation of F9 teratocarcinoma cells and anti-angiogenesis. It is an excellent anti-toxin and highly effective new anti-cancer drug. It also has obvious antioxidant function and has a central nervous system effect. Obvious stability and cooling effect, so it is widely used as a raw material for medicine and cosmetics, and is gaining more and more attention [6-8]. The determination method of ursolic acid includes thin layer scanning method [9 ～ 12], high performance liquid chromatography, etc. [13 ～ 15]. At present, there are few reports on the analysis method of ursolic acid content in Shanxiangyuan, except that Mao Youchang et al. [11] and Wei Fuyou et al. [12] applied TLC to the content of ursolic acid in Shanxiangyuan leaf and Shanxiangyuan lozenge. Perform quantitative analysis. In this paper, a high performance liquid chromatography method for the determination of the content of ursolic acid in Mangosteen is established. The results show that this method is simple and accurate, and it is suitable for the quality control of medicinal materials and preparations containing ursolic acid.
1 Equipment and methods
1.1 Material and instrument samples: Shanxiangyuanye was provided by Nanchang Huayi Pharmaceutical Co., Ltd .; ursolic acid reference substance: purchased from China National Institute for the Control of Pharmaceutical and Biological Products, batch number 742.200111; methanol and acetonitrile are chromatographically pure, and the remaining reagents are analytically pure; HP1050 high performance liquid chromatograph, equipped with HP1050 chromatographic workstation, vacuum degasser, manual sampler, column temperature box.
Figure 1 Chemical structure of ursolic acid
1.2 Preparation of reference substance stock solution Weigh 25.125 mg of ursolic acid standard sample, place it in a 25 ml volumetric flask, dissolve in methanol and dilute to the mark to obtain a standard solution containing ursolic acid 1.005 mg / ml. Precisely transfer 0.5, 2, 4, 8, 10ml of ursolic acid reference stock solution and place them in 10ml volumetric flasks. Dilute to the mark with methanol, and prepare a series of standard working solutions.
1.3 Preparation of sample solution Crush the Shanxiangyuan sample, pass through a 60 mesh sieve, and dry to constant weight at 80 ° C. Precisely weigh 2.0g, place in a Soxhlet extractor, heat and reflux with methanol for 6h, extract the solution with a 0.45μm microporous filter membrane, concentrate and dilute to a 25ml volumetric flask, and use the filtrate as the sample solution.
1.4 Chromatographic conditions HypersiODSC18 column (4.6mm × 250mm; 5μm); mobile phase is acetonitrile: methanol (volume ratio is 68:32); detection wavelength is 210nm; mobile phase flow rate is 1.0ml / min; column temperature is 25 ℃; sample injection The amount is 10ml.
2 results
2.1 Selection of chromatographic conditions After ultraviolet spectrum scanning, the maximum absorption wavelength of ursolic acid is 210nm. Therefore, this experiment uses 210nm as the detection wavelength. Under the "1.4" chromatographic conditions, the retention time of the ursolic acid standard and the sample solution is about 32 minutes, the peak shape is good, and the separation effect of ursolic acid and oleanolic acid is good (see Figures 2 to 3).
2.2 Drawing of standard curve Prepare the standard curve by taking the stock solution prepared according to the method of "1.2". Inject 10 μl of each control stock solution to determine the peak area. Taking the peak area value (Y) as the ordinate and the corresponding injection mass concentration (X) as the abscissa, the regression calculation is performed, and the linear equation is: Y = 102.05X-0.796, R2 = 0.9987. It shows that the injection volume of ursolic acid is linear in the range of 0.50 ～ 10.05mg.
2.3 Precision experiment Take 10ml of ursolic acid reference substance solution (0.1005mg / ml), repeat the injection 5 times, record the peak area, and obtain the relative standard deviation RSD of 1.44%, indicating that the precision of the instrument is good.
2.4 Stability experiment For the same ursolic acid reference solution, 3 injections were performed at 0, 2, 4, 6, 8, and 10h, 10ml / time, and the peak area value was recorded. Its RSD was 0.995%, indicating that the sample solution was Good stability within 10h.
2.5 Repeatability experiment Weigh accurately 2.0g 5 samples of the same batch of Shanxiangyuan, prepare the sample solution according to the method described in "1.3", measure 5 times on the same day, inject 10ml / time, and measure the intra-day precision of the peak area ( (Calculated by RSD) is 1.89%, indicating that the method is reproducible.
2.6 Sample recovery rate experiment Weigh accurately 5 samples of the same batch with known content, each sample is about 2.5g, placed in 5 conical flasks, add 1ml of ursolic acid reference solution (1.005mg / ml) ). Prepare the sample solution according to the method described in item "1.3" to obtain 5 solutions of the same concentration. Analyze according to the chromatographic conditions described in "1.4" and calculate the recovery rate of the sample. The average recovery rate of ursolic acid is 99.28%, RSD = 1.96%. The results are shown in Table 1. Table 1 Experimental results of ursolic acid recovery
2.7 Determination of sample content Take "1.1" item of Shanxiang round leaves provided by Nanchang Huayi Pharmaceutical Co., Ltd., prepare the sample solution according to the method described in "1.3" item, take 3 samples of the same batch of sample solution, and directly inject under the above chromatographic conditions 10μl / time, each sample was injected 3 times, the average value was calculated, and the content of ursolic acid was calculated according to the regression equation to be 1.025mg / g. The results are shown in Table 2. Table 2 The content of ursolic acid in Shanxiang round leaves
3 Discussion
3.1 Selection of extraction conditions According to the solubility of ursolic acid, methanol, absolute ethanol, ethyl acetate, and chloroform were selected as solvents. The samples were subjected to Soxhlet extraction treatment, and the effects of different extraction solvents on the extraction effect were compared. The results showed that methanol was used as the solvent with the highest extraction rate, and the chromatogram peak separation of the methanol extract was good, and it did not interfere with the determination of ursolic acid. Therefore, methanol is used as the extraction solvent.
3.2 Selection of mobile phase According to literature reports [14-16], choose methanol-water (90:10), methanol-0.1% phosphoric acid aqueous solution, acetonitrile-water (75:25), methanol-acetonitrile-water (64:20: 16). Methanol-acetonitrile (68:32) and other mobile phases were measured. The results show that methanol-acetonitrile (68:32) can be used as a mobile phase to obtain a well-dispersed chromatographic peak, and the separation of ursolic acid and oleanolic acid in the supplied product is also high.
The content of ursolic acid was analyzed by high-performance liquid chromatography, the chromatographic conditions were optimized, the standard ursolic acid was determined under the best conditions, a standard curve was made and the content of the extracted ursolic acid sample was determined. The analysis results show that this method is simple, fast, efficient, sensitive and reproducible, and can be used for the quality control of ursolic acid-containing medicinal materials and their preparations.
Customized Automatic Screen Printing Machine
Garment Screen Printing Machine,Automatic Screen Printing Equipment,T Shirt Screen Printing Machine,Screen Printing Printer Machine
KC Printing Machine (Group) Limited , http://www.kcautopm.com