High performance liquid chromatography method for simultaneously determining contents of rutin, gallic acid and ellagic acid in rose extract

Abstract

The invention belongs to the technical field of detection methods, and particularly relates to a high performance liquid chromatography method for simultaneously determining the contents of rutin, gallic acid and ellagic acid in a rose extract. The invention provides a high performance liquid chromatography method for simultaneously determining the contents of rutin, gallic acid and ellagic acid in a rose extract, and the method avoids the problems of high operation difficulty, huge instrument and equipment volume, high price and low universality of a liquid chromatography-tandem mass spectrometry method. Certain technical guidance is provided for studying the use conditions and safety of the three active ingredients in the plant raw material rose extract, monitoring the content of the plant raw materials and performing scientific production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection methods, and particularly relates to a high-performance liquid chromatography method for simultaneously determining the contents of rutin, gallic acid and ellagic acid in rose extract. BACKGROUND

[0002] Rose (Rosa rugosa Thunb.) belongs to the Rosaceae family and is widely planted in China. Its flowers are elegant and fragrant, and have been used in food, cosmetics, medicine and other fields for a long time. Its application in the food field in China can be traced back to the Tang Dynasty. The Food Herbalism recorded that "rose flowers are fragrant and sweet, and can make people feel refreshed". It contains essential amino acids, proteins and other nutrients, and can be used for cake, beverage flavoring and coloring, and the production of special food to improve sensory quality. It was included in the first batch of "List of Items that Can be Used for Health Foods" announced by China in 2002. In the cosmetics field, it has become a popular raw material due to its antioxidant, moisturizing and anti-inflammatory effects, and is commonly found in products such as perfume and cream, which can resist skin oxidative damage, regulate moisture and inflammation. In the field of medicine and health products, the flavonoids and other components in its extract inherit the traditional medicinal value and have been proven to have physiological potential such as regulating blood lipids and enhancing immunity, providing a direction for natural drug development. As a key form of deep development of rose resources, rose extract is usually prepared from dried rose flowers as the starting material, using water, organic alcohol or a mixture of the two as the extraction solvent, followed by extraction by ultrasonic, decoction, reflux and other processes, and then concentrated, impurity-removed and purified.

[0003] The active ingredients of rose extract are very rich, among which flavonoids and polyphenols are the core of antioxidant function. Studies have shown that polyphenolic components such as gallic acid have antioxidant activity. Compared with common antioxidants on the market (such as butylated hydroxyanisole), gallic acid is a natural antioxidant and has no carcinogenicity. Total flavonoids can regulate intestinal flora and show potential in improving sugar and lipid metabolism in functional foods. At the same time, polyphenols and flavonoids have been proven to have effects on various chronic diseases and tumors, including gallic acid, which helps to regulate blood lipids, inhibit platelet aggregation and protect cardiovascular health. In addition, according to the provisions of Rose Flower Formula Granules under the National Drug Standard for Traditional Chinese Medicine Formula Granules, gallic acid, ellagic acid and total flavonoids are listed as index components of rose extract to measure the quality of the product.

[0004] Currently, the detection methods for polyphenols and flavonoids mainly include high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry and spectrophotometry. Among them, liquid chromatography-tandem mass spectrometry has extremely high sensitivity and accuracy in the detection field, but the method is difficult to operate, the instrument equipment is large in size and expensive, and the universality is low. SUMMARY

[0005] In view of the above technical problems, the present application establishes a method for determining the contents of gallic acid, rutin and ellagic acid, so as to study the use and safety of the three active ingredients in the plant raw material rose extract, monitor the content of the plant raw material and provide certain technical guidance for scientific production.

[0006] Specifically includes the following contents: The present application aims to provide a high performance liquid chromatography method for simultaneously determining the contents of rutin, gallic acid and ellagic acid in rose extract, which comprises the following steps: (1) Preparation of standard solution: (2) Sample pretreatment: (3) Chromatographic conditions: the chromatographic column is CAPCELL PAK C18 MG II (5 μm, 4.6 x 250 mm); the mobile phase A is acetonitrile, and the mobile phase B is 0.2% phosphoric acid aqueous solution; the flow rate is 1.0 ml / min; the column temperature is 30°C; the injection volume is 10 μL; the gradient elution program is shown in Table 1 of the specification; the detection wavelength is 273 nm for gallic acid, and 254 nm for rutin and ellagic acid, and the running time is 30 min.

[0007] Preferably, the preparation of the standard solution in step (1) is as follows: rutin, gallic acid and ellagic acid reference substances are weighed respectively, gallic acid and rutin are dissolved in methanol and constant volume, and ellagic acid is dissolved in dimethyl sulfoxide and constant volume to obtain rutin, gallic acid and ellagic acid single standard stock solutions with a concentration of 1.0 mg / L respectively; the rutin, gallic acid and ellagic acid single standard stock solutions are removed and mixed to obtain a mixed standard solution, which is diluted with methanol and constant volume to obtain a standard series solution of rutin, gallic acid and ellagic acid.

[0008] Preferably, the mixed standard solution is as follows: 1.0, 5.0 and 2.5 mL of rutin, gallic acid and ellagic acid single standard stock solutions are removed and mixed, diluted with methanol and constant volume to 25 mL to obtain a mixed standard solution with concentrations of 40, 200 and 100 μg / ml in turn.

[0009] Preferably, the concentration of the gallic acid standard series solution is 0.4, 2.0, 4.0, 8.0, 16 and 32 μg / ml; the concentration of the rutin standard series solution is 2.0, 10.0, 20.0, 40.0, 80.0 and 160.0 μg / ml; and the concentration of the ellagic acid standard series solution is 1.0, 5.0, 10.0, 20.0, 40.0 and 80.0 μg / ml.

[0010] Preferably, the sample pre-treatment of step (2) is: weighing the sample, adding 15% DMSO in methanol solution to prepare a concentration of 0.01 g / ml, ultrasonic extraction, centrifugation, taking the supernatant and passing through a 0.45 μm microporous filter membrane.

[0011] Preferably, the sample pre-treatment of step (2) is: weighing the sample 0.40 g, adding methanol 40 mL, vortex oscillation for 30 s, ultrasonic extraction for 30 min, centrifugation at 5000 r / min for 5 min, taking the supernatant and passing through a 0.45 μm microporous filter membrane.

[0012] Preferably, the ultrasonic parameters are: 20-43 KHz, 200 W.

[0013] The present application has the beneficial effects that: the present application provides a high performance liquid chromatography method for simultaneously determining the contents of rutin, gallic acid and ellagic acid in rose extract, which avoids the problems of high operation difficulty, large size and high price of instrument equipment, and low universality of liquid chromatography tandem mass spectrometry, and provides certain technical guidance for studying the use and safety of the three active ingredients in plant raw material rose extract, and monitoring the content and scientific production of plant raw materials. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Gallic acid standard chromatogram; Figure 2 Rutin and ellagic acid standard chromatogram; Figure 3 Linear relationship diagram of rutin; Figure 4 Linear relationship diagram of gallic acid; Figure 5 Linear relationship diagram of ellagic acid. DETAILED DESCRIPTION

[0015] The scheme described in the present application will be discussed below in combination with specific embodiments, but the scheme described in the present application is not limited to the embodiments described below.

[0016] The instruments and reagents used in the following embodiments are as follows: Waters-2695 type high performance liquid chromatograph, equipped with a diode array detector (Waters Corporation, USA); CAPCELL PAK C18 MG II (5 μm, 4.6*250 mm); Milli-Q IQ 7000 ultrapure water machine (Millipore Corporation, USA); ultrasonic extraction instrument (Kunshan Ultrasonic Instrument Co., Ltd.); vortex mixer (Changzhou Guohua Electrical Appliance Co., Ltd.); electronic balance (Switzerland Mettler Corporation).

[0017] Rutin reference substance (batch number: 100080-202012; purity: 92.2%), Gallic acid reference substance (batch number: 110831-201906; purity: 91.5%), Ellagic acid (batch number: 111959-201903; purity: 92.2%); methanol, acetonitrile, dimethyl sulfoxide (chromatographically pure); phosphoric acid (extra pure); microporous filter membrane (0.45 μm); and experimental water was first-grade water.

[0018] 0.2% Phosphoric acid solution: 1000 mL of first-grade water was measured, and 0.2 mL of phosphoric acid was added. Example 1

[0019] 1. Experimental method 1.1 Preparation of standard solution 10.0 mg of gallic acid, rutin and ellagic acid reference substances were accurately weighed into a 10 mL volumetric flask, and gallic acid and rutin were dissolved with methanol to constant volume, and ellagic acid was dissolved with dimethyl sulfoxide to constant volume, thereby obtaining a gallic acid, rutin and ellagic acid single standard stock solution with a concentration of 1.0 mg / mL.

[0020] 1.0 mL, 5.0 mL and 2.5 mL of the gallic acid, rutin and ellagic acid single standard stock solutions were accurately transferred into the same 25 mL volumetric flask, diluted with methanol to constant volume, and shaken to obtain a mixed standard solution with concentrations of 40 μg / mL, 200 μg / mL and 100 μg / mL, respectively.

[0021] 0.1 mL, 0.5 mL, 1.0 mL, 2.0 mL, 4.0 mL and 8.0 mL of the mixed standard solution were accurately measured into different 10 mL volumetric flasks, diluted with methanol to constant volume, and shaken to obtain a gallic acid standard series solution with concentrations of 0.4 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16 μg / mL and 32 μg / mL, a rutin standard series solution with concentrations of 2.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 40.0 μg / mL, 80.0 μg / mL and 160.0 μg / mL, and an ellagic acid standard series solution with concentrations of 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 40.0 μg / mL and 80.0 μg / mL.

[0022] 1.2 Sample pretreatment 0.40 g of the sample was accurately weighed (accurate to 0.001 g), placed in a 50 mL stoppered cuvette, 40 mL of 15% DMSO methanol solution was added, vortexed for 30 s, the sample and the extraction solvent were fully mixed, ultrasonic extraction was performed for 30 min (working frequency 20-43 KHz, 200 W), and centrifugation was performed at 5000 r / min for 5 min. The supernatant was filtered through a 0.45 μm microporous filter membrane, and was ready for determination.

[0023] 1.3 Chromatographic conditions Chromatographic column: CAPCELL PAK C18 MG II (5 μm, 4.6 x 250 mm); mobile phase: mobile phase A was acetonitrile, mobile phase B was 0.2% phosphoric acid aqueous solution, gradient elution was performed according to Table 1; flow rate was 1.0 ml / min, column temperature was 30 °C, injection volume was 10 μL, detection wavelength: gallic acid was 273 nm, rutin, ellagic acid was 254 nm, and the running time was 30 min.

[0024]

[0025] 1.4 Data processing The data collected by the high performance liquid chromatograph were integrated by Empower 3 software, and the corresponding chromatogram was provided. The data such as recovery rate and relative standard deviation were analyzed and processed by Microsoft Office Excel 2022 software.

[0026] 2 Results and analysis 2.1 Chromatographic condition optimization 2.1.1 Chromatographic column and detection wavelength In this embodiment, the performance, filler, polarity and acid and alkali resistance of the chromatographic column were investigated and compared, and finally it was determined that the Agilent CAPCELL PAK C18 MG II (5 μm, 4.6 x 250 mm) chromatographic column could meet the experimental requirements. The characteristic absorption wavelength and retention time of gallic acid and ellagic acid could be used as important qualitative determination basis. Full wavelength (200 ~ 700 nm) scanning was performed by using a diode array detector, and it was found that gallic acid, rutin and ellagic acid had maximum absorption at 273 nm, 254 nm and 254 nm, respectively. Therefore, 273 nm was determined as the quantitative detection wavelength of gallic acid, and 254 nm was the quantitative detection wavelength of rutin and ellagic acid. Figure 1 is the standard chromatogram of gallic acid, Figure 2 is the standard chromatogram of rutin and ellagic acid.

[0027] 2.1.2 Mobile phase In order to achieve the best separation effect of the chromatographic column, acetonitrile-water and acetonitrile-0.2% phosphoric acid aqueous solution were used as the mobile phase for comparison. It was found that in the acetonitrile-water system, the peak shape of each target compound was slightly poor. In the acetonitrile-0.2% phosphoric acid aqueous solution system, the chromatographic peak of the target compound was sharp and symmetrical, and the separation degree was good, which met the requirements of accurate quantitative analysis. Therefore, acetonitrile-0.2% phosphoric acid aqueous solution was finally selected as the mobile phase.

[0028] 2.2 Standard sample dissolving agent The ellagic acid control was dissolved in methanol, acetonitrile, methanol + dimethyl sulfoxide, acetonitrile + dimethyl sulfoxide and dimethyl sulfoxide, respectively. It was found that only when dimethyl sulfoxide was used as the dissolving agent, the ellagic acid standard solution was still clear and transparent after standing for a period of time, and there was no turbidity. When the gallic acid and rutin controls were dissolved in methanol, acetonitrile, 75% methanol and 75% acetonitrile, respectively, it was found that the solubility of the gallic acid and rutin controls in methanol was the best. Therefore, in order to obtain better extraction efficiency, considering the solubility of the target standard substance, dimethyl sulfoxide was finally selected as the dissolving agent for the ellagic acid control, and methanol was selected as the dissolving agent for the gallic acid and rutin controls.

[0029] 2.3 Pretreatment solvent optimization Methanol, 70% methanol and 15% DMSO methanol solution were used as extractants, respectively, to extract the same sample, and the results are shown in Table 2.

[0030]

[0031] As can be seen from Table 2, the 15% DMSO methanol solution has the best extraction effect on the three components, especially it can significantly improve the extraction amount of gallic acid and rutin, and is suitable as the extraction solvent for the sample. The extraction effect of 70% methanol is better than that of pure methanol, which indicates that the appropriate amount of water can promote the dissolution of polar components, and DMSO can further improve the extraction efficiency due to its polarity and permeability. Therefore, the 15% DMSO methanol solution is finally selected as the extraction liquid for sample pretreatment.

[0032] 2.4 Methodology verification 2.4.1 Linear relationship investigation and detection limit, quantification limit According to the finally optimized method, 10 μL of the mixed standard series solution of gallic acid, rutin and ellagic acid under item “1.1” was precisely taken, and chromatographic analysis was performed under the chromatographic conditions described in “1.3” (chromatographic column: CAPCELL PAK C 18 MGⅡ, 5 μm, 4.6 × 250 mm; mobile phase: A phase was acetonitrile, B phase was 0.2% phosphoric acid aqueous solution, gradient elution program was shown in Table 1; flow rate was 1.0 mL / min; column temperature was 30℃; detection wavelength was 273 nm for gallic acid, and 254 nm for rutin and ellagic acid). The peak area was determined. The concentration (μg / mL) of gallic acid, rutin and ellagic acid control was taken as the abscissa, and the average peak area was taken as the ordinate, and the linear regression program was obtained. The linear curve is shown in FIG. Figures 3-5 The results show that the standard working solution curve r 2 of gallic acid, rutin and ellagic acid of the method is all above 0.99, and the linear relationship of the method is good. The detection limit (3σ) and quantification limit (10σ) of the method are shown in Table 3.

[0033]

[0034] 2.4.2 Stability experiment The rose extract sample was precisely weighed, and the peak area was measured at 0, 1, 2, 4, 8, 12, 24 and 48 h respectively according to the method described in item 1. The relative standard deviations of the retention time and peak area of gallic acid, rutin and ellagic acid were calculated, and the results were all less than 2.0 %, indicating that the test solution had a certain stability within 48 h, and the contents of gallic acid, rutin and ellagic acid would not degrade within 48 h. The results are shown in Table 4.

[0035]

[0036] 2.4.4 Precision According to the method described in item 1, the second point and the fifth point of the mixed standard solution were continuously injected into 6 needles respectively, and the peak area RSD (n=6) was calculated. The RSD was less than 2.0 %, indicating that the instrument precision was good. The results are shown in Table 5.

[0037]

[0038] 2.4.5 Reproducibility Six test sample solutions were prepared from the same sample, and were injected and analyzed under the chromatographic conditions according to the method described in item 1. The peak area was recorded, and the contents of gallic acid, rutin and ellagic acid in the test sample were calculated by the standard curve method to test the reproducibility of the method. The results are shown in Table 6. According to the table, the average contents of gallic acid, rutin and ellagic acid in the test sample were 554.4276, 9459.4392 and 1334.3019 μg / g respectively, and the RSDs were 0.01 %, 0.95 % and 2.03 % respectively, indicating that the experimental reproducibility was good.

[0039]

[0040] 2.4.6 Accuracy The standard addition was in the form of high, medium and low concentrations. The specific standard addition treatment method was as follows: 0.4 g of the blank sample was weighed, and was added with high, medium and low concentrations respectively. The sample pretreatment and detection analysis were carried out according to the test method, and each sample could be detected in parallel for 3 times, and the average value was taken to obtain the standard addition recovery rate. The results showed that the standard addition recovery rate of gallic acid was 93.91 %~104.93 %, the standard addition recovery rate of rutin was 90.74 %~102.96 %, and the standard addition recovery rate of ellagic acid was 96.00~105.79 %. The specific results are shown in Table 7. It can be seen that the recovery rates of the standard addition samples were all between 90.74 % and 105.79 %, indicating that the accuracy of the method could meet the detection requirements.

[0041]

[0042] 2.5 Actual sample determination According to the method described in item 1, 6 batches of rose extract provided by enterprises were determined, and gallic acid, rutin and ellagic acid were detected in 6 batches of samples. The specific data is shown in Table 8.

[0043]

[0044] The present application establishes a high performance liquid chromatography method for simultaneously detecting gallic acid, rutin and ellagic acid, three kinds of polyphenol active ingredients in rose extract. The selection of several conditions such as detection wavelength, reference substance dissolving agent, extraction solvent and chromatographic column is investigated, and the best experimental condition is selected.

[0045] The detection range, recovery rate, precision, stability and specificity of the method can meet the actual detection requirements. The experimental steps of the method are simple and efficient.

Claims

1. A high-performance liquid chromatography method for simultaneously determining the contents of rutin, gallic acid, and ellagic acid in rose extract, characterized in that, The method includes the following steps: (1) Preparation of standard solutions: (2) Sample pretreatment: (3) Chromatographic conditions: The column was CAPCELL PAK C18 MGⅡ (5 μm, 4.6×250 mm); the mobile phase A was acetonitrile, and the mobile phase B was 0.2% phosphoric acid aqueous solution; the flow rate was 1.0 ml / min; the column temperature was 30 ℃; the injection volume was 10 μL; the gradient elution program was as shown in Table 1 of the instruction manual; the detection wavelengths were: gallic acid 273 nm, rutin and ellagic acid 254 nm, and the running time was 30 min.

2. The method as described in claim 1, characterized in that, The preparation of the standard solution in step (1) is as follows: Gallic acid, rutin, and ellagic acid reference standards are weighed separately. Gallic acid and rutin are dissolved and diluted with methanol, and ellagic acid is dissolved and diluted with dimethyl sulfoxide to obtain single standard stock solutions of gallic acid, rutin, and ellagic acid with concentrations of 1.0 mg / mL. The single standard stock solutions of gallic acid, rutin, and ellagic acid are mixed to obtain a mixed standard working solution, which is diluted with methanol and diluted to volume to obtain a series of standard solutions of gallic acid, rutin, and ellagic acid.

3. The method as described in claim 2, characterized in that, The mixed standard working solution is prepared by mixing 1.0, 5.0, and 2.5 mL of gallic acid, rutin, and ellagic acid single standard stock solutions, respectively, diluting with methanol and bringing the volume to 25 mL, to obtain mixed standard working solutions with concentrations of 40, 200, and 100 μg / mL, respectively.

4. The method as described in claim 3, characterized in that, The concentrations of the gallic acid standard series solutions are 0.4, 2.0, 4.0, 8.0, 16, and 32 μg / ml; the concentrations of the rutin standard series solutions are 2.0, 10.0, 20.0, 40.0, 80.0, and 160.0 μg / ml; and the concentrations of the ellagic acid standard series solutions are 1.0, 5.0, 10.0, 20.0, 40.0, and 80.0 μg / ml.

5. The method as described in claim 1, characterized in that, The sample pretreatment in step (2) is as follows: weigh the sample and add 15% DMSO methanol solution to prepare a concentration of 0.01 g / ml, extract by ultrasonication and centrifuge, and take the supernatant to filter through a 0.45 μm microporous membrane.

6. The method as described in claim 5, characterized in that, The sample pretreatment in step (2) is as follows: weigh 0.40 g of sample, add 40 mL of methanol, vortex for 30 s, extract by ultrasound for 30 min, centrifuge at 5000 r / min for 5 min, and filter the supernatant through a 0.45 μm microporous membrane.

7. The method as described in claim 6, characterized in that, The ultrasonic parameters are: 20-43 kHz, 200 W.

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