Method for simultaneously determining contents of bulbus lilii glycoside C and bulbus lilii glycoside F in lily

Abstract

The invention provides a method for simultaneously determining the contents of bulbus lilii glycoside C and bulbus lilii glycoside F in lilies, and relates to the technical field of chromatography. The method comprises the following steps: extracting lily to obtain a to-be-detected sample; and establishing a high performance liquid chromatography to simultaneously determine the contents of the king lily glycoside C and the king lily glycoside F. The method is simple and easy to implement, has accuracy, repeatability and reliability, has a certain reference value for content determination of the bulbus lilii glycoside C and the bulbus lilii glycoside F in the bulbus lilii, and provides a reference for quality control of the bulbus lilii.

Description

Technical Field

[0001] The present invention relates to the technical field of chromatography, and in particular to a method for simultaneously determining the contents of lilyside C and lilyside F in lily. Background Art

[0002] Lily contains steroidal saponins, phenolic acid glyceride compounds, flavonoids, amino acids, alkaloids and other alkanes. Regaloside C and Regaloside F are phenylpropanoid glycerol glucosides. The structural formulas of Regaloside C and Regaloside F are as follows: Figure 1 and Figure 2 shown.

[0003] High-performance liquid chromatography (HPLC) offers high sensitivity, high specificity, and a reasonable degree of accuracy, making it commonly used for content determination in complex samples. Currently, chromatographic techniques for qualitative and quantitative determination of components in lilies primarily focus on the determination of lilyside A, lilyside B, and lilyside C. For example, Hu Wenyan et al. established a method for the determination of lilyside A and lilyside C in lily using HPLC. Yuan Zhiying et al. also developed a method for the determination of lilyside A using ATR-FTIR combined with UPLC-DAD technology for the identification of lily and its analogs. Gu Anna et al. and Zhong Xia et al. both used HPLC methods to determine lilyside B for lily quality control. However, simultaneous chromatographic determination of lilyside C and lilyside F in lily has not yet been reported. Therefore, this study established an HPLC method for the determination of lilyside C and lilyside F in lily and investigated their contents in lilies from different habitats. Summary of the Invention

[0004] Based on this, the present invention provides a method for simultaneously determining the content of lilyside C and lilyside F in lily. The present invention establishes a high-performance liquid chromatography method for simultaneously determining the content of lilyside C and lilyside F. This method is simple and easy to perform, and has accuracy, repeatability, and reliability. It has a certain reference value for the determination of the content of lilyside C and lilyside F in lily, and also provides a reference for the quality control of lily.

[0005] The method for simultaneously determining the contents of lilyside C and lilyside F in lily comprises the following steps:

[0006] (1) Pretreatment of lily raw materials

[0007] The lily raw material is dried, crushed and sieved to obtain lily powder;

[0008] (2) Preparation of lily extract

[0009] The lily powder is added to ethanol and subjected to ultrasonic extraction; the lily powder is then heated under reflux and filtered to obtain a filtrate and a filter residue; the filter residue is added to ethanol and subjected to heating under reflux, filtered, and the two filtrates are combined; the combined filtrate is subjected to reduced pressure distillation to recover the solvent, to obtain a lily extract;

[0010] (3) Preparation of test samples

[0011] The lily extract is added to a methanol aqueous solution for dissolution, and after centrifugation, the supernatant is filtered through a microporous filter membrane to obtain a sample to be tested;

[0012] (4) Preparation of standard solution

[0013] Weigh lilyside C and lilyside F, and dissolve them in methanol to prepare lilyside C standard solution and lilyside F standard solution respectively;

[0014] (5) Liquid chromatography detection

[0015] The sample to be tested is quantitatively detected by high performance liquid chromatography, wherein the chromatographic conditions are as follows:

[0016] The chromatographic column was ZORBAX SB-C18, with specifications of 4.6 mm × 150 mm, 5 μm;

[0017] Column temperature 35°C;

[0018] In the mobile phase, solution A was 0.1 wt% phosphoric acid aqueous solution, and solution B was acetonitrile;

[0019] Elution mode: gradient elution;

[0020] Flow rate: 1.0 mL min -1 ;

[0021] Detection wavelength: 325nm;

[0022] Injection volume: 5 μL.

[0023] Preferably, the particle size of the lily powder is 40 mesh.

[0024] Preferably, the concentration of ethanol in step (2) is 70 wt%.

[0025] Preferably, the ultrasonic extraction time in step (2) is 120 min; and the reflux time is 2 h.

[0026] Preferably, the volume ratio of water to methanol in the methanol aqueous solution in step (3) is 3:7.

[0027] Preferably, the gradient elution program is shown in Table 1:

[0028] Table 1 Gradient elution program

[0029]

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention utilizes high performance liquid chromatography to detect lily alcohol extracts, and by adjusting extraction parameters and chromatographic analysis parameters, accurately detects the content of lilyside C and lilyside F in lily. The present invention is simple and easy to perform, and has accuracy, repeatability, and reliability. It has a certain reference value for determining the content of lilyside C and lilyside F in lily, and also provides a reference for lily quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the chemical structural formula of lilyside C;

[0033] Figure 2 is the chemical structural formula of lilyoside F;

[0034] Figure 3 This is the standard curve of lilyoside C;

[0035] Figure 4 This is the standard curve of Wangliuside F;

[0036] Figure 5 This is the chromatogram of the standard products of lilyoside C and lilyoside F;

[0037] Figure 6 is the chromatogram of the sample to be tested. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the present invention more clearly apparent, preferred embodiments of the present invention are further described in detail below in conjunction with the following examples. The materials and reagents used in the present invention are as follows: Reference substances, Wangbaiheoside C (Batch No.: PSO12325) and Wangbaiheoside F (Batch No.: PS012304), were purchased from Chengdu Pusi Biotechnology Co., Ltd.; 11 lilies from different origins were sourced from Baise, Guangxi; Qingyuan, Lishui, Zhejiang; Yixing, Jiangsu; Ganzhou, Jiangxi; Lanzhou, Gansu; Longshan, Xiangxi; Kunming, Sichuan; Lu'an, Anhui; Aba, Yunnan; Nanyang, Henan; and Longhui, Shaoyang. Methanol, specifically for high-performance liquid chromatography, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; acetonitrile, specifically for high-performance liquid chromatography, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; water was Wahaha purified water; and all other reagents were of analytical grade.

[0039] Example 1

[0040] A method for simultaneously determining the contents of lilyside C and lilyside F in lily comprises the following steps:

[0041] (1) Pretreatment of lily raw materials

[0042] The lily raw material is dried and then crushed through a 40-mesh sieve to obtain lily powder;

[0043] (2) Preparation of lily extract

[0044] The lily powder is added to 70 wt% ethanol and subjected to ultrasonic extraction for 120 minutes; the mixture is then heated under reflux for 2 hours and filtered to obtain a filtrate and a filter residue; the filter residue is added to 70 wt% ethanol and subjected to heating under reflux for 2 hours, filtered, and the two filtrates are combined; the combined filtrate is subjected to reduced pressure distillation to recover the solvent, to obtain a lily extract;

[0045] (3) Preparation of test samples

[0046] The lily extract was added to a methanol aqueous solution (the volume ratio of water to methanol was 3:7) for dissolution, and after centrifugation, the supernatant was filtered through a 0.45 μm microporous filter membrane to obtain a sample to be tested;

[0047] (4) Preparation of standard solution

[0048] Weigh lilyside C and lilyside F, and dissolve them in methanol to prepare lilyside C standard solution and lilyside F standard solution respectively;

[0049] (5) Liquid chromatography detection

[0050] The sample to be tested is quantitatively detected by high performance liquid chromatography, wherein the chromatographic conditions are as follows:

[0051] The chromatographic column was ZORBAX SB-C18, with specifications of 4.6 mm × 150 mm, 5 μm;

[0052] Column temperature 35°C;

[0053] In the mobile phase, solution A was 0.1 wt% phosphoric acid aqueous solution, and solution B was acetonitrile;

[0054] Elution method: gradient elution, the elution program is shown in Table 1;

[0055] Flow rate: 1.0 mL min -1 ;

[0056] Detection wavelength: 325nm;

[0057] Injection volume: 5 μL.

[0058] The present invention uses standard products to conduct chromatographic experiments to draw standard curves of lilyside C and lilyside F, respectively. Figure 3 and Figure 4 shown.

[0059] from Figure 3and Figure 4 The regression equation of lilyside C can be obtained as y=131915x+379041, and the linear range is 0.051~147μg·mL -1 , correlation coefficient r=0.9998; regression equation of lilyside F y=213637x+308459, linear range is 0.050~145μg·mL -1 , correlation coefficient r=0.9993. The experimental results showed that the peak area of ​​royaside C and royaside F had a good linear relationship with their concentration.

[0060] The standards were chromatographically tested, e.g. Figure 5 As shown, the sample solution to be tested is measured under the same chromatographic conditions, and its chromatogram is as shown Figure 6 The results showed that the separation effect of lilioside C and lilioside F in the sample was good compared with other components, and the retention times of lilioside C and lilioside F in the reference solution and the sample solution were consistent.

[0061] By comparing the gradient elution of methanol-water, methanol-0.1% phosphoric acid water, acetonitrile-water, and 0.1% phosphoric acid water-acetonitrile in different proportions, it was shown that the separation effect and peak shape of 0.1% phosphoric acid water-acetonitrile as the mobile phase were better. Different proportion gradients were investigated for this mobile phase, among which 0.1% phosphoric acid water-acetonitrile (0-5 min, 10%-12.5% B; 5-15 min, 12.5%-20% B; 15-20 min, 20%-90% B; 20-24 min, 90%-10% B; 24-29 min, 10%-10% B) did not show peaks when eluting with 0.1% phosphoric acid water-acetonitrile (0-15 min, 3%-10% B; 15- When gradient elution was performed with 0.1% aqueous phosphoric acid-acetonitrile (0-20 min, 10%-90% B; 20-22 min, 90%-10% B; 22-27 min, 10%-10% B), the peak shape of lilyoside F was poor; when gradient elution was performed with 0.1% aqueous phosphoric acid-acetonitrile (0-20 min, 10%-90% B; 20-22 min, 90-10% B; 22-27 min, 10-10% B), the peak shape separation effect of lilyoside C and lilyoside F was better, so this gradient was used as the elution condition.

[0062] The present invention further investigates the precision of the instrument. The specific testing method is to mix standard solutions of lirioside C and lirioside F to obtain a mixed solution, wherein the concentration of lirioside C in the mixed solution is 4.594 μg / mL; the concentration of lirioside F is 4.531 μg / mL. Automatic injection of 5 μL is performed under the chromatographic conditions of Example 1, and the injection is repeated 6 times. The peak areas of lirioside C and lirioside F are recorded, and the RSD values are calculated to investigate the precision of the instrument, as shown in Table 2.

[0063] Table 2 Precision investigation (n=6)

[0064]

[0065] As can be seen from Table 3, the RSD values of lilioside C and lilioside F are 1.62% and 2.48%, respectively, indicating that the precision of the instrument is good.

[0066] Some examples of the present invention also examined the stability of the sample solution. Specifically, 20 g of lily powder from Ganzhou, Jiangxi Province, was accurately weighed into a round-bottom flask. Samples were injected automatically at 0, 2, 4, 6, 8, 12, and 24 hours according to the chromatographic conditions of Example 1. 5 μL of the sample was then injected automatically. The peak areas of lilyside C and lilyside F were recorded, and the RSD values were calculated, as shown in Table 3.

[0067] Table 3 Stability study (n=7)

[0068]

[0069] As can be seen from Table 3, the RSD values of lilioside C and lilioside F at the 7 time points after the test samples were treated were all <2%, indicating that the test sample solutions prepared according to this test method have good stability.

[0070] Some embodiments of the present invention also investigated the repeatability of the samples to be tested. Specifically, 20 g of each of 6 portions of lily powder from Ganzhou, Jiangxi Province was accurately weighed into a round-bottom flask. Chromatographic analysis was performed according to the method of Example 1. Each of the 6 portions of the test solution was continuously injected 3 times, with 5 μL automatically injected each time. The peak areas of lilyside C and lilyside F were recorded, and the RSD values were calculated, as shown in Table 4.

[0071] Table 4 Repeatability study (n=6)

[0072]

[0073] As can be seen from Table 4, the peak area RSD values of the six parallel samples of lilioside C and lilioside F were all <1%, indicating that the test solution prepared by this method had good repeatability.

[0074] Some embodiments of the present invention investigated the recovery rate of sample addition. Specifically, 6 parts of lily powder were removed, and standard products of lilyside C and lilyside F were added according to 100% of the content of lilyside C and lilyside F in the lily sample. The sample solution to be tested was prepared according to this test method, and chromatographic analysis was performed according to the method of Example 1. The peak areas of lilyside C and lilyside F were measured and the recovery rate of sample addition was calculated, as shown in Table 5.

[0075] Table 5: Recovery rate of sample addition (Wangliuside C and Wangliuside F) (n=6)

[0076]

[0077]

[0078] As can be seen from Table 5, the average recoveries of lilioside C and lilioside F were 96.23% and 107.83%, respectively, indicating that the method was accurate and reliable.

[0079] Example 2

[0080] The content of lilyside C and lilyside F in 11 lily samples from different origins was determined by the method of Example 1. The results are shown in Table 6.

[0081] Table 6 Sample content determination results

[0082]

[0083]

[0084] Note: “ / ” means not detected

[0085] As shown in Table 6, the contents of lilies from 11 origins were relatively low (less than 0.4 mg·g -1 ), and except for the content of lilyside C in Guangxi Baise which was lower than that of lilyside F, the content of lilyside C in the other 10 lilies from different production areas was higher than that of lilyside F. Among them, the content of lilyside C was relatively high in the following lilies: -1 ), Xiangxi Longshan lily (0.2762 mg g -1 ), Jiangxi Ganzhou lily (0.2540 mg g -1 ), the contents of lilyside C were relatively low in Shaoyang Longhui lily (0.0119 mg·g -1 ), Guangxi Baise lily (0.0152 mg·g -1 ), Gansu Lanzhou lily (0.0859 mg g -1 The content of lilyoside F is relatively high in: Anhui Lu'an lily (0.1344 mg·g -1 ), Jiangxi Ganzhou lily (0.1247 mg g -1 ), Henan Nanyang lily (0.1242 mg g -1 ), the contents of lilyside F were relatively low in Guangxi Baise lily (0.0244 mg·g -1 ), Gansu Lanzhou lily (0.0744 mg g -1 ), Jiangsu Yixing lily (0.0781 mg g -1 ), among which Wanglioside F was not detected in Shaoyang Longhui lily.

[0086] The results of this experiment indicate that the content of roquetiaside C and roquetiaside F in lilies from different origins varies slightly. This variation is attributed to both natural and human factors. Natural climate conditions, geological environment, and the duration, intensity, and quality of light exposure all influence lily growth. Different regions have distinct cultures and cultivation methods, which also affect lily growth. Differences in the natural environment and cultivation techniques during lily growth directly contribute to differences in the content of lily's active ingredients. Furthermore, the active ingredients in lilies decrease during the processing of lilies, even affecting their composition. Therefore, different processing methods indirectly affect their content. Given the differences in active ingredients in lilies, analyzing these differences can help us further investigate and optimize the growing environment of lily medicinal materials, as well as improve production management, harvesting time, processing methods, and storage conditions, thereby standardizing the quality of lilies in the medicinal material market.

[0087] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining the contents of lilyside C and lilyside F in lily, characterized in that: The following steps are involved: (1) Lily raw material pretreatment The lily raw material is dried, crushed and sieved to obtain lily powder; (2) Preparation of lily extract The lily powder is added to ethanol and subjected to ultrasonic extraction; the lily powder is then heated under reflux and filtered to obtain a filtrate and a filter residue; the filter residue is added to ethanol and subjected to heating under reflux, filtered, and the two filtrates are combined; the combined filtrate is subjected to reduced pressure distillation to recover the solvent, to obtain a lily extract; (3) Preparation of test samples The lily extract is added to a methanol aqueous solution for dissolution, and after centrifugation, the supernatant is filtered through a microporous filter membrane to obtain a sample to be tested; (4) Preparation of standard solution Weigh lilyside C and lilyside F, and dissolve them in methanol to prepare lilyside C standard solution and lilyside F standard solution respectively; (5) Liquid chromatography detection The sample to be tested is quantitatively detected by high performance liquid chromatography, wherein the chromatographic conditions are as follows: The chromatographic column was ZORBAX SB-C18, with specifications of 4.6 mm × 150 mm, 5 μm; Column temperature 35°C; In the mobile phase, solution A was 0.1 wt% phosphoric acid aqueous solution, and solution B was acetonitrile; Elution mode: gradient elution; Flow rate: 1.0 mL min -1 ; Detection wavelength: 325nm; Injection volume: 5 μL.

2. The method for simultaneously determining the contents of lilyside C and lilyside F in lily according to claim 1, characterized in that: The particle size of the lily powder is 40 mesh.

3. The method for simultaneously determining the contents of lilyside C and lilyside F in lily according to claim 1, characterized in that: The concentration of ethanol in step (2) is 70wt%.

4. The method for simultaneously determining the contents of lilyside C and lilyside F in lily according to claim 1, characterized in that: The ultrasonic extraction time in step (2) is 120 min; the reflux time is 2 h.

5. The method for simultaneously determining the contents of lilyside C and lilyside F in lily according to claim 1, characterized in that: The volume ratio of water to methanol in the methanol aqueous solution in step (3) is 3:

7.

6. The method for simultaneously determining the contents of lilyside C and lilyside F in lily according to claim 1, characterized in that: The gradient elution procedure is as follows:

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