Method for determining flavonoid aglycone content of sea buckthorn leaf extract by acid hydrolysis
By converting flavonoid glycosides into aglycones through acid hydrolysis and combining it with high performance liquid chromatography, the accuracy and cost issues of quantitative analysis of flavonoids in sea buckthorn leaves have been solved, and efficient detection of quercetin, kaempferol, and isorhamnetin has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
- Filing Date
- 2026-02-09
- Publication Date
- 2026-06-26
AI Technical Summary
Existing technologies are insufficient for accurately and economically determining the content of flavonoids in sea buckthorn leaves, especially the quantitative analysis of flavonoid glycosides, which suffers from high costs and poor reproducibility.
Acid hydrolysis was used to convert flavonoid glycosides in sea buckthorn leaf extract into corresponding aglycones, and quantitative analysis was performed by high performance liquid chromatography. A C18 column, a specific mobile phase, and a detection wavelength were used to eliminate interference from coexisting components and reduce dependence on reference standards.
This method enables accurate quantification of quercetin, kaempferol, and isorhamnetin in sea buckthorn leaves, improving the specificity and accuracy of detection while reducing detection costs.
Smart Images
Figure CN122282979A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of detection technology, specifically relating to a method for determining the content of flavonoid aglycones in acid-hydrolyzed sea buckthorn leaf extract. Background Technology
[0002] The fruit of Hippophae rhamnoides L. is widely recognized for its high nutritional value, and its leaves are also rich in various bioactive components, showing broad development potential. Studies have shown that flavonoids are among the important functional components of sea buckthorn leaf extract, possessing various biological activities such as antioxidant, anti-inflammatory, immunomodulatory, antitumor, lipid-lowering, and insulin sensitivity-improving effects. However, a systematic, efficient, and low-cost quality control method for sea buckthorn leaf extract is lacking.
[0003] Accurately determining the content of flavonoids in sea buckthorn leaves is a crucial step in evaluating the quality of the raw materials and the efficacy of the products. Currently, the main methods for detecting flavonoids in sea buckthorn leaves include spectroscopic methods, chromatographic methods, and electrochemical analysis methods. Among them, ultraviolet spectrophotometry (UV) is simple to operate and low in cost, but because it is based on the colorimetric reaction of total flavonoids with specific reagents, it is easily affected by other coexisting components in the sample (such as polyphenols and pigments). Furthermore, it usually uses a single reference standard (such as rutin) as the standard for content calculation, resulting in insufficient accuracy and specificity, making it difficult to reflect the true flavonoid composition in the sample. High-performance liquid chromatography (HPLC) can accurately quantify single flavonoid components, with advantages such as high separation efficiency and high sensitivity. However, it faces challenges when directly analyzing flavonoids in sea buckthorn leaves: flavonoids in sea buckthorn leaves mainly exist in various chemically similar glycoside forms (such as isorhamnetin-3-O-rutin glycoside, quercetin glycoside, etc.). These glycoside monomer reference standards are expensive and difficult to obtain, increasing detection costs and technical barriers. At the same time, the chromatographic separation of various glycosides is difficult, resulting in complex method development and poor reproducibility.
[0004] Therefore, there is an urgent need in this field for a method to detect flavonoids in sea buckthorn leaf extract that can balance accuracy, economy, and practicality. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a method for determining the content of flavonoid aglycones in acid-hydrolyzed sea buckthorn leaf extract. The method provides accurate, specific and stable results. Another technical problem to be solved by the present invention is to provide a method for preparing a test solution of sea buckthorn leaf extract rich in quercetin, kaempferol and isorhamnetin. The method uses acid hydrolysis pretreatment to convert the complex flavonoid glycosides in the sea buckthorn leaf extract into their corresponding aglycones.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0007] A method for determining the flavonoid aglycone content in an acid-hydrolyzed sea buckthorn leaf extract includes the following steps:
[0008] 1) The sea buckthorn leaf extract was subjected to acid hydrolysis to obtain a filtrate containing flavonoid aglycones, which was used as the test solution;
[0009] 2) The content of flavonoid aglycones in the test solution and the reference solution was determined by high performance liquid chromatography (HPLC).
[0010] The flavonoid aglycones are quercetin, kaempferol, and isorhamnetin; the acid hydrolysis step is as follows: the sea buckthorn leaf extract is mixed with methanol solution and hydrochloric acid solution, heated under reflux, cooled, methanol is added, shaken well, filtered, and the filtrate is collected.
[0011] Furthermore, the methanol solution is a 70% methanol solution by volume, and the hydrochloric acid solution is a 25% hydrochloric acid solution by volume.
[0012] Furthermore, the temperature of the heating reflux is 100°C, and the reflux time is 2 hours.
[0013] Furthermore, the filter material used in the filtration is a 0.45μm microporous filter membrane.
[0014] Furthermore, the concentrations of quercetin, kaempferol, and isorhamnetin in the reference solution are all 1.4–9.0 μg / mL, and the reference solution is prepared using methanol as the solvent.
[0015] Furthermore, the chromatographic column of the high-performance liquid chromatograph is C18. 18 The mobile phase is a packing material; mobile phase A is a 0.4% phosphoric acid solution, and mobile phase B is methanol; the ratio of mobile phase A to mobile phase B is 50:50; the detection wavelength is 367 nm.
[0016] Furthermore, the theoretical plate number of the high-performance liquid chromatograph, calculated based on the quercetin peak, is not less than 2500.
[0017] Furthermore, the standard curve for quercetin is y = 90.866x - 5.821, R0 2 The value is 0.9995; the standard curve for kaempferol is y = 82.695x + 1.2018, R0. 2 The value is 0.9991; the standard curve for isorhamnetin is y = 77.313x - 0.6901, R0 = 0.9991. 2 It is 0.9996.
[0018] A method for preparing a test solution of sea buckthorn leaf extract rich in quercetin, kaempferol, and isorhamnetin, comprising the following steps: Acid hydrolysis is used to convert flavonoid glycosides in the sea buckthorn leaf extract into quercetin, kaempferol, and isorhamnetin.
[0019] (1) Add 70% methanol solution and 25% hydrochloric acid solution to the sea buckthorn leaf extract, mix well and heat under reflux at 100°C for 2 hours;
[0020] (2) Cool the hydrolysate to room temperature, shake well, and filter through a 0.45 μm microporous membrane to obtain a test solution of sea buckthorn leaf extract rich in quercetin, kaempferol and isorhamnetin.
[0021] Further, the preparation method of the sea buckthorn leaf extract is as follows: sea buckthorn leaves are extracted three times with water as the extraction solvent. The first extraction is carried out at 100°C for 30 minutes, the second extraction is carried out at 100°C for 15 minutes, and the third extraction is carried out at 100°C for 20 minutes. The extracts are combined, concentrated, and refrigerated. The refrigerated sea buckthorn leaf concentrate is stirred and heated to 60°C, maltodextrin is added, and the mixture is stirred and heated to 100°C for 10 minutes to make it uniform. Then, it is spray-dried to obtain the sea buckthorn leaf extract.
[0022] Compared with the prior art, the present invention has the following advantages:
[0023] (1) This invention improves the specificity and accuracy of detection: Compared with the traditional ultraviolet spectrophotometry, this invention transforms the non-specific total determination into aglycone quantitative analysis based on a well-defined chemical structure through hydrolysis pretreatment combined with high-performance liquid chromatography. This method effectively eliminates the interference of coexisting pigments and polyphenols, and the determination results have clear chemometric significance, providing a reliable quantitative basis for quality evaluation.
[0024] (2) This invention overcomes the technical bottleneck of reliance on reference standards: Addressing the problem that direct chromatography requires multiple expensive and difficult-to-obtain flavonoid glycoside reference standards, this invention establishes a standardized acid hydrolysis conversion system to convert complex glycoside mixtures into a limited number of aglycones (mainly isorhamnetin, quercetin, and kaempferol). This innovative strategy reduces the number of reference standards required to 3-4 common aglycones, significantly reducing detection costs while ensuring quantitative accuracy. Attached Figure Description
[0025] Figure 1 This is a chromatogram of the reference standards for quercetin, kaempferol, and isorhamnetin from Example 4 of this application;
[0026] Figure 2 The chromatogram of the test sample in Example 4 of this application;
[0027] Figure 3 This is a linear regression diagram of quercetin in Example 6 of this application;
[0028] Figure 4 This is a linearity curve of kaempferol in Example 6 of this application;
[0029] Figure 5 This is a linear regression diagram of isorhamnetin in Example 6 of this application. Detailed Implementation
[0030] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
[0031] The chemical structural formulas of the three flavonoids in the following examples are as follows:
[0032]
[0033] Example 1
[0034] Sea buckthorn leaves were dried in an oven at 50℃, then pulverized into powder using a grinder and sealed for later use. 100g of the powder was accurately weighed, added to 600mL of water, and refluxed at 100℃ for three extractions, each time adding 600mL of water and extracting for 2 hours. The filtrates were combined. The extract was concentrated under reduced pressure at 60℃, and then dried in a 70℃ water bath until viscous. It was then transferred to a 70℃ vacuum drying oven and dried for 6 hours to obtain lumpy sea buckthorn leaf extract. The lumpy sea buckthorn leaf extract was ground into powder using a fully automated rapid sample grinder, and the yield was calculated. Three batches were prepared, and the results are shown in Table 1, with an average yield of 17.8%.
[0035] Table 1. Yield of sea buckthorn leaf extract in small-scale preparation
[0036]
[0037] Example 2
[0038] Sea buckthorn leaves were dried in an oven at 50℃, then pulverized into powder using a grinder and sealed for later use. 1 kg of sea buckthorn leaf powder was accurately weighed, and 8 L of water was added and refluxed at 100℃ for three extractions, each time adding 8 L of water and extracting for 2 hours. The filtrates were combined. The extract was concentrated under reduced pressure at 60℃, and then dried in a 70℃ water bath until viscous. It was then transferred to a 70℃ vacuum drying oven and dried for 6 hours to obtain lumpy sea buckthorn leaf extract. The lumpy sea buckthorn leaf extract was ground into powder using a fully automated rapid sample grinder, and the yield was calculated. Three batches were prepared, and the results are shown in Table 2, with an average yield of 23.9%.
[0039] Table 2. Yield of sea buckthorn leaf extract in small-scale preparation
[0040]
[0041] Example 3
[0042] 476 kg of dried sea buckthorn leaves were extracted twice, 238 kg each time, according to the extraction tank capacity. The 238 kg of sea buckthorn leaves were extracted three times with water as the extraction solvent: the first time with 4000 L of water, extracted at 100℃ for 30 min; the second time with 1500 L of water, extracted at 100℃ for 15 min; and the third time with 960 L of water, extracted at 100℃ for 20 min. The extracts were combined and concentrated to 600 kg, then temporarily stored in a transfer tank under cold storage. The 600 kg of concentrated sea buckthorn leaf extract was stirred and heated to 60℃, then 6.5 kg of maltodextrin was added, and the mixture was stirred and heated to 100℃ for 10 min to ensure homogeneity. The mixture was then transferred to a buffer tank for spray drying. The spray drying parameters were set as follows: inlet air temperature 215℃, outlet air temperature 80℃. The resulting sea buckthorn leaf extract product had uniform and fine particles with good flowability, yielding 117 kg of sea buckthorn leaf water extract powder, with a yield of 24.6%.
[0043] Example 4
[0044] 1) Instrument and Sample Selection: The liquid chromatograph was an Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA); quercetin reference standard (batch number 47HHRENQ, purity: >95%) was purchased from Shanghai Anaiji Chemical Co., Ltd.; kaempferol reference standard (batch number VGDMRREW, purity: >98%) was purchased from Shanghai Anaiji Chemical Co., Ltd.; isorhamnetin reference standard (batch number 82EXWERH, purity: >97%) was purchased from Shanghai Anaiji Chemical Co., Ltd.
[0045] 2) Set up the liquid chromatography conditions: The chromatographic column is a Poroshell 120 EC-C. 18 Column (4.6 × 100 mm, 2.7 μm); Mobile phase A is 0.4% phosphoric acid solution, mobile phase B is methanol, mobile phase A:mobile phase B ratio is 50:50; detection wavelength is 367 nm; column temperature is 25 ℃; flow rate is 0.5 mL / min; injection volume is 10 μL; solvent is methanol; theoretical plate number calculated based on quercetin peak should not be less than 2500.
[0046] 3) Preparation of sample solution:
[0047] Test solution: Accurately weigh 30 mg of sea buckthorn leaf extract powder from Example 3, add 30 mL of a mixed solution of 70% (v / v) methanol and 25% (v / v) hydrochloric acid (5:1 (v / v)); place in a heating mantle and heat under reflux (100 °C). At 0 h, 0.5 h, 1.0 h, 1.5 h, 2.0 h, and 2.5 h, transfer 2 mL to a 10 mL volumetric flask, cool to room temperature, dilute to the mark with methanol, shake well, filter through a 0.45 μm microporous membrane, and use the filtrate as the test sample.
[0048] Reference solution: A mixed solution of quercetin, kaempferol and isorhamnetin, each containing 9 μg / mL, was precisely prepared using methanol.
[0049] 4) Accurately pipette 10 µL of the test solution and the reference solution into the liquid chromatograph, respectively, and record the chromatograms. The results are shown in Table 3 and... Figure 1-2 As shown.
[0050] Table 3. Test results for different hydrolysis times of the test samples
[0051]
[0052] From Table 3 and Figure 1-2 It can be seen that the sum of the peak areas of quercetin, kaempferol and isorhamnetin reached its highest point after 2 hours of treatment, so hydrolysis for 2 hours was chosen for subsequent experiments.
[0053] Example 5
[0054] 1) Instrument and sample selection: Same as in Example 4.
[0055] 2) Set the liquid chromatography conditions: Same as in Example 4.
[0056] 3) Preparation of sample solution:
[0057] Test solution: Accurately weigh 50 mg of sea buckthorn leaf extract powder from Example 3, add 3, 5, 10, and 20 mL of 25% (v / v) hydrochloric acid solution respectively, and then add 70% (v / v) methanol to 40 mL. Place the solution in a heating mantle at 25 °C. After 2 hours, transfer all hydrolysates to a 50 mL volumetric flask, cool to room temperature, dilute to the mark with methanol, shake well, filter through a 0.45 μm microporous membrane, and use the filtrate as the test sample.
[0058] Reference solution: A mixed solution of quercetin, kaempferol and isorhamnetin, each containing 9 μg / mL, was precisely prepared using methanol.
[0059] 4) Accurately pipette 10µL of the test solution and the reference solution into the liquid chromatograph, record the chromatograms, and the results are shown in Table 4.
[0060] Table 4. Test results of different amounts of hydrochloric acid used for hydrolysis of test samples
[0061]
[0062] As shown in Table 4, the sum of the peak areas of quercetin, kaempferol and isorhamnetin reached its maximum when the amount of hydrochloric acid used was 10 mL. Therefore, the amount of hydrochloric acid used was 10 mL for subsequent experiments.
[0063] Example 6
[0064] The detection method was validated in terms of stability, repeatability, linearity, recovery rate, and content detection.
[0065] 1. Stability
[0066] Test solution: Accurately weigh 50 mg of sea buckthorn leaf extract powder, add 10 mL of 25% hydrochloric acid solution, then add 70% (v / v) methanol to 40 mL, place in a heating mantle and heat under reflux (100℃) for 2 h, then transfer all hydrolysate to a 50 mL volumetric flask, cool to room temperature, dilute to the mark with methanol, shake well, filter through a 0.45 μm microporous membrane, and use the filtrate as the test sample;
[0067] Reference solution: A mixed solution of quercetin, kaempferol and isorhamnetin, each containing 9 μg / mL, was precisely prepared using methanol.
[0068] Take the same test solution and inject 10µL at 0h, 2h, 4h, 8h, 16h and 24h respectively. Record the chromatograms according to the above chromatographic conditions and record the area of the components of quercetin, kaempferol and isorhamnetin in the test sample. The results are shown in Table 5.
[0069] Table 5. Stability test results
[0070]
[0071] Table 5 shows that the RSDs of quercetin, kaempferol, and isorhamnetin were 0.4%, 0.9%, and 0.7%, respectively, indicating that the method has good stability.
[0072] 2. Repeatability
[0073] Test solution: Take the same batch of sea buckthorn leaf extract and prepare 6 test solutions according to the test preparation method.
[0074] Reference solution: Take the solution from the stability section.
[0075] Six test samples were prepared from the same batch of filtrate. 10 μL of each sample was injected, and the chromatograms were recorded under the chromatographic conditions described above. The contents of quercetin, kaempferol, and isorhamnetin in the test samples were calculated. The results are shown in Table 6.
[0076] Table 6 Repeatability test results
[0077]
[0078] As shown in Table 6, the RSDs of quercetin, kaempferol, and isorhamnetin were 1.0%, 0.8%, and 0.7%, respectively, indicating that the method had good reproducibility.
[0079] 3. Linear relationship
[0080] Take appropriate amounts of quercetin, kaempferol, and isorhamnetin, and prepare reference solutions with different concentrations of quercetin, kaempferol, and isorhamnetin per 1 mL using methanol (concentrations are shown in Table 7). Accurately pipette 10 μL of each component and inject it into the high-performance liquid chromatograph (HPLC). Record the chromatograms under the chromatographic conditions described above, and record the total peak area of each component. Plot the injection volume of each component on the x-axis (X) and the peak area of each component on the y-axis (Y). The results are shown in Table 8 and... Figure 3-5 .
[0081] Table 7. Preparation of different concentrations of quercetin, kaempferol, and isorhamnetin
[0082]
[0083] Table 8. Linearity determination results of quercetin, kaempferol and isorhamnetin
[0084]
[0085] From Table 8 and Figure 3-5 It can be seen that quercetin, kaempferol, and isorhamnetin exhibit good linearity within the linear range.
[0086] 4. Detection of quercetin, kaempferol and isorhamnetin content in sea buckthorn leaf extract
[0087] Test solution: Take the same batch of sea buckthorn leaf extract and prepare 3 test solutions according to the test solution preparation method.
[0088] Reference solution: Take the solution from the linear relationship section.
[0089] The above-mentioned test samples and reference standards were injected into a high-performance liquid chromatograph, and the chromatograms were recorded according to the above chromatographic conditions. The contents of quercetin, kaempferol, and isorhamnetin were calculated, and the results are shown in Table 9.
[0090] Table 9. Detection results of quercetin, kaempferol and isorhamnetin content in sea buckthorn leaf extract.
[0091]
[0092] As shown in Table 9, the RSDs of quercetin, kaempferol and isorhamnetin in the sample were 4.9%, 5.1% and 5.1%, respectively, indicating that the method has good reproducibility.
[0093] 5. Recovery rate
[0094] Accurately weigh 50 mg of sea buckthorn leaf extract into three portions (quercetin, kaempferol, and isorhamnetin content were 2.902 mg / g, 4.486 mg / g, and 3.734 mg / g, respectively). Add 600 μL of reference solution (300 μg / mL for each portion) to each portion and prepare the test sample according to the test sample preparation method. Record the chromatograms under the above chromatographic conditions and calculate the recovery rates, as shown in Table 10.
[0095] Table 10 Results of Sample Recovery Test
[0096]
[0097] As shown in Table 10, the detection accuracy of quercetin, kaempferol and isorhamnetin in sea buckthorn leaf extract is good.
[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for determining the flavonoid aglycone content in an acid-hydrolyzed sea buckthorn leaf extract, characterized in that: Includes the following steps: 1) The sea buckthorn leaf extract was subjected to acid hydrolysis to obtain a filtrate containing flavonoid aglycones, which was used as the test solution; 2) The content of flavonoid aglycones in the test solution and the reference solution was determined by high performance liquid chromatography (HPLC). The flavonoid aglycones are quercetin, kaempferol, and isorhamnetin; the acid hydrolysis step is as follows: the sea buckthorn leaf extract is mixed with methanol solution and hydrochloric acid solution, heated under reflux, cooled, methanol is added, shaken well, filtered, and the filtrate is collected.
2. The method for determining the flavonoid aglycone content of acid-hydrolyzed sea buckthorn leaf extract according to claim 1, characterized in that: The methanol solution is a 70% methanol solution by volume, and the hydrochloric acid solution is a 25% hydrochloric acid solution by volume.
3. The method for determining the flavonoid aglycone content of acid-hydrolyzed sea buckthorn leaf extract according to claim 1, characterized in that: The heating and reflux temperature is 100℃, and the reflux time is 2 hours.
4. The method for determining the flavonoid aglycone content of acid-hydrolyzed sea buckthorn leaf extract according to claim 1, characterized in that: The filtration material used is a 0.45μm microporous membrane.
5. The method for determining the flavonoid aglycone content of acid-hydrolyzed sea buckthorn leaf extract according to claim 1, characterized in that: The concentrations of quercetin, kaempferol, and isorhamnetin in the reference solution were all 1.4–9.0 μg / mL, and the reference solution was prepared using methanol as the solvent.
6. The method for determining the flavonoid aglycone content of acid-hydrolyzed sea buckthorn leaf extract according to claim 1, characterized in that: The chromatographic column of the high-performance liquid chromatograph is C10. 18 The mobile phase is a packing material; mobile phase A is a 0.4% phosphoric acid solution, and mobile phase B is methanol; the ratio of mobile phase A to mobile phase B is 50:50; the detection wavelength is 367 nm.
7. The method for determining the flavonoid aglycone content of acid-hydrolyzed sea buckthorn leaf extract according to claim 1, characterized in that: The theoretical plate number of the high-performance liquid chromatograph, calculated based on the quercetin peak, shall not be less than 2500.
8. The method for determining the flavonoid aglycone content of acid-hydrolyzed sea buckthorn leaf extract according to claim 1, characterized in that: The standard curve for quercetin is y = 90.866x - 5.821, R0 2 The value is 0.9995; the standard curve for kaempferol is y = 82.695x + 1.2018, R0. 2 The value is 0.9991; the standard curve for isorhamnetin is y = 77.313x - 0.6901, R0 = 0.9991. 2 It is 0.9996.
9. A method for preparing a test solution of sea buckthorn leaf extract rich in quercetin, kaempferol, and isorhamnetin, characterized in that, The flavonoid glycosides in sea buckthorn leaf extract are converted into quercetin, kaempferol, and isorhamnetin using an acid hydrolysis process, specifically including the following steps: (1) Add 70% methanol solution and 25% hydrochloric acid solution to the sea buckthorn leaf extract, mix well and heat under reflux at 100°C for 2 hours; (2) Cool the hydrolysate to room temperature, shake well, and filter through a 0.45 μm microporous membrane to obtain a test solution of sea buckthorn leaf extract rich in quercetin, kaempferol and isorhamnetin.
10. A method for preparing sea buckthorn leaf extract, characterized in that: Sea buckthorn leaves were extracted three times with water as the extraction solvent. The first extraction was carried out at 100℃ for 30 min, the second extraction was carried out at 100℃ for 15 min, and the third extraction was carried out at 100℃ for 20 min. The extracts were combined, concentrated, and temporarily stored under cold. The concentrated sea buckthorn leaf extract was stirred and heated to 60℃, maltodextrin was added, and the mixture was stirred and heated to 100℃ for 10 min to ensure uniform mixing. The mixture was then spray-dried to obtain sea buckthorn leaf extract powder.