Coreopsis tinctoria flavone extract and preparation method thereof
By combining the ethanol reflux extraction and AB-8 resin purification process with a hot air drying process of 70-80°C, the problem of the existing drying method having a great impact on the flavonoid components of chrysanthemum is solved, and high-quality flavonoid extract preparation is achieved.
Patent Information
- Application Number
- CN202411788942.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-03-14
AI Technical Summary
The existing drying methods have a great impact on the flavonoid components in the chrysanthemum, resulting in significant differences in the quality of the dry products, making it difficult to retain flavonoids to the maximum extent while ensuring the yield.
The ethanol reflux extraction process and AB-8 type macroporous resin purification process were used to extract serry flavonoids, and the extract was processed through a hot air drying process of 70-80°C to maximize the retention of flavonoid components.
It is achieved by minimizing the flavonoid components in the snow chrysanthemum while ensuring the yield rate, and improving the quality of the dry product.
Smart Images
Figure CN119607044A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of flavonoid extraction, in particular to a snow chrysanthemum flavonoid extract and a preparation method thereof. Background Art
[0002] Snow chrysanthemum is the flower of Coreopsis tinctoria Nutt., a plant of the Asteraceae family. It can be used as a tea, which has the effects of clearing away heat and detoxifying, promoting blood circulation and removing blood stasis. It can be used to treat high blood pressure, high blood lipids, gastrointestinal discomfort, dysentery, and sores, boils, and swellings. Recent studies have shown that flavonoids in snow chrysanthemum are its main active ingredients.
[0003] At present, the common drying methods for Chinese medicine extracts mainly include hot air drying, vacuum drying, freeze drying, spray drying, etc. Studies have shown that different drying methods and temperatures have different degrees of influence on flavonoid components, resulting in significant differences in the final quality of the dried products. Ao Fen et al. (2022, Chinese Patent Medicine) found that the transfer rate of quercetin in hot air dried geranium was the highest, and the transfer rate continued to increase with the increase in temperature. The quercetin transfer rate of geranium increased continuously at 40-50°C by vacuum drying and hot air vacuum combined drying, but as the temperature continued to rise, the quercetin transfer rate showed a downward trend. The quercetin transfer rate of freeze-dried geranium was second, and microwave drying was the lowest. Summary of the invention
[0004] The purpose of the present invention is to provide a snow chrysanthemum flavonoids extract and a preparation method thereof, in particular to provide a method for drying the snow chrysanthemum flavonoids extract, which can retain the flavonoids therein as much as possible while ensuring the yield.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a method for preparing a snow chrysanthemum flavonoid extract, comprising the following steps:
[0007] (1) extracting snow chrysanthemum with ethanol solution under reflux, collecting the extract, and concentrating it until there is no alcohol taste, thereby obtaining a crude extract of snow chrysanthemum flavonoids;
[0008] (2) diluting the crude extract of snow chrysanthemum flavonoids and purifying it with AB-8 macroporous resin to obtain a purified snow chrysanthemum flavonoids solution;
[0009] (3) After the purified snow chrysanthemum flavonoids solution is concentrated under reduced pressure, the concentrated solution is collected and hot-air dried to obtain a snow chrysanthemum flavonoids extract.
[0010] Preferably, the concentration of the ethanol solution is 60-70%.
[0011] Preferably, the mass volume ratio of the snow chrysanthemum to the ethanol solution is 1 g: 60-70 mL.
[0012] Preferably, the temperature of the reflux extraction in step (1) is 75-85° C., the time is 20-30 min, and the extraction is repeated 2-4 times.
[0013] Preferably, the dilution is dilution with water, and the loading concentration is 3-4 mg / mL based on the crude drug amount.
[0014] Preferably, during purification with the AB-8 macroporous resin, the sample loading amount is 6 to 7 BV, the loading flow rate is 0.5 to 0.6 BV / h, after adsorption, 1 to 2 BV of purified water is used for impurity removal, the flow rate is 0.5 to 0.6 BV / h, after impurity removal, 45 to 55% ethanol solution is used for desorption, the amount is 5 to 6 BV, the flow rate is 0.5 to 0.6 BV / h, the eluate is collected to obtain a purified snow chrysanthemum flavonoid solution.
[0015] Preferably, the temperature of the reduced pressure concentration is 45-50°C.
[0016] Preferably, the hot air drying temperature is 70-80°C.
[0017] The invention also provides a snow chrysanthemum flavonoids extract obtained according to the preparation method.
[0018] The preparation method of the snow chrysanthemum flavonoid extract provided by the present invention obtains the total flavonoids of snow chrysanthemum through an ethanol reflux extraction process and an AB-8 resin purification process, and adopts a 70-80°C hot air drying process to retain the flavonoid components therein to the maximum extent. The flavonoid content in the crude extract obtained by the ethanol reflux extraction process of the present invention is 12.64%, the transfer rate of flavonoids after resin purification is 86.84%, the extract yield under the 80°C hot air drying process is 15.27%, and the total flavonoid content of snow chrysanthemum is 703.46 mg / mg. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 These are the results of the specificity investigation. A is the mixed reference substance, B is the test substance of snow chrysanthemum flavonoids, 1 is quercetin, 2 is marigold glycoside, and 3 is luteolin.
[0020] Figure 2 This is an overlay of fingerprint spectra of snow chrysanthemum flavonoid samples with different drying processes.
[0021] Figure 3 Cluster heat map of total flavonoids samples of snow chrysanthemum with different drying processes. DETAILED DESCRIPTION
[0022] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0023] Example 1
[0024] 1. Instruments and Materials
[0025] 1.1 Instrument
[0026] Table 1 List of experimental instruments
[0027]
[0028]
[0029] Snow chrysanthemum was purchased from Xinjiang Huacao Pharmaceutical Technology Co., Ltd., batch number: 20171026, and was identified as the capitula of Coreopsis tinctoria Nutt. of the genus Coreopsis in the Asteraceae family.
[0030] Pretreatment and regeneration of macroporous resin: Pretreatment: Take AB-8 macroporous resin, add 95% ethanol and soak for 24 hours, then elute with distilled water after column loading until there is no alcohol smell and it is not turbid when mixed with water. Regeneration: Elute with 4BV of 95% ethanol, then elute with water until there is no alcohol smell. If necessary, elute with 4BV of 95% ethanol, then soak with 4% hydrochloric acid solution for 4 hours, wash with distilled water until neutral, then soak with 4% sodium hydroxide solution for 4 hours, wash with distilled water until neutral.
[0031] 2 Methods and Results
[0032] 2.1 Establishment of the determination method of total flavonoids in snow chrysanthemum
[0033] 2.1.1 Preparation of snow chrysanthemum flavonoids extract
[0034] Weigh 5g of snow chrysanthemum, add 60% ethanol solution according to the material-liquid ratio of 1g:67mL, extract under water bath reflux at 80℃ for 21min, repeat 3 times, combine the extracts to obtain a filtrate. The filtrate is evaporated and concentrated until there is no alcohol taste, and diluted with water to a loading concentration of 4mg / mL (based on the amount of crude drug). Wet-pack AB-8 type macroporous resin 200mL, take 7BV of the above diluted solution for loading, the loading liquid flow rate is 0.6BV / h, after adsorption, remove impurities with 1BV purified water, the flow rate is 0.6BV / h, and finally elute with 5BV of 50% ethanol, the flow rate is 0.6BV / h, collect the eluate, and obtain a purified snow chrysanthemum flavonoids liquid. Take the above purified snow chrysanthemum flavonoids liquid, concentrate under reduced pressure at 50℃, and collect the concentrated liquid when the amount of evaporated solvent is significantly reduced and the liquid does not stick to the wall. The concentrated liquid is dried with hot air at 80℃ and dried to constant weight to obtain the snow chrysanthemum flavonoids extract.
[0035] 2.1.2 Determination of total flavonoids in snow chrysanthemum by UV-Vis method
[0036] (1) Preparation of reference solution
[0037] Take an appropriate amount of rutin reference substance, weigh it accurately, add 60% ethanol to dissolve it, and prepare a reference substance solution with a concentration of 201 μg / mL.
[0038] (2) Preparation of test solution
[0039] Take 20 mg of the snow chrysanthemum flavonoid extract prepared under 2.1.1, accurately weigh it, put it into a 25 mL volumetric flask, add 5 mL of 60% ethanol, ultrasonically treat (power 200 W, frequency 40 kHz) for 10 min, then make up the volume with methanol, shake well, and obtain.
[0040] (3) Determination of total flavonoids in snow chrysanthemum
[0041] The rutin reference solution (concentration of 201 μg / mL) was prepared into 6 reference solutions of different concentrations, and the linear relationship was investigated, and the regression equation was Y=0.0257X-0.0188 (4.02-32.16 μg / mL, r=0.9996). The methodological results showed that the RSD of precision, stability, and repeatability were all less than 1.25%, the average recovery rate was 100.03%, and the RSD was 0.54%. The test solution was taken, 60% ethanol was diluted to 25mL, the triethylamine method was used for color development, the absorbance was measured at 406nm, and the rutin standard curve was substituted to calculate the total flavonoid content of snow chrysanthemum.
[0042] 2.1.3 HPLC determination of the content of index components in total flavonoids of snow chrysanthemum
[0043] (1) Chromatographic conditions
[0044] Yilite SinoChrom ODS-BP C18 chromatographic column (4.6mm×250mm, 5μm), acetonitrile (A)-0.1% phosphoric acid (B) as mobile phase, gradient elution (0-5min, 5%→25%A; 5-18min, 25%→28%A; 18-23min, 28%→44%A; 23-30min, 44%→100%A; 30-35min, 100%A), detection wavelength 370nm, column temperature 30℃, flow rate 0.6mL / min, injection volume 20μL.
[0045] (2) Preparation of reference solution
[0046] Take appropriate amount of mariglycoside, quercetin and luteolin reference substances, weigh accurately, dissolve in methanol, and prepare mixed reference substance solutions with concentrations of 354, 79 and 63 μg / mL, respectively. Filter through a 0.22 μm microporous membrane to obtain the solution.
[0047] (3) Preparation of test solution
[0048] Same as the preparation method of test solution in item 2.1.2 (2). Filter through 0.22μm microporous membrane to obtain the product.
[0049] (4) Specificity inspection
[0050] Take the reference solution and the test solution respectively, inject and analyze, and record the chromatogram ( Figure 1 ). The results showed that the chromatographic peaks had good shape and good separation.
[0051] (5) Determination of the content of mariglycoside, quercetin and luteolin
[0052] Five mixed reference solutions of mariglycoside, quercetin and luteolin (concentrations were 354, 79 and 63 μg / mL, respectively) were prepared and injected according to the chromatographic conditions in (1) to investigate the linear relationship. The regression equations of mariglycoside, quercetin and luteolin were Y=106.55X+17.348 (3.54-354.00 μg / mL, r=0.9998), Y=74.668X-34.196 (0.79-79.00 μg / mL, r=0.9991) and Y=73.221X-13.743 (0.63-63.00 μg / mL, r=0.9997), respectively. The methodological results showed that the RSD of precision of mariglycoside, quercetin and luteolin were all less than 0.64%, the RSD of stability were all less than 1.10%, the RSD of repeatability were all less than 2.90%, and the average recovery rates were 99.49%, 100.30% and 101.60%, respectively, and the RSDs were all less than 2.75%.
[0053] Take the test solution under item (3), inject and analyze it according to the chromatographic conditions in (1), substitute it into the above standard curve, and calculate the content of marigold, quercetin and luteolin.
[0054] 2.2 Drying technology of total flavonoids from snow chrysanthemum and establishment of fingerprint
[0055] According to the method in 2.1.1, 8 g of snow chrysanthemum was prepared as a concentrate and divided into 8 portions, one of which was left untreated (NS1), and the other 7 portions were subjected to 60°C hot air drying (GZ1), 70°C hot air drying (GZ2), 80°C hot air drying (GZ3), 50°C vacuum drying (GZ4), 60°C vacuum drying (GZ5), 70°C vacuum drying (GZ6), and freeze drying (GZ7), respectively, dried to constant weight, and the dry extract was collected.
[0056] 2.2.1 Preparation of test solution
[0057] Take snow chrysanthemum flavonoids obtained by different drying processes, grind them through a No. 9 sieve, and prepare the test solution according to the method in 2.1.2. The concentrate is used to prepare the test solution according to the amount of crude drug.
[0058] 2.2.2 Methodological investigation
[0059] With reference to the 2020 edition of the "Chinese Pharmacopoeia", Peak 8 was used as the reference peak (S) to examine the precision, repeatability and stability; the calculated RSDs of the relative retention times of the common peaks were all less than 1.0%, and the RSDs of the relative peak areas were all less than 4.0%, indicating that the instrument precision and method repeatability were good, and the test solution was stable within 24 hours.
[0060] 2.2.3 Establishment of fingerprint
[0061] Take the test solution of 8 total flavonoids samples of snow chrysanthemum, and analyze them according to the chromatographic conditions under 2.1.3. Import the spectrum into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 Edition), use NS1 as the reference spectrum, use the average method, and the time window is 0.1min. After multi-point calibration and matching with the common peak, generate a reference spectrum (GZR), see Figure 2 A total of 15 common peaks were identified in the fingerprints of 8 snow chrysanthemum total flavonoids samples. Compared with the reference substances, quercetin (peak 5), marigold glycoside (peak 8), and luteolin (peak 15) were identified.
[0062] 2.2.4 Similarity Analysis
[0063] The fingerprints were evaluated for similarity using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition). The results showed that compared with GZR, the similarities of the samples GZ1, GZ2, GZ3, GZ4, GZ5, GZ6, and GZ7 were 0.953, 0.966, 0.980, 0.996, 0.986, 0.994, and 0.998, respectively, with a similarity range of 0.953-0.998; compared with the NS1 spectrum, the similarities of GZ1, GZ2, GZ3, GZ4, GZ5, GZ6, and GZ7 were 0.869, 0.891, 0.917, 0.991, 0.928, 0.994, and 0.966, respectively, with a similarity range of 0.869-0.994, indicating that the composition of snow chrysanthemum flavonoids before and after drying was consistent.
[0064] 2.2.5 Cluster heat map analysis
[0065] The peak areas of the 15 common peaks in the fingerprint were imported into Origin 2021 software, and the common peak areas were standardized. The “average” clustering method was used, and “Euclidean” was used as the metric to generate a cluster heat map (the colors in the figure range from dark red to dark blue, representing the common peak areas from large to small), see Figure 3 .
[0066] The heat map shows that the 15 common peaks can be clustered into 2 categories. Peaks 1, 5, 8, 10, 12, and 2 are clustered into 1 category (category a), including mariglycoside, quercetin, and other components, with a total peak area of about 80% and a relatively high content of the components; the remaining peaks are clustered into 1 category (category b), including luteolin and other components, with a low total peak area and a relatively low content of the components. The 7 snow chrysanthemum total flavonoids samples can be clustered into 4 categories, GZ2 and GZ3 are clustered into 1 category, GZ5 and GZ6 are clustered into 1 category, GZ4 and GZ1 are clustered into 1 category, and GZ7 is clustered into 1 category alone. The contents of mariglycoside and quercetin in samples with the same drying method decreased continuously with decreasing temperature. It is speculated that the decrease in drying temperature prolonged the hydrolysis time of enzymes on flavonoid glycosides, thus leading to a decrease in content. Different drying methods have different retention levels for different components. Hot air drying at 70℃ and 80℃ has a better retention level for type a peak components; vacuum drying at 60℃ and 70℃ has a better retention level for type b peak components; freeze-drying has a relatively balanced retention level for the overall peak components.
[0067] 2.3 AHP-CRITIC-TOPSIS evaluation of drying process
[0068] 2.3.1 Evaluation index determination
[0069] The total flavonoids samples of snow chrysanthemum with different drying processes were taken, and the extract yield, water content, total flavonoids content of snow chrysanthemum, mariglycoside content, quercetin content, and luteolin content were determined according to the extract determination method under item 2201, the drying method under item 0832, and the content determination method under item 2.1 of the general rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 3.
[0070] Table 3 Determination results of total flavonoids samples of snow chrysanthemum with different drying processes
[0071]
[0072]
[0073] 2.3.2 Evaluation Index Determination of Weights
[0074] (1) Determine the index weight (WA) using the AHP method
[0075] The AHP method is a subjective weighting method that combines qualitative and quantitative methods. Based on the 1-9 scale method, the evaluation indicators can be compared and evaluated in pairs, and the judgment matrix of the evaluation indicators can be constructed to realize the dataization of subjective decision-making; the evaluation criteria are shown in Table 4, and the judgment matrix of importance evaluation is shown in Table 5.
[0076] Table 4 Importance evaluation criteria
[0077]
[0078] Table 5 Judgment matrix for evaluation of drying process indicators
[0079]
[0080]
[0081] Substitute the judgment matrix of indicator importance evaluation into formula (1) to calculate the indicator weight:
[0082]
[0083] Note: n is the number of evaluation schemes; m is the number of evaluation indicators; i and j are the vector values of the rows and columns of the matrix respectively. To ensure the scientificity and logic of the indicator weights, the judgment matrix is tested for consistency, see formula (2):
[0084]
[0085] Where CI is the consistency test index; CR is the random consistency ratio; IR is the average random consistency test index, which can be obtained by looking up Table 6.
[0086] Table 6 Average random consistency test index IR lookup table
[0087] n 1 2 3 4 5 6 7 8 9 10 11 RI 0 0 0.52 0.89 1.12 1.26 1.36 1.41 1.46 1.49 1.52
[0088] (2) CRITIC method to determine indicator weights (WC)
[0089] The CRITIC method is an objective weighting method based on the combination of data volatility and indicator correlation. According to the type of evaluation indicator, the original data matrix is dimensionless. The positive indicator processing is shown in formula (3), and the reverse indicator processing is shown in formula (4):
[0090] x′ ij =(x ij -x j min ) / (x j max -x j min ) (3)
[0091] x′ ij =(x j max -x ij ) / (x j max -x j min ) (4)
[0092] According to the volatility of indicator data and the correlation between indicators, the indicator information volume Cj is calculated as shown in formula (5):
[0093]
[0094] Note: rij is the correlation coefficient between indicators; Sj is the standard deviation
[0095] The information volume of each indicator is normalized to obtain the weight of each indicator. The calculation is shown in formula (6):
[0096]
[0097] (3) Composite Weight (W Composite)
[0098] The weights obtained by AHP method and CRITIC method are processed comprehensively to obtain the composite weight (W composite), and the calculation method is shown in formula (7):
[0099]
[0100] The weight coefficients of extract yield, CTF, mariglycoside, quercetin, luteolin and water content were calculated by the above formula, see Table 7.
[0101] Table 7 Weight coefficients of drying process evaluation indicators
[0102]
[0103] 2.3.3AHP-CRITIC-TOPSIS comprehensive evaluation
[0104] (1) Construction and processing of the original matrix
[0105] The extract yield, total flavonoids content of snow chrysanthemum, marisin content, quercetin content, luteolin content, and water content were used as evaluation indicators, and 7 drying processes were used as evaluation schemes to establish the original data matrix (X ij ) n×m After analysis, it is found that the water content is a very small indicator among the seven indicators, and needs to be processed positively and unified into positive trend data, as shown in formula (8); then the matrix is standardized according to formula (9), the indicator dimension is eliminated, and the standardized matrix (Z ij ) n×m , standardized formula (8):
[0106] x ij =x max -x ij (8)
[0107]
[0108] (2) Determine the best solution
[0109] Substitute the composite weights of each evaluation index into the standardized matrix to determine the optimal solution Z + =max(Z1,Z2,···,Zn) and the worst solution Z-=min(Z1,Z2,···,Zn). The best solution Z+=(0.1112,0.2117,0.1966,0.0514); the worst solution Z-=(0.0889,0.1051,0.1587,0.0414).
[0110] (3) Comprehensive ranking
[0111] According to formulas (10) and (11), the distances between the seven evaluation schemes and the optimal scheme (D+) and the distances between the worst scheme (D-) are calculated, and the Euclidean closeness Si of each scheme to the optimal solution is calculated, normalized and comprehensively ranked. The results are shown in Table 8.
[0112]
[0113] S i =D i - / (D i - +D i + ) (11)
[0114] Table 8 Comprehensive evaluation results of drying process
[0115]
[0116] The results showed that the ranking of the scores of the seven drying processes was: GZ3>GZ2>GZ7>GZ6>GZ5>GZ4>GZ1. The scores of 80℃ hot air drying and 70℃ hot air drying were higher, both >0.2, the score of freeze drying was 0.1936, the scores of 70℃ vacuum drying and 60℃ hot air drying were between 0.10 and 0.15, and the scores of 60℃ hot air drying and 50℃ vacuum drying were lower, both <0.05, which was consistent with the results of cluster analysis. Therefore, 80℃ hot air drying was selected as the drying process of snow chrysanthemum flavonoids extract in this experiment.
[0117] It can be seen from the above embodiments that different drying processes and different drying temperatures under the same drying process have different effects on the extract yield, water content and active ingredient content in the process of extracting total flavonoids from snow chrysanthemum. The present invention has found a method suitable for drying total flavonoids extract from snow chrysanthemum.
[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing a snow chrysanthemum flavonoid extract, characterized in that: The steps include: (1) extracting snow chrysanthemum with ethanol solution under reflux, collecting the extract, and concentrating it until there is no alcohol taste, thereby obtaining a crude extract of snow chrysanthemum flavonoids; (2) diluting the crude extract of snow chrysanthemum flavonoids and purifying it with AB-8 macroporous resin to obtain a purified snow chrysanthemum flavonoids solution; (3) After the purified snow chrysanthemum flavonoids solution is concentrated under reduced pressure, the concentrated solution is collected and hot-air dried to obtain a snow chrysanthemum flavonoids extract.
2. The preparation method according to claim 1, characterized in that The concentration of the ethanol solution is 60-70%.
3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the snow chrysanthemum to the ethanol solution is 1g:60-70mL.
4. The preparation method according to claim 3, characterized in that: The temperature of the reflux extraction in step (1) is 75-85° C., the time is 20-30 min, and the extraction is repeated 2-4 times.
5. The preparation method according to claim 4, characterized in that: The dilution is dilution with water, and the loading concentration is 3-4 mg / mL based on the crude drug amount.
6. The preparation method according to claim 5, characterized in that: During the purification by the AB-8 macroporous resin, the sample loading amount is 6-7 BV, the loading flow rate is 0.5-0.6 BV / h, after the adsorption is completed, 1-2 BV of purified water is used for impurity removal, the flow rate is 0.5-0.6 BV / h, after impurity removal, 45-55% ethanol solution is used for desorption, the amount is 5-6 BV, the flow rate is 0.5-0.6 BV / h, and the eluent is collected to obtain a purified snow chrysanthemum flavonoid solution.
7. The preparation method according to claim 6, characterized in that: The temperature of the reduced pressure concentration is 45-50°C.
8. The preparation method according to any one of claims 1 to 7, characterized in that: The temperature of the hot air drying is 70-80°C.
9. A snow chrysanthemum flavonoids extract obtained according to the preparation method according to any one of claims 1 to 8.