CN111346195A - Preparation method of tsaoko total flavonoids - Google Patents
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Abstract
The invention provides a preparation method of tsaoko total flavonoids. Weighing fructus Tsaoko medicinal material powder (sieving with a No. two sieve), extracting with ethanol at 60 vol%, material-liquid ratio of 1:50(g/mL), extraction temperature of 60 deg.C, ultrasonic (power: 160W, frequency: 60kHz) for 60min, vacuum filtering while hot, repeating for 2 times, mixing filtrates, concentrating, and evaporating to obtain fructus Tsaoko total flavone crude product; adding water into the obtained total flavone extract to prepare a water solution with a certain concentration, loading the water solution on pretreated HPD450 macroporous adsorption resin, adsorbing for 12h, eluting with water until effluent is colorless, eluting with 8BV of 60% ethanol, collecting eluate, concentrating and evaporating to dryness to obtain the purified total flavone extract. The purity of the total flavone in the extract is improved by 2.56 times on average after detection and purification. The verification proves that the preparation process conditions are stable and feasible, and the prepared tsaoko amomum fruit total flavone has stable content and higher purity.
Description
Technical Field
The invention particularly relates to a method for extracting, separating and purifying amomum tsao-ko total flavonoids and detecting the total flavonoids.
Background
Amomum tsaoko belongs to the genus Amomum of Zingiberaceae, is an important medicinal and edible Chinese medicinal material, and is mainly produced in Yunnan, Guangzhou, Guizhou and other places. The tsaoko amomum fruit is used as the traditional common spice, and is widely applied to the diet industry because the tsaoko amomum fruit has special and strong spicy flavor, can remove fishy smell and promote appetite; as a traditional medicinal material, the tsaoko amomum fruit has the effects of eliminating phlegm, preventing malaria, strengthening spleen, resisting oxidation and the like. In recent years, scholars at home and abroad have conducted some researches on the tsaoko amomum fruits and have preliminarily understood the chemical components and the biological activity of the tsaoko amomum fruits. The tsaoko contains tsaoko volatile oil, tsaoko polysaccharide, flavonoid and phenolic acid chemical components. At present, the extraction and detection research of the total flavonoids of tsaoko amomum fruits is carried out, but no research report is reported on the adoption of macroporous resin for further separation and purification of the extracted total flavonoids.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of tsaoko total flavonoids.
The preparation method of the tsaoko total flavonoids comprises the following operation steps:
1) extracting the total flavonoids of tsaoko amomum fruits: weighing tsaoko amomum fruit, crushing, and extracting according to the following extraction process: extracting with 30-90% ethanol solution at a ratio of material to liquid of 1:10-1:100(g/mL) at 25-80 deg.C for 30-120min for 1-3 times, mixing filtrates, concentrating, and evaporating to obtain fructus Tsaoko total flavone crude product;
2) and (3) separating and purifying the amomum tsao-ko total flavonoids: adding water into the crude product of the tsaoko fruit total flavonoids in the step 1) to prepare an extract water solution with the mass concentration of 0.5-3mg/mL, loading the extract water solution on macroporous adsorption resin, eluting with water until an effluent liquid is colorless, eluting with 30-80% ethanol with 5-10BV, collecting an eluent, concentrating and evaporating to dryness to obtain the tsaoko fruit total flavonoids.
Wherein, the tsaoko amomum fruit medicinal material in the step 1) is crushed and then screened by a second sieve.
The preferable volume fraction of ethanol in step 1) is 60%.
The preferable feed-liquid ratio in step 1) is 1:50 (g/mL).
The preferred extraction temperature in step 1) is 60 ℃.
In the ultrasonic extraction method in the step 1), the ultrasonic extraction power is 160W, and the frequency is 60 kHz.
The preferable ultrasonic extraction time in the step 1) is 60 min.
The preferred number of repetition operations in step 1) is 2.
Each gram of the crude product of the tsaoko total flavonoids obtained in the step 1) is equal to 73.10g of crude drugs.
The preferable pH value of the flavone extract solution used in the step 2) is 7, and the preferable mass concentration is 1.85 mg/mL.
The macroporous resin used in the step 2) is one of HPD100, HPD300, HPD450, AB-8, X-5 and D101, wherein the preferred type of the resin is HPD 450.
The eluent used in the step 2) has the preferred volume fraction of 60 percent of ethanol and the preferred elution volume of 8 BV.
The purity of the total flavonoids of the tsaoko amomum fruits prepared by the method can be obviously improved.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 HPLC chart of rutin control solution
FIG. 2 HPLC chart of the sample solution
FIG. 3 negative control solution HPLC chart
FIG. 4 static adsorption kinetics curves
FIG. 5 static desorption kinetics curves
FIG. 6 is a graph for examining the concentration of a sample liquid
FIG. 7 pH examination curve of the sample solution
FIG. 8 volume fraction ethanol investigation curves
FIG. 9 ethanol dosage investigation curve
Detailed Description
Instrument and reagent
1 apparatus
High Performance Liquid Chromatography (HPLC) instrument model LC-1260 (Agilent, USA); analytical balance model ME204 (Metler-Torledo instruments Shanghai Co., Ltd.); model BJ-750A functional pulverizer (Deqing Bye Jie electric Co., Ltd.); PHS-320 type acidimeters (medley science and technology ltd); model THZ-320 desk top constant temperature oscillator (shanghai sperm macro laboratory equipment ltd).
2 reagent
The fructus Tsaoko medicinal material is purchased from Sichuan New Green pharmaceutical science and technology development GmbH (batch number: 20180705), and is identified as dry mature fruit of fructus Tsaoko (Amomum tsaoko Crevost et Lemaire) of Amomum genus of Zingiberaceae family by professor Limin of Chengdu Chinese medicine university college of pharmacy.
Rutin control (Doudeman Biotech Co., Ltd., batch No.: MUST-19010210, purity: 99.47%); HPD100, HPD300, HPD450, AB-8, X-5, D101 type macroporous adsorption resin (0.3-1.25 mm, Zhongshan Dong hong chemical Co., Ltd., batch numbers of 20180618, 20181126, 20180626, 20181005, 20180716, 20190111 respectively); the methanol and the acetonitrile are both chromatographically pure, the other reagents are all commercially available analytically pure, and the water is distilled water.
Example 1 extraction of Amomum tsao-ko Total Flavonoids
Weighing appropriate amount of fructus Tsaoko medicinal materials, pulverizing, and sieving with a second sieve. Precisely weighing a certain amount of tsaoko amomum fruit powder according to the following extraction process conditions: extracting with ethanol at 60 vol% and material-liquid ratio of 1:50(g/mL) at 60 deg.C under ultrasonic (power: 160W, frequency: 60kHz) for 60min, vacuum filtering, repeating the extraction for 2 times, mixing filtrates, concentrating, and evaporating to obtain crude product of fructus Tsaoko total flavone (crude product per gram is equal to 73.10g of crude drug).
Example 2 HPLC assay of Total Flavonoids of Tsaoko
1 preparation of a reference substance solution rutin 7.02mg is precisely weighed, placed in a 5mL measuring flask, dissolved by adding a proper amount of methanol and ultrasonic waves (power: 160W, frequency: 60kHz), cooled, added with methanol to complement to scale, shaken up to obtain a rutin reference substance solution with the mass concentration of 1.404mg/mL, and filtered through a 0.45 mu m microporous filter membrane to obtain the rutin reference substance solution.
2 preparing a test solution A proper amount of the crude product of the tsaoko total flavonoids obtained in the example 1 is dissolved by methanol, placed in a 5mL measuring flask, then the volume is determined by the methanol, and the solution is filtered through a 0.45 mu m microporous membrane to obtain the test solution.
3 preparation of negative control solution A negative sample containing no Amomum tsao-ko herbs was prepared by the method of example 1, dissolved in a suitable amount of methanol and fixed in a 5mL volumetric flask, and passed through a 0.45 μm microporous membrane to obtain a negative control solution.
4 chromatographic conditions of a chromatographic column, Eclipse Plus C18(150mm × 3.0.0 mm, 2.7 mu m), a mobile phase of acetonitrile-1% acetic acid aqueous solution (15:85, V/V), a column temperature of 40 ℃, a flow rate of 0.8mL/min, a detection wavelength of 256nm and a sample injection amount of 10 mu L.
The beneficial effects of the present invention are illustrated by the following experimental examples:
(1) specialization inspection
Taking appropriate amount of rutin control, test sample and negative control solution, performing sample injection measurement according to chromatographic conditions, and recording chromatogram. As a result, under the chromatographic conditions, the separation degree of rutin is good, the retention time is about 5.95min, the negative control has no interference to the measurement, and the chromatogram of the rutin control solution, the test solution and the negative control solution is shown in figures 1, 2 and 3 of the attached drawings.
(2) Investigation of linear relationships
Precisely absorbing 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mL of rutin control solution respectively, placing in different 5mL brown measuring bottles, adding methanol to fix the volume, shaking up to obtain standard curve solutions with mass concentrations of 0.028, 0.056, 0.112, 0.168, 0.225 and 0.281mg/mL respectively, filtering by a 0.45-micrometer microporous filter membrane, taking 10 mu L of subsequent filtrate, carrying out sample injection analysis according to the chromatographic conditions in the example 2, and recording a chromatogram. And performing linear regression by taking the rutin mass concentration (x, mg/mL) as an abscissa and taking the peak area (y) as an ordinate to obtain a regression equation y which is 7468.1 x-60.438 (r which is 0.9999). The result shows that the linear range of the rutin detection mass concentration is 0.028-0.281 mg/mL.
(3) Quantitative limit and detection limit
Precisely sucking a proper amount of rutin reference substance standard curve solution with the mass concentration of 0.112mg/mL, diluting by methanol multiple ratio, and calculating the quantitative limit and the detection limit by the signal-to-noise ratio of 10:1 and 3:1 respectively. As a result, the limit of quantitation and the limit of detection were 437.5 and 109.4ng/mL, respectively.
(4) Precision test
Precisely sucking a proper amount of rutin control standard curve solution with the mass concentration of 0.112mg/mL, continuously measuring for 6 times according to the chromatographic conditions in the example 2, and recording the peak area. As a result, the peak area of rutin had RSD of 1.85% (n ═ 6), indicating good precision of the apparatus.
(5) Stability test
Taking the same sample solution, respectively standing at room temperature (25 deg.C) for 0, 2, 4, 6, 8, and 10h, analyzing by sample injection, and recording peak area. As a result, the peak area of rutin was 0.79% in RSD (n ═ 6), indicating that the test solution was stable within 10 hours at room temperature.
(6) Repeatability test
Taking 6 parts of the crude tsaoko total flavone powder, preparing a test solution according to the method in the section of example 2, continuously measuring for 6 times, recording peak areas, and calculating the content of the total flavone in the test solution according to a standard curve. As a result, the average content of total flavonoids in the test sample was 0.0323mg/mL, and RSD was 0.60% (n ═ 6), indicating that the method was excellent in reproducibility.
(7) Sample application recovery test
Taking 6 parts of crude tsaoko fruit total flavone powder with known content, adding 0.056mg/mL rutin reference substance standard solution according to 50%, 100% and 150% of the known content respectively, preparing low, medium and high quality concentration test solution according to the method in the example 2, then carrying out sample injection measurement according to the chromatographic condition in the example 2, recording the peak area and calculating the sample injection recovery rate. As a result, the sample recovery rates of the low, medium, and high-quality sample solutions were 96.24%, 99.75%, and 98.13%, respectively (average sample recovery rate was 98.04%), and RSD were 1.83%, 1.76%, and 1.78%, respectively (n ═ 6).
(8) Determination of content of total flavonoids in tsaoko amomum fruits
Taking a proper amount of tsaoko total flavone crude product powder, preparing a test solution according to the method in the item of the example 2, filtering the test solution by a microporous filter membrane with the diameter of 0.45 mu m, then carrying out sample injection measurement under the chromatographic condition in the item of the example 2, and calculating the content of the total flavone in the solution according to a standard curve.
EXAMPLE 3 macroporous resin type screening
1 static adsorption and static desorption
Pretreating 6 kinds of macroporous resin (HPD100, HPD300, HPD450, AB-8, X-5, and D101), and screening the optimal resin type for purifying fructus Tsaoko total flavone by static adsorption and desorption.
Precisely weighing 1.0g of each of 6 pretreated macroporous adsorption resins, paralleling 3 parts, respectively placing the 6 pretreated macroporous adsorption resins into 50mL triangular bottles with plugs and ground openings, respectively adding 20mL of a tsaoko total flavone aqueous solution, shaking and adsorbing for 24h in a constant temperature oscillator at 25 ℃, performing suction filtration after full adsorption, evaporating the filtrate to dryness, and then performing constant volume to 10mL by using methanol to obtain an adsorption solution, taking the resin which is saturated in adsorption and cleaned and dried, adding 20mL of 75% ethanol, shaking and desorbing for 24h in the constant temperature oscillator at 25 ℃, performing suction filtration on the desorption solution after full desorption, concentrating and drying the filtrate, performing constant volume to 10mL by using methanol to obtain a desorption solution, respectively absorbing 1.0mL of each of the adsorption solution and desorption solution, filtering by using a 0.45μm microporous filter membrane, performing sample injection analysis under the chromatographic condition of the embodiment 2, recording peak areas, calculating the content of the total flavone in each solution, and calculating the adsorption amount, adsorption rate, desorption rate and desorption rate of the 6 macroporous adsorption resins to the tsaoko total flavone and desorption rate according to the following formulas ① - ④.
Specific adsorption capacity ═ c0V0－c1V1)/m………………………………………①
Adsorption rate [ (c)0V0－c1V1)/c0V0]×100％；……………………………②
Specific desorption amount ═ c2V2/m……………………………………………………③
Desorption rate ═ c2V2/(c0V0－c1V1)]×100％。……………………………④
Wherein m is the resin mass (g), c0The mass concentration (mg/mL) of the total flavone in the sample solution is V0Volume of the sample (mL); c. C1The mass concentration (mg/mL) of total flavonoids in the adsorption solution is V1Volume of adsorption solution (mL), c2Is ethanolThe mass concentration (mg/mL) of total flavonoids in the eluate, V2Volume of ethanol eluate (mL).
TABLE 1 results of static adsorption and static desorption of different types of macroporous adsorbent resins (n ═ 3)
| Type of resin | Specific adsorption capacity (mg/g) | Adsorption Rate (%) | Specific desorption amount (mg/g) | Desorption ratio (%) |
| HPD100 | 8.122 | 87.86 | 4.542 | 55.92 |
| HPD300 | 7.463 | 80.49 | 4.056 | 54.35 |
| HPD450 | 7.162 | 77.24 | 5.158 | 72.02 |
| AB-8 | 7.560 | 81.54 | 4.524 | 59.84 |
| X-5 | 5.877 | 63.38 | 3.579 | 60.90 |
| D101 | 6.859 | 73.98 | 3.698 | 53.91 |
As can be seen from Table 1, the adsorption rates of the 6 macroporous adsorption resins are HPD100 > AB-8 > HPD300 > HPD450 > D101 > X-5 from high to low; the desorption rate is sequentially HPD450 > X-5 > AB-8 > HPD100 > HPD300 > D101. Although the adsorption rate was the highest for the HPD100 type, the desorption rate was relatively low, while the desorption rate was the highest for the HPD450 type resin and the adsorption rate was centered. In general terms, HPD450 type macroporous adsorbent resins were selected for further investigation.
2 static adsorption kinetics investigation
Precisely weighing 2g of pretreated HPD450 macroporous adsorption resin, paralleling 3 parts, respectively placing in 50mL ground conical flasks, respectively adding 40mL of tsaoko total flavone aqueous solution, shaking on a constant temperature oscillator at 25 ℃, respectively absorbing 4mL of supernatant at 1, 2, 4, 8, 12 and 24h, concentrating, evaporating, and diluting to 2mL with methanol to obtain adsorption solution for later use. Washing the macroporous resin after 24h adsorption with water, filtering, adding 40mL of 75% ethanol, shaking and desorbing on a constant temperature oscillator at 25 ℃, absorbing 4mL of solution when 1, 2, 4, 8, 12 and 24h respectively, concentrating and evaporating to dryness, and fixing the volume of methanol to 2mL to obtain desorption solution for later use. Respectively sucking the adsorption solution and desorption solution of 1mL at each time point, filtering by a 0.45-micrometer microporous filter membrane, performing sample injection analysis under the chromatographic condition of the embodiment 2, recording peak area, calculating the content of total flavonoids in each solution, and calculating specific adsorption amount and specific desorption amount. The static adsorption and desorption kinetics curves of the macroporous adsorption resin are respectively drawn by taking the specific adsorption amount (mg/g) and the specific desorption amount (mg/g) as ordinate and time (h) as abscissa, and the results are shown in figure 4 and figure 5.
In the static adsorption process, the specific adsorption capacity of the macroporous adsorption resin is in an ascending trend within 1-12 h, and the adsorption is saturated after 12h (see figure 4); during the static desorption process, the specific desorption amount of the macroporous adsorption resin fluctuates obviously within 0-12h, and the equilibrium is basically achieved after 12h (see figure 5). For this purpose, the optimum static adsorption and desorption times were selected to be 12 h.
Example 4 optimization of the purification Process of Tsaoko Total Flavonoids
(1) Inspection of sample loading liquid mass concentration
Precisely measuring 15mL of 5 parts of pretreated HPD450 macroporous adsorption resin, wet-packing the resin into a glass column (diameter: 1.5cm, height: 20cm) with the diameter-height ratio of about 1:13, respectively adding 20mL of tsaoko fruit total flavone aqueous solution with the mass concentration of 0.1159, 0.2318, 0.4636, 0.9272 and 1.8544mg/mL, adsorbing for 12h, discharging an adsorption solution, eluting with a proper amount of water until an effluent is colorless, combining the adsorption solution and the water eluate, concentrating and evaporating to dryness, fixing the volume to 2mL with methanol, precisely absorbing 1mL, filtering by a 0.45 mu m microporous filter membrane, carrying out sample injection analysis under the chromatographic condition of example 2, recording the peak area, calculating the content of the total flavone in each solution according to the method under the example 2, and calculating the adsorption rate according to the formula under the example 3. As a result, when the mass concentration is less than or equal to 0.9272mg/mL, the adsorption rate tends to increase with the increase of the mass concentration of the adsorbent; however, the mass concentration is more than 0.9272mg/mL, and the increase of the mass concentration of the loading liquid has no obvious influence on the adsorption rate and slightly decreases. Considering together, 0.9272mg/mL was determined as the optimal mass concentration of the loading solution, as shown in FIG. 6.
(2) pH examination of sample liquid
Precisely measuring 15mL of 5 parts of pretreated HPD450 macroporous adsorption resin, placing the 15mL of pretreated HPD450 macroporous adsorption resin in a glass column, respectively adding 20mL of 0.9272mg/mL tsaoko fruit total flavone aqueous solution (the pH is adjusted by 1mol/L hydrochloric acid solution or 1mol/L sodium hydroxide solution) with the pH values of 3, 4, 5, 6 and 7, adsorbing for 12 hours, discharging an adsorption solution, eluting with a proper amount of water until an effluent liquid is colorless, combining the adsorption solution and the water eluent, concentrating and evaporating to dryness, fixing the volume to 2mL with methanol, precisely absorbing 1mL, filtering by a 0.45-micrometer microporous filter membrane, carrying out sample injection analysis under the chromatographic condition of example 2, recording the peak area, and calculating the content and the adsorption rate of the total flavone in each solution. As a result, when the pH of the sample solution is 3-6, the adsorption rate tends to increase with the increase of the pH; when the pH value is more than 6, the adsorption rate is rather lowered. For general consideration, pH 6 was chosen as the optimum pH for the loading solution, as detailed in FIG. 7.
(3) Ethanol volume fraction inspection
Precisely measuring 15mL of 5 parts of pretreated HPD450 macroporous adsorption resin, placing the resin in a glass column, respectively adding 20mL of 0.9272mg/mL tsaoko fruit total flavone aqueous solution with the pH value of 6, adsorbing for 12h, eluting with proper amount of water until effluent is colorless, respectively carrying out dynamic desorption with ethanol with volume fractions of 50%, 60%, 70%, 80% and 90%, concentrating and evaporating eluent, fixing the volume to 2mL with methanol, respectively and precisely absorbing 1mL, filtering with a 0.45-micrometer microporous filter membrane, carrying out sample injection analysis under chromatographic conditions in the chromatographic condition of example 2, recording peak area, and calculating the content of the total flavone in each solution. As a result, when the volume fraction of the ethanol is 50-70%, the total flavone eluted by the macroporous adsorption resin tends to increase along with the increase of the volume fraction of the ethanol; when the volume fraction is more than 70%, the total flavone content is rather reduced, which may be related to that the higher the volume fraction of ethanol is, the more impurities are eluted. For comprehensive consideration, 70% was selected as the optimal volume fraction for ethanol elution, as detailed in FIG. 8.
(4) Examination of ethanol elution amount
Precisely measuring 5 parts of pretreated HPD450 macroporous adsorption resin 15mL, placing the resin in a glass column, respectively adding 0.9272mg/mL tsaoko amomum fruit total flavone aqueous solution with pH 6 20mL, adsorbing for 12h, eluting with appropriate amount of water until effluent is colorless, eluting with 70% ethanol at the speed of 1 time of column volume (BV)/h, collecting eluent, collecting 1 part of eluent every 1BV, concentrating and evaporating the eluent, diluting to 2mL with methanol, respectively and precisely absorbing 1mL, filtering by a 0.45-micrometer microporous filter membrane, performing sample injection analysis under the chromatographic condition in example 2, recording peak area, and calculating the total flavone content in each BV eluent. As a result, when the elution volume was > 9BV, there was almost no total flavonoids in the eluate, indicating that the elution was substantially complete. Considering comprehensively, 9BV is selected as the best ethanol elution dosage, as shown in figure 9.
(5) Technical investigation test result of fructus tsaoko total flavonoids
Based on the above test results, the test was performed using the mass concentration (a) of the sample solution, the ph (b) of the sample solution, the volume fraction (C) of ethanol, and the amount (D) of ethanol eluted as factors, and the total flavone content as an evaluation index, and the results are shown in table 2.
TABLE 2 investigation test and results of purification process of fructus Tsaoko total flavone
According to the test and investigation results, the optimal process conditions for purifying the total flavone by the macroporous resin are finally determined as follows: the mass concentration of the sample is 1.8544mg/mL, the pH value of the sample solution is 7, the volume fraction of ethanol is 60%, and the dosage of the eluent is 8 BV. The purity of the purified total flavonoids is improved by 2.56 times through detection, and the average recovery rate is 72.01%, which shows that the HPD450 type macroporous resin can effectively purify the tsaoko total flavonoids.
In conclusion, the invention provides a preparation method of tsaoko fruit total flavonoids, which comprises the following steps: weighing fructus Tsaoko powder (sieved by a No. two sieve), extracting with ethanol at 60 vol%, material-liquid ratio of 1:50(g/mL), extraction temperature of 60 deg.C and ultrasonic (power: 160W, frequency: 60kHz) for 60min, vacuum-filtering while hot, repeating for 2 times, mixing filtrates, concentrating, and evaporating to dryness to obtain fructus Tsaoko total flavone crude product; adding water into the obtained total flavone extract to prepare a water solution with a certain concentration, loading the water solution on pretreated HPD450 macroporous adsorption resin, adsorbing for 12h, eluting with water until effluent is colorless, eluting with 8BV of 60% ethanol, collecting eluate, concentrating and evaporating to dryness to obtain the purified total flavone extract. The purity of the total flavone in the extract is improved by 2.56 times on average after detection and purification. The verification proves that the preparation process conditions are stable and feasible, and the prepared tsaoko amomum fruit total flavone has stable content and higher purity.
Claims (10)
1. A preparation method of tsaoko total flavonoids is characterized in that: the method comprises the following operation steps:
1) extracting the total flavonoids of tsaoko amomum fruits: weighing tsaoko amomum fruit, crushing, and extracting according to the following extraction process: extracting with 30-90% ethanol solution at a ratio of material to liquid of 1:10-1:100(g/mL) at 25-80 deg.C for 30-120min for 1-3 times, mixing filtrates, concentrating, and evaporating to obtain fructus Tsaoko total flavone crude product;
2) and (3) separating and purifying the amomum tsao-ko total flavonoids: adding water into the crude product of the tsaoko fruit total flavonoids in the step 1) to prepare an extract water solution with the mass concentration of 0.5-3mg/mL, loading the extract water solution on macroporous adsorption resin, eluting with water until an effluent liquid is colorless, eluting with 30-80% ethanol with 5-10BV, collecting an eluent, concentrating and evaporating to dryness to obtain the tsaoko fruit total flavonoids.
2. The method of claim 1, wherein: crushing the tsaoko medicinal materials in the step 1) and screening the crushed tsaoko medicinal materials through a second sieve.
3. The method of claim 1, wherein: step 1) the volume fraction of ethanol is 60%.
4. The method of claim 1, wherein: the feed-liquid ratio in step 1) is 1:50 (g/mL).
5. The method of claim 1, wherein: the extraction temperature in step 1) was 60 ℃.
6. The method of claim 1, wherein: in the ultrasonic extraction method of the step 1), the ultrasonic extraction power is 160W, and the frequency is 60 kHz; or the ultrasonic extraction time is 60 min.
7. The method of claim 1, wherein: the number of times of the repeated operation in the step 1) is 2.
8. The method of claim 1, wherein: in the step 2), the preferable mass concentration of the flavone extract solution is 1.85 mg/mL; preferably, the pH of the aqueous extract solution is adjusted to 3 to 8, preferably 7.
9. The method of claim 1, wherein: the macroporous resin used in the step 2) is one of HPD100, HPD300, HPD450, AB-8, X-5 and D101, wherein the preferred type of the resin is HPD 450.
10. The method of claim 1, wherein: the eluent used in the step 2) has the preferred volume fraction of 60 percent of ethanol and the preferred elution volume of 8 BV.
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| CN113318203A (en) * | 2021-05-10 | 2021-08-31 | 文山壮族苗族自治州农业科学院 | Method for extracting tsaoko total flavonoids from tsaoko residues after volatile oil extraction |
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| CN107525862A (en) * | 2017-08-03 | 2017-12-29 | 广西壮族自治区中医药研究院 | Intend preparation and the quality determining method of tsaoko extractive of general flavone |
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| CN107525862A (en) * | 2017-08-03 | 2017-12-29 | 广西壮族自治区中医药研究院 | Intend preparation and the quality determining method of tsaoko extractive of general flavone |
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| 王婉愉等: "响应面法优化乙醇提取草果黄酮的工艺研究", 《中国调味品》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113318203A (en) * | 2021-05-10 | 2021-08-31 | 文山壮族苗族自治州农业科学院 | Method for extracting tsaoko total flavonoids from tsaoko residues after volatile oil extraction |
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