Method for enriching and purifying total flavonoids in lotus leaves by using macroporous resin

Abstract

The invention discloses a method for enriching and purifying total flavonoids in lotus leaves by using macroporous resin, which comprises the following steps: extracting, concentrating, separating and purifying: concentrating an extracting solution obtained in the step of concentrating under reduced pressure to recover ethanol, concentrating until no alcohol smell exists, diluting by using 8-12 times of 20% ethanol until the loading concentration of the macroporous resin is 3.0 mg / mL, and purifying by selecting AB-8 macroporous adsorption resin with the diameter-height ratio of 1: 5, the maximum sample loading amount is 5BV, the sample loading flow rate is 2.0 BV / h, and finally, 70% ethanol of 9BV is used as an eluent and is eluted at the flow rate of 3.0 BV / h to obtain the total flavone extract, and the content of the purified total flavone extract can reach 52.24%; the method is simple in process, low in cost, easy to operate and high in efficiency, the content of the total flavonoids in the obtained extract is high, the optimal process for purifying the total flavonoids of the lotus leaves is provided, and reference is provided for further research on pharmaceutics and pharmacodynamics.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extracts, and particularly relates to a method for enriching and purifying total flavonoids in lotus leaves with macroporous resin. Background Art

[0002] Lotus leaf is the dried leaf of Nelumbo nucifera Gaertn., a plant of the Nymphaeaceae family. It grows in water, ponds or lakes and is widely distributed in the Yangtze River Basin, South my country, North China, Northeast China and other regions. The Compendium of Materia Medica states that "taking lotus leaf can make people thin and weak. Taking it alone can eliminate the floating qi of Yang water." It has the effects of nourishing the kidney, warming the lungs and moistening the intestines. It is mainly used for kidney yang deficiency, soreness of waist and knees, dry intestines and constipation. Modern pharmacological research shows that lotus leaf can nourish the kidney, warm the lungs and moisten the intestines. Lotus leaf has pharmacological activities such as regulating blood lipids, anti-oxidation and anti-aging, anti-inflammatory and antibacterial, sedative and anti-fatigue. The preparations made from it mainly include lotus leaf pills and lotus leaf tablets. Lotus leaf can also be used to make lotus leaf tea and other lotus leaf foods, and is listed in the catalog of medicine and food of the same origin.

[0003] In addition to the common chemical components such as carbohydrates, lipids, proteins, tannins, etc. shared by ordinary plants, lotus leaves also contain two major categories of substances: flavonoids and alkaloids with obvious biological activities and physiological functions. Among them, flavonoids have multiple pharmacological activities such as regulating blood lipids, inhibiting bacteria, and anti-oxidation. Lotus leaf alkaloids have multiple pharmacological effects such as regulating blood lipids, lowering blood sugar, anti-tumor, and anti-cerebral ischemia.

[0004] Macroporous adsorption resin is a kind of organic polymer adsorbent. In terms of adsorption performance, it has many advantages, such as stable physical and chemical properties, insoluble in acid, alkali and organic solvents, good selectivity, and not affected by inorganic substances. In terms of its own characteristics, it has the characteristics of large specific surface area, fast exchange rate, high mechanical strength, low pollution to the extract, and good thermal stability. Compared with other separation technologies, it has the advantages of increasing the relative content of active ingredients, the product does not absorb moisture, short production cycle, convenient resin regeneration, and reusability. Therefore, it has been widely used in the separation and purification of natural products in recent years, especially suitable for the separation and purification of flavonoid chemicals.

[0005] The present invention uses lotus leaf as raw material, takes the adsorption rate and desorption rate of macroporous resin on lotus leaf flavonoids as investigation indicators, selects the best macroporous resin required for enriching total flavonoids in lotus leaf, and optimizes various optimal conditions for enriching total flavonoids in lotus leaf using the resin, such as sample loading amount, adsorption time, eluent, sample loading concentration, sample loading flow rate, elution flow rate, etc., to provide theoretical and practical basis for the extraction, purification and application of total flavonoids in lotus leaf. Summary of the invention

[0006] The object of the present invention is to provide a method for enriching and purifying total flavonoids in lotus leaves using macroporous resin. The method has the advantages of simple process, low cost and high content of total flavonoids in the extract. The technical scheme is as follows: A method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin comprises the following steps: (1) Extraction: The lotus leaves were crushed by a grinder and passed through a 60-mesh sieve. 60% ethanol was added as a solvent at a solid-liquid ratio of 1:40 (g / mL). Ultrasonication was performed for 20 min. After the extraction was completed, the extract was filtered and set aside. (2) Concentration: The extract in the above step is concentrated under reduced pressure to recover ethanol, concentrated until there is no alcohol taste, diluted with 8-12 times 20% ethanol to a loading concentration of 1.0 mg / mL-5.0 mg / mL, purified by adsorption through a macroporous resin at a diameter-to-height ratio of 1:2-1:6 and a loading flow rate of 1.0 BV / h-5.0 BV / h, and then eluted with 50%-90% ethanol at an elution flow rate of 1.0 BV / h-5.0 BV / h to obtain an eluent, wherein the macroporous resin is an XDA-8 macroporous resin; (3) Separation and purification: Collect the eluate obtained in the above steps, recover the solvent under reduced pressure, and dry to obtain the flavonoid extract.

[0007] Furthermore, the ethanol concentration used for elution in step (2) is preferably 70%.

[0008] Furthermore, the concentration of the loading solution in step (2) is preferably 3.0 mg / mL.

[0009] Furthermore, the sample loading flow rate in step (2) is preferably 2.0 BV / h.

[0010] Furthermore, the aspect ratio in step (2) is preferably 1:5.

[0011] Furthermore, the elution flow rate in step (2) is preferably 3.0 BV / h.

[0012] Furthermore, the maximum sample loading volume in step (2) is 5 BV, and the amount of eluent used is 9 BV.

[0013] Furthermore, the macroporous resin in step (2) is pretreated, comprising the following steps: (1) Soak the resin in 2 times the amount of 95% ethanol for 24 hours, stirring continuously to expel the bubbles inside, and stir the resin with a glass rod to form a suspension; (2) Pour the mixture into a resin column pre-filled with a small amount of water while stirring, and at the same time turn on the switch at the bottom of the resin column to avoid bubbles in the resin column; (3) Load the column with 95% ethanol, then wash with 95% ethanol until the effluent is free of white turbidity by adding water (1:5). Then wash with purified water until there is no ethanol smell. Soak in 5% hydrochloric acid for 2 h and wash with pure water until neutral. Then soak in 5% sodium hydroxide solution for 2 h and wash with pure water until neutral. The pretreatment is complete. Filter with filter paper and set aside.

[0014] Furthermore, in the regeneration treatment of the macroporous resin in step (2), the used resin is washed with distilled water until the eluent is colorless, then the resin is soaked in 4% HCl solution for 12 hours, and then the resin is rinsed with 4% HCl solution after column loading, and then washed with distilled water until neutral, and then the resin is soaked in 4% NaOH solution for 12 hours, and then the resin is passed through the column with 4% NaOH solution after column loading, and finally washed with distilled water until neutral.

[0015] The above technical solution can achieve the following beneficial effects: Compared with the prior art, the invention has a simple process, low cost, easy operation and high efficiency. The obtained extract has a high content of total flavonoids, which can reach 50% after purification. It provides an optimal process for purifying total flavonoids from lotus leaves, has great development and utilization value, and provides a reference for further research on formulation and pharmacodynamics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the purification process of total flavonoids from lotus leaves.

[0017] Figure 2 Rutin standard curve Figure 3 This is a trend chart of the effects of different types of macroporous resins on the static adsorption and desorption of total flavonoids in lotus leaves.

[0018] Figure 4 It is the static adsorption kinetic curve of macroporous resin.

[0019] Figure 5 It is the static desorption kinetic curve of macroporous resin.

[0020] Figure 6 This is a trend chart showing the effect of sample solution concentration on resin adsorption.

[0021] Figure 7 This is a trend chart of the effect of the volume fraction of ethanol in the desorption liquid on the desorption rate.

[0022] Figure 8 It is the dynamic leakage curve of XDA-8 resin.

[0023] Fig. 9 This is a trend chart showing the effect of sample flow rate on resin adsorption.

[0024] Fig.10 This is a trend chart showing the effect of diameter-to-height ratio on the dynamic adsorption and desorption of macroporous resin.

[0025] Fig.11 This is a trend chart showing the effect of desorption liquid volume on desorption amount.

[0026] Fig.12 This is a trend chart showing the effect of desorption liquid flow rate on desorption rate. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings: In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously in conjunction with preferred embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0029] (1) Preparation of reference solution Accurately weigh 5 mg of rutin standard, place it in a 25 mL volumetric flask, add 60% ethanol to dissolve, dilute to the mark, shake well, and obtain a 200 μg / mL rutin standard solution.

[0030] (2) Preparation of test solution Crush the lotus leaf and pass it through a 60-mesh sieve. Take 1.0 g of lotus leaf powder, weigh it accurately, add 60% ethanol as solvent at a solid-liquid ratio of 1:40 (g / mL), ultrasonicate for 20 min, make up to 50 mL volumetric flask with 60% ethanol, filter, and obtain the lotus leaf total flavonoids extract for later use.

[0031] (3) Selection of measurement wavelength The NaNO2-AlCl3-NaOH method was used for determination. A 60% ethanol solution was used as blank and rutin was used as reference, and a full scan was performed in the wavelength range of 400-900nm. The results showed that the maximum absorption wavelength of the reference solution was 507nm, and the maximum absorption wavelength of the test solution was 507nm. The ultraviolet spectra and maximum absorption wavelengths of the two were basically the same, so the determination wavelength of the total flavonoids content in lotus leaves was determined to be 507nm.

[0032] (4) Preparation of standard curve Accurately measure 0.0, 0.5, 0.8, 1.0, 1.5 and 2.0 mL of the above standard solution and place them in 10 mL stoppered colorimetric tubes, respectively, then add 0.3 mL of 5% sodium nitrite solution, mix well, let stand for 6 min, then add 0.3 mL of 10% aluminum nitrate solution, mix well, add 4 mL of 4% sodium hydroxide solution after 6 min, mix well, calibrate to the scale line with 60% ethanol, mix well and let stand for 10 min, measure the absorbance at a wavelength of 507 nm, and use concentration C (abscissa) and absorbance A (ordinate) as standard curve to obtain the regression equation y=0.0106x-0.0027, R 2 =0.9998, the results showed that the flavonoid content was in the range of 0-80.56 μg / mL with good linearity. The results are shown in Table 1 and Figure 2 .

[0033] Concentration (μg / mL) Absorbance (Abs) Volume of reference solution added (mL) 0 0.002 0 20.14 0.206 0.5 32.22 0.339 0.8 40.28 0.423 1.0 60.42 0.643 1.5 80.56 0.852 2.0 Table 1 Results of flavonoid content range Example 1: Determination of adsorption and desorption rates of lotus leaf flavonoids by six macroporous resins (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute with 8-12 times 20% ethanol to the required concentration, and refrigerate for later use.

[0034] (2) Separation and purification: Weigh 2.0g of six types of resins after pretreatment, namely macroporous resins AB-8, D101, HPD-400, HPD-100, XDA-8, and NKA-9, and place them in a conical flask. Add 40mL of lotus leaf total flavonoids extract of the required concentration, stir to make it fully contact with the macroporous resin, seal the bottle mouth, place it in a shaker, shake it at 25℃ and 120 r / min for 24h to make it fully adsorbed, filter, measure the absorbance of the solution, substitute it into the regression equation to calculate the corresponding flavonoid concentration, and calculate the static adsorption amount and adsorption rate of each resin. Filter to separate the resin and the adsorbed solution, transfer the filtrate to a test tube, and the resin to a conical flask. The resin was washed twice with distilled water until the eluent was colorless. The residual solution on the surface of the resin was absorbed with filter paper, placed in a conical flask, and 40 mL of 70% ethanol solution was added and placed in a shaker (25 ° C, 120 r / min) for static desorption for 24 h. The resin was filtered out, the total flavonoids concentration in the filtrate was determined, and the desorption amount and desorption rate of each resin were calculated.

[0035] The formula is as follows: Adsorption amount (mg / g) = (C0-C1) × V1 / M Adsorption rate%==(C0-C1) / C0×100 Desorption amount (mg / g) = C2×V2 / M Desorption rate % = C2 / (C0-C1)×100 Where: C0 (mg / mL) is the concentration before adsorption; C1 is the equilibrium concentration (mg / mL); V1 is the volume of the sample (mL); M is the mass of the macroporous resin (g); C2 is the concentration of the eluent (mg / mL); V2 is the volume of the eluent ethanol (mL); M is the mass of the macroporous resin (g) (3) Experimental results: comprehensive adsorption rate and desorption rate. The results are shown in Table 2 and Figure 3 , XDA-8 resin was selected for subsequent dynamic adsorption tests. type Equilibrium solution concentration (mg / mL) Eluent concentration (mg / mL) Adsorption rate% Desorption rate% AB-8 1.132 1.190 63.03 61.61 D101 1.300 1.140 57.55 64.67 HPD400 1.246 1.109 59.33 61.04 HPD100 1.156 0.994 62.26 52.11 XDA-8 0.741 1.621 75.81 69.81 NKA-9 0.975 1.300 68.17 62.27 Table 2 Static adsorption and desorption experimental results of different types of macroporous resins Example 2: Static adsorption kinetics curve (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0036] (2) Separation and purification: Accurately weigh 2.0 g of pretreated resin and 40 mL of lotus leaf total flavonoids extract of the required concentration into a conical flask, stir to make it fully contact with the macroporous resin, and place it in a shaker (25°C, 120 r / min) for 9 h of oscillation adsorption. Take 1 mL of sample solution every 30 minutes with a pipette for measurement, determine its concentration, and calculate its adsorption amount.

[0037] (3) Experimental results: From 0 to 5.5 hours, the adsorption amount gradually increased; from 5.5 to 7 hours, the adsorption amount gradually stabilized; after 7 hours, the adsorption amount began to show a downward trend. The adsorption equilibrium time was selected as 5.5 hours. Figure 4 shown.

[0038] Example 3: Static desorption kinetics curve (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 min. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, and dilute with 8-12 times of 20% ethanol.

[0039] (2) Separation and purification: Filter to separate the resin and the adsorbed solution, transfer the filtrate to a test tube and the resin to a conical flask. Wash the saturated resin with distilled water twice until the eluent is colorless. Use filter paper to absorb the residual solution on the surface of the resin, add it to the conical flask with 40mL of 70% ethanol, and place it in a shaker at (25°C, 120r / min) for 6 hours of shaking and desorption. Take 1mL of sample solution every 30 minutes with a pipette for measurement, determine its concentration, and calculate its desorption amount.

[0040] (3) Experimental results: Within 2 hours, the desorption amount increases with the increase of desorption time; from 2 to 3 hours, the desorption amount increases slowly with the passage of time. After 3 hours, the desorption process is still going on, but the desorption amount does not change significantly, and the desorption reaches equilibrium. The desorption equilibrium time is selected as 2 hours. Figure 5 shown.

[0041] Example 4: Effect of different sample concentrations on the static adsorption effect of macroporous resin (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0042] (2) Separation and purification: Accurately weigh 5 portions of 2.0 g of the pretreated resin and add them into conical flasks with 40 mL (concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL) of lotus leaf total flavonoids extract. Stir to allow them to fully contact with the macroporous resin and place them in a shaker (25°C, 120 r / min) for 5.5 h of oscillation adsorption. Filter and measure the absorbance of the solution. Substitute the solution into the regression equation to calculate the corresponding flavonoid concentration and the adsorption rate.

[0043] (3) Experimental results: With the increase of sample concentration, the adsorption rate of total flavonoids by macroporous resin XDA-8 showed a trend of first increasing and then decreasing. When the solution concentration was 3.0 mg / mL, the adsorption rate of the resin to the solution reached its maximum value. When the sample concentration was low, even if the effluent leaked, the resin could not be completely saturated; the increase in solution concentration was conducive to its diffusion to the resin, increasing the contact rate between the solution and the resin, thereby improving the adsorption rate of the resin; however, when the solution concentration was too high, the resin adsorption was saturated, and the flavonoids flowed out without being adsorbed, resulting in a waste of raw materials and a decrease in the adsorption rate. Therefore, when the solution concentration was 3.0 mg / mL, the resin XDA-8 had the maximum adsorption rate for it, which was the optimal sample concentration. Figure 6 shown.

[0044] Example 5: Effect of different concentrations of desorbent on the static desorption effect of macroporous resin (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0045] (2) Separation and purification: Accurately weigh 2.0g of the pre-treated resin and 40mL of the required concentration of lotus leaf total flavonoids extract were added to a conical flask, stirred to make it fully contact with the macroporous resin, placed in a shaker (25℃, 120 r / min), and adsorbed for 5.5h. Filter to separate the resin and the adsorbed solution. Wash the resin with distilled water until the eluent is colorless. Add 40mL of 50%, 60%, 70%, 80%, and 90% ethanol solutions to desorb the resin column, place in a shaker (25℃, 120 r / min), and desorb for 2h. Filter out the resin, collect the eluent, determine its concentration, and calculate its desorption rate.

[0046] (3) Experimental results: With the increase of ethanol volume fraction, the desorption rate gradually increased. Under the condition of 70% ethanol, the desorption rate reached the maximum value, and after 70%, the desorption rate began to decrease. It may be that as the ethanol concentration increases, its polarity decreases, and the solvent molecules cannot form hydrogen bonds with the XDA-8 resin, making it difficult to replace the flavonoid molecules on the resin, resulting in the difficulty of flavonoid desorption. Figure 7 shown.

[0047] Example 6: Leakage Curve (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0048] (2) Separation and purification: 23.31 g of pretreated XDA-8 macroporous resin was wet loaded onto the column, and the required concentration of lotus leaf total flavonoids extract was added. The sample was loaded at a flow rate of 3 BV / h. The effluent was collected every 1 BV and the flavonoid concentration of the effluent was measured. The leakage point was reached when the concentration of the effluent reached one tenth of the concentration of the loaded solution, and a leakage curve was drawn.

[0049] (3) Experimental results: With the increase of the volume of the effluent, the total flavonoid content in the effluent gradually increased, indicating that the adsorption effect of XDA-8 resin on the total flavonoids of lotus leaves gradually became worse. When the effluent concentration reached 10% of the loading concentration, it was called the leakage point (in the dynamic adsorption process, when the adsorption capacity of the resin column reaches saturation, the target compound begins to leak out of the resin column, and the concentration of the target compound in the effluent begins to increase significantly.) The total flavonoid concentration of the loading solution was 3.044 mg / mL, and the leakage point concentration was 0.304 mg / mL. When the effluent volume was 5 BV, the total flavonoid concentration in the effluent reached 0.304 mg / mL, so it was determined to be the leakage point, and 5 BV was the optimal loading volume. Figure 8 shown.

[0050] Example 7: Effect of different loading flow rates on dynamic adsorption of macroporous resin (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0051] (2) Separation and purification: 23.31 g of pretreated XDA-8 macroporous resin was wet loaded into the column, and 5 BV of lotus leaf total flavonoids extract of the required concentration was added. The resin column was passed through at a speed of 1.0 BV / h, 2.0 BV / h, 3.0 BV / h, 4.0 BV / h, and 5.0 BV / h, respectively. After complete adsorption, the concentration was determined and the adsorption rate was calculated.

[0052] (3) Experimental results: The adsorption rate increases first and then decreases with the increase of the loading speed, and reaches the maximum at 2.0 BV / h. If the loading speed is too fast, the retention time of the loaded liquid in the XDA-8 resin is too short, and the adsorbed liquid cannot be completely absorbed and flows out, which is not conducive to adsorption; when the loading flow rate is too slow, the adsorption rate will also decrease. Therefore, the loading flow rate of 2.0 BV / h is the most appropriate. Fig. 9 shown.

[0053] Example 8: Effects of different diameter-to-height ratios on dynamic adsorption and desorption of macroporous resins (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0054] (2) Separation and purification: 19.11 g, 23.31 g, 28.43 g, 34.67 g and 41.8 g of the pretreated XDA-8 macroporous resin were wet-loaded into the column with diameter-to-height ratios of 1:2, 1:3, 1:4, 1:5 and 1:6, respectively. 60 mL, 90 mL, 120 mL, 150 mL and 180 mL of lotus leaf total flavonoids extract of the required concentration were added and passed through the resin column at a rate of 2.0 BV / h. After complete adsorption, the resin column was washed twice with distilled water until the eluent was colorless. The resin column was then desorbed with 5 BV of 70% ethanol solution at a desorption rate of 2.0 BV / h. The eluent was collected, its concentration was determined, and its adsorption rate and desorption rate were calculated.

[0055] (3) Experimental results: The adsorption rate and desorption rate increase first and then decrease with the increase of the diameter-to-height ratio. When the diameter-to-height ratio is small, it may lead to insufficient adsorption, resulting in poor separation effect and reduced content; when the diameter-to-height ratio is large, the diffusion rate of total flavonoids in the resin column slows down, affecting the adsorption efficiency. During the desorption process, the total flavonoids stay in the resin column for too long, which may cause the total flavonoids to bind too tightly to the resin surface and be difficult to desorb. When the diameter-to-height ratio is 1:5 and the weight of the resin used is 34.67g, the height of the resin layer is moderate and the purification effect is optimal. Fig.10 shown.

[0056] Example 9: Effect of different dosages of desorbent on dynamic elution effect of macroporous resin (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0057] (2) Separation and purification: 34.67 g of pretreated XDA-8 macroporous resin was wet-packed into a column with a diameter-to-height ratio of 1:5, and 5 BV of lotus leaf total flavonoids extract of the required concentration was added. The resin column was passed through at a rate of 2.0 BV / h. After complete adsorption, the resin column was washed twice with distilled water until the eluent was colorless. The resin column was then desorbed with 70% ethanol solution at a desorption rate of 2.0 BV / h. The eluent was collected at 1 BV as a fraction until the eluent was colorless and its concentration was determined.

[0058] (3) Experimental results: As the elution progresses, it reaches the highest value at 1BV. This indicates that the total flavonoids content in the eluent is the highest at this time, and the elution effect is the best at this time. As the elution progresses, the total flavonoids concentration in the eluent continues to decrease. The total flavonoids concentration in the eluent is extremely low when the collected liquid volume is 9-10BV. The total flavonoids concentration in the eluent is basically stable, indicating that the desorption is complete. In addition, if the amount of eluent used is too small, the lotus leaf total flavonoids solution adsorbed on the XDA-8 resin cannot be completely eluted. If the amount of eluent used is too large, it will cause waste. On the whole, it is more appropriate to select an eluent volume of 9BV. Fig.11 shown.

[0059] Example 10: Effect of different desorption flow rates on dynamic elution effect of macroporous resin (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0060] (2) Separation and purification: 34.67 g of pretreated XDA-8 macroporous resin was wet-packed into a column with a diameter-to-height ratio of 1:5, and 5 BV of lotus leaf total flavonoids extract of the required concentration was added. The resin column was passed through at a rate of 2 BV / h. After adsorption was completed, the resin column was washed twice with distilled water until the eluent was colorless. The resin column was then desorbed with 9 BV of 70% ethanol solution at desorption flow rates of 1.0 BV / h, 2.0 BV / h, 3.0 BV / h, 4.0 BV / h, and 5.0 BV / h. The eluents at each elution flow rate were collected, the absorbance was measured, and the desorption rate was calculated.

[0061] (3) Experimental results: With the increase of desorption flow rate, the desorption rate first increases and then decreases. When the flow rate reaches 3BV / h, the desorption rate reaches the maximum. The appropriate increase of flow rate can increase the desorption rate. However, when the elution flow rate is too fast, the eluent stays on the resin XDA-8 for too short a time and cannot fully elute the total flavonoids on it and flows out directly, resulting in poor elution effect. Therefore, the eluent flow rate of 3BV / h is the best flow rate. Fig.12 shown.

[0062] Example 11: Purification process verification experiment (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure until there is no alcohol taste, dilute to the required concentration with 8-12 times 20% ethanol, and refrigerate for later use.

[0063] (2) Separation and purification: Take 3 portions of pretreated XDA-8 resin with a diameter-to-height ratio of 1:5, and load the sample at a flow rate of 2.0 BV / h. After adsorption is completed, wash twice with distilled water until the eluent is colorless, then desorb the resin column with 9 BV of 70% ethanol at a desorption flow rate of 3 BV / h, collect the eluent, determine the concentration, and calculate the desorption rate and adsorption rate respectively.

[0064] (3) Experimental results: The average adsorption rate of total flavonoids in lotus leaves was 87.31%, the RSD value was 0.11%, and the average resolution rate was 53.99%, the RSD value was 1.65%, indicating that the purification process of the present invention is stable and feasible.

[0065] Example 12: Purity determination of total flavonoids in lotus leaves before and after purification (1) Extraction: Crush the lotus leaves with a 60-mesh sieve using a grinder, add 60% ethanol as a solvent at a solid-liquid ratio of 1:40 (g / mL), and sonicate for 20 minutes. After the extraction is completed, filter, concentrate under reduced pressure, and dilute with 8-12 times of 20% ethanol.

[0066] (2) Determination method: Determine the flavonoid concentration of the lotus leaf total flavonoid extract obtained by the optimal extraction process and the lotus leaf total flavonoid eluate obtained by the optimal purification process. Compare the purity difference of lotus leaf total flavonoids before and after purification.

[0067] The lotus leaf total flavonoids extract obtained according to the best extraction process and the lotus leaf total flavonoids eluate obtained according to the best purification process in this experiment were evaporated to extract in a water bath at 85°C, dried in an electric constant temperature drying oven, and the mass of the dried product was measured. 100 mg of the dried lotus leaf total flavonoids powder was taken respectively, accurately weighed, dissolved in 70% ethanol and fixed to 10 mL, 1 mL was taken for each dilution 100 times, the absorbance was measured, and the corresponding flavonoid concentration was calculated by substituting into the regression equation.

[0068] The formula is as follows: Purity (%) = (C × V) / M × 100 Where: C is the concentration of the extract / eluent (mg / ml); V is the volume of the extract / eluent (ml); V1 is the mass of the extract / eluent after drying.

[0069] (3) Experimental results: The results show that as shown in Table 3, the XDA-8 macroporous resin purification in this experiment increased the yield to 52.24%, which may be due to different extraction conditions before purification. The enzymatic extraction was used in the early stage of this experiment, which greatly improved the yield of total flavonoids.

[0070] In modern research, AB-8 resin has been proven to have good adsorption rate and high desorption rate, and is often used for the separation and purification of total flavonoids. In this study, XDA-8 macroporous resin was used for the first time to purify total flavonoids from lotus leaves. Through a systematic study of the adsorption and desorption properties of XDA-8 macroporous resin, we found that the resin has a significant adsorption effect on total flavonoids from lotus leaves, and the desorption rate is also high. Compared with AB-8 resin, XDA-8 macroporous resin has certain advantages in terms of adsorption rate, adsorption capacity and desorption conditions.

[0071] Each resin has its specific adsorption performance and purification effect. In the previous extraction of total flavonoids from lotus leaves, it was found that the highest contents of total flavonoid compounds in lotus leaves were hyperoside and isoquercetin. These two active ingredients have similar polarity, strong polarity or medium polarity. The XDA-8 resin used in this experiment is of medium polarity and has high adsorption and desorption performance for compounds with medium polarity, which makes the XDA-8 resin have better efficiency in adsorbing and desorbing total flavonoid compounds from lotus leaves.

[0072] The properties of the resin itself are also one of the key factors affecting the purification effect of macroporous resin. XDA-8 resin has a higher specific surface area, so in this experiment, XDA-8 resin can effectively adsorb flavonoids than AB-8 resin. See Table 3, which shows that XDA-8 macroporous resin has a better purification effect on lotus leaf total flavonoids.

[0073] In summary, this study used XDA-8 macroporous resin to purify lotus leaf total flavonoids for the first time and achieved remarkable results. The resin has excellent adsorption and desorption properties, providing a new choice for the industrial production and promotion of lotus leaf total flavonoids. type Flavonoid concentration (mg / mL) purity(%) Flavonoid crude extract 2.969 29.69 XDA-8 purified 5.224 52.24 Table 3 shows the purity of total flavonoids in lotus leaves before and after purification. The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.

Claims

1. A method for enriching and purifying total flavonoids in lotus leaves using macroporous resin, characterized in that: The steps include: (1) Extraction: The lotus leaves were crushed by a grinder and passed through a 60-mesh sieve. 60% ethanol was added as a solvent at a solid-liquid ratio of 1:40 (g / mL). Ultrasonication was performed for 20 min. After the extraction was completed, the extract was filtered and set aside. (2) Concentration: The extract in the above step is concentrated under reduced pressure to recover ethanol, concentrated until there is no alcohol taste, diluted with 8-12 times 20% ethanol to a loading concentration of 1.0 mg / mL-5.0 mg / mL, purified by adsorption through a macroporous resin at a diameter-to-height ratio of 1:2-1:6 and a loading flow rate of 1.0 BV / h-5.0 BV / h, and then eluted with 50%-90% ethanol at an elution flow rate of 1.0 BV / h-5.0 BV / h to obtain an eluent, wherein the macroporous resin is an XDA-8 macroporous resin; (3) Separation and purification: Collect the eluate obtained in the above steps, recover the solvent under reduced pressure, and dry to obtain the flavonoid extract.

2. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: The ethanol concentration used for elution in step (2) is preferably 70%.

3. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: The concentration of the sample solution in step (2) is preferably 3.0 mg / mL.

4. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: The sample loading flow rate in step (2) is preferably 2.0 BV / h.

5. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: The diameter-to-height ratio in step (2) is preferably 1:

5.

6. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: The elution flow rate in step (2) is preferably 3.0 BV / h.

7. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: The maximum sample loading volume in step (2) is 5 BV, and the amount of eluent used is 9 BV.

8. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: The step (2) of pre-treating the macroporous resin comprises the following steps: (1) Soak the resin in 2 times the amount of 95% ethanol for 24 hours, stirring continuously to expel the bubbles inside, and stir the resin with a glass rod to form a suspension; (2) Pour the mixture into a resin column pre-filled with a small amount of water while stirring, and at the same time turn on the switch at the bottom of the resin column to avoid bubbles in the resin column; (3) Load the column with 95% ethanol, then wash with 95% ethanol until the effluent is free of white turbidity by adding water (1:5). Then wash with purified water until there is no ethanol smell. Soak in 5% hydrochloric acid for 2 h and wash with pure water until neutral. Then soak in 5% sodium hydroxide solution for 2 h and wash with pure water until neutral. The pretreatment is complete. Filter with filter paper and set aside.

9. The method for enriching and purifying total flavonoids in lotus leaves using a macroporous resin according to claim 1, characterized in that: In the step (2), the macroporous resin is regenerated by washing the used resin with distilled water until the eluent is colorless, then soaking the resin in a 4% HCl solution for 12 hours, eluting with a 4% HCl solution after loading into a column, then washing with distilled water until neutral, then soaking the resin in a 4% NaOH solution for 12 hours, passing the resin with a 4% NaOH solution after loading into a column, and finally washing with distilled water until neutral.

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