Method for extracting total flavonoids from Chinese redbud seeds

Abstract

The invention discloses a method for extracting total flavonoids from Chinese redbud seeds, and belongs to the technical field of natural product extraction and separation. The method adopts a deep eutectic solvent (DES) as an extraction medium, and specifically comprises the following steps: mixing dried Chinese redbud seed powder with the DES, carrying out ultrasonic-assisted extraction, and then carrying out centrifugal separation; and purifying the extracting solution by using macroporous adsorption resin, and eluting by using distilled water and ethanol in sequence to finally obtain the total flavonoids. According to the invention, cyclic utilization of the DES solvent is innovatively realized, DES in the loading effluent and the distilled water eluent is recovered through evaporation, and the DES solvent can be repeatedly used for at least three times. Compared with a traditional ethanol reflux extraction method, the method has the remarkable advantages that the total flavone extraction amount is increased by 3 times, and the method is simple and convenient in operation process, high in safety, environment-friendly and low in cost. The method not only improves the extraction efficiency, but also meets the requirements of modern green processing, and provides a new technical approach for sustainable development of natural products.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction and separation, and specifically relates to a method for extracting total flavonoids from Cercis chinensis seeds by utilizing DES combined with macroporous adsorption resin chromatography column technology. Background Art

[0002] Cercis chinensis is a shrub belonging to the genus Cercis in the Leguminosae family. Its seeds are broadly oblong, 5-6 mm long, dark brown, and shiny. According to the Chinese Materia Medica Dictionary, Cercis seeds can treat coughs and heartaches in pregnant women. The above medicinal value of Cercis seeds mainly comes from the presence of a variety of flavonoids, which are organic compounds with a C6-C3-C6 structure as the basic skeleton. In recent years, researchers have isolated a variety of flavonoids from Cercis seeds, including kaempferol, afudamicin, astragaloside, quercetin, myricetin, and quercitrin.

[0003] At present, most of the seeds of Bauhinia are arbitrarily discarded and not used reasonably, and there are few studies on the extraction of total flavonoids from Bauhinia seeds. Li Hongping, Chen Haiqin, Sun Xiuqing, You Xiaolin and others used ethanol extraction to extract total flavonoids from Bauhinia seeds, and assisted with ultrasound, and the total flavonoid extraction rate was 2.91%. This method has problems such as long extraction time, low extraction efficiency, and the use of a large amount of toxic and flammable organic solvents during the extraction process. Therefore, the development of new green solvents to replace organic solvents to extract total flavonoids from Bauhinia seeds is an urgent problem to be solved.

[0004] DES extraction has the advantages of no pollution and high extraction efficiency. It has been widely studied in recent years. However, its extraction is affected by many factors. Currently, there is no method to extract total flavonoids from Bauhinia seeds using DES. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting total flavonoids from Cercis chinensis seeds in advance, so as to improve the extraction efficiency of total flavonoids from Cercis chinensis seeds.

[0006] Based on the above purpose, the present invention adopts the following technical solutions:

[0007] A method for extracting total flavonoids from Bauhinia seeds, the process is as follows:

[0008] 1) preparing DES: melting a hydrogen bond acceptor and a hydrogen bond donor together to obtain DES, and letting it stand; the hydrogen bond acceptor is at least one of choline chloride, L-proline and betaine; the hydrogen bond donor is at least one of levulinic acid, citric acid, L-malic acid, glycerol, sorbitol and lactic acid; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-3):(1-4);

[0009] 2) DES extraction: the DES obtained in step 1) is mixed with water to form an extraction solvent, and then the dried Cercis chinensis seed powder and the extraction solvent are evenly mixed, and ultrasonic extraction is performed to obtain an extract of total flavonoids;

[0010] 3) Separation of total flavonoids by macroporous adsorption resin: The total flavonoids extract obtained in step 2) is loaded onto a macroporous adsorption resin, eluted with distilled water and ethanol in sequence, and concentrated to obtain total flavonoids.

[0011] Furthermore, the method further comprises step 4) DES recovery: collecting the sample solution and the distilled water eluate in step 3), evaporating the water therein, and reusing the solution to extract total flavonoids from Cercis chinensis seeds, thus completing the recovery of the low eutectic solvent.

[0012] Furthermore, the eutectic treatment conditions in step 1) are: stirring in a water bath at 60-80° C. for 1-3 hours and standing for 8-20 hours.

[0013] Furthermore, the particle size of the dried Cercis chinensis seed powder in step 2) is less than 0.25 mm.

[0014] Furthermore, in the step 2), DES and water are mixed to form an extraction solvent with a moisture content of 10-50v%; the liquid-to-solvent ratio of the extraction solvent to the Cercis chinensis seed powder is 15-30mL / g; and the extraction conditions are: 30-70°C, 100-500W ultrasonic extraction for 15-40min.

[0015] Furthermore, in the step 3), the model of the macroporous adsorption resin is ADS-17, XAD-76, DM301, NKA-9, HPD-1100, D101 or AB-8, and the adsorption separation conditions of the macroporous adsorption resin are: the solid-liquid ratio is 0.33 to 3 g / mL.

[0016] Furthermore, in the step 3), 70v% to 90v% ethanol is used for elution, and the eluate is collected and concentrated to obtain total flavonoids.

[0017] Furthermore, the DES is composed of choline chloride and levulinic acid with a water content of 10 to 30 v%, wherein the molar ratio of choline chloride to levulinic acid is 1:(2 to 4).

[0018] The present invention screened the process of extracting total flavonoids from Bauhinia seeds with water-soluble green DES, and obtained the optimal process conditions: using choline chloride-levulinic acid with a water content of 30v% as an extractant, a solid-liquid ratio of 25mL / g, centrifuging after 28min, and the supernatant contains total flavonoids from Bauhinia seeds (12.85%). Compared with the traditional extraction method of 60v% ethanol reflux (4.35%), the yield of total flavonoids extracted from Bauhinia seeds by DES is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the standard curve of rutin concentration and absorbance;

[0020] Figure 2 The effects of different DES and 60v% ethanol on the extraction rate of total flavonoids from Cercis chinensis seeds;

[0021] Figure 3 Scanning electron micrographs of Cercis chinensis seed powder: (a) untreated; (b) 60v% ethanol reflux extraction; (c) optimal DES extraction;

[0022] Figure 4 The effects of DES molar ratio (a), water content (b), liquid-to-solid ratio (c), extraction temperature (d), and extraction time (e) on the extraction rate of total flavonoids from Cercis chinensis seeds;

[0023] Figure 5 The effect of interaction on the extraction rate of total flavonoids from Cercis chinensis seeds was optimized by response surface method. DETAILED DESCRIPTION

[0024] The following examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional reagent companies unless otherwise specified.

[0025] DES was first proposed in 2001 and is mainly composed of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD). As a new type of solvent, DES has significant advantages such as easy preparation, low cost, low toxicity, low volatility, and high extraction rate. Although there are many reports on the application of DES in the extraction of natural products, there are no reports on the extraction of total flavonoids from Bauhinia seeds.

[0026] DES is a low eutectic mixture composed of hydrogen bond acceptors and hydrogen bond donors in a certain stoichiometric ratio, and its melting point is significantly lower than that of each component substance. As a new type of advanced solvent, DES is a completely biodegradable and biocompatible material. DES combines the characteristics of ionic liquids, has the advantages of low raw material price, good biocompatibility, low volatility, simple operation process, and reusability, which fully embodies the characteristics of green chemistry. The present invention uses different DES to extract total flavonoids from Bauhinia seeds, and compares and studies with traditional experimental methods, completes the initial screening of DES, and optimizes the parameters of DES for extracting total flavonoids from Bauhinia seeds.

[0027] 1 Materials and methods

[0028] 1.1 Materials

[0029] Cercis seeds were collected from the Yanling County planting base of Yanling Zhonglin Garden Engineering Co., Ltd.; all reagents were purchased from Nanjing Angel Biotechnology Co., Ltd. and were of analytical grade.

[0030] 1.2 Instruments

[0031] Thermo Scientific GENESYS 50 UV spectrometer, FEI Nova NanoSEM450 electron microscope.

[0032] 1.3 Methods

[0033] 1.3.1 Preparation of different DES solvents

[0034] The hydrogen bond acceptor and the hydrogen bond donor are mixed uniformly in a certain molar ratio. The hydrogen bond acceptor and the hydrogen bond donor and their molar ratio are shown in Table 1. The mixture is placed in a sealed glass bottle and stirred in a water bath at 500 rpm and 70°C for 1 to 3 hours to obtain a pure uniform liquid. The liquid is then allowed to stand at 30°C for 16 hours and set aside for later use.

[0035] Table 1 Combinations of different DES solvents

[0036]

[0037]

[0038] 1.3.2 Extraction of total flavonoids from Bauhinia seeds

[0039] (1) DES extraction

[0040] The Cercis chinensis seed powder and the above-mentioned DES were mixed evenly at a liquid-to-solid ratio of 24 mL / g, and extracted at 60°C and 250W ultrasound for 20 minutes to obtain an extract of total flavonoids from Cercis chinensis seeds. After the extraction, the mixture was transferred to a centrifuge tube for centrifugal separation at 5000 rpm for 5 minutes to separate the supernatant. 1 mL of the supernatant was taken and the absorbance was measured at a wavelength of 510 nm using a NaNO2-Al(NO3)3-NaOH color development system, and the total flavonoid extraction rate was calculated.

[0041]

[0042] Wherein, the concentration of total flavonoids in the sample solution is C (mg / mL), the volume of the test solution is V (mL), the dilution multiple (the volume multiple of the extract used to determine the absorbance after volume determination, color development and dilution) is N, and the mass of the Cercis seed powder is M (g).

[0043] (2) Traditional solvent ethanol reflux extraction

[0044] Accurately weigh two 1g portions of Bauhinia seed powder, place in a round-bottom flask, add 25mL of 60v% ethanol, heat and reflux for extraction for 1h, cool after extraction, make up the weight with 60v% ethanol, filter, and use the filtrate to determine the amount of total flavonoids.

[0045] 1.3.3 Drawing of standard curve

[0046] Accurately weigh 6.0 mg of rutin reference substance, dissolve it with anhydrous ethanol, make up to volume in a 30 mL volumetric flask, shake well to obtain a standard solution with a concentration of 0.2 mg / mL, and store it in a refrigerator at 4°C for later use. Pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL of rutin standard substance into 25 mL volumetric flasks No. 1 to 8, add 60v% ethanol to 1 mL, and do not add standard substance to the blank control group. Add 30 μL of 20 wt% NaNO2 solution, mix well and react for 3 minutes, add 40 μL of 40 wt% Al(NO3)3 solution, mix well and react for 3 minutes, then add 100 μL of 20 wt% NaOH solution, mix well and react for 10 minutes, and take 200 μL of each tube to measure the absorbance value of each solution at 510 nm to prepare a rutin standard curve, with the mass concentration of rutin as the horizontal axis and the absorbance value as the vertical axis.

[0047] 1.3.4 Determination of total flavonoids content in Cercis chinensis seeds

[0048] Take 1 mL of total flavonoids solution from Cercis chinensis seeds respectively, and determine the total flavonoids content of Cercis chinensis seeds extracted by different methods according to the method shown in 1.3.3. According to the extraction rate of total flavonoids by different DES, it is determined to use the choline chloride-levulinic acid system for the extraction parameter optimization experiment.

[0049] 1.3.5 Parameter Optimization

[0050] On the basis of determining the choline chloride-levulinic acid combination as the target DES, referring to "1.3.2 Extraction of total flavonoids from Bauhinia seeds", the molar ratio of choline chloride-levulinic acid (1:1, 1:2, 1:3, 1:4, 2:1, 3:1), the moisture content of DES (0, 10, 20, 30, 40, 50, 60v%), the liquid-to-solid ratio of DES to Bauhinia seed powder (5, 10, 15, 20, 25, 30mL / g), extraction time (15, 20, 25, 30, 35, 40min), and extraction temperature (30, 40, 50, 60, 70℃) were screened in turn to determine the optimal extraction process parameters.

[0051] 1.3.6 Recovery of total flavonoids from Bauhinia seeds and deep eutectic solvent

[0052] The optimal extraction process parameters optimized in 1.3.5 were used to extract the total flavonoids from Bauhinia seeds, and the extract was purified by AB-8 macroporous adsorption resin, and eluted with distilled water and 90v% ethanol in turn, and the sample solution and the distilled water eluate were collected and concentrated to obtain the recovered DES. The 90v% ethanol eluate was collected and concentrated to obtain the recovered total flavonoids.

[0053] 1.3.7 Reuse of DES

[0054] The DES recovered in 1.3.6 was used to extract the total flavonoids from Cercis chinensis seeds again. The DES was then recovered and used for the next extraction of total flavonoids from Cercis chinensis seeds. This was repeated 5 times in total.

[0055] 2 Results

[0056] 2.1 Drawing of standard curve

[0057] Draw the standard curve according to the method in 1.3.3, the regression equation is Y=9.28561X+0.04328, the correlation coefficient is R 2 =0.99212, indicating that there is a good linear relationship between rutin concentration and absorbance in the range of 0.02-0.14 mg / mL. The standard curve is as follows Figure 1 shown.

[0058] 2.2 Extraction rate of total flavonoids from Bauhinia seeds by different DES and ethanol reflux extraction

[0059] Comparison of total flavonoids concentrations in Cercis chinensis seeds extracted with different DES and ethanol reflux Figure 2 As shown, it can be seen that the total flavonoid extraction rate of choline chloride-acetylcholine is the highest (11.21%), which is significantly different from the traditional 60v% ethanol reflux (4.35%).

[0060] 2.3 Scanning electron microscope images

[0061] The untreated Bauhinia seed powder sample, the Bauhinia seed powder sample after ethanol reflux extraction, and the Bauhinia seed powder sample after optimal DES extraction were analyzed by scanning electron microscopy (instrument model: FEI Nova NanoSEM450). The results are as follows: Figure 3 (a), 3(b) and 3(c). For the dried untreated sample, the surface is relatively intact. After extraction with DES or ethanol reflux, obvious pores and cracks can be seen on the sample surface, and the surface morphology of the sample is destroyed. This damage is more obvious in DES, indicating that DES can more effectively destroy cells, thereby releasing more compounds in the material.

[0062] 2.4 Optimization of DES extraction parameters

[0063] 2.4.1 Choline chloride-levulinic acid molar ratio

[0064] The present invention compares the extraction efficiency of hydrogen bond donor DES with different molar ratios. Figure 4 aIt can be seen that with the increase of the molar ratio of hydrogen bond donor (1:1 to 1:3), the extraction efficiency gradually increased and reached the highest at 1:3, and then the extraction rate gradually decreased. The main reason may be that the excessive hydrogen donors caused the binding force of choline chloride to the total flavonoids of Cercis seeds to decrease. Therefore, the molar ratio of choline chloride to levulinic acid was set at 1:3 for the next optimization experiment.

[0065] 2.4.2 Moisture content

[0066] After the formation of water-soluble DES, many donor hydroxyl chemical groups will be exposed. Therefore, adding appropriate water will form more hydrogen bonds with these groups, making DES more stable. The surface tension of DES is the most important physical property of water content, but too much water will reduce the concentration of DES after the binding groups are saturated, affecting the extraction efficiency. Therefore, the appropriate water content is very important for the extraction effect of DES. Figure 4 b It can be seen that the extraction rate of total flavonoids from Bauhinia seeds is the highest when the water content is 20v%, and the extraction efficiency decreases at 30v% and 40v%, which is due to the decrease in surface tension and the decrease in DES concentration.

[0067] 2.4.3 Material-liquid ratio

[0068] In the extraction of active ingredients from traditional Chinese medicine, different solid-liquid ratios show different extraction efficiencies. In the extraction of total flavonoids from Bauhinia seeds by DES, a too small solid-liquid ratio will lead to the formation of hydrogen bonds between DES molecules, thus failing to fully extract the total flavonoids from Bauhinia seeds. A too large solid-liquid ratio will lead to DES failing to fully bind to the total flavonoids from Bauhinia seeds. Figure 4 c It can be seen that the extraction rate is the highest when the solid-liquid ratio is 25mL / g.

[0069] 2.4.4 Extraction temperature

[0070] like Figure 4 As shown in Figure d, at 60 °C, DES showed the highest total flavonoid extraction rate, and then the extraction rate began to decrease, which may be due to the volatilization of DES with the increase in temperature.

[0071] 2.4.5 Extraction time

[0072] like Figure 4 As shown in Figure e, at 30 min, DES showed the highest total flavonoid extraction rate, and the extraction efficiency decreased slightly with the increase of time from 30 to 40 min.

[0073] 2.5 Optimization of extraction conditions by response surface experiment

[0074] According to the Box-Behnken central composite experimental design principle, the total flavonoids extraction rate of Bauhinia seeds was taken as the response value (Y), the liquid-to-solid ratio was fixed at 25 mL / g, the extraction temperature (A), extraction time (B), and DES moisture content (C) were selected as optimization factors, and the three-factor three-level response surface analysis method was adopted. The experimental factors and level design are shown in Table 2.

[0075] Table 2 Response surface factor design level table

[0076]

[0077] In order to investigate the influence of various factors and their interactions on the extraction efficiency, the present invention uses Design-Expert software to perform multiple regression fitting on the four factors and three levels in the response surface factor design level table to obtain the calculation equation of the total extraction rate:

[0078] R1=-1.09319+0.028213A+0.020254B+0.004150C-0.000023AB+0.000014AC-0.000061BC-0.000228A 2 -0.000290B 2 -0.000049C 2

[0079] The variance analysis of the quadratic polynomial model is shown in Table 3. The model P<0.0001 (extremely significant), indicating that the model can be used to predict the response value. The model lack of fit term is not significant, indicating that the regression equation has a good degree of fit in the entire regression space.

[0080] Table 3 Model ANOVA

[0081]

[0082] The response surface diagram obtained based on the regression equation is as follows: Figure 5 As shown, the relationship between the effects and interactions of various factors and the extraction rate is shown. Through analysis, the optimal extraction conditions for total flavonoids from Cercis chinensis seeds are as follows: temperature 60℃, time 28min, water content 30v%, solid-liquid ratio 25mL / g, and molar ratio of choline chloride-levulinic acid eutectic solution 1:3. Under these conditions, the total flavonoids extraction rate of Cercis chinensis seeds was measured to be 12.85%, which is close to the calculated value of 13.50% by the model, indicating that the optimal extraction conditions obtained by the response surface methodology are accurate and reliable, and have practical application value.

[0083] 2.6 Recovery of total flavonoids from Bauhinia seeds and deep eutectic solvent

[0084] Take 20g of Cercis seed powder and extract it using the optimal extraction process parameters (extraction temperature 60℃, extraction time 28 minutes, water content 30v%, solid-liquid ratio 25mL / g, choline chloride-levulinic acid eutectic solution molar ratio 1:3). Under these conditions, the total flavonoids extraction rate of Cercis seed was 13.17%. The extract was added to AB-8 macroporous adsorption resin (the solid-liquid ratio of macroporous adsorption resin to extract was 3g / mL), and eluted with distilled water (the total volume was about 5 times the volume of the sample solution) and 90v% ethanol (the total volume was about 10 times the volume of the sample solution) in turn. The sample solution and the distilled water eluate were collected and concentrated to obtain the recovered DES. The 90v% ethanol eluate was collected and concentrated to obtain 2.20g of total flavonoids from Cercis seed (extraction rate 11.02%).

[0085] 2.7 Reuse of DES

[0086] The recovered DES and distilled water were mixed into an extraction solvent with a water content of 30v%, which was used for the next extraction of total flavonoids from Cercis chinensis seeds. This process was repeated 5 times in total.

[0087] As shown in Table 4, the DES prepared by the present invention has an extraction efficiency of 80.89% of the original when the total flavonoids of Cercis seeds are extracted for the third time after being recovered, and still has a good extraction effect, indicating that the DES recovered by the AB-8 macroporous resin has good solvent integrity and can be recycled at least 3 times, thereby saving costs to a greater extent.

[0088] Table 4 Extraction effect of DES reused 5 times

[0089]

[0090] Note: Extraction efficiency (%) = (single extraction rate / 13.17%) × 100%, where 13.17% is the extraction rate of total flavonoids obtained by using DES for the first time.

[0091] The present invention screened the process of extracting total flavonoids from Cercis chinensis seeds by DES, and obtained the optimal process conditions for extracting total flavonoids from Cercis chinensis seeds by DES assisted by ultrasound: using choline chloride-levulinic acid with a water content of 30v% as an extractant, a solid-liquid ratio of 25mL / g, and an extraction time of 28min to extract total flavonoids from Cercis chinensis seeds. Compared with the traditional extraction method of 60v% ethanol reflux, DES has a higher efficiency in extracting total flavonoids from Cercis chinensis seeds.

[0092] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the specification, should be within the protection scope of the present invention.

Claims

1. A method for extracting total flavonoids from Cercis chinensis seeds, characterized in that: The process is as follows: 1) Preparation of DES: co-melting a hydrogen bond acceptor and a hydrogen bond donor to obtain DES, and allowing to stand; the hydrogen bond acceptor is at least one of choline chloride, L-proline and betaine; the hydrogen bond donor is at least one of levulinic acid, citric acid, L-malic acid, glycerol, sorbitol and lactic acid; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-3):(1-4); 2) DES extraction: the DES obtained in step 1) is mixed with water to form an extraction solvent, and then the dried Cercis chinensis seed powder and the extraction solvent are evenly mixed, and ultrasonic extraction is performed to obtain an extract of total flavonoids; 3) Separation of total flavonoids by macroporous adsorption resin: The total flavonoids extract obtained in step 2) is loaded onto a macroporous adsorption resin, eluted with distilled water and ethanol in sequence, and concentrated to obtain total flavonoids.

2. The method for extracting total flavonoids from Cercis chinensis seeds according to claim 1, characterized in that: The method further comprises step 4) DES recovery: collecting the sample solution and the distilled water eluate in step 3, evaporating the water therein, and reusing the solution for extracting total flavonoids from Cercis chinensis seeds, thus completing the recovery of the low eutectic solvent.

3. The method for extracting total flavonoids from Cercis chinensis seeds according to claim 1, characterized in that: The eutectic treatment conditions in step 1) are: stirring in a water bath at 60-80° C. for 1-3 hours.

4. The method for extracting total flavonoids from Cercis chinensis seeds according to claim 1, characterized in that: The particle size of the dried Cercis chinensis seed powder in step 2) is less than 0.25 mm.

5. The method for extracting total flavonoids from Cercis chinensis seeds according to claim 1, characterized in that: In the step 2), DES and water are mixed to form an extraction solvent with a water content of 10-30v%; the liquid-to-solvent ratio of the extraction solvent to the Cercis chinensis seed powder is 15-30mL / g; and the extraction conditions are: 30-70°C, 100-500W ultrasonic extraction for 15-40 min.

6. The method for extracting total flavonoids from Cercis chinensis seeds according to claim 1, characterized in that: In the step 3), the model of the macroporous adsorption resin is ADS-17, XAD-76, DM301, NKA-9, HPD-1100, D101 or AB-8, and the adsorption separation conditions of the macroporous adsorption resin are: the solid-liquid ratio is 0.33-3 g / mL.

7. The method for extracting total flavonoids from Cercis chinensis seeds according to claim 1, characterized in that: In the step 3), 70% to 90% ethanol is used for elution, and the ethanol eluate is collected and concentrated to obtain total flavonoids.

8. The method for extracting total flavonoids from Cercis chinensis seeds according to claim 1, characterized in that: The DES is composed of choline chloride and levulinic acid with a water content of 10-30v%, wherein the molar ratio of choline chloride to levulinic acid is 1:(2-4).

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