Tricholoma matsutake polysaccharide and deep eutectic solvent extraction method and application thereof

Abstract

The application provides a tricholoma matsutake polysaccharide and a deep eutectic solvent extraction method and application thereof, and relates to the technical field of plant extraction.The deep eutectic solvent extraction method of the tricholoma matsutake polysaccharide comprises the following steps: preparing a deep eutectic solution: uniformly mixing and dissolving a hydrogen donor and a hydrogen acceptor reagent, and obtaining a transparent solution after adding water; extracting polysaccharide: after drying, crushing and defatting the tricholoma matsutake, the tricholoma matsutake is added into the deep eutectic solution, and an extraction solution is obtained after heating extraction; after purification, alcohol precipitation, dialysis and freeze-drying of the extraction solution, the tricholoma matsutake polysaccharide is obtained. Compared with traditional solvents and prior art, the efficiency of the method for extracting the tricholoma matsutake polysaccharide is significantly higher than that of the traditional water solvent extraction, the protein content of the extract is low, the tricholoma matsutake polysaccharide in the extract is more stable than that extracted by the traditional solvent, the antioxidant activity of the polysaccharide is better than that of the traditional method, the equipment is simple, the production cost is low, the continuous operation can be realized, and the method has strong practicability and operability.

Description

Technical Field

[0001] This application relates to the field of plant extraction technology, and more specifically, to a method and application for extracting matsutake polysaccharide and its deep eutectic solvent. Background Technology

[0002] Matsutake (scientific name: *Tricholoma matsutake* (S. Ito & S. Imai) Singer), also known as pine mushroom, is a mycorrhizal fungus belonging to the genus *Tricholoma* in the family Tricholomataceae. It is a rare and precious natural medicinal fungus found on trees such as pine and oak, and is hailed as the "King of Fungi." It is a Class II endangered protected species in China. Matsutake mushrooms rely on the roots of pine trees for survival. Varieties include *Tricholoma quercetin*, *Tricholoma pseudocarpa*, and *Tricholoma chestnutense*. Matsutake mushrooms are rich in protein, containing 18 amino acids and 14 essential trace elements.

[0003] Choosing the right extraction method is crucial for the study of plant polysaccharides, as different extraction techniques significantly affect polysaccharide yield, physicochemical properties, and bioactivity. Traditional polysaccharide extraction methods include hot water reflux, enzyme-assisted water extraction, ultrasound-assisted water extraction, and microwave-assisted water extraction. Given the complexity and diversity of materials, as well as varying economic costs and environmental impacts, each method has its unique advantages and limitations. Therefore, deep eutectic solvent (DES) extraction technology offers new possibilities for polysaccharide extraction. DES is a biodegradable, recyclable, and easily prepared natural green solvent composed of a hydrogen donor (HBD) and a hydrogen acceptor (HBA), with choline chloride (ChCl) being the most commonly used hydrogen acceptor. Currently, DES has been applied to the extraction of various plant polysaccharides, such as those from kelp, Sargassum, and lotus leaves. Polysaccharides extracted using deep eutectic methods exhibit better antioxidant activity compared to those extracted using traditional methods. However, research on the extraction of fungal polysaccharides is relatively limited.

[0004] Currently, the extraction processes for matsutake polysaccharides mainly include traditional hot water extraction, dilute acid (alkali) extraction, enzymatic extraction, ultrasound-assisted extraction, and enzymatic methods. The extract is settled by adding ethanol, and the resulting solution is filtered to obtain matsutake polysaccharides. During the extraction process, techniques such as ultrasound-assisted extraction and microwave-assisted extraction are used to improve extraction efficiency. However, water extraction, alkali extraction, and enzymatic extraction often result in the co-extraction of other components such as proteins and glycosides, which increases the difficulty of subsequent separation and purification. Although methods such as ultrasound-assisted extraction and microwave-assisted extraction can improve extraction efficiency, they require large-scale equipment, leading to higher investment and costs. These traditional methods generally suffer from long extraction cycles, low extraction rates, significant loss of effective components, and high costs. Furthermore, the toxic organic solvents commonly used in traditional extraction processes (such as dichloromethane, acetonitrile, and methanol) pose a threat to the environment and human health. Therefore, there is an urgent need to develop a new, green, and environmentally friendly extraction method to improve the efficiency of polysaccharide extraction. Summary of the Invention

[0005] The purpose of this application is to provide a convenient and rapid method for extracting polysaccharides from matsutake mushrooms. This method uses a deep eutectic solvent, is environmentally friendly, time-efficient, and uses inexpensive reagents, making it suitable for widespread application.

[0006] Another objective of this application is to provide a matsutake polysaccharide obtained by the above extraction method, which has a high yield, low protein content, and good antioxidant activity.

[0007] Another objective of this application is to provide the use of matsutake polysaccharide in the preparation of a drug for treating testicular damage caused by diabetes, which provides a new direction for the use of matsutake polysaccharide.

[0008] To solve the above-mentioned technical problems, the technical solution adopted in this application is as follows:

[0009] On the one hand, this application provides a method for deep eutectic solvent extraction of matsutake polysaccharides, comprising the following steps:

[0010] S1. Preparation of deep eutectic solution: Mix and dissolve the hydrogen donor and hydrogen acceptor reagents, and add water to obtain a transparent solution;

[0011] S2. Extraction of polysaccharides: After drying, crushing and defatting the matsutake mushrooms, they are added to the above deep eutectic solution and extracted by heating to obtain an extract. The extract is then purified, precipitated with alcohol, dialyzed and freeze-dried to obtain matsutake polysaccharides.

[0012] On the other hand, this application provides a matsutake polysaccharide prepared by the above extraction method.

[0013] In another aspect, this application provides the use of matsutake polysaccharide in the preparation of a drug for treating testicular damage caused by diabetes.

[0014] Compared with the prior art, the embodiments of this application have at least the following advantages or beneficial effects:

[0015] 1. This application provides a deep eutectic solvent system and a method for extracting polysaccharides from matsutake mushrooms. Compared with traditional solvents and existing technologies, the efficiency of this method in extracting matsutake polysaccharides is significantly higher than that of traditional aqueous solvent extraction. Moreover, the protein content produced during the extraction process is low, the matsutake polysaccharides in the extract are more stable than those extracted by traditional solvents, and the polysaccharides are more effective in alleviating testicular damage caused by diabetes than traditional methods. The equipment is simple, the production cost is low, and it can be operated continuously, making it highly practical and operable.

[0016] 2. This application provides a deep eutectic solvent system that primarily uses choline chloride as the hydrogen bond acceptor and 1,2-propylene glycol, 1,4-butanediol, 1,3-butanediol, ethylene glycol, malonic acid, and urea as hydrogen bond donors. It has the following advantages: a) Abundant raw materials at moderate prices. b) Non-toxic and pollution-free. The raw materials in this deep eutectic system are non-toxic and have good biocompatibility. All raw materials are easily degradable, non-volatile, and environmentally friendly. In summary, the deep eutectic solvent system provided in this application has strong practicality. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Here are the three-dimensional and two-dimensional plots of the response surface of the significant interaction item in Embodiment 6 of this application;

[0019] Figure 2 This is a comparison of cell viability between water-extracted matsutake polysaccharide and deep eutectic solvent-extracted matsutake polysaccharide in the treatment of diabetic testicular injury according to Example 7 of this application; Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present application will now be described in detail with reference to specific embodiments.

[0022] A method for deep eutectic solvent extraction of matsutake polysaccharides includes the following steps:

[0023] S1. Preparation of deep eutectic solution: Mix and dissolve the hydrogen donor and hydrogen acceptor reagents, and add water to obtain a transparent solution;

[0024] S2. Extraction of polysaccharides: After drying, crushing and defatting the matsutake mushrooms, they are added to the above deep eutectic solution and extracted by heating to obtain an extract. The extract is then purified, precipitated with alcohol, dialyzed and freeze-dried to obtain matsutake polysaccharides.

[0025] In some embodiments of this application, the molar ratio of the hydrogen donor and the hydrogen acceptor reagent is 1:2; the hydrogen donor is 1,2-propylene glycol, 1,4-butanediol, 1,3-butanediol, ethylene glycol, malonic acid, or urea, preferably malonic acid; the hydrogen acceptor is choline chloride.

[0026] In some embodiments of this application, the dissolution in step S1 is specifically carried out by stirring at a temperature of 70-90°C for 20-50 minutes.

[0027] In some embodiments of this application, the water content of the deep eutectic solution in step S1 is 10-50%.

[0028] In some embodiments of this application, the degreasing of matsutake mushrooms in step S2 above specifically involves soaking dried and pulverized matsutake mushroom powder in ethanol for 20-30 hours.

[0029] In some embodiments of this application, the ratio of defatted matsutake mushroom to deep eutectic solution in step S2 is 1:(10-50), preferably 1:30; the extraction temperature is 70-100℃, preferably 90℃, and the extraction time is 40-60 min.

[0030] In some embodiments of this application, the purification and alcohol precipitation in step S2 above specifically involves: centrifuging the extract at 5000 rpm and collecting the centrifuged liquid; adding 4 times the volume of ethanol to the centrifuged liquid to precipitate the precipitate, and then centrifuging at 6000 rpm and collecting the precipitate.

[0031] In some embodiments of this application, the dialysis in step S2 above specifically involves adding pure water to the centrifuged precipitate to dissolve and dialyze for 24 hours, and the flow rate used for dialysis is 3500 Da.

[0032] A matsutake polysaccharide was prepared by extraction using the deep eutectic solvent extraction method described above.

[0033] Use of a matsutake polysaccharide in the preparation of a drug for treating testicular damage caused by diabetes.

[0034] The features and performance of this application are further described in detail below with reference to the embodiments. Choline chloride, 1,2-propylene glycol, 1,4-butanediol, 1,3-butanediol, ethylene glycol, malonic acid and urea used in this application are all of analytical grade.

[0035] Example 1

[0036] This example explores the optimal hydrogen donor (HBD).

[0037] This embodiment sets up 6 experimental groups, each corresponding to a different HBD reagent, as shown in Table 1. HBA (choline chloride) and HBD reagent are added to a flask at a molar ratio of 1:2 and stirred at 70°C for 30 min. After stirring, water is added to prepare a colorless and transparent solution with a water content of 30%, which is a deep eutectic solvent system.

[0038] Table 1

[0039] experimental group HBD HBA∶HBD shape 1 1,2-Propane glycol 1∶2 Colorless and transparent 2 1,4-Butanediol 1∶2 Colorless and transparent 3 1,3-Butanediol 1∶2 Colorless and transparent 4 Ethylene glycol 1∶2 Colorless and transparent 5 malonic acid 1∶2 Colorless and transparent 6 Urea 1∶2 Colorless and transparent

[0040] Matsutake mushrooms were dried and pulverized, then soaked in ethanol for 24 hours to obtain defatted matsutake powder. The defatted matsutake powder was added to a deep eutectic solvent at a material-to-liquid ratio (W / V = 1:30), and extracted at 90°C for 45 minutes. After extraction, the mixture was centrifuged at 5000 rpm, and the centrifuged liquid was collected. Four times the volume of ethanol was added to the centrifuged liquid to precipitate the precipitate, which was then collected by centrifugation at 6000 rpm. The precipitate was dissolved in pure water and dialyzed for 24 hours. After dialyzing, the precipitate was freeze-dried to obtain crude matsutake polysaccharide. In this embodiment, water was used as a control group. The water extraction group was prepared by adding defatted matsutake powder at a material-to-liquid ratio of 1:30, boiling in water at 100°C for 3 hours, and repeating this process three times. The concentrated extract was collected, and subsequent steps were the same. The yield of crude polysaccharide (x, n = 3) is shown in Table 2.

[0041] Table 2

[0042]

[0043] As can be seen from Tables 1 and 2, the experimental results show that the traditional water extraction rate is around 3.0%, while the deep eutectic solvent system using choline chloride and malonic acid has a better extraction rate than water extraction, and the required steps and time are less than those of water extraction.

[0044] Example 2

[0045] This embodiment investigates the effect of deep eutectic solutions with different water contents on the yield of matsutake polysaccharides.

[0046] Choline chloride and malonic acid were added to a beaker at a molar ratio of 1:2. While heating, water was added by weight (wt%) to prepare deep eutectic solutions with different water contents. Then, pulverized and defatted matsutake mushroom powder was added, and the mixture was heated at 90℃ for 45 min for extraction. After extraction, the mixture was centrifuged at 5000 rpm, and the centrifuged liquid was collected. Four times the volume of ethanol was added to the centrifuged liquid to precipitate the precipitate. After centrifugation at 6000 rpm, the precipitate was collected, dissolved in pure water, and dialyzed for 24 h. After dialyzing, the precipitate was freeze-dried to obtain crude matsutake polysaccharide. Each group was repeated three times. The yield of crude matsutake polysaccharide is shown in Table 3.

[0047] Table 3

[0048] Serial Number HBD Moisture content Crude polysaccharide yield (%) 1 malonic acid 10％ 1.3308±0.2018 2 malonic acid 20％ 2.0918±0.2064 3 malonic acid 30％ 4.2792±0.3391 4 malonic acid 40％ 2.9076±0.2063 5 malonic acid 50％ 2.8195±0.2086

[0049] As can be seen from Table 3, the experimental results show that the extraction efficiency is higher when the water content of the deep eutectic solution is 30%, therefore, the water content is selected to be 30%.

[0050] Example 3

[0051] This embodiment investigates the effect of different extraction times on the yield of matsutake polysaccharides.

[0052] Matsutake mushroom powder was added to a deep eutectic solution with 30% water content (molar ratio of choline chloride to malonic acid 1:2) at a material-to-liquid ratio of 1:30, and extracted by heating to 90℃. After extraction, the mixture was centrifuged at 5000 rpm, and the centrifuged liquid was collected. Four volumes of ethanol were added to the centrifuged liquid to precipitate the precipitate, which was then collected by centrifugation at 6000 rpm. The precipitate was dissolved in pure water and dialyzed for 24 h. After dialyzing, the precipitate was freeze-dried to obtain crude matsutake polysaccharide. The extraction time and the corresponding crude polysaccharide yield (x, n=3) are shown in Table 4.

[0053] Table 4

[0054] Serial Number HBD Extraction time (min) Crude polysaccharide yield (%) 1 malonic acid 15 1.3563±0.2183 2 malonic acid 25 2.3564±0.3784 3 malonic acid 35 3.3999±0.102 4 malonic acid 45 4.1872±0.199 5 malonic acid 55 5.6512±0.2385 6 malonic acid 65 3.502±0.2265

[0055] As can be seen from Table 4, the experimental results show that the extraction efficiency is higher when the extraction time is 55 min, so the extraction time was chosen to be 55 min.

[0056] Example 4

[0057] This embodiment investigates the effect of different extraction heating temperatures on the yield of matsutake polysaccharides.

[0058] Matsutake mushroom powder was added to a deep eutectic solution with 30% water content (molar ratio of choline chloride to malonic acid 1:2) at a material-to-liquid ratio of 1:30 and heated for 55 min. After extraction, the mixture was centrifuged at 5000 rpm, and the centrifuged liquid was collected. Four times the volume of ethanol was added to the centrifuged liquid to precipitate the polysaccharide. After centrifugation at 6000 rpm, the precipitate was collected, dissolved in pure water, and dialyzed for 24 h. After dialyzing, the polysaccharide was freeze-dried to obtain crude matsutake polysaccharide. The extraction temperature and yield of crude polysaccharide were also discussed. As shown in Table 5.

[0059] Table 5

[0060] Serial Number HBD Extraction temperature (°C) Crude polysaccharide yield (%) 1 malonic acid 60 1.9983±0.0406 2 malonic acid 70 3.2229±0.2034 3 malonic acid 80 2.5093±0.2482 4 malonic acid 90 5.526±0.2694 5 malonic acid 100 2.9615±0.0873

[0061] As can be seen from Table 5, the experimental results show that the extraction efficiency is higher when the extraction temperature is 90℃, so the extraction temperature was chosen to be 90℃.

[0062] Example 5

[0063] This embodiment investigates the effect of different liquid-to-solid ratios on the yield of matsutake polysaccharides.

[0064] Matsutake mushroom powder was added to a deep eutectic solution with a 30% water content (molar ratio of choline chloride to malonic acid 1:2) at different material-to-liquid ratios, and extracted at 90℃ for 55 min. After extraction, the mixture was centrifuged at 5000 rpm, and the centrifuged liquid was collected. Four times the volume of ethanol was added to the centrifuged liquid to precipitate the precipitate, which was then collected by centrifugation at 6000 rpm. The precipitate was dissolved in pure water and dialyzed for 24 h. After dialyzing, the crude matsutake polysaccharide was obtained by freeze-drying. (Liquid-to-material ratio and polysaccharide yield are described.) As shown in Table 6.

[0065] Table 6

[0066] Serial Number HBD Liquid-to-solid ratio (V / g) Crude polysaccharide yield (%) 1 malonic acid 10:1 3.0512±0.0573 2 malonic acid 20:1 3.6781±0.2408 3 malonic acid 30:1 5.578±0.2124 4 malonic acid 40:1 4.221±0.1511 5 malonic acid 50:1 3.544±0.1795

[0067] As can be seen from Table 6, the experimental results show that the extraction efficiency is higher when the liquid-to-solid ratio is 30:1, therefore the liquid-to-solid ratio is chosen to be 30:1.

[0068] Example 6

[0069] This embodiment involves 27 sets of experiments, with extraction temperature (°C), time (min), liquid-to-solid ratio (V / g), and water content (%) as influencing factors. The polysaccharide extraction rate is the evaluation index. The design combination and its corresponding experimental results are shown in Table 7 (response surface experimental factor levels).

[0070] Table 7

[0071]

[0072]

[0073] The variance results of the response surface methodology experiment are shown in Table 8:

[0074] Table 8

[0075]

[0076]

[0077] As shown in Table 8, the P-value of the regression equation model is extremely significant, indicating that the model has a good fit and the experimental method is feasible; the P-value of the lack-of-fit term is not significant, indicating that the model error is small; the larger the F-value and the smaller the P-value of a factor, the greater the influence of that factor on the result. Therefore, the order of influence of each factor on the extraction rate of matsutake polysaccharide is: extraction time > liquid-to-solid ratio > water content > extraction temperature.

[0078] A regression equation was established by fitting data to the extraction rate Y of matsutake polysaccharides using extraction temperature (A), extraction time (B), liquid-to-solid ratio (C), and moisture content (D) as independent variables:

[0079] Y=5.46663+0.102135*A+0.394502*B+0.193767*C+-0.177117*D+0.04598*AB+-0.16075*AC+0.149175*AD +0.1795*BC+-0.164025*BD+-0.12375*CD+-0.974469*A^2+-1.40289*B^2+-0.688872*C^2+-1.10022*D^2

[0080] Response surface diagrams of salient interaction terms (3D and 2D, as shown in Figure A) Figure 1 As shown; Figure 1 A: Interaction between temperature and liquid-to-solid ratio; B: Interaction between time and liquid-to-solid ratio; C: Interaction between time and moisture content.

[0081] Response surface methodology (RSM) plots are used to analyze target response values ​​and independent variables. Together, they form a three-dimensional spatial plot. By analyzing the three-dimensional RSM plot, the influence of different factors on the target response value can be significantly reflected, thus obtaining the interaction factors of each factor in the response process. The effectiveness of the influence and extreme values ​​can be determined by analyzing response parameters under different conditions. Response surface experimental plots and contour lines are shown below. Figure 1 As shown, the curves of the interaction terms AC, BC, and BD are relatively steep and have a significant impact on the results.

[0082] The optimal model obtained through response surface methodology is as follows: extraction temperature 90.2℃, extraction time 56 min, liquid-to-solid ratio 31.5 V / g, and water content 29.6%. Three repeated experiments verified that the average polysaccharide extraction rate was 5.4516%, which is close to the predicted value of 5.5155%, demonstrating the high reliability of the extraction process obtained from the response surface methodology.

[0083] Example 7

[0084] This embodiment investigates the therapeutic effect of matsutake polysaccharide on diabetic testicular injury.

[0085] TM4 cells were cultured at 37°C, 5% CO2, and 95% air in DMEM / F12 medium containing 10% FBS and 1% penicillin-streptomycin. When the cells reached 80-90% confluence, they were divided into four groups: a normal control group, a PA model group (PA 400 μM), a water-extracted polysaccharide group, and a deep co-crystallized solvent-extracted polysaccharide group. The PA model group and the treatment group received a combined intervention for 48 hours in DMEM / F12 medium containing a final concentration of 400 μM palmitic acid and the same concentration of matsutake polysaccharide. After the intervention, 100 μL of pre-prepared 10% CCK8 solution was added to each well, and the cells were returned to the incubator. After reacting in the dark for 2 hours, the results were detected using a microplate reader (absorbance 450 nM).

[0086] Figure 2 Comparison of cell viability between water-extracted and deep eutectic solvent-extracted matsutake polysaccharides in the treatment of diabetic testicular injury. (TMP-W: water-extracted matsutake polysaccharides; TMP-DES: deep eutectic solvent-extracted matsutake polysaccharides)

[0087] Depend on Figure 2 The results showed that matsutake polysaccharides extracted by deep eutectic extraction were more effective than those extracted by water in treating diabetic testicular injury.

[0088] Example 8

[0089] This embodiment investigates the comparison of protein content between deep eutectic solvents and traditional water-extracted matsutake polysaccharides.

[0090] Polysaccharide extraction was performed on 1g of matsutake mushroom powder using the same material-to-liquid ratio of 1:30. The protein concentration of the extract was then detected using the protein A280 method with an ultra-micro nucleic acid protein analyzer. The protein concentration comparison results are shown in Table 9.

[0091] Table 9

[0092] Sample Name mg / ml A280 Water extraction method 25.59 25.591 Deep eutectic solvent extraction method 15.018 15.02

[0093] As can be seen from Table 9, the protein content produced during the deep eutectic solvent extraction process is lower. Meanwhile, the equipment indicated nucleic acid contamination after testing of the water extraction sample, while the deep eutectic solvent extraction sample did not show any contamination during testing.

[0094] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A deep eutectic solvent extraction method of Tricholoma matsutake polysaccharides, characterized by, The method comprises the following steps: S1, preparing a deep eutectic solution: mixing and dissolving a hydrogen donor and a hydrogen acceptor reagent, and obtaining a transparent solution after adding water; the molar ratio of the hydrogen donor and the hydrogen acceptor reagent is 1:2; the hydrogen donor is malonic acid, and the hydrogen acceptor is choline chloride; the dissolving is specifically stirring for 20-50 min at a temperature of 70-90 DEG C; the water content of the deep eutectic solution is 30%; S2, extracting polysaccharides: after drying, crushing and defatting the Tricholoma matsutake, the Tricholoma matsutake is added to the deep eutectic solution, and an extract is obtained after heating extraction; the extract is purified, alcohol precipitated, dialyzed and freeze-dried to obtain Tricholoma matsutake polysaccharides; The solid-liquid ratio of the defatted Tricholoma matsutake and the deep eutectic solution is 1: (10-50), the heating extraction temperature is 70-100 DEG C, and the extraction time is 40-60 min; the dialysis is specifically adding pure water to the centrifugal precipitate to dissolve and dialyze for 24 h, and the cut-off volume used in the dialysis is 3500 Da; The Tricholoma matsutake polysaccharides are used for preparing a medicine for treating testicular damage caused by diabetes.

2. The deep eutectic solvent extraction method of tricholoma matsutake polysaccharides according to claim 1, characterized in that, The defatting of the Tricholoma matsutake in the S2 step is specifically soaking the dried and crushed Tricholoma matsutake powder in ethanol for 20-30 h.

3. The deep eutectic solvent extraction method of tricholoma matsutake polysaccharides according to claim 1, characterized in that, The purification and alcohol precipitation in the S2 step are specifically: centrifuging the extract at a speed of 5000 rpm, collecting the centrifugal liquid, adding 4 times the volume of ethanol to the centrifugal liquid to precipitate, and then centrifuging at a speed of 6000 rpm to collect the precipitate.

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