A method for extracting highly active polysaccharides from Grifola frondosa

Abstract

A method for extracting highly active polysaccharides from Grifola frondosa solves the technical problems of the yield and purity of highly active polysaccharides, and the method comprises: extracting lipids and small molecular substances from Grifola frondosa, extracting strong non-polar substances, polar substances, extracting weak non-polar substances, extracting non-polar and polar substances simultaneously, extracting highly active polysaccharides, dissolving highly active polysaccharides in water, evaporating and concentrating highly active polysaccharide aqueous solutions, purifying by gel chromatography, and obtaining highly active polysaccharides with high purity. The beneficial effects are: simple process route, low cost, high efficiency, no destruction of the structure of highly active polysaccharides from Grifola frondosa, high active polysaccharide yield of 7.12%, and high active polysaccharide purity of 99.37%.

Description

Technical Field

[0001] The invention relates to a chemical extraction method, in particular to a method for extracting highly active polysaccharides from Grifola frondosa. Background Art

[0002] Edible fungi generally have large fruits and delicious taste. They are more medicinal than other fungi such as Ganoderma lucidum. They are rare and can be used as both medicine and food. They have a refreshing and refreshing smell, tender and tender flesh, unique flavor and endless aftertaste. The dried product of Grifola frondosa contains 21.4% carbohydrates and 25.6% protein. It has dual economic value for food and medicine, especially a large amount of protein, highly active polysaccharides, minerals and vitamins can be extracted from edible fungi. Grifola frondosa, also known as chestnut mushroom, has 2% edible value and medicinal value (18.86% of which are 18 kinds of amino acids required by the human body), 3.2% fat, 33.7% dietary fiber and 5.1% ash. At the same time, Grifola frondosa is rich in various minerals and vitamins, among which the content of vitamin E is 10 to 20 times higher than that of ordinary edible fungi, and the content of vitamin C is 3 to 5 times that of its peers. It is also rich in carotene and VB [1]. Grifola frondosa is rich in amino acids needed by the human body, such as arginine, lysine, aspartic acid and glutamic acid. Therefore, Grifola frondosa can promote the healthy physical growth and intellectual development of children, help food maintain its freshness, and is beneficial to balanced nutrition.

[0003] In addition to its edible value, many researchers have recently found that highly active polysaccharides extracted from Grifola frondosa have extremely high biological activities, such as anti-tumor[2], antioxidant[3, 4], antibacterial and other activities[5]. High-purity and highly active polysaccharides can be developed into safe drugs without side effects[6]. Grifola frondosa highly active polysaccharides can be used as a natural and potential immunomodulator and as a therapeutic drug to alleviate chemotherapy-induced immunosuppression.

[0004] In recent years, in the prior art, the extraction and separation of highly active polysaccharides with active components in Grifola frondosa is carried out by cold or hot water extraction or alkaline extraction. Although cold water extraction does not destroy the structure of highly active polysaccharides, the yield is very low and the extraction time is long, which makes it impossible to industrialize. See references zhang[7], Li[5] and Chen[8], all of which use hot water extraction and then alcohol precipitation to obtain crude highly active polysaccharides. However, although this method has a higher yield than cold water extraction, it is easy to destroy the structure of highly active polysaccharides in Grifola frondosa, and the yield of highly active polysaccharides is only 2.45%. The purity of highly active polysaccharides extracted by hot water is only about 80-90%. The purity of highly active polysaccharides extracted by alkaline extraction can reach up to 90%. Literature reports that the biological activity of Grifola frondosa highly active polysaccharides is mainly due to the connection mode of highly active polysaccharides and the three-dimensional helical structure of highly active polysaccharides. However, during the hot water or alkali extraction process, the glycopeptide bond may break, the molecular weight or structure changes, and the three-dimensional helical structure is destroyed. Therefore, the biological activity of highly active polysaccharides extracted by this method is relatively low. Although the yield of highly active polysaccharides is increased by hot alkali extraction, the peptide chain and spatial structure of highly active polysaccharides may be destroyed, and the biological activity of highly active polysaccharides is poor. How to invent an extraction method that does not destroy the structure of Grifola frondosa highly active polysaccharides and can improve the yield and purity of highly active polysaccharides is a technical problem that needs to be solved urgently to achieve the medicinal purpose of highly active polysaccharides. Summary of the invention

[0005] In order to achieve the invention objective of not destroying the structure of Grifola frondosa high-activity polysaccharide and improving the yield and purity of the high-activity polysaccharide, the present invention discloses a method for extracting high-activity polysaccharide from Grifola frondosa.

[0006] The method adopted by the present invention to achieve the purpose of the invention is:

[0007] Step 1. Remove impurities from the maitake mushroom, wash it, dry it, and then crush it into less than 200 mesh;

[0008] Step 2. The crushed maitake mushrooms are repeatedly extracted multiple times with a lipid and small molecule substance extracting solution in milliliter volume at a mixing ratio of 1:5-15 by gram weight until all lipids and small molecules in the maitake mushrooms are extracted, and the extracting solution is removed after the extraction treatment to obtain maitake mushroom powder with lipids and small molecules removed, and the residue is recorded as precipitate 1;

[0009] Step 3. The precipitate 1 obtained in step 2 is repeatedly extracted multiple times with the strong non-polar substance extracting solution in milliliters at a ratio of 1:5-15, until all the strong non-polar substances in the precipitate 1 are extracted, and the extracting solution is removed to obtain the precipitate 1 from which the strong non-polar substances are removed, and the residue is recorded as the precipitate 2;

[0010] Step 4. The precipitate 2 obtained in step 3 is repeatedly extracted multiple times by weight using a polar substance extraction solution by volume in milliliters at a ratio of 1:5-15 until all polar substances in the precipitate 2 are extracted, and the extraction solution is removed to obtain the precipitate 2 from which the polar substances are removed, and the residue is recorded as precipitate 3;

[0011] Step 5. Extract the precipitate 3 obtained in step 4 with a weak non-polar substance extracting solution in milliliter volume at a ratio of 1:5-15 by gram weight, and extract the weak non-polar substance in the precipitate 3 for more than 100 hours, remove the extracting solution, and obtain the precipitate 3 with the weak non-polar substance removed, and the residue is recorded as precipitate 4;

[0012] Step 6. The precipitate 4 obtained in step 5 is continuously extracted by gram weight using a non-polar material extracting solution and a polar material extracting solution by milliliter volume at a ratio of 1:1:1 for more than 100 hours to extract both non-polar and non-polar substances in the precipitate 4, and the extracting solution is removed to obtain the precipitate 4 from which both non-polar and non-polar substances are removed, and the residue is recorded as precipitate 5;

[0013] Step 7. Extract the highly active polysaccharide from the precipitate 5 obtained in step 6 by weight in grams and by volume in milliliters of DMSO at a ratio of 1:20, at an extraction temperature of 20-50° C., and for 4-8 hours. Then slowly drip the DMSO containing the highly active polysaccharide into 10V acetone, and after standing for 12 hours, obtain flocculent crystals of crude highly active polysaccharide and a mixed liquid of DMSO and acetone;

[0014] Step 8. Use a centrifuge to separate the flocculent crystals of the highly active polysaccharide from the DMSO and acetone liquids, add the flocculent highly active polysaccharide crystals into purified water, completely dissolve the highly active polysaccharide soluble in water in the purified water, remove the highly active polysaccharide impurities insoluble in water, and obtain purified water containing highly active polysaccharide dissolved in water;

[0015] Step 9. The purified water containing highly active polysaccharides dissolved in water obtained in step 8 is concentrated by rotary evaporation to obtain deionized water, and then freeze-dried to obtain crude highly active polysaccharides;

[0016] Step 10. Purify the crude high-activity polysaccharide obtained in step 9 by gel chromatography to obtain high-purity high-activity polysaccharide.

[0017] The invention has the following beneficial effects: the process route is simple, the cost is low, the efficiency is high, the structure of the Grifola frondosa high-activity polysaccharide is not destroyed, the yield of the high-activity polysaccharide reaches 7.12%, and the purity of the high-activity polysaccharide can reach 99.37%.

[0018] The present invention is described in detail below with reference to the flow chart. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Attached Figure 1 It is a process flow chart of the present invention.

[0020] Attached Figure 2 The following is a comparison table of beneficial effects of the embodiments of the invention and the prior art. DETAILED DESCRIPTION

[0021] Referring to the flow chart, a method for extracting highly active polysaccharides from Grifola frondosa is implemented by the following steps:

[0022] Step 1. Remove impurities from the maitake mushroom, wash it, dry it, and then crush it into less than 200 mesh;

[0023] Step 2. The crushed maitake mushrooms are repeatedly extracted multiple times with a lipid and small molecule substance extracting solution in milliliter volume at a mixing ratio of 1:5-15 by gram weight until all lipids and small molecules in the maitake mushrooms are extracted, and the extracting solution is removed after the extraction treatment to obtain maitake mushroom powder with lipids and small molecules removed, and the residue is recorded as precipitate 1;

[0024] Step 3. The precipitate 1 obtained in step 2 is repeatedly extracted multiple times with the strong non-polar substance extracting solution in milliliters at a ratio of 1:5-15, until all the strong non-polar substances in the precipitate 1 are extracted, and the extracting solution is removed to obtain the precipitate 1 from which the strong non-polar substances are removed, and the residue is recorded as the precipitate 2;

[0025] Step 4. The precipitate 2 obtained in step 3 is repeatedly extracted multiple times by weight using a polar substance extraction solution by volume in milliliters at a ratio of 1:5-15 until all polar substances in the precipitate 2 are extracted, and the extraction solution is removed to obtain the precipitate 2 from which the polar substances are removed, and the residue is recorded as precipitate 3;

[0026] Step 5. Extract the precipitate 3 obtained in step 4 with a weak non-polar substance extracting solution in a milliliter volume at a ratio of 1:5-15 by gram weight, and extract the weak non-polar substance in the precipitate 3 for more than 100 hours, remove the extracting solution, and obtain the precipitate 3 with the weak non-polar substance removed, and the residue is recorded as precipitate 4;

[0027] Step 6. The precipitate 4 obtained in step 5 is continuously extracted by gram weight using a non-polar material extracting solution and a polar material extracting solution by milliliter volume at a ratio of 1:1:1 for more than 100 hours to extract both non-polar and non-polar substances in the precipitate 4, and the extracting solution is removed to obtain the precipitate 4 from which both non-polar and non-polar substances are removed, and the residue is recorded as precipitate 5;

[0028] Step 7. Extract the highly active polysaccharide from the precipitate 5 obtained in step 6 by weight in grams and by volume in milliliters of DMSO at a ratio of 1:20, at an extraction temperature of 20-50° C., and for 4-8 hours. Then slowly drip the DMSO containing the highly active polysaccharide into 10V acetone, and after standing for 12 hours, obtain flocculent crystals of crude highly active polysaccharide and a mixed liquid of DMSO and acetone;

[0029] Step 8. Use a centrifuge to separate the flocculent crystals of the highly active polysaccharide from the DMSO and acetone liquids, add the flocculent highly active polysaccharide crystals into purified water, completely dissolve the highly active polysaccharide soluble in water in the purified water, remove the highly active polysaccharide impurities insoluble in water, and obtain purified water containing highly active polysaccharide dissolved in water;

[0030] Step 9. The purified water containing highly active polysaccharides dissolved in water obtained in step 8 is concentrated by rotary evaporation, and then freeze-dried to obtain crude highly active polysaccharides;

[0031] Step 10. The crude high-activity polysaccharide obtained in step 9 is purified by gel chromatography (agar column analysis) to obtain high-purity and high-activity polysaccharide.

[0032] In the embodiment of the present invention, in step 7 of the method, when DMSO is used to extract the highly active polysaccharides, a microwave generator is placed in the extraction to promote the extraction of the highly active polysaccharides.

[0033] In the embodiment of the present invention, in step 2 of the method, the lipid and small molecule substance extracting liquid is: petroleum ether or cyclohexane.

[0034] In the embodiment of the present invention, in step 3 of the method, the non-polar material extracting liquid is: carbon disulfide or carbon tetrachloride, etc.

[0035] In an embodiment of the present invention, in step 4 of the method, the polar material extraction liquid is: methanol or pyridine.

[0036] In the embodiment of the present invention, in step 5 of the method, the weaker non-polar extracting liquid is acetone or n-propanol.

[0037] In the embodiment of the present invention, in step 6 of the method, the non-polar extracting liquid and the polar extracting liquid are: acetone or carbon disulfide.

[0038] Case 1.

[0039] Weigh 50 g of dried raw material of Grifola frondosa, crush it, and pass it through a 200-mesh sieve. Pretreatment of Grifola frondosa:

[0040] a) The ground Grifola frondosa powder was extracted with 500 ml of petroleum ether (solid-liquid ratio: 1:10) until the lipids and small molecules in the Grifola frondosa were completely removed. The Grifola frondosa powder treated with petroleum ether was recorded as precipitate 1.

[0041] b) Extract the precipitate 1 with 300 ml of carbon disulfide until the extracted carbon disulfide is colorless. The residue after treating the precipitate 1 with carbon disulfide is recorded as precipitate 2.

[0042] c) Extract the precipitate 2 with 300 ml of methanol until the methanol extract is colorless. The residue after treating the precipitate 2 with methanol is recorded as precipitate 3.

[0043] d) The precipitate 3 was extracted with 300 ml of acetone for 5 consecutive days. The residue after the precipitate 3 was treated with acetone was recorded as precipitate 4.

[0044] e) The precipitate 4 was extracted with 500 ml of an equal volume of acetone / carbon disulfide for 5 consecutive days. The residue after treating the precipitate 4 with acetone was recorded as precipitate 5.

[0045] f) The precipitate 5 was extracted with DMSO at a solid-liquid ratio of (1:20), an extraction temperature of 20°C, a microwave power of 80W, and an extraction time of 6h for highly active polysaccharides. Then, DMSO was slowly dripped into 10V acetone, and then allowed to stand overnight. Then, the liquid was poured out after centrifugation at 8000r / min for 30min in a high-speed centrifuge, and deionized water was added to the centrifuge cup to completely dissolve the precipitate. Then, the liquid was poured out and collected after centrifugation at 8000r / min for 30min. The solution was concentrated by rotary evaporation, and freeze-dried after concentration to obtain a crude highly active polysaccharide weighing 5.12g. Then, the highly active polysaccharide was purified by gel chromatography to obtain 3.07g of highly active polysaccharide after purification.

[0046] Case 2.

[0047] The precipitate 5 was extracted with DMSO at a solid-liquid ratio of (1:30), an extraction temperature of 35°C, a microwave power of 120w, and an extraction time of 8h for highly active polysaccharides, and then DMSO was slowly dripped into 10V acetone, and then allowed to stand overnight, and then centrifuged at 8000r / min for 30min using a high-speed centrifuge, and then the liquid was poured out, and deionized water was added to the centrifuge cup to completely dissolve the precipitate, and then centrifuged at 8000r / min for 30min, and then the liquid was poured out and collected, and the solution was concentrated by rotary evaporation, and then freeze-dried after concentration to obtain a highly active polysaccharide weighing 5.43g. Then the highly active polysaccharide was purified by gel chromatography, and the purified highly active polysaccharide was 3.56g.

[0048] Case 3

[0049] The precipitate 5 was extracted with DMSO at a solid-liquid ratio of (1:40), an extraction temperature of 50°C, a microwave power of 160w, and an extraction time of 10h for highly active polysaccharides, and then DMSO was slowly dripped into 10V acetone, and then allowed to stand overnight, and then centrifuged at 8000r / min for 30min using a high-speed centrifuge, and then the liquid was poured out, and deionized water was added to the centrifuge cup to completely dissolve the precipitate, and then centrifuged at 8000r / min for 30min, and then the liquid was poured out and collected, and the solution was concentrated by rotary evaporation, and then freeze-dried after concentration to obtain a highly active polysaccharide weighing 5.37g. Then the highly active polysaccharide was purified by gel chromatography, and the purified highly active polysaccharide was 3.41g.

[0050] Case 4

[0051] The precipitate 5 was extracted with DMSO at a solid-liquid ratio of (1:40), an extraction temperature of 35°C, a microwave power of 160w, and an extraction time of 10h for highly active polysaccharides, and then DMSO was slowly dripped into 10V acetone, and then allowed to stand overnight, and then centrifuged at 8000r / min for 30min using a high-speed centrifuge, and then the liquid was poured out, and deionized water was added to the centrifuge cup to completely dissolve the precipitate, and then centrifuged at 8000r / min for 30min, and then the liquid was poured out and collected, and the solution was concentrated by rotary evaporation, and then freeze-dried after concentration to obtain a highly active polysaccharide weighing 5.43g. Then the highly active polysaccharide was purified by gel chromatography, and the purified highly active polysaccharide was 3.52g.

[0052] Case 5

[0053] The precipitate 5 was extracted with DMSO at a solid-liquid ratio of (1:40), an extraction temperature of 35°C, a microwave power of 120w, and an extraction time of 10h for highly active polysaccharides, and then DMSO was slowly dripped into 10V acetone, and then allowed to stand overnight, and then centrifuged at 8000r / min for 30min using a high-speed centrifuge, and then the liquid was poured out, and deionized water was added to the centrifuge cup to completely dissolve the precipitate, and then centrifuged at 8000r / min for 30min, and then the liquid was poured out and collected, and the solution was concentrated by rotary evaporation, and then freeze-dried after concentration to obtain a highly active polysaccharide weighing 5.41g. Then the highly active polysaccharide was purified by gel chromatography, and the purified highly active polysaccharide was 3.50g.

[0054] The prior art documents of the present invention are as follows:

[0055] [1] H. Horio, M. OHTSURU, Maitake (Grifola frondosa) Improve GlucoseTolerance of Experimental Diabetic Rats, Journal of Nutritional Science &Vitaminology 47(1) 57-63.

[0056] [2] L.M. Xie, M.Y. Shen, Y.Z. Hong, H.D. Ye, L.X. Huang, J.H. Xie,Chemical modifications of polysaccharides and their anti-tumor activities,Carbohyd Polym 229 (2020).

[0057] [3] L. Wang, H.M. Liu, G.Y. Qin, Structure characterization andantioxidant activity of polysaccharides from Chinese quince seed meal, FoodChem 234 (2017) 314-322.

[0058] [4] G.H. Wu, T. Hu, Z.Y. Li, Z.L. Huang, J.G. Jiang, In vitroantioxidant activities of the polysaccharides from Pleurotus tuber-regium(Fr.) Sing., Food Chem 148 (2014) 351-356.

[0059] [5] Q. Li, F.M. Zhang, G.Y. Chen, Y. Chen, W.J. Zhang, G.H. Mao, T.Zhao, M. Zhang, L.Q. Yang, X.Y. Wu, Purification, characterization andimmunomodulatory activity of a novel polysaccharide from Grifola frondosa,Int J Biol Macromol 111 (2018) 1293-1303.

[0060] [6] Y.Q. Xu, F. Cai, Z.Y. Yu, L. Zhang, X.G. Li, Y. Yang, G.J. Liu,Optimisation of pressurised water extraction of polysaccharides fromblackcurrant and its antioxidant activity, Food Chem 194 (2016) 650-658.

[0061] [7] A.Q. Zhang, J.Y. Deng, S.Y. Yu, F.M. Zhang, R.J. Linhardt, P.L.Sun, Purification and structural elucidation of a water-solublepolysaccharide from the fruiting bodies of the Grifola frondosa, Int J BiolMacromol 115 (2018) 221-226.

[0062] [8] P. Chen, H.P. Liu, H.H. Ji, N.X. Sun, Y.Y. Feng, A cold-watersoluble polysaccharide isolated from Grifola frondosa induces the apoptosisof HepG2 cells through mitochondrial passway, Int J Biol Macromol 125 (2019)1232-1241.

Claims

1. A method for extracting highly active polysaccharides from Grifola frondosa, characterized in that: The method is implemented by the following steps: Step 1. Remove impurities from the maitake mushroom, wash it, dry it, and then crush it into less than 200 mesh; Step 2. The crushed maitake mushroom is repeatedly extracted multiple times by gram weight using a lipid and small molecule substance extracting solution at a mixing ratio of 1:5-15 by volume in milliliters until all lipids and small molecules in the maitake mushroom are extracted. After the extraction treatment, the extracting solution is removed to obtain maitake mushroom powder with lipids and small molecules removed. The residue is recorded as precipitate 1. The lipid and small molecule substance extracting solution is: petroleum ether or cyclohexane; Step 3. The precipitate 1 obtained in step 2 is repeatedly extracted multiple times by gram weight using a strong non-polar substance extracting solution at a ratio of 1:5-15 by volume per milliliter until all the strong non-polar substances in the precipitate 1 are extracted, and the extracting solution is removed to obtain the precipitate 1 from which the strong non-polar substances are removed. The residue is recorded as precipitate 2, and the non-polar substance extracting solution is: carbon disulfide or carbon tetrachloride; Step 4. The precipitate 2 obtained in step 3 is repeatedly extracted multiple times by gram weight using a polar substance extraction solution at a ratio of 1:5-15 per milliliter volume until all polar substances in the precipitate 2 are extracted, and the extraction solution is removed to obtain the precipitate 2 from which the polar substances are removed. The residue is recorded as precipitate 3, and the polar substance extraction solution is: methanol or pyridine; Step 5. Extract the precipitate 3 obtained in step 4 by gram weight with a weak non-polar substance extracting solution at a ratio of 1:5-15 per milliliter volume, and extract the weak non-polar substance in the precipitate 3 for more than 100 hours, remove the extracting solution, and obtain the precipitate 3 with the weak non-polar substance removed. The residue is recorded as precipitate 4. The weak non-polar extracting solution is acetone or n-propanol. Step 6. The precipitate 4 obtained in step 5 is continuously extracted by gram weight using equal volumes of acetone / carbon disulfide for more than 100 hours, and the extract is removed to obtain a residue which is recorded as precipitate 5; Step 7. Extract the highly active polysaccharide by weight of the precipitate 5 obtained in step 6 with DMSO in a ratio of 1:20 by volume to milliliter, at an extraction temperature of 20-50° C., and for 4-8 hours. Then slowly drip the DMSO containing the highly active polysaccharide into 10V volume of acetone, and after standing for 12 hours, obtain flocculent crystals of crude highly active polysaccharide and a mixed liquid of DMSO and acetone; Step 8. Use a centrifuge to separate the flocculent crystals of the highly active polysaccharide from the DMSO and acetone liquids, add the flocculent highly active polysaccharide crystals into purified water, completely dissolve the highly active polysaccharide soluble in water in the purified water, remove the highly active polysaccharide impurities insoluble in water, and obtain purified water containing highly active polysaccharide dissolved in water; Step 9. The purified water containing highly active polysaccharides dissolved in water obtained in step 8 is concentrated by rotary evaporation, and then freeze-dried to obtain crude highly active polysaccharides; Step 10. Purify the crude high-activity polysaccharide obtained in step 9 by gel chromatography to obtain high-purity high-activity polysaccharide.

2. The method for extracting highly active polysaccharides from Grifola frondosa according to claim 1, characterized in that: In step 7 of the method, when DMSO is used to extract the highly active polysaccharide, a microwave generator is placed in the extraction solution to promote the extraction of the highly active polysaccharide.

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