CN116854835A - High-immunocompetence acetylated longan water-soluble glucan, and preparation method and application thereof - Google Patents
High-immunocompetence acetylated longan water-soluble glucan, and preparation method and application thereof Download PDFInfo
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- CN116854835A CN116854835A CN202310872096.XA CN202310872096A CN116854835A CN 116854835 A CN116854835 A CN 116854835A CN 202310872096 A CN202310872096 A CN 202310872096A CN 116854835 A CN116854835 A CN 116854835A
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- 235000000235 Euphoria longan Nutrition 0.000 title claims abstract description 73
- 229920001503 Glucan Polymers 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 244000139609 Euphoria longan Species 0.000 title 1
- 240000001008 Dimocarpus longan Species 0.000 claims abstract description 72
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 31
- 230000005965 immune activity Effects 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000006467 substitution reaction Methods 0.000 claims abstract description 13
- 230000021736 acetylation Effects 0.000 claims abstract description 11
- 238000006640 acetylation reaction Methods 0.000 claims abstract description 11
- 210000002540 macrophage Anatomy 0.000 claims abstract description 6
- 230000004048 modification Effects 0.000 claims abstract description 6
- 238000012986 modification Methods 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims abstract description 5
- 235000013305 food Nutrition 0.000 claims abstract description 4
- 125000000218 acetic acid group Chemical class C(C)(=O)* 0.000 claims abstract 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 41
- 229920001282 polysaccharide Polymers 0.000 claims description 28
- 239000005017 polysaccharide Substances 0.000 claims description 28
- 150000004676 glycans Chemical class 0.000 claims description 27
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 238000000108 ultra-filtration Methods 0.000 claims description 15
- 229920002307 Dextran Polymers 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 210000004027 cell Anatomy 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 239000000287 crude extract Substances 0.000 claims description 6
- 238000002390 rotary evaporation Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000003809 water extraction Methods 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 5
- 238000002386 leaching Methods 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 4
- 230000002708 enhancing effect Effects 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 claims description 4
- 230000000397 acetylating effect Effects 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000012456 homogeneous solution Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 150000004075 acetic anhydrides Chemical class 0.000 claims description 2
- 230000001900 immune effect Effects 0.000 claims 1
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 230000002596 correlated effect Effects 0.000 abstract description 2
- 239000000284 extract Substances 0.000 abstract description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 8
- 239000011347 resin Substances 0.000 description 8
- 229920005989 resin Polymers 0.000 description 8
- 238000000746 purification Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 235000013376 functional food Nutrition 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 208000000044 Amnesia Diseases 0.000 description 1
- 208000031091 Amnestic disease Diseases 0.000 description 1
- 241001107116 Castanospermum australe Species 0.000 description 1
- 235000002469 Clitocybe nuda Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 244000290281 Lepista nuda Species 0.000 description 1
- 206010033557 Palpitations Diseases 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- -1 acetyl polysaccharide Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- 230000006986 amnesia Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 235000021279 black bean Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000008975 immunomodulatory function Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 description 1
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 description 1
- 235000008696 isoflavones Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The application relates to the field of foods, in particular to acetylated longan water-soluble glucan with high immune activity, a preparation method and application thereof. The method comprises extracting longan with hot water, dialyzing, ultrafiltering, etc., to obtain the extract with average molecular weight cut-off of longanMw=9.68×10 4 kDa Water-soluble glucan (LP). And then acetic anhydride is used for carrying out acetylation modification, so that derivatives with different degrees of substitution of acetyl are obtained. The RAW264.7 macrophage immune model verifies that the longan glucan with different acetyl substitution degrees shows better immune activity than natural glucan, and the immune activity is positively correlated with the acetylation degree. The preparation method described by the application has simple and reasonable operationThe selected extraction method and reagent improve the solubility of the longan glucan and improve the immunocompetence of the longan glucan.
Description
Technical Field
0001. The application relates to the field of foods, in particular to acetylated longan water-soluble glucan with high immune activity, a preparation method and application thereof.
Background
0002. At present, the preparation of water-soluble polysaccharide in plant raw materials mainly comprises a method of combining solvent extraction and macroporous resin purification. Shu Xu et al patent name "Lepista nuda water-soluble polysaccharide and preparation method and application thereof" (grant number CN 109293791B), and patent name "Tang Meiyu et al patent name" 10/09/2022 "applied by 2018, 9/12" a method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans "(grant number CN 115417848A). Although the polysaccharide can effectively remove impurities in the extracting solution after being purified by macroporous resin, the macroporous resin has the defects of long time consumption, low efficiency, complex operation, easy pollution of the resin, difficult cleaning and the like. It is therefore necessary to isolate water-soluble polysaccharides using a green, efficient process. In recent years, the ultrafiltration membrane technology has the characteristics of simple process, low cost, environmental protection, high product yield, good purity and the like, and has industrialized development prospect in the aspect of purification and enrichment of polysaccharide. Most importantly, the ultrafiltration membrane can separate polysaccharide components with specific molecular weight cut-off from the polysaccharide, thereby providing guidance for the structural analysis of the polysaccharide.
0003. Longan is commonly used in traditional Chinese medicine for promoting blood metabolism of organisms, relieving nerves, relieving insomnia, treating palpitation, amnesia and the like. In recent years, polysaccharides are considered as main components of longan which exert their biological activities, and in particular, as immune-modulating functions in organisms, and have become one of research hotspots in the medical field. However, the longan polysaccharide is not easy to dissolve in water due to the large molecular weight of the longan polysaccharide, so that the further exertion of the biological activity of the polysaccharide is affected.
Disclosure of Invention
The purpose of the application is that: in order to provide the acetylated longan water-soluble glucan with better effect and high immune activity, a preparation method and application thereof, the specific purpose is to achieve a plurality of substantial technical effects in the specific implementation part.
0004. In order to achieve the above purpose, the application adopts the following technical scheme:
scheme one:
the acetylated longan water-soluble glucan with high immune activity is characterized by being an acetylated structure of the longan water-soluble glucan.
0005. The application further adopts the technical scheme that the acetylation structure of the longan water-soluble glucan comprises derivatives with different acetyl substitution degrees of the longan water-soluble glucan.
0006. Scheme II:
a process for preparing the water-soluble dextran of acetylated longan with high immunocompetence features that the water-soluble dextran of acetylated longan is prepared through hot-water extraction, dialysis and ultrafiltration to obtain the molecular weight with Mw=9.68×10 4 The kDa water-soluble glucan (LP) is subjected to acetylation modification by using acetic anhydride, so that derivatives with different degrees of acetyl substitution are obtained.
0007. The application further adopts the technical scheme that the method comprises the following steps:
s1, cleaning fresh longan and drying;
s2, obtaining a longan polysaccharide crude extract by adopting a hot water extraction method;
s3, dialyzing the crude extract, concentrating the dialyzate by adopting rotary evaporation after the dialyzate is finished, and then centrifuging the concentrated solution and obtaining a water-soluble glucan component (LP) by an ultrafiltration membrane technology;
s4, acetylating the water-soluble glucan.
0008. The application has the further technical scheme that S4, acetic anhydride and sodium hydroxide with different concentrations are respectively added to be mutually alternated, and water-soluble glucan is acetylated into three components: ac-LP1, ac-LP2 and Ac-LP3.
0009. The further technical scheme of the application is that the application also comprises S5, and the activity of the cell experiment proves that the acetylated longan glucan has stronger immune activity than LP.
0010. The further technical scheme of the application is that in the step S1, the drying treatment refers to heat pump drying at 65 ℃ for 48 hours; the conditions of hot water leaching in the step S2 are as follows: 80 ℃; extraction time: 3h;
in the step S3, the dialysis condition is that the temperature is 4 ℃ and the dialysis time is 72 hours; the rotary evaporation temperature is 60 ℃; ultrafiltration cut-off average molecular weight mw=9.68×10 4 kDa。
0011. The further technical scheme of the application is that in the step S4, the addition amounts of different acetic anhydride are respectively 1, 4 and 7mL, and the mixture is stirred for 4 hours at 30 ℃; the pH of the reaction system is regulated by 0.5M NaOH to always maintain the pH of the reaction system at 8.0-8.5; after the reaction was completed, the solution was neutralized with 5M HCl to terminate the reaction at pH 7.0, and the solution was put in a cut-off film of Mw 8000Da, dialyzed against distilled water for 48 hours and then directly lyophilized.
0012. The application further adopts the technical proposal that the dextran is separated from the longan by adopting the processes of hot water extraction, dialysis, ultrafiltration, freeze-drying and the like; 100mg of dextran was dissolved in 10mL of distilled water; stirring the mixture with a magnetic stirrer at 30 ℃ overnight until a homogeneous solution is obtained; adjusting the pH value to 9.0 by using 0.5M NaOH solution and keeping the pH value for 10min; the mixture was stirred at 30 ℃ for 4h during which 1, 4 and 7mL of acetic anhydride were added, respectively; simultaneously, 0.5M NaOH was added to the mixture with continuous stirring to maintain the pH at 8.0-8.4; after the reaction was completed, the solution was neutralized with 5M HCl to terminate the reaction at pH 7.0; the solution was loaded in a truncated membrane of Mw 8000Da and directly lyophilized after dialysis against distilled water for 48 hours.
0013. The use of the acetylated longan water-soluble glucan prepared by the preparation method of any one of the above in preparing food or medicine.
0014. The use of an acetylated longan water-soluble glucan as claimed in any preceding claim in the manufacture of a medicament for enhancing macrophage immune activity or enhancing immune activity.
0015. Compared with the prior art, the application adopting the technical scheme has the following beneficial effects: the application adopts ultrafiltration membrane technology to separate and purify longan crude polysaccharide, and the technology not only can intercept polysaccharide components with a certain molecular weight range, but also has simple process and short time consumption, and can greatly save the time (activation, column loading and sample loading and elution for about 3-4 days) required by the traditional macroporous resin purification method. Meanwhile, the macroporous resin has the characteristics of limited sample quantity, low efficiency, complex operation, weakening of purification effect due to easy pollution of the resin, low cost, environmental protection, high product yield, good purity and the like. Meanwhile, the purified longan glucan is subjected to acetylation treatment to different degrees, and the macrophage immune activity verification proves that the acetylated longan glucan with different substitution degrees shows higher immune activity than the natural glucan, so that the experimental result of the application achieves the aim.
Drawings
0016. For further explanation of the application, reference is made to the following further description, taken in conjunction with the accompanying drawings:
FIG. 1 shows longan water-soluble glucan with different degrees of acetyl substitution obtained by the preparation method of the application; 0017. FIG. 2 shows the degree of substitution results for different acetylated longan glucans;
0018. FIG. 3 shows the average molecular weight distribution of acetylated longan dextran of Table 1;
0019. FIG. 4 shows the cell viability results of Table 2, acetylated longan glucan;
0020. FIG. 5 shows the NO content results of acetylated longan dextran.
Description of the embodiments
0021. The present application is further illustrated in the following drawings and detailed description, which are to be understood as being merely illustrative of the application and not limiting the scope of the application.
0022. The patent provides a plurality of parallel schemes, and the different expressions belong to an improved scheme based on a basic scheme or a parallel scheme. Each scheme has its own unique features. In addition, the technical features of the different embodiments of the present application described below may be combined with each other as long as they do not collide with each other. The fixing manner not described herein may be any fixing manner such as screw fixing, bolt fixing or glue bonding.
0023. Chemical modification of polysaccharides is an effective method for improving their biological functions. Wherein, acetylation can expose more hydroxyl groups of the polysaccharide to increase the solubility of the polysaccharide in water, which is beneficial for the polysaccharide to further exert the biological activity. Based on the research basis of the previous acetyl polysaccharide from different sources, the chemical property and the physiological function of the longan polysaccharide can be improved by utilizing the acetyl modification, so that the application of the longan polysaccharide in the functional food and medicine industry is realized. Therefore, the application aims to invent a preparation method for improving the immunocompetence of longan water-soluble glucan. Through searching, no report of separating and preparing immunocompetent polysaccharide from longan by utilizing an ultrafiltration membrane technology and performing acetylation on the immunocompetent polysaccharide is currently seen.
0024. The application aims to solve the problem that the biological activity of natural longan glucan cannot meet the application requirement, and provides a preparation method for improving the immune activity of water-soluble longan glucan.
0025. In order to achieve the above purpose, the technical scheme adopted by the application comprises the following steps:
s1, cleaning fresh longan and drying;
s2, obtaining a longan polysaccharide crude extract by adopting a hot water extraction method;
s3, dialyzing the crude extract, concentrating the dialyzate by adopting rotary evaporation after the dialyzate is finished, and then centrifuging the concentrated solution and obtaining a water-soluble glucan component (LP) by an ultrafiltration membrane technology;
s4, respectively adding acetic anhydride and sodium hydroxide with different concentrations to alternate with each other, and acetylating the water-soluble glucan into three components: ac-LP1, ac-LP2 and Ac-LP3.
0026S 5 shows that the acetylated longan glucan has stronger immune activity than LP through cell experimental activity.
0027. Further, in the step S1, the heat pump is used for drying for 48 hours at the temperature of 65 ℃;
further, the conditions for hot water leaching in the step S2 are as follows: 80 ℃; extraction time: 3h;
further, in the step S3, the dialysis condition is 4 ℃ for 72 hours; the rotary evaporation temperature is 60 ℃; ultrafiltration cut-off average molecular weight mw=9.68×10 4 kDa；
Further, in the step S4, the addition amounts of different acetic anhydrides are 1, 4 and 7mL respectively, and the mixture is stirred for 4 hours at 30 ℃; the pH of the reaction system is regulated by 0.5M NaOH to always maintain the pH of the reaction system at 8.0-8.5; after the reaction was completed, the solution was neutralized with 5M HCl to terminate the reaction (pH 7.0). The solution was loaded in a truncated membrane of Mw 8000Da and directly lyophilized after dialysis against distilled water for 48 hours. Respectively obtaining three acetylated glucan derivatives Ac-LP1, ac-LP2 and Ac-LP3;
further, the cell experiment in step S5 uses a RAW264.7 macrophage immune model.
0028. Compared with the prior art, the application has the following beneficial effects:
the application provides a preparation method of acetylated longan water-soluble glucan with immune activity. The method has simple and convenient operation flow, green and safe; compared with the traditional purification method (macroporous resin), the polysaccharide purification method (ultrafiltration membrane technology) has the characteristics of simple process, low cost, environmental protection, high product yield, good purity and the like; the chemical modification method (acetylation) improves the solubility and immunocompetence of the longan glucan.
The present application will be further described in detail with reference to the drawings and specific examples, which are given for the purpose of illustration only, and the experimental methods used in the following examples are conventional methods unless otherwise specified; materials, reagents and the like used, unless otherwise specified, are commercially available.
1. Preparation of acetylated longan glucan
The dextran is separated from longan by hot water extraction, dialysis, ultrafiltration, freeze-drying and other processes. 100mg of dextran was dissolved in 10mL of distilled water. The mixture was stirred overnight with a magnetic stirrer at 30 ℃ constant temperature until a homogeneous solution was obtained. The pH was adjusted to 9.0 with 0.5M NaOH solution for 10min. The mixture was stirred at 30℃for 4h, during which 1, 4 and 7mL of acetic anhydride were added, respectively. At the same time, 0.5M NaOH was added to the mixture with continuous stirring to maintain the pH at 8.0-8.4. After the reaction was completed, the solution was neutralized with 5M HCl to terminate the reaction (pH 7.0). The solution was loaded in a truncated membrane of Mw 8000Da and directly lyophilized after dialysis against distilled water for 48 hours. Three acetylated glucan derivatives were obtained separately: example 1, example 2, example 3.
2. Determination of the degree of substitution of acetyl groups
20mg of Ac-LPs was dissolved with 10mL of a 0.1M NaOH solution, and then transferred to a 250mL flask with continuous stirring and maintained at 50℃for 2h. Phenolphthalein was used as an indicator and the excess lye was back-titrated with 0.1M HCl until the pink color disappeared, and the acetyl content (%) was calculated as follows:
wherein V is 1 Is the volume (mL) of 0.1M HCl used to titrate the blank; v (V) 2 Is the volume (mL) of 0.1M HCl used to titrate the sample; m is the molar concentration of HCl; w is the mass (g) of the sample.
The Degree of Substitution (DS) is the average number of substituents per glucose unit, and its calculation formula is:
3. cell viability and NO content determination of acetylated longan dextran
The viability of the samples was determined using CCK-8 kit. With a content of 10% FRAW264.7 cells (5X 10) were cultured in DEME complete medium of BS 5 Individual cells/mL) was inoculated into a 96-well plate, and after incubation for 24 hours, the medium was removed and different dextran fractions were set at a concentration of 200 μg/mL as sample groups. Positive control LPS (1 μg/mL), blank (NC), after 24h of treatment, with incomplete medium according to 10:1, adding 100 mu L of CCK-8 solution into each hole, incubating for 1h, and measuring the absorbance of the cells at 450nm by using an enzyme-labeled instrument. Cell viability was calculated using the blank group as 100%. After RAW264.7 cells were cultured for 24 hours in the same manner, cell supernatants (50. Mu.L) were collected for NO determination. The NO content was determined by Griess method and the absorbance was measured at 540nm wavelength of the microplate reader.
0031. In general, the application discloses a preparation method of acetylated longan water-soluble glucan for improving immune activity. The method comprises extracting longan with hot water, dialyzing, ultrafiltering to obtain the extract with average molecular weight of Mw=9.68X10 4 kDa Water-soluble glucan (LP). And then acetic anhydride is used for carrying out acetylation modification, so that derivatives with different degrees of substitution of acetyl are obtained. The RAW264.7 macrophage immune model verifies that the longan glucan with different acetyl substitution degrees shows better immune activity than natural glucan, and the immune activity is positively correlated with the acetylation degree. The preparation method disclosed by the application is simple and reasonable to operate, and the selected extraction method and reagent improve the solubility of the longan glucan and simultaneously improve the immunocompetence of the longan glucan. The research result provides guidance for developing functional foods, health care products and other related industries with immunoregulatory activity.
0032. The foregoing has shown and described the basic principles, principal features and advantages of the application. It will be understood by those skilled in the art that the present application is not limited to the foregoing embodiments, which have been described in the foregoing description merely illustrates the principles of the application, and that various changes and modifications may be made therein without departing from the spirit and scope of the application, which is defined in the appended claims.
Claims (10)
1. The acetylated longan water-soluble glucan with high immune activity is characterized by being an acetylated structure of the longan water-soluble glucan.
2. The high immunocompetent acetylated longan water-soluble glucan of claim 1, wherein the acetylated structure of the longan water-soluble glucan comprises derivatives of longan water-soluble glucan with different degrees of acetyl substitution.
3. The preparation process of high immunological activity acetylated longan water soluble dextran is characterized by separating longan with hot water leaching, dialysis and ultrafiltering to obtain longan with interception average molecular weightMw = 9.68×10 4 The kDa water-soluble glucan (LP) is subjected to acetylation modification by using acetic anhydride, so that derivatives with different degrees of acetyl substitution are obtained.
4. The method for preparing the high-immunocompetence acetylated longan water-soluble glucan according to claim 3, wherein the method is characterized in that,
the method comprises the following steps:
s1, cleaning fresh longan and drying;
s2, obtaining a longan polysaccharide crude extract by adopting a hot water extraction method;
s3, dialyzing the crude extract, concentrating the dialyzate by adopting rotary evaporation after the dialyzate is finished, and then centrifuging the concentrated solution and obtaining a water-soluble glucan component (LP) by an ultrafiltration membrane technology;
s4, acetylating the water-soluble glucan.
5. The method for preparing the high-immunocompetence acetylated longan water-soluble glucan, as claimed in claim 4, is characterized in that S4, acetic anhydride and sodium hydroxide with different concentrations are respectively added to be alternated, and the water-soluble glucan is acetylated into three components: ac-LP1, ac-LP2 and Ac-LP3.
6. The method for preparing the high-immunocompetence acetylated longan water-soluble glucan of claim 5, further comprising the step of performing cell experiment on S5 to prove that the acetylated longan glucan has stronger immunocompetence than LP.
7. The method for preparing the high-immunocompetence acetylated longan water-soluble glucan according to claim 4, wherein the drying treatment in the step S1 is drying by a heat pump at 65 ℃ of 48h;
the conditions of hot water leaching in the step S2 are as follows: 80. the temperature is lower than the temperature; extraction time: 3h;
the dialysis conditions in step S3 were 4℃and 72h; the rotary evaporation temperature is 60 ℃; ultrafiltration cut-off average molecular weight ofMw=9.68×10 4 kDa。
8. The method for preparing the high-immunocompetence acetylated longan water-soluble glucan according to claim 5, wherein in the step S4, the addition amounts of different acetic anhydrides are 1, 4 and 7mL respectively, and the mixture is stirred at 30 ℃ for 4h; the pH of the reaction system is regulated by 0.5M NaOH to always maintain the pH of the reaction system at 8.0-8.5; after the reaction was completed, the solution was neutralized with 5M HCl to terminate the reaction at pH 7.0, and the solution was put in a cut-off film of Mw 8000Da, dialyzed against distilled water for 48h, and then lyophilized directly.
9. The method for preparing the high-immunocompetence acetylated longan water-soluble glucan according to claim 3, wherein the glucan is separated from longan by adopting hot water leaching, dialysis, ultrafiltration, freeze-drying and other processes; dissolving 100mg dextran in 10mL distilled water; stirring the mixture with a magnetic stirrer at 30 ℃ overnight until a homogeneous solution is obtained; adjusting the pH value to 9.0 by using 0.5M NaOH solution and keeping the pH value for 10min; the mixture was stirred at 30 ℃ for 4h, during which 1, 4 and 7mL acetic anhydride were added, respectively; simultaneously, 0.5M NaOH was added to the mixture with continuous stirring to maintain the pH at 8.0-8.4; after the reaction was completed, the solution was neutralized with 5M HCl to terminate the reaction at pH 7.0; the solution is filled inMw8000 In Da cut-off membrane, dialyzing with distilled water 48h and directly lyophilizing。
10. Use of an acetylated longan water-soluble glucan prepared by the method of preparing an acetylated longan water-soluble glucan according to any of claims 3-8 in the preparation of a food or pharmaceutical product; alternatively, the use of an acetylated longan water-soluble glucan according to any of claims 3-8 for the manufacture of a medicament for enhancing macrophage immune activity or enhancing immune activity.
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