Litchi chitosan as well as extraction method and application thereof

Abstract

The invention discloses lychee chitosan as well as an extraction method and application thereof. The lychee shell polysaccharide is successfully separated and purified from lychee shells through a series of processes such as hot water ultrasonic extraction, ethanol precipitation, deproteinization, ion exchange column chromatography and gel column chromatography. The lychee chitosan is heteropolysaccharide and comprises arabinose, galactose, glucose and galacturonic acid, and the molar mass ratio of the arabinose to the galactose to the glucose to the galacturonic acid is (27-29): (13-15): (13-15): (18-19). The lychee shell polysaccharide prepared by the method has good oil holding capacity and hygroscopicity, and due to the characteristics, the lychee shell polysaccharide can be used as a preparation auxiliary material and is widely applied to the fields of medicines, foods, health care products and cosmetics. In addition, the polysaccharide has a remarkable inhibition effect on alpha-amylase and alpha-glucosidase, and due to the characteristic, the polysaccharide has an extremely wide application prospect in the aspect of developing hypoglycemic drugs, functional foods and health-care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharides, and particularly relates to a litchi shell polysaccharide, an extraction method thereof and an application thereof. Background Art

[0002] Litchi (Litchi chinensis Sonn.) belongs to the Sapindaceae family. Its pericarp, i.e., litchi shell, is often discarded as waste during the processing and consumption of litchi products. This not only causes waste of resources but also may pollute the environment. However, studies have shown that litchi shells contain various active ingredients, such as proanthocyanidins, flavonoids, phenolic acids and polysaccharide substances, and have high medicinal value.

[0003] Existing research shows that active polysaccharides have good antioxidant capacity and free radical scavenging ability, and have certain effects on lowering blood pressure, blood lipid, blood sugar, anti-tumor, preventing atherosclerosis, anti-inflammatory and analgesic, enhancing immune ability, etc. In the research on litchi-related polysaccharides, the patent document CN117304360A discloses a method for preparing litchi polysaccharide with a purity of more than 95% and a specific structure by using litchi juice as a raw material, removing monosaccharides and disaccharides by yeast, centrifuging to remove solids to obtain a clear juice, and jointly using nanofiltration, ultrafiltration technology, ion exchange chromatography and Sephadex gel chromatography. The litchi polysaccharide is composed of galactose at a mass percentage of 42.87%, arabinose at 30.43% and glucose at 26.70%, and is connected by α 1,6, α 1,3, α 1,5, with a molecular weight of 1.0 - 2.0×10 5 Da, and its specific structure is shown as follows:

[0004]

[0005] In addition, Yang Bao et al. separated a water-soluble polysaccharide composition from litchi shells by using DEAE Sepharose Fast Flow anion exchange column and G50 Sephadex gel column, and analyzed its structure. The research results showed that the litchi shell polysaccharide was composed of mannose, galactose and a small amount of arabinose, with a molar percentage of 65.6%:33.0%:1.4%. The inter-monosaccharide linkages were composed of 1,2 bond, 1,3 bond and 1,6 bond, with a molar percentage of 8.7%:83.3%:8.0%, but without 1,4 bond. By gel permeation chromatography, the molecular weight of the polysaccharide was 14000D (see Yang Bao, Zhao Mouming, Liu Yang, etc. Isolation and Identification of a Litchi Shell Polysaccharide [J]. Food Science, 2006, (02): 81 - 83.).

[0006] Despite the above-mentioned research, there are still few reports on litchi pericarp polysaccharides at present, especially the research on the fine structure and application of litchi pericarp polysaccharides is even more scarce. Therefore, developing an extraction method for litchi pericarp polysaccharides and deeply studying its fine structure and application are of great significance for making full use of this resource of litchi pericarp and expanding its application prospects in the fields of medicine and so on. Summary of the Invention

[0007] To overcome the defects of the above-mentioned prior art, the present invention provides a litchi pericarp polysaccharide and its extraction method and application.

[0008] In the first aspect of the present invention, a litchi pericarp polysaccharide is provided, and the litchi pericarp polysaccharide contains arabinose, galactose, glucose and galacturonic acid, and their molar mass ratio is (27 - 29):(13 - 15):(13 - 15):(18 - 19).

[0009] In some embodiments of the present invention, the molar mass ratio of arabinose, galactose, glucose and galacturonic acid is 28.01:13.77:14.06:18.55.

[0010] Furthermore, arabinose, galactose, glucose and galacturonic acid can be of any configuration, for example, D-form, L-form, α-D-form, β-D-form, α-L-form, β-L-form.

[0011] Furthermore, arabinose, galactose, glucose and galacturonic acid can be of any conformation, for example, pyranose residue or furanose residue.

[0012] Furthermore, the litchi pericarp polysaccharide contains 1-linked arabinose residues, 5-linked arabinose residues, 1,3-linked arabinose residues, 1,5-linked arabinose residues, 1,3,6-linked galactose residues, 1,4-linked glucose residues and 1,4-linked galacturonic acid residues.

[0013] Furthermore, the litchi pericarp polysaccharide contains 1-linked α-L-arabinose residues, 5-linked α-L-arabinose residues, 1,3-linked α-L-arabinose residues, 1,5-linked α-L-arabinose residues, 1,3,6-linked β-D-galactose residues, 1,4-linked α-D-glucose residues and 1,4-linked α-D-galacturonic acid residues.

[0014] Furthermore, in the litchi shell polysaccharide, the main chain is composed of 1-linked α-L-arabinose residues, 1,4-linked α-D-galacturonic acid residues, 1,3,6-linked β-D-galactose residues, 1,5-linked α-L-arabinose residues and 5-linked α-L-arabinose residues, and the branched chain is composed of 1-linked α-L-arabinose residues, 1,4-linked α-D-glucose residues and 1,3-linked α-L-arabinose residues.

[0015] Furthermore, in the litchi shell polysaccharide, the main chain is composed of α-L-Araf-(1→, →4)-α-D-GalpA-(1→, →3,6)-β-D-Galp-(1→, →5)-α-L-Araf-(1→ and →5)-α-L-Araf, and the branched chain is composed of α-L-Araf-(1→, →4)-α-D-Glcp-(1→, →3)-α-L-Araf-(1→.

[0016] Furthermore, the litchi shell polysaccharide comprises the following structure:

[0017]

[0018] Furthermore, the molecular weight of the litchi shell polysaccharide is 20,000 - 50,000 Da, specifically such as 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50,000 Da.

[0019] In some embodiments of the present invention, the molecular weight of the litchi shell polysaccharide is 38,974 Da.

[0020] In the second aspect of the present invention, a preparation method of litchi shell polysaccharide is provided, and the preparation method includes the step of extracting litchi shells.

[0021] Furthermore, the preparation method includes the step of obtaining crude litchi shell polysaccharide by water extraction and alcohol precipitation.

[0022] Furthermore, the preparation method further includes the step of purifying the crude litchi shell polysaccharide (for example, by ion exchange column chromatography, gel column chromatography).

[0023] In some embodiments of the present invention, the preparation method includes the following steps:

[0024] (1) Water is added to the litchi shell powder for extraction, and the obtained aqueous extract is successively subjected to alcohol precipitation and protein removal to obtain crude litchi shell polysaccharide;

[0025] (2) The crude litchi shell polysaccharide obtained in step (1) is eluted through an ion exchange column to obtain a litchi shell polysaccharide fraction;

[0026] (3) The litchi shell polysaccharide fraction obtained in step (2) is eluted through a gel column to obtain litchi shell polysaccharide.

[0027] Further, in step (1), the material-liquid ratio of the litchi shell powder to water (W / V, mg / mL) is 1:(10 - 30), specifically such as 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, 1:30. In some embodiments of the present invention, the material-liquid ratio is 1:20.

[0028] Further, in step (1), the extraction is carried out under ultrasound, and the ultrasound power is 100 - 1000W, specifically such as 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000W. In some embodiments of the present invention, the ultrasound power is 500W.

[0029] Further, in step (1), the extraction temperature is 70 - 100°C, specifically such as 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100°C. In some embodiments of the present invention, the extraction temperature is 80°C.

[0030] Further, in step (1), the number of extractions is 1 or more times, specifically such as 1, 2, 3, 4, 5 times. In some embodiments of the present invention, the number of extractions is 3 times.

[0031] Further, in step (1), the extraction time for each time is 10 - 60 min, specifically such as 10, 15, 20, 250, 30, 350, 40, 45, 50, 55, 60 min. In some embodiments of the present invention, the extraction time for each time is 25 min.

[0032] Further, in step (1), in the alcohol precipitation step, the alcohol solvent used is ethanol, specifically such as absolute ethanol.

[0033] Further, in step (1), in the alcohol precipitation step, the volume ratio of the alcohol solvent used to the water extract is (1 - 10):1, specifically such as 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. In some embodiments of the present invention, the volume ratio of the alcohol solvent to the water extract is 1.5:1.

[0034] Further, in step (1), in the protein removal step, the protein removal reagent used is an aqueous trichloroacetic acid solution, and its volume percentage concentration is 1 - 10%, specifically such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%. In some embodiments of the present invention, the protein removal reagent used is a 5% aqueous trichloroacetic acid solution.

[0035] Further, in step (1), after the protein removal, it further includes the steps of alcohol precipitation and freeze-drying.

[0036] Further, in step (2), the crude litchi shell polysaccharide obtained in step (1) is dissolved in water, and the concentration of the crude litchi shell polysaccharide after being dissolved in water is 5 - 20 mg / mL, specifically such as 5, 10, 15, 20 mg / mL. In some embodiments of the present invention, the concentration of the crude litchi shell polysaccharide after being dissolved in water is 10 mg / mL.

[0037] Further, in step (2), the ion exchange column is an anion exchange column, such as a DEAE Sephadex FF anion exchange column.

[0038] Further, in step (2), the elution is gradient elution.

[0039] Further, in step (2), the eluent used for elution is water and / or an aqueous sodium chloride solution.

[0040] Further, in step (2), the concentration of the aqueous sodium chloride solution is arbitrarily selected from two or more of 0.001 - 0.30 mol / L (such as 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30 mol / L).

[0041] In some embodiments of the present invention, in step (2), gradient elution is carried out successively with water, a 0.1 mol / L aqueous sodium chloride solution, and a 0.2 mol / L aqueous sodium chloride solution.

[0042] Further, in step (2), the elution flow rate is 1-10 mL / min, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mL / min. In some embodiments of the present invention, the elution flow rate is 4 mL / min.

[0043] Further, in step (2), after the elution, there is also a step of concentrating the eluate and then desalting it, and the desalting is achieved by dialysis using a dialysis bag.

[0044] Further, in step (3), the litchi shell polysaccharide component obtained in step (2) is dissolved in water, and the concentration of the litchi shell polysaccharide component after being dissolved in water is 30-60 mg / mL, specifically 30, 35, 40, 45, 50, 55, 60 mg / mL. In some embodiments of the present invention, the concentration of the litchi shell polysaccharide component after being dissolved in water is 45 mg / mL.

[0045] Further, in step (3), the gel column is a Sephecryl S-400HR gel chromatography column.

[0046] Further, in step (3), the solvent used for elution is water. In some embodiments of the present invention, water elution is carried out for 1.5 times the column volume.

[0047] Further, in step (3), the elution flow rate is 0.1-5 mL / min, specifically 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mL / min. In some embodiments of the present invention, the elution flow rate is 1 mL / min.

[0048] Further, in step (3), after the elution, there is also a step of concentrating the eluate and then freeze-drying it.

[0049] The above preparation method of the present invention can prepare the litchi shell polysaccharide described in the first aspect of the present invention.

[0050] In the third aspect of the present invention, there is provided an application of the litchi shell polysaccharide described in the first aspect of the present invention as an adjuvant.

[0051] Further, the adjuvant is selected from one or more of thickeners, stabilizers (such as colloidal stabilizers), fat substitutes, texture modifiers, gelling agents, humectants, moisturizers, disintegrants, binders, fillers, emulsifiers, suspending agents, antioxidants and bacteriostatic agents.

[0052] Preferably, the adjuvant is selected from one or more of thickeners, stabilizers, fat substitutes, texture modifiers, gelling agents, humectants, moisturizers and disintegrants.

[0053] Further, the litchi shell polysaccharide is added as an auxiliary material to solid preparations, semi-solid preparations, liquid preparations or gas preparations.

[0054] Further, the solid preparations are selected from one or more of tablets, powders, capsules, granules, pills, suppositories, films, microcapsules and freeze-dried preparations.

[0055] Further, the semi-solid preparations are selected from one or more of ointments (such as ointments, creams), pastes and gels.

[0056] Further, the liquid preparations are selected from one or more of solutions, syrups, tinctures, emulsions, suspensions, mixtures, drops, lotions and liniments.

[0057] Further, the gas preparations are selected from one or more of aerosols, sprays, powder aerosols and inhalants.

[0058] Preferably, the litchi shell polysaccharide is added as an auxiliary material to tablets, powders, capsules, granules, ointments, gels, emulsions or suspensions.

[0059] Further, the litchi shell polysaccharide is added as an auxiliary material (additive) to drugs, foods, health products or cosmetics.

[0060] Further, the litchi shell polysaccharide is added to drugs as a thickening agent, stabilizer, gelling agent, hygroscopic agent, humectant, disintegrant, binder, filler, emulsifier, suspending agent, antioxidant or bacteriostatic agent.

[0061] Preferably, the litchi shell polysaccharide is added to drugs as a thickening agent, stabilizer, gelling agent, hygroscopic agent, humectant or disintegrant.

[0062] Further, the litchi shell polysaccharide is added to foods or health products as a thickening agent, stabilizer, fat substitute, texture modifier, gelling agent, hygroscopic agent, humectant, disintegrant, emulsifier, suspending agent, antioxidant or bacteriostatic agent.

[0063] Preferably, the litchi shell polysaccharide is added to foods or health products as a thickening agent, stabilizer, fat substitute, texture modifier, gelling agent, hygroscopic agent, humectant or disintegrant.

[0064] Further, the litchi shell polysaccharide is added to cosmetics as a thickening agent, stabilizer, texture modifier, gelling agent, hygroscopic agent, humectant, emulsifier, suspending agent, antioxidant or bacteriostatic agent.

[0065] Preferably, the litchi shell polysaccharide is added to cosmetics as a thickening agent, stabilizer, texture modifier, gelling agent, hygroscopic agent or humectant.

[0066] In the fourth aspect of the present invention, there is provided an application of the litchi shell polysaccharide described in the first aspect of the present invention in the preparation of hypoglycemic drugs.

[0067] Furthermore, the litchi shell polysaccharide has the effect of lowering blood sugar and can also be used in combination with other active ingredients.

[0068] Furthermore, the hypoglycemic drug can be used for preventing and / or treating diabetes and / or its complications.

[0069] Furthermore, the hypoglycemic drug also includes pharmaceutically acceptable excipients.

[0070] In the fifth aspect of the present invention, there is provided an application of the litchi shell polysaccharide described in the first aspect of the present invention in the preparation of foods or health products.

[0071] Furthermore, the food or health product helps to maintain a healthy blood sugar level (regulating blood sugar and assisting in lowering blood sugar).

[0072] Furthermore, the food or health product also includes excipients acceptable for foods or health products.

[0073] The present invention has the following beneficial effects:

[0074] (1) Through a series of processes such as hot water ultrasonic extraction, ethanol precipitation, protein removal, ion exchange column chromatography, and gel column chromatography, the present invention successfully isolated and purified a litchi shell polysaccharide from litchi shells, and deeply analyzed and identified its molecular weight, monosaccharide composition, chemical structure, etc., clarifying its weight average molecular weight and structural composition, laying a solid foundation for subsequent development and application.

[0075] (2) The litchi shell polysaccharide prepared by the present invention has good oil-holding capacity (about 2.5 g / g) and hygroscopicity (more than 15%), and these characteristics enable it to be used as a formulation excipient and be widely applied in the fields of pharmaceuticals, foods, health products, and cosmetics.

[0076] (3) The litchi shell polysaccharide prepared by the present invention shows significant inhibitory effects on α-amylase and α-glucosidase, and this characteristic makes it show great potential in the field of lowering blood sugar. It can not only be used to develop new hypoglycemic drugs for the treatment of diabetes and its complications, but also be used to develop functional foods or health products to maintain a healthy blood sugar level.

[0077] (4) The litchi shell polysaccharide of the present invention has both medicinal and excipient dual functions, has extremely broad application prospects in the fields of developing hypoglycemic drugs, functional foods, and health products, and can bring significant economic benefits. Description of the Drawings

[0078] Figure 1The ion chromatograms of monosaccharide standards (i.e., the control group) and litchi shell polysaccharide LPPs-1 (i.e., the refined litchi shell polysaccharide fraction) are shown as follows.

[0079] Figure 2 The GPC chromatogram of litchi shell polysaccharide LPPs-1 is shown as follows.

[0080] Figure 3 The Fourier transform infrared spectrum of litchi shell polysaccharide LPPs-1 is shown as follows.

[0081] Figure 4 The scanning electron micrograph of litchi shell polysaccharide LPPs-1 is shown as follows.

[0082] Figure 5 The ultraviolet spectrum of litchi shell polysaccharide LPPs-1 is shown as follows (CK: blank control).

[0083] Figure 6 The 1 1H NMR spectrum of litchi shell polysaccharide LPPs-1 is shown as follows.

[0084] Figure 7 The 13 13C NMR spectrum of litchi shell polysaccharide LPPs-1 is shown as follows.

[0085] Figure 8 The H-H COSY spectrum of litchi shell polysaccharide LPPs-1 is shown as follows.

[0086] Figure 9 The H-H NOESY spectrum of litchi shell polysaccharide LPPs-1 is shown as follows.

[0087] Figure 10 The C-H HSQC spectrum of litchi shell polysaccharide LPPs-1 is shown as follows.

[0088] Figure 11 The C-H HMBC spectrum of litchi shell polysaccharide LPPs-1 is shown as follows.

[0089] Figure 12 The chemical structural formula of litchi shell polysaccharide LPPs-1 is shown as follows.

[0090] Figure 13 The hygroscopicity diagram of litchi shell polysaccharide LPPs-1 is shown as follows.

[0091] Figure 14 The alcohol solubility diagram of litchi shell polysaccharide LPPs-1 is shown as follows.

[0092] Figure 15 The inhibitory effects of litchi shell polysaccharide LPPs-1 on (A) α-amylase activity and (B) α-glucosidase are shown as follows. Detailed implementation manners

[0093] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by those skilled in the technical field to which this invention pertains.

[0094] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0095] In this article, the "refined litchi shell polysaccharide component" and "litchi shell polysaccharide LPPs-1" can be used interchangeably.

[0096] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0097] Example 1: Extraction and purification of litchi shell polysaccharide LPPs-1

[0098] 1. Extraction of crude litchi shell polysaccharide by water extraction and ethanol precipitation

[0099] Add litchi shell powder to distilled water with a volume 20 times that of the material (the unit of the solid-liquid ratio is mg / mL), sonicate at 500W and 80 °C for 25 min, repeat 3 times, collect the extract, then centrifuge at 4000 rpm for 5 min, collect the supernatant, add anhydrous ethanol with a volume 1.5 times that of the supernatant, incubate at 4 °C for 48 h to produce a precipitate, and then centrifuge at 8000g for 10 min to collect the precipitated solid, thus obtaining crude litchi shell polysaccharide (containing impurities). Add pure water to the crude litchi shell polysaccharide (containing impurities) solid to dissolve it, then add an equal volume of 5% trichloroacetic acid, place it at 4 °C for 12 h to precipitate proteins. Then centrifuge and collect the supernatant, add anhydrous ethanol and incubate at 4 °C for 24 h to produce a precipitate. Then take the precipitate for lyophilization to obtain crude litchi shell polysaccharide.

[0100] 2. Ion purification of crude litchi shell polysaccharide

[0101] An appropriate amount of crude polysaccharide from litchi pericarp was dissolved in pure water to prepare a polysaccharide stock solution with a concentration of 10 mg / mL. After centrifugation at 10000 g for 10 min, the supernatant was added to a DEAE Sephadex FF anion exchange column (26×400 mm) at a flow rate of 4 mL / min. Gradient elution was performed successively with pure water and 0.1 M and 0.2 M sodium chloride solutions, and the eluates were collected. The eluates were concentrated and then dialyzed against salt using a dialysis bag. The phenol-sulfuric acid method was used to detect the content and purity of the polysaccharide. The results showed that three polysaccharide fractions from litchi pericarp were separated successively, with purities of 93.5% (eluted with pure water), 67.5% (eluted with 0.1 M sodium chloride solution), and 55.0% (eluted with 0.2 M sodium chloride solution), respectively.

[0102] 3. Gel purification of polysaccharide fractions from litchi pericarp

[0103] The polysaccharide fraction from litchi pericarp eluted with pure water (purity 93.5%) obtained above was dissolved in pure water to prepare a polysaccharide stock solution with a concentration of 45 mg / mL. After centrifugation at 10000 g for 10 min, the supernatant was passed through a Sephecryl S-400HR gel chromatography column for separation and purification at a flow rate of 1 mL / min; elution was performed with pure water for 1.5 column volumes, and the eluates were collected. The eluates were concentrated and freeze-dried. The sulfuric acid-phenol method was used to identify the content and purity of the polysaccharide. The results showed that a purified polysaccharide fraction from litchi pericarp was separated, named litchi pericarp polysaccharide LPPs-1, with a purity of 94.8%.

[0104] Example 2: Structural identification of litchi pericarp polysaccharide LPPs-1

[0105] 1. Monosaccharide composition

[0106] Sample pretreatment: Take a clean chromatographic vial, weigh an appropriate amount of litchi pericarp polysaccharide LPPs-1 sample, add 1 mL of 2 M TFA solution, and heat at 121 °C for 2 h. Pass nitrogen to blow dry the excess TFA. Add 99.99% methanol for washing and then blow dry again. Repeat the washing 2 - 3 times. Add sterile water to dissolve and transfer to a chromatographic vial for on-machine detection.

[0107] Standard sample preparation: Weigh accurately the required standards of fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), and mannuronic acid (Man-UA) (purchased from sigma company), then add water to prepare a single-standard stock solution with a concentration of 10 mg / mL. Then, take an appropriate amount of the single-standard stock solution and mix it to prepare a mixed standard sample with a maximum target concentration of 60 μg / mL, 50 μg / mL, or 40 μg / mL, and prepare a series of standard samples required for injection.

[0108] Use a Thermo ICS 5000+ ion chromatography system and an electrochemical detector to analyze and detect monosaccharide components.

[0109] 2. Molecular weight determination

[0110] Sample pretreatment: Dissolve the litchi pericarp polysaccharide LPPs-1 sample in DMSO solution with a final concentration of 1 mg / mL, and filter it through a filter with a pore size of 0.45 μm before injection for detection.

[0111] Chromatographic conditions: The chromatographic system used is a gel permeation chromatography - differential refractive index - multi-angle laser light scattering system. The liquid phase system is U3000, the differential refractive index detector is Optilab T-rEX, and the laser light scattering detector is DAWN HELEOSⅡ. According to the sample pretreatment, the specific chromatographic column and elution conditions are as follows: Use two gel permeation chromatography columns Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) in series. The column temperature is 45°C, the injection volume is 100 μL, the mobile phase A (0.02% NaN3, 0.1 M NaNO3), the flow rate is 0.6 mL / min, and the elution gradient: isocratic for 75 min.

[0112] 3. Infrared spectrum determination

[0113] Further analyze the functional group composition of litchi pericarp polysaccharide LPPs-1 by Fourier transform infrared spectroscopy. Weigh a small amount of litchi pericarp polysaccharide LPPs-1 and mix it evenly with 200 mg of potassium bromide, then press it into a sheet with a thickness of 1 mm, and then detect it by machine. Use a Nicoleti Z-10 Fourier transform infrared spectrometer for scanning analysis, with an instrument resolution of 4.00 cm - 1, and the scanning range is 4000 - 400 cm - 1, the number of scans: 32 times. The sampling gain is 8.0; the moving mirror speed is 0.4747; the aperture is 80.00; DTGS KBr detector; KBr beam splitter; infrared light source.

[0114] 4. SEM measurement

[0115] Take a small amount of litchi shell polysaccharide LPPs-1 and stick it on the conductive adhesive, then perform gold spraying treatment, and observe its morphology with SEM.

[0116] 5. UV measurement

[0117] Weigh a small amount of litchi shell polysaccharide LPPs-1 sample and dissolve it in pure water to prepare a polysaccharide solution with a concentration of 5 mg / mL. Use a Thermo Fisher (USA) multi-functional microplate reader to perform quantitative analysis on the polysaccharide solution. The microplate is from Corning (USA). It is required that the difference in absorbance between wells in parallel experiments is less than 0.02 at the target wavelength. Scanning start wavelength: 200 nm, end wavelength: 1000 nm, scanning interval: 1 nm. Use pure water as the blank control and measure under the same conditions.

[0118] 6. Bonding structure determination

[0119] Weigh a small amount of litchi shell polysaccharide LPPs-1 sample (about 5 mg), add 1 mL of pure water to dissolve it, then add 1 mL of 100 mg / mL 1-cyclohexyl-2-morpholinoethylcarbodiimide methyl p-toluenesulfonate, and react for 2 h. Add 1 mL of 2 M imidazole, divide the sample into two equal parts, and add 1 mL of 30 mg / ml NaBH4 and 1 mL of 30 mg / mL NaBD4 respectively, and react for 3 h. Add 100 μL of glacial acetic acid to terminate the reaction. After dialysis for 48 h, freeze-dry the sample for subsequent treatment. Dissolve the sample in 500 μL of DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μL of methyl iodide solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex and mix well, centrifuge, and discard the aqueous phase. Repeat the water washing 3 times. Pipette the lower dichloromethane phase and dry it with nitrogen. Add 100 μL of 2 M TFA and react at 121 °C for 90 min. Evaporate to dryness at 30 °C. Add 50 μL of 2 M ammonia water and 50 μL of 1 M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL of acetic acid to terminate the reaction, dry with nitrogen, wash twice with 250 μL of methanol, and dry with nitrogen. Add 250 μL of acetic anhydride, vortex and mix well, and react at 100 °C for 2.5 h. Add 1 mL of water and let it stand for 10 min. Add 500 μL of dichloromethane, vortex and mix well, centrifuge, and discard the aqueous phase. Repeat the water washing 3 times. Take the lower dichloromethane phase to obtain methylated alditol acetate (PMAAs) for GC-MS analysis.

[0120] The analytical instrument used in this experiment was an Agilent 7890A-5977B gas chromatography-mass spectrometry (GC-MS) instrument from Agilent Technologies Inc. (CA, USA), and the model of the automatic sampler was G4567A. The chromatographic system used was an Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), and the chromatographic column was BPX70 (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was high-purity helium. The initial temperature of the column oven was maintained at 140 °C for 2.0 min, and then programmed to increase to 230 °C at a rate of 3 °C / min and held for 3 min. The mass spectrometry system used was a quadrupole mass detection system from Aiglent (USA) (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization source (EI) and a MassHunter workstation. Using the electron impact ionization source (EI), the analytes were detected in the full scan (SCAN) mode, and the mass scan range (m / z) was 50 - 350.

[0121] 7. NMR Spectrometry

[0122] An appropriate amount of litchi shell polysaccharide LPPs-1 was fully dissolved in D2O to prepare a polysaccharide solution with a concentration of 40 mg / mL or higher. The dissolved solution was transferred to an NMR tube, and the added volume was 0.5 mL. The NMR tube was placed in an NMR spectrometer to scan one-dimensional 1H spectra, 13C spectra, and two-dimensional COSY, HSQC, HMBC, and NOESY spectra.

[0123] A Bruker (Germany) 500 MHz NMR spectrometer was used for quantitative analysis of the target compound, and the scanning temperature was 25 °C. A liquid probe QXI 1H / 31P / 13C / 15N 5 mm four-resonance inverse detection probe (Z-gradient, ATM Acc), technical parameters: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating) BBFO 1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradient, ATM) technical parameters: signal-to-noise ratio (1H): 798; resolution (Hz): 0.26 (rotating); signal-to-noise ratio (13C): 328; resolution (Hz): 0.1.

[0124] 8. Results and Analysis

[0125] 8.1 Results of Monosaccharide Composition Analysis

[0126] As Figure 1As shown, litchi shell polysaccharide LPPs-1 is composed of 9 kinds of monosaccharides: fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), galacturonic acid (Gal-UA) and glucuronic acid (Glc-UA), and the molar mass ratio is 1.18:3.84:28.01:13.77:14.06:9.42:10.10:18.55:1.06. The molar mass ratio of arabinose is the highest, followed by galacturonic acid, glucose, and galactose in turn. It is speculated that fucose, rhamnose, xylose, mannose, and glucuronic acid are impurities. Generally speaking, litchi shell polysaccharide LPPs-1 is a heteropolysaccharide, and Ara is the main monosaccharide.

[0127] 8.2 Molecular weight determination results

[0128] The GPC chromatogram of litchi shell polysaccharide LPPs-1 is as Figure 2 shown, which is a single symmetric peak, indicating that litchi shell polysaccharide LPPs-1 is a homogeneous polysaccharide. After measurement, the weight-average molecular weight (Mw) of litchi shell polysaccharide LPPs-1 is 38.974 kDa.

[0129] 8.3 Infrared spectroscopy analysis results

[0130] The infrared spectroscopy results display diagram of litchi shell polysaccharide LPPs-1 is as Figure 3 shown, the absorption band at 3600 - 3200 cm -1 is the stretching vibration absorption peak of -OH, and the absorption peak in this region is the characteristic peak of carbohydrates. Specifically as follows: 3329.91 cm -1 is the stretching vibration absorption peak of O-H, which is the characteristic peak of carbohydrates. The absorption peak at 2931.05 cm -1 belongs to the stretching vibration of C-H. There is an absorption peak at 1015.44 cm -1 which belongs to the stretching vibration of C-O.

[0131] 8.4 Scanning electron microscopy analysis results

[0132] As Figure 4As shown, the surface morphology images of litchi shell polysaccharide LPPs-1 were magnified 500×, 2000×, 5000×, and 10000× respectively. It can be seen from the images that litchi shell polysaccharide LPPs-1 presents an irregular and rough surface layered structure with dense surface cracks. This multi-slit structural characteristic may be the key factor for its water absorption and swelling properties as well as excellent water retention properties, indicating that litchi shell polysaccharide LPPs-1 can be developed and utilized as a disintegrant, hygroscopic agent, or moisturizer. At the same time, litchi shell polysaccharide LPPs-1 has more circular cavities, which may be due to the weak intermolecular attraction caused by the repulsive force in the polysaccharide molecules.

[0133] 8.5 UV Scanning Analysis Results

[0134] As Figure 5 shown, there are no obvious peaks in the UV absorbance at 260 and 280 nm, indicating that no proteins and nucleic acids were detected in litchi shell polysaccharide LPPs-1.

[0135] 8.6 Bonding Structure and NMR Scanning Analysis Results

[0136] Through methylation analysis, litchi shell polysaccharide LPPs-1 mainly contains 10 sugar residues, including t-Ara(f), t-Xyl(p), t-Man(p), 3-Ara(f), 5-Ara(f), 2-Xyl(p), 2-Man(p), 4-Gal(p)-UA, 6-Gal(p), and 3,6-Gal(p), with a molar ratio of 14.06:1.73:2.73:6.60:10.74:1.13:4.43:22.03:3.48:6.56. The molar ratio of glycosidic bonds is consistent with the results of monosaccharide composition analysis. The contents of 4-Gal(p)-UA, t-Ara(f), and 5-Ara(f) account for a relatively large proportion, indicating that the backbone of litchi shell polysaccharide LPPs-1 may be composed of 4-Gal(p)-UA, t-Ara(f), and 5-Ara(f). The analysis results of the bonding structure of the polysaccharide sample are shown in Table 1.

[0137] Table 1 Analysis Results of the Bonding Structure of the Polysaccharide Sample

[0138]

[0139] Multiple signal peaks were identified in the anomeric carbon region of the litchi shell polysaccharide LPPs-1 sample. Combining the cross-peaks in the anomeric region of the 13C NMR spectrum and HSQC spectrum, the anomeric signals present in this sample were determined to be: δ5.02 / 101.63, 5.17 / 109.36, 5.08 / 106.9, 5.0 / 107.41, 4.45 / 103.21, 5.04 / 98.78 ppm, and were denoted as sugar residues A, B, C, D, E, F respectively. Combining the sample's bonding structure (methylation) information, anomeric signals, and comprehensive literature reports, it was speculated that sugar residue A was →4)-α-D-GalpA-(1→, sugar residue B was α-L-Araf-(1→, sugar residue C was →5)-α-L-Araf-(1→, sugar residue D was →3)-α-L-Araf-(1→, sugar residue E was →3,6)-β-D-Galp-(1→, and sugar residue F was →4)-α-D-Glcp-(1→. The 1H and 13C chemical shifts were assigned, and the results are shown in Table 2.

[0140] Table 2 Each sugar residue 1 H and 13 C chemical shifts

[0141]

[0142] Combining one-dimensional NMR and two-dimensional NMR information ( Figures 6 - 11 ) and methylation result analysis, it was deduced that litchi shell polysaccharide LPPs-1 was mainly composed of →4)-α-D-GalpA-(1→ and a small amount of →3,6)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, α-L-Araf-(1→, →5)-α-L-Araf connected to form the main chain, and the branches were mainly composed of α-L-Araf-(1→, →4)-α-D-Glcp-(1→, →3)-α-L-Araf-(1→ connected to each other and then connected to the O-6 position of sugar residue →3,6)-β-D-Galp-(1→, etc. Based on this, the structure of litchi shell polysaccharide LPPs-1 was speculated as Figure 12 shown.

[0143] Example 3: Physicochemical properties of litchi shell polysaccharide LPPs-1

[0144] 1. Oil-holding capacity

[0145] Transfer 0.2 g of litchi shell polysaccharide LPPs-1 into a centrifuge tube and weigh its mass \(W_1\). Pipette 6 mL of soybean oil into a test tube, heat it in a boiling water bath, stir evenly for 20 min, then take it out and cool it to room temperature. Centrifuge at 3000 r / min for 20 min, pour out the supernatant, invert the centrifuge tube on filter paper, and let it stand for 20 min until the oil has drained completely, then weigh its mass \(W_2\). Calculate the difference in mass before and after. Oil holding capacity represents the mass of oil absorbed per gram of sample, and each group is determined in parallel three times. The oil holding capacity (OHC) is calculated according to the following formula:

[0146] OHC = \((W_2 - W_1) / \) sample mass

[0147] Where: \(W_1\) is the total mass of the sample and the centrifuge tube, g; \(W_2\) is the total mass of the sample and the centrifuge tube after the oil has drained completely, g.

[0148] 2. Hygroscopicity

[0149] Weigh 0.2 g of litchi shell polysaccharide LPPs-1 and place it in a dry weighing bottle. Place it in an oven at 25 °C with a saturated NaCl solution. Weigh the sample every 1 h. The hygroscopicity of the sample is expressed by the following formula:

[0150] Hygroscopicity rate (%) = 100(\(M\) n - \(M_0) / M_0\)

[0151] Where: \(M_0\) is the mass of the sample before moisture absorption, g; \(M\) n is the mass after different times of moisture absorption, g.

[0152] 3. Alcohol solubility

[0153] Weigh 0.05 g of litchi shell polysaccharide LPPs-1 each and add them to 10 mL of ethanol solutions with volume fractions of 30%, 40%, 50%, 60%, and 70% respectively. After complete dissolution, centrifuge at 5000 r / min for 20 min. Dry the supernatant to a constant weight at 105 °C. Calculate the solubility (S) of the sample in the ethanol solution according to the following formula:

[0154] S (%) = (\(A / M\)) × 100

[0155] Where: \(A\) is the mass of the dissolved polysaccharide sample, g; \(M\) is the mass of the polysaccharide sample, g.

[0156] 4. α-Amylase and α-Glucosidase Inhibition Activity Tests

[0157] Mix 600 μL of equal amounts of litchi shell polysaccharide LPPs-1 (0, 0.01, 0.04, 0.06, 0.1, 0.2, 0.4, 0.8 mg / mL) with 500 μL of α-amylase solution (0.4 U / mL) and incubate at 37 °C for 10 min. Then, add 600 μL of soluble starch solution (10 mg / mL) to the mixture and incubate for another 5 min. To complete the reaction, introduce 700 μL of DNS into the system. Immediately place the reaction mixture in a 100 °C water bath for 10 min to inactivate α-amylase. Measure the absorbance reading at 540 nm using a UV-visible spectrophotometer.

[0158] Mix 400 μL of litchi shell polysaccharide LPPs-1 at different concentrations with 400 μL of α-glucosidase solution (60 U / mL) and incubate at 37 °C for 10 min. Next, add 400 μL of PNPG solution (2.5 mmol / L) to the mixture and incubate for another 10 min. To terminate the reaction, introduce 550 μL of sodium carbonate solution (0.2 mol / L). Record the absorbance reading at 405 nm.

[0159] All experiments used acarbose as a positive control. Curve fitting analysis was performed using SPSS software to calculate the maximum half-inhibitory concentration values of litchi shell polysaccharide LPPs-1 against the two enzymes. The inhibitory effects of this component on α-amylase and α-glucosidase were quantified using the following formula:

[0160] Inhibition rate (%) = [1 - (A 样品 - A 对照-1 ) / A 对照-2 × 100

[0161] In the formula, the absorbance after reacting only with litchi shell polysaccharide LPPs-1 is used as control - 1, and the absorbance after reacting the enzyme without this component is used as control - 2.

[0162] 5. Results and Analysis

[0163] 5.1 Oil-holding Results

[0164] Studies have shown that polysaccharides have oil-holding properties and can be used as gelling agents, thickening agents, colloidal stabilizers, and fat substitutes in food processing. Oil-holding is the most prominent functional property of the oil-absorbing ability of samples. A higher oil-holding capacity can maintain food flavor and enhance the texture of food (see Xu Yaqin, Liu Ningyue, Li Dalong, et al. Functional properties, structure, and antiglycation activity of blueberry fruit polysaccharides [J]. Food Science, 2020, 41(2): 8 - 14.). The oil-holding property of polysaccharides can guide the use of dosage forms. If the oil-holding capacity is high, it indicates that the molecular weight and viscosity of the polysaccharide are relatively large.

[0165] The results showed that the oil-holding capacities of litchi shell polysaccharide LPPs-1 for three times were 2.56 g / g, 2.64 g / g, and 2.41 g / g respectively, and the average oil-holding capacity was 2.54 ± 0.10 g / g, which was higher than that of commercial gluten (1.19 ± 0.13 g / g) and lower than that of blueberry fruit polysaccharide (4.60 ± 0.45 g / g) (see Xu Yaqin, Liu Ningyue, Li Dalong, etc. Functional properties, structure and antiglycation activity of blueberry fruit polysaccharide [J]. Food Science, 2020, 41(2): 8-14.). This indicates that litchi shell polysaccharide LPPs-1 has a certain oil-holding capacity and can be added to foods as a texture modifier, thickener, and gelling agent to improve the food tissue structure and increase the taste.

[0166] 5.2 Hygroscopicity results

[0167] It can be seen from Figure 13 that as time increases, the hygroscopic rate shows an upward trend. The hygroscopic rate of litchi shell polysaccharide LPPs-1 reaches equilibrium after 12 h, and then shows a gentle trend. Its hygroscopic rate reaches the highest at 36 h, which is 15.73%. Within 12 h, the hygroscopic rate of litchi shell polysaccharide LPPs-1 is higher than that of osmanthus leaf polysaccharide (15.56%), the hygroscopic agent chitosan (11.43%), and glucose (3.17%) (see Chen Zheng, Gao Youjun, Deng Fangfei, etc. Study on microwave extraction of osmanthus leaf polysaccharide and its hygroscopicity, moisture retention and antioxidant properties [J]. New Chemical Materials, 2022, 50(S1): 337-340.). Generally, people do not use cosmetics for more than 12 h, and the hygroscopic rate of litchi shell polysaccharide LPPs-1 can reach % within 12 h, and the hygroscopic rate is higher than that of the commonly used hygroscopic agent chitosan (11.43%), indicating that litchi shell polysaccharide LPPs-1 can be developed and utilized as a natural hygroscopic agent.

[0168] Research shows that high hygroscopicity may be related to the polarity, structural characteristics, and porosity of polysaccharides. After polysaccharides combine with water molecules, a network structure will be formed, which can better retain water (see Qin Yin, Xu Wenhui, Wu Kai, etc. Effects of different extraction methods on the physicochemical properties and biological activities of polygonatum polysaccharide [J / OL]. Modern Food Science and Technology, 1-12 [2024-02-18]). This shows that polysaccharides can also be developed and utilized as moisturizing agents.

[0169] 5.3 Alcohol solubility results

[0170] It can be seen from Figure 14It can be seen that with the increase of ethanol concentration, the solubility of litchi shell polysaccharide LPPs-1 gradually decreases. The alcohol solubility of this polysaccharide in 30% ethanol solution is 23.80%, and it is almost insoluble in 70% ethanol solution. This is because the polarity of the polysaccharide molecule obtained by water extraction is similar to that of water and is soluble in water. Its solubility in water is higher, showing easy solubility in water. Moreover, this polysaccharide contains more uronic acid with hydrophilic groups, which can greatly change the solubility of the polysaccharide.

[0171] 5.4 Test results of α-amylase and α-glucosidase inhibitory activities

[0172] As Figure 15 shown, litchi shell polysaccharide LPPs-1 shows dose-dependent inhibitory activities against both enzymes. When the concentration is in the range of 0.01 - 0.8 mg / mL, the inhibition rate increases significantly (p < 0.05). The half-inhibitory concentrations of litchi shell polysaccharide LPPs-1 against α-amylase and α-glucosidase are 0.16 mg / mL and 0.26 mg / mL respectively. This indicates that litchi shell polysaccharide LPPs-1 has a stronger inhibitory effect on α-amylase. This further shows that litchi shell polysaccharide LPPs-1 has great potential in reducing blood sugar and can be used to treat diabetes or its complications, providing a theoretical basis for the development of new hypoglycemic drugs, functional foods and health products.

[0173] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

[0174] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience and preferences of those skilled in the art.

[0175] The steps of the method listed only in a certain order in the present invention do not constitute any limitation on the order of the method steps.

Claims

1. A litchi chitosan, characterized in that: The litchi chitosan polysaccharide contains arabinose, galactose, glucose and galacturonic acid, and the molar mass ratio thereof is (27-29):(13-15):(13-15):(18-19).

2. The litchi chitosan according to claim 1, characterized in that The molar mass ratio of arabinose, galactose, glucose and galacturonic acid is 28.01:13.77:14.06:18.55; Preferably, in the litchi chitosan, the main chain is composed of α-L-Araf-(1→, →4)-α-D-GalpA-(1→, →3,6)-β-D-Galp-(1→, →5)-α-L-Araf-(1→ and →5)-α-L-Araf, and the side chain is composed of α-L-Araf-(1→, →4)-α-D-Glcp-(1→, →3)-α-L-Araf-(1→.

3. The litchi chitosan according to any one of claims 1 to 2, characterized in that The litchi chitosan polysaccharide comprises the following structure: Preferably, the molecular weight of the litchi chitosan is 20000-50000 Da, preferably 38974 Da.

4. A method for preparing litchi chitosan according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) adding water to litchi shell powder for extraction, and subjecting the obtained water extract to alcohol precipitation and protein removal in sequence to obtain litchi shell crude polysaccharide; (2) eluting the crude litchi shell polysaccharide obtained in step (1) through an ion exchange column to obtain a litchi shell polysaccharide component; (3) The litchi chitosan component obtained in step (2) is eluted through a gel column to obtain litchi chitosan.

5. The preparation method according to claim 4, characterized in that: In step (1), the solid-liquid ratio of the litchi shell powder to water is 1:(10-30); Preferably, in step (1), the extraction is carried out under ultrasound, and the ultrasound power is 100-1000W; Preferably, in step (1), the extraction temperature is 70-100°C; Preferably, in step (1), in the alcohol precipitation step, the alcohol solvent used is ethanol; Preferably, in step (1), in the alcohol precipitation step, the volume ratio of the alcohol solvent used to the water extract is (1-10):

1.

6. The preparation method according to claim 4, characterized in that: In step (2), the ion exchange column is an anion exchange column, preferably a DEAE Sephadex FF anion exchange column; Preferably, in step (2), the elution is gradient elution; Preferably, in step (2), the eluent used for elution is water and / or sodium chloride aqueous solution; Preferably, in step (2), water, 0.1 mol / L sodium chloride aqueous solution and 0.2 mol / L sodium chloride aqueous solution are used sequentially for gradient elution; Preferably, in step (3), the gel column is a Sephecryl S-400HR gel chromatography column; Preferably, in step (3), the solvent used for elution is water.

7. Use of the litchi chitosan as claimed in any one of claims 1 to 3 or the litchi chitosan prepared by the preparation method as claimed in any one of claims 4 to 6 as an auxiliary material.

8. The use according to claim 7, characterized in that: The auxiliary material is selected from one or more of a thickener, a stabilizer, a fat substitute, a texture modifier, a gelling agent, a hygroscopic agent, a humectant and a disintegrant; Preferably, the litchi chitosan polysaccharide is added as an excipient to tablets, powders, capsules, granules, pastes, gels, emulsions or suspensions; Preferably, the litchi chitosan polysaccharide is added as an auxiliary material to medicines, foods, health products or cosmetics.

9. Use of the litchi chitosan as claimed in any one of claims 1 to 3 or the litchi chitosan prepared by the preparation method as claimed in any one of claims 4 to 6 in the preparation of hypoglycemic drugs, foods or health products.

10. The use according to claim 9, characterized in that: The glucose-lowering drug is used to prevent and / or treat diabetes and / or its complications; Preferably, the food or health product helps maintain healthy blood sugar levels.

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