A litchi shell polysaccharide, its extraction method and application

Abstract

This invention discloses a litchi peel polysaccharide, its extraction method, and its applications. The invention successfully isolated and purified a litchi peel polysaccharide from litchi peels using a series of processes including hot water ultrasonic extraction, ethanol precipitation, protein removal, ion exchange column chromatography, and gel column chromatography. The litchi peel polysaccharide is a heteropolysaccharide containing arabinose, galactose, glucose, and galacturonic acid in a molar ratio of (27-29):(13-15):(13-15):(18-19). The litchi peel polysaccharide prepared by this invention exhibits good oil-holding capacity and hygroscopicity, making it suitable as a pharmaceutical excipient for wide application in pharmaceuticals, food, health products, and cosmetics. Furthermore, this polysaccharide shows significant inhibitory effects on α-amylase and α-glucosidase, demonstrating its broad application prospects in the development of hypoglycemic drugs, functional foods, and health products.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide technology, specifically relating to a litchi shell polysaccharide, its extraction method, and its application. Background Technology

[0002] Lychee (Litchi chinensis Sonn.) belongs to the Sapindaceae family. Its peel, or lychee shell, is often disposed of as waste during lychee processing and consumption, which not only wastes resources but may also pollute the environment. However, research shows that lychee shells contain various active ingredients, such as proanthocyanidins, flavonoids, phenolic acids, and polysaccharides, and have high medicinal value.

[0003] Existing research indicates that active polysaccharides possess excellent antioxidant and free radical scavenging capabilities, exhibiting certain effects in lowering blood pressure, blood lipids, and blood sugar; anti-tumor activity; prevention of atherosclerosis; anti-inflammatory and analgesic effects; and enhanced immunity. In research on litchi-related polysaccharides, patent document CN117304360A discloses a method for preparing litchi polysaccharides with a purity exceeding 95% and a specific structure using litchi juice as raw material. This method involves removing monosaccharides and disaccharides with yeast, centrifuging to remove solids to obtain a clear juice, and then combining nanofiltration, ultrafiltration, ion exchange chromatography, and dextran gel chromatography. This litchi polysaccharide is composed of 42.87% galactose, 30.43% arabinose, and 26.70% glucose by mass percentage, linked by α1,6, α1,3, and α1,5, with a molecular weight of 1.0-2.0 × 10⁻⁶. 5 The specific structure of Da is shown in the following formula:

[0004]

[0005] In addition, Yang Bao et al. used a DEAE Sepharose Fast Flow anion exchange column and a G50 dextran gel column to isolate a water-soluble polysaccharide from litchi peel and analyzed its structure. The results showed that the litchi peel polysaccharide was composed of mannose, galactose and a small amount of arabinose, with a molar percentage of 65.6%:33.0%:1.4%. The intermolecular bonds of the monosaccharide molecules were composed of 1,2, 1,3 and 1,6 bonds, with a molar percentage of 8.7%:83.3%:8.0%, but no 1,4 bonds. The molecular weight of the polysaccharide was determined to be 14000D by gel permeation chromatography (see Yang Bao, Zhao Mouming, Liu Yang, et al. Isolation and identification of a litchi peel polysaccharide [J]. Food Science, 2006, (02):81-83.).

[0006] Despite the aforementioned research, current research reports on litchi peel polysaccharides are still limited, especially regarding their fine structure and applications. Therefore, developing an extraction method for litchi peel polysaccharides and conducting in-depth research on their fine structure and applications is of great significance for fully utilizing litchi peel as a resource and expanding its application prospects in medicine and other fields. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides a litchi shell polysaccharide, its extraction method, and its application.

[0008] In a first aspect, the present invention provides a litchi shell polysaccharide comprising arabinose, galactose, glucose and galacturonic acid in a molar mass ratio of (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, the arabinose, galactose, glucose, and galacturonic acid can be of any configuration, such as D-type, L-type, α-D-type, β-D-type, α-L-type, and β-L-type.

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

[0012] Furthermore, the litchi shell 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 shell 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 consists 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, while the side chains consist 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 side chains are 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 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, and 50,000 Da.

[0019] In some embodiments of the present invention, the molecular weight of the litchi shell polysaccharide is 38974 Da.

[0020] In a second aspect, the present invention provides a method for preparing litchi peel polysaccharide, the method comprising the step of extracting litchi peel.

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

[0022] Furthermore, the preparation method also includes a step of purifying the crude polysaccharide from litchi peel (e.g., by ion exchange column chromatography or gel column chromatography).

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

[0024] (1) Add water to litchi peel powder for extraction, and then subject the resulting water extract to alcohol precipitation and protein removal to obtain crude litchi peel polysaccharide.

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

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

[0027] Further, in step (1), the ratio of litchi peel powder to water (w / v, mg / mL) is 1:(10-30), specifically 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, or 1:30. In some embodiments of the present invention, the ratio is 1:20.

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

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

[0030] Further, in step (1), the extraction is performed once or more, specifically 1, 2, 3, 4, or 5 times. In some embodiments of the present invention, the extraction is performed 3 times.

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

[0032] Furthermore, in step (1), the alcohol solvent used in the alcohol precipitation step is ethanol, specifically anhydrous ethanol.

[0033] Further, in step (1), the volume ratio of the alcohol solvent to the water extract in the alcohol precipitation step is (1-10):1, specifically 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 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), the protein removal reagent used in the protein removal step is an aqueous solution of trichloroacetic acid with a volume percentage concentration of 1-10%, specifically 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 solution of trichloroacetic acid.

[0035] Furthermore, in step (1), the process after removing the protein also includes alcohol precipitation and freeze-drying.

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

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

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

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

[0040] Further, in step (2), the concentration of the sodium chloride aqueous solution is selected from two or more of the following: 0.001-0.30 mol / L (e.g., 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 performed sequentially using water, 0.1 mol / L sodium chloride aqueous solution and 0.2 mol / L sodium chloride aqueous 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] Furthermore, in step (2), the elution process further includes a step of concentrating the eluent and then desalting it, which is achieved by dialysis using a dialysis bag.

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

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

[0046] Furthermore, in step (3), the solvent used for elution is water. In some embodiments of the present invention, water is used for elution at 1.5 column volumes.

[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] Furthermore, in step (3), the elution process further includes a step of concentrating the eluent and then freeze-drying it.

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

[0050] A third aspect of the present invention provides an application of the litchi shell polysaccharide described in the first aspect of the present invention as an excipient.

[0051] Furthermore, the excipients are selected from one or more of the following: thickeners, stabilizers (such as colloidal stabilizers), fat substitutes, texture modifiers, gelling agents, hygroscopic agents, humectants, disintegrants, binders, fillers, emulsifiers, suspending agents, antioxidants, and antibacterial agents.

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

[0053] Furthermore, the litchi shell polysaccharide is added as an excipient to solid dosage forms, semi-solid dosage forms, liquid dosage forms, or gaseous dosage forms.

[0054] Furthermore, the solid dosage form is selected from one or more of tablets, powders, capsules, granules, pills, suppositories, films, microcapsules, and lyophilized preparations.

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

[0056] Furthermore, the liquid formulation is selected from one or more of the following: solutions, syrups, tinctures, emulsions, suspensions, mixtures, drops, lotions, and liniments.

[0057] Furthermore, the gaseous formulation is selected from one or more of aerosols, sprays, powders, and inhalants.

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

[0059] Furthermore, the litchi shell polysaccharide is added as an excipient (additive) to pharmaceuticals, food, health products, or cosmetics.

[0060] Furthermore, the litchi shell polysaccharide is added to the drug as a thickener, stabilizer, gelling agent, hygroscopic agent, moisturizer, disintegrant, binder, filler, emulsifier, suspending agent, antioxidant, or antibacterial agent.

[0061] Preferably, the litchi shell polysaccharide is added to the drug as a thickener, stabilizer, gelling agent, hygroscopic agent, moisturizer, or disintegrant.

[0062] Furthermore, the litchi shell polysaccharide is added to food or health products as a thickener, stabilizer, fat substitute, texture modifier, gelling agent, hygroscopic agent, moisturizer, disintegrant, emulsifier, suspending agent, antioxidant, or antibacterial agent.

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

[0064] Furthermore, the litchi shell polysaccharide is added to cosmetics as a thickener, stabilizer, texture modifier, gelling agent, humectant, moisturizer, emulsifier, suspending agent, antioxidant, or antibacterial agent.

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

[0066] In a fourth aspect, the present invention provides the 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 a hypoglycemic effect and can be used in combination with other active ingredients.

[0068] Furthermore, the hypoglycemic drug can be used to prevent and / or treat diabetes and / or its complications.

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

[0070] In a fifth aspect, the invention provides the application of the litchi shell polysaccharide described in the first aspect of the invention in the preparation of food or health products.

[0071] Furthermore, the food or health product mentioned above helps maintain healthy blood sugar levels (regulates blood sugar, assists in lowering blood sugar).

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

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

[0074] (1) This invention successfully isolated and purified a litchi shell polysaccharide from litchi shell through a series of processes such as hot water ultrasonic extraction, ethanol precipitation, protein removal, ion exchange column chromatography and gel column chromatography. Its molecular weight, monosaccharide composition and chemical structure were analyzed and identified in depth, and its weight-average molecular weight and structural composition were clarified, 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%). These properties enable it to be used as a pharmaceutical excipient and widely applied in the fields of pharmaceuticals, food, health products and cosmetics.

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

[0077] (4) The litchi shell polysaccharide of the present invention has both medicinal and excipient functions, and has a very broad application prospect in the development of hypoglycemic drugs, functional foods and health products, and can bring significant economic benefits. Attached Figure Description

[0078] Figure 1The image shows the ion chromatograms of the monosaccharide standard (i.e., the control group) and litchi shell polysaccharide LPPs-1 (i.e., the refined litchi shell polysaccharide component).

[0079] Figure 2 The image shows the GPC chromatogram of litchi shell polysaccharide LPPs-1.

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

[0081] Figure 4 The image shown is a scanning electron microscope image of litchi shell polysaccharide LPPs-1.

[0082] Figure 5 The image shows the ultraviolet spectrum of litchi shell polysaccharide LPPs-1 (CK: blank control).

[0083] Figure 6 The image shows litchi shell polysaccharide LPPs-1. 1 H NMR spectrum.

[0084] Figure 7 The image shows litchi shell polysaccharide LPPs-1. 13 C NMR spectrum.

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

[0086] Figure 9 The image shows the HH NOESY spectrum of litchi shell polysaccharide LPPs-1.

[0087] Figure 10 The CH HSQC spectrum of litchi shell polysaccharide LPPs-1 is shown.

[0088] Figure 11 The CH HMBC spectrum of litchi shell polysaccharide LPPs-1 is shown.

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

[0090] Figure 13 The diagram shows the hygroscopicity of litchi shell polysaccharide LPPs-1.

[0091] Figure 14 The figure shown is a graph of the alcohol solubility of litchi shell polysaccharide LPPs-1.

[0092] Figure 15 The figure shows the inhibitory effects of litchi shell polysaccharide LPPs-1 on (A) α-amylase activity and (B) α-glucosidase activity. Detailed Implementation

[0093] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0094] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

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

[0096] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are 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 polysaccharides from litchi peel by water extraction and alcohol precipitation

[0099] Litchi peel powder was added to 20 times its volume of distilled water (solid-to-liquid ratio in mg / mL), and sonicated at 500W and 80℃ for 25 min, repeated three times. The extract was collected, then centrifuged at 4000 rpm for 5 min, and the supernatant was collected. 1.5 times its volume of anhydrous ethanol was added, and the mixture was incubated at 4℃ for 48 h to produce a precipitate. The precipitate was then centrifuged at 8000g for 10 min, and the solid precipitate was collected, yielding crude litchi peel polysaccharide (containing impurities). Pure water was added to the crude litchi peel polysaccharide (containing impurities) solid to dissolve it, followed by the addition of an equal volume of 5% trichloroacetic acid. The mixture was incubated at 4℃ for 12 h to precipitate the protein. The mixture was then centrifuged, and the supernatant was collected. Anhydrous ethanol was added, and the mixture was incubated at 4℃ for 24 h to produce a precipitate. The precipitate was then freeze-dried to obtain crude litchi peel polysaccharide.

[0100] 2. Ion purification of crude polysaccharides from litchi peel

[0101] An appropriate amount of crude litchi peel polysaccharide was dissolved in pure water to prepare a 10 mg / mL polysaccharide stock solution. After centrifugation at 10000g 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 sequentially with pure water and 0.1 M and 0.2 M sodium chloride solutions, and the eluent was collected. The eluent was concentrated and then dialyzed to remove salts. The content and purity of the polysaccharides were determined using the phenol-sulfuric acid method. The results showed that three litchi peel polysaccharide fractions were sequentially separated, 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 litchi shell polysaccharide components

[0103] The litchi peel polysaccharide fraction (purity 93.5%) obtained by elution with pure water was dissolved in pure water to prepare a 45 mg / mL polysaccharide stock solution. The solution was centrifuged at 10000 g for 10 min, and the supernatant was passed through a Sephecryl S-400HR gel chromatography column at a flow rate of 1 mL / min for separation and purification. Elution was performed with 1.5 column volumes of pure water, and the eluent was collected. The eluent was concentrated and freeze-dried. The content and purity of the polysaccharide were identified using the sulfuric acid-phenol method. The results showed that a purified litchi peel polysaccharide fraction was isolated, named litchi peel polysaccharide LPPs-1, with a purity of 94.8%.

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

[0105] 1. Monosaccharide composition

[0106] Sample pretreatment: Take a clean chromatographic vial, weigh an appropriate amount of litchi peel polysaccharide LPPs-1 sample, add 1 mL of 2M TFA solution, and heat at 121℃ for 2 hours. Purge with nitrogen to dry excess TFA. Wash with 99.99% methanol, then dry again, repeating the washing process 2-3 times. Dissolve in sterile water, transfer to a chromatographic vial, and perform analysis.

[0107] Preparation of standards: Accurately weigh the required 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) standards (purchased from Sigma-Aldrich), add water to prepare a 10 mg / mL standard stock solution. Then, take an appropriate amount of the stock solution and mix the single standard to prepare a standard mixed standard with a maximum index concentration of 60 μg / mL, 50 μg / mL, or 40 μg / mL, and prepare the series of standards required for instrumentation.

[0108] The monosaccharide components were analyzed and detected using a Thermo ICS 5000+ ion chromatography system and an electrochemical detector.

[0109] 2. Molecular weight determination

[0110] Sample pretreatment: The litchi shell polysaccharide LPPs-1 sample was dissolved in DMSO solution to a final concentration of 1 mg / mL, and then filtered through a 0.45 μm filter before being analyzed.

[0111] Chromatographic conditions: The chromatographic system used was a gel chromatography-differential chromatography-multi-angle laser light scattering system. The liquid chromatography system was a U3000, the differential detector was an Optilab T-rEX, and the laser light scattering detector was a DAWN HELEOS II. Based on sample pretreatment, the specific column and elution conditions were as follows: Ohpak SB-805HQ (300×8mm) and Ohpak SB-803HQ (300×8mm) gel size exclusion columns were used in series. The column temperature was 45℃, the injection volume was 100μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6mL / min, and the elution gradient was isocratic for 75min.

[0112] 3. Infrared spectroscopy determination

[0113] The functional group composition of litchi peel polysaccharide LPPs-1 was further analyzed by Fourier transform infrared spectroscopy. A small amount of litchi peel polysaccharide LPPs-1 was weighed and mixed with 200 mg of potassium bromide, then pressed into 1 mm thick sheets. The sheets were then analyzed using a Nicoletti Z-10 Fourier transform infrared spectrometer with a resolution of 4.00 cm⁻¹. - 1. Scanning range is 4000-400cm - 1. Number of scans: 32. Sampling gain: 8.0; Moving mirror speed: 0.4747; Aperture: 80.00; DTGS KBr detector; KBr beam splitter; Infrared light source.

[0114] 4. Scanning electron microscopy determination

[0115] A small amount of litchi shell polysaccharide LPPs-1 was attached to conductive adhesive, sputtered with gold, and its morphology was observed using SEM.

[0116] 5. Ultraviolet Measurement

[0117] A small amount of litchi peel polysaccharide LPPs-1 sample was weighed and dissolved in pure water to prepare a 5 mg / mL polysaccharide solution. The polysaccharide solution was quantitatively analyzed using a Thermo Fisher Scientific (USA) multi-functional microplate reader. Corning (USA) microplates were used, requiring that the absorbance difference between wells in parallel experiments at the target wavelength be less than 0.02. The scan start wavelength was 200 nm, the stop wavelength was 1000 nm, and the scan interval was 1 nm. Pure water was used as a blank control, and measurements were performed under the same conditions.

[0118] 6. Bond structure determination

[0119] Weigh a small amount of litchi peel polysaccharide LPPs-1 sample (approximately 5 mg), dissolve it in 1 mL of pure water, add 1 mL of 100 mg / mL 1-cyclohexyl-2-morpholinoethyl carbodiimide methyl p-toluenesulfonate, and react for 2 h. Add 1 mL of 2 M imidazole, divide the sample into two equal portions, add 1 mL of 30 mg / mL NaBH4 and 1 mL of 30 mg / mL NaBD4 to each portion, and react for 3 h. Terminate the reaction by adding 100 μL of glacial acetic acid. After dialyzing for 48 h, freeze-dry the sample for further processing. Dissolve the sample in 500 μL of DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times. Pipette the lower dichloromethane phase and dry it under nitrogen. Add 100 μL of 2M TFA and react at 121℃ for 90 min. Evaporate to dryness at 30℃. Add 50 μL of 2M ammonia and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL of acetic acid to terminate the reaction, dry under nitrogen, wash twice with 250 μL of methanol, and dry under nitrogen. Add 250 μL of acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h. Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash three times with water. Take the lower dichloromethane phase to obtain methylated aldose acetates (PMAAs), and analyze by GC-MS.

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

[0121] 7. Nuclear magnetic resonance spectroscopy determination

[0122] Dissolve an appropriate amount of litchi peel polysaccharide LPPs-1 thoroughly in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. Transfer the dissolved solution to an NMR tube, adding 0.5 mL. Place the NMR tube in an NMR spectrometer to scan one-dimensional 1H and 13C spectra, and two-dimensional COSY, HSQC, HMBC, and NOESY spectra.

[0123] Quantitative analysis of the target analytes was performed using a Bruker (Germany) 500MHz nuclear magnetic resonance spectrometer at a scanning temperature of 25℃. The liquid probe used was a QXI 1H / 31P / 13C / 15N 5mm quad-resonance reverse detection probe (Z-gradient, ATM Acc), with the following technical parameters: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating). The BBFO 1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradient, ATM) had the following 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] like Figure 1As shown, litchi shell polysaccharide LPPs-1 is composed of nine monosaccharides: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid (Gal-UA), and glucuronic acid (Glc-UA), with a molar ratio of 1.18:3.84:28.01:13.77:14.06:9.42:10.10:18.55:1.06. Arabinose has the highest molar ratio, followed by galacturonic acid, glucose, and galactose. It is speculated that fucose, rhamnose, xylose, mannose, and glucuronic acid are impurities. In summary, litchi shell polysaccharide LPPs-1 is a heteropolysaccharide, with Ara being the dominant monosaccharide.

[0127] 8.2 Molecular weight determination results

[0128] The GPC chromatogram of litchi shell polysaccharide LPPs-1 is shown below. Figure 2 As shown, it is a single symmetrical peak, indicating that litchi shell polysaccharide LPPs-1 is a homogeneous polysaccharide. The weight-average molecular weight (Mw) of litchi shell polysaccharide LPPs-1 was determined to be 38.974 kDa.

[0129] 8.3 Infrared Spectroscopic Analysis Results

[0130] The infrared spectrum of litchi shell polysaccharide LPPs-1 is shown in the figure below. Figure 3 As shown, the absorption band is in the range of 3600-3200 cm⁻¹. -1 This is the absorption peak of the stretching vibration of -OH, and the absorption peak in this region is a characteristic peak of carbohydrates. Specifically: 3329.91 cm⁻¹ -1 This is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. It is located at 2931.05 cm⁻¹. -1 The absorption peak at 1015.44 cm⁻¹ is attributed to the CH stretching vibration. -1 There is an absorption peak at this point, which is attributed to the stretching vibration of CO.

[0131] 8.4 Scanning Electron Microscopy Analysis Results

[0132] like Figure 4As shown, the surface morphology images of litchi peel polysaccharide LPPs-1 were magnified at 500×, 2000×, 5000×, and 10000×, respectively. The images reveal that litchi peel polysaccharide LPPs-1 exhibits an irregular, rough, layered structure with dense surface cracks. This multi-slit structure is likely a key factor in its water absorption, swelling, and excellent water retention properties, indicating that litchi peel polysaccharide LPPs-1 can be developed and utilized as a disintegrant, hygroscopic agent, or humectant. Furthermore, litchi peel polysaccharide LPPs-1 possesses more circular cavities, which may be due to the repulsive forces within the polysaccharide molecules, resulting in weak intermolecular attraction.

[0133] 8.5 Results of Ultraviolet Scanning Analysis

[0134] like Figure 5 As shown, there are no obvious peaks in the ultraviolet absorbance at 260 and 280 nm, indicating that no protein or nucleic acid was detected in litchi shell polysaccharide LPPs-1.

[0135] 8.6 Bonding structure and NMR analysis results

[0136] Methylation analysis revealed that litchi peel 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 high proportion of 4-Gal(p)-UA, t-Ara(f), and 5-Ara(f) indicates that the backbone of litchi peel polysaccharide LPPs-1 is likely composed of 4-Gal(p)-UA, t-Ara(f), and 5-Ara(f). The results of the polysaccharide sample bonding structure analysis are shown in Table 1.

[0137] Table 1. Results of bonding structure analysis of polysaccharide samples

[0138]

[0139] Multiple signal peaks were identified in the anomeric carbon region of the litchi shell polysaccharide LPPs-1 sample. Combining the cross peaks of the anomeric region in the 13CNMR and HSQC spectra, the anomeric signals present in the sample were determined to be: δ 5.02 / 101.63, 5.17 / 109.36, 5.08 / 106.9, 5.0 / 107.41, 4.45 / 103.21, and 5.04 / 98.78 ppm, and were respectively denoted as sugar residues A, B, C, D, E, and F. Based on the sample bonding structure (methylation) information, anodic signals, and comprehensive literature reports, it was deduced that sugar residue A is →4)-α-D-GalpA-(1→), sugar residue B is α-L-Araf-(1→), sugar residue C is →5)-α-L-Araf-(1→, sugar residue D is →3)-α-L-Araf-(1→, sugar residue E is →3,6)-β-D-Galp-(1→, sugar residue F is →4)-α-D-Glcp-(1→). The 1H and 13C chemical shifts were assigned, and the results are shown in Table 2.

[0140] Table 2 Sugar residues 1 H and 13 Chemical shift of C

[0141]

[0142] Combining one-dimensional and two-dimensional NMR information ( Figure 6-11 Based on the analysis of methylation results, it was deduced that the litchi peel polysaccharide LPPs-1 is 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 linked together to form the main chain. The side chains are mainly composed of α-L-Araf-(1→, →4)-α-D-Glcp-(1→, →3)-α-L-Araf-(1→) linked together and attached to the O-6 positions of the sugar residue →3,6)-β-D-Galp-(1→). Based on this, the structure of litchi peel polysaccharide LPPs-1 is inferred as follows: Figure 12 As shown.

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

[0144] 1. Oil retention

[0145] Transfer 0.2g of litchi shell polysaccharide LPPs-1 into a centrifuge tube and weigh it (W1). Pipette 6mL of soybean oil into a test tube, heat in a boiling water bath, and stir evenly for 20 minutes. Remove and cool to room temperature, then centrifuge at 3000 rpm for 20 minutes. Discard the supernatant, invert the centrifuge tube onto filter paper, and let it stand for 20 minutes until the oil has drained completely. Weigh the tube and calculate the difference in mass (W2). Oil holding capacity (OHC) represents the mass of oil absorbed per gram of sample. Each group is measured in triplicate. OHC is calculated using the following formula:

[0146] OHC = (W2 - W1) / sample mass

[0147] In the formula: W1 is the total mass of the sample and centrifuge tube, g; W2 is the total mass of the sample and centrifuge tube after the oil has dried, g.

[0148] 2. Hygroscopicity

[0149] 10.2 g of litchi peel polysaccharide LPPs was weighed and placed in a dry weighing bottle. The bottle was then placed in a saturated NaCl solution in a 25°C oven, and the sample was weighed every hour. The hygroscopicity of the sample is expressed by the following formula:

[0150] Moisture absorption rate (%) = 100 (M n -M0) / M0

[0151] Where: M0 is the mass of the sample before moisture absorption, in g; M n The mass is expressed in grams after different moisture absorption times.

[0152] 3. Alcohol solubility

[0153] Weigh 0.05 g of each litchi peel polysaccharide LPPs-1 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 at 105℃ to constant weight. Calculate the solubility (S) of the sample in the ethanol solution according to the following formula:

[0154] S(%)=(A / M)×100

[0155] In the formula: A is the mass of the dissolved polysaccharide sample, g; M is the mass of the polysaccharide sample, g.

[0156] 4. Inhibitory activity test of α-amylase and α-glucosidase

[0157] 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) were combined with 500 μL of α-amylase solution (0.4 U / mL) and incubated at 37 °C for 10 min. Then, 600 μL of soluble starch solution (10 mg / mL) was added to the mixture, and incubation was repeated for 5 min. To complete the reaction, 700 μL of DNS was introduced into the system. The reaction mixture was immediately placed in a 10 °C water bath for 10 min to inactivate the α-amylase. The absorbance was measured at 540 nm using a UV-Vis spectrophotometer.

[0158] 400 μL of litchi peel polysaccharide LPPs-1 at different concentrations was combined with 400 μL of α-glucosidase solution (60 U / mL) and incubated at 37 °C for 10 min. Next, 400 μL of PNPG solution (2.5 mmol / L) was added to the mixture, and incubation was repeated for another 10 min. To terminate the reaction, 550 μL of sodium carbonate solution (0.2 mol / L) was introduced. The absorbance was recorded at 405 nm.

[0159] Acarbose was used as a positive control in all experiments. SPSS software was used for curve fitting analysis to calculate the maximum half-maximum inhibitory concentration (HIC) of litchi peel polysaccharide LPPs-1 against the two enzymes. The inhibitory effect of this component on α-amylase and α-glucosidase was 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 with litchi shell polysaccharide LPPs-1 is used as control-1, and the absorbance after reacting with the enzyme without this component is used as control-2.

[0162] 5. Results and Analysis

[0163] 5.1 Results on oil retention

[0164] Studies have shown that polysaccharides possess oil-holding capacity and can be used as gelling agents, thickeners, colloidal stabilizers, and fat substitutes in food processing. Oil-holding capacity is the most significant functional characteristic of a sample's oil absorption capacity. Higher oil-holding capacity can maintain food flavor and enhance its texture (see Xu Yaqin, Liu Ningyue, Li Dalong, et al. Functional characteristics, structure and anti-glycation activity of honeysuckle fruit polysaccharide [J]. Food Science, 2020, 41(2):8-14.). The oil-holding capacity of polysaccharides can guide the use of dosage forms. High oil-holding capacity indicates that the polysaccharide has a relatively large molecular weight and viscosity.

[0165] The results showed that the three oil-holding capacities of litchi shell polysaccharide LPPs-1 were 2.56 g / g, 2.64 g / g, and 2.41 g / g, respectively, with an average oil-holding capacity of 2.54 ± 0.10 g / g. This was higher than that of commercial gluten (1.19 ± 0.13 g / g) and lower than that of honeysuckle fruit polysaccharide (4.60 ± 0.45 g / g) (see Xu Yaqin, Liu Ningyue, Li Dalong, et al. Functional characteristics, structure and anti-glycation activity of honeysuckle 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 food as a texture modifier, thickener, and gelling agent to improve food texture and enhance taste.

[0166] 5.2 Hygroscopicity Results

[0167] Depend on Figure 13 It can be seen that the moisture absorption rate increases with time. The moisture absorption rate of litchi shell polysaccharide LPPs-1 reaches equilibrium after 12 hours, and then shows a gradual trend. The moisture absorption rate reaches its highest value of 15.73% at 36 hours. Within 12 hours, the moisture absorption rate of litchi shell polysaccharide LPPs-1 is higher than that of osmanthus leaf polysaccharide (15.56%), hygroscopic agent chitosan (11.43%), and glucose (3.17%) (see Chen Zheng, Gao Youjun, Deng Fangfei, et al. Study on microwave extraction of osmanthus leaf polysaccharide and its moisture absorption, moisturizing and antioxidant properties [J]. New Chemical Materials, 2022, 50(S1):337-340.). Under normal circumstances, people do not use cosmetics for more than 12 hours. However, the moisture absorption rate of litchi shell polysaccharide LPPs-1 can reach % within 12 hours, which is higher than that of chitosan (11.43%), a commonly used hygroscopic agent. This indicates that litchi shell polysaccharide LPPs-1 can be developed and utilized as a natural hygroscopic agent.

[0168] Studies have shown that high hygroscopicity may be related to the polarity, structural characteristics, and porosity of polysaccharides. After polysaccharides combine with water molecules, they form a network structure, which can better retain moisture (see Qin Yin, Xu Wenhui, Wu Kai, et al. Effects of different extraction methods on the physicochemical properties and bioactivity of Polygonatum polysaccharides [J / OL]. Modern Food Science and Technology, 1-12 [2024-02-18]). This indicates that polysaccharides can also be developed and utilized as humectants.

[0169] 5.3 Results of alcohol solubility

[0170] Depend on Figure 14It can be seen that the solubility of litchi shell polysaccharide LPPs-1 gradually decreases with the increase of ethanol concentration. 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 polysaccharide molecules obtained by water extraction have similar polarity to water and are miscible. Its solubility in water is higher, and it is easily soluble in water. In addition, this polysaccharide contains a lot of uronic acid with hydrophilic groups, which can greatly change the solubility of the polysaccharide.

[0171] 5.4 Results of α-Amylase and α-Glucosidase Inhibitory Activity Tests

[0172] like Figure 15 As shown, litchi peel polysaccharide LPPs-1 exhibited dose-dependent inhibitory activity against both enzymes, with a significant increase in inhibition rate (p<0.05) at concentrations ranging from 0.01 to 0.8 mg / mL. The half-maximal inhibitory concentrations (IC50) of litchi peel polysaccharide LPPs-1 against α-amylase and α-glucosidase were 0.16 mg / mL and 0.26 mg / mL, respectively. This indicates that litchi peel polysaccharide LPPs-1 has a stronger inhibitory effect on α-amylase. This further demonstrates the great potential of litchi peel polysaccharide LPPs-1 in lowering blood sugar, and its potential use in treating diabetes or its complications, providing a theoretical basis for the development of novel hypoglycemic drugs, functional foods, and health products.

[0173] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

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

[0175] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. The application of litchi shell polysaccharide in the preparation of α-amylase inhibitors or α-glucosidase inhibitors, characterized in that, The litchi shell polysaccharide contains arabinose, galactose, glucose and galacturonic acid, with a molar mass ratio of (27-29):(13-15):(13-15):(18-19). 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 side chain is composed of α-L-Araf-(1→,→4)-α-D-Glcp-(1→,→3)-α-L-Araf-(1→); The molecular weight of the litchi shell polysaccharide is 38,000-39,000 Da.

2. The application according to claim 1, characterized in that, The molecular weight of the litchi shell polysaccharide is 38974 Da.

3. The application according to claim 1, characterized in that, The litchi shell polysaccharide comprises the following structure: 。 4. The application according to any one of claims 1-3, characterized in that, The preparation method of the litchi shell polysaccharide includes the following steps: (1) Add water to litchi peel powder for extraction, and then subject the resulting water extract to alcohol precipitation and protein removal to obtain litchi peel crude polysaccharide. (2) The crude polysaccharide of litchi shell obtained in step (1) is eluted by an ion exchange column to obtain litchi shell polysaccharide components; (3) The litchi shell polysaccharide component obtained in step (2) is eluted through a gel column to obtain litchi shell polysaccharide.

5. The application according to claim 4, characterized in that, In step (1), the ratio of litchi shell powder to water is 1 mg: (10-30) mL.

6. The application according to claim 4, characterized in that, In step (1), the extraction is performed under ultrasound, and the ultrasound power is 100-1000 W.

7. The application according to claim 4, characterized in that, In step (1), the extraction temperature is 70-100℃.

8. The application according to claim 4, characterized in that, In step (1), the alcohol solvent used in the alcohol precipitation step is ethanol.

9. The application according to claim 4, characterized in that, In step (1), the volume ratio of alcohol solvent to water extract used in the alcohol precipitation step is (1-10):

1.

10. The application according to claim 4, characterized in that, In step (2), the ion exchange column is an anion exchange column.

11. The application according to claim 10, characterized in that, In step (2), the anion exchange column is a DEAESephadex FF anion exchange column.

12. The application according to claim 4, characterized in that, In step (2), the elution is gradient elution.

13. The application according to claim 4, characterized in that, In step (2), the eluent used for elution is water and / or an aqueous solution of sodium chloride.

14. The application according to claim 4, characterized in that, In step (2), gradient elution is performed sequentially using water, 0.1 mol / L sodium chloride aqueous solution and 0.2 mol / L sodium chloride aqueous solution.

15. The application according to claim 4, characterized in that, In step (3), the gel column is a SephecrylS-400HR gel chromatography column.

16. The application according to claim 4, characterized in that, In step (3), the solvent used for elution is water.

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