CN109369824B - Crocodile chondroitin sulfate and preparation method thereof - Google Patents

Crocodile chondroitin sulfate and preparation method thereof Download PDF

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CN109369824B
CN109369824B CN201811272071.1A CN201811272071A CN109369824B CN 109369824 B CN109369824 B CN 109369824B CN 201811272071 A CN201811272071 A CN 201811272071A CN 109369824 B CN109369824 B CN 109369824B
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crocodile
chondroitin sulfate
concentrated solution
cartilage
drying
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CN109369824A (en
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李华亮
陈清西
郑雅惠
熊佑熊
董欣
丁玉梅
张紫然
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Fujian Tuolong Industrial Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass

Abstract

The application discloses crocodile chondroitin sulfate, which comprises D-glucuronic acid, hexosamine and potassium sulfate. The application also provides a preparation method of the crocodile chondroitin sulfate, which comprises the following steps: (1) heating alcohol solvent and crocodile cartilage, drying, and pulverizing; (2) alkali extracting the crocodile cartilage powder crushed in the step (1) by using a sodium hydroxide solution, and precipitating by using ethanol; (3) centrifuging the ethanol precipitation liquid in the step (2) to obtain a crocodile chondroitin sulfate crude product; (4) separating and purifying the crocodile chondroitin sulfate crude product, dialyzing, and performing rotary evaporation to obtain a concentrated solution 1; (5) separating and purifying the concentrated solution 1 by column chromatography, dialyzing, and performing rotary evaporation to obtain a concentrated solution 2; and (3) freeze-drying the concentrated solution 2 to obtain the crocodile chondroitin sulfate. The chondroitin sulfate also has utility in relieving fatigue and enhancing immunity.

Description

Crocodile chondroitin sulfate and preparation method thereof
Technical Field
The application relates to the fields of food health products and medicines, in particular to crocodile chondroitin sulfate and a preparation method thereof. The application also relates to compositions comprising crocodile chondroitin sulfate and the use of crocodile chondroitin sulfate for relieving fatigue and enhancing immunity.
Background
Chondroitin sulfate is one of main components of connective tissues of mammals, is mostly distributed in cartilage, nasal bone, laryngeal bone and the like, is combined with protein, exists in the form of proteoglycan, belongs to glycosaminoglycan substances, and is an acidic mucopolysaccharide. Polysaccharides, which are natural high molecular compounds derived from cell membranes of higher plants and animals and cell walls of microorganisms, are biomacromolecules with wide biological activity, also called polysaccharides, which are one of basic substances for maintaining normal operation of life activities.
Chondroitin sulfate is polymerized from two monosaccharides, and generally, a structural unit is formed by first polymerizing various monosaccharides through glycosidic bonds. Although different chondroitin sulfate molecules have different molecular weights due to different numbers of polymerized structural units, the composition ratio of each monosaccharide in the structural units forming the chondroitin sulfate is fixed, so that different molecules of the same chondroitin sulfate have the same monosaccharide composition ratio. Although some chondroitin sulfates are composed of the same monosaccharide, the properties are different due to different composition ratios of the monosaccharides.
Chondroitin sulfate is a macromolecular heteropolysaccharide formed by polymerizing a disaccharide basic unit combined by glucuronic acid and acetyl galactosamine through beta-1, 3 glycosidic bonds, acidic anion groups with different compositions and quantities are distributed on long-bond molecules of the macromolecular heteropolysaccharide, and most of the acidic anion groups are sulfate groups. According to the difference of chemical composition and structure, it can be classified into A, C, D, E, F, H, wherein the content of chondroitin sulfate A and chondroitin sulfate C is more.
Chondroitin sulfate contains a large amount of carboxyl groups and sulfate groups, and most of them exist in the form of polyanion, so that water molecules with positive charges can be adsorbed, and if chondroitin sulfate is sufficiently supplemented, skin aging can be inhibited, and skin elasticity can be maintained. The natural substances can be used as biochemical medicaments for clinical application and can also be used as dietary supplements for improving related functions of human bodies, and the extraction and application of the natural aminopolysaccharide chondroitin sulfate in animal cartilages become new research hotspots.
Crocodile is an animal of the class of the reptilia, alligata and alligator, and has high economic and medical values. The development and utilization of the animal bone products can fully utilize resources, reduce environmental pollution, improve the added value of animal bone processing byproducts, and have wide application prospect for improving the comprehensive benefits of meat product processing enterprises.
Disclosure of Invention
The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.
The application provides chondroitin sulfate extracted from crocodile bones and a preparation method thereof, and the crocodile chondroitin sulfate extracted by the application plays a role in relieving fatigue and improving immunity.
Specifically, the crocodile chondroitin sulfate is extracted from crocodile bones and comprises 20-30% of D-glucuronic acid, 45-55% of hexosamine and 20-30% of potassium sulfate by mass fraction.
In the present application, the crocodile chondroitin sulfate comprises, by mass fraction, 23-27% of D-glucuronic acid, 48-52% of hexosamine, 22-28% of potassium sulfate.
In the present application, the crocodile chondroitin sulfate comprises 26% of D-glucuronic acid, 50% of hexosamine, and 24% of potassium sulfate by mass fraction.
In the application, the crocodile chondroitin sulfate ranges from 3500 cm to 3000cm-1(3422.21cm-1) O-H stretching vibration; 3000-2800 cm-1(2925.41cm-1) C-H stretching vibration; 3500-3000 cm-1(3422.21cm-1) Is N-H stretching vibration, 1655-1590 cm-1(1620.93cm-1) Is N-H bending vibration, 1420-1400 cm-1(1411.23cm-1) C-N stretching vibration; 1320-1210 cm-1(1252.60cm-1) C-O stretching vibration; 1100-1000 cm-1(1055.81cm-1) Corner vibration for O-H; 930.55cm-1Is asymmetric ring stretching vibration, 719.51cm-1855 + 833cm for symmetric ring stretching vibration-1(852.89cm-1) Is the C-H variable angle vibration of alpha-end group epimerization.
In the present application, theCrocodile chondroitin sulfate is 3422.21cm-1O-H stretching vibration; 2925.41cm-1C-H stretching vibration; 3422.21cm-1Is N-H stretching vibration, 1620.93cm-1Is N-H bending vibration, 1411.23cm-1C-N stretching vibration; 1252.60cm-1C-O stretching vibration; 1055.81cm-1Corner vibration for O-H; 930.55cm-1Is asymmetric ring stretching vibration, 719.51cm-1Is symmetrically and telescopically vibrated, 852.89cm-1Is the C-H variable angle vibration of alpha-end group epimerization.
The application also provides a preparation method of the crocodile chondroitin sulfate, which comprises the following steps:
(1) heating alcohol solvent and crocodile cartilage, drying, and pulverizing;
(2) alkali extracting the crocodile cartilage powder crushed in the step (1) by using a sodium hydroxide solution, centrifuging, adjusting the pH of the obtained supernatant to 6.8-7.2 by using hydrochloric acid, adding ethanol, and precipitating overnight;
(3) centrifuging the ethanol precipitation liquid in the step (2), removing the upper layer liquid, and vacuum-drying the lower layer precipitate to obtain a crocodile chondroitin sulfate crude product;
(4) preparing the crocodile chondroitin sulfate crude product into aqueous solution, separating and purifying by using an ion exchange chromatography column, collecting eluent 1 at an elution peak, dialyzing by using a dialysis bag to obtain dialysate 1, and performing rotary evaporation to obtain concentrated solution 1;
(5) separating and purifying the concentrated solution 1 by column chromatography, collecting eluent 2 at an elution peak, dialyzing by a dialysis bag to obtain dialysate 2, and performing rotary evaporation to obtain concentrated solution 2; and (3) freeze-drying the concentrated solution 2 to obtain the crocodile chondroitin sulfate.
In this application, the crocodile mentioned is Siamese crocodile artificial breeding son second generation, and crocodile breeds and raises the license: the Chinese fishing water field domesticating and propagating letters 2012 and 5019.
In the present application, in step (1), the alcohol includes any one or more of methanol, ethanol, benzyl alcohol, and ethylene glycol.
In this application, in step (1), heating is carried out at 50 to 60 ℃.
In this application, in step (1), heating is carried out for 30 to 35 minutes.
In this application, in step (1), heating is carried out at 50 to 60 ℃ for 30 to 35 minutes.
In this application, in step (1), the crocodile cartilage comprises the rib and bone joints of crocodile.
In the application, in the step (1), the ratio of the volume of the alcohol to the weight of the crocodile cartilage is 1L: 50-200 g.
In the present application, in the step (2), the mass fraction of the sodium hydroxide solution is 2% to 10%.
In the present application, in the step (2), the mass ratio of the crocodile cartilage powder to the sodium hydroxide solution is 1:6 to 1: 14.
In the application, in the step (2), the crocodile cartilage powder is subjected to alkali extraction in a water bath at 45 +/-5 ℃ for 2.5-3.5 h.
In the present application, in step (2), the alkali extraction is carried out at a temperature in the range of 30 ℃ to 50 ℃.
In the present application, in step (2), the crocodile cartilage powder is subjected to alkali extraction in a water bath for 2-4 h.
In the application, in the step (2), ethanol is added, so that the volume concentration of the ethanol in an ethanol precipitation system is 30-70%.
In the present application, in step (4), the concentration of the crocodile chondroitin sulfate crude aqueous solution is 1 g/ml. Separating and purifying by using a Q-Sepharose-F-F ion exchange chromatography column, wherein the initial buffer solution is a Tris-HCl buffer solution with pH of 8.0 and 0mol/L NaCl, and the eluent is a Tris-HCl buffer solution with pH of 8.0 and 1mol/L NaCl.
In this application, in step (4), separation and purification are carried out using Sephadex G-200 column chromatography.
The present application also provides a composition comprising said crocodile chondroitin sulfate, said composition further comprising a pharmaceutically acceptable additive.
In the present application, the pharmaceutically acceptable additives include diluents, lubricants, dispersants, antioxidants, preservatives and the like. The pharmaceutically acceptable additives are suggested from pharmacy, treford, people health press, 6 th edition.
The application also provides application of the crocodile chondroitin sulfate in relieving fatigue.
The application also provides application of the crocodile chondroitin sulfate in improving immunity.
In the present application, the crocodile chondroitin sulfate can be applied to a human body by injection, oral administration, or the like.
In the present application, the crocodile chondroitin sulfate can be administered 1-3 times per day, and each administration dose can be 10-100mg/kg body weight.
Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. The objectives and other advantages of the application may be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
The accompanying drawings are included to provide a further understanding of the claimed subject matter and are incorporated in and constitute a part of this specification, illustrate embodiments of the subject matter and together with the description serve to explain the principles of the subject matter and not to limit the subject matter.
FIG. 1 is a UV spectrum of chondroitin sulfate standard;
FIG. 2 is a graph of the ultraviolet spectrum of crocodile chondroitin sulfate extracted in the present application;
FIG. 3 is an infrared spectrum of crocodile chondroitin sulfate extracted in the present application;
FIG. 4 shows crocodile chondroitin sulfate extracted by the present application1An H-NMR spectrum;
FIG. 5 is a graph showing the effect of different concentrations of NaOH solutions on crocodile chondroitin sulfate content;
FIG. 6 is a graph illustrating the effect of different feed liquid ratios on crocodile chondroitin sulfate content;
FIG. 7 is a graph illustrating the effect of different temperatures on crocodile chondroitin sulfate content;
FIG. 8 is a graph showing the effect of different extraction time solutions on crocodile chondroitin sulfate content;
FIG. 9 shows the isolation of crocodile chondroitin sulfate by Q-Sepharose-F-F ion exchange resin column chromatography;
FIG. 10 shows Sephadex G-200 column chromatography for separation and purification of crocodile chondroitin sulfate;
fig. 11 is a graph of the effect of crocodile chondroitin sulfate extracted herein on swimming time of mice, where the number indicates direct significance of the group, p < 0.05, p < 0.01;
fig. 12 is a graph of the effect of crocodile chondroitin sulfate on Con a-induced T lymphocyte proliferation as extracted herein, wherein the number indicates direct significance of the group, p < 0.05, p < 0.01;
fig. 13 is a graph of the effect of the extracted chondroitin crocodile sulphate on LPS-induced B lymphocyte proliferation, wherein the number indicates direct significance of the groups, p < 0.05, and p < 0.01.
Detailed Description
To make the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.
The instrumentation used in the examples included:
ultraviolet spectrophotometer DU650 type BECKMAN (USA)
Infrared spectrometer American THORLABS
Nuclear magnetic resonance apparatus REFLEXTM III BRUKER
HPLC 2695 Waters Co
Visible spectrophotometer (Shanghai spectrometer Co., Ltd., type 722)
Example 1: preparation and purification of crocodile chondroitin sulfate
Heating methanol and de-fatted crocodile cartilage at a ratio of 1L to (100 + -2) g at 50-60 deg.C for 30 + -5 min, oven drying the heated crocodile cartilage in an oven, and pulverizing with a pulverizer (XL-30C, Asahan mechanical equipment Limited, Guangzhou) to obtain crocodile cartilage powder 5 g.
Example 1.1: influence of NaOH solutions with different concentrations on content of crocodile chondroitin sulfate
Respectively weighing 5 parts of equivalent crocodile cartilage powder, respectively adding 2%, 4%, 6%, 8% and 10% NaOH solutions according to the mass ratio of the feed liquid to 1: 10, shaking on a shaking table for 1h, placing in a water bath kettle at 40 ℃ for extraction for 3h, centrifuging at 8000rpm for 10 +/-2 min, discarding the precipitate, collecting the supernatant, adjusting the pH to 6.8 with hydrochloric acid, adding absolute ethyl alcohol until the volume concentration of an alcohol precipitation system is 60%, shaking uniformly, and precipitating overnight; centrifuging at 8000rpm for 10 + -2 min, removing supernatant, collecting precipitate, washing the precipitate with anhydrous ethanol twice, and vacuum drying at 65 deg.C for 4 hr to obtain dried product, i.e. crocodile chondroitin sulfate crude product.
Respectively weighing 10mg of crocodile chondroitin sulfate crude products obtained by equivalent NaOH solutions with different concentrations, and fixing the volume to 25mL by using distilled water; respectively sucking 0.5mL of equivalent solution into a test tube, placing on ice, respectively adding 5.0mL of sodium tetraborate-concentrated sulfuric acid, shaking uniformly, heating for 10min, taking out, and rapidly cooling to room temperature; respectively adding 200 μ L of carbazole reagent, shaking, heating for 15min, taking out, and rapidly cooling to room temperature; a visible spectrophotometer (Shanghai spectrometer, Ltd., model 722) measures absorbance at a wavelength of 530nm, using distilled water as a blank.
The results show that, as shown in fig. 5, the extraction content of crocodile chondroitin sulfate is in negative correlation with the concentration of the NaOH solution, and the extraction content of crocodile chondroitin sulfate reaches nearly 40% when the concentration of the NaOH solution is 2%.
Example 1.2:influence of different feed liquid ratios on crocodile chondroitin sulfate content
Respectively weighing 5 parts of crocodile cartilage powder with the same amount, respectively adding 2% NaOH solution according to the material-liquid ratio of 1:6, 1: 8, 1: 10, 1: 12 and 1:14, shaking on a shaking table for 1h, placing in a 40 ℃ water bath pot for extraction for 3h, centrifuging at 8000rpm for 10 +/-2 min, discarding the precipitate, collecting the supernatant, adjusting the pH to 6.8, adding absolute ethyl alcohol until the concentration of an alcohol precipitation system is 60%, shaking uniformly, and precipitating overnight; centrifuging at 8000rpm for 10 + -2 min, removing supernatant, collecting precipitate, washing the precipitate with anhydrous ethanol twice, and vacuum drying at 65 deg.C for 4 hr to obtain dried product, i.e. crocodile chondroitin sulfate crude product.
Respectively weighing 10mg of crocodile chondroitin sulfate crude products obtained by equivalent amount and different material-liquid ratios, and fixing the volume to 25mL by using distilled water; respectively sucking 0.5mL of solution into a test tube, placing the test tube on ice, respectively adding 5.0mL of sodium tetraborate-concentrated sulfuric acid, shaking uniformly, heating for 10min, taking out and rapidly cooling to room temperature; respectively adding 200 μ L of carbazole reagent, shaking, heating for 15min, taking out, and rapidly cooling to room temperature; the absorbance was measured at 530nm using distilled water as a blank.
The results are shown in fig. 6, the influence of different feed liquid ratios on the content of crocodile chondroitin sulfate, and the feed-liquid ratio of 1: 8 obtains the highest extraction content of crocodile chondroitin sulfate.
Example 1.3:effect of different temperatures on crocodile chondroitin sulfate content
Respectively weighing 5 parts of equivalent cartilage powder, respectively adding 2% NaOH solution according to the material-liquid ratio of 1: 8, shaking for 1h on a shaking table, respectively placing in 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ water bath kettle for extracting for 3h, centrifuging at 8000rpm for 10 +/-2 min, discarding the precipitate, collecting the supernatant, adjusting the pH to 6.8, adding absolute ethyl alcohol until the concentration of an alcohol precipitation system is 60%, shaking uniformly, and precipitating overnight; centrifuging at 8000rpm for 10 + -2 min, removing supernatant, collecting precipitate, washing the precipitate with anhydrous ethanol twice, and vacuum drying at 65 deg.C for 4 hr to obtain dried product, i.e. crocodile chondroitin sulfate crude product.
Respectively weighing 10mg of crocodile chondroitin sulfate crude products obtained at the same amount and different temperatures, and fixing the volume to 25mL by using distilled water; respectively sucking 0.5mL of the solution into a test tube, placing the test tube on ice, respectively adding 5.0mL of sodium tetraborate-concentrated sulfuric acid, shaking uniformly, heating for 10min, taking out and rapidly cooling to room temperature; respectively adding 200 μ L of carbazole reagent, shaking, heating for 15min, taking out, and rapidly cooling to room temperature; the absorbance was measured at 530nm using distilled water as a blank.
The results show that, as shown in fig. 7, the extraction temperature of 45 ℃ is the highest, and the extraction rate of crocodile chondroitin sulfate reaches 60%.
Example 1.4:effect of solutions at different extraction times on crocodile chondroitin sulfate content
Respectively weighing 5 parts of equivalent cartilage powder, respectively adding 2% NaOH solution according to the material-liquid ratio of 1: 8, shaking for 1h on a shaking table, placing in a 45 ℃ water bath kettle for extraction for 2h, 2.5h, 3h, 3.5h and 4h, centrifuging at 8000rpm for 10 +/-2 min, discarding the precipitate, collecting the supernatant, adjusting the pH to 6.8, adding absolute ethyl alcohol until the concentration of an alcohol precipitation system is 60%, shaking uniformly, and precipitating overnight; centrifuging at 8000rpm for 10 + -2 min, removing supernatant, collecting precipitate, washing the precipitate with anhydrous ethanol twice, and vacuum drying at 65 deg.C for 4 hr to obtain dried product, i.e. crocodile chondroitin sulfate crude product.
Respectively weighing 10mg of crocodile chondroitin sulfate crude products obtained in equal amount and different extraction time, and fixing the volume to 25mL by using distilled water; respectively sucking 0.5mL of solution into a test tube, placing the test tube on ice, respectively adding 5.0mL of sodium tetraborate-concentrated sulfuric acid, shaking uniformly, heating for 10min, taking out and rapidly cooling to room temperature; respectively adding 200 μ L of carbazole reagent, shaking, heating for 15min, taking out, and rapidly cooling to room temperature; the absorbance was measured at 530nm using distilled water as a blank.
The result shows that, as shown in fig. 8, the influence of the solution on the content of the crocodile chondroitin sulfate in different extraction time is realized, and the content of the crocodile chondroitin sulfate is highest and reaches 45% after the extraction time is further 3.5 hours.
Example 1.5:Q-Sepharose-F-F ion exchange resin column chromatography separation of crocodile chondroitin sulfate
Preparing the gel and an initial buffer solution (20mmol/L Tris-HCl buffer solution) into a homogenate according to the mass ratio of 3: 1; assembling a chromatographic column, draining all prepared homogenate along the inner wall of the column by using a glass rod, pouring the homogenate into the column, opening a rubber tube below the column to enable a gel column to freely settle in the column, and connecting an elution column when a column bed is stable; opening the constant flow pump, allowing the initial buffer solution to flow through at a flow rate 1.33 times the flow rate when in use, stabilizing the column bed, and balancing the buffer solution with at least 3 times the column volume; dissolving 10mg of crocodile chondroitin sulfate crude product in a buffer solution, fully dissolving, and adding the crude product solution along the tube wall by using a liquid transfer gun; after the sample loading is finished, opening a constant flow pump for elution, eluting by using 2 times of column volume of Tris-HCl eluent containing 0mol/L NaCl, eluting by using 1mol/L of NaCl Tris-HCl eluent at the flow rate of 1mL/min, collecting one tube per 4mL, respectively measuring the content of crocodile chondroitin sulfate and the content of protein by using a concentrated sulfuric acid-carbazole method and an ultraviolet absorption method, and drawing an elution curve; collecting the eluate at the elution peak of the crocodile chondroitin sulfate, dialyzing by a dialysis bag, and performing rotary evaporation to obtain concentrated solution.
The result shows that the absorption value peak and the protein absorption peak of the crocodile chondroitin sulfate are staggered as shown in fig. 9, which shows that the crocodile chondroitin sulfate has high purity and a good extraction process.
Example 1.6:sephadex G-200 column chromatography separation and purification of crocodile chondroitin sulfate
Soaking the gel dry powder in 60% ethanol for swelling completely, and degassing by air suction for later use; assembling a chromatographic column, draining all prepared homogenate along the inner wall of the column by using a glass rod, pouring the homogenate into the column, opening a rubber tube below the column to enable a gel column to freely settle in the column, and connecting an elution column when a column bed is stable; opening a constant flow pump, and allowing an initial buffer solution to flow through the constant flow pump at a flow rate of 0.5mL/min, so that the column bed is stable, and the buffer solution with at least 3 times of column volume is balanced; dissolving 10mg of crocodile chondroitin sulfate crude product in a buffer solution, fully dissolving, filtering crocodile chondroitin sulfate solution prepared by an ion exchange column by using a filter membrane of 0.22 mu m, and adding the crude product solution along the tube wall by using a liquid transfer gun; after the sample loading is finished, opening a constant flow pump for elution, eluting with 2 times of column volume of Tris-HCl eluent containing 0.1mol/LNaCl, collecting one tube every 4mL at the flow rate of 0.5mL/min, respectively measuring the content of crocodile chondroitin sulfate and the content of protein by a concentrated sulfuric acid-carbazole method and an ultraviolet absorption method, and drawing an elution peak; combining and collecting the eluent at the elution peak of the crocodile chondroitin sulfate, dialyzing by a dialysis bag, performing rotary evaporation to obtain concentrated solution, and performing freeze drying to obtain the crocodile chondroitin sulfate. The separation is shown in FIG. 10, and the high content of crocodile chondroitin sulfate is mainly concentrated in 4-8 tubes.
Example 1.7:component determination in crocodile chondroitin sulfate
In the experiment, D-glucuronic acid is used as a standard sample to determine the content of glucuronic acid in crocodile chondroitin sulfate, N-acetyl-D-galactosamine is used as a standard sample to determine the content of hexosamine in crocodile chondroitin sulfate, potassium sulfate is used as a standard sample to determine the content of potassium sulfate in crocodile chondroitin sulfate, and the obtained standard sampleQuasi curve R2Greater than 0.99.
TABLE 1 Standard Curve plotting
Figure BDA0001845429170000091
Crocodile chondroitin sulfate sample assay results are shown in table 2. According to the standard curve, the content of D-glucuronic acid in the sulfated cartilage sample is 26.05% + -0.01, the content of hexosamine is 49.93% + -0.01, and the content of potassium sulfate is 24.02% + -0.01. The experiment result shows that uronic acid exists in the crocodile chondroitin sulfate, so that the crocodile chondroitin sulfate shows acidic sugar property and is an acidic sugar.
TABLE 2 results of sample measurement
Figure BDA0001845429170000092
Example 2: bioactivity of crocodile chondroitin sulfate
Example 2.1: determination of physical fatigue relieving function of crocodile chondroitin sulfate
(1) Grouping of experimental mice and drug preparation: dissolving crocodile chondroitin sulfate in dH2O, wherein the concentration of the crocodile chondroitin sulfate in a low-dose group is 12.5mg/mL, the concentration of the crocodile chondroitin sulfate in a medium-dose group is 25mg/mL, and the concentration of the crocodile chondroitin sulfate in a high-dose group is 37.5 mg/mL; the positive control group was red ginseng powder (purchased from Sichuan biosciences, Inc., Sichuan), the concentration was 25mg/mL, and the blank control group was dH 2O. Each group of reagents was sterilized by filtration through a 0.22 μm filter. .
(2) The mice were subjected to gavage treatment:
gavage is carried out once a day for 5 consecutive days on Monday to Friday, and 200 μ L of the solution is gavaged once every time for continuous treatment for four weeks.
(3) Exhaustive swimming experiment of mice
After the last gastric lavage is finished, all mice open the stomach for 20 hours, then the mice are placed in a swimming box, the water depth is not less than 30cm, the water temperature is 25 +/-1.0 ℃, the mice are enabled to swim continuously until the mice can not float out of the water surface within 7s for ventilation, and the mice die, and the time from swimming to death, namely the time of the mice spent swimming, is recorded.
As shown in fig. 11, all mice in the experimental group and the positive control group swim longer than the mice in the blank control group. In the mice of the medium-dose group, the dosage of crocodile chondroitin sulfate is the same as that of the red ginseng powder given by the positive control group, but the swimming time of the mice is twice that of the mice of the positive control group. When the intake of crocodile chondroitin sulfate is increased, the swimming time is prolonged continuously. The mice in the high dose group had a 40.68% higher exhaustion swimming time (5.37 + -1.14 h) than the low dose group (3.82 + -0.62 h), indicating that the swimming ability of the mice is directly proportional to the amount of crocodile chondroitin sulfate ingested, with a significant dose effect between the two.
Example 2.2: immunologic function determination of crocodile chondroitin sulfate
Proliferation of T and B lymphocytes by crocodile chondroitin sulfate
Preparation of spleen cells: killing the mouse by removing the neck, dissecting and taking out the spleen, placing the spleen into complete RPMI-1640 culture solution, repeatedly washing and crushing the complete RPMI-1640 culture solution, filtering and sterilizing the mixture by using a filter membrane with the diameter of 0.22 mu m, centrifuging the mixture in a centrifuge for 5min at the rotating speed of 2000rpm at 4 ℃, discarding the supernatant, adding 3mL of Red blood cell lysis buffer to lyse Red blood cells, adding 3mL of complete RPMI-1640 culture solution after 5min to terminate the reaction, centrifuging the mixture for 5min at the rotating speed of 2000rpm at 4 ℃, discarding the supernatant, then adding the complete RPMI-1640 culture solution, centrifuging the mixture, repeatedly washing the mixture twice, and finally preparing the complete RPMI-1640 culture solution into the culture solution with the concentration range of 2 x 106cfu/mL of spleen cell suspension for use.
Determination of the rate of T cell and lipopolysaccharide LPS-induced proliferation of T cells induced by phaseolin a (con a) and B cells induced by chondroitin crocodile sulphate: respectively putting 100 mu L of spleen cell suspension into a 96-hole culture plate, respectively adding 95 mu L of culture medium containing crocodile chondroitin sulfate with different concentrations and 5 mu L of Con A/LPS, continuously culturing for 24h and 48h, then taking out, adding 5 mg/mL/20 mu L of MTT solution into each hole, placing the MTT solution into a CO2 incubator for reaction for 4h, taking out and centrifuging for 5min, rotating the speed of 2000rpm, discarding supernatant at 4 ℃, respectively adding 150 mu L of DMSO into each hole, fully dissolving at 37 ℃, measuring absorbance by using a microplate reader, measuring the wavelength at 570nm, repeating the experiment for three times, and taking the average value to draw a concentration effect chart.
This experiment investigated the proliferative response of mouse B lymphocytes to the stimulation of their mitogen LPS in an in vitro environment containing a solution of crocodile chondroitin sulfate. As shown in the results of fig. 13, the experimental group with low, medium and high concentrations, to which LPS and crocodile chondroitin sulfate were simultaneously added, had a significant increase in the level of B lymphocyte proliferation, and had a dose effect with the concentration of crocodile chondroitin sulfate, compared to the blank group; compared with the experimental group, the positive control group only added with LPS has a slightly lower T lymphocyte proliferation level than the experimental group, which indicates that the crocodile chondroitin sulfate has a certain promotion effect on the cellular immunity in the immune system of the mouse.
Although the embodiments disclosed in the present application are described above, the descriptions are only for the convenience of understanding the present application, and are not intended to limit the present application. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the appended claims.

Claims (12)

1. Crocodile chondroitin sulfate is extracted from crocodile cartilage and comprises 20-30% of D-glucuronic acid, 45-55% of hexosamine and 20-30% of potassium sulfate by mass,
wherein the crocodile chondroitin sulfate is prepared by a process comprising the steps of:
(1) heating alcohol solvent and crocodile cartilage, drying, and pulverizing;
(2) alkali extracting the crocodile cartilage powder crushed in the step (1) by using a sodium hydroxide solution, centrifuging, adjusting the pH of the obtained supernatant to 6.8-7.2 by using hydrochloric acid, adding ethanol, and precipitating overnight;
(3) centrifuging the ethanol precipitation liquid in the step (2), removing the upper layer liquid, and vacuum-drying the lower layer precipitate to obtain a crocodile chondroitin sulfate crude product;
(4) preparing the crocodile chondroitin sulfate crude product into aqueous solution, separating and purifying by using a Q-Sepharose-F-F ion exchange chromatography column, collecting eluent 1 at an elution peak, dialyzing by using a dialysis bag to obtain dialysate 1, and performing rotary evaporation to obtain concentrated solution 1, wherein the initial buffer is a Tris-HCl buffer with pH8.0 and 0mol/L NaCl, and the eluent is a Tris-HCl buffer with pH8.0 and 1mol/L NaCl;
(5) separating and purifying the concentrated solution 1 by Sephadex G-200 column chromatography, eluting by Tris-HCl eluent containing 0.1mol/L NaCl, collecting eluent 2 at an elution peak, dialyzing by a dialysis bag to obtain dialysate 2, and performing rotary evaporation to obtain concentrated solution 2; and (3) freeze-drying the concentrated solution 2 to obtain the crocodile chondroitin sulfate.
2. The crocodile chondroitin sulphate of claim 1 wherein the composition, in mass fraction, is 23-27% D-glucuronic acid, 48-52% hexosamine and 22-28% potassium sulphate.
3. The crocodile chondroitin sulfate of claim 1 wherein the crocodile chondroitin sulfate comprises, in mass fractions, 26% D-glucuronic acid, 50% hexosamine, and 24% potassium sulfate.
4. A method of preparing crocodile chondroitin sulfate as claimed in any one of claims 1 to 3, comprising:
(1) heating alcohol solvent and crocodile cartilage, drying, and pulverizing;
(2) alkali extracting the crocodile cartilage powder crushed in the step (1) by using a sodium hydroxide solution, centrifuging, adjusting the pH of the obtained supernatant to 6.8-7.2 by using hydrochloric acid, adding ethanol, and precipitating overnight;
(3) centrifuging the ethanol precipitation liquid in the step (2), removing the upper layer liquid, and vacuum-drying the lower layer precipitate to obtain a crocodile chondroitin sulfate crude product;
(4) preparing the crocodile chondroitin sulfate crude product into aqueous solution, separating and purifying by using a Q-Sepharose-F-F ion exchange chromatography column, collecting eluent 1 at an elution peak, dialyzing by using a dialysis bag to obtain dialysate 1, and performing rotary evaporation to obtain concentrated solution 1, wherein the initial buffer is a Tris-HCl buffer with pH8.0 and 0mol/L NaCl, and the eluent is a Tris-HCl buffer with pH8.0 and 1mol/L NaCl;
(5) separating and purifying the concentrated solution 1 by Sephadex G-200 column chromatography, eluting by Tris-HCl eluent containing 0.1mol/L NaCl, collecting eluent 2 at an elution peak, dialyzing by a dialysis bag to obtain dialysate 2, and performing rotary evaporation to obtain concentrated solution 2; and (3) freeze-drying the concentrated solution 2 to obtain the crocodile chondroitin sulfate.
5. The production method according to claim 4, wherein in the step (2), the mass fraction of the sodium hydroxide solution is 2% to 10%.
6. The preparation method according to claim 4, wherein in the step (2), the mass ratio of the crocodile cartilage powder to the sodium hydroxide solution is 1:6-1: 14.
7. The preparation method of claim 4, wherein in the step (2), the crocodile cartilage powder is subjected to alkali extraction in a water bath at 45 ℃ ± 5 ℃ for 2.5-3.5 h.
8. The production method according to claim 4, wherein, in the step (2), the alkali extraction is performed at a temperature in the range of 30 ℃ to 50 ℃.
9. The preparation method according to claim 4, wherein in the step (2), the crocodile cartilage powder is alkali-extracted in water bath for 2-4 h.
10. A composition comprising the crocodile chondroitin sulfate of any of claims 1-3, further comprising a pharmaceutically acceptable additive.
11. Use of crocodile chondroitin sulfate according to any of claims 1-3 for the preparation of a medicament for alleviating fatigue.
12. Use of crocodile chondroitin sulfate according to any of claims 1-3 for the preparation of a medicament for enhancing immunity.
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