CN109320626B

CN109320626B - Aloe polysaccharide and preparation method and application thereof

Info

Publication number: CN109320626B
Application number: CN201811167205.3A
Authority: CN
Inventors: 伍曾利
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-10-08
Filing date: 2018-10-08
Publication date: 2020-06-23
Anticipated expiration: 2038-10-08
Also published as: CN109320626A

Abstract

The invention belongs to the technical field of plant extracts, and discloses aloe polysaccharide and a preparation method and application thereof. The aloe polysaccharide is prepared from fresh aloe leaves as a raw material by the processes of pretreatment, pulping, colloid milling, double-enzyme hydrolysis, solid-liquid separation, concentration, drying and the like. The extraction yield, the polysaccharide content and the viscosity of the aloe polysaccharide prepared by the preparation method are obviously higher than those of the prior art. Experiments show that the aloe polysaccharide can enhance the immune function of the aloe polysaccharide, has the function of improving the nonspecific phagocytic function of a mouse reticuloendothelial system, and has obvious effect of resisting mouse SI₁₈₀Effect of A, either on solid tumors SI₁₈₀Whether A is ascites type SI₁₈₀A has strong antagonistic action. Therefore, the aloe polysaccharide can be used for preparing the anti-tumor and immunity-enhancing medicines.

Description

Aloe polysaccharide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of plant extracts, and particularly relates to aloe polysaccharide and a preparation method and application thereof.

Background

Aloe (Aloe vera) is a perennial evergreen fleshy herb plant of the genus Aloe of the family Liliaceae, distributed in tropical and subtropical regions, has over 600 varieties at present, is a widely used natural plant integrating edible, medicinal, cosmetic and ornamental functions, and has been used for detoxifying, treating constipation and the like for thousands of years in folks. Modern pharmacological experiments prove that the active ingredients in the aloe have more than ten pharmacological actions of sterilizing, diminishing inflammation, resisting cancer, promoting wound healing, enhancing the immunity of the organism and the like. Researches show that the aloe is rich in various bioactive substances and is widely applied to the fields of food, beauty treatment, health care, medicine and the like. Aloe is complex in chemical composition, and mainly contains anthraquinone substances, polysaccharides, amino acids, organic acids, vitamins, minerals and microelements. The aloe polysaccharide is the main bioactive substance in aloe gel, and has the effects of regulating immunity, resisting tumor, resisting radiation, resisting aging, inhibiting bacteria, diminishing inflammation, preventing and treating AIDS, and enhancing immunity.

At present, many methods for extracting aloe polysaccharides are reported at home and abroad, and the traditional methods for extracting aloe polysaccharides include a water extraction method, an acid extraction method, an alkali extraction method, an enzyme extraction method and the like. Chinese patent No. CN02159725.1 adopts pectinase enzymolysis, ultrafiltration and freeze-drying techniques to extract aloe polysaccharides, but the method has low yield, long processing time and high cost, and cannot be used for large-scale production. Chinese patent No. CN201010505190.4 adopts a technical means of pectinase enzymolysis and conventional drying to produce aloe polysaccharide, the content of the aloe polysaccharide is more than 15%, but the method also has the problems of low yield, high cost and the like in the extraction of the aloe polysaccharide. Chinese patent No. CN201410036579.7 adopts pectinase enzyme extraction technology, but the method has high aloe polysaccharide yield, but the aloe polysaccharide product has low viscosity and is unstable. Therefore, the existing extraction method of aloe polysaccharide generally has the defects of long time consumption, low yield, low viscosity and instability.

Disclosure of Invention

In view of the above, the present invention provides an aloe polysaccharide and a preparation method and application thereof, aiming at the problems of the prior art.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

a method for preparing Aloe polysaccharide comprises pretreating folium Aloe, pulping, colloid milling, adding cellulase and pectinase into the pulp for enzymolysis; carrying out solid-liquid separation after enzyme deactivation of the enzymatic hydrolysate, collecting liquid, heating, adding activated carbon for removing glycoside and decoloring, filtering by a plate frame, and collecting clear liquid; concentrating the clear liquid, and drying to obtain Aloe gel polysaccharide.

Wherein the pretreatment is to clean and disinfect fresh leaves of freshly picked aloe.

Preferably, the fresh aloe leaves are fresh and plump fresh aloe leaves for more than two years.

Preferably, the pretreatment is to wash fresh aloe leaves with water and soak the fresh aloe leaves in 0.1 percent sodium chlorate solution for 1 hour for disinfection.

Preferably, the beating is double-pass beating.

Further, the preparation method provided by the invention is used for grinding treatment by a colloid mill after pulping. Aloe polysaccharide exists in aloe leaf tissue cell, and is difficult to dissolve out, and the mechanical action of colloid mill can accelerate the dissolution of intracellular solute.

The preparation method of the invention adds cellulase and pectinase into the pulp for double enzymolysis. The cellulase can break cell wall of Aloe to facilitate dissolution of Aloe polysaccharide. Pectinase is easy for later processing by destroying aloe gel.

Preferably, the addition amount of the cellulase is 0.2-2.0 per mill of the aloe juice, and the addition amount of the pectinase is 0.2-1.0 per mill of the aloe juice. In some embodiments, the cellulase is added in an amount of 0.2% and the pectinase is added in an amount of 0.3% of the aloe vera slurry.

Preferably, the enzymolysis temperature is 50 +/-5 ℃, and the enzymolysis time is 20-60 min. In some embodiments, the enzymatic time is 30 min.

The preparation method of the invention also comprises the step of inactivating enzyme after the enzymolysis is finished. In some embodiments, the enzyme inactivation is specifically enzyme inactivation by heating to above 90 ℃ for 5-10 min. In some embodiments, the inactivating is at 90 ℃ for 5 min.

Further, the preparation method of the invention carries out solid-liquid separation on the enzymolysis liquid after enzymolysis, collects the separated liquid, adds active carbon to heat for carrying out the glucoside removal and the decoloration treatment.

In some embodiments, the solid-liquid separation is separation via a horizontal centrifuge.

Preferably, the adding amount of the activated carbon is 0.5-1.0% of the weight of collected liquid after solid-liquid separation; the heating is carried out to 70-80 ℃; the time for removing glycoside and decolorizing is 20-60 min. In some embodiments, the collected liquid is heated to 70 deg.C, 0.63% activated carbon is added to maintain 70 deg.C, and stirring is carried out for 30min to remove glycoside and decolorize.

The preparation method comprises the steps of carrying out plate-frame filtration on enzymatic hydrolysate after the enzymatic hydrolysate is subjected to glycoside removal and decoloration by activated carbon, and then collecting clear liquid, concentrating and drying.

Preferably, the concentration is carried out by performing first concentration by using a vacuum concentrator, performing ultrafiltration by using an ultrafiltration membrane of 100000 daltons, collecting the trapped fluid, and performing second concentration by using the vacuum concentrator. Wherein the ultrafiltration after the first concentration ensures the molecular weight and viscosity of the aloe polysaccharides.

In some embodiments, the first concentration temperature is below 70 ℃ and is concentrated to a feed BX value of 20 ° to 30 °; the second concentration temperature is below 70 ℃, and the concentration is carried out until the BX value is above 40 degrees.

In some embodiments, the drying is vacuum belt dryer drying at 60 ℃ for 60 min.

In some embodiments, the method of preparation further comprises the step of sieving after pulverization after drying. In some embodiments, the post-crushing sieving is a 100-mesh and 140-mesh sieve after crushing,

the invention also provides the aloe polysaccharide prepared by the preparation method.

In one embodiment, the invention adopts an immune hypofunction model to detect the influence of the aloe polysaccharide on the organ weight and the phagocytic function of mononuclear macrophages, and the result shows that the aloe polysaccharide can enhance the immune function and has the effect of improving the nonspecific phagocytic function of the reticuloendothelial system of mice. Therefore, the invention provides the application of the aloe polysaccharide in preparing the medicine for enhancing the immunity.

In one embodiment, the present invention contemplates the aloe polysaccharides of the present invention on SI in mice₁₈₀A, the results show that aloe polysaccharide has significant SI resistance in mice₁₈₀Effect of A, either on solid tumors SI₁₈₀Whether A is ascites type SI₁₈₀A has strong antagonistic action. Therefore, the invention provides the application of the aloe polysaccharide in preparing anti-tumor drugs.

According to the technical scheme, the invention provides aloe polysaccharide and a preparation method and application thereof. The aloe polysaccharide is prepared from fresh aloe leaves as a raw material by the processes of pretreatment, pulping, colloid milling, double-enzyme hydrolysis, solid-liquid separation, concentration, drying and the like. The extraction yield, the polysaccharide content and the viscosity of the aloe polysaccharide prepared by the preparation method are obviously higher than those of the prior art. Experiments show that the aloe polysaccharide can enhance the immune function of the aloe polysaccharide, has the function of improving the nonspecific phagocytic function of a mouse reticuloendothelial system, and has obvious effect of resisting mouse SI₁₈₀Effect of A, either on solid tumors SI₁₈₀Whether A is ascites type SI₁₈₀A has strong antagonistic action. Therefore, the aloe polysaccharide can be used for preparing the anti-tumor and immunity-enhancing medicines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a schematic diagram of a viscometer of the Ubbelohde capillary type, FIG. 1. the main tube; 2. a wide tube; 3. a side tube; 4. bending the pipe;

A. measuring the ball; B. a reservoir; C. a buffer ball; D. hanging a horizontal reservoir; E. a capillary tube; m is₁、m₂An annular measurement line.

Detailed Description

The invention discloses aloe polysaccharide and a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, all the raw materials or auxiliary materials used in the examples of the present invention are commercially available products, and are commercially available.

Example 1:

picking 1 ton fresh and plump aloe leaves for more than two years, cleaning the fresh and plump aloe leaves by tap water, soaking the fresh and plump aloe leaves in 0.1 percent sodium chlorate solution for 1 hour, and airing the sodium chlorate solution for later use;

pulping fresh folium Aloe with double-channel pulping machine, grinding with colloid mill, heating the pulp to 50 deg.C, adding 200g cellulase and 300g pectinase for enzymolysis at 50 + -5 deg.C for 30min, inactivating enzyme at 90 deg.C for 5 min;

carrying out solid-liquid separation on the enzymolysis liquid by a horizontal centrifuge to obtain 820kg of liquid;

heating the liquid to 70 deg.C, adding 5.2kg of activated carbon, maintaining at 70 deg.C, stirring for 30min, and filtering with plate frame to obtain 650kg of clear liquid (BX is 1.0 deg.C, pH is 4.42);

performing first concentration on the clear liquid by using a vacuum concentration unit to obtain 22kg of first concentrated solution (BX is 26 ℃ and pH is 4.31);

ultrafiltering the first concentrated solution with 100000 dalton ultrafiltration membrane to obtain 13kg of retentate (BX 23 deg.C, pH 4.28), and performing second concentration with vacuum concentration unit to obtain 5.4kg of second concentrated solution (BX 45 deg.C, pH 4.25);

drying the second concentrated solution with vacuum belt drier at 60 deg.C for 60min, and pulverizing with 100-mesh 140-mesh sieve to obtain 2.4kg of Aloe polysaccharide.

Example 2:

pulping fresh folium Aloe with double-channel pulping machine, grinding with colloid mill, heating the pulp to 55 deg.C, adding 180g cellulase and 180g pectinase for enzymolysis at 55 deg.C for 20min, inactivating enzyme at 90 deg.C for 8 min;

carrying out solid-liquid separation on the enzymolysis liquid by a horizontal centrifuge to obtain 800kg of liquid;

heating the liquid to 70 deg.C, adding 4.4kg of activated carbon, maintaining at 70 deg.C, stirring for 20min, and filtering with plate frame to obtain 620kg of clear liquid (BX 0.8 deg.C, pH 4.51);

subjecting the clear solution to first concentration by vacuum concentrator to obtain 21.5kg of first concentrated solution (BX 25 deg.C, pH 4.46);

ultrafiltering the first concentrated solution with 100000 dalton ultrafiltration membrane to obtain 12.2kg of retentate (BX 22.6 deg.C, pH 4.42), and concentrating by vacuum concentration unit to obtain 4.8kg of second concentrated solution (BX 50 deg.C, pH 4.38);

drying the second concentrated solution with vacuum belt drier at 60 deg.C for 60min, and pulverizing with 100-mesh and 140-mesh sieve to obtain 2.1kg of Aloe polysaccharide.

Example 3:

pulping fresh folium Aloe with double-channel pulping machine, grinding with colloid mill, heating the pulp to 55 deg.C, adding 1800g cellulase and 900g pectinase for enzymolysis at 55 deg.C for 60min, inactivating enzyme at 90 deg.C for 5 min;

carrying out solid-liquid separation on the enzymolysis liquid by a horizontal centrifuge to obtain 830kg of liquid;

heating the liquid to 70 deg.C, adding 8.3kg of activated carbon, maintaining at 70 deg.C, stirring for 60min, and filtering with plate frame to obtain 630kg of clear liquid (BX is 1.2 deg.C, pH is 4.38);

performing first concentration on the clear liquid by using a vacuum concentration unit to obtain 24kg of first concentrated solution (BX is 27 ℃, and the pH is 4.32);

ultrafiltering the first concentrated solution with 100000 dalton ultrafiltration membrane to obtain retentate 18kg (BX 25 deg.C, pH 4.29), and concentrating by vacuum concentration unit to obtain second concentrated solution 6.8kg (BX 55 deg.C, pH 4.26);

drying the second concentrated solution with vacuum belt drier at 60 deg.C for 60min, and pulverizing with 100-mesh 140-mesh sieve to obtain aloe polysaccharide 3.2 kg.

Example 4:

pulping fresh folium Aloe with double-channel pulping machine, grinding with colloid mill, heating the pulp to 50 deg.C, adding 900g cellulase and 450g pectinase for enzymolysis at 50 deg.C for 30min, inactivating enzyme at 90 deg.C for 5 min;

performing solid-liquid separation on the enzymolysis solution by using a horizontal centrifuge to obtain 840kg of liquid;

heating the liquid to 70 deg.C, adding 6.3kg of activated carbon, maintaining at 70 deg.C, stirring for 30min, and filtering with plate frame to obtain 680kg of clear liquid (BX is 1.0 deg.C, pH is 4.56);

subjecting the clear solution to first concentration by vacuum concentrator to obtain 23.5kg of first concentrated solution (BX 25 deg.C, pH 4.52);

ultrafiltering the first concentrated solution with 100000 dalton ultrafiltration membrane to obtain 20.4kg of retentate (BX 23 deg.C, pH 4.49), and concentrating by vacuum concentration unit to obtain 8.5kg of second concentrated solution (BX 45 deg.C, pH 4.45);

drying the second concentrated solution with vacuum belt drier at 60 deg.C for 60min, and pulverizing with 100-mesh and 140-mesh sieve to obtain aloe polysaccharide 3.1 kg.

Detection example:

the results of examining the aloe polysaccharides obtained in example 1 and example 1 with application numbers CN02159725.1, CN201010505190.4, example 1 and CN201410036579.7, respectively, for extraction rate, polysaccharide content and viscosity are shown in table 1.

Wherein the calculation formula of the extraction rate of the aloe polysaccharide is as follows:

the extraction rate of aloe polysaccharide is the total amount of actually extracted aloe polysaccharide/total amount of aloe polysaccharide (obtained by measuring the content of aloe polysaccharide after peeling by phenol-sulfuric acid method).

The detection method of the polysaccharide content comprises the following steps:

the measurement is carried out by an ultraviolet-visible spectrophotometry (the general rule of the four parts of the Chinese pharmacopoeia 2015 edition is less than the general rule of 0401).

Preparation of control solutions: a measuring flask of 20mg to 100mL of mannose dried to a constant mass at 105 ℃ is precisely weighed, and the volume is fixed by water for later use as a reference solution.

Preparation of a test solution: dissolving 10g of test sample in 30ml of water, adding 45ml of 90% ethanol, filtering the precipitate to obtain a clear solution, adding 230ml of 95% ethanol, filtering to obtain a precipitate, and drying at 105 ℃ to constant weight M0 g. Precisely measuring 0.06g of precipitate with constant weight, adding water to dissolve, transferring into a 100mL measuring flask, and fixing the volume to the scale to obtain a test solution.

The determination method comprises the following steps: respectively taking 2mL of each of the test solution and the reference solution, placing the test solution and the reference solution in a test tube with a plug, then adding 1.0mL of 5% phenol, shaking uniformly, adding 5.0mL of concentrated sulfuric acid, shaking vigorously and mixing uniformly, immediately placing in a boiling water bath for heating, timing by shaking automatically, reacting for 20min accurately, moving the test tube to an ice bath for cooling, and then placing at room temperature. Determining absorbance (A) at 490nm by the method, calculating polysaccharide content in water with the same color development operation as blank, and calculating polysaccharide content of the product not less than 8.5% in terms of mannose.

The viscosity was measured as follows:

inspection is performed according to (Chinese pharmacopoeia 2015 edition four parts < general rule 0633 >; viscometry).

The intrinsic viscosity should be above 20 ml/g.

Taking about 0.5g of a test sample, precisely weighing, placing in a conical flask with a plug, precisely adding 100ml of water, and shaking to dissolve to obtain a test sample solution; filtering with No. 3 vertical melting glass funnel, discarding the primary filtrate (1ml), taking the continuous filtrate (not less than 7ml), injecting into B along the inner wall of tube 2 of clean and dry Ubbelohde capillary viscometer (figure 1), vertically fixing the viscometer in a constant temperature water bath, making the liquid level of the water bath higher than the middle part of ball C, standing for 15 minutes, connecting tube ports 1 and 3 with a latex tube respectively, clamping the rubber tube of tube port 3, exhausting air from tube port 1, making the liquid level of the sample solution rise to the middle part of ball C slowly, opening tube port 3, opening tube port 1 again, making the sample solution fall down naturally in the tube, accurately recording the liquid level with stopwatch, and determining line m₁Is lowered to the measuring line m₂The time of outflow of (c). The measurement was repeated 2 times without reloading the sample, the difference between the flow times of the two measurements not exceeding. + -. 0.5% of the mean value. The average of the two times was taken as the flow-out time (T) of the test solution. Let-off No. 3 plumbThe solvent filtered by the molten glass funnel is operated in the same way, the measurement is repeated for 2 times, the measured values of the two times are the same, and the average value is the outflow time (T) of the solvent₀)。

The intrinsic viscosity is calculated as follows:

characteristic viscosity number

Formula (III) η_rIs T/T₀(ii) a c is the concentration of the test solution, g/ml.

Table 1: the Aloe polysaccharide obtained by the above methods has detection results

The results in Table 1 show that the aloe polysaccharide products obtained in examples 1-4 of the present invention have higher extraction yield, polysaccharide content and viscosity than the products obtained in the examples of the prior 3 patent methods, wherein the polysaccharide content is as high as 38.3%, which is 1 times higher than the products of the prior patents.

Test example 1: test for enhancing immunity function of active aloe polysaccharide

1. Test materials

1.1 test samples

Aloe polysaccharide prepared by the processes of inventive example 1 and comparative examples (application numbers CN02159725.1, CN201010505190.4 and CN201410036579.7) was used as a test sample.

1.2 test dose

The amount of the test sample used in example 1 and the comparative example was 2.4g/d, and the average amount was 40 mg/(kg. d) in terms of 60kg of adult body weight, i.e., the amount of the test sample was 40 mg/(kg. d).

In example 1, the test samples were set to 40, 80 and 160 mg/(kg. d) in the low, medium and high 3 dose groups.

1.3 Experimental animals

Kunming mice, 18-22 g, provided by the Experimental animal center of Guangzhou Chinese medicine university, are normally bred for 3 days and then are tested.

1.4 Main instruments and reagents

UV-2501 ultraviolet-visible spectrophotometer (Shimadzu, Japan); BS110S electronic balance (Sartorius corporation); XZC-5A mouse diving tower instrument (jiangsu seons biotechnology limited); d-galactose; a SOD reagent; an MDA reagent; coomassie brilliant blue reagent; cyclophosphamide is used for injection.

2. Test method

2.1 establishment of immune hypofunction model and group administration

108 Kunming mice, each half of which is male and female, were randomly divided into a blank control group, a negative control group, a high, medium and low dose group of example 1, a comparative example 1 group (application No. CN02159725.1 patent), a comparative example 2 group (application No. CN201010505190.4 patent), and a comparative example 3 group (application No. CN201410036579.7 patent), each group consisting of 12 mice. Except for the blank control group, the other groups of mice were injected with cyclophosphamide injection at 50.0mg/kg in the abdominal cavity on days 1, 3 and 5. And (3) performing modeling, and simultaneously performing intragastric administration on the test sample according to the corresponding dose, performing intragastric administration on the blank control group and the negative control group for 1 time every day by using distilled water with the corresponding volume, and continuously performing intragastric administration for 14 days.

2.2 Observation index

1h after the last administration, the mice were weighed, 25% Chinese ink physiological saline solution was injected into the tail vein of 10.0mL/kg, 2 minutes and 12 minutes after the injection of the Chinese ink, 2-3 drops of blood were collected from the retroorbital venous plexus of the mice by using a capillary glass tube, and 20.0. mu.l of the blood was dissolved in 2.0mL of 0.1% Na₂CO₃In the solution, the mixture was shaken up, and the absorbance at 680nm was measured under 721 type spectrophotometry, and the phagocytosis index K was calculated according to the following formula. And (5) dissecting spleen and thymus at the same time, and calculating thymus and spleen coefficients.

The experimental data adopts a one-factor variance analysis for comparison among multiple groups, a t-test analysis method for experimental data, and differences among the groups are compared, and the data are expressed by x +/-s.

3. Results

The influence of aloe polysaccharides on organ weight and phagocytic function of mononuclear macrophages was analyzed by comparison, and the results are shown in Table 2.

TABLE 2 Effect of Aloe polysaccharides on immunocompromised mice (x. + -. s)

Note: compared with the blank control group, the composition of the composition,^*P<0.01; compared with the negative control group, the test results show that,^##P<0.01，^#P<0.05。

the results show that the thymus coefficient, spleen coefficient and carbon clearance index K of the mice in the negative control group are obviously reduced compared with the blank group (P < 0.01); moreover, each dose group of the aloe polysaccharide test sample prepared in example 1 can obviously improve spleen coefficient and carbon particle clearance index (P <0.01 or 0.05) of mice with low immunity caused by cyclophosphamide, and the high and medium dose groups can obviously improve thymus coefficient (P <0.01 or 0.05) of mice with low immunity, which suggests that the aloe polysaccharide test sample prepared in example 1 can enhance the immune function and has the function of improving nonspecific phagocytic function of reticuloendothelial system of mice.

Under the experimental condition that cyclophosphamide forms immunosuppression on mice, the carbon clearance index of each dose group of mice of the aloe polysaccharide test sample prepared in example 1 is obviously higher than that of a negative control group, namely the mononuclear phagocyte function is obviously enhanced, which indicates that the aloe polysaccharide test sample can enhance the nonspecific immune function of an organism. Meanwhile, the aloe polysaccharide test sample can increase the thymus coefficient and the spleen coefficient of a mouse.

The function of aloe polysaccharide was evaluated from immunomodulation, and it was demonstrated that aloe polysaccharide could play a role in enhancing immune system function.

The results of the tests on the immunity enhancing function of the aloe polysaccharides prepared in examples 2 to 4 of the present invention were similar to the aloe polysaccharides prepared in example 1, and the aloe polysaccharides prepared in examples 2 to 4 were all able to significantly improve the spleen coefficient, the carbon clearance index and the thymus coefficient (P <0.01 or 0.05) of mice with hypoimmunity caused by cyclophosphamide, which suggests that the aloe polysaccharide test samples prepared in examples 2 to 4 could enhance the immune function and have the effect of enhancing the nonspecific phagocytic function of the reticuloendothelial system of mice.

Test example 2: anti-tumor function test of aloe active polysaccharide

1. Test drugs:

sample preparation: aloe polysaccharide prepared by the processes of inventive example 1 and comparative examples (application numbers CN02159725.1, CN201010505190.4 and CN201410036579.7) was used as a test sample.

Comparison products: cyclophosphamide.

2. Animal(s) production

NIH₆₁₅Pure mouse.

3. Tumor strain

SI₁₈₀Mice a were provided by the institute for blood, chinese medical science.

4. Experimental methods and results

Aloe polysaccharide to mouse S₁₈₀Influence of (2)

Taking NIH pure line mice with weight of 20 +/-2 g and dual purposes of male and female, inoculating according to the method of the national anti-tumor medicament in-vivo screening regulation, and inoculating SI with good tumor growth in 6-9 days₁₈₀A, ascites is taken out, and diluted with sterilized normal saline in a ratio of l to 4 to prepare tumor cell suspension (5 × 10)⁶Cells/ml). Respectively in an amount of 0.2ml_RInoculating to the right axilla of the mouse or inoculating to the abdominal cavity of the mouse.

After 24h, randomly dividing the test sample into a blank control group, a cyclophosphamide control group, a sample group of the invention example 1, a sample group of the comparative example 1 (CN02159725.1 patent example 1 sample), a sample group of the comparative example 2 (CN201010505190.4 patent example 1 sample) and a sample group of the comparative example 3 (CN201410036579.7 patent example sample), wherein 10 samples in each group are respectively administrated by intraperitoneal injection or subcutaneous injection once a day, the dose of aloe polysaccharide is 60mg/kg, the dose of cyclophosphamide is 60mg/kg, the control group is administrated by physiological saline with the same volume for 10d continuously, mice inoculated with subcutaneous injection are killed the next day after drug withdrawal, tumor blocks are taken out and weighed, and the tumor inhibition rate is calculated. The number of days of life was observed after the mice inoculated with the abdominal cavity had stopped taking the drug, and the life extension rate was calculated. The results are shown in tables 3 and 4 below.

Table 3: aloe polysaccharide docking mouse SI under seed coat₁₈₀Influence of A

Note: compared with the blank control group, the composition of the composition,^*P<0.01。

table 4: aloe polysaccharide to mice SI inoculated with abdominal cavity₁₈₀Influence of A

as can be seen from the above test results, aloe polysaccharides have significant SI resistance against mice₁₈₀Effect of A, either on solid tumors SI₁₈₀Whether A is ascites type SI₁₈₀A has strong antagonistic action and better effect than the comparative product.

The aloe polysaccharides obtained in examples 2 to 4 of the present invention were tested for their antitumor activity according to the above-mentioned method, and the results were similar to those of example 1, and all of the aloe polysaccharides obtained in examples 2 to 4 had a significant SI resistance against mice₁₈₀Effect of A, either on solid tumors SI₁₈₀Whether A is ascites type SI₁₈₀A has strong antagonistic action and better effect than the comparative product.

Claims

1. A method for preparing Aloe polysaccharide comprises pretreating folium Aloe, pulping, colloid milling, adding cellulase and pectinase into the pulp for enzymolysis; carrying out solid-liquid separation after enzyme deactivation of the enzymatic hydrolysate, collecting liquid, heating, adding activated carbon for removing glycoside and decoloring, filtering by a plate frame, and collecting clear liquid; concentrating the clear liquid, and drying to obtain aloe gel polysaccharide; the concentration is specifically that ultrafiltration is carried out by an ultrafiltration membrane of 100000 daltons after first concentration is carried out by a vacuum concentrator, and second concentration is carried out by the vacuum concentrator after trapped fluid is collected; the first concentration temperature is below 70 ℃, and the concentration is carried out until the BX value of the feed liquid is 20-30 degrees.

2. The preparation method of claim 1, wherein the pretreatment is water cleaning, and disinfection is carried out by soaking in 0.1% sodium chlorate solution for 1 hour.

3. The method of manufacturing of claim 1, said beating being a two pass beater beating.

4. The preparation method according to claim 1, wherein the cellulase is added in an amount of 0.2-2.0 ‰ of aloe vera serum, and the pectinase is added in an amount of 0.2-1.0 ‰; the enzymolysis temperature is 50 +/-5 ℃, and the enzymolysis time is 20-60 min.

5. The production method according to claim 1, wherein the solid-liquid separation is separation by a horizontal centrifuge; the heating is carried out to 70-80 ℃; the adding amount of the activated carbon is 0.5-1.0% of the weight of the collected liquid after solid-liquid separation; the time for removing glycoside and decolorizing is 20-60 min.

6. The method according to claim 5, wherein the second concentration temperature is 70 ℃ or lower and the concentration is carried out until the BX value is 40 ° or higher.

7. The method as claimed in claim 1, wherein the drying is carried out by a vacuum belt dryer at 60 ℃ for 60min, and the powder is crushed through a 100-mesh and 140-mesh screen.

8. Aloe polysaccharide obtainable by a process according to any one of claims 1 to 7.

9. The use of aloe polysaccharides according to claim 8 for the preparation of a medicament for anti-tumour and enhancing immunity.