An immunopotentiator containing low molecular weight glucuronoxylomannan

Abstract

The present application relates to the technical field of biological medicine, disclose a kind of containing low molecular weight glucuronan-xylan immunopotentiator.The immunopotentiator described in the present application uses low molecular weight glucuronan-xylan as active substance, wherein the average molecular weight of low molecular weight glucuronan-xylan is 0.7-0.9kDa.Compared with the existing glucuronan-xylan, the present application has a lower molecular weight and a narrower molecular weight range, which makes the immunopotentiator containing low molecular weight glucuronan-xylan prepared by the present application have good solubility and smaller viscosity, so that glucuronan-xylan can be better absorbed by immune cells and better reflect the effect of improving immunity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an immune enhancer containing low molecular weight glucuronic acid-xylammonan. Background Technology

[0002] Tremella, a fungus belonging to the genus Tremella in the family Tremellaceae, has the effects of nourishing yin and moistening the lungs, clearing heat and benefiting the stomach, replenishing qi and blood, strengthening the heart and body, improving immunity, and fighting cancer. Modern medical clinical verification has shown that the active polysaccharides in Tremella can enhance human immunity, anti-aging, anti-thrombosis, and anti-mutation effects.

[0003] Conventionally extracted Tremella polysaccharides are mostly macromolecular structures with high viscosity, resulting in low extraction rates and difficulty in absorption and biological activity. Therefore, how to extract low molecular weight Tremella polysaccharides has become a technical problem that urgently needs to be solved by those skilled in the art.

[0004] In existing technologies, acid hydrolysis can effectively break the glycosidic bonds in polysaccharide molecules, thereby forming oligosaccharides. However, acid hydrolysis of polysaccharides is mostly carried out by strong acids, which lack selectivity and cannot maintain the basic structure of the original polysaccharide, resulting in significant structural damage. Moreover, the acid hydrolysis conditions and the quality of the depolymerization products are difficult to control. Therefore, improving acid hydrolysis to prepare low molecular weight Tremella fuciformis polysaccharides and applying them to the preparation of immune enhancers has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide an immune enhancer with glucuronic acid-xylammonan as the active substance, which can make the polysaccharide better absorbed by the body, thereby effectively exerting the immune activity of the polysaccharide.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] One of the technical solutions of this invention:

[0008] An immune enhancer containing low molecular weight glucuronic acid-xylamtan, wherein the immune enhancer uses low molecular weight glucuronic acid-xylamtan as the active substance;

[0009] The low molecular weight glucuronic acid-xylammonan has an average molecular weight of 0.7–0.9 kDa.

[0010] This invention uses glucuronic acid-xylammonan with an average molecular weight of 0.7-0.9 kDa as an immune-enhancing active substance. It has a low molecular weight and a narrow molecular weight range, has good solubility, and can be well absorbed by immune cells, thereby achieving an immune-enhancing effect.

[0011] Furthermore, the preparation method of the low molecular weight glucuronic acid-xylammonan includes the following steps:

[0012] Step 1: Wash the white fungus, dry it, and grind it with water to obtain white fungus paste;

[0013] Step 2: Decolorize the tremella slurry obtained in Step 1, and then sonicate the decolorized tremella slurry to obtain a crude extract of tremella polysaccharides.

[0014] Step 3: Mix the crude extract of Tremella polysaccharide obtained in Step 2 with acid and extract at low temperature to obtain Tremella polysaccharide extract.

[0015] Step 4: The tremella polysaccharide extract obtained in Step 3 is subjected to ion exchange chromatography with gradient elution of sodium chloride solution to obtain components TFP1 to TFP10.

[0016] Step 5: Combine components TFP3 and TFP7 obtained in Step 4 and perform ion exchange chromatography with gradient elution using ammonium bicarbonate solution to obtain components TFPS1 to TFPS3, wherein TFPS1 is the low molecular weight glucuronic acid-xylan.

[0017] Furthermore, in step one, the drying is vacuum freeze drying, specifically drying at a temperature of -10℃ to -20℃ and a pressure of 10 to 20 Pa until the moisture content is less than 20%.

[0018] Preferably, the vacuum freeze-drying temperature is -15°C and the pressure is 15Pa.

[0019] Furthermore, in step one, the water milling specifically involves mixing the dried tremella with water at a ratio of 1g:10-20mL and then grinding it at room temperature for 10-15 minutes.

[0020] As a preferred method, the white fungus and water are mixed at a ratio of 1g:15mL and then ground at room temperature for 10 minutes.

[0021] Furthermore, in step two, the decolorization process specifically involves adding activated carbon to the tremella slurry, and filtering out the activated carbon after the tremella slurry has no obvious color.

[0022] Furthermore, in step two, the ultrasonic treatment specifically involves ultrasonic treatment at a temperature of 30–60°C and an ultrasonic frequency of 1 MHz for 20–40 minutes.

[0023] Furthermore, in step three, the acid is a mixture of 15% hydrochloric acid and 35% acetic acid at a volume ratio of 1:2.

[0024] Furthermore, in step three, the volume ratio of the crude extract of Tremella fuciformis polysaccharide to acid is 1:(3-9); preferably, the volume ratio is 1:6.

[0025] Furthermore, in step three, the low-temperature extraction specifically refers to extraction at a temperature of 10–20°C for 1–3 hours;

[0026] Preferably, the optimal low-temperature extraction conditions are extraction at 15°C for 2 hours;

[0027] Furthermore, in step four, the ion exchange chromatography specifically involves chromatographic fractionation using a DEAE-Bi o Ge l Agarose FF ion column;

[0028] Furthermore, in step four, the gradient elution with sodium chloride solution specifically involves eluting with sodium chloride solutions of concentrations of 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, and 1M in sequence.

[0029] Furthermore, in step five, the ion exchange chromatography specifically involves chromatographic fractionation using a DEAE-Bi o Ge l Agarose FF ion column;

[0030] Furthermore, in step five, the gradient elution of the ammonium bicarbonate solution specifically involves eluting with sodium bicarbonate solutions of concentrations of 0.3M, 0.6M, and 0.9M in sequence.

[0031] This invention utilizes ultrasound and acid to extract polysaccharides from Tremella fuciformis. Ultrasound effectively extracts polysaccharides from Tremella fuciformis, while acid breaks glycosidic bonds, causing high molecular weight polysaccharides to degrade into oligosaccharides, thereby obtaining glucuronic acid-xylo-glycan with an average molecular weight of 0.7-0.9 kDa.

[0032] This invention uses mixed acids as the extraction acid. By combining strong and weak acids, it can effectively break glycosidic bonds (mainly through the strong acid) to form oligosaccharides without excessively damaging them (because the weak acid cannot completely ionize, it can provide a certain amount of active hydrogen to the strong acid, and at the same time stabilize the total active hydrogen content in the acid system through ionization balance, reducing the excessive damage to glycosidic bonds). This achieves technical effects that a single acid cannot achieve, thereby increasing the content of low molecular weight glucuronic acid-xylo-glycan and ultimately enhancing immune capabilities.

[0033] In this invention, the pretreatment of Tremella fuciformis (i.e., step one) and the extraction of Tremella fuciformis polysaccharides (steps two and three) are both controlled at relatively low temperatures. This is because excessively high temperatures will cause Tremella fuciformis polysaccharides to become inactive, and lower temperatures are conducive to the degradation of polysaccharides into oligosaccharides, while higher temperatures make it difficult to achieve the formation of glucuronic acid-xylammonan with an average molecular weight of 0.7-0.9 kDa.

[0034] The second technical solution of this invention:

[0035] The above-mentioned immune enhancer containing low molecular weight glucuronic acid-xylammonan is used in the preparation of immune-enhancing drugs.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] The glucuronic acid-xylamtan prepared by this invention has an average molecular weight of 0.7-0.9 kDa, which is lower and narrower than existing glucuronic acid-xylamtan. This results in the immune enhancer containing low molecular weight glucuronic acid-xylamtan prepared by this invention having good solubility and low viscosity, thus enabling glucuronic acid-xylamtan to be better absorbed by immune cells and better exert its immune-enhancing effect. Attached Figure Description

[0038] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0039] Figure 1 This is a flowchart illustrating the preparation of low molecular weight glucuronic acid-xylammonan according to the present invention. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0041] In the following examples and comparative examples, the room temperature is 25±2℃.

[0042] Example 1

[0043] Preparation of low molecular weight glucuronic acid-xylammonan

[0044] 1) Wash 10g of white fungus with distilled water and dry it at a temperature of -15℃ and a pressure of 15Pa until the moisture content is less than 20%. Then take 1g of the dried white fungus and mix it with 15mL of water. Grind it at room temperature for 10min to obtain white fungus slurry.

[0045] 2) Add 0.5g of block activated carbon to the tremella slurry obtained in step 1), and filter out the activated carbon after the tremella slurry has no obvious color.

[0046] 3) The decolorized tremella slurry from step 2) was ultrasonically treated at 30°C and 1MHz for 40 minutes to obtain crude tremella polysaccharide extract.

[0047] 4) Mix 15 mL of crude extract of Tremella polysaccharide obtained in step 3) with 15 mL of hydrochloric acid with a mass fraction of 15% and 30 mL of acetic acid with a mass fraction of 35%, and extract at 15°C for 2 hours to obtain Tremella polysaccharide extract.

[0048] 5) The Tremella polysaccharide extract obtained in step 4) was loaded onto a DEAE-BioGel Agarose FF ion exchange column and eluted with sodium chloride solutions of concentrations of 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M and 1M to obtain components TFP1 to TFP10;

[0049] 6) Combine components TFP3 and TFP7 obtained in step 5), load them onto a DEAE-BioGel Agarose FF ion exchange column, and elute with sodium bicarbonate solutions of concentrations of 0.3M, 0.6M and 0.9M respectively to obtain components TFPS1 to TFPS3.

[0050] High performance liquid chromatography analysis of TFPS1 obtained in Example 1 showed that the polysaccharide in TFPS1 was polymerized from glucuronic acid, xylose and mannose as monosaccharides (i.e., the polysaccharide was glucuronic acid-xylo-glycan), with an average molecular weight of 0.897 kDa.

[0051] Analysis showed that the yield of low molecular weight glucuronic acid-xylammonan was 63.1%.

[0052] Example 2

[0053] Preparation of low molecular weight glucuronic acid-xylammonan

[0054] Same as Example 1, except that:

[0055] Step 3) is to sonicate the decolorized tremella slurry from step 2) at 50°C and 1MHz for 25 minutes to obtain a crude extract of tremella polysaccharides.

[0056] Step 4) involves mixing 15 mL of crude extract of Tremella fuciformis polysaccharide obtained in step 3) with 30 mL of 15% hydrochloric acid and 60 mL of 35% acetic acid, and extracting at 15°C for 2 hours to obtain Tremella fuciformis polysaccharide extract.

[0057] High performance liquid chromatography analysis of TFPS1 obtained in Example 2 showed that the polysaccharide in TFPS1 was polymerized from glucuronic acid, xylose and mannose as monosaccharides (i.e., the polysaccharide was glucuronic acid-xylo-glycan), with an average molecular weight of 0.702 kDa.

[0058] Analysis showed that the yield of low molecular weight glucuronic acid-xylammonan was 66.4%.

[0059] Example 3

[0060] Preparation of low molecular weight glucuronic acid-xylammonan

[0061] Same as Example 1, except that:

[0062] Step 3) is to sonicate the decolorized tremella slurry from step 2) at a temperature of 60℃ and an ultrasonic frequency of 1MHz for 20 minutes to obtain a crude extract of tremella polysaccharide.

[0063] Step 4) involves mixing 15 mL of crude extract of Tremella fuciformis polysaccharide obtained in step 3) with 45 mL of 15% hydrochloric acid and 90 mL of 35% acetic acid, and extracting at 15°C for 2 hours to obtain Tremella fuciformis polysaccharide extract.

[0064] High performance liquid chromatography analysis of TFPS1 obtained in Example 3 showed that the polysaccharide in TFPS1 was polymerized from glucuronic acid, xylose and mannose as monosaccharides (i.e., the polysaccharide is glucuronic acid-xylo-glycan), with an average molecular weight of 0.825 kDa.

[0065] Analysis showed that the yield of low molecular weight glucuronic acid-xylammonan was 64.7%.

[0066] Comparative Example 1

[0067] Preparation of glucuronic acid-xylammonan

[0068] Same as Example 2, except that:

[0069] Step 1) is as follows: Wash 10g of white fungus with distilled water and dry it at 50℃ until the water content is less than 20%. Then take 1g of the dried white fungus and mix it with 15mL of water. Grind it at room temperature for 10min to obtain white fungus slurry.

[0070] High performance liquid chromatography analysis revealed that the polysaccharide in TFPS1 was glucuronic acid-xylamtan, with an average molecular weight of 0.711 kDa, and the yield of low molecular weight glucuronic acid-xylamtan was 58.7%.

[0071] Comparative Example 2

[0072] Preparation of glucuronic acid-xylammonan

[0073] Same as Example 2, except that in step 3), the temperature of ultrasonic treatment is 90°C.

[0074] High performance liquid chromatography analysis revealed that the polysaccharide in TFPS1 was glucuronic acid-xylamtan, with an average molecular weight of 0.698 kDa, and the yield of low molecular weight glucuronic acid-xylamtan was 60.3%.

[0075] Comparative Example 3

[0076] Preparation of glucuronic acid-xylammonan

[0077] Same as Example 2, except that in step 4), the extraction temperature is 40°C.

[0078] High performance liquid chromatography analysis revealed that the polysaccharide in TFPS1 was glucuronic acid-xylamtan, with an average molecular weight of 0.705 kDa, and the yield of low molecular weight glucuronic acid-xylamtan was 59.5%.

[0079] Comparing Comparative Examples 1-3 with Example 2, it can be found that increasing the pretreatment temperature and extraction temperature does not affect the average molecular weight of glucuronic acid-xylaman, but it can significantly reduce the yield of glucuronic acid-xylaman.

[0080] Comparative Example 4

[0081] Same as Example 2, except that step 3) is omitted, that is, the decolorized tremella slurry is directly subjected to acid extraction.

[0082] High performance liquid chromatography analysis revealed that the polysaccharide in TFPS1 was glucuronic acid-xylamtan, with an average molecular weight of 1.039 kDa, and the yield of low molecular weight glucuronic acid-xylamtan was 31.2%.

[0083] Comparative Example 5

[0084] Same as Example 2, except that step 4) is omitted, that is, the crude extract of Tremella polysaccharide is directly subjected to ion column chromatography.

[0085] High performance liquid chromatography analysis revealed that the polysaccharide in TFPS1 was glucuronic acid-xylamtan, with an average molecular weight of 1.589 kDa, and the yield of low molecular weight glucuronic acid-xylamtan was 27.6%.

[0086] Comparing Comparative Examples 4-5 with Example 2, it can be found that omitting ultrasonic extraction or acid extraction leads to an increase in molecular weight and a decrease in the yield of low molecular weight glucuronic acid-xylan. Therefore, the present invention utilizes a combined extraction method of ultrasonic extraction and acid extraction to obtain low molecular weight (0.7-0.9 kDa) glucuronic acid-xylan with a high yield (around 65%).

[0087] Comparative Example 6

[0088] Same as Example 2, except that step 4) is performed first and then step 3), that is, acid extraction is performed first and then ultrasonic extraction is performed.

[0089] High performance liquid chromatography analysis revealed that the polysaccharide in TFPS1 was glucuronic acid-xylamtan, with an average molecular weight of 0.726 kDa, and the yield of low molecular weight glucuronic acid-xylamtan was 30.5%.

[0090] Comparing Comparative Example 6 with Example 2, it can be found that although the molecular weight of polysaccharide did not change significantly after changing the order of ultrasonic extraction and acid extraction, the yield was greatly reduced. This is because acid extraction first causes many glycosidic bonds to break, thus destroying the polysaccharide structure. Therefore, ultrasound plays a role in protecting glycosidic bonds to a certain extent.

[0091] Immunity Enhancement Test:

[0092] (I) Adaptation culture of test mice

[0093] Fifty three-month-old ICR mice, weighing 20±2g, were randomly selected and acclimatized in the laboratory (temperature 20℃) with a complete feed for 7 days, with free access to water and no drugs.

[0094] (ii) Test substance

[0095] The low molecular weight glucuronic acid-xylo-glucan prepared in Example 2 (hereinafter referred to as low-GXM), the glucuronic acid-xylo-glucan prepared in Comparative Example 4 (hereinafter referred to as GXM-1), the glucuronic acid-xylo-glucan prepared in Comparative Example 5 (hereinafter referred to as GXM-2), and the glucuronic acid-xylo-glucan prepared in Comparative Example 6 (hereinafter referred to as GXM-3);

[0096] (III) Feeding Experiment

[0097] Fifty mice were randomly divided into five groups of 10 each. Four groups of mice were given 10 mL of different test substances by gavage daily, while one group was given 10 mL of distilled water as a control. The mice were given the test substances for 45 days. During this period, the mice had free access to food and water and were not given any drugs.

[0098] (iv) Determination of organ / body weight ratio

[0099] After the feeding experiment, five mice were randomly selected from each group, sacrificed by cervical dislocation, weighed, and the spleen and thymus were removed, washed with physiological saline, weighed, and the thymus / body weight ratio and spleen / body weight ratio were calculated.

[0100] The measurement results are shown in Table 1:

[0101] Table 1 Organ / Body Weight Ratio

[0102] Low-GXM GXM-1 GXM-2 GXM-3 control group Thymus / body weight ratio (%) 1.83 1.65 1.62 1.76 1.59 Spleen / body weight ratio (%) 2.72 2.41 2.38 2.57 2.31

[0103] As shown in Table 1, low-GXM, GXM-1, GXM-2, and GXM-3 can all improve the organ-to-body weight ratio to some extent. Among them, low-GXM can improve the organ-to-body weight ratio more effectively than GXM-1, GXM-2, and GXM-3, indicating that it has a better ability to improve immunity. GXM-1 and GXM-2 are not significantly different from the control group, indicating that they do not have a significant effect on improving immunity. GXM-3 is better than GXM-1 and GXM-2 and has a certain effect on improving immunity, but it is inferior to low-GXM.

[0104] (V) Assay of macrophage phagocytic function

[0105] 1. Three days before the end of the feeding experiment, five mice were randomly selected and placed in an isolation box. One mL of 6% soluble starch broth was injected into the abdominal cavity of each mouse.

[0106] 2. After the feeding experiment, each mouse was injected intraperitoneally with 0.5 mL of 1% chicken red blood cell suspension and the abdomen was gently massaged. 30 min after injection, the mice were sacrificed, peritoneal fluid was collected and smeared, and Wright staining was performed. The smears were then observed under an oil immersion microscope.

[0107] Under the microscope, the nuclei of phagocytes appear blue, while the cytoplasm of phagocytes appears red and the nuclei appear blue. Count 100 chicken red blood cells and calculate the phagocytosis rate.

[0108] Phagocytosis rate (%) = (Number of chicken red blood cells phagocytosed) / 100 × 100%

[0109] The measurement results are shown in Table 2:

[0110] Table 2. Measurement of phagocytic function

[0111] Low-GXM GXM-1 GXM-2 GXM-3 control group Phagocytosis rate (%) 88.57 75.69 73.81 79.5 71.37

[0112] As shown in Table 2, low-GXM, GXM-1, GXM-2, and GXM-3 can all improve macrophage phagocytic function to some extent. Among them, low-GXM can more effectively improve macrophage phagocytic function than GXM-1, GXM-2, and GXM-3, indicating that it has a better ability to improve immunity. GXM-1 and GXM-2 are not significantly different from the control group, indicating that their ability to improve immunity is relatively poor. GXM-3 is better than GXM-1 and GXM-2, and has a certain effect on improving immunity, but it is inferior to low-GXM.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An immunopotentiator comprising a low molecular weight glucuronoxylomannan, characterized in that, The immune enhancer uses low molecular weight glucuronic acid-xylamtan as the active substance; the average molecular weight of the low molecular weight glucuronic acid-xylamtan is 0.7-0.9 kDa; The preparation method of the low molecular weight glucuronic acid-xylammonan includes the following steps: Step 1: Wash the white fungus, dry it, and grind it with water to obtain a white fungus paste; the drying temperature is -10℃ to -20℃. Step 2: Decolorize the tremella slurry obtained in Step 1, and then sonicate the decolorized tremella slurry to obtain a crude extract of tremella polysaccharide; the sonication is performed at a temperature of 30-60℃ and an ultrasonic frequency of 1MHz for 20-40 minutes. Step 3: Mix the crude extract of Tremella polysaccharide obtained in Step 2 with acid and extract at low temperature (10-20°C) to obtain Tremella polysaccharide extract; the acid is a mixture of 15% hydrochloric acid and 35% acetic acid at a volume ratio of 1:2; the volume ratio of the crude extract of Tremella polysaccharide to the acid is 1:(3-9). Step four: The Tremella polysaccharide extract obtained in step three is subjected to ion exchange chromatography with gradient elution using sodium chloride solution to obtain components TFP1 to TFP10; the ion exchange chromatography is performed using a DEAE-BioGelAgarose FF ion column for chromatography fractionation, and the gradient elution using sodium chloride solution is performed sequentially with sodium chloride solutions of concentrations of 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, and 1M. Step 5: Combine components TFP3 and TFP7 obtained in Step 4 and perform ion exchange chromatography with gradient elution using ammonium bicarbonate solution to obtain TFPS1 to TFPS3, wherein TFPS1 is the low molecular weight glucuronic acid-xylo-glycan; the ion exchange chromatography is performed using a DEAE-BioGelAgarose FF ion column for chromatography fractionation, and the gradient elution using ammonium bicarbonate solution is performed by sequentially eluting with sodium bicarbonate solutions of concentrations of 0.3M, 0.6M, and 0.9M.

2. The application of the immunostimulant containing low molecular weight glucuronic acid-xylammonan as described in claim 1 in the preparation of immunostimulant drugs.

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