Bamboo red polysaccharide hb-0.1 with immunocompetence, extraction and purification method and application thereof

Abstract

The application discloses bamboo red polysaccharide HB-0.1 with immunocompetence, an extraction and purification method and application thereof. The bamboo red polysaccharide HB-0.1 is mainly composed of mannose, glucose and galactose, and the preparation steps are as follows: 1) crushing bamboo red fungus stipe, stirring with water, concentrating supernatant to obtain concentrated solution, adding anhydrous ethanol to the concentrated solution, alcohol precipitation at 4 DEG C overnight, centrifugation, water dialysis in a dialysis bag, and freeze-drying to obtain bamboo red fungus crude polysaccharide; 2) subjecting bamboo red fungus crude polysaccharide components to anion exchange column chromatography separation, collecting and concentrating elution components; subjecting the elution components to gel column purification, collecting eluent, and freeze-drying to obtain a powder sample of bamboo red polysaccharide HB-0.1. The bamboo red fungus is a medicinal fungus unique to bamboo forests, and the bamboo red polysaccharide HB-0.1 obtained through extraction, separation and purification can significantly reduce overproliferation of fruit fly intestinal stem cells and reduce ROS production, and has good immunoenhancing activity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural medicine, and particularly relates to a bamboo red polysaccharide HB-0.1 with immunocompetence and an extraction and purification method and application. BACKGROUND

[0002] Hypocrella Bambusae is a kind of unique traditional medicine of many nationalities, belongs to Hypocreaceae bamboo small Hypocrea, and is used as medicine with dry perithecia. Hypocrella Bambusae mainly parasitizes on arrow bamboo in plateau areas. It is widely used in traditional medicine and has the effects of detoxification, itching relief, and sore healing and muscle regeneration. With the development of molecular biology research and improvement of fermentation technology, more and more researches are devoted to exploring the biosynthesis mechanism of active ingredients in Hypocrella Bambusae, genetic resources of innovative strains, and improvement of utilization efficiency and the like.

[0003] Polysaccharide is a kind of natural high molecular compound, is an indispensable component of all living organisms, and is an important immune enhancer. Polysaccharide with a specific structure can enhance the immunity of the body and improve the resistance of the body to pathogenic microorganisms and tumors. At present, the researches on Hypocrella Bambusae mainly focus on bamboo red fungus and the like, and bamboo red fungus polysaccharide is also an important active ingredient in Hypocrella Bambusae. Therefore, it is of great significance for the deep development of Hypocrella Bambusae to extract, separate and purify polysaccharide of Hypocrella Bambusae and explore the active ingredients.

[0004] SPECIFICATION

[0005] In view of the problems in the prior art, the application is designed to provide a bamboo red polysaccharide HB-0.1 with immunocompetence and an extraction and purification method and application.

[0006] The application is implemented by the following technical solutions:

[0007] The bamboo red polysaccharide HB-0.1 with immunocompetence is mainly composed of mannose, glucose and galactose.

[0008] The extraction and purification method of the bamboo red polysaccharide HB-0.1 with immunocompetence comprises the following steps:

[0009] 1) After the dried Hypocrea bambusae perithecium is crushed by a ball mill, pure water is added at a ratio of 1:50, and stirring extraction is performed for 3h. After the solution is cooled to room temperature, the supernatant is filtered to remove other solid impurities. The supernatant is added to a distillation flask and further concentrated to one-tenth of the original volume by rotary evaporation to obtain a concentrated solution. The concentrated solution is transferred to a transparent beaker and anhydrous ethanol is added. The solution is placed in a refrigerator at 4 DEG C and alcohol precipitation is performed overnight. After centrifugation, the supernatant is obtained, and the supernatant is dialyzed by flowing water for more than 48h by using a 1500Da dialysis bag to remove small salt molecules. After freeze-drying, Hypocrea bambusae crude polysaccharide is obtained.

[0010] 2) The bamboo red fungus crude polysaccharide component obtained in step 1) is subjected to Q Sepharose Fast Flow anion exchange column chromatography, and elution components are collected and concentrated by a rotary evaporator and reserved;

[0011] 3) The elution components collected in step 2) are subjected to Sephacryl S-200 gel column purification again, and eluate is collected and freeze-dried to obtain a powder sample of bamboo red polysaccharide HB-0.1.

[0012] Preferably, in step 1), the temperature of the pure water is 96℃, and the added amount of anhydrous ethanol is 5 times the volume of the concentrated solution.

[0013] Preferably, in step 2), the bamboo red fungus crude polysaccharide component is prepared into a polysaccharide solution of 50 mg / mL with deionized water, and after centrifugation, the supernatant is sampled to a Q Sepharose Fast Flow ion exchange column, and gradient elution is performed with 0, 0.1, 0.25, and 0.5 mol / L NaCl solutions, respectively, at an elution flow rate of 1.5 ml / min, and the elution components of the 0.1 mol / L NaCl solution are collected.

[0014] Preferably, in step 3), before the Sephacryl S-200 gel column purification again, the elution components concentrated in step 2) are dissolved into a solution with deionized water, and after low-speed centrifugation to remove insoluble impurities, sampling is performed, and the elution flow rate is 0.5 mL / min.

[0015] The bamboo red polysaccharide HB-0.1 prepared by the above extraction and purification method is used in the preparation of an immune enhancer.

[0016] Preferably, the immune enhancer is an intestinal immune enhancer.

[0017] The bamboo red polysaccharide HB-0.1 obtained by taking the bamboo forest unique medicinal fungus bamboo red as a raw material through extraction, separation and purification can increase the levels of ROS, NO, TNF-α, IL-1β, IL-6 and IL-12 secreted by macrophages, can significantly reduce the overproliferation of intestinal stem cells of fruit flies and reduce the production of ROS, and has good immune enhancing activity. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 QFF column chromatography elution diagram of bamboo red fungus crude polysaccharide;

[0019] Figure 2 Gel chromatogram of bamboo red fungus polysaccharide HB0.1;

[0020] Figure 3 Pre-column derivatization high performance liquid chromatogram of HB0-2PMP;

[0021] Figure 4High-performance gel size exclusion chromatography for HB-0.1;

[0022] Figure 5 The infrared spectrum of HB-0.1;

[0023] Figure 6 The effect of HB-0.1 on the survival rate of intestinal macrophages;

[0024] Figure 7 The effect of HB-0.1 on macrophage phagocytic activity;

[0025] Figure 8 The effect of HB-0.1 on the phagocytosis of red latex beads by macrophages;

[0026] Figure 9 The effect of HB-0.1 on ROS secretion by RAW264.7 macrophages;

[0027] Figure 10 The effect of HB-0.1 on the secretion of various cytokines by RAW264.7 cells;

[0028] Figure 11 The effect of HB-0.1 on Drosophila intestinal stem cells under DSS damage (A: NC; B: HB-0.25%; C: HB-0.5%; D: HB-1%).

[0029] Figure 12 The effect of HB-0.1 on ROS secretion in the intestine of Drosophila under DSS damage. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments in order to better understand the technical solution.

[0031] Example

[0032] The dried *Rhododendron simsii* stroma was pulverized using a ball mill and extracted with pure water at 96°C for 3 hours at a material-to-liquid ratio of 1:50. After the solution cooled to room temperature, the supernatant was filtered to remove other solid impurities. The supernatant was added to a distillation flask and further concentrated to one-tenth of the original volume by rotary evaporation. The concentrate was transferred to a transparent beaker and 5 times the volume of anhydrous ethanol was added. The mixture was placed in a refrigerator at 4°C for alcohol precipitation overnight. The supernatant was collected by centrifugation and dialyzed with running water using a 1500 Da dialysis bag for more than 48 hours to remove small molecules of salts. After freeze-drying, crude polysaccharide of *Rhododendron simsii* was obtained.

[0033] Q Sepharose Fast Flow anion exchange column chromatography to purify polysaccharides. The freeze-dried crude polysaccharide of bamboo red fungus was dissolved in deionized water to prepare a polysaccharide solution of 50 mg / mL. After centrifugation, the supernatant was collected. The supernatant containing the polysaccharide sample was eluted with 0, 0.1, 0.25, and 0.5 mol / L NaCl solutions (flow rate of 1.5 ml / min), and then the sugar content of the sample eluent was detected by the H2SO4-phenol method. After 200 uL of phenol was added and shaken, 1.5 mL of concentrated sulfuric acid was added for sufficient reaction. The absorbance was measured at 490 nm by ultraviolet spectrophotometry. The main components of bamboo red fungus polysaccharide were collected and concentrated by rotary evaporation for standby use. The QFF column chromatography elution diagram of the crude polysaccharide of bamboo red fungus is shown in Figure 1 The gel chromatogram of bamboo red fungus polysaccharide HB-0.1 is shown in Figure 2 .

[0034] The concentrated HB-0.1 component was further purified by Sephacryl S-200 gel column. The bamboo red fungus polysaccharide sample was dissolved in deionized water, and insoluble impurities were removed by low-speed centrifugation (or filtration of the polysaccharide sample with a water phase filter). The constant flow pump was set at a flow rate of 0.5 mL / min. After collection, the sugar content of the eluted sample was detected by the sulfuric acid-phenol method, the experimental data was recorded, and the elution curve was drawn. Finally, the eluent containing the polysaccharide sample was freeze-dried for standby use.

[0035] The PMP pre-column derivatization high-performance liquid chromatogram of HB-0.1 is shown in Figure 3 From Figure 3 it can be seen that the purified polysaccharide of bamboo red fungus is composed of mannose, galactose, and glucose, among which the content of glucose is the highest. The specific content of each monosaccharide is shown in Table 1.

[0036] Table 1: Monosaccharide composition of bamboo red fungus polysaccharide

[0037]

[0038] The high-performance gel exclusion chromatography result of HB-0.1 is shown in Figure 4 . After substituting the retention time into the standard regression curve equation of polysaccharide, it can be known that the molecular weight of HB-0.1 is 116 kDa. The infrared spectrum of HB-0.1 is shown in Figure 5 , and the GC-MS data analysis is shown in Table 2.

[0039] Table 2: GC-MS data analysis of HB-0.1

[0040]

[0041] In the HB-0.1 fraction, the mannose linkages are mainly composed of Manp(1→ and →6)Manp(1→), with a small amount of →4)Manp(1→. The glucose linkages are Glcp(1→ and →3,6)Glcp(1→). Based on the proportion of each polysaccharide structure in the GC-MS spectrum, it is inferred that the main linkage of HB-0.1 is Manp(1→ and →3,6)Glcp(1→).

[0042] Structural analysis indicates that HB-0.1 is likely a neutral heteropolysaccharide with complex branching. Its core structure is a (1→6)-α-D-Manp skeleton, and its side chains consist of (1→6)-α-D-Galf-(1→2)-β-D-Galf linked by terminal β-D-Galf(1→), (1→3)-α-D-Glcp-(1→4)-α-D-Glcp-(1→2)-α-D-Galf linked by terminal β-D-Glcp(1→), and terminal β-D-Glcp(1→).

[0043] Experimental Example

[0044] Effects of *Rhododendron molle* polysaccharide on intestinal macrophage survival: The effect of different concentrations of HB-0.1 on intestinal macrophage survival was determined using the CCK-8 assay. When intestinal macrophages were in the logarithmic growth phase, cells were gently pipetted with a small amount of phosphate buffer solution until they detached from the cell wall, and an appropriate amount of complete culture medium was added to adjust the cell density to 6 × 10⁶ cells / year. 4 Cells were administered at a concentration of [number] cells / mL until fully adhered to the plate. Different concentrations of HB-0.1 solution were added to the experimental groups, while 2 μg / mL LPS was added to the positive control group. Repeat experiments were performed in each group to ensure accurate and reliable results. After drug administration, the 96-well plates were incubated for 24 hours. The culture medium was then discarded, and 150 μL of pre-diluted CCK-8 solution was added to each well. After incubation for 1 hour, the absorbance of each well was measured, and cell viability was calculated based on the measured values. Results are as follows: Figure 6 As shown, by Figure 6 It can be seen that as the concentration of polysaccharides increases, the survival rate of macrophages in the intestine also gradually increases, but the growth trend gradually decreases; the survival rate of HB-0.1 at concentrations of 200 μg / mL and 400 μg / mL is roughly the same, which indicates that high doses of HB-0.1 have little effect on the survival rate of macrophages.

[0045] Effects of *Rhododendron molle* polysaccharide on the phagocytic capacity of intestinal macrophages: When the adherent area of ​​intestinal macrophages exceeded 80% of the bottom area of ​​the culture flask and they were in the logarithmic growth phase, macrophages were aspirated from the culture flask using a Pasteur dropper, and an appropriate amount of complete culture medium was added to adjust the cell density to 8 × 10⁻⁶ cells / year. 4The cell plate was incubated in the incubator overnight. The next day, the drug administration treatment was performed, different concentrations of polysaccharide HB-0.1 solution were added to the experimental group, LPS was selected as the positive control group, and repeated experimental holes were set in each group to ensure the accuracy and reliability of the experimental results. After the cell plate was incubated for 24 h, 150 μL of neutral red was added to each well for incubation again, and the OD value was measured at 540 nm by an enzyme label instrument after 2 h. The experimental results were recorded and the phagocytosis index was calculated. The results are shown in Figure 7 As can be seen from Figure 7 : when the drug concentration of HB-0.1 polysaccharide component is 50-200 μg / mL, the phagocytic activity of macrophages shows a growth trend to varying degrees. When the drug concentration of HB-0.1 reaches 200 μg / mL, the phagocytic activity of macrophages is the largest. When the concentration of HB-0.1 increases to 400 μg / mL, the phagocytic activity of macrophages begins to decrease, which indicates that high-dose HB-0.1 may have a certain inhibitory effect on the phagocytic activity of macrophages.

[0046] To prove Figure 7 the conclusion, the effect of HB-0.1 polysaccharide at different concentrations on the phagocytosis of macrophages to red latex beads was experimentally verified, and the results are shown in Figure 8 As can be seen from Figure 8 : with the increase of the concentration of polysaccharide, the phagocytosis of macrophages to fluorescent latex beads increases, and the fluorescence intensity increases, indicating that the phagocytic activity of macrophages increases, which shows that the polysaccharide has an activating effect on macrophages.

[0047] Effect of bamboo red fungus polysaccharide on the secretion of active oxygen level of intestinal macrophages: the effect of different concentrations of HB-0.1 on the secretion of ROS of intestinal macrophages was measured by fluorescence probe method (DCFH-DA). Intestinal macrophages were inoculated in a 96-well plate, and the density of macrophages was adjusted to 6×10 4 / mL using complete culture medium. 100 μL of complete culture medium containing macrophages was added to each well of the 96-well plate, 150 μL of phosphate buffer was added to the edge of each well of the experimental group, and the plate was incubated overnight until the macrophages were completely adhered. The culture medium was discarded. Different concentrations of HB-0.1 solution were added to the experimental group, LPS was selected as the positive control group, and repeated experimental holes were set in each group to ensure the accuracy and reliability of the experimental results. After incubation in a CO2 incubator for 24 hours, the upper culture medium was gently aspirated with a needle tube, and 10 μM of DCFH-DA was added to each well for incubation again for 35 minutes. After incubation, the fluorescence intensity of each well of the cell plate was detected using a multifunctional enzyme label instrument, and the experimental data was recorded. The results are shown in Figure 9 As can be seen from Figure 9It can be seen that compared with the blank group, polysaccharide HB-0.1 of each concentration promoted RAW264.7 macrophages to secrete ROS to different degrees, and the relative fluorescence intensity of ROS reached the highest value of the experimental group at 200 μg / mL.

[0048] Effect of bamboo red mushroom polysaccharide on secretion of cytokines by intestinal macrophages:

[0049] Cell pretreatment: when the adherent area of intestinal macrophages exceeded 80% of the bottom area of the bottle and was in the logarithmic growth phase, the cells were gently blown into a suspension state, and the concentration of macrophages in each well was adjusted to 9×10 4

[0050] Cytokine secretion detection: the cell supernatant stored in a-20°C freezer was thawed into a solution at room temperature, and then centrifuged at low speed to prepare for use. The subsequent experimental operation was carried out according to the instructions of the ELISA kit, and the detection contents included tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-12 (IL-12). The results are shown in Figure 10 Figure 10 It can be seen that when the concentration of HB-0.1 polysaccharide was 50-200 μg / mL, the secretion of IL-1β, IL-6, IL-12, and TNF-α and other cytokines by macrophages showed different degrees of growth trend. When the concentration of HB-0.1 reached 200 μg / mL, the secretion of various inflammatory factors reached the highest value, and the secretion of inflammatory factors began to decrease with the increase of the concentration of HB-0.1 to 400 μg / mL, which indicated that high-dose HB-0.1 might have a certain inhibitory effect on the secretion of cytokines by macrophages. However, for IL-12, the secretion increased to the highest value when the concentration of HB-0.1 was 400 μg / mL.

[0051] Feeding DSS to fruit flies can cause intestinal stem cells to overproliferate and differentiate, and the damaged cells interact with the complex environment in the intestine, thereby triggering a series of inflammatory responses, which are similar to human ulcerative colitis. Figure 11 As shown in the results, feeding fruit flies with different concentrations of bamboo red polysaccharide HB-0.1 after SDS-induced intestinal injury can dose-dependently inhibit the overproliferation of precursor cells induced by SDS. The principle may be to avoid the overproliferation of ISC cells and maintain the intestinal homeostasis of fruit flies through the negative feedback regulation mechanism of the JNK-MAPK signaling pathway.​​

[0052] The Effect of HB-0.1 on ROS Secretion in the Drosophila Intestine After Pathogen Infection: The Drosophila intestine not only plays a role in digesting and absorbing nutrients but also exerts a crucial immune function. When the Drosophila intestine comes into contact with external pathogens, harmful bacteria, or other stimuli, the intestinal epithelial cells produce reactive oxygen species (ROS) to resist pathogen invasion. However, while ROS can participate in and promote the immune response of intestinal cells, excessive ROS in the Drosophila intestinal environment can severely damage intestinal health when levels exceed a certain threshold. Excessive ROS can also stimulate intestinal stem cells, leading to excessive proliferation and differentiation, thereby affecting intestinal homeostasis.

[0053] This study used DHE staining to investigate whether the polysaccharide HB-0.1 from the fungus *Rhododendron molle* in Drosophila can prevent excessive proliferation of intestinal stem cells by reducing ROS secretion. DHE can be oxidized by ROS in Drosophila intestinal cells, producing red fluorescence. The relative intensity of the red fluorescence indicates the intracellular ROS level. Results are as follows: Figure 12 As shown, bamboo red polysaccharide HB-0.1 can reduce ROS production in a dose-dependent manner, thereby preventing excessive proliferation of intestinal stem cells (ISCs). In summary, the underlying mechanism may be: HB-0.1 activates the JNK-MAPK signaling pathway and, through the negative feedback regulation mechanism of JNK-MAPK, inhibits the excessive secretion of reactive oxygen species (ROS) by Drosophila intestinal epithelial stem cells, thus preventing excessive proliferation of ISCs, maintaining intestinal homeostasis in Drosophila, and prolonging the lifespan of Drosophila after SDS-induced damage.

Claims

1. Bamboo red polysaccharide HB- with immunomodulatory activity The extraction and purification method for 0.1 is characterized by comprising the following steps: 1) After pulverizing the dried bamboo red fungus stroma with a ball mill, pure water was added at a material-to-liquid ratio of 1:50 and stirred for 3 hours. After the solution cooled to room temperature, the supernatant was filtered to remove other solid impurities. The supernatant was added to a distillation flask and further concentrated to one-tenth of the original volume by rotary evaporation to obtain a concentrated solution. The concentrated solution was transferred to a transparent beaker and anhydrous ethanol was added. The solution was placed in a refrigerator at 4°C for alcohol precipitation overnight. The supernatant was centrifuged and dialyzed with running water for more than 48 hours using a 1500 Da dialysis bag to remove small molecules of salts. After freeze-drying, the crude polysaccharide of bamboo red fungus was obtained. 2) The crude polysaccharide fraction of *Rhizopus spp.* obtained in step 1) was subjected to Q Sepharose Fast Flow anion exchange column chromatography, eluted with gradients of 0, 0.1, 0.25, and 0.5 mol / L NaCl solutions, and the eluted fraction of 0.1 mol / L NaCl solution was collected and concentrated by rotary evaporator for later use. 3) The eluent from the 0.1 mol / L NaCl solution collected in step 2) was purified again using a Sephacryl S-200 gel column. The eluent was collected and freeze-dried to obtain a powder sample of bamboo red polysaccharide HB-0.

1.

2. The extraction and purification method of bamboo red polysaccharide HB-0.1 with immunomodulatory activity as described in claim 1, characterized in that... In step 1), the temperature of the pure water is 96℃, and the amount of anhydrous ethanol added is 5 times the volume of the concentrated liquid.

3. The extraction and purification method of bamboo red polysaccharide HB-0.1 with immunomodulatory activity as described in claim 1, characterized in that... In step 2), the crude polysaccharide component of *Rhizopus spp.* was prepared into a 50 mg / mL polysaccharide solution using deionized water. After centrifugation, the supernatant was aspirated and loaded onto a Q Sepharose Fast Flow ion exchange column. The gradient elution flow rate was 1.5 ml / min.

4. The method for extracting and purifying the immunologically active bamboo red polysaccharide HB-0.1 as described in claim 1, characterized in that... In step 3), before further purification with the Sephacryl S-200 gel column, the concentrated elution fraction from step 2) is dissolved in deionized water to form a solution. After removing insoluble impurities by low-speed centrifugation, the sample is loaded and eluted at a flow rate of 0.5 mL / min.

5. The application of bamboo red polysaccharide HB-0.1 obtained by the extraction and purification method according to any one of claims 1-4 in the preparation of immune enhancers.

6. The application as described in claim 5, characterized in that, The immune enhancer is an intestinal immune enhancer.

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