Ganoderma lucidum spore polysaccharide as well as extraction method and application thereof

Abstract

The invention discloses ganoderma lucidum spore polysaccharide as well as an extraction method and application thereof, and belongs to the technical field of edible mushroom extraction. According to the method, ganoderma leuconostoc spores are used as raw materials, polysaccharide in the ganoderma leuconostoc spores is extracted through a three-phase extraction method, the optimal extraction conditions of the polysaccharide are determined by setting a single factor experiment and response surface optimization, and the optimal process conditions are as follows: the material-liquid ratio is 1g: 20mL, the ratio of an extracting solution to tert-butyl alcohol is 1: 1.9, the oscillation temperature is 55 DEG C, and the extraction time is 2-3 hours. The optimization method provided by the invention is high in model fitting degree, accurate and effective, and can be used for optimizing the ganoderma leuconostoc spore polysaccharide extraction process. The physicochemical properties and pharmacological functions of the purified ganoderma leukemia spore polysaccharide are further analyzed, and experimental results show that the ganoderma leukemia spore polysaccharide provided by the invention has outstanding anti-inflammatory, anti-oxidation and anti-aging effects. According to the invention, the application of the ganoderma leucocontextum spores in preparation of medicines, health-care foods and nutritional supplements is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus extraction, in particular to ganoderma lucidum spore polysaccharide and an extraction method and application thereof. Background Art

[0002] Ganoderma leucocontextum is a new variety of Ganoderma found by Li Taihui's research group at Guangdong Institute of Microbiology in Linzhi, Tibet, my country in 2014. It is currently widely grown in Tibet, my country, and is also known as "Tibetan Ganoderma", "Tibetan Ganoderma" and "Tibetan White Ganoderma". Ganoderma leucocontextum belongs to the tri-lineal mycelial system, and the hyphae are all colorless. The cap of Ganoderma leucocontextum is semicircular or fan-shaped, the stipe is dark red after maturity, the flesh is white, the edge is white and gradually turns dark red or reddish brown inward, there is thick cork or cork near the stipe, and the surface is brown. Due to the unique natural environment in Tibet, Ganoderma leucocontextum has a high content of active ingredients such as polysaccharides and triterpenes, and is regarded as a high-end Ganoderma variety. Ganoderma spores, as an important component of Ganoderma, have a variety of biologically active ingredients and broad application prospects. Through further research and development, the application of Ganoderma spores in tumor treatment, immune regulation, anti-oxidation and liver protection will be more widely developed.

[0003] Anti-inflammatory means reducing or eliminating the body's inflammatory response through drugs or other methods. Inflammation is a defensive response of the body to various damaging factors (such as infection, trauma, chemicals, etc.), which manifests as symptoms such as local redness, swelling, heat, pain, and dysfunction. The inflammatory response helps to remove damaging factors and repair damaged tissues, but excessive inflammatory response may lead to tissue damage and disease. The purpose of anti-inflammatory treatment is to reduce inflammatory symptoms, prevent tissue damage, and promote recovery.

[0004] There are many different methods for extracting polysaccharides, which usually affect the type, physicochemical properties and biological activity of the final polysaccharide extraction product. The current polysaccharide extraction methods include: traditional hot water extraction, shock wave assisted extraction, microwave assisted extraction, enzyme assisted extraction, etc. Three-phase partitioning (TPP) is an alternative technology to traditional extraction and separation. It is simple and efficient and has been used to effectively extract and separate various bioactive compounds from natural resources, such as polysaccharides, proteins, enzymes, enzyme inhibitors, edible oils and small molecules. Polysaccharides have also been extracted from fresh fruits and vegetables. This technology can be easily carried out on a large scale at room temperature and can be used directly with crude extracts or suspensions. At present, the three-phase separation technology has not been applied to the extraction of polysaccharides from white meat Ganoderma lucidum spores, and the extraction process has not been optimized. Summary of the invention

[0005] The purpose of the present invention is to provide a ganoderma lucidum spore polysaccharide and an extraction method and application thereof to solve the problems existing in the above-mentioned prior art. In this experiment, a three-phase extraction method is used to extract white-fleshed ganoderma lucidum spore polysaccharide, and a response surface experimental design (Box-Behnken experimental design) is used for optimization on the basis of a single factor experiment to obtain the optimal extraction conditions of the polysaccharide. The anti-inflammatory activity of white-fleshed ganoderma lucidum spore polysaccharide is studied by a pro-inflammatory cytokine inhibition test, providing important data for the evaluation and development and utilization of the medical and health care functions of white-fleshed ganoderma lucidum spores.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for extracting polysaccharide from white-fleshed ganoderma spores, comprising the following steps:

[0008] Take the white-fleshed Ganoderma lucidum spores, mix them with water, extract them, and collect the filtrate by suction;

[0009] Add ammonium sulfate and tert-butanol to the filtrate, shake, centrifuge, collect the lower layer solution, dialyze to remove salt, and freeze-dry to obtain the white-fleshed Ganoderma lucidum spore polysaccharide.

[0010] Preferably, the extraction time is 4 hours and the temperature is 80° C.; the solid-liquid ratio of the white-fleshed Ganoderma lucidum spores to water is 1 g:(5-60) mL.

[0011] Preferably, in the system after adding ammonium sulfate to the filtrate, the mass fraction of the ammonium sulfate is 10%-50%; and the volume ratio of the filtrate to the tert-butanol is 1:(0.5-7).

[0012] Preferably, the shaking time is 4 hours and the temperature is 20°C-100°C.

[0013] The invention also provides white-fleshed ganoderma spore polysaccharide obtained according to the extraction method.

[0014] The present invention also provides an application of the white-fleshed Ganoderma lucidum spore polysaccharide in the preparation of anti-inflammatory drugs.

[0015] The present invention also provides an anti-inflammatory drug, the active ingredient of which includes the white-fleshed Ganoderma lucidum spore polysaccharide.

[0016] The present invention also provides an application of the white-fleshed Ganoderma lucidum spore polysaccharide in preparing anti-oxidation and / or anti-aging drugs.

[0017] The present invention also provides an anti-oxidation and / or anti-aging drug, wherein the active ingredient of the anti-oxidation and / or anti-aging drug comprises the white-fleshed Ganoderma lucidum spore polysaccharide.

[0018] The present invention also provides a process optimization method for the extraction method, characterized in that it includes the steps of optimizing the solid-liquid ratio of the white-fleshed Ganoderma lucidum spores to water, the concentration of the ammonium sulfate, the ratio of the tert-butanol to the filtrate, and the oscillation temperature according to the response surface experimental design principle.

[0019] The present invention discloses the following technical effects:

[0020] The response surface experimental design showed that the optimal process conditions were: solid-liquid ratio: 1:20 (g / mL), the ratio of extract to tert-butanol was 1:1.9 (v / v), and the oscillation temperature was 55°C. Under these conditions, the yield of polysaccharides from white-fleshed Ganoderma lucidum spores was 5.3%±0.027%, which was consistent with the predicted value (5.432%). This shows that the model of this experiment has a high degree of fitting, is accurate and effective, and can be used to optimize the extraction process of polysaccharides from white-fleshed Ganoderma lucidum spores.

[0021] Compared with the traditional extraction method, three-phase separation provides a simple, effective and environmentally friendly method to extract polysaccharides from white-fleshed Ganoderma lucidum spores. The inventor optimized the extraction process of white-fleshed Ganoderma lucidum spore polysaccharide (GLSP) and finally purified GLSP-A1. The biological activity evaluation showed that GLSP-A1 has good anti-inflammatory, antioxidant and anti-aging effects. The present invention promotes the application of this precious ingredient in anti-aging diseases and also promotes the application of white-fleshed Ganoderma lucidum spores in medicines and nutritional supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The present invention is a process flow chart for extracting polysaccharides from white-fleshed Ganoderma lucidum spores;

[0024] Figure 2 The statistical diagram of the effect of different single-factor experiments on the yield of polysaccharide from white-fleshed Ganoderma lucidum spores; (A) is the solid-liquid ratio; (B) is the ammonium sulfate concentration; (C) is the ratio of the extract to tert-butanol; (D) is the oscillation temperature;

[0025] Figure 3The response surface analysis results are shown in FIG. 1 ; wherein (A) is the response surface of the solid-liquid ratio and the ratio of the extract to tert-butanol; (B) is the response surface of the solid-liquid ratio and the oscillation temperature; (C) is the response surface of the ratio of the extract to tert-butanol and the oscillation temperature; (D) is the contour line of the solid-liquid ratio and the ratio of the extract to tert-butanol; (E) is the contour line of the solid-liquid ratio and the oscillation temperature; (F) is the contour line of the ratio of the extract to tert-butanol and the oscillation temperature;

[0026] Figure 4 The liquid phase chromatograms of the polysaccharide of the spores of white meat Ganoderma lucidum; wherein, (A) is the chromatogram of GLSP-A and GLSP-B obtained after the spore polysaccharide of white meat Ganoderma lucidum was dialyzed and freeze-dried; (B) is the chromatogram of GLSP-A1 after GLSP-A was purified by gel permeation column;

[0027] Figure 5 The figures are statistical diagrams of the experimental results of anti-inflammatory activity of GLSP-A1; (A) is cell viability; (B) is IL-6 expression level; (C) is IL-1β expression level;

[0028] Figure 6 The fluorescence images and statistical graphs of the experiment on the effect of GLSP-A1 on the level of intracellular reactive oxygen species; (A) is the fluorescence image of the control group; (B) is the fluorescence image of GLSP-A1 treatment; (C) is the fluorescence image of VC treatment; (D) is the statistical graph of the results, ** indicates P < 0.01;

[0029] Figure 7 The fluorescence images and statistical graphs of the experiment on the effect of GLSP-A1 on the level of intracellular lipofuscin; (A) is the fluorescence image of the control group; (B) is the fluorescence image of the GLSP-A1 treatment group; (C) is the fluorescence image of the VC treatment group; (D) is the statistical graph of the results, * indicates P<0..05, ** indicates P<0.01. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The flowchart of the method for extracting polysaccharide from white-fleshed Ganoderma lucidum spores of the present invention is as follows Figure 1 The process is summarized as follows: taking white-fleshed Ganoderma lucidum spores, mixing them with water, decocting and extracting, collecting the filtrate by suction filtration; adding ammonium sulfate and tert-butyl alcohol to the filtrate, shaking, centrifuging, collecting the lower layer solution, dialyzing to remove salt, and freeze-drying to obtain white-fleshed Ganoderma lucidum spore polysaccharide.

[0036] Example 1

[0037] 1 Experimental part

[0038] 1.1 Experimental instruments and drugs

[0039] Spores of Ganoderma lucidum were obtained from Milin Red Sun Tibetan Medicinal Materials Technology Development Co., Ltd., Milin County, Tibet, China. Cell culture medium (RAW264.7 mouse macrophages) was obtained from Wuhan Puli Biotechnology Co., Ltd. (Wuhan, China), and qPCR reagents (#Q711-02) were obtained from Nanjing Vazyme Biotechnology Co., Ltd. (Nanjing, China). Lipopolysaccharide, LPS (#L2880) and dexamethasone, Dex (#D1756) were obtained from Merck & Co., Ltd. (Darmstadt, Germany). All other chemicals and solvents were of laboratory grade and used as received.

[0040] 1.2 Experimental methods

[0041] 1.2.1 Extraction of polysaccharides from white-fleshed Ganoderma lucidum spores

[0042] The three-phase extraction method was used to decoct the white-fleshed Ganoderma lucidum spores with distilled water and then the filtrate was filtered out. Experiments were carried out according to four factors: solid-liquid ratio (g / mL), mass fraction of ammonium sulfate (%), ratio of tert-butyl alcohol to extract (mL / mL) and oscillation temperature (℃). The lower layer was collected and dialyzed and freeze-dried to obtain the crude polysaccharide GLSP from the white-fleshed Ganoderma lucidum spores, and the polysaccharide yield was calculated.

[0043] 1.2.2 Single Factor Experiment

[0044] (1) Effect of solid-liquid ratio on polysaccharide yield

[0045] Take 0.5 g of sample and extract at a solid-liquid ratio of 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, and 1:60 under the conditions of ammonium sulfate concentration of 20% (the mass fraction in the total system after adding ammonium sulfate, the same below), extraction solution: tert-butanol = 1:3, and oscillation temperature of 40°C. The polysaccharide yield was calculated to investigate the effect of the solid-liquid ratio on the polysaccharide yield.

[0046] (2) Effect of ammonium sulfate concentration on polysaccharide yield

[0047] Take 0.5g sample, under the conditions of solid-liquid ratio of 1:20, extraction solution: tert-butanol = 1:3, and oscillation temperature of 40°C, extract with ammonium sulfate concentration of 10%, 20%, 30%, 40%, and 50%, respectively, calculate the polysaccharide yield, and investigate the effect of ammonium sulfate concentration on the polysaccharide yield.

[0048] (3) Effect of the ratio of extract and tert-butyl alcohol on polysaccharide yield

[0049] Take 0.5g sample, under the conditions of solid-liquid ratio of 1:20, ammonium sulfate concentration of 30%, and oscillation temperature of 40℃, extract with the volume ratio of extract to tert-butanol of 1:7, 1:5, 1:3, 1:1, and 1:0.5 respectively, calculate the polysaccharide yield, and investigate the effect of the volume ratio of extract to tert-butanol on the polysaccharide yield.

[0050] (4) Effect of oscillation temperature on polysaccharide yield

[0051] Take 0.5g sample, under the conditions of solid-liquid ratio of 1:20, ammonium sulfate concentration of 30%, extracting solution: tert-butanol = 1:1, extract at oscillation temperatures of 20℃, 40℃, 60℃, and 80℃ respectively, calculate the polysaccharide yield, and investigate the effect of oscillation temperature on the polysaccharide yield.

[0052] 1.2.3 Response surface optimization experiment

[0053] According to the results of the single factor experiment, a Box-Behnken experimental design was conducted on the three factors that affect the yield of polysaccharides from white-fleshed Ganoderma lucidum spores: the ratio of solid to liquid (A1), the ratio of extract to tert-butyl alcohol (A2), and the shaking temperature (A3). The factor coding and level are shown in Table 1.

[0054] Table 1 Factor coding and level settings for response surface experiments

[0055]

[0056] 1.2.4 Purification of crude polysaccharide from white-fleshed Ganoderma lucidum spores

[0057] GLSP was redissolved in 50 mL of ultrapure water and eluted on a DEAE-52 anion column (5.5×50 cm) with two column volumes of ultrapure water and 0.1-0.7 M NaCl solution at a flow rate of 2 mL / min. The total carbohydrate content of the eluted fractions was determined using the phenol-sulfuric acid technique (8 mL / tube). Test tubes with the same elution peak were collected. The fractions obtained from ultrapure water were then further purified on an Experdex 75 gel permeation column (2.6×60 cm). The elution peak was obtained when the column was eluted with ultrapure water at a flow rate of 1.0 mL / min.

[0058] Purified Ganoderma lucidum spore polysaccharide (GLSP-A1) was obtained by dialysis and freeze-drying.

[0059] 1.2.5 Cell viability assay

[0060] The cell counting kit (CCK) reduction colorimetric assay was used to assess cell viability.

[0061] RAW264.7 mouse macrophages were inoculated in 96-well plates. Next, different amounts of GLSP-A1 (0, 0.5, 1, and 2 μg / mL) were applied. After incubation for 24 h, 10 μL of CCK was applied to each well and kept for 4 h. After removing the supernatant, 100 μL of dimethyl sulfoxide was used to dissolve the formazan crystals. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices Corporation, San Jose, CA, USA). The experiment was performed 3 times, and cell viability was expressed as a percentage.

[0062] 1.2.6 Cytokine assay

[0063] Each group included a drug-treated group (0.5, 1.2 mg / mL GLSP-A1 + 1 μg / mL lipopolysaccharide (LPS)), an LPS-stimulated group (1 μg / mL), and a normal control group (no LPS stimulation). The dexamethasone group (DEX, 1 μM) was used as a positive control. After 4 h of induction with 1 μg / mL LPS, RNA was collected and cDNA was prepared by real-time fluorescence quantitative PCR according to the Vazyme method.

[0064] 1.2.7 Cultivation of Caenorhabditis elegans

[0065] All C. elegans were grown on nematode growth medium (NGM) plates and inoculated with E. coli OP50 at 20°C. E. coli were cultured at 37°C for 12 h. Eggs were harvested using bleach solution and washed using M9 buffer. After 48 h of synchronization, L4 nematodes were prepared for subsequent assays.

[0066] 1.2.8 Determination of reactive oxygen species (ROS) levels

[0067] The synchronized L4 nematodes were divided into a blank group, a 1 g / mL GLSP-A1 administration group, and a 1 g / mL VC positive control group. After 48 h of incubation after administration, the nematodes were treated with 10 mmol / mL H2O2 for 15 min. All worms were collected and washed three times using M9 buffer. To analyze the ROS content, the worms were exposed to 10 mM DCFH-DA fluorescent probe dye solution at 37 °C for 30 min. Fluorescence microscopy and ImageJ software were used to determine and examine the relative intensity of ROS fluorescence.

[0068] 1.2.9 Lipofuscin accumulation assay

[0069] Synchronized L4 nematodes were screened onto NGM plates containing 1 mg / mL GLSP-A1 and OP50, and the control group was treated with 1 mg / mL VC as a positive control. Two days after administration, the nematodes were treated with 10 mmol / mL H2O2 for 15 min and then transferred to a centrifuge tube containing M9 buffer. The centrifuge tube containing the nematodes was centrifuged at 3000 rpm for 2 min to precipitate the nematodes. After discarding the supernatant, the nematodes were placed on a 2% agarose plate. They were examined by fluorescence microscopy. Their shapes were observed in the field and dark field, and then photographed; fluorescence was counted using ImageJ. Statistical analysis of light intensity was performed using Graphpad Pism 8.

[0070] 2 Results and discussion

[0071] 2.1 Single factor test results

[0072] 2.1.1 Effect of solid-liquid ratio on polysaccharide yield

[0073] The extraction of GLSP first increased and then decreased in the solute to solvent ratio range of 1:5 to 1:60 (g / mL). This may be due to the fact that the mass transfer driving force of polysaccharides reaches its maximum at 1:20 (g / mL), see Figure 2 Middle (A).

[0074] 2.1.2 Effect of ammonium sulfate concentration on polysaccharide yield

[0075] When the mass fraction of (NH4)2SO4 is 10%-30%, the salting-out effect is a positive driving force for the increase in GLSP production. When the mass fraction of (NH4)2SO4 is 40%-50%, the excess (NH4)2SO4 exacerbates the salting-out effect, and the production of GLSP gradually decreases. Figure 2 Middle (B).

[0076] 2.1.3 Effect of the ratio of extract and tert-butyl alcohol on polysaccharide yield

[0077] When the ratio of slurry to tert-butyl alcohol was reduced from 1:7 to 1:0.5, an effective synergistic effect was produced between tert-butyl alcohol and (NH4)2SO4, thereby improving the extraction rate of GLSP. The extraction rate of GLSP reached the highest when the ratio was 1:1, see Figure 2 Middle (C).

[0078] 2.14 Effect of oscillation temperature on polysaccharide yield

[0079] The extraction rate of GLSP gradually increased from 40℃ to 60℃, and then decreased. This is because as the temperature of the three-phase separation system increased, a large number of hydroxyl groups in the polysaccharide molecules were exposed, forming more hydrogen bonds, thereby increasing the hydrophilicity of the polysaccharide and causing the concentration of GLSP in the bottom phase to increase. Figure 2 Middle (D).

[0080] 2.2 Response surface optimization experimental results

[0081] 2.2.1 Model and ANOVA

[0082] Taking the yield of polysaccharides from white-fleshed Ganoderma lucidum spores as the response value, the Box-Behnken experimental design principle was applied, and the solid-liquid ratio (A), the ratio of the extract and tert-butanol (B) and the oscillation temperature (C) were selected as the influencing factors to obtain the experimental results shown in Table 2. The variance analysis of the data model for the yield of polysaccharides from white-fleshed Ganoderma lucidum spores extracted by three-phase extraction is shown in Table 3.

[0083] Table 2 Response surface experiment scheme and experimental results

[0084]

[0085] Table 3 Analysis of variance

[0086]

[0087]

[0088] Note: * indicates extremely significant difference. ***p<0.001, **p<0.01, *p<0.05.

[0089] The smaller the p value, the more significant the model. The coefficient of determination (R 2 ) is 0.9908, indicating that only 0.92% of the total variation cannot be explained by the model. The adjusted determination coefficient (Adj-R 2 ) was 0.9789, indicating that the model had high significance. The interaction coefficients (AC) and (BC) were significant (P<0.05), the coefficient of variation (CV=4.81) was low, the lack of fit (p value=0.9289) was not significant, and the model was fitted, indicating that the model had good accuracy and reliability.

[0090] 2.2.2 Response surface plot analysis

[0091] Figure 3 Middle (A)- Figure 3 (F) Use Design-Expert 13 software to draw the contour lines and response surfaces of each factor according to the model. The shape of the contour line is a powerful indicator, which can intuitively reveal the strength of the interaction effect between two or more factors. By analyzing the contour lines, it can be found that when there is a strong interaction between two factors, the contour lines will be elliptical. The stronger the interaction between them, the more contact points of the elliptical contour lines will increase. This phenomenon is called the "amplification effect" in physics. On the contrary, if the contour lines are circular, it means that the interaction between the two factors is relatively weak, or the degree of independence of the two factors is relatively high. In addition, the strength of the interaction between factors can also be shown by the shape of the response surface. The more obvious the interaction between the two factors, the steeper the corresponding response surface. A steep response surface may imply a two-way, nonlinear interaction pattern, while a flat surface may reveal a unidirectional, linear influence. From Figure 3 It can be seen that the response surfaces of AC and BC are steeper, and the contour lines are obviously elliptical, indicating that the interaction between the factors is significant, which is consistent with the results of the regression equation analysis.

[0092] 2.2.3 Optimal Condition Verification

[0093] The optimal extraction process parameters of Ganoderma lucidum spore polysaccharide predicted by Design-Expert 13 software are: solid-liquid ratio of 1:21.126 (g / mL), ratio of extracting liquid to tert-butyl alcohol of 1:1.945 (v / v), and oscillation temperature of 54.136°C. Under these conditions, the maximum extraction rate of polysaccharide predicted by the three-phase separation model is 5.432%.

[0094] The actual conditions are: solid-liquid ratio is 1:20 (g / mL), the ratio of extracting liquid to tert-butyl alcohol is 1:1.9 (v / v), and the shaking temperature is 55°C. The experimental extraction rate is 5.3%±0.027% (n=3), which is close to the predicted value (5.432%). This shows that the response surface experimental design (Box-Behnken experimental design) is an accurate and decisive method for optimizing the process conditions of three-phase separation.

[0095] 2.3 Purification of Ganoderma lucidum spore polysaccharide

[0096] After elution of GLSP with ultrapure water and 0.1 M NaCl on a DEAE-52 anion exchange column, GLSP-A and GLSP-B were obtained after dialysis and lyophilization, see Figure 4 The yields were 30% and 17.8%, respectively, and the total sugar contents were 84.5% and 43.54%, respectively. GLSP-A was further purified in a gel permeation column Expertex 75 (2.6×60 cm) to obtain GLSP-A1, see Figure 4 Middle (B).

[0097] Table 4 Total sugar content, uronic acid content and protein content of GLSP and GLSP-A1

[0098]

[0099] Note: Nd: Not Detected

[0100] 2.4 Anti-inflammatory activity of GLSP-A1

[0101] Small peptides or glycoproteins called cytokines synthesized and secreted by various tissue cells (mainly immune cells). In addition to being able to mediate cell-to-cell interactions, cytokines also perform a variety of biological functions, including controlling immune responses, wound healing, cell proliferation and differentiation, cell development, and tumor growth and expansion. Under LPS stimulation, macrophages release a series of inflammatory mediators and proteins, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10).

[0102] like Figure 5 As shown in Figure 2, we evaluated the anti-inflammatory efficacy of GLSP-A1 in RAW264.7 macrophages by quantifying IL-6 and IL-1β expression. DEX and GLSP-A1 significantly reduced the expression of IL-6 and IL-1β compared with the LPS group. Cell viability was not affected by GLSP-A1 in the dose range of 0.5 to 2 g / mL (P>0.05), see Figure 5 Middle (A). When the concentration of GLSP-A1 increased from 0.5 to 2 g / mL, the expression levels of IL-6 and IL-1β decreased, and the anti-inflammatory effect was enhanced. Figure 5 (B) and (C).

[0103] 2.5 Antioxidant and anti-aging activity of GLSP-A1

[0104] 2.5.1 GLSP-A1 reduces ROS accumulation in C. elegans

[0105] ROS are closely related to the aging of organisms. On the one hand, excessive reactive oxygen species can cause cell damage and aging. On the other hand, appropriate reactive oxygen species can act as signal molecules and participate in a variety of physiological processes in cells, including cell growth, differentiation and apoptosis. DCFH-DA (2',7'-dichlorofluorescein diacetate) is a commonly used fluorescent probe for detecting intracellular reactive oxygen species (ROS) levels.

[0106] like Figure 6 As shown in (A)-(D), the ROS fluorescence accumulation level of nematodes treated with 1 mg / mL GLSP-A1 and 1 mg / mL VC was reduced compared with the control. It is worth noting that the ROS level of GLSP-A1 at a concentration of 1 mg / mL was significantly lower than that of the control by 49.34% (**p<0.05). The results show that the polysaccharides from the spores of white meat Ganoderma lucidum reduce the accumulation of ROS in nematodes, thereby delaying the degree of nematode aging.

[0107] 2.5.2 GLSP-A1 reduces lipofuscin accumulation in C. elegans

[0108] Lipofuscin is the residue of organelle fragments and lipid peroxidation products that have not been completely digested in autophagic lysosomes. The accumulation of lipofuscin increases with age and is commonly found in nerve, myocardial, liver and other tissue cells, as well as forming age spots on the skin surface. Its accumulation is closely related to the aging process, and reducing the accumulation of lipofuscin may help delay aging. Under a fluorescence microscope, lipofuscin in nematodes can be observed to show spontaneous blue fluorescence. Compared with the control, the fluorescence accumulation level of lipofuscin in nematodes treated with 1mg / mL GLSP-A1 and 1mg / mLVC was reduced, as shown in Figure 2. Figure 7 As shown in (A)-(D), compared with the blank group, the lipofuscin level of 1 mg / mL GLSP-A1 was significantly reduced by 33.15% (**p<0.05).

[0109] 3. Conclusion

[0110] The experimental results show that the solid-liquid ratio, ammonium sulfate concentration, the ratio of the extract to tert-butanol concentration and the oscillation temperature have an effect on the yield of polysaccharides from white-fleshed Ganoderma lucidum spores, among which the solid-liquid ratio, the ratio of the extract to tert-butanol concentration and the oscillation temperature have the greatest influence. The optimal process conditions obtained by response surface experimental design are solid-liquid ratio: 1:20 (g / mL), the ratio of the extract to tert-butanol is 1:1.9 (v / v), and the oscillation temperature is 55℃. Under these conditions, the yield of polysaccharides from white-fleshed Ganoderma lucidum spores is 5.3%±0.027%, which is consistent with the predicted value (5.432%). This shows that the model of this experiment has a high degree of fitting, is accurate and effective, and can be used to optimize the extraction process of polysaccharides from white-fleshed Ganoderma lucidum spores.

[0111] Compared with traditional extraction methods, three-phase separation provides a simple, effective and environmentally friendly method to extract polysaccharides from white-fleshed Ganoderma lucidum spores. We optimized the extraction process of GLSP and purified GLSP-A1. Bioactivity evaluation showed that GLSP-A1 has good anti-inflammatory, antioxidant and anti-aging effects. The present invention promotes the application of this precious ingredient in anti-aging diseases and promotes the application of white-fleshed Ganoderma lucidum spores in medicines and nutritional supplements.

[0112] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting polysaccharides from white-fleshed Ganoderma lucidum spores, characterized in that: The following steps are involved: Take the white-fleshed Ganoderma lucidum spores, mix them with water, extract them, and collect the filtrate by suction; Add ammonium sulfate and tert-butanol to the filtrate, shake, centrifuge, collect the lower layer solution, dialyze to remove salt, and freeze-dry to obtain the white-fleshed Ganoderma lucidum spore polysaccharide.

2. The extraction method according to claim 1, characterized in that The extraction time is 4 hours and the temperature is 80° C.; the solid-liquid ratio of the white-fleshed Ganoderma lucidum spores to water is 1 g: (5-60) mL.

3. The extraction method according to claim 1, characterized in that In the system after adding ammonium sulfate to the filtrate, the mass fraction of the ammonium sulfate is 10%-50%; the volume ratio of the filtrate to the tert-butyl alcohol is 1:(0.5-7).

4. The extraction method according to claim 1, characterized in that The shaking time is 4 hours and the temperature is 20°C-100°C.

5. A polysaccharide from white-fleshed Ganoderma lucidum spores obtained according to the extraction method according to any one of claims 1 to 4.

6. Use of the white-fleshed Ganoderma lucidum spore polysaccharide as claimed in claim 5 in the preparation of anti-inflammatory drugs.

7. An anti-inflammatory drug, characterized in that: The active ingredient of the anti-inflammatory drug includes the white-fleshed Ganoderma lucidum spore polysaccharide described in claim 5.

8. Use of the white-fleshed Ganoderma lucidum spore polysaccharide as claimed in claim 5 in the preparation of anti-oxidation and / or anti-aging drugs.

9. An anti-oxidation and / or anti-aging drug, characterized in that: The active ingredient of the antioxidant and / or anti-aging drug includes the white-fleshed Ganoderma lucidum spore polysaccharide according to claim 5.

10. A process optimization method for the extraction method according to claim 1, characterized in that: The method comprises the steps of optimizing the solid-liquid ratio of the white-fleshed ganoderma spores to water, the concentration of the ammonium sulfate, the ratio of the tert-butyl alcohol to the filtrate and the oscillation temperature according to the response surface experimental design principle.

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