Ganoderma leucoproctum polysaccharide and preparation method thereof

Abstract

The invention relates to the technical field of natural compounds, in particular to ganoderma leukemia polysaccharide and a preparation method thereof. The ganoderma leukemia polysaccharide comprises galactose, glucose and mannose, and the molar ratio of the galactose to the mannose to the glucose is (1.5-3.5): (15-30): (65-85); the ganoderma leukemia polysaccharide comprises:-> 6)-Glcp-(1-> dominant residues; (-> 6)-Manp-(1->,-> 4)-Glcp-(1-> residue; and-> 4, 6)-Manp-(1->,-> 3, 6)-Galp-(1->. The ganoderma leukemia polysaccharide has anti-oxidation and detoxification capabilities, can avoid neurodegenerative diseases, reduce lipid accumulation and prolong the service life, and also has high-temperature resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of natural compounds, in particular to white-fleshed Ganoderma lucidum polysaccharide and a preparation method thereof. Background Art

[0002] With the onset of aging, the risk of developing age-associated diseases (AAD) increases, accounting for approximately 70% of global deaths. Aging is a growing concern. White Ganoderma (Ganoderma lucidum) is a new species of Ganoderma discovered in 2014. It originates primarily from the Sichuan-Tibet region and is rich in active substances, making it a high-quality local Ganoderma. It has neuroprotective, lipid-lowering, and immune-enhancing properties. However, the specific active ingredient in white Ganoderma that possesses antioxidant properties remains unknown, as does the structure and effects of the polysaccharide in white Ganoderma lucidum.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The present invention aims to provide a white-fleshed Ganoderma lucidum polysaccharide and a preparation method thereof. The present invention provides a white-fleshed Ganoderma lucidum polysaccharide having antioxidant and detoxification capabilities, which can prevent neurodegenerative diseases, reduce lipid accumulation, increase lifespan, and also have high temperature resistance.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a white-fleshed Ganoderma lucidum polysaccharide, which comprises galactose, glucose and mannose, wherein the molar ratio of the galactose, the mannose and the glucose is (1.5-3.5): (15-30): (65-85);

[0007] The white-fleshed Ganoderma lucidum polysaccharide comprises: →6)-Glcp-(1→ dominant residue;

[0008] →6)-Manp-(1→, →4)-Glcp-(1→ residues; and

[0009] →4,6)-Manp-(1→, →3,6)-Galp-(1→.

[0010] In an optional embodiment, the white-fleshed Ganoderma lucidum polysaccharide is a honeycomb structure of stacked block crystals.

[0011] In an optional embodiment, the white-fleshed Ganoderma lucidum polysaccharide contains α-configuration glycosidic bonds and β-configuration glycosidic bonds.

[0012] In an optional embodiment, the white-fleshed Ganoderma lucidum polysaccharide comprises: →4)-α-D-Glcp-(1→;

[0013] →6)-α-D-Manp-(1→;

[0014] →6)-α-D-Glcp-(1→;

[0015] →4,6)-β-D-Manp-(1→;

[0016] →3,6)-β-D-Galp-(1→.

[0017] In an optional embodiment, the molecular weight of the white flesh Ganoderma lucidum polysaccharide is (10-20)×10 4 Da.

[0018] In an optional embodiment, the primary structural unit of the white-fleshed Ganoderma lucidum polysaccharide is as follows:

[0019]

[0020] In a second aspect, the present invention provides a method for preparing the white-fleshed Ganoderma lucidum polysaccharide according to the aforementioned embodiment, comprising:

[0021] DEAE-52 cellulose column and Sephadex gel column were used to separate and purify the crude polysaccharide from Ganoderma lucidum.

[0022] In an optional embodiment, the method comprises: separating and purifying the second water-soluble polysaccharide from the four water-soluble polysaccharides obtained after separation and purification by the DEAE-52 cellulose column using the Sephadex gel column.

[0023] In an optional embodiment, when a DEAE-52 cellulose column is used for separation and purification, a linear gradient elution is performed using 5-100% of a 1 mol / L sodium chloride solution;

[0024] When using a Sephadex gel column for separation and purification, elution is performed with water.

[0025] In an optional embodiment, the preparation steps of the crude polysaccharide of white-fleshed Ganoderma lucidum include: mixing white-fleshed Ganoderma lucidum and water and heating them for extraction, then filtering and collecting the filtrate, and then removing the protein in the filtrate, then removing the solvent, and then mixing with an adsorption resin, adjusting the pH and filtering, and then filtering and drying.

[0026] The present invention has the following beneficial effects: The present invention provides a white-fleshed Ganoderma lucidum polysaccharide with antioxidant and detoxifying capabilities, anti-aging properties, and the ability to increase lifespan and prevent neurodegenerative diseases. Furthermore, the white-fleshed Ganoderma lucidum polysaccharide reduces lipid accumulation, thereby extending lifespan. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the elution curve of the DEAE-52 cellulose column provided in Example 1 of the present invention;

[0029] Figure 2 This is the elution curve of the Sephadex gel column provided in Example 1 of the present invention;

[0030] Figure 3 Infrared spectra provided for characterization in embodiments of the present invention;

[0031] Figure 4 A diagram showing the monosaccharide determination results in the characterization provided in an embodiment of the present invention;

[0032] Figure 5 A scanning electron microscope microscopic feature map provided in the characterization of the embodiment of the present invention;

[0033] Figure 6 This is a graph showing the effect of Ganoderma lucidum polysaccharide on the lifespan of N2 Caenorhabditis elegans provided in Experimental Example 1 of the present invention;

[0034] Figure 7 This is a graph showing the effect of Ganoderma lucidum polysaccharide on the reproduction of N2 Caenorhabditis elegans provided in Experimental Example 1 of the present invention;

[0035] Figure 8 This is a graph showing the effect of Ganoderma lucidum polysaccharide on the pharyngeal pump of N2 Caenorhabditis elegans provided in Experimental Example 1 of the present invention;

[0036] Figure 9 This is a graph showing the effect of Ganoderma lucidum polysaccharide on the movement of N2 Caenorhabditis elegans provided in Experimental Example 1 of the present invention;

[0037] Figure 10 This is a graph showing the effect of Ganoderma lucidum polysaccharide on lipofuscin and body length in N2 Caenorhabditis elegans provided in Experimental Example 1 of the present invention;

[0038] Figure 11 This is a graph showing the effect of Ganoderma lucidum polysaccharide on the stress resistance of N2 Caenorhabditis elegans provided in Experimental Example 1 of the present invention;

[0039] Figure 12 This is a graph showing the effect of Ganoderma lucidum polysaccharide on the stress resistance of CF1038 Caenorhabditis elegans provided in Experimental Example 1 of the present invention;

[0040] Figure 13 This is a graph showing the antioxidant test results of Ganoderma lucidum polysaccharide on HT22 cells provided in Experimental Example 1 of the present invention;

[0041] Figure 14 This is a graph showing the effects of Ganoderma lucidum polysaccharide on SOD, MDA and ROS in HT22 cells provided in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0042] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0043] In a first aspect, an embodiment of the present invention provides a white-fleshed Ganoderma lucidum polysaccharide, which includes galactose, glucose and mannose, and is mainly composed of galactose, glucose and mannose.

[0044] Specifically, the molar ratio of galactose, the mannose and the glucose is (1.5-3.5):(15-30):(65-85); for example, any value between (1.5-3.5):(15-30):(65-85) such as 2.64:21.51:75.85, 1.5:15:65, 3.5:30:85, 2.0:25:80 and 3.2:17.5:70.3.

[0045] The polysaccharide of white flesh Ganoderma lucidum includes: →6)-Glcp-(1→ dominant residue; →6)-Manp-(1→, →4)-Glcp-(1→ residue; and →4,6)-Manp-(1→, →3,6)-Galp-(1→.

[0046] Furthermore, the molecular weight of the polysaccharide of white flesh Ganoderma lucidum is (10-20)×10 4 Da. For example, 10×10 4 Da, 12×10 4 Da, 15×10 4 Da, 16.2513×10 4 Da, 18×10 4 Da, 19×10 4 Da and 20×10 4 Da etc. (10-20)×10 4 Any value between Da.

[0047] The polysaccharide of white-fleshed Ganoderma lucidum contains α-configuration glycosidic bonds and β-configuration glycosidic bonds. Specifically, the polysaccharide of white-fleshed Ganoderma lucidum includes: →4)-α-D-Glcp-(1→;→6)-α-D-Manp-(1→;→6)-α-D-Glcp-(1→;→4,6)-β-D-Manp-(1→ and →3,6)-β-D-Galp-(1→).

[0048] The primary structural unit of white-fleshed Ganoderma lucidum polysaccharide is as follows:

[0049]

[0050] Furthermore, the white-fleshed Ganoderma lucidum polysaccharide is a honeycomb structure of stacked block crystals.

[0051] In a second aspect, an embodiment of the present invention provides a method for preparing white-fleshed Ganoderma lucidum polysaccharide, comprising: mixing white-fleshed Ganoderma lucidum and water and heating them for extraction, filtering and collecting the filtrate, and then removing the protein in the filtrate, then removing the solvent, and then mixing with an adsorption resin, adjusting the pH and filtering, and then filtering and drying.

[0052] Specifically, crude white Ganoderma lucidum powder and ethanol are mixed and cold-soaked to remove lipids, filtered, and the residue is dried. The white Ganoderma lucidum powder and water are mixed and stirred in a water bath at 60-90°C. The filtrate is filtered to obtain a filtrate, concentrated under reduced pressure, and centrifuged for 3 minutes to remove fine powder precipitates. The supernatant is deproteinized using the sevage method, followed by rotary evaporation to remove residual reagents. A macroporous adsorption resin is added, the pH is adjusted to 2.5-3.5, and filtered. The resulting solution is filtered through an ultrafiltration membrane, the filtrate is dried, water is added, and ethanol is added to a concentration of 60-80%. The solution is then refrigerated at 4°C, centrifuged, and the precipitate is removed and freeze-dried to form crude white Ganoderma lucidum polysaccharide.

[0053] DEAE-52 cellulose column and Sephadex gel column were used to separate and purify the crude polysaccharide from Ganoderma lucidum.

[0054] Specifically, the crude polysaccharide from Ganoderma lucidum was separated and purified using a DEAE-52 cellulose column, wherein four water-soluble polysaccharides were obtained by linear gradient elution using a 5-100% 1 mol / L sodium chloride solution. Subsequently, a second water-soluble polysaccharide was separated and purified using the Sephadex gel column, this time using water for elution.

[0055] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0056] Example 1

[0057] The embodiment of the present invention provides a method for preparing white-fleshed Ganoderma lucidum polysaccharide, comprising:

[0058] Take a certain amount of crude white Ganoderma lucidum powder, cold-soak it in 95% ethanol for 24 hours (to remove lipids), filter it, and dry the residue at 70°C. Add the powder to a certain amount of distilled water at room temperature and soak for 30 minutes. Stir it in an 80°C water bath for 12 hours. Filter the filtrate, concentrate it under reduced pressure, and centrifuge it at 10,000 rpm for 30 minutes to remove fine powder precipitates. Deproteinize the supernatant using the sevage method (dichloromethane:n-butanol in a ratio of 4:1, reagent to polysaccharide solution in a ratio of 1:3, shake it on a shaker at 240 rpm for 1 hour, remove it and let it stand for 10 minutes, collect the supernatant, and repeat this process until no protein precipitates. Remove any residual reagents by rotary evaporation at 60°C, add AB-8 macroporous adsorption resin (1:10 weight ratio to crude drug), adjust the pH to 3 with dilute HCl, shake it for 120 minutes, and filter it. The obtained solution was filtered through a 3500Da ultrafiltration membrane for 24 hours. The filtrate was dried by rotary filtration and water was added to 50 ml. Ethanol was added to make the alcohol concentration 80%. The solution was placed in a 4°C refrigerator and centrifuged at 4000r for 10 minutes. The precipitate was taken out and freeze-dried to form crude polysaccharide.

[0059] The crude polysaccharide is dissolved in an appropriate amount of eluent, and the solution is filtered through a 0.22 μm filter membrane. The filtered polysaccharide solution is passed through a DEAE-52 cellulose column and eluted with a 5-100% linear gradient of 1 mol / L sodium chloride (PBS buffer is added at a concentration of 0.01 M) at a flow rate of 1, resulting in four eluates. The second polysaccharide solution obtained by elution is concentrated under reduced pressure and then dialyzed for 48 hours on a 1000 Da dialysis membrane. The polysaccharide is removed and freeze-dried to form a polysaccharide powder. The polysaccharide powder is passed through a Sephadex gel column using water as the eluent at a flow rate of 1, and the corresponding fractions are collected and freeze-dried. The polysaccharide elution curves of the polysaccharide after passing through two elution columns (10 mL per tube for the DEAE column, a total of 150 tubes; 5 mL per tube for the Sephadex column, a total of 120 tubes) are compared to obtain the desired white-fleshed Ganoderma lucidum polysaccharide.

[0060] The elution curve of the DEAE-52 cellulose column is shown in Figure 1 , the elution curve of Sephadex gel column can be found in Figure 2 .

[0061] according to Figure 1 It can be seen that four fragments can be separated after elution of the polysaccharide extract of white flesh Ganoderma lucidum, among which the most abundant one is water-soluble neutral polysaccharide (see Figure 1 Peak 1), 3 acidic polysaccharide fragments (see Figure 2 Peaks 2, 3, and 4 can be separated into three acidic polysaccharide fragments based on their polarity. The elution curve shows that the neutral polysaccharide elutes first from the Ganoderma lucidum polysaccharide extract, consistent with its solubility characteristics. The product from Peak 2 is then eluted subsequently.

[0062] according to Figure 2It can be seen that the elution curve has a main peak with a slight tail and a secondary peak. The main peak (peak 1) is collected, concentrated and then freeze-dried.

[0063] Comparative Example

[0064] The red Ganoderma lucidum was treated by the preparation method provided in Example 1 to obtain red Ganoderma lucidum polysaccharide, which is hereinafter recorded as pharmacopoeial Ganoderma lucidum polysaccharide.

[0065] Determination of Ganoderma lucidum polysaccharide content

[0066] The Ganoderma lucidum polysaccharides in Example 1 and the comparative example were determined with reference to the polysaccharide detection method under the Ganoderma lucidum content determination item in the 2020 edition of the "Chinese Pharmacopoeia".

[0067] The test showed that the content of the pharmacopoeia Ganoderma lucidum polysaccharide provided in the comparative example was 91.7%, and the content of the white-fleshed Ganoderma lucidum polysaccharide provided in Example 1 was 90.4%.

[0068] Characterization

[0069] The white flesh Ganoderma lucidum polysaccharide provided in Example 1 was characterized as follows:

[0070] (1) Protein content determination

[0071] The structure of the polysaccharide from white flesh Ganoderma lucidum was analyzed by ultraviolet spectroscopy. The results showed that the UV spectrum of the polysaccharide from white flesh Ganoderma lucidum had no absorption peak at 260nm, indicating that it did not contain nucleic acid substances; and no absorption peak at 280nm, indicating that it did not contain protein.

[0072] (2) Fourier transform infrared spectroscopy functional group analysis

[0073] The structure of Ganoderma lucidum polysaccharide was analyzed by Fourier transform infrared spectroscopy. Figure 3 , it can be seen that at 1033cm -1 The peaks appearing nearby are the common resonance absorption peaks of pyranose ring and hydroxyl group, 875cm -1 and 917cm -1 The absorption peaks at the bottom are the characteristic absorption peaks of α-type glycosidic bond and β-type glycosidic bond.

[0074] (3) Determination of monosaccharide composition

[0075] Methods: A 1 mg / mL solution of Ganoderma lucidum polysaccharide sample was prepared in a headspace vial. 2.0 mL of 2.0 mol / L trifluoroacetic acid was added and the solution was quickly sealed. Hydrolysis was performed at 110°C for 5 h. The sample was removed, cooled to room temperature, and the pH was adjusted to 7.0 with 5 mol / L NaOH. The sample was transferred to a 5 mL volumetric flask, brought to volume, and shaken thoroughly for later use. Appropriate amounts of nine monosaccharide reference substances (D-mannose, rhamnose, glucuronic acid, galacturonic acid, D-anhydroglucose, D-galactose, arabinose, fucose, and xylose) were weighed and dissolved in water to prepare the corresponding reference substance stock solutions. The solution was then serially diluted to create mixed reference substance solutions of varying concentrations.

[0076] Take 200 μL of the monosaccharide mixed standard solution and the polysaccharide hydrolysis solution respectively in a 10 mL centrifuge tube, add 200 μL of 0.5 mol / L NaOH solution, mix well, then add 500 μL of 0.5 mol / L PMP methanol solution, mix well, derivatize in a 70 ° C water bath for 1 hour, take out, cool to room temperature, add 200 μL of 0.5 mol / L HCl solution, mix well, add 1.0 mL of dichloromethane, vortex for 1 minute, and refrigerate centrifuge at 8000 r / min for 5 minutes, discard the lower layer liquid, repeat 3 times, remove the excess PMP solution, take the supernatant to 2 mL, and filter with a 0.22 μm microporous filter membrane to obtain the corresponding monosaccharide derivatives.

[0077] Chromatographic column: Shiseido CAPCELL PAK C18 column (4.6 mm × 250 mm, 5 μm); detection wavelength: 249 nm; column temperature: 30°C; injection volume: 10 μL; flow rate: 1.0 mL / min; mobile phase: A: acetonitrile, B: 0.1 mol / L phosphate buffer solution (pH 6.75), B:A: 84:16 (v / v).

[0078] Results see Figure 4 ,in, Figure 4 A in the middle is a monosaccharide mixed standard. Figure 4 B is the monosaccharide component of white flesh Ganoderma lucidum polysaccharide. Figure 4 It can be seen that after the white flesh Ganoderma lucidum polysaccharide was hydrolyzed and derivatized, it was separated by HPLC. The results were compared with the standard and it was found that there were mainly three types of polysaccharides, namely mannose, glucose and galactose.

[0079] (4) Analysis of methylated glycosidic bonds

[0080] Methods: 2-3 mg of Ganoderma lucidum polysaccharide sample was weighed and placed in a glass reaction bottle. 1 mL of anhydrous DMSO was added, followed by rapid addition of NaOH powder, which was sealed and dissolved under ultrasound. 1 mL of iodomethane was then added and the mixture was reacted in a magnetically stirred water bath at 30°C for 60 minutes. Finally, 2 mL of ultrapure water was added to terminate the methylation reaction.

[0081] A methylated sample of white-fleshed Ganoderma lucidum polysaccharide was hydrolyzed in 1 mL of 2M trifluoroacetic acid for 90 minutes and then evaporated to dryness on a rotary evaporator. The residue was reduced in 2 mL of double-distilled water and 60 mg of sodium borohydride for 8 hours, neutralized with glacial acetic acid, rotary evaporated, and oven-dried at 101°C. Acetylation was then performed by adding 1 mL of acetic anhydride and reacting at 100°C for 1 hour. The mixture was cooled, and 3 mL of dichloromethane was added. The reaction was concentrated under reduced pressure and evaporated to dryness. This process was repeated four to five times to remove excess acetic anhydride. The acetylated product was dissolved in 3 mL of dichloromethane and transferred to a separatory funnel. A small amount of distilled water was added and thoroughly shaken, and the upper aqueous layer was removed. This process was repeated four times. The dichloromethane layer was dried with an appropriate amount of anhydrous sodium sulfate, brought to a volume of 10 mL, and placed in a liquid chromatography vial. The acetylated product was analyzed by gas chromatography-mass spectrometry.

[0082] GC-MS analysis conditions: RXI-5SIL MS column 30m×0.25mm×0.25μm; programmed temperature conditions: starting temperature 120℃, heating to 250℃ / min at 3℃ / min; hold for 5min; inlet temperature 250℃, detector temperature 250℃ / min, carrier gas helium, flow rate 1mL / min.

[0083] After the hydroxyl group disappears, the glycosidic bond is derivatized by hydrolysis, and the ion fragments of different sugar alcohol acetyl groups in the polysaccharide are analyzed by GC-MS. The results are shown in Table 1.

[0084] Table 1 Methylation analysis results of Ganoderma lucidum polysaccharide

[0085]

[0086] According to Table 1, this polysaccharide is composed of at least three monosaccharides, namely galactose, glucose, and mannose, most of which are glucose, with only a small amount of galactose. This polysaccharide group contains →6)-Glcp-(1→ dominant residues, →4)-Glcp-(1→, →6)-Manp-(1→ residues, and →3,6)-Galp-(1→, →4,6)-Manp-(1→ branch residues.

[0087] (5) Scanning electron microscope microscopic feature analysis

[0088] The morphology of Ganoderma lucidum polysaccharide was analyzed by scanning electron microscopy. Figure 5 ,according to Figure 5 It can be seen that the surface of white-fleshed Ganoderma lucidum polysaccharide is rough and porous, presenting a honeycomb structure, which is composed of crystalline blocks of different sizes.

[0089] (6) Relative molecular weight determination

[0090] The molecular weight of the white meat Ganoderma lucidum polysaccharide sample was determined by gel permeation chromatography. The results showed that the weight average molecular weight of the white meat Ganoderma lucidum polysaccharide was 16.2513x10 4 Da.

[0091] (7) Structural analysis

[0092] Based on the above test results, the primary structural unit of white flesh Ganoderma lucidum polysaccharide is deduced as follows:

[0093]

[0094] Experimental Example 1-Anti-aging Study

[0095] The white-fleshed Ganoderma lucidum polysaccharide extract provided in Example 1 and the pharmacopoeial Ganoderma lucidum polysaccharide of the comparative example were used as research objects. Five doses of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.5 mg / mL, and 5.0 mg / mL were administered to N2 type Caenorhabditis elegans. Lifespan experiments, reproduction experiments, pharyngeal pumping and bending experiments, lipofuscin level determination, heat shock experiments, ultraviolet radiation experiments, and antioxidant experiments were performed on the nematodes to explore the effects of white-fleshed Ganoderma lucidum polysaccharide on the resistance to adverse environments and anti-aging of the Caenorhabditis elegans model.

[0096] (1) Effects of Ganoderma lucidum polysaccharides on the lifespan of N2 Caenorhabditis elegans

[0097] Age-synchronized young adult worms (L4 stage) were transferred to nematode growth medium (NGM) (containing FUdR) containing a 100% E. coli solution and appropriate doses of Ganoderma lucidum polysaccharides (0 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.5 mg / mL, and 5.0 mg / mL). Ten worms were placed per plate, for a total of 50 worms. Worms were transferred to new experimental plates every other day. Worms that could not be moved by gentle prodding with a needle were counted as dead. During the counting process, any worms that burrowed into the agar, escaped from the agar, or died were immediately removed.

[0098] Results see Figure 6 ,in, Figure 6 A in the middle is the effect of white-fleshed Ganoderma lucidum polysaccharide on the lifespan of N2 Caenorhabditis elegans. Figure 6Figure B shows the effect of pharmacopoeia Ganoderma lucidum polysaccharide on the lifespan of N2 C. elegans. The maximum lifespan of N2 C. elegans in the control group reached 22 days, with a median survival of 15 days. White Ganoderma lucidum polysaccharide at a dose of 1.0 mg significantly extended the lifespan of C. elegans, with a median survival of 17.5 days and a maximum lifespan of 41 days. After 15 days, the survival rates of the 1.0 mg and 2.5 mg white Ganoderma lucidum polysaccharide groups were significantly improved compared to the control group. The maximum lifespan of nematodes in the pharmacopoeia Ganoderma lucidum group was 30 days, with a median survival period that was not significantly different from that of the control group. This indicates that white Ganoderma lucidum has a stronger lifespan-extending effect than pharmacopoeia Ganoderma lucidum, with a 16.7% increase in median survival and a maximum lifespan that is 86.4% longer than that of the control group.

[0099] (2) Effects of Ganoderma lucidum polysaccharides on the reproduction of N2 Caenorhabditis elegans

[0100] During the reproductive period of age-synchronized young adults, three nematodes from each group were transferred from the medium containing Ganoderma lucidum extract to freshly treated plates without FUdR. The original plates were placed at 20°C to allow viable eggs to hatch and then stored at 4°C for scoring. The number of eggs laid and the number of eggs ovulated by each nematode during the reproductive period (five days) were calculated and the average of three experiments was used.

[0101] Results see Figure 7 ,in Figure 7 A in the middle is the effect of white-fleshed Ganoderma lucidum polysaccharide on the reproduction level of N2 Caenorhabditis elegans. Figure 7 Middle B shows the effect of pharmacopoeial Ganoderma lucidum polysaccharide on the reproduction level of N2 Caenorhabditis elegans (B) (n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0102] according to Figure 7 It can be seen that both Ganoderma lucidum polysaccharides have the effect of promoting egg laying and delaying the reproductive period in N2 Caenorhabditis elegans, and the effect of Pharmacopoeia Ganoderma lucidum polysaccharide is stronger than that of white Ganoderma lucidum polysaccharide. However, at high doses, Pharmacopoeia Ganoderma lucidum polysaccharide may advance the reproductive period of nematodes. White Ganoderma lucidum polysaccharide has a tendency to prolong the egg laying period of nematodes.

[0103] (3) Effects of Ganoderma lucidum polysaccharides on the pharyngeal pump of N2 Caenorhabditis elegans

[0104] On the fifth day of life (the day of the first intervention with Ganoderma lucidum extract was considered the first day of life), three individuals of age-synchronized young adults were transferred to corresponding unseeded plates, and the pumping rate (the number of pharyngeal blood pumping times within 30 seconds) was measured under a dissecting microscope.

[0105] Results see Figure 8 ,in, Figure 8 Middle A indicates the number of pharyngeal pumping times of N2 Caenorhabditis elegans within 30 seconds under the action of white meat Ganoderma lucidum polysaccharide at different times; Figure 8 Middle B indicates the number of pharyngeal pumping times of N2 Caenorhabditis elegans within 30 seconds under the action of pharmacopoeial Ganoderma lucidum polysaccharide at different times (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0106] according to Figure 8 It can be seen that after the administration of white-fleshed Ganoderma lucidum polysaccharide, the nematode pharyngeal pumping frequency increased, reaching the highest on the third day of administration. Except for the 0.1mg and 5.0mg doses, the other drug-administered groups were significantly different from the control group. However, there was no significant difference between the groups on the 15th day. The nematode pharyngeal pumping frequency of the pharmacopoeial Ganoderma lucidum group reached the highest on the 5th day. Except for 0.1mg, the other four doses were significantly different from the control group. The 2.5mg group was still able to significantly change the nematode pharyngeal pumping frequency on the 15th day. It can be seen that white-fleshed Ganoderma lucidum polysaccharide has a promoting effect on the feeding ability of nematodes, and the difference appears earlier than the pharmacopoeial Ganoderma lucidum group.

[0107] (4) Effects of Ganoderma lucidum polysaccharides on the movement of N2 Caenorhabditis elegans

[0108] After administering the drug to young adults of the same age, three nematodes were selected from each group and the number of body bends and swings of the nematodes were observed under a microscope. A complete swing of the head and neck to one side was counted as one bend. The body swing frequency of the nematodes within 30 seconds was observed.

[0109] Results see Figure 9 ,in, Figure 9 Middle A is the number of body bending times of N2 Caenorhabditis elegans within 30 seconds under the action of white meat Ganoderma lucidum polysaccharide at different times; Figure 9 Middle B shows the number of body bending times of N2 Caenorhabditis elegans within 30 seconds under the action of pharmacopoeial Ganoderma lucidum polysaccharide at different times (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0110] according to Figure 9 As shown, after administration of white-fleshed Ganoderma lucidum polysaccharide, the frequency of nematode body bending increased, reaching its peak on the third day of administration. All four dose groups showed significant differences from the control group. The 1.0 mg group still showed significant differences on the 17th day. The pharyngeal pumping frequency of the nematodes reached its highest level on the fifth day in the Pharmacopoeia Ganoderma lucidum group, with the 2.5 mg group showing the greatest significance.

[0111] (5) Effects of Ganoderma lucidum polysaccharides on lipofuscin and body length in N2 Caenorhabditis elegans

[0112] Five days after the extract was added, the nematodes were immobilized on a 2% agarose pad containing 0.5% NaN₃ and observed using a fluorescence microscope under red excitation light (Ex / Em 546 / 600 nm). Body length and fluorescence intensity were quantified using Image J software. (Nematodes were collected five days after exposure, washed three times with M9 Bufrost, and transferred to a glass slide containing 30 μL of 5 mM levamisole hydrochloride.)

[0113] Results see Figure 10 ,in, Figure 10 Middle A shows the effects of white-fleshed Ganoderma lucidum polysaccharide and pharmacopoeial Ganoderma lucidum polysaccharide on the lipofuscin level in Caenorhabditis elegans; Figure 10 Middle B shows the effects of white-fleshed Ganoderma lucidum polysaccharide and pharmacopoeial Ganoderma lucidum polysaccharide on the body length of Caenorhabditis elegans (B) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0114] according to Figure 10 The body length measurement results show that both white-fleshed Ganoderma lucidum polysaccharide and pharmacopoeia Ganoderma lucidum polysaccharide have an effect on increasing the body length of nematodes, and the effect of white-fleshed Ganoderma lucidum polysaccharide is significantly stronger than that of pharmacopoeia Ganoderma lucidum polysaccharide. The results show that white-fleshed Ganoderma lucidum polysaccharide can effectively promote the growth of nematodes, and this effect is stronger than that of pharmacopoeia Ganoderma lucidum.

[0115] (6) Effects of Ganoderma lucidum polysaccharides on stress resistance of N2 Caenorhabditis elegans

[0116] Synchronized cultures were performed as described above. L4 adults were incubated for 5 days on 35 mm NGM / OP50 plates containing Ganoderma lucidum extract and control plates containing 50 μM FUDR. All experiments were repeated three times.

[0117] Heat shock test: 5 days after administration, approximately 30 C. elegans were transferred to fresh NGM / OP50 plates and incubated at 35°C. They were monitored every 2 h thereafter until death (no movement upon pin pricking).

[0118] UV irradiation test: 30 nematodes were treated with the extract for 5 days and then irradiated with a 254 nm UV bulb (1500 Jm-2), and the number of surviving and dead worms was monitored.

[0119] t-BHP-induced oxidative stress test: 5 days after the extract intervention, the worms were placed in a culture dish containing 2 mM t-BHP oxidant and incubated at 20°C, and the death of the nematodes was monitored.

[0120] Results see Figure 11 ,in, Figure 11 A in the middle represents the effect of Ganoderma lucidum polysaccharide on the high temperature resistance of N2 Caenorhabditis elegans; Figure 11 B in the middle is the effect of resistance to t-BHP oxidation; Figure 11 Middle C represents the effect of resistance to ultraviolet radiation (C) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0121] according to Figure 11 The results show that after being administered Ganoderma lucidum polysaccharide, the survival rate of C. elegans in high-temperature, oxidative, and ultraviolet environments was significantly higher than that of the control group. In the antioxidant experiment, the survival rate of nematodes at the 5.0 mg dose was not significantly different from that of the control group. This may be because the high concentration of this dose caused some damage to the nematodes, masking the antioxidant effect of the drug on the nematodes.

[0122] (7) Effects of Ganoderma lucidum polysaccharides on stress resistance of CF1038 Caenorhabditis elegans

[0123] CF1038 nematodes (DAF-16 gene defective) were treated in the same manner as (6) and their ability to resist adverse environmental conditions after administration was determined.

[0124] Results see Figure 12 ,in, Figure 12 Middle A shows the effect of Ganoderma lucidum polysaccharide on the lifespan of CF1038 Caenorhabditis elegans; Figure 12 Middle B is the effect of Ganoderma lucidum polysaccharide on the high temperature resistance of CF1038 Caenorhabditis elegans; Figure 12 Middle C shows the effect of Ganoderma lucidum polysaccharide on the antioxidant capacity of CF1038 Caenorhabditis elegans; Figure 12 Middle D shows the effect of Ganoderma lucidum polysaccharide on the UV resistance of CF1038 Caenorhabditis elegans (n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0125] according to Figure 12 At a dose of 2.5 mg of Ganoderma lucidum polysaccharide, the lifespan of CF1038 C. elegans was not significantly different from that of the control group. However, the survival rate was significantly higher when exposed to 35°C, t-BHP oxidative inducer, and UV light. These results indicate that Ganoderma lucidum polysaccharide can significantly enhance the stress resistance of CF1038 nematodes.

[0126] Experimental Example 2

[0127] Antioxidant effect

[0128] Methods: The cells were cultured in 96-well plates and divided into three groups when the cell concentration was about 60%, namely, blank group (HT22 cells were cultured normally without any treatment), negative control group (cultured at a concentration of 55 μM t-BHP without drug administration), and experimental group (pretreated with 0.5 mg / mL, 1.0 mg / mL, 2.5 mg / mL, and 4.0 mg / mL of Ganoderma lucidum polysaccharide extract). After culturing at 37°C for 12 hours, t-BHP was added to the negative group and experimental group, and an equal amount of blank reagent was added to the blank group. After treatment at 37°C for 1 hour, the cells were harvested and the cell viability was detected using the MTT method.

[0129] Results see Figure 13 ,according to Figure 13 It can be seen that the cell survival rate was significantly reduced after administration of t-BHP oxidative inducer, while at a dose of 1.0 mg of white meat Ganoderma lucidum polysaccharide, the survival rate of HT22 cells in an oxidative environment was significantly improved. The results showed that white meat Ganoderma lucidum polysaccharide had a significant antioxidant effect on HT22 cells.

[0130] Study on the Effect of Ganoderma Lucidum Polysaccharide on MDA, SOD, CAT and ROS Levels in HT22 Cells

[0131] Methods: Referring to the above antioxidant experimental method, the cells were incubated at 37°C for 12 hours. After the culture was completed, the malondialdehyde content detection kit, catalase content detection kit, superoxide dismutase content detection kit and reactive oxygen species fluorescence probe kit were used for detection, and ROS fluorescence detection was performed using flow cytometry.

[0132] Results see Figure 14 ,in, Figure 14 Middle A shows the effect of Ganoderma lucidum polysaccharide on MAD level in HT22 cells; Figure 14 Middle B is the effect of Ganoderma lucidum polysaccharide on SOD level in HT22 cells; Figure 14 Effects of Ganoderma lucidum polysaccharide on CAT levels in HT22 cells; Figure 14 Effects of D-white Ganoderma lucidum polysaccharide on ROS levels in HT22 cells (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3).

[0133] according to Figure 14 It can be seen that white-fleshed Ganoderma lucidum polysaccharide can prevent the increase of MDA level and the decrease of SOD level in HT22 cells, but has no significant effect on CAT level. At the same time, white-fleshed Ganoderma lucidum polysaccharide can significantly reduce the level of ROS in HT22 cells. This shows that white-fleshed Ganoderma lucidum polysaccharide has a significant antioxidant capacity on HT22 cells, especially affecting the levels of SOD, MDA and ROS in cells.

[0134] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A white-fleshed Ganoderma lucidum polysaccharide, characterized in that: It comprises galactose, glucose and mannose, wherein the molar ratio of the galactose, the mannose and the glucose is (1.5-3.5): (15-30): (65-85); The white-fleshed Ganoderma lucidum polysaccharide comprises: →6)-Glcp-(1→ dominant residue; →6)-Manp-(1→, →4)-Glcp-(1→ residues; and →4,6)-Manp-(1→, →3,6)-Galp-(1→.

2. The white flesh Ganoderma lucidum polysaccharide according to claim 1, characterized in that The white-fleshed Ganoderma lucidum polysaccharide is a honeycomb structure of stacked block crystals.

3. The white flesh Ganoderma lucidum polysaccharide according to claim 1, characterized in that The white-fleshed ganoderma lucidum polysaccharide contains α-configuration glycosidic bonds and β-configuration glycosidic bonds.

4. The white flesh Ganoderma lucidum polysaccharide according to claim 1, characterized in that The white flesh Ganoderma lucidum polysaccharide comprises: →4)-α-D-Glcp-(1→; →6)-α-D-Manp-(1→; →6)-α-D-Glcp-(1→; →4,6)-β-D-Manp-(1→; →3,6)-β-D-Galp-(1→.

5. The white flesh Ganoderma lucidum polysaccharide according to claim 1, characterized in that The molecular weight of the white flesh Ganoderma lucidum polysaccharide is (10-20)×10 4 Da.

6. The white flesh Ganoderma lucidum polysaccharide according to claim 1, characterized in that The primary structural unit of the white-fleshed Ganoderma lucidum polysaccharide is as follows:

7. A method for preparing the white flesh Ganoderma lucidum polysaccharide according to claim 1, characterized in that: include: DEAE-52 cellulose column and Sephadex gel column were used to separate and purify the crude polysaccharide from Ganoderma lucidum.

8. The preparation method according to claim 7, characterized in that include: The four water-soluble polysaccharides obtained after separation and purification by the DEAE-52 cellulose column are separated and purified by the Sephadex gel column for separating and purifying the second water-soluble polysaccharide.

9. The preparation method according to claim 7 or 8, characterized in that When using a DEAE-52 cellulose column for separation and purification, a linear gradient elution was performed using a 5-100% 1 mol / L sodium chloride solution; When using a Sephadex gel column for separation and purification, elution is performed with water.

10. The preparation method according to claim 7 or 8, characterized in that: The preparation steps of the crude polysaccharide of white-fleshed Ganoderma lucidum include: mixing white-fleshed Ganoderma lucidum and water, heating and extracting, filtering and collecting the filtrate, removing protein from the filtrate, removing the solvent, mixing with adsorption resin, adjusting pH and filtering, and then filtering and drying.

Images