Ganoderma lucidum polysaccharide and application thereof in preparation of immunoregulation and / or anti-lung cancer product

Abstract

The present application provides a ganoderma lucidum polysaccharide and its application in the preparation of immunomodulatory and / or anti-lung cancer products, wherein the ganoderma lucidum is WXJ-M624, which was preserved in Guangdong Microbial Culture Collection Center on November 7, 2019, and the preservation number is GDMCC NO: 60889. The present application researches and finds that compared with other strains of ganoderma lucidum, the polysaccharide crude extract obtained by water extraction and alcohol precipitation of ganoderma lucidum WXJ-M624 has significant immunomodulatory activity. Further, the present application uses water extraction and fractional alcohol precipitation and other methods to separate small molecule polysaccharide from ganoderma lucidum WXJ-M624, and the molecular weight of the prepared small molecule polysaccharide is 14.05 kDa. The small molecule polysaccharide has good anti-lung cancer effect, and shows more excellent lung cancer inhibition effect than macromolecular weight ganoderma lucidum polysaccharide, which provides a new choice for the development of anti-lung cancer products in clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural extracts, in particular, to a ganoderma lucidum polysaccharide and its application in the preparation of an immunomodulatory and / or anti-lung cancer product. BACKGROUND

[0002] Ganoderma lucidum polysaccharide is a kind of polysaccharide extracted from ganoderma lucidum (Ganoderma lucidum , one of the most important active ingredients, has the effects of improving body immunity, antioxidant, anti-aging, anti-tumor, etc., and has high application value.

[0003] Polysaccharide is a high molecular polymer formed by connecting ketose and aldose through α- or β-glycosidic bond, with a molecular weight of several thousand to one million, and has the characteristics of non-toxic and harmless, different structures, various types, and extensive activity. Studies have shown that the molecular weight is closely related to the anti-tumor activity of plant polysaccharides. For example, some literature reports that when different molecular weight components of grifola frondosa polysaccharide are used to inhibit human lung cancer cells, it is found that the inhibition rate of large molecular weight grifola frondosa polysaccharide components on human lung cancer cells is significantly higher than that of other small molecular weight components; but another study shows that when different molecular weight porphyra polysaccharides are used to inhibit human gastric cancer cell line SGC7901 and human lung cancer cell line 95D, low molecular weight porphyra polysaccharide has a stronger inhibitory effect on human gastric cancer cell line SGC7901 than high molecular weight porphyra polysaccharide, but for human lung cancer cell line 95D, the effect of two different molecular weight porphyra polysaccharides is not much different (Research Progress of Molecular Weight Influence on Polysaccharide Biological Activity, Publication Date: September 8, 2023).

[0004] In addition, the composition and ratio of monosaccharides are also closely related to the function of polysaccharides. Studies have reported that ginseng crude polysaccharide (GP-c) has a molecular weight of 1177.66 kDa. Monosaccharide composition analysis shows that GP-c is composed of Glc (45.4%), GalA (31.8%), Rha (8.1%), Ara (7.6%), Gal (6.3%), Man (0.4%), and GlcA (0.4%). Ginseng pectin (another polysaccharide component isolated from ginseng crude polysaccharide, GP-a) has a molecular weight of 972.82 kDa. Monosaccharide composition analysis shows that GP-a is mainly composed of Glc (7.6%), GalA (62.0%), Rha (10.5%), Ara (11.5%), Gal (7.6%), Man (0.2%), and GlcA (0.6%). Although GP-c and GP-a contain the same types of monosaccharides, differing only in proportion, their effects on tumors differ significantly: GP-c significantly inhibits melanoma growth and improves the gut microbiota ecosystem; while GP-a promotes melanoma proliferation and exacerbates gut dysbiosis (PMID: 36607268). This demonstrates that even with the same types of monosaccharides, different proportions can lead to completely opposite functions in polysaccharides.

[0005] Related studies have shown that the higher the molecular weight of Ganoderma lucidum polysaccharides, the higher their biological activity (PMID: 38104078). Regarding the application of Ganoderma lucidum polysaccharides in anti-lung cancer research, according to Chinese invention patent CN103073651A and prior art (PMID: 33011264), 0.3 mg / mL Ganoderma lucidum polysaccharides (molecular weight approximately 1000 kDa) and 2 mg / mL Ganoderma lucidum polysaccharides (molecular weight less than 10 kDa) achieved similar inhibitory effects on A549 lung cancer cells. This may indicate that higher molecular weight Ganoderma lucidum polysaccharides have a better anti-lung cancer effect than lower molecular weight Ganoderma lucidum polysaccharides. However, due to differences in Ganoderma lucidum strains, origins, and extraction methods, the biological activity of the obtained Ganoderma lucidum polysaccharide extracts or components varies considerably.

[0006] Ganoderma lucidum WXJ-M624 is the fruiting body of a high-polysaccharide, low-spore variety of Ganoderma lucidum, which was naturally selected from wild Ganoderma lucidum by Infinitus (China) Company Limited and the Institute of Microbiology, Guangdong Academy of Sciences. It is the Ganoderma lucidum species listed in Chinese invention patent CN112662566A. Ganoderma lucidumGanoderma lucidum polysaccharide WXJ-M624 (accession number GDMCC NO: 60889, deposited at Guangdong Provincial Center for Microbial Culture Collection on November 7, 2019) is currently unknown in terms of its polysaccharide composition and anti-lung cancer effects. Developing Ganoderma lucidum polysaccharide products with high anti-lung cancer activity based on WXJ-M624 is of great significance for the development and utilization of WXJ-M624 and the development of anti-lung cancer products. Summary of the Invention

[0007] To overcome the aforementioned defects and shortcomings in the prior art, the present invention provides a Ganoderma lucidum polysaccharide and its application in the preparation of immunomodulatory and / or anti-lung cancer products.

[0008] The first objective of this invention is to provide the use of Ganoderma lucidum or Ganoderma lucidum polysaccharides in the preparation of immunomodulatory and / or anti-lung cancer products, wherein the Ganoderma lucidum is Ganoderma lucidum WXJ-M624.

[0009] The second objective of this invention is to provide a Ganoderma lucidum WXJ-M624 polysaccharide.

[0010] The third objective of this invention is to provide a small molecule polysaccharide, Ganoderma lucidum WXJ-M624.

[0011] A fourth objective of this invention is to provide the application of the above-mentioned Ganoderma lucidum WXJ-M624 polysaccharide and / or Ganoderma lucidum WXJ-M624 small molecule polysaccharide in the preparation of immunomodulatory and / or anti-lung cancer products.

[0012] A fifth objective of this invention is to provide a product for immunomodulation and / or anti-lung cancer treatment.

[0013] This invention claims protection for the following:

[0014] The application of Ganoderma lucidum or Ganoderma lucidum polysaccharides in the preparation of immunomodulatory and / or anti-lung cancer products, wherein the Ganoderma lucidum is Ganoderma lucidum WXJ-M624;

[0015] The Ganoderma lucidum WXJ-M624 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 7, 2019, with accession number GDMCC NO: 60889, and is the Ganoderma lucidum described in Chinese invention patent CN112662566A. Ganoderma lucidum M624.

[0016] A polysaccharide from Ganoderma lucidum WXJ-M624, which is a crude extract of polysaccharide obtained by water extraction and alcohol precipitation of Ganoderma lucidum WXJ-M624;

[0017] The Ganoderma lucidum WXJ-M624 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 7, 2019, with accession number GDMCC NO: 60889, and is the Ganoderma lucidum described in Chinese invention patent CN112662566A.Ganoderma lucidum M624.

[0018] A Ganoderma lucidum WXJ-M624 small molecule polysaccharide, which is prepared by the following steps:

[0019] S1. The Ganoderma lucidum WXJ-M624 raw material is subjected to water extraction treatment, and then the supernatant A is obtained by solid-liquid separation. Ethanol is added to the supernatant A until the volume fraction of ethanol in the system reaches 35-45%. After alcohol precipitation, the supernatant B is separated.

[0020] S2. Add ethanol to the supernatant B until the volume fraction of ethanol in the system reaches 65-75%, and separate the precipitate after alcohol precipitation;

[0021] S3. Redissolve the precipitate, separate it using a membrane with a molecular weight cutoff of 3500 Da, collect the retentate, add ethanol to the retentate until the volume fraction of ethanol in the system reaches 35-45%, and separate the supernatant C after alcohol precipitation.

[0022] S4. Add ethanol to the supernatant C until the volume fraction of ethanol in the system reaches 65-75%. After alcohol precipitation, separate the precipitate to obtain Ganoderma lucidum WXJ-M624 small molecule polysaccharide.

[0023] The Ganoderma lucidum WXJ-M624 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 7, 2019, with accession number GDMCC NO: 60889, and is the Ganoderma lucidum described in Chinese invention patent CN112662566A. Ganoderma lucidum M624.

[0024] Preferably, the monosaccharide component of the Ganoderma lucidum WXJ-M624 small molecule polysaccharide includes galactose, glucose and mannose, wherein the molar ratio of galactose, glucose and mannose is (0.271~0.311):(0.578~0.618):(0.030~0.070).

[0025] More preferably, the molar ratio of galactose, glucose and mannose is 0.291:0.598:0.050.

[0026] Preferably, the Ganoderma lucidum WXJ-M624 raw material is the fruiting body of Ganoderma lucidum.

[0027] Preferably, in step S1, the water extraction method is as follows: Ganoderma lucidum WXJ-M624 is mixed with water and extracted at 95-105℃ for 0.5-1.5 h.

[0028] More preferably, in step S1, the water extraction method is as follows: Ganoderma lucidum WXJ-M624 raw material is mixed with water and extracted at 100°C for 1 h.

[0029] Preferably, the mass-to-volume ratio of the Ganoderma lucidum WXJ-M624 raw material to water is 1 g: (45-55) mL.

[0030] More preferably, the mass-to-volume ratio of the Ganoderma lucidum WXJ-M624 raw material to water is 1 g: 50 mL.

[0031] Preferably, in step S1, the solid-liquid separation method is centrifugation at 7000-9000 rpm for 25-35 min.

[0032] More preferably, in step S1, the solid-liquid separation method is centrifugation at 8000 rpm for 30 min.

[0033] Preferably, in step S1, ethanol is added to the supernatant A until the volume fraction of ethanol in the system reaches 40%.

[0034] Preferably, in step S1, the method for separating the supernatant B after alcohol precipitation is as follows: let it stand at 3-5°C for 45-50 h, and then centrifuge at 7000-9000 rpm for 15-25 min.

[0035] More preferably, in step S1, the method for separating the supernatant B after alcohol precipitation is as follows: let it stand at 4°C for 48 h, and then centrifuge at 8000 rpm for 20 min.

[0036] Preferably, in step S2, ethanol is added to the supernatant B until the volume fraction of ethanol in the system reaches 70%.

[0037] Preferably, in step S3, the separation method using a membrane with a molecular weight cutoff of 3500 Da is dialysis.

[0038] More preferably, in step S3, the dialysis time is 45 to 50 hours.

[0039] More preferably, in step S3, the dialysis time is 48 hours.

[0040] Preferably, in step S3, ethanol is added to the retentate until the volume fraction of ethanol in the system reaches 40%.

[0041] Preferably, in step S4, ethanol is added to the supernatant C until the volume fraction of ethanol in the system reaches 70%.

[0042] The above-mentioned Ganoderma lucidum WXJ-M624 polysaccharide and / or Ganoderma lucidum WXJ-M624 small molecule polysaccharide are used in the preparation of immunomodulatory and / or anti-lung cancer products.

[0043] A product for immunomodulation and / or anti-lung cancer treatment, comprising Ganoderma lucidum WXJ-M624 polysaccharide and / or any of the aforementioned Ganoderma lucidum WXJ-M624 small molecule polysaccharides.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention provides a Ganoderma lucidum polysaccharide and its application in the preparation of immunomodulatory and / or anti-lung cancer products. The Ganoderma lucidum is Ganoderma lucidum WXJ-M624, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 7, 2019, with accession number GDMCC NO: 60889. This invention found that, compared with other strains of Ganoderma lucidum, the crude polysaccharide extract obtained from Ganoderma lucidum WXJ-M624 by water extraction and alcohol precipitation has significant immunomodulatory activity. Further research revealed that the small-molecule polysaccharide of Ganoderma lucidum WXJ-M624 has a better anti-lung cancer effect and exhibits a superior lung cancer inhibitory effect compared to large-molecule Ganoderma lucidum polysaccharides, providing a new option for the development of clinical anti-lung cancer products. Attached Figure Description

[0046] Figure 1 The images are of Ganoderma lucidum samples from different origins listed in Table 1; A: WXJ-M624, B: WXJ-Zhejiang (WXJ-ZJ), C: YW-Sichuan (YW-SC), D: WXJ-Shaanxi (WXJ-SX), E: YW-Anhui (YW-AH), F: YW-Zhejiang (YW-ZJ), G: YW-Shandong (YW-SD), H: YW-Jilin (YW-JL).

[0047] Figure 2 This is a technical roadmap for the preparation of Ganoderma lucidum polysaccharide components of different molecular weights (WXJ-M624).

[0048] Figure 3 The images show the morphology of MP (high molecular weight polysaccharide component) and SP (low molecular weight polysaccharide component) from Ganoderma lucidum.

[0049] Figure 4 HP-GPC spectra of a series of dextran standards with different molecular weights.

[0050] Figure 5 This is the standard curve for dextran.

[0051] Figure 6 HP-GPC spectra of Ganoderma lucidum high molecular weight polysaccharide component MP and low molecular weight polysaccharide component SP.

[0052] Figure 7 The ion chromatograms are for monosaccharide standards and Ganoderma lucidum low molecular weight polysaccharide component SP.

[0053] Figure 8 Flowchart for animal modeling, drug administration, and sampling.

[0054] Figure 9 The graph shows the changes in body weight of mice in each group.

[0055] Figure 10 The image shows the tumor size of mice in each group.

[0056] Figure 11 * Tumor weight of mice in each group P <0.05.

[0057] Figure 12 The values ​​represent changes in tumor volume and tumor inhibition rate in mice; A: Tumor volume change, B: Tumor inhibition rate. Detailed Implementation

[0058] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0059] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0060] The Ganoderma lucidum WXJ-M624 used in this embodiment was deposited on November 7, 2019, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO: 60889, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. It is the Ganoderma lucidum described in Chinese Invention Patent CN112662566A. Ganoderma lucidum M624.

[0061] Example 1: In vitro immunomodulatory activity of Ganoderma lucidum polysaccharides from different origins

[0062] I. Experimental Methods

[0063] 1. Materials

[0064] Information on Ganoderma lucidum samples from different origins is shown in Table 1, provided by Infinitus (China) Company Limited (WXJ) and Guangdong Yuewei Edible Fungus Technology Co., Ltd. (YW), respectively.

[0065] Table 1 Information on Ganoderma lucidum Samples

[0066]

[0067] 2. Preparation of Ganoderma lucidum polysaccharide extract

[0068] For each type of Ganoderma lucidum sample in Table 1, 10 to 30 fruiting bodies were randomly selected. For each fruiting body, appropriate amounts of tissue were cut from different positions on the edge, middle, and stipe of the cap, so that a total of 500 g of each type of Ganoderma lucidum fruiting body was obtained. The samples were then mixed and crushed.

[0069] Accurately weigh 1.0000 g of pulverized Ganoderma lucidum fruiting body, add 50 mL of distilled water, heat under reflux for 1 h, filter while hot, place the filtrate in an evaporating dish, wash the container several times with a small amount of distilled water, combine the washings and add them to the evaporating dish, evaporate to dryness on a water bath, dissolve the residue in 5 mL of distilled water, place in a 50 mL centrifuge tube, slowly add 25 mL of anhydrous ethanol, stir constantly, let stand for 1 h, centrifuge at 4000 rpm / min for 20 min, collect the precipitate, wash with 10 mL of anhydrous ethanol, centrifuge at 4000 rpm / min for 20 min, collect all the precipitate and heat in an oven at 35℃ for 2 h to evaporate the ethanol. Add 20 mL of distilled water to reconstitute and dialyze, freeze-dry the dialyzed solution to obtain Ganoderma lucidum polysaccharide extract.

[0070] 3. Cell activation and culture

[0071] The cryopreservation tubes containing mouse RAW 264.7 macrophages were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath (to ensure cell viability, the cryopreservation material needs to be completely thawed in about 1 minute), and the cells were then revived and passaged.

[0072] Mouse RAW 264.7 macrophages were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 1% penicillin and antibiotics, v / v) in a cell culture incubator at 37°C and 5% CO2.

[0073] 4. Measurement of cellular NO release

[0074] Griess reagent preparation:

[0075] Solution A (sulfonamide): Weigh 2.0000 g of sulfonamide powder into a 100 mL beaker, add about 50 mL of 4% phosphoric acid solution (v / v), stir thoroughly with a glass rod to dissolve, transfer to a 100 mL brown volumetric flask, rinse the beaker and glass rod three times with a small amount of 4% phosphoric acid solution (v / v), transfer all the washings to the volumetric flask, make up to volume, transfer to a brown reagent bottle, and store at 4℃.

[0076] Solution B (naphthylethylenediamine): Weigh 0.2000 g of naphthylethylenediamine powder into a 100 mL beaker, add approximately 50 mL of distilled water, and stir thoroughly with a glass rod to dissolve. Transfer the solution to a 100 mL brown volumetric flask. Rinse the beaker and glass rod three times with a small amount of distilled water, and transfer all the washings to the volumetric flask. Make up to volume. Transfer to a brown reagent bottle and store at 4°C.

[0077] Mouse RAW 264.7 macrophages were divided into groups of 4.0 × 10⁻⁶ cells. 5200 μL of cells / mL was seeded into 96-well plates and cultured for 24 h in a cell culture incubator at 37℃ and 5% CO2. The old culture medium was aspirated, and culture medium containing different concentrations (25–800 μg / mL) of Ganoderma lucidum polysaccharide extract prepared in step 2 was added. 1 μg / mL LPS was used as a positive control. Cultured for another 24 h, then 100 μL of the old culture medium was added to a new 96-well plate, followed by 100 μL of Griess reagent (V). A液 V B液 The mixture was prepared at a ratio of 1:1, and after incubation in the dark for 10 min, the absorbance at 542 nm was measured using a microplate reader. A standard curve was constructed using the NO content, and the amount of NO released from the cell culture supernatant was calculated.

[0078] 5. Measurement of cytokine secretion levels

[0079] Mouse RAW 264.7 macrophages were divided into groups of 4.0 × 10⁻⁶ cells. 5 200 μL of cells / mL were seeded into 96-well plates and cultured for 24 h in a cell culture incubator at 37°C and 5% CO2. The old culture medium was aspirated, and culture medium containing different concentrations (25–400 μg / mL) of Ganoderma lucidum polysaccharide extract prepared in step 2 was added. 1 μg / mL LPS was used as a positive control. The cells were cultured for another 24 h. 150 μL of cell culture supernatant was aspirated from each well into a centrifuge tube and stored at -80°C for later use. After thawing, the secretion of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) was measured according to the ELISA kit instructions.

[0080] II. Experimental Results

[0081] 1. Effects of Ganoderma lucidum polysaccharide samples from different origins on NO secretion in mouse macrophage RAW 264.7 cells.

[0082] Compared with the blank control group (NO secretion level of 0.17 ± 0.11 μmol / L), NO secretion in mouse macrophage RAW 264.7 cells was significantly increased after treatment with 1 μg / mL LPS (NO secretion level of 24.40 ± 0.53 μmol / L). P The result was <0.01, indicating that LPS greatly promoted the secretion of NO by macrophages.

[0083] Within the experimental sample concentration range, the NO secretion of the Ganoderma lucidum sample group increased compared with the blank control group (Table 2). The NO-promoting effect of seven Ganoderma lucidum polysaccharide samples (WXJ-M624, WXJ-Zhejiang, WXJ-Shaanxi, YW-Sichuan, YW-Zhejiang, YW-Anhui, and YW-Jilin) ​​on macrophages increased with increasing polysaccharide concentration and was significantly higher than that of the blank control group. P<0.01), among which the Ganoderma lucidum polysaccharide sample WXJ-M624 showed the most significant promoting effect; while the Ganoderma lucidum polysaccharide sample YW-Shandong showed a trend of promoting NO synthesis by macrophages at low concentrations and inhibiting it at high concentrations.

[0084] Table 2. Effects of Ganoderma lucidum polysaccharide samples on NO secretion in mouse macrophage RAW 264.7 cells ( ±S, μmol / L)

[0085]

[0086] Note: Results are expressed as mean ± standard deviation. In the same row, compared with the WXJ-M624 group, *** p <0.001, **** p <0.0001.

[0087] 2. Effects of Ganoderma lucidum polysaccharide samples from different origins on TNF-α secretion in mouse macrophage RAW 264.7 cells.

[0088] The results are shown in Table 3. Compared with the blank control group (TNF-α secretion level of 0.45±0.01 ng / mL), all sample groups in the three concentration ranges and the LPS positive control group (TNF-α secretion level of 168.34±6.31 ng / mL) significantly promoted the production of TNF-α by macrophages. P <0.01), and the TNF-α secretion of all sample groups increased with increasing sample concentration. Among them, the WXJ-M624 group had the highest TNF-α secretion at a concentration of 400 μg / mL, which was 200.87±3 ng / mL.

[0089] Table 3. Effects of Ganoderma lucidum polysaccharide samples on TNF-α secretion in mouse macrophage RAW 264.7 cells ( ±S, ng / mL)

[0090]

[0091] Note: Results are expressed as mean ± standard deviation. In the same row, compared with the WXJ-M624 group, **** p <0.0001.

[0092] 3. Effects of Ganoderma lucidum polysaccharide samples from different origins on IL-6 secretion in mouse macrophage RAW 264.7 cells.

[0093] The results are shown in Table 4. The IL-6 secretion level in the LPS-positive control group was 17.88 ± 0.81 ng / mL, which was significantly different from that in the blank control group (IL-6 secretion level was 0.0032 ± 0.0023 ng / mL).P <0.01). Within the experimental concentration range (25, 100, 400 μg / mL), all Ganoderma lucidum polysaccharide samples significantly promoted IL-6 secretion. When the sample concentration was 400 μg / mL, the Ganoderma lucidum polysaccharide sample WXJ-M624 showed the highest IL-6 secretion, at 25.34 ± 0.44 ng / mL. Among the other samples, the Ganoderma lucidum polysaccharide samples WXJ-Zhejiang, WXJ-Shaanxi, YW-Zhejiang, YW-Shandong, and YW-Jilin showed relatively weak stimulatory effects on macrophages and secreted less IL-6.

[0094] Table 4. Effects of Ganoderma lucidum polysaccharide samples on IL-6 secretion in mouse RAW 264.7 macrophages ( ±S, ng / mL)

[0095]

[0096] Note: Results are expressed as mean ± standard deviation. In the same row, compared with the WXJ-M624 group, **** p <0.0001.

[0097] In conclusion, compared with Ganoderma lucidum polysaccharides from other origins, Ganoderma lucidum polysaccharides from WXJ-M624 can better promote the secretion of NO, TNF-α and IL-6 by macrophages, and have better immunomodulatory activity.

[0098] Example 2 Preparation of Ganoderma lucidum polysaccharide components with different molecular weights (WXJ-M624)

[0099] I. Experimental Methods

[0100] according to Figure 2 The specific experimental steps for preparing Ganoderma lucidum polysaccharides of different molecular weights are as follows:

[0101] 10 kg of Ganoderma lucidum fruiting bodies (i.e., Ganoderma lucidum WXJ-M624) were pulverized, and water was added at a material-to-liquid ratio of 1:50. Extraction was carried out at 100℃ for 1 h, followed by concentration to 20 L (extraction and preparation were commissioned to the production workshop of Guangdong Yuewei Edible Fungus Technology Co., Ltd.). The concentrate was centrifuged at 8000 rpm for 30 min, and the supernatants were combined and then subjected to fractional alcohol precipitation.

[0102] (1) Add anhydrous ethanol slowly and quickly to the supernatant until the ethanol concentration is 40% (v / v). Let it stand at 4℃ for 48 h. The sample is viscous and in a gel state. Centrifuge at 8000 rpm / min for 20 min to obtain precipitate A and supernatant A.

[0103] (2) Add an appropriate amount of water to precipitate A obtained in step (1) and concentrate under reduced pressure at 55℃ to evaporate the ethanol. Redissolve in water and perform secondary alcohol precipitation: add anhydrous ethanol until the concentration of ethanol in the system is 40% (v / v), let stand at 4℃ for 48 h. The sample is still viscous. Centrifuge at 8000 rpm / min for 20 min. The precipitate and supernatant cross-reduction, and precipitate B is obtained. Redissolve precipitate B in water, filter through a 0.22 μm filter membrane, and perform ultrafiltration using a 100,000 molecular weight ultrafiltration membrane. Collect the inner liquid, concentrate, and freeze-dry to obtain the Ganoderma lucidum high molecular weight polysaccharide component MP ( G.lucidum Macromolecular Polysaccharide);

[0104] (3) Add anhydrous ethanol to the supernatant A obtained in step (1) until the concentration of ethanol in the system is 70% (v / v), and separate to obtain precipitate C. Redissolve precipitate C in water, dialyze (3500 Da) for 48 h, collect the dialysate, and concentrate. Then add anhydrous ethanol dropwise to the concentrate until the concentration of ethanol in the system is 40% (v / v), perform alcohol precipitation, and separate to obtain supernatant B. Add anhydrous ethanol dropwise to supernatant B until the concentration of ethanol in the system is 70% (v / v), and separate to obtain precipitate D. Redissolve precipitate D in water, freeze-dry, and obtain Ganoderma lucidum low molecular weight polysaccharide component SP ( G.lucidum Small molecular Polysaccharide).

[0105] Take 10 g each of Ganoderma lucidum high molecular weight polysaccharide component MP and Ganoderma lucidum low molecular weight polysaccharide component SP into blue-capped bottles for visual observation.

[0106] II. Experimental Results

[0107] The morphology of Ganoderma lucidum polysaccharide components with different molecular weights as follows: Figure 3 As shown, the appearance of Ganoderma lucidum high molecular weight polysaccharide component MP is a fluffy cotton-like structure with a soft texture; while the appearance of Ganoderma lucidum low molecular weight polysaccharide component SP is a fine granular crystal, similar to fine sand, with no obvious adhesion between the particles.

[0108] Example 3: HP-GPC determination of Ganoderma lucidum polysaccharide fractions of different molecular weights (WXJ-M624)

[0109] I. Experimental Methods

[0110] 0.0050 g of a series of dextran standards with different molecular weights, as well as the Ganoderma lucidum high molecular weight polysaccharide component MP and Ganoderma lucidum low molecular weight polysaccharide component SP prepared in Example 2, were accurately weighed and prepared into 10 mg / mL solutions with 0.1 M sodium nitrate. The solutions were then filtered through a 0.22 μm microporous membrane and injected for analysis.

[0111] Use TSKgel G6000PW XL Dextran agarose gel column (7.8 mm × 300 mm, 10 μm) and TSKgel G3000PW XL HP-GPC spectra of Ganoderma lucidum high molecular weight polysaccharide component MP and low molecular weight polysaccharide component SP were determined by tandem dextran agar gel columns (7.8 mm × 300 mm, 10 μm) under the following chromatographic elution conditions:

[0112] Detector: Differential detector (RID-20A);

[0113] Mobile phase: 0.1 M sodium nitrate;

[0114] Flow rate: 0.5 mL / min;

[0115] Injection volume: 20 μL;

[0116] Column temperature: 35℃;

[0117] Molecular weight determination software: HP-GPC software.

[0118] II. Experimental Results

[0119] HP-GPC spectra of dextran standards with different molecular weights are as follows: Figure 4 As shown in the HP-GPC chromatograms of a series of dextran standard solutions with different molecular weights, the peak times for molecular weights of 12 kDa, 25 kDa, 50 kDa, 80 kDa, 150 kDa, 270 kDa, 410 kDa, and 670 kDa are 36.88 min, 35.10 min, 33.75 min, 32.78 min, 31.64 min, 30.79 min, 30.48 min, and 29.81 min, respectively. Peak area integration was performed using HP-GPC software, and the different molecular weights and peak times were imported into the HP-GPC software to obtain the standard curves for a series of dextran solutions with different molecular weights.

[0120] Y = -0.002231778X 3 +0.2381318X 2 -8.657084X+111.4076 (where X is the peak elution time min, Y is log(Mw), R) 2 = 0.9989795), its R 2 =0.99, indicating good linearity ( Figure 5 ).

[0121] HP-GPC spectra of Ganoderma lucidum high molecular weight polysaccharide component MP and Ganoderma lucidum low molecular weight polysaccharide component SP are as follows: Figure 6As shown in Table 5, the weight-average molecular weight (Mw) of the high molecular weight polysaccharide component MP and the low molecular weight polysaccharide component SP of Ganoderma lucidum were determined based on the dextran standard curve.

[0122] Table 5. Weight-average molecular weight (Mw) and relative peak area percentage of MP and SP

[0123]

[0124] from Figure 6 As shown in Table 5, the high molecular weight polysaccharide component MP and the low molecular weight polysaccharide component SP of Ganoderma lucidum were effectively separated, with Mw values ​​of ≥670 kDa and 14.05 kDa, respectively. However, due to the high viscosity and poor flowability of the high molecular weight polysaccharide component MP, it formed a gel after alcohol precipitation, making complete precipitation difficult by centrifugation. Furthermore, the solution became viscous after reconstitution, making it impossible to remove the residual low molecular weight polysaccharide component by dialysis. Therefore, ultrafiltration was used for separation. However, due to the limitations of ultrafiltration membrane separation, a small amount of low molecular weight polysaccharide component MP remained (approximately 15.36% of the relative peak area), presenting as two small peaks. Peak 1 had an Mw of 14.20 kDa and a relative peak area of ​​approximately 5.54%, while peak 2 had an Mw of 1.35 kDa and a relative peak area of ​​approximately 9.82%.

[0125] The above results indicate that the low molecular weight polysaccharides remaining in the high molecular weight polysaccharide component MP of Ganoderma lucidum are few and have a low relative concentration. Peaks 1 and 2 can achieve a certain degree of separation, exhibiting a double peak rather than a broad single peak. HP-GPC results show that two polysaccharide components with high (≥670 kDa) and low (14.05 kDa) molecular weights were successfully separated from Ganoderma lucidum M624 using fractional alcohol precipitation, dialysis, and ultrafiltration techniques.

[0126] Example 4 Monosaccharide composition analysis of Ganoderma lucidum low molecular weight polysaccharide component SP

[0127] I. Experimental Methods

[0128] The monosaccharide composition of the low molecular weight polysaccharide component SP from Ganoderma lucidum was determined by high performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD).

[0129] 1. Reagent preparation:

[0130] 15 mM NaOH solution: Weigh 0.60 g NaOH, add ultrapure water to dissolve it completely, make up to 1 L, and filter through a 0.22 μm (aqueous) microporous membrane to obtain a 15 mM NaOH solution;

[0131] Mixed solution of 15 mM NaOH and 100 mM sodium acetate (NaOAc): Weigh 0.60 g NaOH and 8.20 g NaOAc, add ultrapure water to dissolve them completely, make up to 1 L, and filter through a 0.22 μm (aqueous) microporous membrane to obtain a mixed solution of 15 mM NaOH and 100 mM NaOAc.

[0132] Preparation of standard solutions: Take 15 monosaccharide standards: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid (GalA), glucuronic acid (GlcA), galactosyl aminohydrochloride (GalN), glucosamine hydrochloride (GlcN), guluronic acid (GulA), and mannuronic acid (ManA) to prepare standard stock solutions.

[0133] Quantitative calculation method: Precisely prepare concentration standards from the stock solutions of each monosaccharide as a mixed standard. Determine the mass of different monosaccharides using an absolute quantification method, and calculate the molar ratio based on the molar mass of the monosaccharides.

[0134] C (Standard) / A (Standard) = C (Sample) / A (Sample)

[0135] Where C (standard) is the concentration of the standard, A (standard) is the peak area of ​​the standard, C (sample) is the concentration of the sample, and A (sample) is the peak area of ​​the sample.

[0136] Sample preparation: Accurately weigh 5 mg of Ganoderma lucidum low molecular weight polysaccharide fraction SP and place it in an ampoule. Add 2 mL of 3 M TFA and hydrolyze at 120 °C for 3 h. After acid hydrolysis, dry the sample with nitrogen evaporation apparatus and add 5 mL of water to vortex mix. Pipette 50 µL, add 950 µL of deionized water, centrifuge at 12000 rpm for 5 min, and filter the supernatant through a 0.22 µm microporous membrane for HPAEC-PAD analysis.

[0137] Chromatographic conditions:

[0138] Column: Dionex Carbopac TM PA20 (3 mm × 150 mm);

[0139] Mobile phases: Phase A: H2O; Phase B: 15 mM NaOH; Phase C: a mixed solution of 15 mM NaOH and 100 mM NaOAc.

[0140] Flow rate: 0.3 mL / min;

[0141] Injection volume: 25 µL;

[0142] Column temperature: 30℃;

[0143] Detector: Electrochemical detector;

[0144] The elution gradient is shown in Table 6:

[0145] Table 6 Elution gradient conditions

[0146]

[0147] II. Experimental Results

[0148] The Ganoderma lucidum low molecular weight polysaccharide component SP is mainly composed of two monosaccharides, galactose (Gal) and glucose (Glu), and also contains trace amounts of mannose (Man). The molar ratio of Gal, Glu, and Man is 0.291:0.598:0.050, indicating that the Ganoderma lucidum low molecular weight polysaccharide component SP may be mannogalactoglucan. Figure 7 ).

[0149] Example 5: Inhibitory effect of different molecular weight WXJ-M624 Ganoderma lucidum polysaccharide components on A549 lung cancer cells

[0150] I. Experimental Methods

[0151] 1. Intervention drug formulation

[0152] Weigh 100 mg of the Ganoderma lucidum high molecular weight polysaccharide component MP and the Ganoderma lucidum low molecular weight polysaccharide component SP prepared in Example 2, add sterile water, and prepare MP stock solution of 200 mg / mL and SP stock solution of 100 mg / mL, respectively. Dilute the MP stock solution and SP stock solution with complete culture medium to prepare working solutions with concentrations of 0.8 mg / mL, 2 mg / mL, and 5 mg / mL, respectively.

[0153] 2. Cell culture and drug treatment

[0154] A549 lung cancer cells were seeded into culture flasks and cultured to the logarithmic growth phase. The old culture medium was then discarded, and the cells were gently washed twice with sterile PBS. 3 mL of trypsin-EDTA digestion solution was added, and the cells were incubated at 37°C for 1–3 min. Digestion was terminated by adding 5 mL of serum-containing complete culture medium. The cells were gently pipetted and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded, and 2 mL of complete culture medium was added. The cells were resuspended by gently pipetting 30 times to prepare a single-cell suspension.

[0155] Add 20 μL of single-cell suspension to a cell counting chamber and count the cells using a cell counter. Dilute the cell suspension to 5 × 10⁶ cells / mL with complete culture medium. 3Add 100 μL of cell suspension to each well of a 96-well plate. Set up a blank control group (add only 100 μL of complete culture medium, without adding cell suspension). Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere completely.

[0156] Cells were randomly divided into a negative control group and an experimental group, and each group was treated as follows:

[0157] Negative control group: Discard the old culture medium and add 100 μL of complete culture medium;

[0158] Experimental group: Discard the old culture medium and add MP working solution and SP working solution with concentrations of 0.8 mg / mL, 2 mg / mL and 5 mg / mL prepared in step 1, respectively;

[0159] After returning the 96-well plate to the incubator and incubating for 24 h, add 100 μL of culture medium containing CCK8 reagent to each well. Incubate at 37°C in the dark for 0.5–4 h, then measure the absorbance of each well at 450 nm, record the OD value of each well, and calculate the cell viability.

[0160] Cell viability (%) = (OD value of experimental group - OD value of blank control) / (OD value of negative control - OD value of blank control) × 100%.

[0161] II. Experimental Results

[0162] The results showed that, compared with the macromolecular polysaccharide component MP, the low molecular weight polysaccharide component SP exhibited a superior inhibitory effect on A549 lung cancer cells in a dose-dependent manner (Table 7).

[0163] Table 7 Survival rate of A549 lung cancer cells ( ±S, %

[0164]

[0165] Example 6: Tumor inhibitory effect of different molecular weight WXJ-M624 Ganoderma lucidum polysaccharide components on lung cancer mice.

[0166] I. Experimental Methods

[0167] 1. LLC cell culture, activation, and preparation of cell suspensions

[0168] Remove the LLC cell cryovials from liquid nitrogen and quickly place them in a 37°C water bath to thaw. After thawing, centrifuge the cryovials at 900 rpm for 5 min. Discard the supernatant to obtain the cell pellet. Add 1 mL of DMEM high-glucose medium (containing 10% FBS, 1% penicillin antibiotics, v / v) to the cryovials to resuspend the cells. Then transfer the cell suspension to a cell culture flask, add 6 mL of culture medium, and place the flask in a cell culture incubator for activation culture at 37°C and 5% CO2.

[0169] After cells were cultured to the logarithmic growth phase, the cells were digested with 0.25% EDTA trypsin solution (v / v), centrifuged, collected, and resuspended. The prepared cell suspension was then added dropwise to a cell counting plate, and cell counting was performed using a cell counter. The cell density was adjusted to 1.5 × 10⁻⁶. 6 ( / mL) is used for animal modeling.

[0170] 2. Animal modeling and drug administration

[0171] according to Figure 8 The specific experimental steps for animal modeling, drug administration, and sampling are as follows:

[0172] C57BL / 6 mice were acclimatized for one week in a temperature- and humidity-controlled SPF-grade animal barrier. The environment was maintained at 20–26°C and 40–70% relative humidity, with 12-hour light / dark cycles, according to the facility environment requirements specified in the national standard GB14925-2010. After acclimatization, the mice were randomly divided into 6 groups:

[0173] Blank group: fed only according to standard feeding procedures, without modeling or drug administration;

[0174] Model group: Solvent (Wahaha purified water) used to prepare the sample with the same volume.

[0175] Medium-dose group of Ganoderma lucidum high molecular weight polysaccharide (MP-M): 200 mg / kg of Ganoderma lucidum high molecular weight polysaccharide component MP prepared in Example 2 by gavage;

[0176] High-dose group of Ganoderma lucidum high molecular weight polysaccharide (MP-H): MP component of Ganoderma lucidum prepared in Example 2 by gavage 400 mg / kg;

[0177] Medium-dose group of Ganoderma lucidum low molecular weight polysaccharide (SP-M): Ganoderma lucidum low molecular weight polysaccharide component SP prepared in Example 2 was administered by gavage at a dose of 200 mg / kg.

[0178] High-dose group of Ganoderma lucidum low molecular weight polysaccharides (SP-H): Ganoderma lucidum low molecular weight polysaccharide component SP prepared in Example 2 was administered by gavage at a dose of 400 mg / kg.

[0179] After 7 days of continuous administration, the LLC cell suspension prepared in step 1 was subcutaneously inoculated into the back of mice at a cell seeding density of 1.5 × 10⁻⁶ cells / day. 6 Each animal was administered via gavage, and then the prescribed dose was continued daily for 21 days.

[0180] During the experiment, mice were provided with sufficient feed and water. Their coat color, activity level, and health status were observed and recorded daily. Mice were weighed on the first day of the gavage experiment, and then every 3 days, as well as on the last day of the gavage experiment (days 1, 5, 9, 13, 17, 21, 25, and 28). Starting two weeks after modeling, tumor volume was measured every 3 days (days 21, 25, and 28). The formula for calculating tumor volume is as follows:

[0181] Tumor volume = (tumor length × tumor width) 2 ) / 2

[0182] Mice were administered the drug continuously for 28 days. The day after the drug administration ended, mice were sacrificed, and peripheral blood, spleen (an immune organ), and tumor tissue were collected. The spleen and tumor tissue were weighed. The spleen was preserved in the tissue preservation solution MACS, while the tumor tissue was preserved using two methods: 10% neutral formalin (NBF) and at -80℃. The tumor inhibition rate of each experimental group was calculated.

[0183] Tumor inhibition rate (%) = (Tumor weight in model group - Tumor weight in experimental group) / Tumor weight in model group × 100%

[0184] II. Experimental Results

[0185] 1. Effects of Ganoderma lucidum high molecular weight polysaccharide component MP and Ganoderma lucidum low molecular weight polysaccharide component SP on mouse body weight

[0186] The changes in body weight of mice in each group during gavage are shown in Table 8 and Figure 9 As shown, the weight of mice in each group showed a steady growth trend in the first 9 days; by the fourth week (day 28), some experimental groups showed differences in the rate of weight gain, but no significant differences were observed overall, suggesting that the experimental intervention did not produce significant toxic effects.

[0187] Table 8. Changes in body weight (g) of mice in each group during gavage.

[0188]

[0189] 2. Inhibitory effects of Ganoderma lucidum high molecular weight polysaccharide component MP and Ganoderma lucidum low molecular weight polysaccharide component SP on the growth of LLC tumors in mice.

[0190] The results are as follows Figure 10 , Figure 11As shown in Table 9, under the same feeding conditions, the average weights of tumor tissues in Model, MP-M, MP-H, SP-M, and SP-H mice were (0.95±0.35) g, (0.84±0.47) g, (0.87±0.45) g, (0.62±0.23) g, and (0.72±0.36) g, respectively.

[0191] Table 9. Tumor weight (g) of mice in each group

[0192]

[0193] Note: Compared to the Model group, * P <0.05.

[0194] Compared with the model, both SP-M and SP-H were able to inhibit the growth of LLC tumors in mice (Table 10), with tumor inhibition rates of 34.74% and 31.94%, respectively (Table 11). The inhibitory effect of SP-M on the growth of LLC tumors in mice was significantly different. P <0.05)( Figure 12 ).

[0195] Table 10. Changes in tumor volume in the Model group and each experimental group within 28 days.

[0196]

[0197] Table 11 Tumor inhibition rate of mice in each experimental group

[0198]

[0199] The results showed that the main active component of Ganoderma lucidum polysaccharide WXJ-M624 in inhibiting LLC cell-induced mouse tumors was the low molecular weight polysaccharide component SP. Under oral administration, both high- and medium-dose SP treatments showed an inhibition rate of over 30% against LLC tumors in mice; compared with the model group, SP-M exhibited a significantly different inhibition rate against LLC tumor growth in mice. In conclusion, the low molecular weight polysaccharide component SP from Ganoderma lucidum has positive effects in the prevention and treatment of lung cancer.

[0200] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A small molecule polysaccharide of Ganoderma lucidum WXJ-M624, characterized in that, The Ganoderma lucidum WXJ-M624 small molecule polysaccharide was prepared by the following steps: S1. The Ganoderma lucidum WXJ-M624 raw material is subjected to water extraction treatment, and then the supernatant A is obtained by solid-liquid separation. Ethanol is added to the supernatant A until the volume fraction of ethanol in the system reaches 35-45%. After alcohol precipitation, the supernatant B is separated. S2. Add ethanol to the supernatant B until the volume fraction of ethanol in the system reaches 65-75%, and separate the precipitate after alcohol precipitation; S3. Redissolve the precipitate, separate it using a membrane with a molecular weight cutoff of 3500 Da, collect the retentate, add ethanol to the retentate until the volume fraction of ethanol in the system reaches 35-45%, and separate the supernatant C after alcohol precipitation. S4. Add ethanol to the supernatant C until the volume fraction of ethanol in the system reaches 65-75%. After alcohol precipitation, separate the precipitate to obtain Ganoderma lucidum WXJ-M624 small molecule polysaccharide. The Ganoderma lucidum WXJ-M624 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 7, 2019, with accession number GDMCC NO: 60889; The monosaccharide components of the Ganoderma lucidum WXJ-M624 small molecule polysaccharide include galactose, glucose and mannose, wherein the molar ratio of galactose, glucose and mannose is (0.271~0.311):(0.578~0.618):(0.030~0.070).

2. The Ganoderma lucidum WXJ-M624 small molecule polysaccharide according to claim 1, characterized in that, In step S1, the water extraction process is as follows: Ganoderma lucidum WXJ-M624 raw material is mixed with water and extracted at 95-105℃ for 0.5-1.5 h.

3. The Ganoderma lucidum WXJ-M624 small molecule polysaccharide according to claim 2, characterized in that, The mass-to-volume ratio of the Ganoderma lucidum WXJ-M624 raw material to water is 1 g: (45-55) mL.

4. The Ganoderma lucidum WXJ-M624 small molecule polysaccharide according to claim 1, characterized in that, In step S1, the solid-liquid separation method is centrifugation at 7000-9000 rpm for 25-35 min.

5. The Ganoderma lucidum WXJ-M624 small molecule polysaccharide according to claim 1, characterized in that, In step S1, the method for separating the supernatant B after alcohol precipitation is as follows: let it stand at 3-5℃ for 45-50 h, and then centrifuge at 7000-9000 rpm for 15-25 min.

6. The Ganoderma lucidum WXJ-M624 small molecule polysaccharide according to claim 1, characterized in that, In step S3, the separation method using a membrane with a molecular weight cutoff of 3500 Da is dialysis, and the dialysis time is 45-50 h.

7. The Ganoderma lucidum WXJ-M624 small molecule polysaccharide according to claim 1, characterized in that, The Ganoderma lucidum WXJ-M624 raw material is the fruiting body of Ganoderma lucidum WXJ-M624.

8. The use of the Ganoderma lucidum WXJ-M624 small molecule polysaccharide according to any one of claims 1 to 7 in the preparation of anti-lung cancer products.

9. A product for treating lung cancer, characterized in that, It contains the Ganoderma lucidum WXJ-M624 small molecule polysaccharide as described in any one of claims 1 to 7.

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