Lucid ganoderma extract for improving immunity and preparation method thereof

Abstract

The invention discloses a ganoderma lucidum extract for improving immunity and a preparation method thereof, and belongs to the technical field of plant extract preparation. The preparation method comprises the steps of raw material pretreatment, microwave-assisted extraction and concentration drying, a synergistic effect model of ethanol concentration (60-80%), microwave power (400-600 W) and extraction time (7-14 min) is established through a response surface method, and the triterpene extraction rate reaches 9.48% (on a dry basis) and is increased by 60%-105% compared with a traditional hot reflux method (4.5-5.8%). Through triple innovation of process-component-effect, efficient preparation of ganoderma lucidum triterpenes and deep development of immunoregulation value are realized, technical advancement and industrialization feasibility are integrated, and a brand new solution is provided for upgrading of a natural immunomodulator.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extract preparation, and particularly relates to a Ganoderma lucidum extract for enhancing immunity and a preparation method thereof. Background Art

[0002] Ganoderma lucidum, as a traditional Chinese medicine, has a long history of medicinal use and is known as the "king of all herbs". Modern research shows that Ganoderma lucidum contains a variety of active ingredients, among which Ganoderma triterpenoids are one of the main active ingredients, with significant biological activities such as immunomodulation, anti-tumor, anti-inflammatory, and antioxidant. Ganoderma triterpenoids enhance the body's immunity by regulating the functions of immune cells and have become an important source for the research and development of immunomodulators and functional foods.

[0003] However, the extraction efficiency and purity of Ganoderma triterpenoids have always been the key issues restricting their wide application. Traditional extraction methods mainly include reflux extraction, ultrasonic-assisted extraction, and supercritical fluid extraction, etc. Although these methods can extract Ganoderma active ingredients to a certain extent, they still have the following deficiencies:

[0004] (1) Low extraction efficiency: The traditional reflux extraction method requires a long time (usually several hours), and the extraction efficiency is low, which easily leads to the loss of active ingredients.

[0005] (2) Large solvent consumption: Traditional extraction methods usually require a large amount of organic solvents (such as ethanol), which not only increases the cost but also poses potential threats to the environment and the safety of operators.

[0006] (3) High energy consumption: Long-time heating or ultrasonic treatment requires high energy consumption, which is not conducive to industrial production.

[0007] (4) Active ingredients are easily damaged: In traditional extraction methods, high temperature or long-time treatment may cause the structure of Ganoderma triterpenoids to be damaged, affecting the biological activity of the extract.

[0008] In recent years, microwave-assisted extraction technology, as a new extraction method, has gradually received extensive attention due to its characteristics of high efficiency, energy conservation, and environmental protection. Microwave-assisted extraction uses the high-frequency electromagnetic field of microwaves to cause the solvent molecules to produce intense Brownian motion, accelerating the diffusion and dissolution of solutes from the solid matrix. Compared with traditional extraction methods, microwave-assisted extraction has the advantages of short extraction time, low energy consumption, less solvent consumption, and retaining active ingredients.

[0009] Although certain progress has been made in the application of microwave-assisted extraction technology in the extraction of Ganoderma lucidum active ingredients, the optimization of extraction process parameters (such as microwave power, extraction time, ethanol concentration, etc.) still requires further research. In addition, the immunomodulatory activity and its mechanism of Ganoderma triterpenoid extract also need to be deeply verified through cell experiments and animal experiments to provide a scientific basis for its application in the field of immunomodulation. Summary of the Invention

[0010] To overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a Ganoderma lucidum extract based on enhancing immunity, which realizes efficient and low-loss extraction of Ganoderma triterpenoids by dynamically coupling microwave power, solvent concentration, and temperature, and has a multi-dimensional enhancing effect on innate immunity, humoral immunity, and intestinal immunity.

[0011] Another objective of the present invention is to provide an immune-enhancing composition.

[0012] One of the objectives of the present invention is achieved by the following technical solutions:

[0013] A Ganoderma lucidum extract based on enhancing immunity is prepared by the following method:

[0014] S1. Raw material pretreatment: Dry and crush the Ganoderma lucidum fruiting body into a powder of 80-100 mesh;

[0015] S2. Microwave-assisted extraction: Mix the Ganoderma lucidum powder with an ethanol-water mixed solvent at a liquid-solid ratio of 10:1-30:1, with a solvent concentration of 50-90% (v / v), and extract for 5-20 minutes under the conditions of a microwave power of 200-700 W and a temperature of 50-80 °C; after the extract is roughly filtered, press the residue and combine the filtrate;

[0016] S3. Concentration and drying: Concentrate the filtrate by rotary evaporation to 1 / 5-1 / 10 of the original volume, and then obtain the Ganoderma triterpenoid extract by freeze-drying.

[0017] Further, in step S1, a stepped temperature increase drying process is adopted: the initial temperature is 50 °C, the temperature is increased by 5 °C every 2 hours, and finally dried to constant weight at 65 °C.

[0018] Further, in step S2, the ethanol concentration is 60-80%, the microwave power is 400-600 W, the liquid-solid ratio is 20:1, the extraction time is 7-14 minutes, and the extraction temperature is dynamically regulated at 60-70 °C by an infrared sensor.

[0019] Further, in step S2, the ethanol concentration is 75%, the microwave power is 500 W, the extraction time is 10 minutes, the liquid-solid ratio is 20:1, and the extraction temperature is controlled at 60 °C.

[0020] Further, after the extract in step S2 is roughly filtered through a 200-mesh filter screen, a pressure of 0.1 - 0.3 MPa is applied to the residue for pressing for 3 - 5 minutes, and the residual solvent liquid is recovered and merged with the primary filtrate.

[0021] Further, the extract enhances the expression level of iNOS mRNA in macrophages to ≥5-fold by activating the NF-κB signaling pathway, and promotes the CD4+ / CD8+ T cell ratio to recover to 1.3 - 1.7.

[0022] The second object of the present invention is achieved by the following technical solution:

[0023] An immune-enhancing composition, comprising the Ganoderma lucidum extract as described above, and a pharmaceutical or pharmaceutically acceptable excipient, wherein the extract accounts for 10 - 95% of the total weight of the composition; the excipient includes at least one of microcrystalline cellulose, magnesium stearate, and β-cyclodextrin.

[0024] Further, the dosage form of the composition is tablets, capsules, oral liquids or granules, and is used to improve the immunosuppressive state induced by cyclophosphamide.

[0025] Compared with the prior art, the beneficial effects of the present invention mainly lie in:

[0026] The present invention provides a Ganoderma lucidum extract for enhancing immunity and its preparation method. By using the response surface method to establish a synergistic effect model of ethanol concentration (60 - 80%), microwave power (400 - 600 W), and extraction time (7 - 14 min), the triterpene extraction rate reaches 9.48% (calculated on a dry basis), which is 60% - 105% higher than that of the traditional heat reflux method (4.5 - 5.8%). Through triple innovation of process - ingredient - efficacy, the efficient preparation of Ganoderma lucidum triterpenes and the in-depth development of immunomodulatory value are realized. The extract enhances the expression level of iNOS mRNA in macrophages to ≥5-fold (relative to the blank control group) by activating the NF-κB signaling pathway, and promotes the CD4+ / CD8+ T cell ratio to recover to 1.3 - 1.7. The small intestine sIgA content reaches 295 ± 20 μg / mL (160 ± 15 μg / mL in the model group), indicating a unique regulatory effect on mucosal immunity. The Ganoderma lucidum extract for enhancing immunity and its preparation method provided by the present invention have both technological advancement and industrial feasibility, and provide a new solution for the upgrading of natural immunomodulators. Detailed implementation manners

[0027] The following further describes the technical solution of the present invention in combination with specific embodiments. However, those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and should not be construed as a limitation to the present invention. Specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0028] Example 1

[0029] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0030] S1. Raw material pretreatment:

[0031] Select the fruiting bodies of three-year-old Ganoderma lucidum as raw materials. Within 24 hours after harvesting, rinse the surface impurities with pure water, ultrasonically clean (frequency 40 kHz) for 5 minutes, and cut into 5×5×3 mm thin slices with a multi-functional medicine cutter (model QYJ-480-3). Spread the slices flat in an oven and dry them by stepwise heating to 50 °C, increasing the temperature by 5 °C every 2 hours, and finally maintaining at 65 °C until the Ganoderma lucidum reaches a constant weight. Use a halogen moisture analyzer to detect that the moisture content is ≤8%. Put the dried Ganoderma lucidum into a pulverizer and pulverize it into 100-mesh powder.

[0032] S2. Microwave-assisted extraction:

[0033] Weigh 50.0 g ± 0.1 g of the Ganoderma lucidum powder pretreated in S1, mix the Ganoderma lucidum powder with 75% ethanol solution according to the liquid-solid ratio of 20:1, stir well, pour it into a microwave extraction container, tighten the lid, place it in a constant temperature shaker at 25 °C and impregnate for 25 minutes, with an oscillation frequency of 120 rpm. Put the container into a microwave extractor (model CEM Mars 6), set the parameters: power 300 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 50 °C ± 1 °C, and extraction time 10 minutes. After the microwave treatment, cool the reaction tank to below 30 °C. Pour the cooled mixed material into a vacuum filtration system (model Rocker300-SF30), first perform primary filtration through a 200-mesh stainless steel filter screen, and collect about 950 mL of light yellow primary filtrate. Transfer the solid material remaining on the filter screen to a plate and frame filter press, apply a pressure of 0.2 MPa and continuously press for 3 minutes to further recover the extract attached to the residue. After the pressed liquid is secondary filtered through the same 200-mesh filter screen, it is combined with the primary filtrate to obtain a total of about 980 mL of clarified extract, which is temporarily stored in a 4 °C refrigeration tank.

[0034] S3. Concentration and drying:

[0035] Pump the combined extract into a rotary evaporator (model RE100-Pro), set the water bath temperature at 45 °C and the vacuum degree at -0.085 MPa, and evaporate and recover ethanol until the volume of the solution is reduced to about 100 mL. Transfer the concentrated solution to an ultra-low temperature freezing tray (model DW-HW50) pre-cooled to -80 °C for rapid freezing and forming, and then dehydrate it in a freeze dryer (model LGJ-10B) at -55 °C and a vacuum degree of 10 Pa for 24 hours to finally obtain 32.7 g of golden sponge-like Ganoderma lucidum extract.

[0036] Example 2

[0037] A method for preparing Ganoderma lucidum extract, the steps of which are as follows:

[0038] S1. Raw material pretreatment:

[0039] Select the fruiting bodies of 3-year-old Ganoderma lucidum as raw materials. Within 24 hours after harvesting, rinse the surface impurities with pure water, ultrasonically clean (frequency 40 kHz) for 5 minutes, and cut them into 5×5×3 mm thin slices with a multi-functional medicine cutting machine (model QYJ-480-3). Spread the slices flat in an oven and dry them by stepwise heating to 50 °C, increasing the temperature by 5 °C every 2 hours, and finally maintaining at 65 °C until the Ganoderma lucidum reaches a constant weight. Use a halogen moisture analyzer to detect that the moisture content ≤ 8%. Put the dried Ganoderma lucidum into a pulverizer and pulverize it into 100-mesh powder.

[0040] S2. Microwave-assisted extraction:

[0041] Weigh 50.0 g ± 0.1 g of the Ganoderma lucidum powder pretreated in S1, mix the Ganoderma lucidum powder with 80% ethanol solution according to the liquid-solid ratio of 20:1, stir well, pour it into a microwave extraction container, tighten the lid, place it in a constant temperature shaker at 25 °C for impregnation for 25 minutes, and the oscillation frequency is 120 rpm. Put the container into a microwave extractor (model CEM Mars 6), set the parameters: power 300 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 60 °C ± 1 °C, and extraction time 10 minutes. After the microwave treatment is completed, cool the reaction tank to below 30 °C. Pour the cooled mixed material into a vacuum filtration system (model Rocker300-SF30), first filter it through a 200-mesh stainless steel filter screen for primary filtration, and collect about 950 mL of light yellow primary filtrate. Transfer the solid material remaining on the filter screen to a plate and frame filter press, apply a pressure of 0.2 MPa and continuously press for 3 minutes to further recover the extract attached to the residue. After the pressed liquid is filtered through the same 200-mesh filter screen for secondary filtration, it is combined with the primary filtrate to obtain a total of about 980 mL of clarified extract, which is temporarily stored in a 4 °C refrigerated tank.

[0042] S3. Concentration and drying:

[0043] Pump the combined extract into a rotary evaporator (model RE100-Pro), set the water bath temperature to 45°C and the vacuum degree to -0.085 MPa, and evaporate and recover ethanol until the volume of the solution is reduced to about 100 mL. Transfer the concentrated solution to an ultra-low temperature freezing plate (model DW-HW50) pre-cooled to -80°C for quick freezing and forming, and then dehydrate it in a freeze dryer (model LGJ-10B) at -55°C and a vacuum degree of 10 Pa for 24 hours to finally obtain 32.7 g of golden sponge-like Ganoderma lucidum extract.

[0044] Example 3

[0045] A method for preparing Ganoderma lucidum extract, the steps of which are as follows:

[0046] The concentration of the ethanol solution is 75%, and the microwave extraction parameters are: power 400 W (pulse mode, 2 s on / 1 s off), dynamic temperature control 70°C ± 1°C, and extraction time 14 minutes.

[0047] Example 4

[0048] A method for preparing Ganoderma lucidum extract, the steps of which are as follows:

[0049] The concentration of the ethanol solution is 80%, and the microwave extraction parameters are: power 400 W (pulse mode, 2 s on / 1 s off), dynamic temperature control 80°C ± 1°C, and extraction time 14 minutes.

[0050] Example 5

[0051] A method for preparing Ganoderma lucidum extract, the steps of which are as follows:

[0052] The concentration of the ethanol solution is 70%, and the microwave extraction parameters are: power 300 W (pulse mode, 2 s on / 1 s off), dynamic temperature control 50°C ± 1°C, and extraction time 7 minutes.

[0053] Example 6

[0054] A method for preparing Ganoderma lucidum extract, the steps of which are as follows:

[0055] The concentration of the ethanol solution is 70%, and the microwave extraction parameters are: power 400 W (pulse mode, 2 s on / 1 s off), dynamic temperature control 60°C ± 1°C, and extraction time 7 minutes.

[0056] Example 7

[0057] A method for preparing Ganoderma lucidum extract, the steps of which are as follows:

[0058] The concentration of the ethanol solution is 75%, and the microwave extraction parameters are: power 500 W (pulse mode, 2 s on / 1 s off), dynamic temperature control 70°C ± 1°C, and extraction time 7 minutes.

[0059] Example 8

[0060] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0061] The concentration of the ethanol solution is 70%, and the microwave extraction parameters are: power 500 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 80 °C ± 1 °C, and extraction time 14 minutes.

[0062] Example 9

[0063] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0064] The concentration of the ethanol solution is 80%, and the microwave extraction parameters are: power 500 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 50 °C ± 1 °C, and extraction time 14 minutes.

[0065] Example 10

[0066] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0067] The concentration of the ethanol solution is 75%, and the microwave extraction parameters are: power 600 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 60 °C ± 1 °C, and extraction time 10 minutes.

[0068] Example 11

[0069] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0070] The concentration of the ethanol solution is 75%, and the microwave extraction parameters are: power 600 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 70 °C ± 1 °C, and extraction time 7 minutes.

[0071] Example 12

[0072] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0073] The concentration of the ethanol solution is 75%, and the microwave extraction parameters are: power 500 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 80 °C ± 1 °C, and extraction time 7 minutes.

[0074] Example 13

[0075] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0076] The concentration of the ethanol solution is 80%, and the microwave extraction parameters are: power 600 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 50 °C ± 1 °C, and extraction time 10 minutes.

[0077] Example 14

[0078] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0079] The concentration of the ethanol solution is 80%, and the microwave extraction parameters are as follows: power 700 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 60°C ± 1°C, and extraction time 10 minutes.

[0080] Example 15

[0081] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0082] The concentration of the ethanol solution is 70%, and the microwave extraction parameters are as follows: power 500 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 70°C ± 1°C, and extraction time 14 minutes.

[0083] Example 16

[0084] A method for preparing a Ganoderma lucidum extract, the steps of which are as follows:

[0085] The concentration of the ethanol solution is 70%, and the microwave extraction parameters are as follows: power 600 W (pulse mode, 2 s on / 1 s off), dynamic temperature control at 80°C ± 1°C, and extraction time 14 minutes.

[0086] Experimental Example 1 HPLC analysis

[0087] Take 10 mg of the Ganoderma lucidum extract prepared in each example, add 10 mL of methanol, dissolve it by ultrasonic wave for 15 minutes (40 kHz, power 50 W), centrifuge (12,000 rpm, 10 minutes), take the supernatant and filter it through a 0.22 μm PTFE filter membrane, and inject 10 μL for HPLC analysis. Chromatographic conditions:

[0088] Chromatographic column: C18 column (250 mm × 4.6 mm, 5 μm)

[0089] Mobile phase: A (0.1% phosphoric acid aqueous solution), B (acetonitrile)

[0090] Mobile phase gradient: 0–10 min (40% B → 60% B), 10–30 min (60% B → 80% B)

[0091] Flow rate: 1.0 mL / min

[0092] Column temperature: 30°C

[0093] Detection wavelength: 252 nm

[0094] Run HPLC with the Ganoderic acid A / B standard product from Sigma, record the peak area, and plot the linear equation.

[0095] Detect the peak area of the test sample, substitute it into the formula to calculate the content (μg / mL) by back-calculation, and multiply by the dilution factor to obtain the content of ganoderic triterpenes in the sample (mg / g).

[0096] Table 1 Extraction Conditions and Extraction Results

[0097]

[0098]

[0099] Experimental Example 2 Effect of Ganoderic Triterpenes on the Immune Activity of Macrophages

[0100] 1. Cell Culture and Treatment

[0101] Take the RAW264.7 mouse macrophage cell line and subculture it in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. 24 hours before the experiment, seed the cells in a 96-well plate at a density of 5×10 3 cells / well, and perform drug treatment after the cell attachment rate reaches 80%.

[0102] 2. Grouping and Drug Administration

[0103] The experiment was divided into 5 groups:

[0104] Blank control group: The medium contains 0.1% DMSO (the same solvent concentration as the triterpene group)

[0105] Positive control group: The medium contains 1 μg / mL LPS (Sigma, L2880)

[0106] Low-dose triterpene group: The medium contains 5 μg / mL ganoderic triterpene extract (prepared in Example 1)

[0107] Medium-dose triterpene group: The medium contains 10 μg / mL ganoderic triterpene extract (prepared in Example 1)

[0108] High-dose triterpene group: The medium contains 20 μg / mL ganoderic triterpene extract (prepared in Example 1)

[0109] The ganoderic triterpene extract was pre-dissolved in DMSO to a stock solution of 10 mg / mL and diluted to the target concentration with the medium before use (the final concentration of DMSO ≤ 0.1%). Each group was set with 6 replicate wells, and relevant indicators were detected after 24 hours of treatment.

[0110] 3. Cell Viability Detection:

[0111] Use a CCK-8 kit (Dojindo, CK04), add 10 μL of the reagent to each well, incubate at 37°C for 2 hours, and then measure the absorbance at 450 nm (OD450) with an enzyme-linked immunosorbent assay reader.

[0112] 2. Determination of NO release amount:

[0113] Collect the cell supernatant and operate according to the instructions of the Griess kit (Beyotime, S0021): Take 50 μL of the supernatant and mix it with an equal volume of Griess reagent, react in the dark at room temperature for 10 minutes, measure the absorbance at 540 nm, and calculate the NO concentration through the sodium nitrite standard curve.

[0114] 3. Detection of cytokines:

[0115] Use ELISA kits (IL-6: Abcam, ab222503; TNF-α: R&D Systems, DY410) to quantify the levels of IL-6 and TNF-α in the supernatant, and perform standard product dilution and sample detection according to the instructions.

[0116] 4. Gene expression analysis:

[0117] Use the TRIzol method to extract total cellular RNA, reverse transcribe it into cDNA (Takara, RR047A), and detect the expression of iNOS and COX-2 mRNA by real-time fluorescence quantitative PCR (qRT-PCR, ABI QuantStudio 5). Primer sequences:

[0118] iNOS F: 5'-CTGCAGCACTTGGATCAGGAACCTG-3' (SEQ ID NO.1)

[0119] iNOS R: 5'-GGGAGTAGCCTGTGTGCACCTGGAA-3' (SEQ ID NO.2)

[0120] COX-2 F: 5'-CACTACATCCTGACCCACTT-3' (SEQ ID NO.2)

[0121] COX-2 R: 5'-ATGCTCCTGCTTGAGTATGT-3' (SEQ ID NO.4)

[0122] β-actin F: 5'-GGCTGTATTCCCCTCCATCG-3' (SEQ ID NO.5)

[0123] β-actin R: 5'-CCAGTTGGTAACAATGCCATGT-3' (SEQ ID NO.6)

[0124] Table 2 Effects of Ganoderma triterpenoids on the immune activity of macrophages

[0125]

[0126]

[0127] The experimental results are shown in Table 2. Ganoderma triterpenoids can enhance the immune activity of macrophages in a dose-dependent manner. The 20 μg / mL dose group was close to the LPS-stimulated effect in terms of NO release, pro-inflammatory cytokine secretion, and iNOS expression, and there was no significant cytotoxicity (OD450 was not different from the blank group, p > 0.05). Immunofluorescence showed that the nuclear translocation rate of NF-κB p65 in the triterpenoid treatment group was 3.2 times higher than that in the blank group (p < 0.001), confirming that it exerts an immunomodulatory effect by activating the NF-κB pathway.

[0128] Test Example 3 Immunopotentiating Effect of Ganoderma Triterpenoids on Immunosuppressed Mice

[0129] 1. Establishment of animal model

[0130] SPF-grade male BALB / c mice (6 - 8 weeks old, 18 - 22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 6 groups (n = 10) after 1 week of adaptive feeding:

[0131] Normal control group: gavaged with normal saline, no drug administration

[0132] Model group: intraperitoneally injected with cyclophosphamide (CTX, 80 mg / kg) for 3 consecutive days and gavaged with normal saline

[0133] Low-dose triterpenoid group: intraperitoneally injected with cyclophosphamide (CTX, 80 mg / kg) for 3 consecutive days and gavaged with 50 mg / kg triterpenoids.

[0134] Medium-dose triterpenoid group: intraperitoneally injected with cyclophosphamide (CTX, 80 mg / kg) for 3 consecutive days and gavaged with 100 mg / kg triterpenoids.

[0135] High-dose triterpenoid group: intraperitoneally injected with cyclophosphamide (CTX, 80 mg / kg) for 3 consecutive days and gavaged with 200 mg / kg triterpenoids.

[0136] Positive control group: CTX treatment + ginsenoside (50 mg / kg, Solarbio)

[0137] 2. Sample collection and detection

[0138] The mice were sacrificed on the 18th day for the following analysis:

[0139] Immune organ index: The weights of the spleen and thymus were precisely weighed, and the organ index (mg / g body weight) was calculated.

[0140] Blood immune indexes: Blood was collected from the tail vein and collected in an EDTA anticoagulant tube. The total white blood cell count and lymphocyte ratio were detected using a blood routine analyzer (model Sysmex XN-1000). Serum was separated, and the levels of IgG, IgM, IL-2, and TNF-α in the serum were detected by ELISA method (IgG: Abcam, ab157718; IgM: Cusabio, CSB-E11109m).

[0141] Cell immune analysis: The spleen was taken to prepare a single cell suspension, and the CD4+ / CD8+ T cell subset was detected using a flow cytometer (BD FACSCanto II) (antibodies: BioLegend, 100406 / 100708).

[0142] Macrophage phagocytic function: India ink diluted 1:5 (0.1 mL / 10 g) was injected into the tail vein. Blood was taken from the orbital cavity 2 minutes later, and the absorbance (680 nm) was measured to calculate the phagocytic index K value.

[0143] Intestinal sIgA detection: Intestinal contents were taken, washed with PBS, centrifuged, and the supernatant was taken. The sIgA ELISA kit (Cusabio, CSB-E08314m) was used for quantitative detection.

[0144] Table 3 Immunopotentiating effects of ganoderic triterpenoids on immunosuppressed mice

[0145]

[0146]

[0147] The results are shown in Table 3. CTX-induced immunosuppression: Humoral immunity: In the 200 mg / kg group, serum IgG was restored to 95% of the normal level (760 vs 800 μg / mL, p>0.05); Cellular immunity: The CD4+ / CD8+ ratio was close to that of the normal group (1.5 vs 1.6, p>0.05); Intestinal immunity: The recovery rate of the small intestine sIgA level reached 95.2% (295 vs 310 μg / mL). Each dose of triterpenoids showed a dose-dependent increase, and some indicators were close to the effects of the positive group (ginsenoside group).

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. The basic principles and main features of the present invention have been described in the above specific implementation manners. On the basis of the present invention, some modifications or replacements can be made, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the present invention claimed.

Claims

1. A Ganoderma lucidum extract for enhancing immunity, characterized in that Prepared by the following method: S1. Raw material pretreatment: Dry and crush the Ganoderma lucidum fruiting body into a powder of 80 - 100 mesh; S2. Microwave-assisted extraction: Mix the Ganoderma lucidum powder with an ethanol-water mixed solvent at a liquid-solid ratio of 10:1 - 30:1, with the solvent concentration of 50 - 90% (v / v), and extract for 5 - 20 minutes under the conditions of a microwave power of 200 - 700 W and a temperature of 50 - 80 °C; After the extract is roughly filtered, press the residue and combine the filtrates; S3. Concentration and drying: Concentrate the filtrate by rotary evaporation to 1 / 5 - 1 / 10 of the original volume, and then obtain the Ganoderma triterpenoid extract by freeze-drying.

2. The ganoderma lucidum extract according to claim 1, wherein: In step S1, a stepwise temperature-rising drying process is adopted: the initial temperature is 50 °C, the temperature is raised by 5 °C every 2 hours, and finally dried to constant weight at 65 °C.

3. The Ganoderma lucidum extract according to claim 1, characterized in that: In step S2, the ethanol concentration is 60 - 80%, the microwave power is 400 - 600 W, the liquid-solid ratio is 20:1, the extraction time is 7 - 14 minutes, and the extraction temperature is dynamically controlled at 60 - 70 °C by an infrared sensor.

4. The Ganoderma lucidum extract according to claim 1, wherein: In step S2, the ethanol concentration is 75%, the microwave power is 500 W, the extraction time is 10 minutes, the liquid-solid ratio is 20:1, and the extraction temperature is controlled at 60 °C.

5. The Ganoderma lucidum extract according to claim 1, wherein: After the extract in step S2 is roughly filtered through a 200-mesh filter screen, apply a pressure of 0.1 - 0.3 MPa to the residue and press for 3 - 5 minutes, recover the solvent residual liquid and combine it with the initial filtrate.

6. The Ganoderma lucidum extract according to claim 1, wherein: The said extract can, by activating the NF-κB signaling pathway, increase the expression level of iNOS mRNA in macrophages to ≥5 times, and promote the ratio of CD4+ / CD8+ T cells to recover to 1.3 - 1.

7.

7. An immune-enhancing composition, characterized in that: Comprising the Ganoderma lucidum extract according to any one of claims 1 - 6, and a pharmaceutical or pharmaceutically acceptable excipient, wherein the extract accounts for 10 - 95% of the total weight of the composition; the excipient includes at least one of microcrystalline cellulose, magnesium stearate, and β-cyclodextrin.

8. The composition according to claim 7, wherein: The dosage form of the said composition is tablets, capsules, oral liquids or granules, and is used to improve the immunosuppressive state induced by cyclophosphamide.

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