Ganoderma lucidum spore oil component emulsion, preparation method thereof and application in anti-tumor and antioxidant products

Abstract

The present invention discloses a ganoderma spore oil component emulsion, a preparation method thereof and an application thereof in anti-tumor and antioxidant products, belonging to the field of pharmaceuticals. The preparation method is as follows: (1) Softening, emulsifying and breaking the wall of ganoderma spores to obtain an emulsion and spore wall shells; (2) Concentrating the emulsion to obtain concentrated solution A; (3) After initially enzymolyzing the spore wall shells with complex enzyme A, adding complex enzyme B for secondary enzymolysis, drying the filter residue and then extracting to obtain ganoderma spore oil; (4) Crushing ganoderma fruiting bodies, adding a solvent for soaking extraction, and concentrating the filtrate to obtain concentrated solution B; (5) Mixing concentrated solution A, ganoderma spore oil and concentrated solution B to obtain the ganoderma spore oil component emulsion. The complex enzyme A includes cellulase and chitinase, and the complex enzyme B includes papain, bromelain and α-amylase. The ganoderma spore oil component emulsion is prepared by the above preparation method. Compared with the prior art, the ganoderma spore oil component emulsion of the present invention has better anti-tumor and antioxidant effects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and particularly to a Ganoderma lucidum spore oil component emulsion, a preparation method thereof, and applications in anti-tumor and antioxidant products. Background Art

[0002] Ganoderma lucidum spore oil is an oily lipid extracted from broken Ganoderma lucidum spores by supercritical carbon dioxide fluid extraction technology, integrating various active ingredients of Ganoderma lucidum spores. Its main components include saturated fatty acids, unsaturated fatty acids, and a small amount of Ganoderma acids, etc., which have functions such as antioxidant and anti-cerebral ischemia. Literature: Guo Yuan, Zhou Yajie, Feng Peng, etc. Research progress on the anti-tumor activity mechanism of Ganoderma lucidum spore oil [J]. Smart Healthcare, 2018, 4(27): 49 - 51. records the research status of Ganoderma lucidum spore oil in terms of anti-tumor activity. The book: Shao Xingjun, Ding Dehua. Health Comes from the Source Ecosystem [M]. Shanghai: Shanghai Scientific and Technical Publishers, 2008. also records the anti-cancer efficacy of Ganoderma lucidum spore oil.

[0003] For related products containing Ganoderma lucidum spore oil, the prior art such as patent CN112043731A discloses a preparation method of a nanoemulsion of whole Ganoderma lucidum spore oil rich in Ganoderma acids, including the following steps: (1) Using whole Ganoderma lucidum spore powder as the raw material, after dry pressing and one-step granulation, it is placed in a supercritical CO2 extraction kettle for supercritical CO2 extraction to obtain light yellow whole Ganoderma lucidum spore oil rich in Ganoderma acids; (2) The whole Ganoderma lucidum spore oil is then made into a nanoemulsion of whole Ganoderma lucidum spore oil together with an auxiliary agent. It is pointed out in this invention that the whole Ganoderma lucidum spore oil is rich in the effective ingredient Ganoderma acid with anti-tumor effects, and the content of its representative component Ganoderma acid B is more than 3 times that of pure Ganoderma lucidum spore oil. The process of this invention is simple and convenient; the quality of the product is stable, and quality control is convenient, and it can be used in pharmaceuticals and foods.

[0004] Another example is patent CN113150867A, which discloses a preparation method of a Ganoderma lucidum extraction oil rich in Ganoderma triterpenes. This method is to granulate Ganoderma lucidum fine powder and broken Ganoderma lucidum spore powder separately to obtain Ganoderma lucidum fruit body granules and Ganoderma lucidum spore powder granules, and then dry the two obtained granules successively, mix them and carry out carbon dioxide supercritical extraction. Finally, the content of Ganoderma triterpenes in the prepared extraction oil is higher than that in Ganoderma lucidum spore oil, and it has significant anti-tumor pharmacological activity. However, there is still room for optimization in the preparation methods of Ganoderma lucidum spore oil-related products in the above patents, and the efficacy of the prepared products in terms of anti-tumor also needs to be improved. In view of the problems existing in Ganoderma lucidum spore oil component emulsions in the prior art, it is very necessary to find a Ganoderma lucidum spore oil component emulsion with better effects. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a Ganoderma lucidum spore oil component emulsion, a preparation method thereof, and applications in anti-tumor and antioxidant products.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides a method for preparing a Ganoderma lucidum spore oil component emulsion, comprising the following steps:

[0008] (1) Soften the Ganoderma lucidum spores, homogenize and emulsify to break the cell walls, and filter to obtain an emulsion and spore wall shells;

[0009] (2) Concentrate the emulsion to obtain concentrated liquid A;

[0010] (3) Enzymatically hydrolyze the spore wall shells with complex enzyme A. After the first enzymatic hydrolysis, add complex enzyme B for secondary enzymatic hydrolysis, inactivate the enzyme to obtain an enzymatic hydrolysis mixture, perform solid-liquid separation, dry the filter residue and then extract to obtain Ganoderma lucidum spore oil;

[0011] (4) Crush the Ganoderma lucidum fruiting bodies, add a solvent to soak, extract and concentrate to obtain concentrated liquid B;

[0012] (5) Mix concentrated liquid A, Ganoderma lucidum spore oil and concentrated liquid B to obtain a Ganoderma lucidum spore oil component emulsion.

[0013] In some embodiments, the softening in step (1) is soaking overnight with water, and the addition amount of water is 3 - 20 times the mass of the Ganoderma lucidum spores; preferably 5 - 10 times.

[0014] In some embodiments, the emulsifying and wall-breaking in step (1) is: using water as a solvent and performing high-pressure homogenization; preferably, the high-pressure homogenization is: the homogenization pressure is 60 - 120 MPa, the homogenization temperature is 30 - 70 °C, and the homogenization is performed at least once.

[0015] In some embodiments, the concentration in step (2) is concentrating to 10 - 40 of the weight of the Ganoderma lucidum spores, preferably 15 - 35%.

[0016] In some embodiments, the complex enzyme A in step (3) is 0.3 - 0.5% of the mass of the spore wall shells, and the complex enzyme B is 0.8 - 1% of the mass of the spore wall shells. Preferably, the complex enzyme A is 0.4% of the mass of the spore wall shells, and the complex enzyme B is 0.9% of the mass of the spore wall shells.

[0017] In some embodiments, the complex enzyme A in step (3) includes cellulase and chitinase, preferably, the weight ratio of cellulase to chitinase is (1 - 2):(2 - 3); the complex enzyme B includes papain, bromelain and α - amylase, preferably, the weight ratio of papain, bromelain and α - amylase is (2 - 4):(2 - 4):1.

[0018] In some embodiments, the conditions for the primary enzymatic hydrolysis in step (3) are as follows: temperature 40 - 65°C, time 1 - 5 h, pH 5 - 5.5.

[0019] In some embodiments, the conditions for the secondary enzymatic hydrolysis in step (3) are as follows: temperature 30 - 50°C, time 1 - 5 h, pH 6 - 7.

[0020] In some embodiments, the conditions for enzyme inactivation in step (3) are as follows: temperature 90 - 100°C, time 10 - 15 min, pressure 0.2 - 0.5 MPa.

[0021] In some embodiments, the extraction in step (3) is CO2 supercritical extraction. Preferably, the conditions for the extraction are as follows: extraction pressure 10 - 45 MPa, temperature 25 - 50°C.

[0022] In some embodiments, the weight of the Ganoderma lucidum spores in step (1) is the same as that of the Ganoderma lucidum fruiting body in step (4).

[0023] In some embodiments, the solvent in step (4) includes ethanol and / or propylene glycol. Preferably, the addition amount of the solvent is 5 - 20 times the weight of the Ganoderma lucidum fruiting body; more preferably 8 - 10 times.

[0024] In some embodiments, the soaking in step (4) is carried out at 25 - 30°C for more than 0.5 h; preferably 1 - 3 h.

[0025] In some embodiments, the extraction in step (4) is ultrasonic extraction for 0.5 - 2 h; preferably, the power of the ultrasonic extraction is 300 - 500 W.

[0026] In some embodiments, the concentration in step (4) is concentrated to 10 - 30% of the weight of the Ganoderma lucidum fruiting body; preferably 15 - 25%.

[0027] In some embodiments, the mass ratio of the concentrated liquid A, Ganoderma lucidum spore oil, and concentrated liquid B in step (5) is 1:1 - 6:1; preferably 1:3 - 6:1.

[0028] The solid-liquid separation involved in the steps of the present invention is a conventional operation means in the art, and its purpose is to separate the liquid phase and the solid phase, such as centrifugation, filtration, or pressure filtration.

[0029] The concentration involved in the steps of the present invention is a conventional operation means in the art, and its purpose is to reduce the water content in the liquid, such as evaporation concentration, membrane separation concentration, freeze concentration, extraction concentration, vacuum concentration. Preferably, the concentration is vacuum concentration, and the temperature is 50 - 80°C.

[0030] The drying involved in the steps of the present invention is a conventional drying method in the art, and the purpose is to control the water content not exceeding 10%. For example, spray drying, freeze drying, vacuum drying, baking or air drying. Preferably, the drying is spray drying, and the temperature is 30-70 °C.

[0031] In a second aspect, the present invention provides a Ganoderma lucidum spore oil component emulsion prepared by the above preparation method.

[0032] In a third aspect, the present invention provides the application of the above Ganoderma lucidum spore oil component emulsion in the preparation of anti-tumor and antioxidant products.

[0033] In a fourth aspect, the present invention provides an anti-tumor and antioxidant drug, comprising the above Ganoderma lucidum spore oil component emulsion.

[0034] The technical effects achieved by the present invention are:

[0035] Through the improvement of the preparation method, the Ganoderma lucidum spore oil component emulsion prepared by the present invention has better anti-tumor and antioxidant effects compared with the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Effects of concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1 on the activity of H9C2 cells. Compared with the model group, **P < 0.01.

[0037] Figure 2 Effects of concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1 on ABTS of H9C2 cells with oxidative stress injury. Compared with the normal group, *P < 0.05, compared with the model group, #P < 0.05.

[0038] Figure 3 Effects of concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1 on the expression level of Bcl-2 / Bax gene. Compared with the control group, *P < 0.05, compared with the model group, ***P < 0.001.

[0039] Figure 4 Effects of concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1 on the expression level of Caspase gene. Compared with the control group, **P < 0.01, compared with the model group, ****P < 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0040] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0041] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention.

[0042] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and the two endpoints themselves can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0043] It is worth noting that the raw materials used in the present invention are all ordinary commercially available products, so their sources are not specifically limited. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0044] Cellulase CAS: 9012-54-8, enzyme activity 500,000 U / g, chitinase CAS: 9001-06-3, enzyme activity 100,000 U / g, papain CAS: 9001-73-4, enzyme activity 1,000,000 U / g, bromelain CAS: 9001-00-7, enzyme activity 200,000 U / g, α-amylase CAS: 9000-90-2, enzyme activity 1,000,000 U / g. Unless otherwise specified, the temperature mentioned is room temperature, 20 - 25°C. The water content of the dried product in this embodiment is 5 - 8%. The concentration in this embodiment is carried out under reduced pressure (-0.08 mpa) at 60°C.

[0045] Example 1 A preparation method of Ganoderma lucidum spore oil component emulsion

[0046] The preparation method is as follows:

[0047] (1) Add 1 kg of Ganoderma lucidum spores to 7 times the amount of water of Ganoderma lucidum spores and soak overnight. Homogenize, emulsify, and break the wall (pressure 80 MPa, temperature 50°C, homogenize 2 times), and then filter to obtain an emulsion and spore wall shells.

[0048] (2) Concentrate the emulsion under reduced pressure to 30% of the weight of Ganoderma lucidum spores to obtain concentrated liquid A.

[0049] (3) Add compound enzyme A to the spore wall shell and enzymatically hydrolyze it at 50 °C for 2 h. After the initial enzymatic hydrolysis at pH 5 - 5.5, add compound enzyme B and perform secondary enzymatic hydrolysis at 45 °C for 3 h, with pH 6 - 7. Inactivate the enzyme at a temperature of 90 °C and a pressure of 0.2 MPa for 10 min, and filter the obtained enzymatic hydrolysis mixture. The obtained filter residue is dried and then subjected to CO2 supercritical extraction at a pressure of 22 MPa and a temperature of 35 °C to obtain ganoderma spore oil;

[0050] The compound enzyme A is 0.4% of the mass of the spore wall shell, and the compound enzyme B is 0.9% of the mass of the spore wall shell;

[0051] The compound enzyme A is a cellulase and a chitinase with a weight ratio of 3:5; the compound enzyme B is a papain, a bromelain, and an α - amylase with a weight ratio of 3:3:1.

[0052] (4) After crushing 1 kg of ganoderma fruit bodies, add ethanol (volume fraction 95%) in an amount 9 times that of the ganoderma fruit bodies, soak at 25 °C for 1 h, and then perform ultrasonic extraction (power 400 W) for 1 h. Filter, and concentrate the filtrate to 20% of the weight of the ganoderma fruit bodies to obtain concentrated solution B;

[0053] (5) Mix concentrated solution A, ganoderma spore oil, and concentrated solution B in a weight ratio of 1:5:1 to obtain a ganoderma spore oil component emulsion.

[0054] Example 2 A preparation method of a ganoderma spore oil component emulsion

[0055] The preparation method is as follows:

[0056] (1) Soak 1 kg of ganoderma spores in water in an amount 3 times that of the ganoderma spores overnight, and perform homogenization emulsification and cell wall breaking (pressure 120 MPa, temperature 30 °C, homogenization 2 times), and then filter to obtain an emulsion and spore wall shell;

[0057] (2) Concentrate the emulsion under reduced pressure to 10% of the weight of the ganoderma spores to obtain concentrated solution A;

[0058] (3) Add compound enzyme A to the spore wall shell and enzymatically hydrolyze it at 65 °C for 1 h. After the initial enzymatic hydrolysis at pH 5 - 5.5, add compound enzyme B and perform secondary enzymatic hydrolysis at 50 °C for 1 h, with pH 6 - 7. Inactivate the enzyme at a temperature of 90 °C and a pressure of 0.2 MPa for 10 min, and filter the obtained enzymatic hydrolysis mixture. The obtained filter residue is dried and then subjected to CO2 supercritical extraction at a pressure of 45 MPa and a temperature of 25 °C to obtain ganoderma spore oil;

[0059] The compound enzyme A is 0.5% of the mass of the spore wall shell, and the compound enzyme B is 0.8% of the mass of the spore wall shell;

[0060] The composite enzyme A is a cellulase and a chitinase with a weight ratio of 2:3; the composite enzyme B is a papain, a bromelain and an α-amylase with a weight ratio of 4:2:1.

[0061] (4) After crushing 1 kg of Ganoderma lucidum fruiting bodies, add ethanol (volume fraction 95%) at 20 times the amount of Ganoderma lucidum fruiting bodies, soak at 25 °C for 1 h, then extract by ultrasonic (power 300 W) for 2 h, filter, and concentrate the filtrate to 30% of the weight of Ganoderma lucidum fruiting bodies to obtain concentrated liquid B;

[0062] (5) Mix concentrated liquid A, Ganoderma lucidum spore oil and concentrated liquid B in a weight ratio of 1:1:1 to obtain a Ganoderma lucidum spore oil component emulsion.

[0063] Example 3 A preparation method of a Ganoderma lucidum spore oil component emulsion

[0064] The preparation method is as follows:

[0065] (1) Soak 1 kg of Ganoderma lucidum spores in water at 20 times the amount of Ganoderma lucidum spores overnight, homogenize and emulsify to break the cell wall (pressure 60 MPa, temperature 70 °C, homogenize 2 times), filter to obtain an emulsion and spore wall shells;

[0066] (2) Concentrate the emulsion under reduced pressure to 40% of the weight of Ganoderma lucidum spores to obtain concentrated liquid A;

[0067] (3) Add composite enzyme A to the spore wall shells, enzymatically hydrolyze at 40 °C for 5 h, after the first enzymatic hydrolysis at pH 5 - 5.5, add composite enzyme B, perform secondary enzymatic hydrolysis at 30 °C for 5 h, pH 6 - 7, inactivate the enzyme at a temperature of 90 °C and a pressure of 0.2 MPa for 10 min, filter the obtained enzymatic hydrolysis mixture, dry the obtained filter residue, and perform CO2 supercritical extraction at a pressure of 10 MPa and a temperature of 50 °C to obtain Ganoderma lucidum spore oil;

[0068] The composite enzyme A is 0.3% of the mass of the spore wall shells, and the composite enzyme B is 1% of the mass of the spore wall shells;

[0069] The composite enzyme A is a cellulase and a chitinase with a weight ratio of 1:2; the composite enzyme B is a papain, a bromelain and an α-amylase with a weight ratio of 2:4:1.

[0070] (4) After crushing 1 kg of Ganoderma lucidum fruiting bodies, add ethanol (volume fraction 95%) at 5 times the amount of Ganoderma lucidum fruiting bodies, soak at 30 °C for 0.5 h, then extract by ultrasonic (power 500 W) for 0.5 h, filter, and concentrate the filtrate to 10% of the weight of Ganoderma lucidum fruiting bodies to obtain concentrated liquid B;

[0071] (5) Mix concentrated liquid A, Ganoderma lucidum spore oil and concentrated liquid B in a weight ratio of 1:6:1 to obtain a Ganoderma lucidum spore oil component emulsion.

[0072] Comparative Example 1 Preparation method of a Ganoderma lucidum spore oil component emulsion

[0073] The difference between this comparative example and Example 1 is only that: cellulase and pectinase with a weight ratio of 3:5 are used to replace Complex Enzyme A, and papain is used to replace Complex Enzyme B.

[0074] The remaining steps are the same as those in Example 1.

[0075] Comparative Example 2 Preparation method of a Ganoderma lucidum spore oil component emulsion

[0076] The difference between this comparative example and Example 1 is only that: cellulase is used to replace Complex Enzyme A, and bromelain and α-amylase with a weight ratio of 3:1 are used to replace Complex Enzyme B.

[0077] The remaining steps are the same as those in Example 1.

[0078] Comparative Example 3 Preparation method of a Ganoderma lucidum spore oil component emulsion

[0079] The difference between this comparative example and Example 1 is only that: cellulase and chitinase with a weight ratio of 3:1 are used to replace Complex Enzyme A; papain, bromelain and α-amylase with a weight ratio of 6:1:3 are used to replace Complex Enzyme B.

[0080] The remaining steps are the same as those in Example 1.

[0081] Comparative Example 4 Preparation method of a Ganoderma lucidum spore oil component emulsion

[0082] The difference between this comparative example and Example 1 is only that: in step (5), the mass ratio of concentrated solution A, Ganoderma lucidum spore oil and concentrated solution B is 1:1:6.

[0083] The remaining steps are the same as those in Example 1.

[0084] Antitumor efficacy evaluation

[0085] Effect Example 1 Determination of the antitumor activity of the Ganoderma lucidum spore oil component emulsion at the 2D level

[0086] 1. Experimental instruments and reagents

[0087] The microplate reader is SpectraMax iD3 (MD Company); the ultra-micro ultraviolet-visible spectrophotometer is NanoDrop one (Thermo Company); the automatic cell counter is CountessⅡ (Thermo Company); McCoy's 5A medium; penicillin-streptomycin; 0.25% trypsin is purchased from Corning Company; fetal bovine serum (FBS) is purchased from Sorfa Company; the HCT116 cells used in the experiment are purchased from Enzyme Research Biology;

[0088] CCK8 Detection Kit (Cat. No. C0046).

[0089] 2. Experimental Methods

[0090] The HCT116 cells in the bottle were blown with 5 mL of DMEM medium, transferred to a 15 mL centrifuge tube, and centrifuged at 1000 rpm for 5 min. The cells were counted and the cells were plated in a 96-well plate at a cell density of 10,000 / well. After 24 hours, the concentrate A, Ganoderma lucidum spore oil and concentrate B were added to the cells at appropriate concentrations, and 4 blank groups were set up in each plate. Six concentrations were set for each extract and Ganoderma lucidum spore oil component emulsion (concentrate A, Ganoderma lucidum spore oil and concentrate B were mixed in a weight ratio of 1:1:1), and the sample concentrations were set to 100, 50, 25, 12.5, 6.25, and 3.125 (unit: μg / mL), respectively, and 4 replicates were set for each group. Incubate at 37°C for 24 hours. Detect according to the instructions of the CCK8 detection kit. Discard the supernatant and add 100 μL of the prepared CCK8 working solution (culture medium: CCK8 reagent = 9:1) to each well. Protect from light, incubate at 37°C for 2 h, and then detect using an enzyme-labeled instrument (detection wavelength: 450 nm). Finally, calculate the IC50 value of each drug group.

[0091] 3. Results

[0092] Table 1 2D tumor cell killing effects of concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1

[0093] Sample Name IC50 (μg / mL) Concentrate A / Ganoderma lucidum spore oil / Concentrate B 57.85 Ganoderma lucidum spore oil mixed emulsion 35.61

[0094] The experimental results showed that concentrate A and Ganoderma lucidum spore oil had no good cell killing effect at the highest dosage concentration, and the IC50 value could not be calculated. The IC50 value of concentrate B was 57.85μg / mL, and the IC50 value of Ganoderma lucidum spore oil mixed emulsion was 35.61μg / mL, which were much lower than those of the single-dose group.

[0095] Effect Example 2 Determination of the antitumor activity of Ganoderma lucidum spore oil emulsion based on 2D level

[0096] Experimental method: The tumor cell proliferation experiment was carried out by CCK-8 method to compare the effects of concentrated solution B, concentrated solution A and Ganoderma lucidum spore oil used alone and in combination on tumor cell proliferation. The experimental method was the same as that of effect example 1.

[0097] Table 2 Proliferation inhibition rate of HCT116 cells by concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1

[0098] Concentrate B (μg / mL) Concentrate A (μg / mL) Ganoderma lucidum spore oil (μg / mL) Inhibition rate / % 50 0 0 45.37 0 50 0 21.61 0 0 50 12.63 25 25 0 61.35 25 0 25 58.36 0 25 25 20.29 16.7 16.7 16.7 79.37

[0099] The experimental results show that: after fixing the total administration concentration at 50 μg / mL, the inhibition rates of concentrated solution A or ganoderma spore oil alone or in combination did not change significantly. When concentrated solution B was combined with concentrated solution A or ganoderma spore oil, the inhibition rate increased slightly. However, when the three were used in combination, the inhibitory effect on the proliferation of HCT116 cells increased significantly.

[0100] Effect Example 3: Determination of the anti-tumor activity of the ganoderma spore oil component emulsion based on the 2D level

[0101] Experimental method: The tumor cell proliferation experiment was carried out by the CCK-8 method to compare the effects of the ganoderma spore oil component emulsions prepared in different examples and comparative examples on tumor cell proliferation. The experimental method was the same as that in Effect Example 1.

[0102] Table 3: Proliferation inhibition rates of the ganoderma spore oil component emulsions prepared in different examples and comparative examples on HCT116 cells

[0103] Concentration (μg / mL) Inhibition rate / % Example 1 50 79.37 Example 2 50 71.21 Example 3 50 69.39 Comparative Example 1 50 43.21 Comparative Example 2 50 39.16 Comparative Example 3 50 41.28 Comparative Example 4 50 47.05 Commercially available Ganoderma lucidum spore oil 50 46.39

[0104] The experimental results show that: after fixing the total administration concentration at 50 μg / mL, the ganoderma spore oil component emulsions provided in Examples 1-3 had a good inhibitory effect on the proliferation of HCT116 cells. However, the ganoderma spore oil component emulsions provided in Comparative Examples 1-4 had a significantly worse inhibitory effect on the proliferation of HCT116 cells. This indicates that the change of enzymes and the ratio of concentrated solution B, concentrated solution A and ganoderma spore oil in the preparation process have a greater impact on the inhibitory effect on the proliferation of HCT116 cells.

[0105] Evaluation of antioxidant efficacy

[0106] Effect Example 4: Determination of the cell activity of the ganoderma spore oil component emulsion

[0107] 1. Experimental instruments and reagents

[0108] The microplate reader was SpectraMax iD3 (MD Company); the H9C2 cells used in the experiment were purchased from Enzyme Research Biology. The CCK8 detection kit (product number: C0046) and the total antioxidant capacity detection kit (product number: S0119) were purchased from Beyotime.

[0109] 2. Experimental method

[0110] The H9C2 cells in the flask were blown down with 6 mL of DMEM / F12 medium and transferred to a 15 mL centrifuge tube, and centrifuged at 1000 rpm for 5 min. Concentrate A, Ganoderma spore oil, concentrate B, and the Ganoderma spore oil component emulsion were added to the cells at appropriate concentrations, and 4 blank wells were additionally set for each plate. A total of seven concentrations were set for the experimental groups, namely 20, 5, 2.5, 1.25, 0.625, 0.3125 (unit: mg / mL), and 4 replicate wells were set for each group. After incubation at 37 °C for 24 h, cell viability was detected according to the instructions of the CCK8 detection kit: discard the supernatant, and add 100 μL of the prepared CCK8 working solution (medium: CCK8 reagent = 9:1) to each well. After incubation at 37 °C for 3 h in the dark, detection was performed using an enzyme-linked immunosorbent assay (detection wavelength: 450 nm). The IC50 of the Ganoderma matrix extract was calculated based on the detection results, and the safe administration concentration of the Ganoderma spore powder extract was determined.

[0111] 3. Results

[0112] The experimental results showed that when the concentration of concentrate A was higher than 2.5 mg / mL, the cell survival rate began to decrease, the IC50 was 8.289 mg / mL, and the concentration was set at 2.5 mg / mL; when the concentration of Ganoderma spore oil was higher than 2.5 mg / mL, the cell survival rate began to decrease, the IC50 was 4.808 mg / mL, and the concentration was set at 2.5 mg / mL; when the concentration of concentrate B was higher than 0.025 mg / mL, the cell survival rate began to decrease, the IC50 was 0.3632 mg / mL, and the concentration was set at 0.025 mg / mL; when the concentration of the Ganoderma spore oil mixed emulsion was higher than 5 mg / mL, the cell survival rate began to decrease, the IC50 was 18.47 mg / mL, and the concentration was set at 5 mg / mL.

[0113] Table 4 Cell viability determination of concentrate A, Ganoderma spore oil, concentrate B, and the Ganoderma spore oil component emulsion prepared in Example 1

[0114]

[0115] Effect Example Five: Anti-H2O2 Oxidative Stress Damage Effect of the Ganoderma Spore Oil Component Emulsion on H9C2 Cardiomyocytes

[0116] Experimental method: H9C2 cells with good growth status and a confluence of 90% were inoculated into cell culture plates and cultured at 37°C under 5% CO2 conditions. The cells were divided into a blank control group (conventional culture), a model group (conventional culture until the cells reached 80%, and then used to establish a model. Modeling conditions: add 250 μmol / L H2O2 for 4 h of intervention), and drug administration groups with low, medium, and high doses (after the cells in conventional culture reached 80%, after pretreatment with different doses of drugs for 24 h, add 250 μmol / L H2O2 to stimulate the cells for 4 h). After culturing the cells for 4 h, the cells were lysed and samples from each group were collected. According to the total antioxidant capacity detection kit from Beyotime, the antioxidant activities of the above samples were measured.

[0117] The results are as Figure 1 shown in Table 5. Compared with the blank control group (CON), the cell viability of the model group (H2O2) was significantly decreased (P < 0.01). Compared with the model group (H2O2), concentrated solution B could not improve the antioxidant activity (P > 0.05), while concentrated solution A, ganoderma spore oil, and the emulsion of ganoderma spore oil components could all significantly increase the cell viability (P < 0.01). Among them, the emulsion of ganoderma spore oil components had the strongest anti-H2O2 oxidative stress damage effect on H9C2 cardiomyocytes.

[0118] Table 5 Effects of concentrated solution A, ganoderma spore oil, concentrated solution B, and the emulsion of ganoderma spore oil components prepared in Example 1 on the activity of H9C2 cells

[0119]

[0120] Effect Example Six: Evaluation of the total antioxidant activity of the emulsion of ganoderma spore oil components

[0121] The effects of each drug on ABTS of H9C2 cells with oxidative stress damage were measured using a kit. The results are shown in Table 6 and Figure 2 as follows. Compared with the control group, the antioxidant activity of the cells in the model group was significantly decreased. Compared with the model group, the emulsion of ganoderma spore oil components significantly increased the antioxidant activity.

[0122] Table 6 Effects of concentrated solution A, ganoderma spore oil, concentrated solution B, and the emulsion of ganoderma spore oil components prepared in Example 1 on ABTS of H9C2 cells with oxidative stress damage

[0123]

[0124] Effect Example Seven: Effects of the emulsion of ganoderma spore oil components on the expression of apoptosis-related mRNA in H9C2 cells

[0125] Experimental method: The expression levels of Bcl-2, Bax, and Caspase-3 genes were detected by RealTime PCR.

[0126] The Bcl-2 family proteins are divided into three subfamilies: Bcl-2, Bax, and BH3. Among them, Bcl-2 is an anti-apoptotic protein, and Bax is a pro-apoptotic protein. They are key factors in cell growth. The Caspase family plays an essential role in apoptosis, and Caspase-3 is the executor of Caspase.

[0127] The results are shown in Table 7 and Figure 3 - Figure 4 as follows. Compared with the blank group (CON), the expression of Bcl-2 / Bax in the model group was significantly down-regulated (P<0.01); compared with the model group (H2O2), the Ganoderma lucidum spore oil component emulsion could significantly up-regulate the expression of Bcl-2 / Bax, indicating that cell apoptosis could be inhibited by up-regulating the expression of Bcl-2 / Bax. Compared with the blank group, the expression of Caspase-3 in the model group was significantly up-regulated; compared with the model group, the Ganoderma lucidum spore oil component emulsion significantly down-regulated the expression of Caspase-3, indicating that cell apoptosis might be inhibited by down-regulating the expression of Caspase-3. Table 7 Effects of the concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B, and Ganoderma lucidum spore oil component emulsion prepared in Example 1 on the expression of apoptosis factors in H9C2 cells induced by H2O2

[0128]

[0129] Effect Example Eight: Evaluation of the total antioxidant activity of the Ganoderma lucidum spore oil component emulsion

[0130] The effects of each drug on ABTS of H9C2 cells with oxidative stress injury were measured using a kit. The results are shown in Table 8.

[0131] The experimental results showed that after fixing the total drug concentration at 5 mg / mL, the Ganoderma lucidum spore oil component emulsions provided in Examples 1-3 all significantly improved the antioxidant activity. However, the Ganoderma lucidum spore oil component emulsions provided in Comparative Examples 1-4 did not significantly improve the antioxidant activity. It indicates that the change of enzymes and the ratio of the concentrated solution A, concentrated solution B, and Ganoderma lucidum spore oil in the preparation process have a great influence on the antioxidant activity of the Ganoderma lucidum spore oil component emulsion.

[0132] Table 8 Effects of the Ganoderma lucidum spore oil component emulsions prepared in different examples and comparative examples on ABTS of H9C2 cells with oxidative stress injury

[0133]

[0134] Note: Promotion rate (%) = (experimental group - model group) ÷ (blank control group - model group) × 100%.

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A preparation method of a ganoderma spore oil component emulsion, characterized in that: It includes the following steps: (1) Soften Ganoderma lucidum spores, homogenize and emulsify to break the cell walls, and perform solid-liquid separation to obtain an emulsion and spore wall shells; (2) Concentrate the emulsion to obtain concentrated liquid A; (3) Enzymatically hydrolyze the spore wall shells with complex enzyme A. After the primary enzymatic hydrolysis, add complex enzyme B for secondary enzymatic hydrolysis, inactivate the enzyme, obtain an enzymatic hydrolysis mixture, perform solid-liquid separation, extract the dried filter residue, and obtain Ganoderma lucidum spore oil; (4) Crush Ganoderma lucidum fruiting bodies, add a solvent to soak and extract, perform solid-liquid separation, and concentrate the filtrate to obtain concentrated liquid B; (5) Mix concentrated liquid A, Ganoderma lucidum spore oil, and concentrated liquid B to obtain a Ganoderma lucidum spore oil component emulsion; In step (3), the complex enzyme A is cellulase and chitinase, and the complex enzyme B is papain, bromelain, and α-amylase; the weight ratio of cellulase to chitinase is (1-2):(2-3), and the weight ratio of papain, bromelain, and α-amylase is (2-4):(2-4):1; In step (5), the mass ratio of concentrated liquid A, Ganoderma lucidum spore oil, and concentrated liquid B is 1:1-6:

1.

2. The preparation method according to claim 1, characterized in that: The weight of the Ganoderma lucidum spores in step (1) is the same as that of the Ganoderma lucidum fruiting bodies in step (4).

3. The preparation method according to claim 1, wherein: In step (2), the concentration is to concentrate to 10-40% of the weight of Ganoderma lucidum spores. In step (3), the complex enzyme A is 0.3-0.5% of the mass of the spore wall shells, the complex enzyme B is 0.8-1% of the mass of the spore wall shells. In step (4), the concentration is to concentrate to 10-30% of the weight of Ganoderma lucidum fruiting bodies.

4. The preparation method according to claim 3, characterized in that: In step (2), the concentration is to concentrate to 15-35% of the weight of Ganoderma lucidum spores. In step (4), the concentration is to concentrate to 15-25% of the weight of Ganoderma lucidum fruiting bodies.

5. The preparation method according to claim 1, characterized in that: The conditions for the primary enzymatic hydrolysis in step (3) are: temperature 40-65°C, time 1-5 h, pH 5-5.5; The conditions for the secondary enzymatic hydrolysis in step (3) are: temperature 30-50°C, time 1-5 h, pH 6-7; The extraction in step (3) is CO2 supercritical extraction.

6. The preparation method according to claim 1, characterized in that: The solvent in step (4) includes ethanol and / or propylene glycol; The extraction is ultrasonic extraction.

7. A Ganoderma lucidum spore oil component emulsion prepared by the preparation method according to any one of claims 1-6.

8. Use of a Ganoderma lucidum spore oil component emulsion prepared by the preparation method according to any one of claims 1-6 in the preparation of anti-colorectal cancer drugs.

9. Use of a Ganoderma lucidum spore oil component emulsion prepared by the preparation method according to any one of claims 1-6 in the preparation of antioxidant products.

10. A pharmaceutical composition for anti-colorectal cancer and antioxidant, characterized in that, It includes a Ganoderma lucidum spore oil component emulsion prepared by the preparation method according to any one of claims 1-6.

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