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

Abstract

The invention discloses a ganoderma lucidum spore oil component emulsion, a preparation method thereof and application of the ganoderma lucidum spore oil component emulsion in anti-tumor and anti-oxidation products, and belongs to the field of medicines.The preparation method comprises the steps that 1, ganoderma lucidum spores are softened, emulsified and subjected to wall breaking, and emulsion and spore wall shells are obtained; (2) concentrating the emulsion to obtain a concentrated solution A; (3) performing primary enzymolysis on the spore wall shells by using a compound enzyme A, adding a compound enzyme B for secondary enzymolysis, drying filter residues, and extracting to obtain ganoderma lucidum spore oil; (4) crushing the ganoderma lucidum sporocarp, adding a solvent for soaking and extracting, and concentrating the filtrate to obtain a concentrated solution B; (5) mixing the concentrated solution A, the ganoderma lucidum spore oil and the concentrated solution B to obtain a ganoderma lucidum spore oil component emulsion; the compound enzyme A comprises cellulase and chitinase, and the compound enzyme B comprises papain, bromelain and alpha-amylase. The ganoderma lucidum spore oil component emulsion is prepared by the preparation method. Compared with the prior art, the ganoderma lucidum spore oil component emulsion disclosed by the invention has better anti-tumor and anti-oxidation effects.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to a ganoderma lucidum spore oil component emulsion, a preparation method thereof and application thereof in anti-tumor and anti-oxidation 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. It integrates multiple active ingredients of Ganoderma lucidum spores. Its main components are saturated fatty acids, unsaturated fatty acids and a small amount of ganoderic acid, etc., which have antioxidant and anti-cerebral ischemia effects. Literature: Guo Yuan, Zhou Yajie, Feng Peng, et al. Research progress on the anti-tumor activity mechanism of Ganoderma lucidum spore oil [J]. Smart Health, 2018, 4 (27): 49-51. Records the research status of Ganoderma lucidum spore oil in terms of anti-tumor activity. Books: Shao Xingjun, Ding Dehua. Health comes from source ecology [M]. Shanghai: Shanghai Science and Technology Press, 2008. It also records the anti-cancer effect of Ganoderma lucidum spore oil.

[0003] Regarding the related products containing Ganoderma lucidum spore oil, the prior art, such as patent CN112043731A, discloses a method for preparing a whole Ganoderma lucidum spore oil nanoemulsion rich in ganoderic acid, comprising the following steps: (1) using whole Ganoderma lucidum spore powder as raw material, granulating it by dry pressing, and placing it in a supercritical CO 2 Extraction kettle, supercritical CO 2 Extraction to obtain light yellow whole Ganoderma lucidum spore oil rich in ganoderic acid; (2) the whole Ganoderma lucidum spore oil is then prepared into whole Ganoderma lucidum spore oil nanoemulsion together with an auxiliary agent. The invention points out that whole Ganoderma lucidum spore oil is rich in ganoderic acid, an effective ingredient with anti-tumor effect, and the content of its representative ingredient ganoderic acid B is more than 3 times that of pure Ganoderma lucidum spore oil. The process of the invention is simple and convenient; the quality of the product is stable, and the quality control is convenient, and it can be used in medicine and food.

[0004] For another example, patent CN113150867A discloses a method for preparing a ganoderma lucidum extract oil rich in ganoderma lucidum triterpenoids, wherein the method comprises first granulating ganoderma lucidum fine powder and broken wall ganoderma lucidum spore powder separately to obtain ganoderma lucidum fruiting body particles and ganoderma lucidum spore powder particles, then drying the obtained two particles in turn, mixing and subjecting them to supercritical extraction with carbon dioxide, and finally making the ganoderma lucidum triterpenoid content of the obtained extract oil higher than that of ganoderma lucidum triterpenoid content in ganoderma lucidum spore oil, and having significant anti-tumor pharmacological activity. However, the preparation method of ganoderma lucidum spore oil-related products in the above patents still has room for optimization, and the efficacy of the obtained products in anti-tumor aspects also needs to be improved. In view of the problems existing in the ganoderma lucidum spore oil component emulsion in the prior art, it is very necessary to find a ganoderma lucidum spore oil component emulsion with better effect. Summary of the invention

[0005] The present invention aims at solving the problems existing in the prior art and provides a ganoderma lucidum spore oil component emulsion, a preparation method thereof and application thereof in anti-tumor and anti-oxidation products.

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

[0007] In one aspect, the present invention provides a method for preparing a ganoderma lucidum spore oil component emulsion, comprising the following steps:

[0008] (1) softening the ganoderma lucidum spores, homogenizing and emulsifying them to break the spore wall, and filtering them to obtain an emulsion and spore wall shells;

[0009] (2) concentrating the emulsion to obtain a concentrated solution A;

[0010] (3) hydrolyzing the spore wall shell with compound enzyme A, adding compound enzyme B after the initial hydrolysis, performing secondary hydrolysis, inactivating the enzyme, obtaining an enzymatic hydrolysis mixture, separating the solid from the liquid, drying the filtered residue, and extracting to obtain Ganoderma lucidum spore oil;

[0011] (4) crushing the Ganoderma lucidum fruiting bodies, adding a solvent for immersion extraction, and concentrating to obtain a concentrated solution B;

[0012] (5) The concentrated solution A, the ganoderma spore oil and the concentrated solution B are mixed to obtain a ganoderma spore oil component emulsion.

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

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

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

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

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

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

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

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

[0021] In some embodiments, the extraction in step (3) is CO 2 Supercritical extraction, preferably, the extraction conditions are: extraction pressure 10-45MPa, temperature 25-50°C.

[0022] In some embodiments, the weight of the Ganoderma lucidum spores in step (1) and the Ganoderma lucidum fruiting bodies in step (4) are the same.

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

[0024] In some embodiments, the soaking in step (4) is 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 0.5-2h; preferably, the power of the ultrasonic extraction is 300-500W.

[0026] In some embodiments, the concentration in step (4) is to concentrate 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 solution A, the Ganoderma lucidum spore oil and the concentrated solution 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 method in the art, and its purpose is to achieve separation of liquid and solid phases, such as centrifugation, filtration or filter pressing.

[0029] The concentration involved in the steps of the present invention is a conventional operation method 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, and vacuum concentration. Preferably, the concentration is vacuum concentration at a temperature of 50-80°C.

[0030] The drying involved in the steps of the present invention is a conventional drying method in the art, the purpose of which is to control the water content to not exceed 10%, such as spray drying, freeze drying, vacuum drying, oven drying or air drying, preferably, the drying is spray drying at a temperature of 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 use of the above-mentioned 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-mentioned Ganoderma lucidum spore oil component emulsion.

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

[0035] The present invention improves the preparation method to prepare the ganoderma spore oil component emulsion. Compared with the prior art, the ganoderma spore oil component emulsion of the present invention has better anti-tumor and anti-oxidation effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 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 activity of H9C2 cells, compared with the model group, **P<0.01.

[0037] Figure 2 The 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 ABTS of H9C2 cells damaged by oxidative stress were compared with the normal group, *P<0.05, and compared with the model group, #P<0.05.

[0038] Figure 3 The 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 level of Bcl-2 / Bax gene were compared with the control group *P<0.05, and compared with the model group ***P<0.001.

[0039] Figure 4The 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 level of Caspase gene were **P<0.01 compared with the control group, and ****P<0.0001 compared with the model group. DETAILED DESCRIPTION

[0040] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways 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 scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0042] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

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

[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 million U / g, bromelain CAS: 9001-00-7, enzyme activity 200,000 U / g, α-amylase CAS: 9000-90-2, enzyme activity 1 million U / g, unless otherwise specified, the temperature is room temperature, 20-25°C, the moisture content of the dried product in this embodiment is 5-8%, and the concentration in this embodiment is concentrated under reduced pressure (-0.08 MPa) at 60°C.

[0045] Example 1 A method for preparing a ganoderma lucidum spore oil component emulsion

[0046] The preparation method is:

[0047] (1) adding 1 kg of Ganoderma lucidum spores to 7 times the amount of water as the Ganoderma lucidum spores and soaking overnight, homogenizing and emulsifying to break the spore wall (pressure 80 MPa, temperature 50° C., homogenizing twice), filtering to obtain an emulsion and spore wall shell;

[0048] (2) concentrating the emulsion under reduced pressure to 30% of the weight of the Ganoderma lucidum spores to obtain a concentrated solution A;

[0049] (3) Adding compound enzyme A to the spore wall shell, performing enzymolysis at 50°C for 2 h, pH 5-5.5, after the initial enzymolysis, adding compound enzyme B, performing secondary enzymolysis at 45°C for 3 h, pH 6-7, inactivating the enzyme at 90°C and 0.2 MPa for 10 min, filtering the obtained enzymolysis mixture, drying the obtained filter residue, and subjecting the obtained filter residue to CO 200 at a pressure of 22 MPa and a temperature of 35°C. 2 Supercritical extraction to obtain Ganoderma lucidum 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 complex enzyme A comprises cellulase and chitinase in a weight ratio of 3:5; and the complex enzyme B comprises papain, bromelain and α-amylase in a weight ratio of 3:3:1.

[0052] (4) After crushing 1 kg of Ganoderma lucidum fruiting body, adding 9 times the amount of ethanol (volume fraction 95%) of the Ganoderma lucidum fruiting body, soaking at 25° C. for 1 hour, ultrasonic extraction (power 400 W) for 1 hour, filtering, and concentrating the filtrate to 20% of the weight of the Ganoderma lucidum fruiting body to obtain a concentrated solution B;

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

[0054] Example 2 A method for preparing a ganoderma lucidum spore oil component emulsion

[0055] The preparation method is:

[0056] (1) adding 1 kg of Ganoderma lucidum spores to 3 times the amount of water as the Ganoderma lucidum spores and soaking overnight, homogenizing and emulsifying to break the spore wall (pressure 120 MPa, temperature 30° C., homogenizing twice), filtering to obtain an emulsion and spore wall shell;

[0057] (2) concentrating the emulsion under reduced pressure to 10% of the weight of the Ganoderma lucidum spores to obtain a concentrated solution A;

[0058] (3) Adding compound enzyme A to the spore wall shell, performing enzymolysis at 65°C for 1 hour, pH 5-5.5, after the initial enzymolysis, adding compound enzyme B, performing secondary enzymolysis at 50°C for 1 hour, pH 6-7, inactivating the enzyme at 90°C and 0.2 MPa for 10 minutes, filtering the obtained enzymolysis mixture, drying the obtained filter residue, and subjecting the obtained filter residue to CO 2 at 45 MPa and 25°C. 2 Supercritical extraction to obtain Ganoderma lucidum 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 complex enzyme A is cellulase and chitinase in a weight ratio of 2:3; the complex enzyme B is papain, bromelain and alpha-amylase in a weight ratio of 4:2:1.

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

[0062] (5) The concentrated solution A, the Ganoderma lucidum spore oil and the concentrated solution B were mixed in a weight ratio of 1:1:1 to obtain a Ganoderma lucidum spore oil component emulsion.

[0063] Example 3 Preparation method of a ganoderma lucidum spore oil component emulsion

[0064] The preparation method is:

[0065] (1) adding 1 kg of Ganoderma lucidum spores to 20 times the amount of water as the Ganoderma lucidum spores and soaking overnight, homogenizing and emulsifying to break the spore wall (pressure 60 MPa, temperature 70° C., homogenizing twice), filtering to obtain an emulsion and spore wall shell;

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

[0067] (3) Adding compound enzyme A to the spore wall shell, performing enzymolysis at 40°C for 5 h, pH 5-5.5, after the initial enzymolysis, adding compound enzyme B, performing secondary enzymolysis at 30°C for 5 h, pH 6-7, inactivating the enzyme at 90°C and 0.2 MPa for 10 min, filtering the obtained enzymolysis mixture, drying the obtained filter residue, and subjecting the obtained filter residue to CO 2 at 10 MPa and 50°C. 2 Supercritical extraction to obtain Ganoderma lucidum spore oil;

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

[0069] The complex enzyme A is cellulase and chitinase in a weight ratio of 1:2; the complex enzyme B is papain, bromelain and alpha-amylase in a weight ratio of 2:4:1.

[0070] (4) After crushing 1 kg of Ganoderma lucidum fruiting body, add 5 times the amount of ethanol (volume fraction 95%) of the Ganoderma lucidum fruiting body, 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 the Ganoderma lucidum fruiting body to obtain concentrated solution B;

[0071] (5) The concentrated solution A, the Ganoderma lucidum spore oil and the concentrated solution B were mixed 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 that cellulase and pectinase in a weight ratio of 3:5 are used instead of compound enzyme A, and papain is used instead of compound 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 only difference between this comparative example and Example 1 is that cellulase is used instead of complex enzyme A, and bromelain and α-amylase in a weight ratio of 3:1 are used instead of 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 that: the complex enzyme A is replaced by cellulase and chitinase in a weight ratio of 3:1; and the complex enzyme B is replaced by papain, bromelain and α-amylase in a weight ratio of 6:1:3.

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

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

[0084] Anti-tumor efficacy evaluation

[0085] Effect Example 1: Determination of the antitumor activity of Ganoderma lucidum spore oil emulsion based on 2D level

[0086] 1. Experimental instruments and reagents

[0087] The microplate reader was SpectraMax iD3 (MD Company); the ultra-micro-volume UV-visible spectrophotometer was NanoDrop one (Thermo Company); the fully automatic cell counter was CountessⅡ (Thermo Company); McCoy's 5A culture medium; penicillin-streptomycin; 0.25% trypsin were purchased from Corning Company; fetal bovine serum (FBS) was purchased from Sorfa Company; HCT116 cells used in the experiment were purchased from Yumeiyan Biological;

[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 showed that after a fixed total drug concentration of 50 μg / mL, there was no significant change in the inhibition rate of concentrate A or Ganoderma lucidum spore oil when used alone or in combination. The inhibition rate was slightly increased when concentrate B was used in combination with concentrate A or Ganoderma lucidum spore oil. However, when the three were used in combination, the proliferation inhibitory effect on HCT116 cells was significantly increased.

[0100] Effect Example 3 Determination of the antitumor activity of Ganoderma lucidum spore oil emulsion based on 2D level

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

[0102] Table 3 Proliferation inhibition rate of Ganoderma lucidum 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 the total administration concentration is fixed at 50 μg / mL, the emulsion of the ganoderma spore oil component provided in Examples 1-3 has a good inhibitory effect on the proliferation of HCT116 cells. However, the emulsion of the ganoderma spore oil component provided in Comparative Examples 1-4 has a significantly worse inhibitory effect on the proliferation of HCT116 cells. This indicates that the changes in the enzymes during the preparation process and the ratio of the concentrate B, concentrate A and ganoderma spore oil have a greater impact on the inhibitory effect on the proliferation of HCT116 cells.

[0105] Antioxidant efficacy evaluation

[0106] Effect Example 4: Cell Activity Determination of Ganoderma Lucidum Spore Oil Component Emulsion

[0107] 1. Experimental instruments and reagents

[0108] The microplate reader was SpectraMax iD3 (MD Company); H9C2 cells used in the experiment were purchased from Enzyme Research Biotechnology. CCK8 detection kit (Cat. No.: C0046) and total antioxidant capacity detection kit (Cat. No.: S0119) were purchased from Bio-Tech.

[0109] 2. Experimental Methods

[0110] The H9C2 cells in the bottle were blown with 6mL DMEM / F12 culture medium, transferred to a 15mL centrifuge tube, and centrifuged at 1000rpm for 5min. Concentrate A, Ganoderma lucidum spore oil, concentrate B, and Ganoderma lucidum spore oil component emulsion were added to the cells at appropriate concentrations, and 4 blank groups were set up in each plate. There were seven concentrations in the experimental group, namely 20, 5, 2.5, 1.25, 0.625, and 0.3125 (unit: mg / mL), and 4 replicates were set up in each group. After incubation at 37℃ for 24h, cell activity was detected according to the instructions of the CCK8 detection kit: the supernatant was discarded, and 100μL of the prepared CCK8 working solution (culture medium: CCK8 reagent = 9:1) was added to each well. Protect from light, incubate at 37℃ for 3h, and then detect with an enzyme reader (detection wavelength: 450nm). According to the test results, the IC50 of the Ganoderma lucidum matrix extract was calculated, and the safe administration concentration of the Ganoderma lucidum 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 decline, the IC50 was 8.289 mg / mL, and the concentration was set to 2.5 mg / mL; when the concentration of Ganoderma lucidum spore oil was higher than 2.5 mg / mL, the cell survival rate began to decline, the IC50 was 4.808 mg / mL, and the concentration was set to 2.5 mg / mL; when the concentration of concentrate B was higher than 0.025 mg / mL, the cell survival rate began to decline, the IC50 was 0.3632 mg / mL, and the concentration was set to 0.025 mg / mL; when the concentration of Ganoderma lucidum spore oil mixed emulsion was higher than 5 mg / mL, the cell survival rate began to decline, the IC50 was 18.47 mg / mL, and the concentration was set to 5 mg / mL.

[0113] Table 4 Cell activity determination of concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1

[0114]

[0115] Effect Example 5: Anti-H effect of Ganoderma lucidum spore oil emulsion on H9C2 cardiomyocytes 2 O 2 Oxidative stress damage

[0116] Experimental method: H9C2 cells with good growth status and 90% confluence were inoculated into cell culture plates at 37°C and 5% CO. 2 The cells were cultured under the conditions and divided into a blank control group (conventional culture) and a model group (the conventional culture cells were used to establish the model after reaching 80%. The modeling conditions were: adding 250 μmol / L H 2 O 2 intervention for 4 hours), low, medium and high doses (after the cells reached 80% in conventional culture, different doses of drugs were pretreated for 24 hours in advance, and a concentration of 250 μmol / L H 2 O 2 Stimulate the cells for 4 hours), and after culturing the cells for 4 hours, lyse the cells and collect the samples from each group. According to the Bio-Time Total Antioxidant Capacity Detection Kit, the antioxidant activity of the above samples was determined.

[0117] The results are as follows Figure 1 As shown in Table 5, compared with the blank control group (CON), the model group (H 2 O 2 ) cell viability was significantly decreased (P<0.01). 2 O 2 ), concentrated solution B could not improve the antioxidant activity (P>0.05), concentrated solution A, Ganoderma lucidum spore oil and Ganoderma lucidum spore oil component emulsion could significantly improve cell viability (P<0.01), among which Ganoderma lucidum spore oil component emulsion had the strongest effect on anti-H2O2 oxidative stress injury in H9C2 cardiomyocytes.

[0118] Table 5 Effects of concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and Ganoderma lucidum spore oil component emulsion prepared in Example 1 on H9C2 cell activity

[0119]

[0120] Effect Example 6: Evaluation of the Total Antioxidant Activity of Ganoderma Lucidum Spore Oil Emulsion

[0121] The kit was used to measure the effects of each drug on ABTS of H9C2 cells damaged by oxidative stress. The results are shown in Table 6 and Figure 2 As shown. Compared with the control group, the antioxidant activity of cells in the model group was significantly reduced. Compared with the model group, the emulsion of Ganoderma lucidum spore oil fraction significantly increased the antioxidant activity.

[0122] Table 6 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 damaged by oxidative stress

[0123]

[0124] Effect Example 7: Effect of Ganoderma lucidum spore oil emulsion on apoptosis-related mRNA expression in H9C2 cells

[0125] Experimental method: RealTime PCR was used to detect the expression levels of Bcl-2, Bax, and Caspase-3 genes.

[0126] Bcl-2 family proteins are divided into three subfamilies: Bcl-2, Bax, and BH3. Bcl-2 is an inhibitor of apoptosis, and Bax is a promoter of apoptosis. 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 Compared with the blank group (CON), the expression of Bcl-2 / Bax in the model group was significantly downregulated (P<0.01); compared with the model group (H 2 O 2 ), the emulsion of the components of Ganoderma lucidum spore oil can significantly up-regulate the expression of Bcl-2 / Bax, indicating that it can inhibit apoptosis 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 emulsion of the components of Ganoderma lucidum spore oil significantly down-regulated the expression of Caspase-3, indicating that it may inhibit apoptosis by down-regulating the expression of Caspase-3. Table 7 Effects of the concentrated solution A, Ganoderma lucidum spore oil, concentrated solution B and the emulsion of the components of Ganoderma lucidum spore oil prepared in Example 1 on H 2 O 2 Effect of induced apoptosis factor expression on H9C2 cells

[0128]

[0129] Effect Example 8: Evaluation of the Total Antioxidant Activity of Ganoderma Lucidum Spore Oil Component Emulsion

[0130] The kit was used to measure the effects of each drug on ABTS of H9C2 cells damaged by oxidative stress. The results are shown in Table 8.

[0131] The experimental results show that after the total administration concentration is fixed at 5 mg / mL, the emulsions of the ganoderma spore oil component provided in Examples 1-3 all significantly improve the antioxidant activity. However, the emulsions of the ganoderma spore oil component provided in Comparative Examples 1-4 do not significantly improve the antioxidant activity. This indicates that the changes in the enzymes during the preparation process and the ratio of the concentrate A, concentrate B and ganoderma spore oil have a greater impact on the antioxidant activity of the ganoderma spore oil component emulsion.

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

[0133]

[0134] Note: Improvement 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 to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a ganoderma lucidum spore oil component emulsion, characterized in that: The following steps are involved: (1) softening the ganoderma lucidum spores, homogenizing and emulsifying the spores, breaking the spore wall, and separating the solid from the liquid to obtain an emulsion and a spore wall shell; (2) concentrating the emulsion to obtain a concentrated solution A; (3) hydrolyzing the spore wall shell with compound enzyme A, adding compound enzyme B after the initial hydrolysis, performing secondary hydrolysis, inactivating the enzyme, obtaining an enzymatic hydrolysis mixture, separating the solid from the liquid, drying the filtered residue, and extracting to obtain Ganoderma lucidum spore oil; (4) crushing the Ganoderma lucidum fruiting body, adding a solvent for soaking and extraction, separating the solid and liquid, and concentrating the filtrate to obtain a concentrated solution B; (5) mixing the concentrated solution A, the ganoderma spore oil and the concentrated solution B to obtain a ganoderma spore oil component emulsion; In step (3), the complex enzyme A comprises cellulase and chitinase, and the complex enzyme B comprises papain, bromelain and α-amylase.

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

3. The preparation method according to claim 1, characterized in that: The concentration in step (2) is concentrated to 10-40% of the weight of the Ganoderma lucidum spores, the complex enzyme A in step (3) is 0.3-0.5% of the weight of the spore wall shell, the complex enzyme B is 0.8-1% of the weight of the spore wall shell, and the concentration in step (4) is concentrated to 10-30% of the weight of the Ganoderma lucidum fruiting body.

4. The preparation method according to claim 5, characterized in that: The concentration in step (2) is to concentrate to 15-35% of the weight of the Ganoderma lucidum spores, and the concentration in step (4) is to concentrate to 15-25% of the weight of the Ganoderma lucidum fruiting body.

5. The preparation method according to claim 1, characterized in that: In step (3), 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; The conditions of the initial enzymatic hydrolysis in step (3) are: temperature 40-65° C., time 1-5 h, pH 5-5.5; The conditions of the secondary enzymatic hydrolysis in step (3) are: temperature 30-50°C, time 1-5h, 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) comprises ethanol and / or propylene glycol; The extraction is ultrasonic extraction.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the concentrated solution A, the ganoderma lucidum spore oil and the concentrated solution B in step (5) is 1:1-6:

1.

8. The ganoderma lucidum spore oil component emulsion prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the Ganoderma lucidum spore oil component emulsion prepared by the preparation method according to any one of claims 1 to 7 in the preparation of anti-tumor and antioxidant products.

10. An anti-tumor and anti-oxidant pharmaceutical composition, characterized in that: The invention comprises a ganoderma lucidum spore oil component emulsion prepared by the preparation method according to any one of claims 1 to 7.

Images