Anti-tumor composition and preparation method thereof

Abstract

The invention provides an anti-tumor composition and a preparation method thereof, and belongs to the technical field of natural medicines. Comprising the following steps: (1) washing black ginseng and lucid ganoderma, carrying out ultrahigh-pressure low-temperature wall breaking treatment, and grinding to obtain wall-broken powder; (2) adding the wall-broken powder into water, adding a compound enzyme for enzymolysis, deactivating the enzyme, filtering, dialyzing filtrate, and intercepting black ginseng and lucid ganoderma active liquid with the molecular weight of 2-5kDa; (3) adding zinc salt into the black ginseng and lucid ganoderma active liquid with the molecular weight of 2-5kDa, stirring for reaction, dialyzing and freeze-drying to obtain an active component chelated with Zn; and (4) carrying out surface modification on the active component chelated with Zn to obtain the anti-tumor composition. According to the anti-tumor composition disclosed by the invention, the targeting capability of the substance on dendritic cells and macrophages is improved, and then proliferation of a large number of specific tumor killing Car-T cells is induced, so that the clinical prevention and treatment effects of tumor vaccines are remarkably enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and in particular to an anti-tumor composition and a preparation method thereof. Background Art

[0002] In recent years, the screening of anti-tumor functional ingredients and the study of their mechanism of action have been the focus of research in the biomedical field. In China, due to the rapid increase in the incidence of tumors, the development prospects of the anti-tumor drug market are very broad. At the same time, due to the limitations of current drug efficacy, the new anti-tumor drug market is in urgent need of vigorous development, and the development of anti-tumor drugs has become a hot topic in the pharmaceutical industry. Many anti-tumor drugs used in clinical practice are obtained through tracking, separation, screening, structural modification and chemical synthesis of natural anti-tumor lead compounds. Using enzymes, receptors, genes and other new molecular targets can provide experimental basis and ideas for studying the targets of anti-cancer drugs. There are also many methods for extracting active substances, and the most commonly used methods include ultrafiltration centrifugation, thin layer chromatography, preparative chromatography preparative separation and other methods. The gradual screening of effective ingredients by separation and purification methods and the determination of mechanisms such as cell apoptosis, cytotoxicity and cell signal transduction have been research hotspots in recent years. Summary of the invention

[0003] The purpose of the present invention is to propose an anti-tumor composition and a preparation method thereof, to improve the targeting ability of the substance to dendritic cells and macrophages, thereby inducing the proliferation of a large number of specific tumor-killing CAR-T cells, thereby significantly enhancing the clinical therapeutic efficacy of tumor vaccines. It has been verified that the anti-tumor composition has a good in vitro and in vivo inhibitory effect on tumor cells.

[0004] The technical solution of the present invention is achieved in this way:

[0005] The present invention provides a method for preparing an antitumor composition, comprising the following steps:

[0006] (1) washing black ginseng and ganoderma lucidum, subjecting them to ultra-high pressure and low temperature cell wall breaking treatment, and grinding them to obtain cell wall broken powder;

[0007] (2) adding the broken wall powder into water, adding compound enzyme to hydrolyze, inactivate the enzyme, add glucose, ferment, filter, dialyze the filtrate, and retain the black ginseng and ganoderma active liquid with a molecular weight cutoff of 2-5 kDa;

[0008] (3) adding zinc salt to the active liquid of black ginseng and Ganoderma lucidum with a molecular weight of 2-5 kDa, stirring for reaction, dialyzing, and freeze-drying to obtain an active component chelating Zn;

[0009] (4) The Zn-chelated active component is surface-modified to obtain an anti-tumor composition.

[0010] As a further improvement of the present invention, the mass ratio of black ginseng to ganoderma lucidum in step (1) is 15-20:3-5, the pressure of the ultra-high pressure and low temperature wall breaking treatment is 2-3 atmospheres, the temperature is 30-35°C, and the time is 1-2h.

[0011] As a further improvement of the present invention, the mass ratio of the cell wall-breaking powder, complex enzyme, glucose and water in step (2) is 100:3-5:20-25:500-700.

[0012] As a further improvement of the present invention, the composite enzyme in step (2) is neutral protease and fructase, with a mass ratio of 5-7:1-2, and the enzymatic hydrolysis temperature is 40-50°C and the time is 1-3h.

[0013] As a further improvement of the present invention, the fermentation bacteria in step (2) is Bacillus subtilis, the fermentation conditions are 45-50°C, 100-150r / min, and the fermentation culture is 24-36h.

[0014] As a further improvement of the present invention, in step (3), the mass ratio of the black ginseng and Ganoderma lucidum active liquid with a molecular weight of 2-5 kDa and the zinc salt is 100:1-2, and the zinc salt is selected from at least one of zinc chloride, zinc sulfate, and zinc nitrate; the temperature of the stirring reaction is 35-45°C, the time is 0.5-1h, and the pore size of the dialysis bag used for dialysis is 2-5 kDa.

[0015] As a further improvement of the present invention, the method for surface modification of the active component chelating Zn in step (4) is as follows: solanine and paclitaxel are placed in a polyethylene glycol solution, the active component chelating Zn is added, the reaction is heated and stirred, and the supernatant is removed by centrifugation. The obtained precipitate is the anti-tumor composition.

[0016] As a further improvement of the present invention, the mass ratio of the active components of solanine, paclitaxel and chelated Zn is 3-5:1-2:100, and the temperature of the heating and stirring reaction is 50-60° C. and the time is 2-4 hours.

[0017] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0018] (1) washing 15-20 parts by weight of black ginseng and 3-5 parts by weight of ganoderma lucidum, subjecting them to ultra-high pressure and low temperature cell wall breaking treatment at 2-3 atmospheres, a temperature of 30-35° C., a time of 1-2 h, and grinding for 1-2 h to obtain cell wall broken powder;

[0019] (2) adding 100 parts by weight of the broken cell wall powder to 500-700 parts by weight of water, adding 3-5 parts by weight of a complex enzyme, performing enzymolysis at 40-50° C. for 1-3 hours, inactivating the enzyme, adding 20-25 parts by weight of glucose, fermenting, using Bacillus subtilis as the fermentation bacteria, fermenting at 45-50° C., 100-150 r / min, fermenting and culturing for 24-36 hours, filtering, dialyzing the filtrate for 2-4 hours, and obtaining a black ginseng and ganoderma lucidum active liquid with a molecular weight cutoff of 2-5 kDa;

[0020] The complex enzyme is neutral protease and fructase, with a mass ratio of 5-7:1-2;

[0021] (3) adding 1-2 parts by weight of zinc salt to 100 parts by weight of black ginseng and Ganoderma lucidum active liquid with a molecular weight of 2-5 kDa, stirring and reacting at 35-45° C. for 0.5-1 h, dialyzing for 2-4 h, and freeze-drying to obtain an active component chelating Zn;

[0022] (4) 3-5 parts by weight of solanine and 1-2 parts by weight of paclitaxel are added to 200 parts by weight of polyethylene glycol solution, and 100 parts by weight of an active component chelating Zn is added. The mixture is heated to 50-60° C., stirred for reaction for 2-4 hours, and the supernatant is removed by centrifugation. The resulting precipitate is the anti-tumor composition.

[0023] The present invention further protects an antitumor composition prepared by the above preparation method.

[0024] The present invention has the following beneficial effects: black ginseng and ganoderma lucidum contain rich active substances such as polysaccharides, proteins, amino acids, saponins, etc., and have good antioxidant, anti-inflammatory, anti-tumor and other activities. The anti-tumor composition prepared by the present invention uses black ginseng and ganoderma lucidum as raw materials, and after liquid nitrogen freezing treatment, black ginseng and ganoderma lucidum powder with broken cell walls is simply and conveniently obtained, added into water, and after enzymatic hydrolysis by composite enzyme (neutral protease and pectinase), the active substances with a molecular weight cutoff of 2-5kDa, including protein peptides, oligosaccharides, glycoprotein peptides, saponins and other active substances, are chelated with Zn ions to obtain the active component of chelated Zn, and the surface is modified by solanine and paclitaxel to improve the targeting ability of the substance for dendritic cells and macrophages, thereby inducing the proliferation of a large number of specific tumor-killing Car-T cells, thereby significantly enhancing the clinical prevention and treatment efficacy of tumor vaccines. It has been verified that the anti-tumor composition has a good in vitro and in vivo inhibitory effect on tumor cells. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] This embodiment provides a method for preparing an antitumor composition, which specifically comprises the following steps:

[0028] (1) 15 parts by weight of black ginseng and 3 parts by weight of ganoderma lucidum were washed, subjected to ultra-high pressure and low temperature cell wall breaking treatment at 2 atmospheres, a temperature of 30° C., a time of 1 h, and ground for 1 h to obtain cell wall-broken powder;

[0029] (2) adding 100 parts by weight of the broken cell wall powder to 500 parts by weight of water, adding 3 parts by weight of a complex enzyme, performing enzymolysis at 40° C. for 1 hour, inactivating the enzyme, adding 20 parts by weight of glucose, fermenting, using Bacillus subtilis as the fermentation bacteria, fermenting at 45° C., 100 r / min, fermenting and culturing for 24 hours, filtering, dialyzing the filtrate for 2 hours, and obtaining a black ginseng and ganoderma lucidum active liquid with a molecular weight cutoff of 2-5 kDa;

[0030] The complex enzyme is neutral protease and fructase, with a mass ratio of 5:1;

[0031] (3) adding 1 part by weight of zinc nitrate to 100 parts by weight of the black ginseng and Ganoderma lucidum active liquid with a molecular weight of 2-5 kDa, stirring and reacting at 35° C. for 0.5 h, dialyzing with a dialysis bag with a pore size of 5 kDa for 2 h, and freeze-drying to obtain an active component chelated with Zn;

[0032] (4) 3 parts by weight of solanine and 1 part by weight of paclitaxel are added to 200 parts by weight of polyethylene glycol solution, and 100 parts by weight of an active component chelating Zn is added. The mixture is heated to 50° C. and stirred for 2 hours. The supernatant is removed by centrifugation to obtain an antitumor composition.

[0033] Example 2

[0034] This embodiment provides a method for preparing an antitumor composition, which specifically comprises the following steps:

[0035] (2) washing 20 parts by weight of black ginseng and 5 parts by weight of ganoderma lucidum, subjecting them to ultra-high pressure and low temperature cell wall breaking treatment at 3 atmospheres, a temperature of 35° C., a time of 2 h, and grinding for 2 h to obtain cell wall-broken powder;

[0036] (2) adding 100 parts by weight of the broken cell wall powder to 700 parts by weight of water, adding 5 parts by weight of a complex enzyme, performing enzymolysis at 50° C. for 3 hours, inactivating the enzyme, adding 25 parts by weight of glucose, fermenting, using Bacillus subtilis as the fermentation bacteria, fermenting at 50° C., 150 r / min, fermenting and culturing for 36 hours, filtering, dialyzing the filtrate for 4 hours, and obtaining a black ginseng and ganoderma lucidum active liquid with a molecular weight cutoff of 2-5 kDa;

[0037] The complex enzyme is neutral protease and fructase, with a mass ratio of 7:2;

[0038] (3) adding 2 parts by weight of zinc sulfate to 100 parts by weight of the black ginseng and Ganoderma lucidum active liquid with a molecular weight of 2-5 kDa, stirring at 45° C. for 1 hour, dialyzing with a dialysis bag with a pore size of 5 kDa for 24 hours, and freeze-drying to obtain an active component chelated with Zn;

[0039] (4) 5 parts by weight of solanine and 2 parts by weight of paclitaxel are added to 200 parts by weight of polyethylene glycol solution, and 100 parts by weight of an active component chelating Zn is added. The mixture is heated to 60° C. and stirred for 4 hours. The supernatant is removed by centrifugation to obtain an antitumor composition.

[0040] Example 3

[0041] This embodiment provides a method for preparing an antitumor composition, which specifically comprises the following steps:

[0042] (1) 17 parts by weight of black ginseng and 4 parts by weight of ganoderma lucidum were washed, subjected to ultra-high pressure and low temperature cell wall breaking treatment at 2.3 atmospheres, a temperature of 32° C., a time of 1.5 h, and ground for 1.5 h to obtain cell wall-broken powder;

[0043] (2) adding 100 parts by weight of the broken cell wall powder to 600 parts by weight of water, adding 4 parts by weight of a complex enzyme, performing enzymolysis at 45° C. for 2 hours, inactivating the enzyme, adding 22 parts by weight of glucose, fermenting, using Bacillus subtilis as the fermentation bacteria, fermenting at 47° C., 125 r / min, fermenting and culturing for 30 hours, filtering, dialyzing the filtrate for 3 hours, and obtaining a black ginseng and ganoderma lucidum active liquid with a molecular weight cutoff of 2-5 kDa;

[0044] The complex enzyme is neutral protease and fructase, with a mass ratio of 6:1.5;

[0045] (3) adding 1.5 parts by weight of zinc chloride to 100 parts by weight of the black ginseng and Ganoderma lucidum active liquid with a molecular weight of 2-5 kDa, stirring at 40° C. for 1 hour, dialyzing with a dialysis bag with a pore size of 5 kDa for 3 hours, and freeze-drying to obtain an active component chelated with Zn;

[0046] (4) 4 parts by weight of solanine and 1.5 parts by weight of paclitaxel are added to 200 parts by weight of polyethylene glycol solution, and 100 parts by weight of an active component chelating Zn is added. The mixture is heated to 55° C. and stirred for reaction for 3 hours. The supernatant is removed by centrifugation to obtain an antitumor composition.

[0047] Example 4

[0048] Compared with Example 3, the difference is that the complex enzyme is a neutral protease.

[0049] Example 5

[0050] Compared with Example 3, the difference is that the complex enzyme is fructose enzyme.

[0051] Comparative Example 1

[0052] Compared with Example 3, the difference is that step (2) is not performed.

[0053] Comparative Example 2

[0054] Compared with Example 3, the difference is that step (3) is not performed.

[0055] Comparative Example 3

[0056] Compared with Example 3, the difference is that solanine is not added in step (4).

[0057] Comparative Example 4

[0058] Compared with Example 3, the difference is that paclitaxel is not added in step (4).

[0059] Comparative Example 5

[0060] Compared with Example 3, the difference is that step (4) is not performed.

[0061] Test Example 1

[0062] HepG2, HCT-116, Hela, and A549 cells in the logarithmic growth phase were digested with trypsin to prepare a cell suspension and the cell concentration was adjusted to 5 × 10 4 / mL, inoculate the cells in a 96-well plate, 100 μL per well, and culture in an incubator at 37°C, 5% CO2, and saturated humidity for 24 hours. After the cells adhere to the wall, add the anti-tumor composition prepared in Examples 1-5 and Comparative Examples 1-5 (at a concentration of 1 μg / mL), 5 replicate wells per group, and set a zeroing well and a control well. The cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity for 24 hours. Pour out the cell culture medium, add 100 μL of fresh culture medium to each well, 10uL of MTT, incubate at 37°C for 4 hours, centrifuge for 10 minutes, discard the culture medium, add 150 μL of dimethyl sulfoxide to each well, and shake for 15 minutes. Measure the absorbance value at 490nm with an enzyme marker.

[0063] Cell growth inhibition rate (%) = (A 对照孔 -A 实验孔 ) / A 对照孔 X100%

[0064] The results are shown in Table 1.

[0065] Table 1

[0066]

[0067] It can be seen from the above table that the anti-tumor compositions prepared in Examples 1-3 of the present invention have good in vitro tumor cell inhibition effects.

[0068] Test Example 2

[0069] MC38 cells were counted and prepared at a density of 1×10 7 The cell suspension of 100 cells / mL was subcutaneously injected into the right leg of C57BL / 6 female mice to establish the model. After 7 days, the tumor was observed to be successful. The mice with successful modeling were divided into Example 1-5 group, Comparative Example 1-5 group and model group, with 6 mice in each group; the Example 1-5 group and the Comparative Example 1-5 group were gavaged with 1 mg / g of the prepared anti-tumor composition, and the model group mice were gavaged with 1 mg / g of 0.9% NaCl, once a day, and the mice in each group were treated for 2 consecutive weeks.

[0070] Before the first administration (recorded as day 0) and after the end of the experiment (recorded as day 14), the long diameter and short diameter of the tumor of each group of mice were measured and their volume was calculated. The formula is: volume = (long diameter × short diameter) 2 ) / 2.

[0071] The relative tumor volume (RTV) calculation formula is: RTV = V t / V0, V0 is the tumor volume measured for the first time in each group, V t is the tumor volume at each measurement.

[0072] The evaluation index of antitumor activity is relative tumor proliferation inhibition rate (%) = (1-RTV实验组 / RTV 模型组 )×100%

[0073] The results are shown in Table 2.

[0074] Table 2

[0075] Group Relative tumor proliferation inhibition rate (%) Model Group / Example 1 69.43 Example 2 70.12 Example 3 71.07 Example 4 50.25 Example 5 48.78 Comparative Example 1 40.09 Comparative Example 2 44.29 Comparative Example 3 34.18 Comparative Example 4 30.92 Comparative Example 5 26.78

[0076] It can be seen from the above table that the anti-tumor compositions prepared in Examples 1-3 of the present invention have a good effect of inhibiting tumor growth.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an antitumor composition, characterized in that: The following steps are involved: (1) washing black ginseng and ganoderma lucidum, subjecting them to ultra-high pressure and low temperature cell wall breaking treatment, and grinding them to obtain cell wall broken powder; (2) adding the broken wall powder into water, adding compound enzyme to hydrolyze, inactivate the enzyme, add glucose, ferment, filter, dialyze the filtrate, and retain the black ginseng and ganoderma active liquid with a molecular weight cutoff of 2-5 kDa; (3) adding zinc salt to the black ginseng and Ganoderma lucidum active liquid with a molecular weight of 2-5 kDa, stirring for reaction, dialyzing, and freeze-drying to obtain an active component chelating Zn; (4) The Zn-chelated active component is surface-modified to obtain an anti-tumor composition.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of black ginseng to ganoderma lucidum is 15-20:3-5, the pressure of the ultra-high pressure and low temperature wall breaking treatment is 2-3 atmospheres, the temperature is 30-35°C, and the time is 1-2h.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the cell wall-breaking powder, the complex enzyme, glucose and water in step (2) is 100:3-5:20-25:500-700.

4. The preparation method according to claim 1, characterized in that: The composite enzyme in step (2) is neutral protease and fructase, with a mass ratio of 5-7:1-2, and the enzymatic hydrolysis temperature is 40-50° C. and the time is 1-3 h.

5. The preparation method according to claim 1, characterized in that: The fermentation bacteria in step (2) is Bacillus subtilis, and the fermentation conditions are 45-50° C., 100-150 r / min, and the fermentation culture is 24-36 hours.

6. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of the black ginseng and ganoderma active liquid with a molecular weight of 2-5 kDa and the zinc salt is 100:1-2, and the zinc salt is selected from at least one of zinc chloride, zinc sulfate, and zinc nitrate; the stirring reaction temperature is 35-45°C, the time is 0.5-1h, and the dialysis bag pore size used in the dialysis is 2-5 kDa.

7. The preparation method according to claim 1, characterized in that: The method for surface modification of the active component chelating Zn in step (4) is as follows: put solanine and paclitaxel into a polyethylene glycol solution, add the active component chelating Zn, heat and stir to react, centrifuge to remove the supernatant, and the obtained precipitate is the anti-tumor composition.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the active components of solanine, paclitaxel and chelated Zn is 3-5:1-2:100, and the temperature of the heating and stirring reaction is 50-60° C. and the time is 2-4 hours.

9. The preparation method according to claim 1, characterized in that: The specific steps include: (1) washing 15-20 parts by weight of black ginseng and 3-5 parts by weight of ganoderma lucidum, subjecting them to ultra-high pressure and low temperature cell wall breaking treatment at 2-3 atmospheres, a temperature of 30-35° C., a time of 1-2 h, and grinding for 1-2 h to obtain cell wall broken powder; (2) adding 100 parts by weight of the broken cell wall powder to 500-700 parts by weight of water, adding 3-5 parts by weight of a complex enzyme, performing enzymolysis at 40-50° C. for 1-3 hours, inactivating the enzyme, adding 20-25 parts by weight of glucose, fermenting, using Bacillus subtilis as the fermentation bacteria, fermenting at 45-50° C., 100-150 r / min, fermenting and culturing for 24-36 hours, filtering, dialyzing the filtrate for 2-4 hours, and obtaining a black ginseng and ganoderma lucidum active liquid with a molecular weight cutoff of 2-5 kDa; The complex enzyme is neutral protease and fructase, with a mass ratio of 5-7:1-2; (3) adding 1-2 parts by weight of zinc salt to 100 parts by weight of black ginseng and Ganoderma lucidum active liquid with a molecular weight of 2-5 kDa, stirring and reacting at 35-45° C. for 0.5-1 h, dialyzing for 2-4 h, and freeze-drying to obtain an active component chelating Zn; (4) 3-5 parts by weight of solanine and 1-2 parts by weight of paclitaxel are added to 200 parts by weight of polyethylene glycol solution, and 100 parts by weight of an active component chelating Zn is added. The mixture is heated to 50-60° C., stirred for reaction for 2-4 hours, and the supernatant is removed by centrifugation. The resulting precipitate is the anti-tumor composition.

10. An antitumor composition prepared by the preparation method according to any one of claims 1 to 9.

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