Application of annona squamosa extract in preparation of product for inhibiting myeloblastoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an annona squamosa extract in preparation of a product for inhibiting myeloblastoma. Through a combined process of multi-strain fermentation and ultrasonic-assisted extraction, anti-cancer components in the annona squamosa can be efficiently extracted, beneficial metabolites generated by microorganisms in the fermentation process can have a synergistic effect with natural components in the annona squamosa, and the anti-cancer activity of the extract is further enhanced; a mixture of choline chloride and glycerol is used as a solvent in the extraction process, so that the use of an organic solvent is avoided, the risks of environmental pollution and solvent residue are reduced, and compared with the traditional ethanol extraction, the extraction efficiency is higher; two-stage filtration and ion exchange resin column purification are adopted, thalli, suspended solids, macromolecular impurities and inorganic ions in fermentation liquor can be effectively removed, and the purity of the extract is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to an application of an Annona custardii extract in preparing a product for inhibiting medulloblastoma. Background Art

[0002] Medulloblastoma (MB) is one of the most common malignant tumors in the central nervous system of children, with high invasiveness and recurrence rate. Traditional treatments include surgical resection, radiotherapy and chemotherapy, but these methods are often accompanied by serious side effects and have limited efficacy for recurrent tumors. Therefore, finding new and more effective treatment strategies is an important direction of current research.

[0003] In recent years, with the in-depth study of the medicinal value of natural products, more and more natural plant extracts have been used in the development of anti-tumor drugs. Natural products have attracted widespread attention due to their wide sources, relatively small toxic side effects, and multi-target mechanisms of action. However, some natural product extracts are easily degraded during storage or use, affecting their stability and efficacy. The oral absorption rate of the active ingredients of many natural products (such as flavonoids) is low, resulting in limited bioavailability in the body, which limits their anti-tumor effects. Many natural plant extracts have complex ingredients, and the active ingredients have not been fully identified, making it difficult to achieve precise efficacy regulation. In addition, although some natural products have certain anti-tumor activity, their mechanism of action is relatively single, making it difficult to cope with the complexity and heterogeneity of tumor cells. Therefore, the development of a natural plant extract that can effectively inhibit medulloblastoma has important scientific significance and application value. Summary of the invention

[0004] The purpose of the present invention is to provide an Annona fruit extract which can effectively inhibit medulloblastoma and has high efficiency, stability and high bioavailability.

[0005] The present invention provides an application of an Annona fruit extract in preparing a product for inhibiting medulloblastoma. The preparation method of the Annona fruit extract comprises the following steps: A first bacterial agent is inoculated into the sugar apple pulp for primary fermentation to obtain a fermentation stock solution A; the first bacterial agent is Bacillus megaterium ( Bacillus megaterium ) and Paenibacillus jelly-like Paenibacillus mucilaginous ) mixed bacterial agent; inoculating the second bacterial agent in the fermentation stock solution A, performing secondary fermentation, and obtaining the fermentation stock solution B; the second bacterial agent is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens); the fermentation liquid B is filtered at the primary and secondary levels, and a composite solvent is added for ultrasonic-assisted extraction to obtain an extract; the composite solvent is a mixture of choline chloride and glycerol; the extract is purified by an ion exchange resin column, and the custard apple extract is obtained after concentration.

[0006] Preferably, the number of viable bacteria of the first bacterial agent is ≥ 200 million / mL; and the ratio of the number of viable bacteria of Bacillus megaterium to that of Paenibacillus gelatinosa is 1:1-2.

[0007] Preferably, the viable bacterial count of the second bacterial agent is ≥ 200 million / mL.

[0008] Preferably, the addition amount of the first bacterial agent is 1% to 5% of the total mass of the custard apple pulp; the addition amount of the second bacterial agent is 0.1‰ to 1‰ of the total mass of the fermentation stock solution A.

[0009] Preferably, the temperature of the primary fermentation is 25°C to 35°C, and the time is 24 to 48 hours.

[0010] Preferably, the temperature of the secondary fermentation is 30-40°C and the time is 12-24 hours.

[0011] Preferably, the pore size of the filter membrane for the primary filtration is 2-5 μm, and the pore size of the filter membrane for the secondary filtration is 0.22 μm or 0.45 μm.

[0012] Preferably, the frequency of the ultrasound-assisted extraction is 20-40 kHz, the power is 300-500 W, and the processing time is 15-30 min.

[0013] Preferably, the molar ratio of choline chloride to glycerol is 1:2.

[0014] Preferably, the volume ratio of the fermentation liquid B to ethanol is 5-10:1.

[0015] Beneficial effects of the present invention: Medulloblastoma is one of the most common malignant brain tumors in children, and traditional treatment methods have many limitations. The custard apple extract of the present invention has significant anti-tumor activity, is naturally derived, and has few side effects, and is expected to provide a new, safe and effective drug option for the treatment of medulloblastoma.

[0016] The present invention can efficiently extract anti-cancer components in sugar apple, such as annona and diterpenoid compounds, through a combined process of multi-strain fermentation and ultrasound-assisted extraction; the fermentation process can decompose cell walls and release more active components, while ultrasound-assisted extraction further improves the extraction efficiency, ensuring a higher content of effective components in the extract; the reaction conditions in the fermentation and ultrasound-assisted extraction stages are mild, avoiding the destruction of heat-sensitive components by high temperature or strong acid and alkali treatment, and retaining the biologically active components in the sugar apple extract.

[0017] The present invention adopts natural fermentation strains and green solvents for extraction (a mixture of choline chloride and glycerol), thus avoiding the use of organic solvents and reducing the risks of environmental pollution and solvent residues; at the same time, the fermentation process can fully utilize the nutrients in the sugar apple fruit and improve resource utilization. The present invention ensures the stability and repeatability of the fermentation process by precisely controlling the fermentation temperature, time and amount of bacterial agent added.

[0018] During the fermentation process, microorganisms can metabolize and produce some beneficial metabolites, which may synergize with the natural components in custard apple to further enhance the anti-cancer activity of the extract. For example, the fermentation process may promote the generation or conversion of active ingredients such as annona and improve their bioavailability.

[0019] The invention adopts two-stage filtration and ion exchange resin column purification, which can effectively remove bacteria, suspended solids, macromolecular impurities and inorganic ions in the fermentation liquid and significantly improve the purity of the extract.

[0020] Ultrasonic-assisted extraction was performed using a mixed solvent of choline chloride and glycerol. This composite solvent had good solubility and biocompatibility, and could effectively extract the active ingredients in custard apple while avoiding the toxicity problem of traditional organic solvents.

[0021] The process steps of the invention are clear, the operation is simple, and it is suitable for large-scale industrial production. The combined process of two-stage filtration and ion exchange resin column purification can efficiently remove impurities, ensure the stable quality of the extract, and is suitable for the development of medicines or health products. DETAILED DESCRIPTION

[0022] In order to further illustrate the present invention, the Annona extract with anti-tumor efficacy and the preparation method thereof provided by the present invention are described in detail below in combination with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Unless otherwise specified, all substances used in the examples of the present invention are conventional commercially available products.

[0024] Example 1 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0025] Bacillus megaterium and Paenibacillus gelatinus were mixed at a live bacterial count ratio of 1:1, inoculated into the sugar apple pulp, the inoculation amount was 2% of the total mass of the sugar apple pulp, and fermented at 28°C for 30 hours to obtain fermentation stock solution A.

[0026] The fermentation solution A was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 1‰ of the total mass of the fermentation solution A. The fermentation was carried out at 35°C for 16 hours to obtain the fermentation solution B.

[0027] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0028] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0029] Example 2 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0030] Bacillus megaterium and Paenibacillus gelatinus were mixed at a live bacterial count ratio of 1:2, inoculated into the sugar apple pulp, the inoculation amount was 3% of the total mass of the sugar apple pulp, and fermented at 28°C for 30 hours to obtain fermentation stock solution A.

[0031] The fermentation solution A was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 1‰ of the total mass of the fermentation solution A. The fermentation was carried out at 35°C for 16 hours to obtain the fermentation solution B.

[0032] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0033] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0034] Example 3 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0035] Bacillus megaterium and Paenibacillus gelatinus were mixed at a live bacterial count ratio of 1:1, inoculated into the sugar apple pulp, the inoculation amount was 1% of the total mass of the sugar apple pulp, and fermented at 28°C for 48 hours to obtain fermentation stock solution A.

[0036] The fermentation solution A was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 1‰ of the total mass of the fermentation solution A. The fermentation was carried out at 32°C for 24 hours to obtain the fermentation solution B.

[0037] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0038] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0039] Example 4 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0040] Bacillus megaterium and Paenibacillus gelatinus were mixed at a live bacterial count ratio of 1:1, inoculated into the sugar apple pulp, the inoculation amount was 5% of the total mass of the sugar apple pulp, and fermented at 28°C for 24 hours to obtain fermentation stock solution A.

[0041] The fermentation solution A was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 1‰ of the total mass of the fermentation solution A. The fermentation was carried out at 40°C for 12 hours to obtain the fermentation solution B.

[0042] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0043] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0044] Comparative Example 1 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0045] Bacillus megaterium and Paenibacillus gelatinus were mixed at a live bacterial count ratio of 1:1, inoculated into the sugar apple pulp, the inoculation amount was 2% of the total mass of the sugar apple pulp, and fermented at 28°C for 30 hours to obtain fermentation stock solution A.

[0046] The fermentation solution A was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 1‰ of the total mass of the fermentation solution A. The fermentation was carried out at 35°C for 16 hours to obtain the fermentation solution B.

[0047] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. 70% v / v ethanol was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0048] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0049] Comparative Example 2 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0050] Bacillus megaterium and Paenibacillus gelatinus were mixed at a live bacterial count ratio of 1:1, inoculated into the sugar apple pulp, the inoculation amount was 2% of the total mass of the sugar apple pulp, and fermented at 28°C for 30 hours to obtain fermentation stock solution A.

[0051] The fermentation liquid A was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0052] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0053] Comparative Example 3 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0054] Bacillus megaterium was inoculated into the sugar apple pulp at an inoculation amount of 2% of the total mass of the sugar apple pulp, and fermented at 28°C for 30 hours to obtain fermentation stock solution A.

[0055] The fermentation solution A was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 1‰ of the total mass of the fermentation solution A. The fermentation was carried out at 35°C for 16 hours to obtain the fermentation solution B.

[0056] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0057] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0058] Comparative Example 4 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0059] The jelly-like Bacillus was inoculated into the sugar apple pulp, and the inoculation amount was 2% of the total mass of the sugar apple pulp. The fermentation was carried out at 28°C for 30 hours to obtain the fermentation stock solution A.

[0060] The fermentation solution A was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 1‰ of the total mass of the fermentation solution A. The fermentation was carried out at 35°C for 16 hours to obtain the fermentation solution B.

[0061] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0062] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0063] Comparative Example 5 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0064] The custard apple pulp was inoculated with a bacterial agent of Bacillus amyloliquefaciens at an inoculation amount of 2% of the total mass of the custard apple pulp, and fermented at 35°C for 16 hours to obtain a fermentation stock solution B.

[0065] The fermentation liquid B was filtered through a 2 μm filter membrane for primary filtration and then filtered through a 0.22 μm filter membrane for secondary filtration. A mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 was added for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0066] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0067] Comparative Example 6 The Annona extract was prepared as follows: Fresh, disease-free sugar apple fruits are selected, cleaned and crushed to obtain sugar apple pulp.

[0068] The custard apple pulp was added into a mixed solvent with a molar ratio of choline chloride to glycerol of 1:2 for ultrasonic-assisted extraction. The extraction frequency was 40 kHz, the power was 400 W, and the processing time was 20 min to obtain an extract.

[0069] The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

[0070] Test Example 1 The effective components in the custard apple extracts prepared in the examples and comparative examples were quantitatively analyzed by high performance liquid chromatography (HPLC). The results are shown in Table 1.

[0071] Table 1 Content of active ingredients in custard apple extract (mg / g)

[0072] As shown in Table 1, the content of effective ingredients in the custard apple extracts prepared in Examples 1 to 4 is much higher than that in the comparative example, which shows that the method provided by the present invention has higher extraction efficiency.

[0073] Test Example 2 The custard apple extract prepared in Example 1 was diluted to 20 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL respectively for use.

[0074] Medulloblastoma adherent tumor cells (D283 Med cells, purchased from Wuhan Sean Biotechnology Co., Ltd., catalog number SNL-634) in the logarithmic growth phase were obtained and digested with trypsin for 3-5 min. RPMI 1640 culture medium containing 1% FBS was added to prepare a single cell suspension. The cells were counted using a hemocytometer and the cell concentration was adjusted to 5 × 10 4 / mL.

[0075] The cells were added to a 96-well culture plate, 100 μL per well, and cultured in a 37°C constant temperature incubator for 24 h. They were then divided into a blank control group, a 20 μg / mL group, a 50 μg / mL group, a 100 μg / mL group, and a 200 μg / mL group. Four parallel wells were set up in each group and treated as follows: Blank control group: add 180µL PBS; 20 μg / mL group: 180 μL of 20 μg / mL Annona extract was added; 50 μg / mL group: 180 μL of 50 μg / mL Annona extract was added; 100 μg / mL group: 180 μL of 100 μg / mL Annona extract was added; 200μg / mL group: 180µL of 200μg / mL Annona extract was added.

[0076] Then the cells were co-cultured with the extract for 48 h, 100 μL of 1 mg / mL MTT solution was added to each well, and the culture was continued for 4 h. The MTT solution was discarded, and 200 μL of DMSO was added to each well. The cells were shaken on a shaker for 30 min. The absorbance was measured at 570 nm using a microplate reader, and the percentage of cytotoxicity was calculated according to the following formula. The results are shown in Table 2.

[0077] Cytotoxicity percentage (%) = (OD value control well - OD value drug well) / OD value control well × 100% Table 2 Cytotoxicity of Annona extract against medulloblastoma cells

[0078] As shown in Table 2, the Annona extract provided by the present invention has a significant inhibitory effect on the proliferation of medulloblastoma in a concentration-dependent manner.

[0079] Test Example 3 Fifty C57 / BL6 female nude mice aged 7 to 8 weeks, weighing 15-20 g, were randomly divided into a normal group, a model group, a positive control group, a treatment group according to Example 1, and a comparative group, with 10 mice in each group. Except for the normal group, the nude mice in the other groups were injected intraventricularly with 0.3 mL of 4×10 7 / mL human medulloblastoma cell suspension (D341 Med cells, purchased from Guangzhou Kefan Biotechnology Co., Ltd.), 2 hours after injection, the normal group and the model group were gavaged with 2mL of normal saline, the positive control group was gavaged with 2mL of a 6.0mg / mL solution made of commercially available cisplatin, the Example 1 treatment group was gavaged with 2mL of a 200μg / mL solution of guava extract prepared in Example 1 of the present invention, and the comparative examples 1 to 6 treatment groups were gavaged with 2mL of a 200μg / mL solution of guava extract prepared in comparative examples 1 to 6 of the present invention, twice a day, for 14 consecutive days, after 14 days, the experiment was terminated, the mice were killed, the tumor tissues were removed, and the tumor weight was weighed. The tumor inhibition rate was calculated, and the results are shown in Table 3.

[0080] Volume = 0.5 × length × width 2 Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of treatment group) / tumor weight of model group × 100% Table 3 Tumor inhibition rate of extracts in each group

[0081] As shown in Table 3, the guava extract prepared by the method provided by the present invention has a higher tumor inhibition rate for medulloblastoma and a higher anticancer activity.

[0082] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of an Annona fruit extract in preparing a product for inhibiting medulloblastoma, characterized in that: The preparation method of the custard apple extract comprises the following steps: A first bacterial agent is inoculated into the sugar apple pulp for primary fermentation to obtain a fermentation stock solution A; the first bacterial agent is Bacillus megaterium ( Bacillus megaterium ) and Paenibacillus jelly-like Paenibacillus mucilaginosus ) of mixed bacterial agents; A second bacterial agent is inoculated into the fermentation liquid A to perform secondary fermentation to obtain the fermentation liquid B; the second bacterial agent is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ); The fermentation liquid B is subjected to primary filtration and secondary filtration, and a composite solvent is added to perform ultrasonic-assisted extraction to obtain an extract; the composite solvent is a mixture of choline chloride and glycerol; The extract is purified by an ion exchange resin column and concentrated to obtain the custard apple extract.

2. The use according to claim 1, characterized in that: The viable bacterial count of the first bacterial agent is ≥ 200 million / mL; the viable bacterial count ratio of the Bacillus megaterium to the Bacillus gelatinosa is 1:1-2.

3. The use according to claim 1, characterized in that: The viable bacterial count of the second bacterial agent is ≥ 200 million / mL.

4. The use according to claim 1, characterized in that: The addition amount of the first bacterial agent is 1% to 5% of the total mass of the custard apple pulp; the addition amount of the second bacterial agent is 0.1‰ to 1‰ of the total mass of the fermentation stock solution A.

5. The use according to claim 1, characterized in that: The temperature of the primary fermentation is 25°C-35°C, and the time is 24-48 hours; the temperature of the secondary fermentation is 30-40°C, and the time is 12-24 hours.

6. The use according to claim 1, characterized in that: The pore size of the filter membrane for the primary filtration is 2-5 μm, and the pore size of the filter membrane for the secondary filtration is 0.22 μm or 0.45 μm.

7. The use according to claim 1, characterized in that: The frequency of the ultrasonic-assisted extraction is 20-40 kHz, the power is 300-500 W, and the processing time is 15-30 min.

8. The use according to claim 1, characterized in that: The molar ratio of choline chloride to glycerol is 1:

2.

9. The use according to claim 1, characterized in that: The volume ratio of the fermentation liquid B to ethanol is 5-10:1.

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