Flos magnoliae chloroform part extract, preparation method thereof and application of flos magnoliae chloroform part extract in resisting tumors and reversing tumor drug resistance

Abstract

The invention discloses a flos magnoliae chloroform part extract, a preparation method thereof and application of the flos magnoliae chloroform part extract in resisting tumors and reversing tumor drug resistance. According to the method disclosed by the invention, the magnolia flower methanol extract, especially the chloroform extract thereof, is efficiently extracted by adopting operation means of extracting and separating natural products such as solvent extraction and extraction, and experiments prove that the magnolia flower chloroform extract has the activity of inhibiting the growth of tumor cells in vitro, so that the magnolia flower chloroform extract has the anti-tumor activity. Meanwhile, the chloroform part of flos magnoliae also can inhibit the expression of EGFR TKI drug-resistant pathway related proteins, so that the tumor drug resistance can be reversed. According to the invention, the raw material channel of the anti-tumor medicine is expanded, the application of the magnolia flower is expanded, the magnolia flower is developed into a new raw material of the anti-tumor medicine, and the medicinal value of the magnolia flower can be obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product chemistry, and particularly relates to a chloroform extract of magnolia officinalis and a preparation method thereof, and application thereof in anti-tumor and reversal of tumor drug resistance. Background Art

[0002] The incidence of malignant tumors continues to increase, and has become the main cause of death from disease. Among them, lung cancer is the number one cause of death. At present, the main treatments for malignant tumors are radiotherapy and chemotherapy, but radiotherapy and chemotherapy have large side effects in clinical practice and are prone to drug resistance, which reduces the clinical efficacy. Therefore, the development of new low-toxic, high-efficiency and safe drugs has become an urgent need for the treatment of tumors. Traditional Chinese medicine has a long history. Many Chinese medicines have anti-tumor activity themselves, and can regulate immune function to inhibit tumors, with an overall therapeutic effect. Finding lead compounds with high-efficiency and low-toxicity anti-tumor activity from natural medicines has always been a hot topic in anti-cancer drug research at home and abroad, and effective active monomers and anti-cancer compound preparations of effective parts of Chinese herbal medicines have important clinical application value.

[0003] Magnolia is a plant of the Magnoliaceae family, the dried flower buds of Magnolia biondii Pamp. Magnolia Magnoliadenudata Desr. or Magnolia sprengeri Pamp. Harvested in late winter and early spring before the flowers open, the branches and stems are removed, and dried in the shade. It dispels wind and cold and clears the nasal passages. It is used for headaches caused by wind and cold, allergic rhinitis, and sinusitis. At present, the main active chemical components of Magnolia include: 1) volatile oil components (enols, esters); 2) lignan components (lignans); 3) water-soluble components (glycosides, esters, phenolic acids and alkaloids). The latest research shows that Magnolia has pharmacological activities such as anti-inflammatory, anti-allergic, and neuroprotective. The research related to its pharmacological effects is mostly focused on crude extracts such as water extracts, alcohol extracts and volatile oils.

[0004] At present, there is no literature report on the pharmacological activity of the methanol extract of Magnolia biondii, especially the extract from the chloroform part. Summary of the invention

[0005] The invention aims to provide a chloroform extract of magnolia flos odoratus and a preparation method thereof and application thereof in anti-tumor and reversing tumor drug resistance.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The invention discloses a preparation method of a chloroform part extract of magnolia, which comprises the following steps: drying magnolia, crushing it, adding methanol for extraction, evaporating methanol from the filtrate to obtain a methanol extract, dissolving it in water, extracting it with n-hexane, chloroform, ethyl acetate and n-butanol in sequence, and finally selecting the concentrated and dried chloroform part as the anti-tumor effective component extract of magnolia.

[0007] Specifically, a method for preparing a chloroform extract of magnolia flos odoratus comprises the following steps: 1) crushing the raw material magnolia, cooling it, and collecting it to obtain magnolia powder; 2) Extracting the magnolia powder with pure methanol by reflux, and then filtering and rotary evaporating to obtain the magnolia methanol extract; 3) The methanol extract of Magnolia officinalis is concentrated and dried to remove methanol to obtain a total extract, which is then stirred with water to form a suspension, and then extracted with n-hexane for several times. The remaining aqueous solution after the n-hexane extraction is decompressed to remove the residual n-hexane, and then extracted with chloroform for several times, the chloroform extracts are combined, the solvent is recovered under reduced pressure, and dried to obtain the chloroform extract of Magnolia officinalis.

[0008] Preferably, in step 1), the crushing is performed for 1 min, and the cooling is performed for 5 min, and the crushing and cooling operations are repeated three times. The original magnolia medicinal material is a flower bud, and the machine will generate heat during the crushing process. To prevent the high temperature from affecting the activity of the medicinal material, the crushing and cooling process needs to be repeated three times to ensure the activity of the effective ingredients of the medicinal material.

[0009] Preferably, in step 2), the material-liquid ratio of magnolia powder to pure methanol is 1 g: (20-50) mL.

[0010] Preferably, in step 2), the reflux extraction is performed at 65-75°C for 2-4 h, which is repeated 3 times.

[0011] Preferably, in step 3), drying is performed in a rotary vacuum drying oven at 50° C.; water 10 to 20 times the volume of the total extract is added and stirred to form a suspension.

[0012] Preferably, in step 3), the extract is performed at least twice with an equal volume of n-hexane and at least twice with an equal volume of chloroform; the drying treatment is performed in a vacuum dryer at 30°C to a constant weight to obtain the chloroform extract of magnolia floret.

[0013] The invention also discloses a magnolia chloroform part extract prepared by the preparation method, wherein the magnolia chloroform part is a magnolia anti-tumor effective component extract.

[0014] The invention also discloses the application of the extract of chloroform part of magnolia flos in preparing anti-tumor drugs.

[0015] The tumor is various solid tumors or leukemia; Furthermore, the tumors include lung cancer, breast cancer, cervical cancer, liver cancer and colon cancer.

[0016] Furthermore, the present invention provides the use of an extract of effective anti-tumor components of magnolia in the preparation of anti-tumor drugs. Lung cancer NCI-H1299 and NCI-H460 cells, breast cancer MCF-7 cells, cervical cancer Hela cells, liver cancer Bel-7404 cells and colon cancer LoVo cells were used as test tumor strains, and the anti-tumor activity experiment was carried out with the ability to inhibit tumor cell proliferation as the activity index. The experimental results showed that in the in vitro cytotoxicity experiment, the methanol extract of magnolia can inhibit the proliferation of tumor cells and has a cytotoxic effect. Moreover, among the five effective parts of n-hexane, ethyl acetate, chloroform, n-butanol and water, the anti-tumor activity of the chloroform part is the most obvious, and it can be used to prepare anti-tumor therapeutic drugs.

[0017] Furthermore, the extract of the chloroform part of magnolia can be used in the preparation of anti-tumor drugs with reduced toxicity and enhanced efficacy, drugs for preventing tumors, drugs for enhancing the efficacy of other anti-tumor drugs, drugs for sensitizing the drug sensitivity of tumor cells, or drugs for improving the body's immunity.

[0018] The invention also discloses the application of the extract of the chloroform part of magnolia flos in preparing a tumor drug resistance reversal agent.

[0019] Preferably, the tumor drug is a tyrosine kinase inhibitor, and the extract from the chloroform part of magnolia can reverse the drug resistance of this type of drug.

[0020] Furthermore, tyrosine kinase inhibitors are tinib drugs, such as afatinib, imatinib, dasatinib, ametinib, gefitinib, nilotinib, sunitinib and osimertinib.

[0021] The present invention also discloses an anti-tumor drug composition, which is composed of the extract of the chloroform part of magnolia officinalis and a pharmaceutically acceptable auxiliary material, and is in the form of a preparation suitable for pharmaceutical use; The preparation forms include tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, lozenges, granules, electuary preparations, pills, powders, pastes, pills, suspensions, solutions, injections, powder injections, freeze-dried powder injections, suppositories, ointments, hard plasters, creams, sprays, drops, dropping pills and patches.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts solvent extraction, extraction and other natural product extraction and separation operation means, and for the first time, the methanol extract of magnolia, especially the chloroform extract, is extracted efficiently. Through comparative analysis, it can be concluded that the relevant effect of magnolia methanol extract on cancer cells is stronger than that of ethanol extract and water extract. In particular, the chloroform extract of magnolia has been proved to have the activity of inhibiting tumor cell growth in vitro, so it has anti-tumor activity. At the same time, the chloroform part of magnolia can also inhibit the expression of proteins related to the EGFR TKI resistance pathway, thereby reversing tumor resistance. The combined use of the chloroform extract of magnolia and small molecule targeted drugs (such as the tinib drugs used in the experiment) can significantly reverse the resistance of small molecule targeted drugs, thereby better exerting its anti-tumor effect. The scheme of the present invention expands the raw material channels of anti-tumor drugs, expands the use of magnolia, and makes magnolia promising to develop into a new raw material for anti-tumor drugs, which can significantly improve the medicinal value of magnolia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The graph shows the inhibitory effect of the chloroform fraction of the methanol extract of Magnolia officinalis on the proliferation of lung cancer NCI-H1299 and NCI-H460 cells, breast cancer MCF-7 cells, cervical cancer Hela cells, liver cancer Bel-7404 cells and colon cancer LoVo cells; Figure 2 This is the KEGG pathway enrichment analysis diagram of potential targets of magnolia in treating lung cancer; Figure 3 This is the effect of the chloroform part of Magnolia officinalis on the expression of proteins related to the EGFR TKI resistance pathway. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0025] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0026] The present invention is further described in detail below in conjunction with the accompanying drawings: The method for extracting the effective anti-tumor components of Magnolia officinalis disclosed in the present invention comprises the following steps: (1) Magnolia officinalis was used as the raw material. The medicinal material Magnolia officinalis was dried and crushed, methanol was added, and heated under reflux at 65-75 °C for 3 h. This was repeated three times, filtered, and the extracts were combined to obtain the extract.

[0027] (2) After the extract of step (1) is concentrated to remove methanol, a total extract is obtained, and 10-20 times the volume of water is added and stirred into a suspension, and the water extract is extracted with n-hexane in a volume ratio of 1:1, and the extraction is performed twice, and the extracts are combined. The n-hexane is recovered, concentrated by a rotary evaporator, and dried in a vacuum dryer at 40°C to obtain the n-hexane fraction.

[0028] (3) The water layer after the n-hexane extraction was extracted with chloroform in a volume ratio of 1:1, and the extraction was performed twice, and the extracts were combined. The chloroform was recovered, concentrated by a rotary evaporator, and dried in a vacuum dryer at 30°C to obtain the chloroform fraction.

[0029] (4) The aqueous layer solution extracted with n-hexane and chloroform was extracted with ethyl acetate in a volume ratio of 1:1, and the extraction was performed twice, and the extracts were combined. The ethyl acetate was recovered, concentrated by a rotary evaporator, and dried in a vacuum dryer at 30°C to obtain the ethyl acetate fraction.

[0030] (5) The aqueous layer extracted with n-hexane, chloroform and ethyl acetate was extracted with n-butanol in a volume ratio of 1:1, and the extraction was performed twice, and the extracts were combined. The n-butanol was recovered and dried in a vacuum dryer at 60°C to obtain the n-butanol fraction.

[0031] (6) The water extract is concentrated and dried by freeze-drying to obtain a water fraction.

[0032] The above five polar parts are the extracts of effective components of Magnolia officinalis.

[0033] The present invention further provides the use of an extract of effective anti-tumor components of magnolia in the preparation of anti-tumor drugs. Lung cancer cells, breast cancer cells, cervical cancer cells, liver cancer cells and colon cancer cells were used as test tumor strains, and the ability to inhibit tumor cell proliferation was used as an activity index to conduct anti-tumor activity experiments. The experimental results showed that in an in vitro cytotoxicity experiment, the methanol extract of magnolia can inhibit the proliferation of tumor cells and has a cytotoxic effect. Moreover, among the five effective polar parts of n-hexane, ethyl acetate, chloroform, n-butanol and water, the chloroform part has the most obvious anti-tumor activity, and can be used to prepare anti-tumor therapeutic drugs.

[0034] Example 1: Extraction of anti-tumor active ingredients from Magnolia officinalis Put the magnolia into a grinder, grind for 1 min, cool for 5 min, repeat three times, collect the powder, mark and set aside. Take 20 g of the powder of this product, add 400 mL of methanol, 400 mL of 70% ethanol, and 400 mL of water, respectively, and heat to reflux at 70 ° C for 3 hours. Filter, concentrate in a rotary evaporator to reduce the volume to 20 mL, dry in a rotary vacuum drying oven at 50 ° C, and make an extract for use. Use an analytical balance to accurately weigh and calculate the yield. After preparing a 50 mg / mL mother solution, filter it with a 0.2 μm filter and store it in a 4 ° C refrigerator for use. The MTT assay was used to detect the effects of magnolia extract on the proliferation of lung cancer NCI-H1299 and NCI-H460 cells, breast cancer MCF-7 cells, cervical cancer Hela cells, liver cancer Bel-7404 cells, and colon cancer LoVo cells. 4 After 48 hours of drug action, MTT solution diluted 10 times with serum-free medium (final concentration of 0.5 mg / mL) was added, incubated in an incubator for 4-6 hours, and then DMSO was added. The cells were shaken on a decolorizing shaker for 15 minutes. The absorbance (OD) value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay, and the IC value of each extract was calculated using GraphPad Prism software. 50 The experimental results are shown in Figure 1 And Table 1. It can be seen that the methanol extract of Magnolia officinalis is superior to the 70% ethanol extract and water extract, and has the most significant inhibitory effect on the proliferation of lung cancer NCI-H1299.

[0035] Table 1. IC values ​​of different extracts from Magnolia officinalis for antitumor activity 50 value

[0036] Example 2: Separation of anti-tumor active ingredients from Magnolia officinalis After the methanol extract of Magnolia officinalis is dried, the total extract is obtained, and distilled water is added to stir evenly to form a suspension, which is then put into a separating funnel, and an equal volume of n-hexane is added to extract twice, and the n-hexane extracts are combined, recovered under reduced pressure, and dried in a vacuum dryer at 40°C to constant weight to obtain the n-hexane part. After the remaining aqueous solution is decompressed to remove the residual n-hexane, it is extracted twice with an equal volume of chloroform, and the chloroform extracts are combined, recovered under reduced pressure, and dried in a vacuum dryer at 30°C to constant weight to obtain the chloroform part. After the remaining aqueous layer solution is decompressed to remove the residual chloroform, it is extracted twice with an equal volume of ethyl acetate, and the ethyl acetate extracts are combined, recovered under reduced pressure, and dried in a vacuum dryer at 30°C to constant weight to obtain the ethyl acetate part. Extract twice with an equal volume of n-butanol, combine the n-butanol extracts, recover under reduced pressure, and dry in a vacuum dryer at 60°C to constant weight to obtain the n-butanol part. Finally, the water extract is concentrated and dried by freeze drying to obtain the water part.

[0037] The MTT experimental method was used to detect the effect of the extracts from the effective parts of Magnolia officinalis on the proliferation of lung cancer cells NCI-H1299. The cells were seeded in 96-well plates (1×10 4 After 48 hours of drug action, MTT solution diluted 10 times with serum-free medium (final concentration of 0.5 mg / mL) was added, incubated in an incubator for 4-6 hours, and then DMSO was added. The cells were shaken on a decolorizing shaker for 15 minutes. The absorbance (OD) value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay, and the IC value of each extraction site was calculated using GraphPad Prism software. 50 The experimental results are shown in Table 2. It can be seen that the five parts of Magnolia floridula extract have good tumor inhibition effects, and the chloroform part has the most obvious anti-tumor effect.

[0038] Example 3: Mouse tumor model experiment of effective components of Magnolia officinalis NCI-H1975 and HCC827AR cells were cultured at 2×10 7 The cells were suspended in sterile saline at a density of 100 / mL and inoculated subcutaneously in the right axilla of nude mice, 0.2 mL per mouse.

[0039] The mice were randomly divided into groups of 6 male mice in each group in the second week after inoculation, and the drugs were administered according to the concentration gradient designed according to the median lethal dose, and the drugs were administered for 12 days or 21 days according to the scheme. After the administration, the mice were killed, the tumors were removed and weighed, and the inhibition rate of the Chinese herbal medicine extract on the growth of the nude mouse transplanted tumor was calculated. The results are shown in Tables 2 and 3 below.

[0040] The calculation formula of tumor inhibition rate is as follows:

[0041] The experimental data were expressed as mean ± standard error (Mean ± SEM) and analyzed using SPSS 19.0 statistical software. P < 0.05 indicated a significant difference.

[0042] Table 2. Animal test results of extracts from chloroform parts of Magnolia officinalis

[0043] Compared with the negative control group, ** P< 0.01, **** P< 0.0001 Table 3. Animal test results of extracts from chloroform parts of Magnolia officinalis

[0044] Compared with the negative control group,* P< 0.05,** P< 0.01, **** P< 0.0001 When used specifically, the extract of the chloroform part of the magnolia of the present invention can be combined with a pharmaceutically acceptable carrier or excipient as a medicinal ingredient as an anti-tumor drug. The present invention has found that the extract of the effective component of magnolia has a good effect in treating breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer and other cancers. The Chinese medicine extract provided by the present invention can be used to prepare drugs for treating breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer and other cancers. This discovery has not been reported so far. The large amount of experimental data in the present invention provides an experimental basis for preparing the magnolia extract that inhibits tumor growth.

[0045] Example 4 Enrichment analysis of potential targets of Magnolia officinalis in treating lung cancer Active ingredients of magnolia flowers were collected by searching the China National Knowledge Infrastructure (CNKI) and PubMed databases. The active ingredients were collected and the targets of each ingredient were summarized and integrated through swisstarget prediction.ch. The key targets were uploaded to the STRING (https: / / cn.string-db.org / ) database, and the species was selected as Homo sapiens. The minimum interaction threshold was set to high confidence (high confidence 0.4). The PPI information of key targets was obtained, and free targets were eliminated. The obtained data was imported into Cytoscape 3.9.1 software to establish a visual PPI network. The core genes screened by PPI were imported into metascape for KEGG analysis. From KEGG pathway enrichment analysis Figure 2 It can be seen that the active ingredients of Magnolia officinalis regulate the resistance pathway of tyrosine kinase inhibitors (EGFR-TKIs).

[0046] Example 5 Experiment on the effect of chloroform fraction of Magnolia officinalis on the expression of proteins related to EGFR TKI resistance pathway The cells treated with chloroform parts of Magnolia officinalis were lysed in RIPA buffer containing phosphatase / protease inhibitors. Protein concentration was quantified using the BCA assay. The protein samples were subjected to SDS-PAGE and transferred to PVDF membranes, which were then blocked with 5% skim milk and incubated with the designated primary antibodies at 4 °C overnight. The membranes were then incubated with secondary antibodies, and protein expression levels were observed using an ECL kit. The results are shown in Figure 3 As shown, according to the results of protein immunoblotting, the chloroform site of Magnolia biondii can affect the expression of proteins related to the EGFR / PI3K / AKT pathway, which indicates that it has the potential to reverse tumor resistance.

[0047] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing an extract from the chloroform part of Magnolia flos odoratissimum, characterized in that: The following steps are involved: 1) crushing the raw material magnolia, cooling it, and collecting it to obtain magnolia powder; 2) Extracting the magnolia powder with pure methanol by reflux, and then filtering and rotary evaporating to obtain the magnolia methanol extract; 3) The methanol extract of Magnolia officinalis is concentrated and dried to remove methanol to obtain a total extract, which is then stirred with water to form a suspension, and then extracted with n-hexane for several times. The remaining aqueous solution after the n-hexane extraction is decompressed to remove the residual n-hexane, and then extracted with chloroform for several times, the chloroform extracts are combined, the solvent is recovered under reduced pressure, and dried to obtain the chloroform extract of Magnolia officinalis.

2. The method for preparing the extract of chloroform part of Magnolia flos florida according to claim 1, characterized in that: In step 1), the powder was crushed for 1 min and cooled for 5 min, and the crushing and cooling operations were repeated three times.

3. The method for preparing the extract of chloroform part of Magnolia flos florida according to claim 1, characterized in that: In step 2), the material-liquid ratio of magnolia powder to pure methanol is 1 g: (20~50) mL.

4. The method for preparing the extract of chloroform part of Magnolia flos florida according to claim 1, characterized in that: In step 2), the reflux extraction is performed at 65-75 °C for 2-4 h and repeated 3 times.

5. The method for preparing the extract of chloroform part of Magnolia flos florida according to claim 1, characterized in that: In step 3), drying is performed in a rotary vacuum drying oven at 50°C; water 10 to 20 times the volume of the total extract is added and stirred to form a suspension.

6. The method for preparing the extract of chloroform part of Magnolia flos florida according to claim 1, characterized in that: In step 3), extract at least twice with an equal volume of n-hexane and extract at least twice with an equal volume of chloroform; the drying treatment is to dry in a vacuum dryer at 30°C to constant weight to obtain the chloroform extract of magnolia floret.

7. The extract of chloroform part of magnolia flos prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the extract from the chloroform part of Magnolia flos florida according to claim 7 in the preparation of anti-tumor drugs, characterized in that: The tumor is various solid tumors or leukemia.

9. Use of the extract of chloroform part of magnolia biondii according to claim 7 in preparing a tumor drug resistance reversal agent.

10. An anti-tumor pharmaceutical composition, characterized in that: The extract of the chloroform part of magnolia flower as claimed in claim 7 is added with pharmaceutically acceptable excipients and is in the form of a preparation suitable for pharmaceutical use; The preparation forms include tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, lozenges, granules, electuary preparations, pills, powders, pastes, pills, suspensions, solutions, injections, powder injections, freeze-dried powder injections, suppositories, ointments, hard plasters, creams, sprays, drops, dropping pills and patches.

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