CN106334108B - Medicinal composition of functional red yeast active ingredient and docosahexaenoic acid and application thereof in preparing antitumor medicament - Google Patents
Medicinal composition of functional red yeast active ingredient and docosahexaenoic acid and application thereof in preparing antitumor medicament Download PDFInfo
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Abstract
The invention relates to the technical field of medicines, and discloses a medicinal composition of functional red yeast active ingredients and docosahexaenoic acid and application thereof in preparing an anti-tumor medicament. The medicinal composition comprises a functional red yeast rice extract and docosahexaenoic acid, wherein the functional red yeast rice extract takes lovastatin as a main active ingredient. The anti-tumor effect of the composition is better than that of each component when the components are used independently, and the inhibition effect on cells is more obvious, so that the combined medicine can be used for treating tumors.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a medicinal composition of functional red yeast active ingredients and docosahexaenoic acid and application thereof in preparing antitumor medicaments.
Background
Malignant tumors are a serious threat to human health and life. In recent years, the incidence of tumors in China is on the rising trend year by year. Therefore, anti-tumor research has become a significant hotspot in many subject research fields such as life sciences, medicine and pharmacology nowadays.
One of the biggest problems faced by the current clinical treatment of tumors is that tumor cells gradually generate drug resistance to therapeutic drugs, thereby causing treatment failure. The reasonable combined medication according to the action mechanism of the antitumor drug and the proliferation kinetics of tumor cells is one of the important progresses in the field of tumor chemotherapy in recent years and is also one of the effective methods for overcoming the drug resistance of the tumor. The combination of the anti-tumor drugs with different mechanisms of action can enhance the curative effect and reduce the dosage, greatly reduce the inevitable toxic and side effects brought by the chemotherapeutic drugs, provide a more effective and advanced scheme for the clinical treatment of tumors and achieve the aim of relieving the pain of patients. Compared with single use of anti-tumor drugs, the combined drug has the advantages of reducing the toxic and side effects of chemotherapy, increasing the curative effect of chemotherapy, avoiding or reducing the occurrence of tumor drug resistance and the like, thereby becoming one of the main means for treating tumors at home and abroad in clinic for half a century. One of the main guiding principles of combined medication is to combine drugs with different mechanisms to kill tumors from different targets and different aspects, so as to achieve the purpose of treating tumors.
The red rice is prepared from rice and Monascus purpureusMonascus spp.The natural product prepared by solid state fermentation is popular with domestic and foreign consumers because of having good function of reducing blood fat. The main metabolite Lovastatin (Monacolin K) of red yeast is the main drug effect component, and can inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which is a rate-limiting enzyme in the synthesis process of cholesterol, thereby regulating fat metabolism. HMG-CoA reductase is the rate-limiting enzyme in the early stage of cholesterol biosynthesis, and lovastatin competitively inhibits HMG-CoA reductase, resulting in a decrease in mevalonate synthesis and thus inhibition of cholesterol biosynthesis.
Disclosure of Invention
In order to solve the technical problems, the invention provides a medicinal composition of functional red yeast active ingredients and docosahexaenoic acid and application thereof in preparing antitumor medicaments. The medicinal composition comprises a functional red yeast rice extract and docosahexaenoic acid which take lovastatin as main active ingredients, and the combined application of the functional red yeast rice extract and the docosahexaenoic acid can generate a synergistic anti-tumor effect, so that the tumor can be more effectively treated, and meanwhile, the toxic and side effects are reduced.
The specific technical scheme of the invention is as follows: a pharmaceutical composition of functional red rice active ingredient and docosahexaenoic acid comprises functional red rice extract and docosahexaenoic acid (DHA) containing lovastatin as main active ingredients. Wherein the functional red rice extract and the docosahexaenoic acid can be in any proportion.
The inventor finds that the lovastatin can inhibit the growth of various tumor cells including breast cancer cells, prostate cancer cells, colon cancer cells and the like in vitro by influencing a cholesterol synthesis pathway through long-term research. Docosahexaenoic acid (DHA) is an unsaturated fatty acid very important to the human body, and belongs to an important member of an omega-3 unsaturated fatty acid family. Marine fish, some algae or lower fungi are an important source of DHA, and DHA-containing Lovaza capsules are currently approved by the U.S. Food and Drug Administration (FDA) as dietary supplements to reduce triglyceride levels in adult patients.
The anti-tumor functional red yeast rice extract and DHA pharmaceutical composition is characterized in that the functional red yeast rice extract with lovastatin as a main active component has an anti-tumor effect and can also enhance the anti-tumor effect of DHA. Wherein the functional red yeast rice extract taking lovastatin as the main active component is used in combination with DHA to achieve the effect of synergistically inhibiting the proliferation of tumor cells.
Further, the functional red yeast extract with lovastatin as the main active ingredient is extracted from functional red yeast; the docosahexaenoic acid is naturally extracted or chemically synthesized and modified.
Further, the docosahexaenoic acid and the functional red yeast rice extract are used as a combined preparation for simultaneous, separate or sequential use.
Furthermore, the functional red yeast rice is a natural product prepared by taking rice as a raw material and performing solid state fermentation on the rice by using monascus fungi.
Furthermore, the functional red yeast rice extract with lovastatin as the main active component has the lovastatin content of 0-99.99 percent.
Further, the functional red yeast rice extract with lovastatin as the main active ingredient is obtained by extracting functional red yeast rice as a raw material by using a solvent, wherein the solvent is selected from water, ethanol, methanol, ethyl acetate, dichloromethane, petroleum ether, ethanol water (0-100%, preferably 40-80%), methanol water (0-100%, preferably 40-80%) and the like.
Further, the functional red yeast extract with lovastatin as main active ingredient is obtained by heating extraction, soaking extraction or primary column chromatography.
Further, the functional red yeast rice extract and the docosahexaenoic acid are jointly prepared into the following medicines: powder, tablet, granule, capsule, solution, powder for injection, emulsion, liposome, suspension, gel, spray, aerosol, powder spray, lotion, liniment, ointment, plaster, paste, patch, suppository, drop or dripping pill.
The invention also discloses the application of the medicinal composition of the functional red yeast active ingredient and the docosahexaenoic acid in preparing an anti-tumor medicament.
Further, the tumor diseases to be treated include benign tumors and malignant tumors. Including any one of leukemia, lymphoma, skin cancer, sarcoma, breast cancer, hepatocarcinoma, lung cancer, glioma or glioblastoma.
Compared with the prior art, the invention has the beneficial effects that: the medicinal composition is subjected to in-vitro cell toxicity experiments of human breast cancer MCF-7 and MDA-MB-231 cells, and the results show that in the MCF-7 and MDA-MB-231 cell experiments, the functional red yeast extract taking lovastatin as the main active ingredient can obviously enhance the cell toxicity of DHA and has more obvious inhibition effect on cells than the functional red yeast extract or DHA is used alone, and the apoptosis detection experiment results show that the functional red yeast extract and DHA can cooperatively induce breast cancer MCF-7 and MDA-MB-231 cells to apoptosis, so that the combined administration of the functional red yeast extract and DHA can enhance the anti-tumor effect of the functional red yeast extract.
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FIG. 1: the functional red yeast rice extract and DHA are used together to inhibit the proliferation of breast cancer cells MCF-7;
FIG. 2: the functional red yeast extract and DHA are used together to inhibit the proliferation of breast cancer cells MDA-MB-231;
FIG. 3: the functional red yeast rice extract and DHA are used together to induce the apoptosis of the breast cancer cells MCF-7;
FIG. 4: the functional red yeast extract and DHA are used together to induce breast cancer cell MDA-MB-231 to die;
FIG. 5: the functional red yeast rice extract and DHA are used together to induce the cracking of apoptosis-related protein PARP.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: cell proliferation assay
And (3) adopting an SRB colorimetric method to investigate the effect of the functional red yeast extract and DHA combined medicine on the in-vitro inhibition of the proliferation capacity of the breast cancer cells, and calculating the inhibition rate.
The experimental method comprises the following steps:
1) tumor cells were seeded at 3000-.
2) After 24 hours, different concentrations of functional red yeast rice extract and DHA were added (half-diluted, set at 6 concentrations), and the corresponding mixed solution, each concentration was repeated for 3 wells.
3) After 72 hours, 50 μ L of 4 deg.C precooled TCA solution (30%, w/v) was added per well to fix the cells to a final concentration of 10% TCA solution. Standing for 5 min, fixing in a refrigerator at 4 deg.C for 1h, taking out, washing with deionized water for 5 times, and air drying at room temperature. After the 96-well plate is dried at room temperature, 70 microliter of SRB dye solution (prepared by 1% acetic acid) of 0.4% (w/v) is added into each well, the dye solution is poured out after dyeing for 30 min, the dye solution is washed for 4 times by 1% (v/v) acetic acid, unbound dye is removed, and the plate is dried at room temperature. The dye bound to the cellular protein was solubilized with 100. mu.L of unbuffered Tris-base lye (10mM, pH =10.5), shaken on a horizontal shaker for 20min, and the light absorption was measured using a microplate reader at 515 nm.
4) The cell survival rate of the control group is 100% by taking 0 mu mol/L of the drug, and the rest groups are as follows: survival rate (absorbance for each concentration group/absorbance for control group) x 100% was compared to it and plotted.
The experimental results are as follows:
as shown in fig. 1 and fig. 2, the functional red yeast extract inhibited the growth of breast cancer cells, and when used in combination with DHA, the inhibition was significantly enhanced (solid squares).
Example 2: apoptosis assay
Adopting PI single staining and Annexin-V/PI double staining methods to investigate the capacity of DHA and functional red yeast extract for promoting the apoptosis of tumor cells in vitro by combining the medicines.
The experimental method comprises the following steps:
1) tumor cells were seeded at 300000-500000 cells/well in 6-well plates;
2) after 24 hours, adding DHA and functional red yeast rice extract with corresponding concentration and the mixed solution respectively;
3) after 24 hours, cells were collected and stained with Annexin-V for 15 minutes at room temperature in the dark;
4) then PI is used for dyeing for 5 minutes at room temperature in a dark place;
5) 10000 cells collected by flow cytometry for analysis to obtain early apoptosis (PI)-AV+) And late apoptosis (PI)+AV+) Percentage, the two were added to give the total apoptosis rate.
The experimental results are as follows:
as shown in FIG. 3 and FIG. 4, DHA and functional red yeast rice extract are used together to increase the ability of inducing apoptosis of breast cancer cells MCF-7 and MDA-MB-231. The apoptosis rate was significantly increased in the combination group compared to the single group.
Example 3: immunoblot analysis (Western Blot)
The change condition of an important protein PARP of an apoptosis pathway is examined by adopting a Western Blot method, so that the protein level reflects the capability of combining a functional red yeast rice extract and DHA for inducing apoptosis.
The experimental method comprises the following steps:
1) tumor cells were seeded at 300000-500000 cells/well in 6-well plates;
2) after 24 hours, adding DHA and functional red yeast rice extract with corresponding concentrations and mixed solution respectively;
3) after the drug acts for 6, 12 and 24 hours, the cells are collected, washed by PBS, and added with 50 mul of precooled cell lysate for lysis at 4 ℃ for 40 min;
4) after protein quantification, adding 40 mu g of sample into an equivalent loading buffer solution, performing electrophoresis by 10% SDS-PAGE, and transferring to a PVDF membrane by using an electric transfer instrument;
5) PARP and GAPDH specific antibodies were added and incubated overnight at 4 ℃. The secondary antibody is fluorescently marked and incubated for 1h at room temperature. The results were analyzed after scanning.
The experimental results are as follows:
as shown in figure 5, the combination of DHA and functional red yeast rice extract significantly caused the cleavage of PARP, an important protein in the MCF-7 apoptosis pathway.
Example 4: functional red yeast extract extraction
Soaking 100g of functional red rice in ethyl acetate (mass volume ratio of 1: 4) at room temperature for 24 hr, repeating for 3 times, respectively pouring out supernatant, filtering with gauze, mixing the 3 times of leachate, concentrating to obtain ethyl acetate extract, and finally obtaining 4.7 g of ethyl acetate extract.
Example 5
A pharmaceutical composition comprises functional red rice extract and docosahexaenoic acid at a mass ratio of 5:5, wherein the functional red rice extract contains lovastatin as main active component.
The functional red yeast extract with lovastatin as main active ingredient is extracted from functional red yeast; the docosahexaenoic acid is naturally extracted.
The functional red yeast rice is a natural product which is prepared by taking rice as a raw material and performing solid state fermentation on monascus fungi.
The functional red yeast rice extract with lovastatin as main active ingredient has lovastatin content of 50%.
Example 6
A pharmaceutical composition comprises functional red rice extract and docosahexaenoic acid at a mass ratio of 1:9, wherein the functional red rice extract contains lovastatin as main active component.
The functional red yeast extract with lovastatin as main active ingredient is extracted from functional red yeast; the docosahexaenoic acid is chemically synthetically modified.
The functional red yeast rice is a natural product which is prepared by taking rice as a raw material and performing solid state fermentation on monascus fungi.
The lovastatin content of the functional red yeast extract with the lovastatin as the main active component is 99.99 percent.
Example 7
A pharmaceutical composition comprises functional red rice extract and docosahexaenoic acid at a mass ratio of 9:1, wherein the functional red rice extract contains lovastatin as main active component.
The functional red yeast extract with lovastatin as main active ingredient is extracted from functional red yeast; the docosahexaenoic acid is naturally extracted.
The functional red yeast rice is a natural product which is prepared by taking rice as a raw material and performing solid state fermentation on monascus fungi.
The lovastatin content of the functional red yeast extract with the lovastatin as the main active component is 1 percent.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (6)
1. A functional red yeast active ingredient and docosahexaenoic acid pharmaceutical composition is characterized in that: the drug effect substance of the pharmaceutical composition consists of a functional red yeast extract and docosahexaenoic acid, wherein the functional red yeast extract takes lovastatin as a main active component; the pharmaceutical composition is a medicament for treating breast cancer;
wherein, the breast cancer cells are MCF-7 cells, and the cell concentration combination of the functional red yeast rice extract and the docosahexaenoic acid is one of the following three groups:
docosahexaenoic acid 160 mu M, functional red yeast extract 53.9389 mu g/mL;
docosahexaenoic acid 180 μ M, functional red rice extract 60.6813 μ g/mL;
200 mu M of docosahexaenoic acid and 67.4237 mu g/mL of functional red yeast extract;
the extraction method of the functional red yeast rice extract comprises the following steps: soaking 100g of functional red rice in 400mL of ethyl acetate at normal temperature for 24 hours, repeating for 3 times, respectively pouring out the supernatant, filtering with gauze, mixing the obtained leachate for 3 times, and concentrating to obtain the functional red rice extract.
2. A functional red yeast active ingredient and docosahexaenoic acid pharmaceutical composition is characterized in that: the drug effect substance of the pharmaceutical composition consists of a functional red yeast extract and docosahexaenoic acid, wherein the functional red yeast extract takes lovastatin as a main active component; the pharmaceutical composition is a medicament for treating breast cancer;
wherein, the breast cancer cell is MDA-MB-231 cell, and the cell concentration combination of the functional red yeast rice extract and the docosahexaenoic acid is one of the following three groups:
docosahexaenoic acid 80 μ M, functional red rice extract 6.74 μ g/mL;
docosahexaenoic acid 90 μ M, functional red rice extract 7.59 μ g/mL;
docosahexaenoic acid 100 μ M, functional red rice extract 8.43 μ g/mL;
the extraction method of the functional red yeast rice extract comprises the following steps: soaking 100g of functional red rice in 400mL of ethyl acetate at normal temperature for 24 hours, repeating for 3 times, respectively pouring out the supernatant, filtering with gauze, mixing the obtained leachate for 3 times, and concentrating to obtain the functional red rice extract.
3. The pharmaceutical composition of functional red yeast active ingredient and docosahexaenoic acid according to claim 1 or 2, wherein the functional red yeast extract having lovastatin as the main active ingredient is extracted from functional red yeast; the docosahexaenoic acid is naturally extracted or chemically synthesized and modified.
4. The pharmaceutical composition of functional red yeast active ingredient and docosahexaenoic acid according to claim 1 or 2, wherein the functional red yeast is a natural product prepared by solid state fermentation of rice as raw material using monascus as fungus.
5. The pharmaceutical composition of functional red yeast rice active ingredient and docosahexaenoic acid according to claim 1 or 2, wherein the functional red yeast rice extract and docosahexaenoic acid are made into the following forms of drugs together: powder, tablet, granule, capsule, solution, powder for injection, emulsion, liposome, suspension, gel, spray, aerosol, powder spray, lotion, liniment, ointment, plaster, paste, patch, suppository, drop or dripping pill.
6. The use of the pharmaceutical composition of functional red yeast rice active ingredient and docosahexaenoic acid as claimed in any one of claims 1-5, wherein the tumor is breast cancer.
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