CN115677634B - Red yeast yellow pigment with anticancer function - Google Patents

Red yeast yellow pigment with anticancer function Download PDF

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CN115677634B
CN115677634B CN202110583103.5A CN202110583103A CN115677634B CN 115677634 B CN115677634 B CN 115677634B CN 202110583103 A CN202110583103 A CN 202110583103A CN 115677634 B CN115677634 B CN 115677634B
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yellow pigment
mycelium
monascus
monascus yellow
phase
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CN115677634A (en
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李牧
段雅丽
易志强
刘佳玮
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Huazhong Agricultural University
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Huazhong Agricultural University
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Abstract

The invention belongs to the field of compounds, and particularly relates to monascus yellow pigment with an anticancer function. The specific technical scheme is as follows: a new monascus yellow pigment Monascin A is provided. Experiments prove that the invention has stronger anticancer function and great potential of becoming an anticancer drug.

Description

Red yeast yellow pigment with anticancer function
Technical Field
The invention belongs to the field of compounds, and particularly relates to monascus yellow pigment with an anticancer function.
Background
Natural products are the main sources of new drugs, such as anticancer, anti-inflammatory, antibacterial, etc. Therefore, the development of natural products with anticancer function is of great importance. The natural product can be used as drug or precursor substance for preparing anticancer drug or related research. Currently, about 50% of anticancer drugs are derived from natural products. Numerous studies have shown that the anticancer effect of natural compounds is versatile and mainly comprises: directly inhibit cancer cell growth, resist oxidation and free radical, block cancer cell division cycle, induce tumor cell apoptosis, inhibit protein kinase activity, inhibit signal transduction, inhibit topoisomerase, etc. Therefore, the discovery of new anticancer natural compounds is of great importance.
Disclosure of Invention
The invention aims to provide monascus yellow pigment with an anticancer function.
In order to achieve the aim of the invention, the invention adopts the following technical scheme: a monascus yellow pigment has a molecular formula: c (C) 15 H 18 O 4 The molecular structural formula is as follows:
correspondingly, the monascus yellow pigment preparation method is characterized in that monascus strain M7 is prepared by fermentation, and monascus strain M7 is preserved in a laboratory of collection and preservation focus of agricultural microorganism resources, wherein the preservation number is: CCAM No.070120.
Preferably, the method for extracting monascus yellow pigment after fermentation comprises the following steps: soaking mycelium obtained after fermentation of the monascus strain M7 by HCl, extracting the mycelium by using an organic solvent to obtain a water phase-organic phase two-phase solution, separating a water phase solution, removing pigments in the organic phase, drying, and removing the organic solution.
Preferably, the organic solvent is a mixed solution of ethyl acetate and acetone.
Preferably, the method for extracting monascus yellow pigment after fermentation comprises the following steps: after the mycelium is soaked in HCl, the mycelium is washed once by clear water, the mycelium is collected by filtration, and then the mycelium is mixed with an organic solvent.
Preferably, the organic solvent is mixed with the mycelium to obtain a mixture, the mixture is heated to 60 ℃, the mixture is subjected to shaking extraction, and the mycelium is removed by filtration through nylon filter cloth to obtain the aqueous-organic phase two-phase solution.
Preferably, the method for removing pigments in the organic phase is as follows: adding macroporous resin into the organic phase, and removing pigment by vibration adsorption.
Preferably, the method for extracting monascus yellow pigment after fermentation comprises the following steps: after the organic solution was removed, the obtained product was redissolved with methanol to obtain a methanol solution, and the methanol solution was separated and purified by a semi-preparative liquid chromatograph.
Correspondingly, the monascus yellow pigment is applied to the preparation of anticancer drugs.
The invention has the following beneficial effects: the invention discovers and purifies a novel compound Monascin A from monascus with homology of traditional food and medicine. The molecular structure is determined by NMR and LC-MS, and experiments prove that the medicine has stronger anticancer and antibacterial functions and great potential of becoming an anticancer antibacterial medicine.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of Monospin A;
FIG. 2 is a nuclear magnetic resonance carbon spectrum of Monospin A.
Detailed Description
The invention provides a new monascus yellow pigment Monascin A, C 15 H 18 O 4 The molecular structural formula is as follows:
the Monospin A has a strong anticancer function.
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Embodiment one: preparation, extraction and identification of Monospin A
1. Monascus strain M7 (collection and preservation of agricultural microbial resources from the Ministry of agriculture, accession number: CCAM No.070120, first published in Extremophiles (2016) 20:451-459, cloning, expression and characterization of a novel cold-active and organic solvent-tolerant esterase from Monascus ruber M). Incidentally, the inventors tried various Monascus rubers, and Monascin A was only found and extracted in M7 at present.
The monascus strain M7 was inoculated onto a fresh PDA medium, cultured at 30 ℃ for 10 days, and monascus mycelia were washed with sterile water to obtain monascus spore liquid. Diluting Monascus spore liquid to 1×10 with sterile water 5 And each mL. Taking 2mL of diluted monascus spore liquid, inoculating into 30mL of fresh PDB culture medium, and heating at 30deg.CCulturing at 150rpm for 15 days, filtering with nylon filter screen, discarding filtrate, and collecting mycelium.
2. Mycelium is soaked in 0.1mol/L HCl solution at 30 ℃ for 1h, then filtered, and the filtrate is discarded to obtain mycelium. Washing the mycelium once with clear water, removing HCl adsorbed on the surface of the mycelium, and filtering to collect the mycelium. Mixing with wet mycelium with organic solvent (ethyl acetate: acetone=3:1, volume ratio) to obtain mixture; the ratio of organic solvent to wet mycelium was 30mL:1g of wet mycelium. The mixture was heated to 60℃and extracted with shaking at 50rpm for 3 hours. The mycelium was removed by filtration through nylon filter cloth to give a two-phase aqueous-organic solution. The two-phase solution was separated using a separating funnel, and the aqueous phase was discarded to obtain an organic phase. Then, 3g of a wet macroporous resin (model LSA-7) was added to about 30mL of the obtained organic solvent, and the mixture was shaken at 30℃and 50rpm to adsorb most of the dye molecules by the resin. Then filtering to remove resin particles to obtain organic solvent, concentrating the organic solvent in the organic phase to 1/10 by vacuum distillation, and using anhydrous Na 2 SO 4 The water was removed, and then the whole organic solution was distilled off under reduced pressure to obtain a solid. The obtained solid was dissolved with methanol to obtain a methanol solution.
3. Separating and purifying the methanol solution by a semi-preparative liquid chromatograph. Semi-preparative liquid chromatograph model was Waters, U.S.A., semi-preparative column was InertsilODS-3 (250 mm. Times.10 mm,5 μm). The prepared sample solution was added to the sample inlet at a sample injection amount of 100 μl, a flow rate of 3mL/min, methanol and water as mobile phases, and an initial mobile phase to methanol: water=1:1, column temperature of 25 ℃, PDA detector. And determining a product peak through the retention time, and collecting an eluting component in time to obtain the Monospin A sample with the purity of more than 95%. The sample was dried with a nitrogen blower to give 84 μg of yellow solid powder.
4. The structure of the compound is identified by using NMR (nuclear magnetic resonance) and GC-MS (gas chromatography-mass spectrometry) technologies, a nuclear magnetic resonance hydrogen spectrum is shown in figure 1, and a nuclear magnetic resonance carbon spectrum is shown in figure 2.
Embodiment two: monospin A anticancer effect display
1. And the effect of inhibiting the growth of cancer cells is displayed.
Liver cancer cell line QGY-7701 and lung cancer cell line SPC-A-1 were selected as subjects for testing Monospin A inhibition of cancer cell growth experiments. Two cancer cell lines were inoculated into RPMI-1640 medium (available from Semer Feishul technologies (China)) at 37℃and 5% CO 2 Cells were collected by centrifugation in a constant temperature incubator until log phase, resuspended in buffer, and added to 96-well plates at 180 μl (about 6000 cells/well) per well. Placing the 96-well plate in a constant temperature incubator at 37deg.C with 5% CO 2 Culturing was performed under the same conditions for 14 hours, and then different concentrations of Monospin A (5, 10, 15 or 20. Mu.M) were added to the different wells, respectively, and culturing was continued under the same conditions for 24 hours.
In each well, 100. Mu.L of cell-free supernatant was carefully removed, and 90. Mu.L of fresh serum medium and 10. Mu.L of MTT reagent were added, and the incubation was continued for 4 hours after mixing well. After removing 110. Mu.L of the cell-free supernatant again, 110. Mu.L of Formazan reagent was added thereto, and the mixture was shaken at a low speed for 10 minutes to complete mixing. Finally, the absorbance of each well was measured at 490nm, and the concentration inhibiting the growth of 50% cancer cells, i.e., half inhibition concentration (IC 50), was calculated. The results show that Monospin A has an IC50 of 11.10 μm for liver cancer cell line QGY-7701 and an IC50 of 12.54 μm for lung cancer cell line SPC-A-1, and shows strong cancer cell inhibition.
2. Animal experiments with anticancer effect show.
SD rats used, weighing about 200g, purchased from Shanghai Laek laboratory animals Co., ltd, were induced to develop liver tumors after one week of adaptive feeding, starting with water containing Diethylnitrosamine (DEN). The following groupings were randomly made for 10 rats with insignificant differences in each group, selected from DEN cancer-induced rats for 14 weeks: (1) control group (blank group, no drug); (2) Monospin A group (2 mg/kg). The administration is carried out once a week along with the feeding of the feed for four weeks. At week 19, each group of rats was sacrificed and tissue specimens were obtained. According to liver anatomy results, the number of tumors (tumor mass > 0.5cm in diameter) in rat liver of Monospin A group was smaller than that of control group, about 60% of control group, and the tumor volume was smaller, about 50% of control group. Monospin A was demonstrated to significantly inhibit tumor growth in rats.
Embodiment III: monospin A antibacterial effect display
For bacteria, a loop of bacteria solution was picked from the glycerol-preserving tube with an inoculating loop and inoculated into 10mL of LB medium. Then, the strain was placed in an incubator and cultured with shaking at 37℃and 180rpm for 18 hours to place the strain in the logarithmic phase of growth. Then 1mL of the bacterial liquid was diluted with fresh LB medium, and the absorbance (OD) of the bacterial liquid at 620nm was measured 620 ) Adjusting the concentration of the bacterial liquid to make OD 620 Between 0.09 and 0.11, the concentration of the bacterial liquid is about 5 multiplied by 10 8 CFU/mL. For the filamentous fungi, the strains are inoculated on PDA culture medium and cultured for 5-10 days at 30 ℃ until the colony diameter is more than 2cm. Then inoculating the pre-cultured strain to a fresh PDA culture medium, culturing for 5-10 days at 30 ℃, and washing mycelium with sterile water to obtain spore liquid. Diluting the spore liquid with sterile water, and detecting spore liquid concentration under microscope with blood cell counting plate to about 1×10 5 And each mL.
198. Mu.L of bacterial or filamentous fungal spore solution was added to one well of a 96-well plate using a sterile pipette, then 2. Mu.L of Monospin A solution (2.0 mg/mL) was added to the first well and mixed well with shaking, the Monospin A concentration in the well being 20. Mu.g/mL. Using this dilution method, 5 treatments were set at Monospin A concentrations of 20, 10, 5, 2.5 and 1.25. Mu.g/mL, respectively. And a negative control group was set: 2. Mu.L of methanol was added to 198. Mu.L of the bacterial or spore solution. After the 96-well plate for the bacterial inhibition test was placed in an incubator at 37℃for 24 hours, absorbance was measured at 620nm using a microplate reader, and the minimum Monospin A concentration when the microbial growth was completely inhibited and no longer grown was the Minimum Inhibitory Concentration (MIC) of Monospin A. As a result, monascin A had MIC's for E.coli, pseudomonas aeruginosa, staphylococcus aureus, and Salmonella of 11.2 μg/mL, 20.1 μg/mL, 17.4 μg/mL, and 5.8 μg/mL, respectively; MIC for Aspergillus flavus, aspergillus fumigatus, aspergillus nidulans was 30.3. Mu.g/mL, 26.8. Mu.g/mL, and 25.4. Mu.g/mL, respectively. Has obvious effect of inhibiting the growth of bacteria and fungi.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications, variations, alterations, substitutions made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the design of the present invention.

Claims (8)

1. A monascus yellow pigment, which is characterized in that: the molecular formula is: c (C) 15 H 18 O 4 The molecular structural formula is as follows:
2. the method for preparing monascus yellow pigment as claimed in claim 1, wherein: the monascus strain M7 is prepared by fermentation, and the monascus strain M7 is preserved in a laboratory of collection and preservation focus of agricultural microorganism resources of the Ministry of agriculture, wherein the preservation number is: CCAM No.070120.
3. The method for preparing monascus yellow pigment according to claim 2, wherein: after fermentation, the method for extracting the monascus yellow pigment comprises the following steps: soaking mycelium obtained after fermentation of the monascus strain M7 by HCl, extracting the mycelium by using an organic solvent to obtain a water phase-organic phase two-phase solution, separating and removing water phase liquid, removing pigment impurities in the organic phase, drying, and removing the organic solution; the organic solvent is a mixed solution of ethyl acetate and acetone.
4. A process for the preparation of monascus yellow pigment according to claim 3, wherein: after fermentation, the method for extracting the monascus yellow pigment comprises the following steps: after the mycelium is soaked in HCl, the mycelium is washed once by clear water, the mycelium is collected by filtration, and then the mycelium is mixed with an organic solvent.
5. A process for the preparation of monascus yellow pigment according to claim 3, wherein: mixing the organic solvent with the mycelium to obtain a mixture, heating the mixture to 60 ℃, carrying out shaking extraction, and filtering with nylon filter cloth to remove the mycelium to obtain the aqueous phase-organic phase two-phase solution.
6. A process for the preparation of monascus yellow pigment according to claim 3, wherein: the method for removing the pigment in the organic phase comprises the following steps: adding macroporous resin into the organic phase, and removing pigment by vibration adsorption.
7. A process for the preparation of monascus yellow pigment according to claim 3, wherein: after fermentation, the method for extracting the monascus yellow pigment comprises the following steps: after the organic solution was removed, the obtained product was redissolved with methanol to obtain a methanol solution, and the methanol solution was separated and purified by a semi-preparative liquid chromatograph.
8. The use of monascus yellow pigment according to claim 1 for the preparation of anticancer drugs, characterized in that: the medicine is an anti-liver cancer medicine or an anti-lung cancer medicine.
CN202110583103.5A 2021-07-21 2021-07-21 Red yeast yellow pigment with anticancer function Active CN115677634B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101862324B (en) * 2010-04-13 2012-05-30 福州大学 Application of monascus color components and derivatives thereof in fighting cancers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101862324B (en) * 2010-04-13 2012-05-30 福州大学 Application of monascus color components and derivatives thereof in fighting cancers

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Cloning, expression and characterization of a novel cold‑active and organic solvent‑tolerant esterase from Monascus ruber M7;Hailun Guo et al.;《Extremophiles》;第20卷;451-459 *
液态发酵红曲黄色素粗提物色素组分的分离纯化与鉴定;徐菲菲 等;《食品与发酵工业》;第46卷(第1期);91-98 *
红曲菌菌株 M7 的生物学特性研究;王小莉 等;《中国热带医学》;第5卷(第6期);1349-1350 *

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