Microbial conversion product of bavachalcone as well as preparation method and application of microbial conversion product

Abstract

The invention provides a microbial conversion product of bavachalcone as well as a preparation method and application thereof, and belongs to the technical field of microbial pharmacy. According to the preparation method of the microbial conversion product of the bavachalcone, disclosed by the invention, the bavachalcone is subjected to microbial conversion reaction by sequentially inoculating and co-culturing Absidia caerulea and Mucor hiemalis which have structure specificity and regioselectivity; the new compound 2 '-O-glucosyl-4'-O-methyl bavachalcone which is difficult to prepare through traditional chemical synthesis is obtained, and compared with a traditional chemical method, the method provided by the invention has the advantages that the steps are simplified, the separation efficiency is high, and the yield is relatively high and can be up to 40%. Compared with a substrate, the prepared microbial conversion product is higher in activity and better in water solubility, has a good effect of inhibiting melanoma and breast cancer cells, can be used as a chemical therapeutic agent for preventing and treating related diseases, and has a good research and development prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial pharmaceutical manufacturing, and more specifically, relates to a microbial transformation product of psoralea corylifolia chalcone and a preparation method and application thereof. Background Art

[0002] Psoralea corylifolia L. is the dried mature fruit of the leguminous plant Psoralea corylifolia L. (Pharmacopoeia of the People's Republic of China (Part I), 2020, 195). Its main function is to tonify kidney yang and strengthen bones. It has a history of clinical application in my country for thousands of years and is used in many traditional Chinese medicine formulas, such as Bu Gu Zhi San, Ba Bao San, and Bu Yang Wan. In recent years, its anti-tumor, anti-inflammatory, antibacterial, anti-platelet, antioxidant and glycosidase inhibition effects have been clinically confirmed, and it is a traditional Chinese medicine with broad development prospects.

[0003] Psoralea corylifolia chalcone (bavachalcone) is one of the main active ingredients of Psoralea corylifolia. Previous studies have shown that psoralea corylifolia chalcone belongs to the class of isopentenyl flavonoid compounds. The structure of psoralea corylifolia chalcone was first isolated and identified from the extract of Psoralea corylifolia. Due to its poor water solubility, it is easily soluble in weakly polar organic solvents such as acetone and ethyl acetate, which affects the formation of its preparations and drug development. Subsequently, a variety of active psoralea corylifolia chalcone derivatives, such as psoralen B and 4'-O-methyl psoralea corylifolia chalcone, were successively isolated from Psoralea corylifolia. In addition, a series of chemical reaction modifications were also carried out on psoralea corylifolia derivatives to obtain more efficient psoralea corylifolia chalcone derivatives (European Journal Of Medicinal Chemistry, 2015, 92, 439-448).

[0004] Although a variety of structurally rich Psoralea corylifolia chalcone derivatives have been obtained through plant isolation and chemical synthesis, the C-2' and C-4' of this skeleton-type compound are low-activity sites. Given the limitations of the chemically variable sites in its structure, it is difficult to simultaneously modify the C-2' and C-4' sites using organic chemistry methods, and there is no literature report on this. Summary of the invention

[0005] The object of the present invention is to provide a microbial transformation product of psoralea corylifolia chalcone and a preparation method and use thereof, so as to prepare a psoralea corylifolia chalcone derivative with stronger activity, better water solubility and better suitability for pharmaceutical preparations through a method of sequential microbial inoculation and co-culture transformation.

[0006] To achieve the above object, the first aspect of the present invention provides a microbial transformation product of psoralea corylifolia chalcone, the chemical structure of which is shown below:

[0007]

[0008] The second aspect of the present invention provides a method for preparing a microbial transformation product of psoralea corylifolia chalcone, comprising the following steps:

[0009] S1, inoculating a first microorganism in a system containing a liquid culture medium and psoralea chalcone to perform a first transformation culture;

[0010] S2, then, inoculating a second microorganism into the system to perform a second transformation culture;

[0011] S3, separating and purifying the product obtained after the second transformation culture to obtain a microbial transformation product of psoralea corylifolia chalcone;

[0012] The first microorganism is Absidia caerulea KCTC 6936 or Mucorhiemalis KCTC 26779, and the second microorganism is Absidia caerulea KCTC 6936 or Mucor hiemalis KCTC 26779. The first microorganism is different from the second microorganism.

[0013] Furthermore, the temperature of the first transformation culture is 25-28°C, the rotation speed is 180-200 rpm, and the time is 24-120 hours.

[0014] Furthermore, the temperature of the second transformation culture is 25-28°C, the rotation speed is 180-200 rpm, and the time is 48-120 hours.

[0015] Furthermore, the first microorganism is Absidia caerulea KCTC 6936, and the second microorganism is Mucor hiemalis KCTC 26779.

[0016] Furthermore, the step S1 includes the following steps:

[0017] preparing the liquid culture medium;

[0018] Inoculate the seed liquid of the first microorganism at 2-3% of the volume of the liquid culture medium, and culture at 25-28° C. and 180-200 rpm for 24-48 hours;

[0019] The psoralea corylifolia chalcone solution is added at 1-2% of the volume of the liquid culture medium, and the transformation culture is carried out at 25-28° C. and 180-200 rpm for 24-96 hours.

[0020] Furthermore, the step S1 includes the following steps:

[0021] Adding psoralea corylifolia chalcone solution into the culture container at 1-2% of the volume of the liquid culture medium;

[0022] Next, the liquid culture medium inoculated with the seed solution of the first microorganism is added to the culture container to obtain a mixed system; the inoculation amount of the seed solution of the first microorganism is 2-3% of the volume of the liquid culture medium;

[0023] The mixed system is cultured at 25-28° C. and 180-200 rpm for 24-96 h.

[0024] Furthermore, the psoralea corylifolia chalcone solution is an ethanol solution with a concentration of 20 mg / mL psoralea corylifolia chalcone.

[0025] Furthermore, in step S2, the inoculation amount of the seed liquid of the second microorganism is 4-6% of the volume of the liquid culture medium.

[0026] Furthermore, the seed liquid of the first microorganism is prepared by the following method: inoculating the hyphae of the first microorganism into the liquid culture medium, and culturing at 25-28° C. and 180-200 rpm for 24-48 hours;

[0027] The seed liquid of the second microorganism is prepared by the following method: inoculating the hyphae of the second microorganism into the liquid culture medium, and culturing at 25-28° C. and 180-200 rpm for 24-48 hours.

[0028] Furthermore, the liquid culture medium is potato glucose liquid culture medium.

[0029] Furthermore, in step S3, the separation and purification includes the following steps:

[0030] The fermentation liquid obtained after the second transformation culture is filtered, the filtrate is collected, extracted with ethyl acetate, and evaporated to dryness under reduced pressure to obtain a crude extract;

[0031] The crude extract is separated and purified by semi-preparative liquid chromatography.

[0032] The third aspect of the present invention provides an application of a microbial transformation product of psoralea corylifolia chalcone in the preparation of a drug for inhibiting human malignant melanoma cells A375P or human breast cancer cells MCF-7.

[0033] Compared with the prior art, the present invention has the following technical effects:

[0034] The invention discloses a method for preparing a microbial transformation product of psoralea chalcone, wherein the blue abscission mold Absidia caerulea and the permafrost mold Mucor hiemalis with structural specificity and regional selectivity are sequentially inoculated and co-cultured to carry out a microbial transformation reaction on the psoralea chalcone, thereby obtaining a new compound 2'-O-glucosyl-4'-O-methyl psoralea chalcone which is difficult to prepare by traditional chemical synthesis, and the method of the invention has simplified steps, high separation efficiency and high yield compared with traditional chemical methods, which can reach up to 40%. The method of the invention utilizes the blue abscission mold Absidia caerulea and the permafrost mold Mucor hiemalis to sequentially inoculate and co-culture to introduce a glucose group at the 2'-OH position of the psoralea chalcone and a methyl group at the 4'-OH position, and the methylation and glucosylation of the two sites can improve the drugability and water solubility of the psoralea chalcone.

[0035] The microbial transformation product (psoralea chalcone derivative) prepared by the present invention has stronger activity and better water solubility than the substrate, and the prepared microbial transformation product has a good effect of inhibiting melanoma and breast cancer cells, can be used as a chemotherapeutic agent for preventing and treating related diseases, and has good research and development prospects. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] In the present invention, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0038] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0039] The weight of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also indicate the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the mass described in the description of the embodiments of the present invention may be μg, mg, g, kg, or other mass units known in the biochemical field.

[0040] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the embodiments of the present invention, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0041] The embodiment of the present invention provides a microbial transformation product of psoralea corylifolia chalcone and a preparation method thereof, comprising the following steps:

[0042] S1, inoculating a first microorganism in a system containing a liquid culture medium and psoralea chalcone to perform a first transformation culture;

[0043] S2, then, inoculating a second microorganism into the system to perform a second transformation culture;

[0044] S3, separating and purifying the product obtained after the second transformation culture to obtain a microbial transformation product of psoralea corylifolia chalcone;

[0045] Wherein, the first microorganism is Absidia caerulea KCTC 6936 or Mucor hiemalis KCTC 26779, the second microorganism is Absidia caerulea KCTC 6936 or Mucor hiemalis KCTC 26779, and the first microorganism is different from the second microorganism. Preferably, the first microorganism is Absidia caerulea KCTC 6936, and the second microorganism is Mucorhiemalis KCTC 26779. Inoculating in this order can obtain a higher yield of microbial conversion products of bone fat chalcone.

[0046] In the above step S1, the liquid culture medium of the embodiment of the present invention is a liquid culture medium suitable for the growth of Absidiacaerulea KCTC 6936 and Mucor hiemalis KCTC 26779. In a specific embodiment, the liquid culture medium of the embodiment of the present invention is a potato glucose liquid culture medium, which can be prepared by the following method: take fresh potatoes, peel them, cut them into small pieces, then add water and boil them for 20-30 minutes, then filter them with gauze, cool the filtrate, add water to make up the volume, then add glucose and stir until dissolved, divide them into portions and sterilize them by high pressure at 118°C, then cool them for later use.

[0047] In the embodiment of the present invention, the temperature of the first transformation culture is 25-28° C., the rotation speed is 180-200 rpm, and the time is 24-120 h.

[0048] Specifically, step S1 of the embodiment of the present invention further includes the following steps:

[0049] Prepare liquid culture medium;

[0050] The seed solution of the first microorganism is inoculated at 2-3% of the volume of the liquid culture medium, and cultured at 25-28° C. and 180-200 rpm for 24-48 hours; the seed solution of the first microorganism can be prepared by the following method: inoculating the hyphae of the first microorganism into the liquid culture medium, and culturing at 25-28° C. and 180-200 rpm for 24-48 hours;

[0051] The psoralea chalcone solution is added at 1-2% of the volume of the liquid culture medium, and the transformation culture is carried out at 25-28°C and 180-200 rpm for 24-96 hours. The psoralea chalcone solution used in the embodiment of the present invention is an ethanol solution of psoralea chalcone with a concentration of 20 mg / mL.

[0052] Alternatively, step S1 implemented in the present invention further includes the following steps:

[0053] Adding a psoralea chalcone solution to a culture container at 1-2% of the volume of the liquid culture medium; the psoralea chalcone solution used in the embodiment of the present invention is an ethanol solution of psoralea chalcone with a concentration of 20 mg / mL;

[0054] Next, a liquid culture medium inoculated with a seed solution of the first microorganism is added to the culture container to obtain a mixed system; the inoculation amount of the seed solution of the first microorganism is 2-3% of the volume of the liquid culture medium;

[0055] The mixed system was cultured at 25-28°C and 180-200 rpm for 24-96 h.

[0056] In the above step S2, the temperature of the second transformation culture of the embodiment of the present invention is 25-28°C, the rotation speed is 180-200rpm, and the time is 48-120h. The inoculation amount of the seed liquid of the second microorganism is 4-6% of the volume of the liquid culture medium. The seed liquid of the second microorganism can be prepared by the following method: inoculating the hyphae of the second microorganism into the liquid culture medium, and culturing for 24-48h at 25-28°C and 180-200rpm.

[0057] In the above step S3, the separation and purification includes the following steps: filtering the fermentation liquid obtained after the second transformation culture, collecting the filtrate, extracting with ethyl acetate and evaporating under reduced pressure to obtain a crude extract; separating and purifying the crude extract by semi-preparative liquid chromatography to obtain a fermentation product: 2'-O-glucosyl-4'-O-methyl psoralea corylifolia chalcone, the purity of which reaches more than 95% as detected by high performance liquid chromatography, and the structure is shown below:

[0058]

[0059] The transformation product has the following physical and chemical data: light yellow amorphous solid powder. The molecular formula is C 27 H 32 O9; UV(c0.10MeOH)λ max :374nm; IR:ν max :3269,2971,1615,1516,1442,1370,1323,1228cm -1 ; HRFDMS (m / z): 500.20457 [M] + (calcd for 500.20408). 1H-NMR(500MHz,DMSO-d6)δ:7.74(1H,J=15.4Hz,H-β),7.63(2H,d,J=8.4Hz,H-2,6),7.51(1H,d,J=15.4Hz,H-α), 7.40(1H,s,H-6'),6.89(1H,s,H-3'),6.81(2H,d,J=8.4Hz,H-3,5),5.22(1H,t,J=7.2Hz,H-2”),5.19(1H,d,J=7. 7Hz,H-1"'),3.87(3H,s,4'-OMe),3.76(1H,m,H-6"'a),3.45(1H,m,H-6"'b),3.44(1H,m,H-5"'),3.36(1H,m,H- 3”'),3.31(1H,m,H-2”’),3.20(2H,d,J=7.2Hz,H-1”),3.14(1H,m,H-4”’),1.68(3H,s,H-4”),1.66(3H,s,H-5”); 13 C-NMR(150MHz,DMSO-d6)δ:189.0(C=O),161.0(C-4'),159.7(C-4),156.6(C-2'),141.5(C-α) ,131.8(C-3”),130.6(C-2,6),130.5(C-6’),126.1(C-1),124.0(C-β),122.9(C-5’),122.3(C- 2"),121.1(C-1'),115.9(C-3,5),100.8(C-1"'),99.0(C-3'),77.6(C-5"'),77.0(C-3"'),73 .6(C-2”'),70.0(C-4”’),60.8(C-6”’),55.8(4’-OMe),27.3(C-1”),25.5(C-5”),17.6(C-4”).

[0060] The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to an embodiment of the present invention can selectively prepare 2'-O-glucosyl-4'-O-methyl psoralea corylifolia chalcone by only one combined fermentation. Due to the specificity and selectivity of the microbial transformation, the generation of by-products is reduced, thereby effectively improving the yield, which can be as high as 40%.

[0061] The method for preparing a microbial transformation product of psoralea chalcone of an embodiment of the present invention uses Absidia caerulea and Mucor hiemalis with structural specificity and regional selectivity to sequentially inoculate and co-cultivate to carry out a microbial transformation reaction on psoralea chalcone, and obtains a new compound 2'-O-glucosyl-4'-O-methyl psoralea chalcone that is difficult to prepare by traditional chemical synthesis, and the method of the present invention is simplified in steps, has high separation efficiency, and has a high yield, which can reach up to 40%. The method of the present invention uses Absidia caerulea and Mucor hiemalis to sequentially inoculate and co-cultivate to introduce a glucose group at the 2'-OH position of psoralea chalcone and a methyl group at the 4'-OH position, and the methylation and glucosylation of these two sites can improve the drugability and water solubility of psoralea chalcone.

[0062] The microbial transformation product (psoralea chalcone derivative) prepared in the embodiment of the present invention is more active and more water-soluble than the substrate, and the activity is evaluated using an in vitro activity screening system. It is found that the microbial transformation product prepared in the embodiment of the present invention has a good inhibitory effect on melanoma and breast cancer cells, can be used as a chemotherapeutic agent for the prevention and treatment of related diseases, and has good research and development prospects.

[0063] The following is a description of a microbial transformation product of psoralea corylifolia chalcone according to an embodiment of the present invention and its preparation method and use through a plurality of specific embodiments.

[0064] Example 1

[0065] Embodiment 1 of the present invention provides a microbial transformation product of psoralea corylifolia chalcone and a preparation method thereof, comprising the following steps:

[0066] (1) Preparation of potato glucose liquid medium: Take 200g of fresh potatoes, peel them, and cut them into 1cm 3 Add 5 times the weight of water and boil for 30 minutes, then filter with gauze, let the filtrate cool, add water to 1L, add 20g glucose and stir until dissolved, divide into portions and sterilize at 118℃ for 15 minutes, then let cool for later use.

[0067] (2) Preparation of potato dextrose agar medium: add 1 wt % agar to the liquid medium prepared in step (1), heat to dissolve, dispense and sterilize by high pressure at 118° C. for 15 minutes, then cool for later use.

[0068] (3) Recovery of strains: The production strains were Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779, which grew well on potato dextrose agar (PDA). Mycelia of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown on agar were streaked onto potato dextrose agar, respectively, and cultured in a constant temperature incubator at 25-28°C for 5 days to obtain test tube strains.

[0069] (4) Obtaining seed solution: The mycelium of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown in test tubes were inoculated into triangular flasks, each containing liquid potato glucose medium, at a culture temperature of 25-28°C, a rotation speed of 180-200 rpm, and a culture time of 24 h to obtain seed solution.

[0070] (5) Preparation of substrate: Dissolve the substrate psoralea corylifolia chalcone in an appropriate amount of ethanol to prepare a 20 mg / mL psoralea corylifolia chalcone ethanol solution.

[0071] (6) Transformation of substrate: a) Inoculate the seed solution of Absidia caerulea KCTC 6936 at 2% of the volume of the liquid culture medium into a freshly prepared potato glucose liquid culture medium, and culture the medium at 25-28° C. and 180-200 rpm for 24 h. Then, add an ethanol solution of the transformation substrate psoralea chalcone at 1% of the volume of the liquid culture medium, and culture the medium at 25-28° C. and 180-220 rpm for 72 h. b) Inoculate the seed solution of Mucorhiemalis KCTC 26779 at 5% of the volume of the liquid culture medium into the liquid culture medium, and continue to culture the medium at 25-28° C. and 180-200 rpm for 48 h.

[0072] (7) Treatment of the fermentation broth: The fermentation broth obtained in step (6) was filtered through a Buchner funnel and the filtrate was collected. The filtrate was extracted three times with ethyl acetate in a 1:1 volume ratio. The ethyl acetate layers were combined and evaporated to dryness under reduced pressure to obtain a crude extract.

[0073] (8) Isolation and purification of the conversion product: The crude extract obtained in step (7) was separated by semi-preparative liquid chromatography to obtain the fermentation product: 2'-O-glucosyl-4'-O-methylpsoralea corylifolia chalcone, the purity of which reached 96% as determined by high performance liquid chromatography, and the yield was 40%.

[0074] Example 2

[0075] Embodiment 2 of the present invention provides a microbial transformation product of psoralea corylifolia chalcone and a preparation method thereof, comprising the following steps:

[0076] (1) Preparation of potato glucose liquid medium: Take 200g of fresh potatoes, peel them, and cut them into 1cm 3 Add 5 times the weight of water and boil for 20 minutes, then filter with gauze, let the filtrate cool, add water to 1L, add 20g glucose and stir until dissolved, divide into portions and sterilize at 118℃ for 15 minutes, then let cool for later use.

[0077] (2) Preparation of potato dextrose agar medium: add 1 wt % agar to the liquid medium prepared in step (1), heat to dissolve, dispense and sterilize by high pressure at 118° C. for 15 minutes, then cool for later use.

[0078] (3) Recovery of strains: The production strains were Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779, which grew well on potato dextrose agar (PDA). Mycelia of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown on agar were streaked onto potato dextrose agar, respectively, and cultured in a constant temperature incubator at 25-28°C for 7 days to obtain test tube strains.

[0079] (4) Obtaining seed solution: The mycelium of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown in test tubes were inoculated into triangular flasks, each containing liquid potato glucose medium, at a culture temperature of 25-28°C, a rotation speed of 180-200 rpm, and a culture time of 48 h to obtain seed solution.

[0080] (5) Preparation of substrate: Dissolve the substrate psoralea corylifolia chalcone in an appropriate amount of ethanol to prepare a 20 mg / mL psoralea corylifolia chalcone ethanol solution.

[0081] (6) Transformation of substrate: a) Inoculate the seed solution of Mucor hiemalis KCTC26779 into a freshly prepared potato glucose liquid medium at 2% of the volume of the liquid medium, and culture at 25-28° C. and 180-200 rpm for 24 h. Then, add an ethanol solution of the transformation substrate psoralea chalcone at 2% of the volume of the liquid medium, and culture at 25-28° C. and 180-220 rpm for 48 h. b) Inoculate the seed solution of Absidiacaerulea KCTC 6936 into the liquid medium at 4% of the volume of the liquid medium, and continue to culture at 25-28° C. and 180-200 rpm for 120 h.

[0082] (7) Treatment of the fermentation broth: The fermentation broth obtained in step (6) was filtered through a Buchner funnel and the filtrate was collected. The filtrate was extracted three times with ethyl acetate in a 1:1 volume ratio. The ethyl acetate layers were combined and evaporated to dryness under reduced pressure to obtain a crude extract.

[0083] (8) Isolation and purification of the conversion product: The crude extract obtained in step (7) was separated by semi-preparative liquid chromatography to obtain the fermentation product: 2'-O-glucosyl-4'-O-methylpsoralea corylifolia chalcone, the purity of which reached 95% as determined by high performance liquid chromatography, and the yield was 27%.

[0084] Example 3

[0085] Embodiment 3 of the present invention provides a microbial transformation product of psoralea corylifolia chalcone and a preparation method thereof, comprising the following steps:

[0086] (1) Preparation of potato glucose liquid medium: Take 200g of fresh potatoes, peel them, and cut them into 1cm 3 Add 5 times the weight of water and boil for 20 minutes, then filter with gauze, let the filtrate cool, add water to 1L, add 20g glucose and stir until dissolved, divide into portions and sterilize at 118℃ for 15 minutes, then let cool for later use.

[0087] (2) Preparation of potato dextrose agar medium: add 1 wt % agar to the liquid medium prepared in step (1), heat to dissolve, dispense and sterilize by high pressure at 118° C. for 15 minutes, then cool for later use.

[0088] (3) Recovery of strains: The production strains were Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779, which grew well on potato dextrose agar (PDA). Mycelia of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown on agar were streaked onto potato dextrose agar, respectively, and cultured in a constant temperature incubator at 25-28°C for 7 days to obtain test tube strains.

[0089] (4) Obtaining seed solution: The mycelium of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown in test tubes were inoculated into triangular flasks, each containing liquid potato glucose medium, at a culture temperature of 25-28°C, a rotation speed of 180-200 rpm, and a culture time of 48 h to obtain seed solution.

[0090] (5) Preparation of substrate: Dissolve the substrate psoralea corylifolia chalcone in an appropriate amount of ethanol to prepare a 20 mg / mL psoralea corylifolia chalcone ethanol solution.

[0091] (6) Conversion of substrate: a) adding an ethanol solution of the conversion substrate psoralea chalcone to a culture container at 2% of the volume of the liquid culture medium; b) adding a potato glucose liquid culture medium inoculated with a seed solution of Absidia caerulea KCTC 6936 to the culture container to obtain a mixed system; the inoculation amount of the seed solution of Absidia caerulea KCTC 6936 is 3% of the volume of the liquid culture medium; culturing the mixed system at 25-28° C. and 180-200 rpm for 48 h; c) inoculating a seed solution of Mucor hiemalis KCTC 26779 to the liquid culture medium at 4% of the volume of the liquid culture medium, and continuing the transformation culture at 25-28° C. and 180-200 rpm for 120 h.

[0092] (7) Treatment of the fermentation broth: The fermentation broth obtained in step (6) was filtered through a Buchner funnel and the filtrate was collected. The filtrate was extracted three times with ethyl acetate in a 1:1 volume ratio. The ethyl acetate layers were combined and evaporated to dryness under reduced pressure to obtain a crude extract.

[0093] (8) Isolation and purification of the conversion product: The crude extract obtained in step (7) was separated by semi-preparative liquid chromatography to obtain the fermentation product: 2'-O-glucosyl-4'-O-methylpsoralea corylifolia chalcone, the purity of which reached 95% as determined by high performance liquid chromatography, and the yield was 32%.

[0094] Example 4

[0095] Embodiment 4 of the present invention provides a microbial transformation product of psoralea corylifolia chalcone and a preparation method thereof, comprising the following steps:

[0096] (1) Preparation of potato glucose liquid medium: Take 200g of fresh potatoes, peel them, and cut them into 1cm 3 Add 5 times the weight of water and boil for 20 minutes, then filter with gauze, let the filtrate cool, add water to 1L, add 20g glucose and stir until dissolved, divide into portions and sterilize at 118℃ for 15 minutes, then let cool for later use.

[0097] (2) Preparation of potato dextrose agar medium: add 1 wt % agar to the liquid medium prepared in step (1), heat to dissolve, dispense and sterilize by high pressure at 118° C. for 15 minutes, then cool for later use.

[0098] (3) Recovery of strains: The production strains were Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779, which grew well on potato dextrose agar (PDA). Mycelia of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown on agar were streaked onto potato dextrose agar, respectively, and cultured in a constant temperature incubator at 25-28°C for 7 days to obtain test tube strains.

[0099] (4) Obtaining seed solution: The mycelium of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown in test tubes were inoculated into triangular flasks, each containing liquid potato glucose medium, at a culture temperature of 25-28°C, a rotation speed of 180-200 rpm, and a culture time of 48 h to obtain seed solution.

[0100] (5) Preparation of substrate: Dissolve the substrate psoralea corylifolia chalcone in an appropriate amount of ethanol to prepare a 20 mg / mL psoralea corylifolia chalcone ethanol solution.

[0101] (6) Conversion of substrate: a) adding an ethanol solution of the conversion substrate psoralea chalcone to a culture container at 2% of the volume of the liquid culture medium; b) adding a potato glucose liquid culture medium inoculated with a seed solution of Mucor hiemalis KCTC 26779 to the culture container to obtain a mixed system; the inoculation amount of the seed solution of Mucor hiemalis KCTC 26779 is 3% of the volume of the liquid culture medium; culturing the mixed system at 25-28° C. and 180-200 rpm for 48 h; c) inoculating a seed solution of Absidia caerulea KCTC 6936 to the liquid culture medium at 6% of the volume of the liquid culture medium, and continuing the transformation culture at 25-28° C. and 180-200 rpm for 120 h.

[0102] (7) Treatment of the fermentation broth: The fermentation broth obtained in step (6) was filtered through a Buchner funnel and the filtrate was collected. The filtrate was extracted three times with ethyl acetate in a 1:1 volume ratio. The ethyl acetate layers were combined and evaporated to dryness under reduced pressure to obtain a crude extract.

[0103] (8) Isolation and purification of the conversion product: The crude extract obtained in step (7) was separated by semi-preparative liquid chromatography to obtain the fermentation product: 2'-O-glucosyl-4'-O-methylpsoralea corylifolia chalcone, the purity of which reached 95% as determined by high performance liquid chromatography, and the yield was 22%.

[0104] Example 5

[0105] Embodiment 5 of the present invention provides a microbial transformation product of psoralea corylifolia chalcone and a preparation method thereof, comprising the following steps:

[0106] (1) Preparation of potato glucose liquid medium: Take 200g of fresh potatoes, peel them, and cut them into 1cm 3 Add 5 times the weight of water and boil for 30 minutes, then filter with gauze, let the filtrate cool, add water to 1L, add 20g glucose and stir until dissolved, divide into portions and sterilize at 118℃ for 15 minutes, then let cool for later use.

[0107] (2) Preparation of potato dextrose agar medium: add 1 wt % agar to the liquid medium prepared in step (1), heat to dissolve, dispense and sterilize by high pressure at 118° C. for 15 minutes, then cool for later use.

[0108] (3) Recovery of strains: The production strains were Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779, which grew well on potato dextrose agar (PDA). Mycelia of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown on agar were streaked onto potato dextrose agar, respectively, and cultured in a constant temperature incubator at 25-28°C for 5 days to obtain test tube strains.

[0109] (4) Obtaining seed solution: The mycelium of Absidia caerulea KCTC 6936 and Mucor hiemalis KCTC 26779 grown in test tubes were inoculated into triangular flasks, each containing liquid potato glucose medium, at a culture temperature of 25-28°C, a rotation speed of 180-200 rpm, and a culture time of 24 h to obtain seed solution.

[0110] (5) Preparation of substrate: Dissolve the substrate psoralea corylifolia chalcone in an appropriate amount of ethanol to prepare a 20 mg / mL psoralea corylifolia chalcone ethanol solution.

[0111] (6) Transformation of substrate: a) Inoculate the seed solution of Absidia caerulea KCTC 6936 into a freshly prepared potato glucose liquid medium at 2% of the volume of the liquid medium, and culture at 25-28° C. and 180-200 rpm for 24 h. Then, add an ethanol solution of the transformation substrate psoralea chalcone at 2% of the volume of the liquid medium, and culture at 25-28° C. and 180-200 rpm for 96 h. b) Inoculate the seed solution of Mucorhiemalis KCTC 26779 into the liquid medium at 5% of the volume of the liquid medium, and continue to culture at 25-28° C. and 180-200 rpm for 72 h.

[0112] (7) Treatment of the fermentation broth: The fermentation broth obtained in step (6) was filtered through a Buchner funnel and the filtrate was collected. The filtrate was extracted three times with ethyl acetate in a 1:1 volume ratio. The ethyl acetate layers were combined and evaporated to dryness under reduced pressure to obtain a crude extract.

[0113] (8) Isolation and purification of the conversion product: The crude extract obtained in step (7) was separated by semi-preparative liquid chromatography to obtain the fermentation product: 2'-O-glucosyl-4'-O-methylpsoralea corylifolia chalcone, the purity of which reached 96% as determined by high performance liquid chromatography, and the yield was 35%.

[0114] In vitro toxicity experiments were conducted on the microbial transformation products and substrates (psoralea chalcone) prepared in Example 1 of the present invention: human melanoma (A375P) and human breast adenocarcinoma cells (MCF-7) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin, and incubated in a constant temperature and humidity incubator at 37°C and 5% CO2. The cells were diluted to 5×10 4 cells / mL concentration, inoculated in a 96-well plate, and 100 μL of cell suspension was added to each well. After 24 hours of culture, the culture medium was removed, and different concentrations of test compounds were added to the test wells. After another 48 hours of incubation, the culture medium was removed, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and the culture was continued for 4 hours, the culture medium was discarded, and the blue-purple crystalline formazan was completely dissolved in 200 μL of dimethyl sulfoxide. The absorbance was read at 490 nm using an ELISA reader, and the IC of the compound was calculated using GraphPad Prism8 50 The activity results are shown in Table 1, where norzelaminaldehyde and 5-fluorouracil are positive control drugs.

[0115] Table 1. Cytotoxicity results of psoralea corylifolia chalcone and its microbial transformation products

[0116]

[0117] As can be seen from Table 1 above, compared with the substrate psoralea corylifolia chalcone, the transformation product 2'-O-glucosyl-4'-O-methyl psoralea corylifolia chalcone obtained after microbial transformation has a better inhibitory effect on human melanoma (A375P) and human breast adenocarcinoma cells (MCF-7), indicating that the microbial transformation product prepared in the embodiment of the present invention is more active than the substrate, has a good inhibitory effect on melanoma and breast cancer cells, can be used as a chemotherapeutic agent for the prevention and treatment of related diseases, and has a good research and development prospect.

[0118] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the attached claims.

Claims

1. A microbial transformation product of psoralea corylifolia chalcone, characterized in that: Its chemical structure is shown below:

2. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to claim 1, wherein: The following steps are involved: S1, inoculating a first microorganism in a system containing a liquid culture medium and psoralea chalcone to perform a first transformation culture; S2, then, inoculating a second microorganism into the system to perform a second transformation culture; S3, separating and purifying the product obtained after the second transformation culture to obtain a microbial transformation product of psoralea corylifolia chalcone; The first microorganism is Absidia caerulea KCTC 6936 or Mucorhiemalis KCTC 26779, and the second microorganism is Absidia caerulea KCTC 6936 or Mucor hiemalis KCTC 26779. The first microorganism is different from the second microorganism.

3. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to claim 2, wherein: The temperature of the first transformation culture is 25-28°C, the rotation speed is 180-200 rpm, and the time is 24-120 hours; or / or, The temperature of the second transformation culture is 25-28°C, the rotation speed is 180-200 rpm, and the time is 48-120 hours.

4. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to claim 2, wherein: The first microorganism is Absidia caerulea KCTC 6936, and the second microorganism is Mucor hiemalis KCTC 26779.

5. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to claim 2, wherein: The step S1 comprises the following steps: preparing the liquid culture medium; Inoculate the seed liquid of the first microorganism at 2-3% of the volume of the liquid culture medium, and culture at 25-28° C. and 180-200 rpm for 24-48 hours; The psoralea corylifolia chalcone solution is added at 1-2% of the volume of the liquid culture medium, and the transformation culture is carried out at 25-28° C. and 180-200 rpm for 24-96 hours.

6. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to claim 2, wherein: The step S1 comprises the following steps: Adding psoralea corylifolia chalcone solution into the culture container at 1-2% of the volume of the liquid culture medium; Next, the liquid culture medium inoculated with the seed solution of the first microorganism is added to the culture container to obtain a mixed system; the inoculation amount of the seed solution of the first microorganism is 2-3% of the volume of the liquid culture medium; The mixed system is cultured at 25-28° C. and 180-200 rpm for 24-96 h.

7. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to claim 2, characterized in that: In the step S2, the inoculation amount of the seed liquid of the second microorganism is 4-6% of the volume of the liquid culture medium.

8. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to claim 2, characterized in that: The seed liquid of the first microorganism is prepared by the following method: inoculating the hyphae of the first microorganism into the liquid culture medium, and culturing at 25-28° C. and 180-200 rpm for 24-48 hours; The seed liquid of the second microorganism is prepared by the following method: inoculating the hyphae of the second microorganism into the liquid culture medium, and culturing at 25-28° C. and 180-200 rpm for 24-48 hours.

9. The method for preparing a microbial transformation product of psoralea corylifolia chalcone according to any one of claims 2 to 8, characterized in that: The liquid culture medium is potato glucose liquid culture medium; and / or, In step S3, the separation and purification includes the following steps: The fermentation liquid obtained after the second transformation culture is filtered, the filtrate is collected, extracted with ethyl acetate, and evaporated to dryness under reduced pressure to obtain a crude extract; The crude extract is separated and purified by semi-preparative liquid chromatography.

10. Use of a microbial transformation product of psoralea corylifolia chalcone as claimed in claim 1 in the preparation of a drug for inhibiting human malignant melanoma cells A375P or human breast cancer cells MCF-7.

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