WO2017071577A1

WO2017071577A1 - Bioconversion method for curcumin, and product and use thereof

Info

Publication number: WO2017071577A1
Application number: PCT/CN2016/103359
Authority: WO
Inventors: 李晓帆; 王立岩
Original assignee: 深圳大学
Priority date: 2015-10-26
Filing date: 2016-10-26
Publication date: 2017-05-04
Also published as: CN105274152B; CN105274152A

Abstract

Provided are a bioconversion method for curcumin, and a product and a use thereof. The method comprises: using Fusarium sp. (fungi) to perform microbial fermentation of curcumin; collecting a fermentation product; and employing multiple chromatography techniques to separate and purify target products. The microbial fermentation is carried out indoors by using a rice solid medium in static culture for 6-7 weeks. The process of separating and purifying the target products comprises: using a reagent to dissolve the collected fermentation product; and after mixing the dissolved fermentation product with a silica gel, performing column chromatography to obtain resulting products. Also disclosed are structural formulas of the resulting products. The resulting products can protect red blood cells better than curcumin, and can be used to produce curcuminoid drugs and health-enhancing foods.

Description

Biotransformation method, product and application of curcumin

Technical field

The invention belongs to the fields of biotechnology and chemistry, and in particular relates to a biotransformation method, product and application of curcumin.

Background technique

Curcuma longa L. is a perennial herb of the genus Curcuma. Its dry roots are called turmeric root powder and can be used for medicinal purposes. The main bioactive components of turmeric for medicinal use are curcumin and volatile oils. The former has the functions of lowering blood fat, anticoagulation, anti-oxidation, anti-cancer, anti-microbial, etc., while the latter mainly plays an anti-inflammatory, anti-bacterial and anti-cough effect. The clinical application of curcumin has been extensive, and it is widely used in the treatment of tumors, which can exert its anticancer effect by inducing the differentiation and apoptosis of malignant tumor cells and the inhibitory effects on various stages of tumor growth. A large number of studies have proved that curcumin has a variety of pharmacological effects. Curcumin utilizes phenolic hydroxyl groups to capture free radicals, which can alleviate the protective effects of radiation-induced liver injury and oxidative damage; play an anti-tumor role by regulating cell cycle, inducing apoptosis of tumor cells, and regulating gene expression; -2, IL-4, IL-8, TNF-α and other inflammatory factors expression plays an anti-inflammatory role, curcumin also has antiviral, antibacterial and other effects.

The deficiency of curcumin is that its structure is unstable and its water solubility is very poor, and it is easily degraded in living organisms. These characteristics lead to the extremely low bioavailability of curcumin, especially the protection of red blood cells needs to be further enhanced, even in There is no activity in many anti-tumor experiments, which greatly hinders the further study of curcumin as a drug candidate.

Summary of the invention

The technical problem to be solved by the present invention is to provide a method for biotransformation of curcumin, which aims to solve the problem of low bioactivity and bioavailability of curcumin existing in the prior art.

The present invention is achieved by a method for biotransformation of curcumin comprising the steps of:

(1) microbial fermentation of curcumin using Fusarium sp. fungi;

(2) collecting fermentation products;

(3) Separating and purifying the fermentation product.

Further, the microbial fermentation conditions are: rice solid medium (rice 70 g, sea salt 3.6 g, 120 ml distilled water), static culture, room temperature fermentation for 6 to 7 weeks; final concentration of curcumin in rice medium is about 4 ×10 ^-4 M.

Further, the microbial fermentation process is: first, the Fusarium sp. fungus is activated, then inoculated on the medium, and the treated curcumin is added to the surface of the medium and allowed to stand for culture. The specific activation process may be to activate the deposited strain on a 28 ° C plate PDA medium (potato dextrose agar medium), and inoculate an appropriate amount into the fungal seed medium (containing glucose 2%, maltose 1%, peptone). 1%, yeast extract 0.5%, sea salt 3%), cultured at 28 ° C, 220 r / min for 2d.

Further, the curcumin is treated before the microbial fermentation, wherein the curcumin is dissolved in DMSO, then filtered through a microporous membrane, and the filtered curcumin is added to the sterile water and uniformly mixed. After the backup.

Further, the process of collecting the fermentation product is: ultrasonically extracting the product after fermentation with ethyl acetate, concentrating to dryness under reduced pressure, and extracting three times to obtain a product.

Further, the process of separating and purifying the target product is: first, the collected fermentation product is dissolved with a reagent, and then mixed with silica gel, followed by column chromatography to obtain each product. Specifically, the fermentation product may be dissolved in an appropriate amount of cyclohexane and ethyl acetate as a reagent, and mixed with silica gel (200-300 mesh), followed by column chromatography and eluted with cyclohexane/ethyl acetate. The fraction was eluted with a gradient, and each fraction was collected to obtain each product.

Further, the product comprises two new compounds, respectively

Compound 3:

Compound 7:

Preferably, the reagent is a mixed reagent of cyclohexane and ethyl acetate.

The present invention also provides a product obtained by the above method, in addition to the above two new compounds, further comprising:

Compound 4:

Compound 5:

Compound 6:

Further, the

compounds

5 and 6 have a stronger protective effect on erythrocyte hemolysis than the curcumin, and the

compounds

3 and 4 have stronger protective effects on the red blood cells, and the protective effect of the compound 7 is weaker than the curcumin.

The invention also provides the use of the product obtained by the above curcumin biotransformation method, and the obtained product can be used for preparing a medicament, a health food or a cosmetic.

Compared with the prior art, the present invention has the beneficial effects of using the Fusarium sp. fungus to ferment curcumin to obtain five curcumin derivatives, including two new compounds, and the obtained curcumin derivative has more curcumin than the curcumin. Strong red blood cell protection activity. The deficiency of curcumin is that its structure is unstable and its water solubility is extremely poor, and it is easily degraded in living organisms. These characteristics lead to the extremely low bioavailability of curcumin, and it is inactive even in many anti-tumor experiments, which greatly hinders Curcumin was further studied as a drug candidate. The method of the invention overcomes the problem that the curcumin has low bioavailability in the drug development and weak protection against red blood cells, and further promotes curcumin as a candidate drug, and widely uses curcumin such as health food and cosmetics. Development and so on have great significance.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the separation of compounds 3-7 provided in an embodiment of the present invention.

2 is an HPLC result of curcumin, crude curcumin extract, and compound 3-7 provided by an embodiment of the present invention.

3 is a graph showing test results of erythrocyte protective activity test of curcumin and compound 3-7 provided by an embodiment of the present invention. Spectrum.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The effective microbial transformation of curcumin has been a great application prospect. The unique living environment of the ocean makes it possible for marine microbes not only to synthesize compounds with complex molecular structures, but also to synthesize specific enzymes for structural modification of foreign compounds. The present invention combines the results of previous experiments, and uses the marine fungus W-6 (determined as Fusarium sp. fungi) to transform curcumin, thereby obtaining curcumin derivatives with higher bioavailability and better pharmacological activity. After the erythrocyte hemolysis protection activity of curcumin derivatives, it was found that curcumin derivatives showed stronger red blood cell protection than curcumin. This method has great significance for the wide application of curcumin.

The deep sea fungus was collected from the South China Sea mud sample at a water depth of 2,460 meters (112°12.188′E, 17°59.887′N) and identified by the laboratory as Fusarium sp. fungus (accession number EU926232.1).

Example 1 Fermentation, Extraction of Fermentation Products

The fermentation conditions were: rice solid medium, static culture, fermentation temperature at room temperature, and fermentation time of 40 days. Pre-fermentation strains were stored in a -80 ° C refrigerator.

The deposited strain was activated on a 28 ° C plate PDA medium (potato dextrose agar medium), and inoculated with an inoculating loop to inoculate a medium containing 100 ml of fungal seed medium (containing glucose 2%, maltose 1%, peptone 1%, yeast extract). 0.5 ml, sea salt 3%) in a 250 ml Erlenmeyer flask, cultured at 28 ° C, 220 r / min for 2 d, and then inoculated 5 ml of seed solution into 56 rice-containing medium (rice 70g, sea salt 3.6g, 120ml distilled water) In a 1L Erlenmeyer flask, in order to make the final concentration of each bottle of rice medium 4×10 ^-4 M, it is necessary to add 10.3 mg of curcumin by calculation (dissolved in DMSO, then filtered through a 0.22 μm microporous membrane filter). The curcumin was added to 5 ml of sterile water and finally uniformly added to the surface of the rice medium, and allowed to stand at room temperature for 40 days.

Each of the cultured products was ultrasonically extracted with 400 mL of ethyl acetate, and concentrated to dryness under reduced pressure, and then extracted three times to obtain 38.8 g of ethyl acetate extract.

Example 2 separation and purification of the target product

The above ethyl acetate extract is taken, dissolved in a proper amount of a 1:1 mixture of cyclohexane and ethyl acetate, and mixed with silica gel (200-300 mesh), and then subjected to column chromatography to obtain cyclohexane/acetic acid B. The ester was eluted as a gradient and the fractions were collected. After TLC analysis (thin layer analysis), the fractions were combined to finally obtain 10 components, respectively Fr-1 to Fr-10. Among them, Fr-7 was subjected to ODS column chromatography to obtain Fr-7-1, Fr-7-6 and Fr-7-7. Using semi-preparative HPLC (high performance liquid chromatography) (Pro C18, 10 × 250 mm I.D. 5μ, 40% → 100% acetonitrile plus 1% acetic acid / 25min gradient elution, 2.5 mL / min, 210 nm detection) on Fr-7 -6 purification and purification gave Compound 4 (10.2 mg, t _R = 10.4 min), Compound 5 (6.4 mg, t _R = 9.8 min), and Compound 6 (2.5 mg, t _R = 9.4 min). Purification and purification of Fr-7-7 by semi-preparative HPLC (40%→100% acetonitrile plus 1% acetic acid/25 min gradient elution, 2.5 mL/min, 210 nm) gave compound 3 (31.0 mg, t _R = 12.0min).

Fr-8 was subjected to Sephadex LH-20 open-column chromatography, and 1:1 gradient of chloroform/methanol as an eluent was used to obtain Fr-8-1 and Fr-8-2. Among them, Fr-8-2 was subjected to ODS column chromatography to obtain Fr-8-2-4 and Fr-8-2-6. Purification and purification of Fr-8-2-6 by semi-preparative HPLC (50%→100% acetonitrile plus 1% acetic acid/30 min gradient elution, 2.5 mL/min, 210 nm) gave compound 7 (10.5 mg, t _R =8.6min). The specific separation process is shown in Figure 1. The HPLC results for compound 3-7 are shown in Figure 2.

Example 3 Red blood cell protection activity test

The 6× ^{10 8} /mL concentration of red blood cell solution was incubated at 37 ° C for 5 min, and a 1.5 mL centrifuge tube was taken. The red blood cell solution was incubated with the compound at 37 ° C for 30 min, and then added with AAPH (azobisisobutyl hydrazine hydrochloride) solution. After hemolysis for 60 min, the centrifuge tube was finally removed and centrifuged at 3000 rpm for 2 min. 200 μL of the supernatant (2 parallel wells per sample) was added to a transparent 96-well plate, and the absorbance was measured at 545 nm. The protection ratio (%) was calculated from the absorbance values, and the results are shown in Fig. 3.

After the above activity tests, the measured IC ₅₀ 3 Compound (half inhibitory concentration) values of 8.32μM, Compound ₅₀ of IC 4 is 20.66μM, ₅₀ of compound IC 5 is 2.79μM, compound IC 6 5.02 ₅₀ value μM, Compound 7 had an IC ₅₀ value of 62 μM. In the previous experiment, the IC ₅₀ value of curcumin was 37.76 μM. It can be seen that compounds 5 and 6 have a strong protective effect on erythrocyte hemolysis compared with curcumin.

Compounds

3 and 4 have stronger protective effects on red blood cells, and compound 7 has a weaker protective effect than curcumin.

In summary, the Fusarium sp. fungus is used for microbial fermentation of curcumin, and the obtained curcumin derivative contains various compounds, and each curcumin derivative has superior medicinal value relative to curcumin. Helps to study curcumin analogue drugs in the future. The specific uses and effects of the various compounds prepared by the present invention include: preparation of compounds, pharmaceuticals, diagnostic and therapeutic agents for diseases, foods, health products, biological pesticides, biological fertilizers, sewage treatment, and improvement of product quality and yield. Improvement, etc., have broad application prospects.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims

A method for biotransformation of curcumin, characterized by comprising the steps of:

(1) microbial fermentation of curcumin using Fusarium sp. fungi;

(2) collecting fermentation products;

(3) Separating and purifying the fermentation product.
The method for biotransformation of curcumin according to claim 1, wherein the microbial fermentation condition is: rice solid medium, static culture, and room temperature fermentation for 6 to 7 weeks;
The method for biotransformation of curcumin according to claim 2, wherein the curcumin is treated before the microbial fermentation; the treatment is: dissolving curcumin in DMSO, and then using a microporous membrane After filtration, the filtered curcumin is added to sterile water, mixed well and used.
The method for biotransformation of curcumin according to claim 3, wherein the microorganism is fermented by first activating the Fusarium sp. fungus, then inoculating the medium, and then adding the treated curcumin to the microorganism. The surface of the medium was allowed to stand for culture.
A product obtained by the biotransformation method of curcumin according to any one of claims 1 to 3, wherein the product comprises one or more of the following compounds, and the structural formula of the compound is as follows:
Use of the product obtained by the biotransformation method of curcumin according to any one of claims 1 to 4, characterized in that the product is used for the preparation of a medicament, a health food or a cosmetic.