A curcumin / dihydrocurcumin reductase mutant and its application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a curcumin / dihydrocurcumin reductase mutant and its application. The curcumin / dihydrocurcumin reductase mutant screened in the present invention has a maximum catalytic efficiency of up to 1641 mM-1S-1 in the reaction of catalyzing the preparation of tetrahydrocurcumin from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione. The highest catalytic efficiency is 8.16 times that of the initial curcumin / dihydrocurcumin reductase, greatly improving the catalytic efficiency and significantly increasing the production efficiency of tetrahydrocurcumin, providing a new method for the preparation of tetrahydrocurcumin.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a curcumin / dihydrocurcumin reductase mutant and its application. Background Art

[0002] Curcumin is a well-known yellow food coloring, which has functions such as protecting vascular health and regulating blood sugar levels. Tetrahydrocurcumin is one of the main reduction metabolites of curcumin. Since the solubility, stability, and bioavailability of tetrahydrocurcumin are all superior to those of curcumin, and their pharmacological activities are similar, the market demand for tetrahydrocurcumin has been increasing in recent years.

[0003] Currently, the synthesis of tetrahydrocurcumin mainly relies on chemical methods. The common routes are as follows: 1. Using the heavy metal catalyst Pd / C to hydrogenate curcumin to tetrahydrocurcumin; 2. By acylating curcumin to diacetylcurcumin, in the presence of a catalyst and a solvent, using hydrogen or a hydrogen donor to reduce it to a diacetyltetrahydrocurcumin intermediate, and then obtaining tetrahydrocurcumin through deacetylation. The above chemical methods often have problems such as the use of heavy metal catalysts, large environmental pollution, and difficult product purification; compared with chemical methods, the synthesis of tetrahydrocurcumin by bioenzymatic methods has advantages such as safety, high efficiency, low cost, and environmental friendliness, but there are few relevant literatures disclosed at present.

[0004] Therefore, if a bioenzyme with stronger catalytic ability can be developed for the production of tetrahydrocurcumin, it will greatly improve the industrial preparation efficiency of tetrahydrocurcumin and is of great significance to the market development of tetrahydrocurcumin. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a curcumin / dihydrocurcumin reductase mutant and its application. Starting from the gene EcCurA from Escherichia coli, the curcumin / dihydrocurcumin reductase KcCurA was determined, and further a curcumin / dihydrocurcumin reductase mutant with high activity was obtained, which showed good catalytic effects in the catalytic reaction of preparing tetrahydrocurcumin using 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin) as a substrate.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides a curcumin / dihydrocurcumin reductase mutant capable of efficiently catalyzing the reaction of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione to prepare tetrahydrocurcumin; in addition, the amino acid sequence, nucleotide sequence, recombinant expression plasmid, genetic engineering strain and preparation method thereof corresponding to the curcumin / dihydrocurcumin reductase mutant, and the application of the curcumin / dihydrocurcumin reductase mutant in synthesizing tetrahydrocurcumin are also within the protection scope of the present invention.

[0008] The curcumin / dihydrocurcumin reductase mutant provided by the present invention is based on the EcCurA gene from Escherichia coli first reported in the literature in 2011 as the starting gene. Through BLAST analysis of the NCBI database, the enzyme activity and solubility test confirmed that it is from Cossackia Kosakonia cowanii The curcumin / dihydrocurcumin reductase KcCurA was isolated and its mutants were screened to identify the key sites affecting the enzymatic properties of the initial curcumin / dihydrocurcumin reductase, and curcumin / dihydrocurcumin reductase mutants with high activity were obtained.

[0009] Wherein, the curcumin / dihydrocurcumin reductase mutant comprises:

[0010] (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having at least one of the following sites of the amino acid sequence in (a): R264, V265, P266, L267, L268, M269, A270, T271, I272, L273, K274, K275 or Q286, which has undergone amino acid mutation and has the function of curcumin / dihydrocurcumin reductase; or (c) a protein having more than 80% homology with the above amino acid sequence defined in any one of (a) and (b) and having the function of curcumin / dihydrocurcumin reductase.

[0011] Furthermore, the amino acid mutation in (b) is selected from at least one of the following:

[0012] R264A, R264L, R264F, R264V, R264C, R264I, R264M, V265A, V265L, V265F, V265G, P266A, P266L, P266F, P266G, P266Y, P266W, L267A, L267F, L268A, L268F, M269A, M269L, M269F, A270L, A270F, T271A, T271L, T271F, I272A, I272L, I272F, L273A, L273F, K274A, K274L, K274F, K274V, K274C, K274I, K274M, K275A, K275L, K275F, K275V, K275C, K275I, K275M, K275Y, K275W, Q286A, Q286L, Q286F, Q286Y, Q286W。

[0013] Furthermore, the amino acid mutation in (b) is selected from any one of the following:

[0014] R264A, R264L, R264F, R264V, R264C, R264I, R264M, V265A, V265L, V265F, V265G, P266A, P266L, P266F, P266G, P266Y, P266W, L267A, L267F, L268A, L268F, M269A, M269L, M269F, A270L, A270F, T271A, T271L, T271F, I272A, I272L, I272F, L273A, L273F, K274A, K274L, K274F, K274V, K274C, K274I, K274M, K275A, K275L, K275F, K275V, K275C, K275I, K275M, K275Y, K275W, Q286A, Q286L, Q286F, Q286Y, Q286W, K274L + R264I, K274L + R264L, K274L + V265A, K274L + V265L, K274L + P266A, K274L + P266L, K274L + P266F, K274L + P266Y, K274L + P266G, K274L + P266I, K274L + K275L, K274L + K275F, K274L + Q286F, K274L + Q286Y, K274L + P266F + R264I, K274L + P266F + R264L, K274L + P266F + V265A, K274L + P266F + V265L, K274L + P266F + K275L, K274L + P266F + K275F, K274L + P266F + Q286F, K274L + P266F + Q286Y, K274L + P266F + Q286Y + R264I, K274L + P266F + Q286Y + R264L, K274L + P266F + Q286Y + V265A, K274L + P266F + Q286Y + V265L, K274L + P266F + Q286Y + K275L or K274L + P266F + Q286Y + K275F.

[0015] In a second aspect of the present invention, there is provided a method for preparing the above curcumin / dihydrocurcumin reductase mutant, and the preparation method includes the following steps: site-directed mutagenesis of the initial curcumin / dihydrocurcumin reductase encoding gene, construction of an expression vector, transformation of a protein expression host bacterium, and induction of protein expression.

[0016] Furthermore, the present invention obtained the coding gene of the mutant by molecular cloning technology, and induced expression by constructing a 6×His fusion expression vector of the mutant gene and introducing it into the genetic engineering bacterium BL21(DE3) to obtain the mutant enzyme protein. Using the mutant as a catalyst and 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as a substrate, an enzymatic catalysis reaction was carried out under appropriate conditions. The results showed that the catalytic efficiency of the mutant provided by the present invention for synthesizing tetrahydrocurcumin was increased by 8.13 times compared with the wild-type curcumin / dihydrocurcumin reductase, and it has great application potential and value in the industrial production of tetrahydrocurcumin.

[0017] All the mutation sites mentioned above in the present invention and the genes encoding the curcumin / dihydrocurcumin reductase mutants with any combination mutations are the key contents to be protected by the present invention. All the modifications made on the said genes are within the key protection scope of the present invention. In addition, the expression cassette, vector or recombinant bacterium containing the said gene also falls within the technical scope protected by the present invention.

[0018] Similarly, the catalyst containing the above-mentioned curcumin / dihydrocurcumin reductase mutant is also the technical content protected by the present invention.

[0019] Furthermore, the present invention also provides the application of the above-mentioned curcumin / dihydrocurcumin reductase mutant or the catalyst containing the above-mentioned curcumin / dihydrocurcumin reductase mutant in the preparation of tetrahydrocurcumin. Specifically, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione is used as a substrate for a catalytic reaction to synthesize tetrahydrocurcumin.

[0020] The specific application is as follows: Using the above-mentioned curcumin / dihydrocurcumin reductase mutant or the catalyst containing the above-mentioned curcumin / dihydrocurcumin reductase mutant, with 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as a substrate, a catalytic reaction is carried out under the conditions of 25~40°C and pH = 5.0~8.0 to synthesize tetrahydrocurcumin.

[0021] Furthermore, the concentration of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione is 0.025 mM.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) The highly active curcumin / dihydrocurcumin reductase mutant obtained by screening in the present invention realizes the efficient catalysis of producing tetrahydrocurcumin using 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as a raw material, significantly improving the production efficiency of tetrahydrocurcumin and providing a new method for preparing tetrahydrocurcumin.

[0024] (2) In the reaction of the curcumin / dihydrocurcumin reductase mutant obtained by screening in the present invention for catalyzing the preparation of tetrahydrocurcumin from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, the highest catalytic efficiency can reach 1641 mM -1 S -1 , and its highest catalytic efficiency is 8.16 times that of the initial curcumin / dihydrocurcumin reductase, greatly improving the catalytic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the reaction principle for the synthesis of tetrahydrocurcumin by using the curcumin / dihydrocurcumin reductase mutant in the present invention to catalyze 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione;

[0026] Figure 2 is the detection of the generation of tetrahydrocurcumin from the catalytic substrate 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione by KcCurA using high performance liquid chromatography. SPECIFIC EMBODIMENTS

[0027] In order to enable those skilled in the art to better understand the present invention, the present invention will be further elaborated below in combination with specific embodiments.

[0028] The main reagents and consumables used in the present invention:

[0029] Phanta Max Super-Fidelity DNA polymerase P505 was purchased from Nanjing Novoprotein Scientific Co., Ltd.; FastDigest DpnI DNA endonuclease was purchased from ThermoFisher Scientific; the pET28(a+) plasmid is a known Escherichia coli expression vector with a vector size of 5369 bp, a T7 promoter, a vector tag N-6×His, and a vector resistance of Kanamycin, and was purchased from Novogen; E. coli DH5α competent cells and BL21 (DE3) competent cells were both purchased from Angyu Biotechnology Co., Ltd.; agar powder was purchased from Solarbio; tryptophan and yeast extract were both purchased from OXOID.

[0030] LB medium

[0031] Each 100 mL of LB medium contains: 1 g of tryptone, 0.5 g of yeast extract, and 1 g of NaCl, with a pH of 7.0 - 7.2.

[0032] Preparation method: Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of distilled water without adjusting the pH value, and sterilize by autoclaving at 121 °C for 20 min. If preparing a solid medium, add 1.8 g of agar powder to 100 mL of the medium.

[0033] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be commercially obtained or prepared according to the conventional methods in the art, and the specification is laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, and relevant experimental manuals or manufacturer's instructions can be referred to.

[0034] Example 1 Preparation of Curcumin / Dihydrocurcumin Reductase Mutant

[0035] I. Design of Curcumin / Dihydrocurcumin Reductase Mutant

[0036] Through gene mining, the gene of curcumin / dihydrocurcumin reductase KcCurA was screened from the NCBI database. The full-length open reading frame of this gene is 1038 bp, and the curcumin / dihydrocurcumin reductase encoded by it consists of 345 amino acids. Its amino acid sequence (345 aa) is shown in SEQ ID NO:1.

[0037] The nucleotide sequence encoding this curcumin / dihydrocurcumin reductase is shown in SEQ ID NO:2.

[0038] In this example, by the method of homology modeling, molecular docking was performed on the substrate 1,7 - bis(4 - hydroxy - 3 - methoxyphenyl) - 1,6 - heptadiene - 3,5 - dione according to the structure of curcumin / dihydrocurcumin reductase KcCurA. The docking results were analyzed, and 13 residues near the active center that might affect the catalytic properties of the protein were selected. These residues include R264, V265, P266, L267, L268, M269, A270, T271, I272, L273, K274, K275, or Q286. Site - directed mutagenesis was performed on these residues to obtain mutants with higher enzyme activity, that is, the mutants provided by the present invention, which exhibit better catalytic performance and enzyme activity performance.

[0039] II. Obtaining of Curcumin / Dihydrocurcumin Reductase Mutant Gene

[0040] The obtained curcumin / dihydrocurcumin reductase mutants can have their genes obtained by total gene synthesis or molecular cloning methods.

[0041] Using the KcCurA gene of curcumin / dihydrocurcumin reductase obtained by total gene synthesis in our laboratory, the gene of curcumin / dihydrocurcumin reductase mutants was obtained by PCR method. The specific steps are as follows:

[0042] 1. Obtaining the full gene of initial curcumin / dihydrocurcumin reductase

[0043] Our laboratory has synthesized curcumin / dihydrocurcumin reductase KcCurA. The above gene was synthesized by Nanjing Genscript Biotechnology Co., Ltd. and ligated into plasmid pET28a(+).

[0044] 2. Site-directed mutagenesis of curcumin / dihydrocurcumin reductase

[0045] (1)Design of site-directed mutagenesis primers

[0046] (2)Site-directed mutagenesis of curcumin / dihydrocurcumin reductase

[0047] Using the pET28a(+) plasmid containing the initial curcumin / dihydrocurcumin reductase gene as a template, and using the upstream primer and downstream primer containing the mutation site designed according to the template, the gene of curcumin / dihydrocurcumin reductase mutants was amplified according to the following PCR system and procedure.

[0048] PCR system: Phanta Max Super-Fidelity DNA polymerase P520 20 μL, template plasmid 1 μL, upstream primer -F(10 μM) 1 μL, downstream primer -R(10 μM) 1 μL, dd H2O 2 μL.

[0049] The PCR procedure is as follows: a. Pre-denaturation at 98℃ for 30 s; b. Denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 72℃ for 40 s, for 32 cycles; c. Extension at 72℃ for 5 min, cooling to 4℃. Use DpnI enzyme to digest the template plasmid containing the initial curcumin / dihydrocurcumin reductase gene, and digest at 37℃ for 30 min.

[0050] (3) Transformation of DH5α competent cells

[0051] The digestion product of the pET28a(+) plasmid containing the curcumin / dihydrocurcumin reductase mutants obtained in (2) was transformed into DH5α competent cells.

[0052] Place the DH5α competent cells on ice. After the cells thaw, add 10 μL of the plasmid solution, place it on ice for 30 min, heat shock at 42°C for 45 s, then let it stand on ice for 2 min. Add 700 μL of sterile LB liquid medium, and culture it in a shaker at 37°C and 200 rpm for 1 h. Centrifuge at 4000 rpm for 1 min, discard 600 μL of the supernatant, resuspend the precipitated bacteria with the remaining supernatant, and spread all of it on an LB plate medium containing Kana resistance (50 μg / mL). Incubate it upside down at 37°C overnight.

[0053] (4) Screening of positive clones

[0054] Pick a single colony from the LB plate and inoculate it into an LB liquid medium containing Kana resistance (50 μg / mL). Culture it overnight in a shaker at 37°C and 200 rpm. After preserving the bacteria (final concentration of sterile glycerol is 15%), send it to ACD (China, Suzhou) company for sequencing to identify whether the initial curcumin / dihydrocurcumin reductase has mutated successfully.

[0055] 3. Heterologous expression of curcumin / dihydrocurcumin reductase mutant 6×His fusion protein

[0056] (1) Transformation of protein expression competent cells

[0057] Take out the BL21(DE3) competent cells from the -80°C ultra-low temperature freezer and place them on ice. After the cells thaw, add 1 μL of the pET28a(+) plasmid containing the curcumin / dihydrocurcumin reductase mutant to each type of competent cell, place it on ice for 30 min, heat shock at 42°C for 45 s, then place it on ice for 2 min. Add 700 μL of sterile LB liquid medium, culture it in a shaker at 37°C and 200 rpm for 1 h. Pipette 200 μL of the cultured bacterial liquid and spread it on an LB plate medium containing Kana resistance (50 μg / mL). Incubate it upside down at 37°C overnight.

[0058] (2) Preservation of curcumin / dihydrocurcumin reductase mutant expression strains

[0059] Pick a single colony from the LB plate and inoculate it into a test tube containing an LB liquid medium with Kana resistance (50 μg / mL). Culture it overnight in a shaker at 37°C and 200 rpm to be used as the expression strain of the curcumin / dihydrocurcumin reductase mutant. Add sterile glycerol with a final volume concentration of 15% to the bacterial liquid and store it in the -80°C ultra-low temperature freezer for a long time.

[0060] (3) Protein expression

[0061] Inoculate the overnight-cultured test tube bacterial liquid into 50 mL of sterile LB liquid medium at an inoculation amount of 1%, with a final concentration of kanamycin of 50 μg / mL. Culture at 37 °C and 200 rpm for about 2.5 h. When OD600 = 0.6 - 0.8, add sterile IPTG with a final concentration of 0.1 mM, and induce culture at 28 °C and 200 rpm for 12 h. Then centrifuge at 8000 rpm for 8 min to collect the bacterial cells.

[0062] Example 2 Synthesis of tetrahydrocurcumin using the curcumin / dihydrocurcumin reductase mutant provided by the present invention

[0063] In this example, the schematic diagram of the reaction principle for synthesizing tetrahydrocurcumin by catalyzing 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione with curcumin / dihydrocurcumin reductase mutant (NADPH) is shown in the appendix Figure 1 as follows. The specific operation is as follows:

[0064] Sequentially add 10 μL of purified enzyme solution, 10 μL of substrate 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, and 20 μL of coenzyme NADPH into a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and make up the system to 200 μL with buffer solution. React at 30 °C.

[0065] Enzyme activity assay:

[0066] The enzyme activity assay system for curcumin / dihydrocurcumin reductase is as follows:

[0067] 160 μL of phosphate buffer (100 mM, pH 6.0), 20 μL of NADPH (1.5 mM), 10 μL of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (0.5 mM), and 10 μL of purified mutant enzyme solution. Add the mixed system into a 96-well plate, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the decrease in absorbance at 430 nm within 6 min at 30 °C. Calculate the amount of curcumin / dihydrocurcumin reductase consumed in the reaction according to the standard curve.

[0068] One enzyme activity unit U involved in the present invention is defined as: the amount of enzyme required to consume 1 μmol of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione within 1 minute.

[0069] The initial curcumin / dihydrocurcumin reductase and mutant enzyme activities are shown in Table 1.

[0070] Table 1 Initial curcumin / dihydrocurcumin reductase and mutant enzyme activities

[0071] Type of enzyme Specific activity (U / mg) <![CDATA[Conversion number (S -1 )]]> Michaelis constant (Mm) <![CDATA[Catalytic efficiency (mM -1 S -1 )]]> Relative catalytic efficiency Initial flavin / dihydrocurcumin reductase 2.29±0.04 5.18±0.46 0.03±0.004 201.89±50.42 1.00 K274L 5.95±0.31 14.31±0.63 0.03±0.002 507.38±59.86 2.51 K274L / P266F 7.44±0.09 15.53±1.24 0.01±0.002 1141.07±265.65 5.65 K274L / P266F / Q286Y 6.31±0.19 12.12±0.42 0.01±0.000 1641.28±214.81 8.12 K274L / P266F / Q286Y / V265L 6.14±0.00 12.75±1.04 0.01±0.001 1115.08±267.35 5.52

[0072] Example 3 The crude enzyme solutions of curcumin / dihydrocurcumin reductase KcCurA and alcohol dehydrogenase ADH were used for the enzymatic catalysis to prepare tetrahydrocurcumin.

[0073] The catalytic reaction system was as follows: 20 g / L of the crude enzyme solution of KcCurA, 20 g / L of the crude enzyme solution of ADH, 20 g / L of curcumin, 0.2 g / L of NADP + , 2.5% (v / v) isopropanol, 1% of Tween 80. The above 20 mL of the reaction solution was placed in a 50 mL round-bottom flask and magnetically stirred in a constant temperature water bath at 38 °C for 24 h.

[0074] The catalytic reaction solution was used for HPLC analysis to obtain the yield of tetrahydrocurcumin. The production of tetrahydrocurcumin in the obtained product was detected by high performance liquid chromatography (HPLC). The results of HPLC are shown in the appendix Figure 2 as follows. Figure 2 It can be seen that the retention time of tetrahydrocurcumin was 8.03 min, and it was basically completely converted into tetrahydrocurcumin.

[0075] The specific activities of curcumin / dihydrocurcumin reductases and mutants with other amino acid sequences obtained by the method of the present invention were verified. The mutant with the highest catalytic efficiency was 8.16 times that of the initial curcumin / dihydrocurcumin reductase. The experimental results showed that the catalytic efficiencies of the other positive mutant enzymes were all higher than 201 mM - 1 S -1 , indicating that the above mutants had good catalytic effects in the reaction of catalyzing the synthesis of tetrahydrocurcumin from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione.

[0076] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A curcumin / dihydrocurcumin reductase mutant, characterized in that: Mutations were made at the following positions of the amino acid sequence shown in SEQ ID NO: 1: K274L+P266F+Q286F.

2. The coding gene of the curcumin / dihydrocurcumin reductase mutant according to claim 1.

3. The gene encoding the curcumin / dihydrocurcumin reductase mutant according to claim 2, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the coding gene according to claim 2 or 3.

5. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria is obtained by transforming the recombinant expression vector described in claim 4 into a host microorganism.

6. A catalyst, characterized in that The active ingredient of the catalyst comprises the curcumin / dihydrocurcumin reductase mutant according to claim 1.

7. Use of the catalyst according to claim 6 in the preparation of tetrahydrocurcumin.

8. The use according to claim 7, characterized in that Tetrahydrocurcumin was prepared from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as substrate.

9. Use of the curcumin / dihydrocurcumin reductase mutant according to claim 1 in the preparation of tetrahydrocurcumin.

10. The use according to claim 9, characterized in that Tetrahydrocurcumin was prepared from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as substrate.

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