A preparation method and application of kojic acid derivative ketoconazole C

Abstract

The present invention discloses a preparation method and application of a kojic acid derivative, tromethamine C, and relates to the technical field of preparation and application of kojic acid derivatives. The structural formula of the compound is #imgabs0#. Subsequent studies have found that tromethamine C has significant antioxidant activity and EC50 activity against ABTS and DPPH free radicals. 50 The inhibitory activity of kojic acid against tyrosinase was stronger than that of kojic acid, with IC 50 The concentrations of kojic acid were 10.8±1.9μM and 8.9±1.5μM, respectively. Even at 10μM, kojic acid exhibited significant anti-browning effects in fresh-cut fruit and fruit juice. Kojic acid provides an effective precursor compound for the development of anti-browning food additives, potentially contributing to meeting social needs, reducing production costs, and increasing market value. This also lays a foundation for further research into the efficacy of kojic acid, drug development, and future applications.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation and application of kojic acid derivatives, and in particular to a preparation method of a kojic acid derivative, tromethamine C, and application thereof. Background Art

[0002] Oxidative browning is a common spoilage phenomenon that occurs during the processing of foods such as fresh-cut fruits, juices, vegetables, and seafood. Annually, fresh-cut fruits lose approximately half their value due to oxidative browning, with significant losses also associated with juices and vegetables. Oxidative browning can occur through two mechanisms: enzymatic and non-enzymatic. Enzymatic browning is caused by the oxidation of polyphenols in food by enzymes such as polyphenol oxidase and peroxidase. Tyrosinase inhibitors or antioxidants are often added to foods prone to browning to extend their shelf life. However, long-term consumption of chemical anti-browning agents poses certain health risks. Therefore, the research and development of natural, effective, and safe antioxidants or anti-browning agents has long been a hot topic in the food and chemical industries.

[0003] Kojic acid, chemically known as 5-hydroxy-2-hydroxymethyl-4-pyrone, exhibits significant tyrosinase inhibitory activity and is an ideal new food antioxidant and industrial raw material. Furthermore, it can inhibit melanin production in the skin and is widely used in medicine and cosmetics. Therefore, identifying natural and highly effective kojic acid derivatives with outstanding anti-browning properties, clarifying the strength and mechanism of these anti-browning effects, and improving the application value of these compounds as food additives are urgent challenges for those skilled in the art.

[0004] The present invention separates a kojic acid derivative from Aspergillus versicolor. Studies have shown that the compound has good antioxidant and anti-food browning activities. However, there are no relevant reports on the compound and its use. Summary of the Invention

[0005] (1) Technical problems solved

[0006] The present invention is based on at least one of the above-mentioned technical problems. The present invention provides a preparation method and application of a kojic acid derivative, troconazole C, which has an anti-browning effect. This compound provides an effective precursor compound for the development of food additives with anti-browning effect, and also lays a material foundation for further applications.

[0007] (2) Technical solution

[0008] In order to provide an effective precursor compound for the development of the above-mentioned anti-browning food additive and lay a material foundation for the next step of application, the present invention provides the following technical solution: a method for preparing kojic acid derivative tromethamine C, comprising the following steps:

[0009] (1) Preparation of PDA solid culture medium: 100-300 g fresh potatoes, 500-1500 mL tap water, 12-24 g agar, boil to dissolve, and then sterilize under high pressure at 100-120°C for 10-50 min to obtain PDA solid culture medium.

[0010] (2) Preparation of PDB liquid culture medium: 20 g potato, 2 g sucrose, 100 mL tap water and 2 g agar, followed by mixing, dissolving, pH correction, clarification and filtration, packaging and high-pressure sterilization at 100-120°C for 15-30 min to obtain PDB liquid culture medium.

[0011] (3) Activation of Aspergillus versicolor: streak the Aspergillus versicolor to revive it, and inoculate it into the PDA solid medium prepared in step (1), and activate it in an incubator at 28°C for 5 days;

[0012] (4) Cultivation of seed solution: using a scalpel to cut a small piece of colony from the PDA medium in step (3), inoculate it into the PDB liquid medium in step (2), and culture it on a shaker at 28°C and 160 rpm for 5 days to obtain seed solution;

[0013] (5) Cultivation and fermentation: The seed solution in step (4) is then inoculated into a rice solid culture medium, and the culture is statically maintained in a constant temperature and humidity chamber at 28° C. for 30 days to obtain a rice fermentation product;

[0014] (6) Obtaining ethyl acetate extract: taking the rice fermentation product obtained in step (5), adding 400 mL of ethyl acetate for every 200 g of the fermentation product, and cold-immersing extraction 2 to 3 times, each cold-immersing extraction for 15 to 21 hours, and combining the extracts, and concentrating under reduced pressure until there is no ethyl acetate taste, to obtain ethyl acetate extract;

[0015] (7) Crude fraction: The ethyl acetate extract obtained in step (6) was subjected to normal phase column chromatography and then macroporous resin column chromatography to obtain a crude kojic acid derivative after overnight adsorption and elution.

[0016] (8) Fine purification: The target subcomponent in the crude kojic acid obtained in step (7) is separated and purified by high performance liquid chromatography, dissolved by heating, filtered, and finally cooled to obtain a pure kojic acid derivative.

[0017] Preferably, the ethyl acetate extraction reagent in step (6) can also be replaced with dichloromethane or anhydrous ethanol extraction reagent.

[0018] Preferably, in the normal phase column chromatography elution in step (7), 200-300 mesh silica gel is used as column chromatography silica gel.

[0019] Preferably, the elution gradient of the macroporous resin column chromatography in step (7) is 10% to 100% methanol-water.

[0020] Preferably, the method for heating and dissolving the crude kojic acid in step (8) is: using 40% ethanol-water to heat to 80°C for dissolution and then filtering, and the target subcomponent is 50% to 90% methanol-water segment.

[0021] Preferably, the HPLC eluent in step (8) is methanol-water with a volume ratio of methanol to water of 55% to 85%, and the detection wavelength is 210 nm.

[0022] Preferably, the kojic acid derivative kojone C is used in the preparation of a food additive precursor with anti-browning effect.

[0023] Preferably, the kojic acid derivative kojone C is used in the preparation of a food additive precursor that resists enzymatic browning.

[0024] Preferably, the kojic acid derivative tromethamine C is used in the preparation of a food additive precursor for preventing enzymatic browning induced by tyrosinase.

[0025] Preferably, the EC of the kojic acid derivative tromethamine C for scavenging ABTS and DPPH free radicals is 50 The inhibitory activity against tyrosinase was stronger than that of kojic acid, and the IC 50 The concentrations of 10.8±1.9μM and 8.9±1.5μM, respectively, are used in the preparation of food additive precursors for preventing enzymatic browning induced by tyrosinase.

[0026] (3) Beneficial effects

[0027] Compared with the prior art, the present invention provides a method for preparing a kojic acid derivative, tromethamine C, and its application, which has the following beneficial effects:

[0028] 1. The preparation method and application of the kojic acid derivative tromethamine C. A new tricyclic kojic acid derivative tromethamine C was proposed. Subsequent studies have found that tromethamine C has significant antioxidant activity and EC for scavenging ABTS and DPPH free radicals. 50 The inhibitory activity of kojic acid against tyrosinase was stronger than that of kojic acid, with IC 50 The concentrations of 10.8±1.9μM and 8.9±1.5μM respectively. 10μM of tromethamine C has obvious anti-browning effect on fresh-cut fruits and juices.

[0029] 2. The preparation method and application of a kojic acid derivative, troconazole C, propose a new tricyclic kojic acid derivative, troconazole C. Troconazole C can provide an effective precursor compound for the development of food additives with anti-browning effects, which has a certain promoting effect on meeting social needs, reducing production costs, and increasing market value as well as the application value of such compounds as food additives. It also lays a material foundation for further efficacy research and drug development of kojic acid and its next application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the structural formula of the trexone C compound of the present invention;

[0031] Figure 2 is the key HMBC signal of trexone C of the present invention;

[0032] Figure 3 This is a high-resolution mass spectrum of troxenone C of the present invention;

[0033] Figure 4 For the present invention, 1 H-NMR spectrum;

[0034] Figure 5 For the present invention, 13 C-NMR spectrum;

[0035] Figure 6 HSQC diagram of troxone C of the present invention;

[0036] Figure 7 is the HMBC diagram of troxerone C of the present invention;

[0037] Figure 8 ROESY diagram of troxone C of the present invention;

[0038] Figure 9 is the structural formula of the compound in Example 2 of the present invention;

[0039] Figure 10 In Example 2 of the present invention 1 H and 13 C NMR data table;

[0040] Figure 11 This is the experimental data table in Example 3 of the present invention;

[0041] Figure 12 This is the experimental data table in Example 4 of the present invention;

[0042] Figure 13 This is the experimental data table in Example 5 of the present invention;

[0043] Figure 14 This is the experimental data table in Example 6 of the present invention. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] Example 1 Fermentation and extraction of bacterial strains

[0046] The present invention provides a technical solution: a method for preparing a kojic acid derivative, tromethamine C. Aspergillus versicolor was purchased from the American Type Culture Collection (ATCC) with accession number ATCC 28286. The strain was revived by streaking, inoculated onto PDA solid culture medium, and activated in a 28°C incubator for 5 days. A small colony fragment was excised from the PDA culture medium with a scalpel and inoculated into PDB liquid culture medium. The culture was shaken and incubated on a shaker at 28°C and 160 rpm for 5 days to obtain a seed solution. The seed solution was then inoculated into rice solid culture medium and incubated in a 28°C constant temperature and humidity chamber for 30 days. The rice fermentation product was extracted twice by adding 400 mL of ethyl acetate to every 200 g of the fermentation product. The combined extracts were concentrated under reduced pressure until the ethyl acetate flavor was eliminated to obtain an ethyl acetate extract. The ethyl acetate extraction reagent can also be replaced with dichloromethane or anhydrous ethanol.

[0047] Example 2 Isolation and Identification of Trixone C

[0048] The present invention provides a technical solution: a method for preparing kojic acid derivative tromethamine C, comprising subjecting an extract to normal phase column chromatography elution using 200-300 mesh silica gel as the column chromatography silica gel, followed by adsorption overnight using macroporous resin column chromatography, and elution with a 10%, 30%, 50%, 70%, 90%, and 100% methanol-water gradient. Each gradient is combined and spin-dried until liquid-free to obtain six subfractions. The macroporous resin column chromatography elution gradient is 10%-100% methanol-water, and the fourth subfraction (70%) is separated and purified using semi-preparative high performance liquid chromatography, using a methanol-water elution solvent with a methanol-water volume ratio of 55%-85% and a detection wavelength of 210 nm. The mobile phase is 65% MeOH, the flow rate is 4.0 mL / min, the detection wavelength is 210 nm, and the peak elution time is 30.5-31.9 min. The peak was collected, dissolved by heating, filtered, and finally cooled to obtain 6.5 mg of a white solid. The crude kojic acid was dissolved by heating to 80°C in 40% ethanol-water and then filtered. The target subfraction was 50% to 90% methanol-water. The white solid was subjected to high-resolution mass spectrometry and one-dimensional and two-dimensional nuclear magnetic resonance analysis. The results are as follows: Figure 2-8, determine the structure of the compound as shown in the accompanying drawings Figure 9 High resolution mass spectrum [M+H]+m / z 421.1481, calculated value 421.1493 (C 21 H 25 O9), the compound formula is C 21 H 24 O9. 1 H and 13 C NMR data as shown in the attached figure of the specification Figure 10 As shown in Table 1. Proton and carbon spectra, combined with HSQC signal assignment, indicate the presence of one olefinic methine (C-3), two methines (C-13 and C-16), one hydroxymethyl (C-22), one methylene (C-17), and five methyl groups (C-18, C-19, C-20, C-21, and C-23). Eleven remaining quaternary carbon signals are further distinguished as four carbonyls, four oxygen-linked aromatic quaternary carbons, one aromatic quaternary carbon, and two oxygen-linked quaternary carbons. Kojic acid fragments are confirmed by HMBC signals from H-22 to C-2 / C-3 and from H-3 to C-2 / C-4 / C-5. The cyclohexenone fragment was confirmed by HMBC signals from H-16 to C-10 / C-15 / C-18, H3-19 to C-15 / C-16 / C-17, H3-20 to C-9 / C-10 / C-11, and H3-23 to C-7 / C-8 / C-9. The two structural fragments of kojic acid and cyclohexenone were connected by a -CH-CH3 fragment, as confirmed by HMBC signals from H-13 to C-5 / C-7 / C-11 / C-12 / C-14 and H3-21 to C-12 / C-13 / C-14.

[0049] Example 3 Free radical scavenging experiment 1

[0050] The present invention provides a technical solution: an application of a kojic acid derivative, trolox C, ABTS working solution (Biyuntian Biotechnology Co., Ltd.), 100 μL of oxidant solution and 100 μL of ABTS solution are mixed, reacted at room temperature in the dark for 16 hours, and diluted with 80% ethanol to an absorbance of 0.65 to 0.75 at 734 nm. The compound solution is diluted to 0.01 to 3.00 mM. In a 96-well plate, 180 μL of ABTS working solution is first added to each well, followed by 20 μL of compound solution. 80% ethanol is added to the blank group, and the vitamin E analog trolox is used as a positive control. The ABTS test is reacted at room temperature in the dark for 6 minutes, and the absorbance at 734 nm is measured. Free radical scavenging ability (%) = [(A1–A2) / A1×100], and the above experiments are repeated three times. The results are shown in the attached figure of the specification. Figure 11 As shown in Table 2. The results showed that tromethamine C had significant antioxidant activity and the EC 50 It is 39.5±2.8μM.

[0051] Example 4 Free Radical Scavenging Experiment 2

[0052] The present invention provides a technical solution: an application of a kojic acid derivative, trolox C, a DPPH working solution (Shanghai McLean Biochemical Reagent Co., Ltd.), and a 0.30 mM DPPH 80% ethanol solution, which must be prepared and used immediately. The compound solution is diluted to 0.01-3.00 mM. In a 96-well plate, 100 μL of DPPH working solution and 100 μL of compound solution are first added to each well. 80% ethanol is added to the blank group, and the vitamin E analog trolox is used as a positive control. The DPPH test is carried out at room temperature in the dark for 30 minutes, and the absorbance at 517 nm is measured. Free radical scavenging ability (%) = [(A1–A2) / A1×100], and the above experiments are repeated three times. The results are shown in the attached figure of the specification. Figure 12 The results are shown in Table 3. The results show that tromethamine C has certain antioxidant activity and its EC 50 It is 84.8±4.6μM.

[0053] Example 5 Tyrosinase inhibition experiment 1

[0054] The present invention provides a technical solution: an improved method for testing the monophenolase activity of a kojic acid derivative, tropolone C, using the tyrosinase-catalyzed L-tyrosine (L-Tyr) oxidation rate method. Using 0.2 M phosphate buffer (PBS) as the solvent, mushroom tyrosinase was prepared at 200 U / mL. A 220 μL reaction system was used, divided into four groups: blank group A (120 μL PBS), blank control group B (90 μL PBS and 30 μL mushroom tyrosinase), sample background control group C (20 μL sample and 100 μL PBS), and sample group D (20 μL sample, 70 μL PBS, and 30 μL mushroom tyrosinase). The reaction was incubated in a 30°C incubator for 10 minutes. Then, 100 μL of 0.5 mmol / L tyrosine was added to each well. After a further 20 minutes of incubation, the absorbance was measured at 475 nm. The inhibition rate of tyrosinase was calculated according to the formula (inhibition rate = [(BA)-(DC)] / (BA) × 100%), and the IC was calculated using SPSS software. 50 The inhibitory activity of kojic acid on tyrosinase is stronger than that of kojic acid, IC 50 10.8±1.9μM, as shown in the attached figure of the specification Figure 13 As shown in Table 4.

[0055] Example 6 Tyrosinase inhibition experiment 2

[0056] The present invention provides a technical solution: an improved method for testing the diphenolase activity of a kojic acid derivative, troperone C, using the tyrosinase-catalyzed levodopa (L-DOPA) oxidation rate method. Using 0.2M phosphate buffer (PBS) as the solvent, mushroom tyrosinase was prepared at a concentration of 200 U / mL. A 220 μL reaction system was used and divided into four groups: blank group A (120 μL PBS), blank control group B (90 μL PBS and 30 μL mushroom tyrosinase), sample background control group C (20 μL sample and 100 μL PBS), and sample group D (20 μL sample, 70 μL PBS, and 30 μL mushroom tyrosinase). The reaction was incubated in a 30°C incubator for 10 minutes. Then, 100 μL of 0.5 mmol / L L-DOPA was added to each well. After a further 20 minutes of incubation, the absorbance was measured at 475 nm. The inhibition rate of tyrosinase was calculated according to the formula (inhibition rate = [(BA)-(DC)] / (BA) × 100%), and the IC was calculated using SPSS software. 50 The inhibitory activity of kojic acid on tyrosinase is stronger than that of kojic acid, IC 50 8.9±1.5μM, as shown in the attached figure of the specification Figure 14 As shown in Table 5.

[0057] Example 7 Anti-browning effect of fresh-cut apples

[0058] The present invention provides a technical solution: a method for using a kojic acid derivative, tromethamine C. Fresh, uniformly ripe, sized, and colored Red Fuji apples are selected, free of mechanical damage and pests. Pre-cooled apples are sterilized and cleaned with sodium hypochlorite solution, then quickly rinsed with sterile water and cut into pieces. Using sterilized knives, the apples are cut into 8 mm × 5 mm × 3 mm pieces within a sterile operating table. The pieces are then divided into two portions, each weighing 100 g. A control group is soaked in sterile ultrapure water for 60 seconds, while experimental groups are soaked in tromethamine C solutions of varying concentrations for 60 seconds. Equal amounts are then packaged in three polyethylene ziplock bags and stored in a refrigerator at 4°C for 10 days. Measurements are taken every 2 hours for the first 12 hours and every 2 days thereafter. Results show that the control group exhibits significant browning after 4 hours, while the addition of 10 μM tromethamine C results in significant browning after 12 hours, demonstrating that 10 μM tromethamine C exhibits a significant anti-browning effect on fruit.

[0059] Example 8 Anti-browning effect of apple juice

[0060] The present invention provides a technical solution: an application of a kojic acid derivative, tromethamine C. Apples are washed, peeled, cored, and cut into 100g pieces. 100mL of distilled water is added, and tromethamine C solutions of varying concentrations are added. The juice is quickly pulped, filtered through filter paper, and its optical density (OD) at 420nm is immediately measured. The remainder is poured into a petri dish. The juice is then left at room temperature (25°C) to allow for full oxidation and browning. The absorbance is measured after 6 hours, 12 hours, 24 hours, and 48 hours. Results show that the control apple juice exhibits significant browning after 6 hours, while the addition of 10μM tromethamine C takes 12 hours to show significant browning. This demonstrates that 10μM tromethamine C exhibits a significant anti-browning effect on juice.

[0061] In summary, the present invention proposes a novel tricyclic kojic acid derivative, troxenone C. Subsequent studies have found that troxenone C has significant antioxidant activity, with EC50 values of 39.5±2.8μM and 84.8±4.6μM for scavenging ABTS and DPPH free radicals, respectively. Troxenone C has stronger tyrosinase inhibitory activity than kojic acid, and IC 50 The concentrations of kojic acid were 10.8±1.9μM and 8.9±1.5μM, respectively. Even at 10μM, kojic acid exhibited significant anti-browning effects in fresh-cut fruit and fruit juice. Kojic acid provides an effective precursor compound for the development of anti-browning food additives, potentially contributing to meeting social needs, reducing production costs, and increasing market value. This also lays a foundation for further research into the efficacy of kojic acid, drug development, and future applications.

[0062] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing kojic acid derivative troxone C, characterized in that: The following steps are involved: (1) Preparation of PDA solid culture medium: 100-300 g fresh potatoes, 500-1500 mL tap water, 12-24 g agar, boil to dissolve, and then sterilize under high pressure at 100-120°C for 10-50 min to obtain PDA solid culture medium; (2) Preparation of PDB liquid culture medium: 20 g potato, 2 g sucrose, 100 mL tap water and 2 g agar, then mix, dissolve, adjust pH, clarify and filter, package and sterilize under high pressure at 100-120°C for 15-30 min to obtain PDB liquid culture medium; (3) Activation of Aspergillus versicolor: The Aspergillus versicolor purchased from the American Type Culture Collection with the accession number ATCC 28286 was streaked and revived, and inoculated into the PDA solid medium in step (1), and activated in an incubator at 28°C for 5 days; (4) Cultivation of seed solution: Use a scalpel to cut a small piece of colony from the PDA medium in step (3), inoculate it into the PDB liquid medium in step (2), and culture it on a shaker at 28°C and 160 rpm for 5 days to obtain seed solution; (5) Cultivation and fermentation: The seed solution in step (4) is then inoculated into a rice solid culture medium, and the culture is statically placed in a constant temperature and humidity chamber at 28° C. for 30 days to obtain a rice fermentation product; (6) Obtaining ethyl acetate extract: taking the rice fermentation product obtained in step (5), adding 400 mL of ethyl acetate for every 200 g of the fermentation product, cold soaking extraction 2 to 3 times, each cold soaking extraction for 15 to 21 hours, and combining the extracts, and concentrating under reduced pressure until there is no ethyl acetate taste, to obtain ethyl acetate extract; (7) Crude extraction: The ethyl acetate extract obtained in step (6) was subjected to normal phase column chromatography, and then macroporous resin column chromatography was used for overnight adsorption and gradient elution with 10%, 30%, 50%, 70%, 90%, and 100% methanol-water to obtain a crude kojic acid derivative. (8) Separation and purification: The target subcomponent in the crude kojic acid obtained in step (7) is separated and purified by high performance liquid chromatography, dissolved by heating, filtered, and finally cooled to obtain a pure kojic acid derivative, the structural formula of which is: 。 2. The method for preparing a kojic acid derivative, troxone C, according to claim 1, wherein: In step (6), the ethyl acetate extraction reagent is replaced with dichloromethane or anhydrous ethanol extraction reagent.

3. The method for preparing a kojic acid derivative, troxone C, according to claim 2, characterized in that: In the normal phase column chromatography elution in step (7), 200-300 mesh silica gel is used as column chromatography silica gel.

4. The method for preparing a kojic acid derivative, troxone C, according to claim 3, characterized in that: The heating and dissolution method of the crude kojic acid in step (8) is: using 40% ethanol-water to heat to 80°C to dissolve and then filter, and the target subfraction is 50%~90% methanol-water section.

5. The method for preparing a kojic acid derivative, troxone C, according to claim 3, characterized in that: In step (8), the HPLC eluent is methanol-water with a volume ratio of methanol to water of 55% to 85%, and the detection wavelength is 210 nm.

6. Use of the troxenone C prepared by the method for preparing the kojic acid derivative troxenone C according to any one of claims 1 to 5 in preparing a food additive precursor for preventing enzymatic browning induced by tyrosinase.

Images