Two polyketide compounds derived from mangrove fungi, their preparation methods, and their applications in whitening products

Abstract

The present invention relates to the technical fields of microbial fermentation and daily chemical industry, and particularly to two polyketide compounds derived from mangrove fungi, their preparation methods, and their use in whitening products. The structural formulas of the two polyketide compounds are shown in Formula (I) and Formula (II). The preparation method comprises the following steps: activating Aspergillus sp. GXNUW29 in PDA culture medium, then inoculating it into a liquid culture medium, and culturing it on a shaking table to obtain a seed culture solution; inoculating the strain in the seed culture solution into a rice fermentation medium, culturing it statically to obtain fungal mycelium; extracting the fungal mycelium three times with methanol, extracting it with ethyl acetate, and concentrating the extract to obtain an extract; and separating and purifying the obtained extract using column chromatography and recrystallization techniques to obtain the polyketide compound shown in Formula (II). The two polyketide compounds (I) and (II) obtained by the present invention have tyrosinase inhibitory activity and can be used to prepare whitening cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation and daily chemical industry, and in particular to two polyketide compounds derived from mangrove fungi, a preparation method thereof, and application thereof in whitening products. Background Art

[0002] Mangroves are complex ecosystems found in tropical and subtropical intertidal estuaries, supporting a diverse array of microorganisms, including fungi, actinomycetes, bacteria, cyanobacteria, algae, and protozoa. Mangrove fungi represent the second largest ecological group within the marine fungal community and have become a prolific source of novel marine natural products. As part of the mangrove microbiome, mangrove fungi play an important role in providing novel lead metabolites.

[0003] The amount of melanin in the skin is directly related to skin darkening, melasma, and hyperpigmentation. Skin whitening methods primarily focus on the production, transfer, and metabolism of melanin. Tyrosinase is the rate-limiting enzyme in melanin synthesis. Its activity directly determines the rate of melanin synthesis and its accumulation in the skin, making it one of the main causes of skin darkening and hyperpigmentation. Inhibiting tyrosinase activity has been a long-standing goal in skin health research and cosmetics, as it reduces melanin synthesis, thereby achieving skin whitening. It is estimated that approximately 15% of global investment in skin whitening agents is spent, with Asia leading the way. According to a report by SIRONA Biochemicals, the Asia-Pacific region spends approximately $13 billion on skincare and cosmetics. In India alone, spending on whitening creams and skincare products is estimated to have reached $432 million in 2010. Many tyrosinase inhibitors, such as hydroquinone, arbutin, kojic acid, azelaic acid, L-ascorbic acid, ellagic acid, and tranexamic acid, have been used as skin-lightening agents. However, they all have drawbacks. Arbutin is chemically unstable and releases hydroquinone, which decomposes into benzene metabolites, potentially toxic to bone marrow. Kojic acid's use in cosmetics is restricted due to its carcinogenicity and instability during storage. L-ascorbic acid is heat-sensitive and easily degraded. Therefore, the search for a safe and effective tyrosinase inhibitor is essential.

[0004] Polyketides are common secondary metabolites of mangrove fungi and are considered attractive and promising pharmaceuticals. Mangrove-derived polyketides exhibit various biological activities, including antimicrobial, cytotoxic, and tyrosinase inhibition. Many drugs in medicine belong to the polyketide class, such as doxorubicin, tetracycline, and lovastatin. my country's abundant mangrove resources have long been a natural treasure trove for drug screening. Therefore, developing mangrove-derived fungal resources has both theoretical and practical application value. Summary of the Invention

[0005] The object of the present invention is to address the above-mentioned problems and provide two polyketide compounds derived from mangrove fungi, their preparation methods and their applications in whitening products.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Two polyketide compounds derived from mangrove fungi, the structural formulas of the two polyketide compounds are shown in formula (I) and formula (II):

[0008]

[0009] The two polyketide compounds (I) and (II) described in the present invention are isolated and obtained from the solid fermentation product of the mangrove fungus Aspergillus sp. GXNU-W29. The mangrove fungus Aspergillus sp. GXNU-W29 was deposited in the Guangdong Provincial Microbiological Culture Collection on June 6, 2024, with the deposit number GDMCC No. 60789.

[0010] Specifically, the preparation method of the two polyketone compounds comprises the following steps:

[0011] (1) Strain activation and seed culture: The mangrove fungus strain Aspergillus sp. GXNUW29 was activated in PDA culture medium, and the activated strain was inoculated into a liquid culture medium and cultured on a shaking platform to obtain a seed culture solution;

[0012] (2) Fermentation culture: The strain in the seed culture solution is transferred into the rice fermentation medium and cultured statically to obtain fungal mycelium;

[0013] (3) Extraction: The fungal mycelium obtained in step (2) was extracted three times with methanol, extracted with ethyl acetate, and the extract was concentrated to obtain an extract;

[0014] (4) Separation and recrystallization: The extract was separated by column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate (0%-100%) as the eluent, and then the same components were combined until 8 components were obtained; the second component was recrystallized from methanol to obtain a polyketone compound as shown in formula (I); the third component was recrystallized from methanol-ethyl acetate to obtain a polyketone compound as shown in formula (II).

[0015] In the above preparation method, preferably, the PDA culture medium used for the bacterial activation in step (1) is composed of 2-5 g agar, 20-40 g glucose, and 1-2 liters of potato liquid.

[0016] In the above preparation method, preferably, the liquid culture medium used for culturing the bacteria in step (1) comprises 20-40 g of glucose and 1-2 liters of potato liquid.

[0017] In the above preparation method, preferably, the shaking culture conditions in step (1) are a shaking speed of 80-150 rpm and a culture temperature of 25-30° C. for 3-10 days.

[0018] In the above preparation method, preferably, the rice culture medium used for the fermentation culture in step (2) is obtained by mixing rice and seawater in a mass volume ratio of 1.0 g:1.0 ml.

[0019] In the above preparation method, preferably, the conditions for the static culture in step (2) are to control the temperature to 25-30° C. and the culture time to 30-50 days.

[0020] In the above preparation method, preferably, the column chromatography separation technology in step (4) is a normal phase silica gel column chromatography technology, wherein the normal phase silica gel column chromatography uses ethyl acetate and petroleum ether as eluents, and the ratios are 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60 respectively; and the recrystallization is a room temperature dissolution and crystallization technology.

[0021] The two polyketide compounds (I) and (II) derived from the mangrove fungi have tyrosinase inhibitory activity and can be used in the preparation of whitening cosmetics.

[0022] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0023] 1. The present invention is the first to use mangrove fungus Aspergillus sp. GXNU-W29 to obtain two polyketide compounds with structural formulas as shown in formula (I) and (II). These compounds have significant tyrosinase inhibitory activity. The results of the tyrosinase inhibitory activity screening test show that the IC of polyketide (I) is 50 The value is 21.50 μM, and the IC value of polyketide (II) is50 The value was 20.20 μM, which was better than the positive control ascorbic acid (IC 50 =33.70μM). In addition, polyketone (I) and polyketone (II) also showed good DPPH free radical scavenging ability, EC 50 The values ​​were 19.59 μM and 12.23 μM, respectively, which were better than the positive control BHA (EC 50 =23.12 μM) and ascorbic acid (EC 50 =20.03 μM). It can be seen that the polyketide (I) and polyketide (II) prepared by the present invention have an inhibitory effect on tyrosinase, the rate-limiting enzyme in melanin synthesis, and can be considered as potential lead compounds for the development of tyrosinase inhibitors, with the potential for application in the preparation of whitening products.

[0024] 2. The present invention utilizes mangrove fungus Aspergillus sp. GXNU-W29 to prepare polyketide compounds, which has a simple preparation process, a short production cycle, low cost, no pollution to the environment, and is easy to mass-produce, which is beneficial to subsequent research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the process of separating and purifying the extract according to the present invention. DETAILED DESCRIPTION

[0026] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0027] 1. Preparation Example

[0028] Example 1

[0029] Isolation and Identification of Mangrove Fungus Aspergillus sp. GXNU-W29

[0030] 1. Materials: Fungal samples were obtained from leaves of Aegiceras corniculata, a mangrove plant, collected in Beihai, Guangxi Zhuang Autonomous Region.

[0031] 2. After culturing, isolating, and identifying the strain, a pure fungal strain was obtained and identified as Aspergillus sp. GXNU-W29. The mangrove fungus Aspergillus sp. GXNU-W29 was deposited with the Guangdong Provincial Microbiological Culture Collection on June 6, 2024, with the deposit number GDMCC No. 60789. The depository's address is 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0032] Example 2

[0033] The preparation method of two polyketone compounds comprises the following steps:

[0034] (1) Strain activation and seed culture: The mangrove fungus strain Aspergillus sp. GXNUW29 was activated in a PDA medium composed of 5 g agar, 20 g glucose, and 1 liter potato liquid; the activated strain was inoculated into a liquid culture medium composed of 20 g glucose and 1 liter potato liquid; the culture was performed on a shaker at a speed of 80 rpm and 25° C. for 3 days to obtain a seed culture solution;

[0035] (2) Fermentation culture: The strain in the seed culture solution was transferred into a solid rice fermentation medium, wherein the solid rice fermentation medium was a mixture of rice and seawater in a mass volume ratio of 1.0 g:1.0 ml, and the culture was allowed to stand at 25° C. for 30 days to obtain fungal mycelium;

[0036] (3) Extraction: After fermentation, the fungal mycelium obtained in step (2) was extracted three times with methanol, the extract was suspended in water, and then extracted with ethyl acetate, and the extract was concentrated to obtain an extract (17 g);

[0037] (4) Separation and recrystallization: According to the attached Figure 1 The extract is separated and purified using a process. The extract is first separated by normal phase silica gel column chromatography, using ethyl acetate and petroleum ether as eluents in ratios of 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60, followed by combining the same components to obtain eight fractions. The second fraction is recrystallized from methanol to obtain a polyketone compound represented by formula (I). The third fraction is recrystallized from methanol-ethyl acetate to obtain a polyketone compound represented by formula (II).

[0038] Example 3

[0039] The preparation method of two polyketone compounds comprises the following steps:

[0040] (1) Strain activation and seed culture: The mangrove fungus strain Aspergillus sp. GXNUW29 was activated in a PDA medium composed of 2 g agar, 25 g glucose, and 2 liters of potato liquid; the activated strain was inoculated into a liquid culture medium composed of 25 g glucose and 2 liters of potato liquid; and the culture was performed on a shaker at a speed of 150 rpm and 30° C. for 10 days to obtain a seed culture solution;

[0041] (2) Fermentation culture: The strain in the seed culture solution was transferred into a solid rice fermentation medium, wherein the solid rice fermentation medium was a mixture of rice and seawater in a mass volume ratio of 1.0 g:1.0 ml, and the culture was allowed to stand at 30° C. for 50 days to obtain fungal mycelium;

[0042] (3) Extraction: After fermentation, the fungal mycelium obtained in step (2) was extracted three times with methanol, the extract was suspended in water, and then extracted with ethyl acetate, and the extract was concentrated to obtain an extract (17 g);

[0043] (4) Separation and recrystallization: Figure 1 The extract is separated and purified using a process. The extract is first separated by normal phase silica gel column chromatography, using petroleum ether and ethyl acetate as eluents in ratios of 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60, followed by combining the same components to obtain eight fractions. The second fraction is recrystallized from methanol to obtain a polyketone compound represented by formula (I). The third fraction is recrystallized from methanol-ethyl acetate to obtain a polyketone compound represented by formula (II).

[0044] 2. Confirmation of Compounds

[0045] The polyketone compound obtained above was dissolved in deuterated chloroform and deuterated methanol, and identified by nuclear magnetic resonance spectroscopy (NMR) and other methods to obtain the following test data:

[0046] Polyketide I: Pale yellow oil, easily soluble in methanol. HRESI: 262.1441[M+H] + , molecular formula is C 15 H 19 NO3. 1 H NMR (600 MHz, CD3OD) 1 H NMR (600 MHz, CD3OD) δ H 6.88(1H,dd,J=7.5,H-4),7.05(1H,t,J=7.5,H-5),6.65(1H,dd,J=15.7,6.8,H-10),5.07(1H,d,J=15.7,H-9) ,2.33(2H,m,H-11),3.88(3H,s,OCH3,H-15),1.27(2H,m,H-12),1.12(3H,t,J=6.80,H-13),1.48(3H,s,H-14); 13 C NMR (150 MHz, CD3OD) δ C109.3(C-1),195.3(C-2),139.5(C-3),119.4(C-4),129.3(C-5),116.6(C-6),147.8(C-7),153.1( C-8),129.3(C-9),134.7(C-10),55.9,(C-15),36.3(C-11),23.6(C-12),17.5(C-13),14.0(C-14).

[0047] Polyketide II: Pale yellow oil, easily soluble in methanol. RESI: 234.1491[M+H] + , molecular formula is C 14 H 19 NO2. 1 H NMR (600 MHz, CD3OD) δ H 4.20(1H,q,J=7.3,H-1),3.79(1H,ddd,J=6.9,7.5,2.1,H-3),3.48(1H,m, H-4),6.70(1H,ddd,J=15.5,7.6,2.2,H-5),6.12(1H,dd,J=15.5,2.5,H-6 ),4.25(1H,d,J=7.5,H-8),5.59(1H,m,H-9),5.66(1H,m,H-10),2.03(2H, m,H-11),1.41(2H,m,H-12),0.85(3H,t,H-13),1.26(3H,d,J=7.3,H-14); 13 C NMR (150 MHz, CD3OD) δ C 65.1(C-1),211.2(C-2),53.5(C-3),41.3(C-4),149.0(C-5),128.1(C-6),192.0(C-7),6 8.5(C-8),127.1(C-9),131.9(C-10),34.6(C-11),24.3(C-12),13.8(C-13),19.5(C-14).

[0048] After identification, the structural formulas of the two polyketone compounds are shown in formula (I) and (II):

[0049]

[0050] 3. Experiment on the inhibitory effect of two polyketide compounds on tyrosinase activity:

[0051] Tyrosinase inhibition experiments of the two polyketide compounds obtained in Examples 2 and 3:

[0052] 1. Materials:

[0053] 1.1 Ascorbic acid; tyrosinase, L-tyrosine, potassium dihydrogen phosphate buffer solution.

[0054] 1.2 Tyrosinase inhibition experiment:

[0055] The compound samples were dissolved in 50 mM potassium dihydrogen phosphate buffer solution (pH = 6.5) and the reaction mixture was prepared according to the methods of Curto et al. and Nerya et al. (Curto EV, Kwong C, et al. Biochemical Pharmacology, 1999, 57, 663-672.; Nerya O, Vaya J, Musa R, et al. J. Agric. Food Chem. 2003, 51, 1201-1207.) were modified to test tyrosinase inhibition in 96-well plates at concentrations of 166.6, 83.8, 41.6, 20.8, and 10.4 μg / mL. The sample solution (70 μL) was mixed with tyrosinase (30 μL, phosphate buffer solution at 333 units / mL). The mixture was incubated at room temperature (37°C) for 10 minutes, and then the substrate, L-tyrosine (110 μL, 2 mM), was added. The reaction mixture was incubated at room temperature (37°C) for an additional 30 minutes. The absorbance was measured at a wavelength of 492 nm. Ascorbic acid was used as a positive control, and the experiment was repeated three times. The percentage of inhibition of tyrosinase activity was calculated as follows:

[0056] Inhibition percentage = (AB) / A×100 [(A: absorbance without test sample (control); B: absorbance with test sample]

[0057] 2. Test results

[0058] The experimental results showed that compounds (I) and (II) could significantly and effectively inhibit the activity of tyrosinase. The inhibitory effects of the two polyketide compounds on tyrosinase were IC 50 The values ​​(μM) are shown in Table 1.

[0059] Table 1 Inhibitory effect of compounds on tyrosinase

[0060]

[0061] 4. DPPH radical scavenging ability experiment of two polyketide compounds

[0062] DPPH free radical scavenging ability experiment of the two polyketide compounds obtained in Examples 2 and 3:

[0063] 1. Materials:

[0064] 1.1DPPH, BHA, ascorbic acid

[0065] 1.2 DPPH free radical scavenging ability experiment

[0066] The free radical scavenging ability of compounds I-II was determined using the method described by Blios in 1985 (Blois MS. Nature 1958, 181, 1199-1200.). A reaction mixture consisting of compound sample / positive control (at concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.63 μg / mL) and a solution of the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was incubated in the dark for 30 minutes, and then the absorbance was measured at a wavelength of 517 nm. BHA and ascorbic acid were used as positive controls. All tests were repeated three times.

[0067] Antioxidant activity percentage AA% = [(control group absorbance - sample absorbance) / control group absorbance] × 100

[0068] 2. Test results

[0069] The test results show that compounds (I) and (II) have good free radical scavenging ability. The DPPH free radical scavenging ability of the two polyketone compounds is shown in Table 2.

[0070] Table 2 DPPH free radical scavenging ability of compounds

[0071]

[0072] Therefore, the compounds (I) and (II) with a polyketide structure provided by the present invention have a good inhibitory effect on tyrosinase, the rate-limiting enzyme in melanin synthesis, and have good free radical scavenging ability. They have great development value and good application prospects. They can be used to prepare tyrosinase inhibitors and are applied in whitening cosmetics.

[0073] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Two polyketide compounds derived from mangrove fungi, characterized in that: The structural formulas of the two polyketone compounds are shown in formula (I) and formula (II): 。 2. The method for preparing two polyketide compounds derived from mangrove fungi according to claim 1, characterized in that: The two polyketides (I) and (II) are isolated from mangrove fungi Aspergillus sp. GXNU-W29 solid fermentation isolated from mangrove fungi Aspergillus sp. GXNU-W29 was deposited in Guangdong Provincial Microbiological Culture Collection on June 6, 2024, with the deposit number GDMCC No. 60789; The following steps are involved: (1) Activation of fungi and seed culture: The mangrove fungus strain Aspergillus sp. GXNUW29 was activated in PDA medium, and the activated strain was inoculated into liquid culture medium and cultured on a shaking platform to obtain a seed culture solution; (2) Fermentation culture: The strain in the seed culture solution is inoculated into the rice fermentation medium and cultured statically to obtain fungal mycelium; (3) Extraction: extracting the fungal mycelium obtained in step (2) three times with methanol, extracting with ethyl acetate, and concentrating the extract to obtain an extract; (4) Separation and recrystallization: The extract was separated by normal phase silica gel column chromatography, and gradient elution was performed using ethyl acetate and petroleum ether as eluents, with the ratios of 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60, respectively; the same components were then combined to obtain 8 components; the second component was recrystallized from methanol to obtain a polyketone compound as shown in formula (I); the third component was recrystallized from methanol-ethyl acetate, and the recrystallization was a room temperature dissolution followed by crystallization technique to obtain a polyketone compound as shown in formula (II).

3. The preparation method according to claim 2, wherein: The composition of the PDA culture medium used for the activation of the bacteria in step (1) is as follows: 2-5 g agar, 20-40 g glucose, and 1-2 liters of potato liquid.

4. The preparation method according to claim 2, wherein: The composition of the liquid culture medium used for the bacterial culture in step (1) is as follows: 20-40 grams of glucose and 1-2 liters of potato liquid.

5. The preparation method according to claim 2, wherein: The shaking culture conditions in step (1) are a shaking speed of 80-150 rpm and a culture temperature of 25-30°C for 3-10 days.

6. The preparation method according to claim 2, wherein: The rice culture medium used for the fermentation culture in step (2) is obtained by mixing rice and seawater in a mass volume ratio of 1.0 g: 1.0 ml.

7. The preparation method according to claim 2, characterized in that: The conditions for the static culture in step (2) are to control the temperature to 25-30°C and the culture time to 30-50 days.

8. Use of the two polyketide compounds (I) and (II) derived from mangrove fungi according to claim 1 in the preparation of whitening cosmetics.

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