Use of Soybean Extract for Anti-ultraviolet Damage and Reducing Pigmentation

Abstract

The present invention provides a use of a Diaporthe caulivora extract for preparing a composition for resisting ultraviolet damage and reducing pigmentation. The present invention also provides a novel compound separated from the Diaporthe caulivora extract and a composition containing the compound.

Description

[0001] This application is a divisional application of the invention patent application with application number 202011344831.2, application date November 26, 2020, and invention name "Use of soybean Aspergillus extract for resisting UV damage and reducing pigmentation". Technical Field

[0002] The present invention relates to the use of Diaporthe caulivora extract in preparing a composition for resisting ultraviolet damage and reducing pigmentation. Background Art

[0003] Ultraviolet (UV) in solar radiation has a great impact on the skin of organisms. Excessive exposure to ultraviolet light not only causes the skin to darken, but also causes the surface layer of the skin to absorb ultraviolet light and produce free radicals. If too many free radicals accumulate in the body, oxidative stress will occur, making the defense system unable to balance and regulate, leading to cell damage. Although some natural substances have been proven to have the effects of ultraviolet protection and melanin inhibition, they are almost all plant-derived extracts or compositions. As for the application of microorganisms in skin care products, most developers currently use exogenous bacteria (such as Ganoderma lucidum and Auricularia auricula), and there is little research on endophytic bacteria.

[0004] Diaporthe caulivora is a species of Diaporthe caulivora, a plant pathogen that often infects soybeans. According to past literature reviews, its chemical composition and biological activities have not been deeply explored.

[0005] Although previous literature has verified that Diaporthe caulivora has anti-inflammatory activity, it has not been confirmed that Diaporthe caulivora extract can be used to prevent skin damage caused by ultraviolet rays, nor has it been confirmed that Diaporthe caulivora extract can be used to inhibit melanin production or reduce pigmentation.

[0006] In view of the deficiencies of the prior art, the inventors, after careful experiments and research, and with perseverance, finally conceived the present case "Use of Diaporthe caulivora extract for resisting UV damage and reducing pigmentation", which can overcome the deficiencies of the prior art. The following is a brief description of the present case. Summary of the invention

[0007] Unless specifically defined otherwise, the terms used in this description shall have the ordinary and common meanings known to those skilled in the art.

[0008] The present invention isolates endophytes from the leaves of Neolitsea daibuensis: Diaporthe caulivora, and develops it into a composition with anti-ultraviolet damage and pigmentation reduction effects. The composition can prevent the skin from being damaged by ultraviolet rays and has the effect of whitening the skin. It can be used alone or as an active additive for medicines, cosmetics, or skin care products. The Diaporthe caulivora of the present invention is classified and named as Diaporthe caulivora, and was deposited in the German Bio-Collection Center (DSMZ) on October 23, 2020, and its deposit number is: DSM33674.

[0009] The present invention uses solid fermentation products and liquid fermentation products obtained by solid and liquid fermentation of soybean fungus (Diaporthe caulivora) as experimental targets. The solid fermentation products are soaked in 95% ethanol and concentrated under reduced pressure to obtain ethanol extracts, which are separated and purified by column chromatography to obtain compounds 1-18. Among them, compounds 1, 2, 5, 6, 9, 10, 11, 12, 13, 14 and 15 are novel compounds, and compound 3 is a compound isolated from natural products for the first time. The soybean fungus (Diaporthe caulivora) extract and the 18 compounds are subjected to cell safety tests to confirm that they are safe for cells. The light protection test shows that the soybean fungus (Diaporthe caulivora) extract has an ultraviolet protection effect and can inhibit the increase of active oxygen species caused by ultraviolet irradiation. In addition, in the melanin inhibition test, it was found that the soybean fungus (Diaporthe caulivora) extract has the effect of inhibiting tyrosinase and melanin, so that it has a good effect of reducing pigmentation. The compounds isolated from the Diaporthe caulivora extract were tested for activity, and it was found that the compounds had the effect of resisting ultraviolet damage, and also had the effect of inhibiting tyrosinase and melanin. The present invention confirms that the Diaporthe caulivora extract and its secondary metabolites have the activity of resisting ultraviolet damage and reducing pigmentation. This experimental result can be used for the development and promotion of whitening, sunscreen, and anti-aging products in related industries such as cosmetics or skin care products, or for pharmaceuticals to prevent or treat discomfort or diseases such as excessive pigmentation and skin aging caused by excessive exposure to ultraviolet rays.

[0010] Therefore, the present invention provides a use of a Diaporthe caulivora extract for preparing a sunscreen and whitening composition. The term "sunscreen and whitening composition" used in the present invention refers to a composition having an effect of resisting ultraviolet damage and reducing pigmentation. In one embodiment, the sunscreen and whitening composition can protect cells from ultraviolet damage, inhibit the increase of reactive oxygen species caused by ultraviolet rays, and inhibit the generation of tyrosinase and melanin. The aforementioned sunscreen and whitening composition can be used alone as a pharmaceutical, cosmetic, or skin care product, or as an active additive for pharmaceuticals, cosmetics, or skin care products (such as lotions, essences, emulsions, and sunscreens, etc.). The aforementioned sunscreen and whitening composition can be used to prevent or treat discomfort or diseases (such as excessive pigmentation, skin aging, etc.) or light damage states (such as erythema, scaling, edema, thickness changes, sunburn, suppressed immune response, tumorigenesis, or any combination thereof) caused by excessive exposure to ultraviolet rays, and for preventing or treating discomfort or diseases caused by excessive pigmentation, including but not limited to freckles, chloasma, striae of pregnancy, senile plaques, and melanoma. As can be seen from the above, the sunscreen and whitening composition of the present invention has the effect of anti-skin aging in addition to sunscreen and whitening, and can be used as an active additive in sunscreen, whitening, anti-skin aging products or sunscreen, whitening, anti-skin aging products. In one embodiment, the sunscreen and whitening composition is used to prepare topical cosmetics or skin preparations. In one embodiment, the Diaporthe caulivora extract is an extract obtained by extracting solid fermentation products of Diaporthe caulivora with a solvent, and the solvent may be an organic solvent, including but not limited to ethanol. In another embodiment, the Diaporthe caulivora extract is an extract obtained by liquid-liquid partitioning of equal volumes of solvent and liquid fermentation products of Diaporthe caulivora, and the solvent may be an organic solvent, including but not limited to ethanol.In one embodiment, the Diaporthe caulivora extract comprises at least one compound selected from the group consisting of caulivotrioloxin A, caulivotrioloxin B, caulivotrioloxin C, caulivotrioloxin D, caulivotrioloxin E, caulivotrioloxin F, caulibysin A, caulibysin B, diapopyrone, diapophthalide A, and diapophthalide B.

[0011] The present invention also provides a compound isolated from an extract of Diaporthe caulivora, which is selected from the group consisting of caulivotrioloxin A, caulivotrioloxin B, caulivotrioloxin C, caulivotrioloxin D, caulivotrioloxin E, caulivotrioloxin F, caulibysin A, caulibysin B, diapopyrone, diapophthalide A, and diapophthalide B.

[0012] The present invention further provides a composition comprising at least one of the aforementioned compounds. In one embodiment, the composition has an effect of protecting against UV damage. In another embodiment, the composition has an effect of reducing pigmentation.

[0013] The composition of the present invention may be formed into a cream, ointment, emulsion, lotion, serum, paste or mousse. It may be applied to the skin in the form of an aerosol as required, or in a solid form, such as a stick. The composition of the present invention may also be applied to any pharmaceutical, skin care, or cosmetic preparation in the form of a topical preparation, in particular an aqueous, aqueous-alcoholic or oily solution, an oil-soluble in water or water-soluble in oil or multiple emulsions, aqueous or oily gels, liquid, pasty or solid anhydrous products. A person skilled in the art will carefully select the adjuvant, the type and amount of the adjuvant being considered in accordance with the properties of the compound or extract of the present invention, and will not or substantially not be adversely affected by the conceived addition.

[0014] The composition of the present invention may also contain auxiliary agents commonly used in skin care products, cosmetics or dermatological preparations, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, sunscreens, pigments, odor absorbers and dyes. The amount of these various auxiliary agents is a known amount in the field under consideration, for example: 0.01 to 20% of the total weight of the composition. According to their nature, these auxiliary agents can be delivered to the fat phase, the water phase, the lipid vesicles.

[0015] When the composition of the present invention is an emulsion, the proportion of the fatty phase can range from 5 to 80% by weight, for example from 5 to 50% by weight, relative to the total weight of the composition. The oil, emulsifier and co-emulsifier used in the composition in the form of an emulsion are selected from agents known for use in the field under consideration. The emulsifier and co-emulsifier are present in the composition in a proportion ranging from 0.3 to 30% by weight, for example from 0.5 to 20% by weight, relative to the total weight of the composition.

[0016] The oil raw materials that can be used in the present invention can be, for example, mineral oils such as liquid petroleum, vegetable oils such as avocado oil and soybean oil, animal oils such as lanolin, synthetic oils, perhydrosqualene, silicone oil, cyclomethicone, fluorinated oil, or perfluoropolyether. Fatty alcohols such as cetyl alcohol, fatty acids, and waxes such as carnauba wax and ozokerite can also be used as fatty substances.

[0017] The raw materials of emulsifiers and auxiliary emulsifiers that can be used in the present invention include, for example, fatty acid esters of polyethylene glycol (such as PEG-20 stearate) and fatty acid esters of glycerol (such as glyceryl stearate). The raw materials of hydrophilic gelling agents that can be used in the present invention include, for example, carboxyvinyl polymers, carbomers, acrylic acid copolymers of acrylate / alkyl acrylate copolymers, polyacrylamide, polysaccharides, natural gums, and clay. The raw materials of lipophilic gelling agents that can be used in the present invention include, for example, modified clays of bentonite, metal salts of fatty acids, hydrophobic silica, and polyethylene.

[0018] Active agents that can be used in the present invention may be, for example, polyols, vitamins, keratolytic agents and / or descaling tablets, anti-inflammatory agents, calmants and mixtures thereof, depigmenting agents (e.g., kojic acid), and derivatives thereof. Lipophilic or hydrophilic UV screeners may also be used in the composition of the present invention, such as benzene-1,4-bis(3-methylene-10-camphorsulfonic acid), 2-ethylhexyl α-cyano-β,β-diphenylacrylate or octocrylen, butyl methoxydibenzoylmethane, octyl methoxycinnamate and / or titanium oxide and zinc oxide.

[0019] Cosmetic powders useful in the present invention generally contain fillers, pigments, and pearls. Suitable fillers include silica, surface-treated silica, alumina, surface-treated alumina, talc and surface-treated talc, zinc stearate, mica and surface-treated mica, kaolin, nylon powder such as Orgasol™, polyethylene powder, Teflon™, starch, boron nitride, lauroyl lysine, copolymer microspheres, cross-linked polymethacrylate copolymers, and silicone resin microspheres, and the like.

[0020] Description of biological material deposit

[0021] Classification name: Diaporthe caulivora

[0022] Depository: German Biological Collection Center (Leibniz-Institut DSMZ-German Collection of Microorganisms and Cell Cultures GmbH)

[0023] Depository Abbreviation: DSMZ

[0024] Address: Inhoffenstraβe 7B, 38124 Braunschweig, GERMANY Date of deposit: October 23, 2020

[0025] Deposit number: DSM33674. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .Preparation process of the extract of solid fermentation product of Diaporthe caulivora and its active extract layer. Solvent A is water; solvent B is ethyl acetate. DC-S-EtOH is the ethanol extract of solid fermentation product of Diaporthe caulivora; DC-S-EA is the ethyl acetate dissolving layer; DC-SW is the water dissolving layer.

[0027] Figure 2 .Preparation process of extract of Diaporthe caulivora liquid fermentation product and its active extract layer. Solvent A is water; solvent B is ethyl acetate. Liquid fermentation product is liquid-liquid partitioned with ethyl acetate (v / v, 1:1) to obtain DC-L-EA as ethyl acetate soluble layer; DC-LW as water soluble layer.

[0028] Figure 3.Separation process of ethanol extract of solid-state fermentation product of Diaporthe caulivora. D is dichloromethane; M is methanol; * is a new compound; ** is a compound isolated from natural products for the first time.

[0029] Figure 4 .Structure diagram of compounds from Diaporthe caulivora.

[0030] Figure 5 (A) Safety analysis of solid and liquid fermentation products of Diaporthe caulivora and their active extracts at a concentration of 30 μg / mL. (B) Safety analysis of solid and liquid fermentation products of Diaporthe caulivora and their active extracts at a concentration of 300 μg / mL.

[0031] Figure 6 (A) Safety analysis of compounds 1-9 isolated from solid-state fermentation of Diaporthe caulivora at a concentration of 10 μM. (B) Safety analysis of compounds 1-9 isolated from solid-state fermentation of Diaporthe caulivora at a concentration of 50 μM. **: P<0.01 indicates a significant difference from the control group irradiated with ultraviolet light.

[0032] Figure 7 .The photoprotective effect of solid and liquid fermentation products of Diaporthe caulivora and its active extracts on cells damaged by ultraviolet light (concentrations of 30 and 300 μg / mL). ##: P<0.01 indicates a significant difference from the control group without ultraviolet light exposure; **: P<0.01 indicates a significant difference from the control group with ultraviolet light exposure; Qu (quercetin) and atRA (all-trans retinoic acid) are positive controls.

[0033] Figure 8 .The photoprotective effect of compound 1-9 from solid-state fermentation of Diaporthe caulivora on cells damaged by ultraviolet light (at concentrations of 10 and 50 μM). ##: P<0.01 indicates a significant difference from the control group without ultraviolet light exposure; **P<0.01 indicates a significant difference from the control group with ultraviolet light exposure; Qu (quercetin) and atRA (all-trans retinoic acid) are positive control groups.

[0034] Fig. 9.Inhibitory effect of solid and liquid fermentation products of Diaporthe caulivora and its active extracts on intracellular reactive oxygen species induced by ultraviolet light (concentrations of 30 and 300 μg / mL). ##: P<0.01 indicates a significant difference from the control group without ultraviolet light exposure; **: P<0.01 indicates a significant difference from the control group with ultraviolet light exposure; Qu (quercetin) and atRA (all-trans retinoic acid) are positive controls.

[0035] Fig.10 .Inhibitory effects of compounds 1-9 isolated from solid-state fermentation of Diaporthe caulivora on intracellular reactive oxygen species induced by ultraviolet light (at concentrations of 10 and 50 μM). ##: P < 0.01 indicates a significant difference from the control group without ultraviolet light exposure; **: P < 0.01 indicates a significant difference from the control group with ultraviolet light exposure. Qu (quercetin) and atRA (all-trans retinoic acid) were positive controls.

[0036] Fig.11 .Inhibitory effect of ethyl acetate extract of solid-state fermentation product of Diaporthe caulivora (DC-S-EA) on ultraviolet-induced COX-2 protein expression.

[0037] Fig.12 Safety analysis of solid and liquid fermentation products of Diaporthe caulivora and its active extract layer (300 μg / mL) and isolated compounds 1-9 (50 μM) in mouse melanoma cells. *: P < 0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P < 0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid are positive control groups. Cpd: compound.

[0038] Fig.13 Safety analysis of solid and liquid fermentation products of Diaporthe caulivora and its active extracts and compound 1 in mouse melanoma cells. *: P<0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P<0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid are positive control groups. Cpd: compound.

[0039] Fig.14 .Tyrosinase inhibition effect of solid and liquid fermentation products of Diaporthe caulivora and their active extracts. *: P < 0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P < 0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were used as positive controls.

[0040] Fig.15 .Tyrosinase inhibitory effects of compounds 1-9 isolated from solid-state fermentation of Diaporthe caulivora. *: P < 0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P < 0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid are positive controls. Cpd: compound.

[0041] Fig.16 .Tyrosinase inhibition effect of solid and liquid fermentation products of Diaporthe caulivora and their active extracts and compound 1. *: P < 0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P < 0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid are positive controls. Cpd: compound.

[0042] Fig.17 Melanin inhibition effect of solid and liquid fermentation products of Diaporthe caulivora and their active extracts. *: P < 0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P < 0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were used as positive controls.

[0043] Fig.18 Melanin inhibition effect of compounds 1-9 isolated from solid-state fermentation of Diaporthe caulivora. *: P < 0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P < 0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojicacid are positive controls. Cpd: Compound.

[0044] Fig.19 Melanin inhibition effect of solid and liquid fermentation products of Diaporthe caulivora and their active extracts and compound 1. *: P < 0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P < 0.01 indicates a significant difference from the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid are positive controls.

[0045] Fig. 20 Effects of compound 1 on tyrosinase and tyrosinase-related proteins. Arbutin (1 mM) and kojic acid (1 mM) were used as positive control groups.

[0046] Fig.21 .UVB phototoxicity evaluation of compound 1. ##P<0.01 indicates a significant difference from the control group without UVB irradiation. **P<0.01 indicates a significant difference from the control group with UVB irradiation. DETAILED DESCRIPTION

[0047] The present invention may be implemented in different forms and is not limited to the examples mentioned in the following text. The following examples are only representative of the different aspects and features of the present invention.

[0048] Example 1. Preparation process of solid and liquid fermentation products of Diaporthe caulivora and their active extract layers. Cut the leaves of Litsea cubeba into pieces of about 7×7 mm using a scalpel. 2 A total of 15 pieces were placed in a centrifuge tube and sterilized with 95% alcohol for 30 seconds, then surface sterilized with 3.5% NaClO for 2 minutes, and then sterilized with 95% alcohol for 30 seconds, drained on filter paper, and directly placed on MEA (malt extract agar) medium (2% malt extract medium), cultured at room temperature, and after mycelium grew out of the tissue, separated and purified, the mycelium tip was cut and transplanted on the MEA plate, and stored after culturing at 25°C for 14 days. All isolated strains were frozen and stored, and the mycelium and agar were cut into 0.5 cm 2 Transfer several pieces of different sizes to a cryovial containing 0.5 mL of antifreeze protectant and store at -80°C. Take them out and reactivate them when needed for experiments. Plant II was used to extract DNA from fungal hyphae. The ITS1 / ITS4 primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) was used for PCR reaction to amplify the ITS1-5.8S-ITS2 sequence (SEQ ID NO: 3), which was closest to Diaporthe caulivora in the sequence analysis results on NCBI GenBank.

[0049] After washing the rice, soak it in 0.2% Haibao (alkali enhancer) and 0.2% tartaric acid overnight, filter out excess water, pack it into 100g per can, sterilize it under high temperature and high pressure, and add 15ml of nutrient solution (0.2% Haibao, 0.2% NH4Cl, 0.2% yeast extract) before inoculation to serve as a solid culture medium for Diaporthe caulivora. When inoculating Diaporthe caulivora in the solid culture medium, place a piece of bacteria that has grown on a 9 cm plate in 100ml sterile water and break it up, inoculate 10ml per can, stir evenly, and then culture it at 25°C in the dark for 14 days. The liquid culture medium is prepared by adding 30g of corn starch (after adding corn starch, add 2.5% α-amylase and microwave heating to gelatinize the starch), 10g of corn steep liquor, 5g of yeast extract, and 2g of hipo to 1.0L of deionized water (pH=6), and then dividing it into 150mL / 500mL conical flasks. When inoculating Diaporthe caulivora in the liquid culture medium, place a piece of bacteria that covers a 9-cm plate in 100ml of sterile water and break it up. Then inoculate 10mL into the liquid culture medium in a sterile operating room. After inoculation, place it on an oscillator (100rpm) in a 25°C culture room and culture it for 14 days. The extraction process of Diaporthe caulivora solid fermentation product (DC-S) can be referred to Figure 1 ; The extraction process of Diaporthe caulivora liquid fermentation product (DC-L) can be referred to Figure 2 .

[0050] Solid-state fermentation products Figure 1 As shown, DC-S was soaked in ethanol for three days, twice in total, and the extract after soaking was concentrated under reduced pressure to obtain an ethanol extract (DC-S-EtOH, 45.0 g). After liquid-liquid partitioning (liquid-liquid partition) using water and ethyl acetate (v / v, 1:1), different polarity extraction layers were obtained. Figure 2As shown, DC-L and ethyl acetate (v / v, 1:1) were subjected to liquid-liquid partition to obtain extraction layers of different polarities.

[0051] Example 2: Analysis of the extracts of solid and liquid fermentation products of Diaporthe caulivora and their activity

[0052] Solid fermentation product part: DC-S (1.4 kg) was soaked in 2000 ml ethanol at room temperature for two times for three days each time. The extract after soaking was concentrated under reduced pressure to obtain 45.0 g ethanol extract (DC-S-EtOH). After liquid-liquid partition using water and ethyl acetate (v / v, 1:1), a water-soluble layer (DC-SW, 11.0 g) and an ethyl acetate-soluble layer (DC-S-EA, 1.1 g) were obtained.

[0053] Liquid fermentation product part: DC-L (900 ml) and ethyl acetate (v / v, 1:1) were subjected to liquid-liquid partition to obtain a water-soluble layer (DC-LW, 6.1 g) and an ethyl acetate-soluble layer (DC-L-EA, 147.3 mg).

[0054] Example 3, Diaporthe caulivora solid fermentation products contained in the analysis of compounds reference Figure 3 The solid fermentation product of Diaporthe caulivora was soaked in 95% ethanol for three days each time for a total of three times. The extract after soaking was concentrated under reduced pressure to obtain an ethanol extract. The components were separated and purified by column chromatography, and a total of 18 compounds were obtained (reference Figure 4), including 11 new compounds: caulivotrioloxin A (compound 1), caulivotrioloxin B (compound 2), caulivotrioloxin E (compound 5), caulivotrioloxin D (compound 6), caulibysin A (compound 9), caulivotrioloxin C (compound 10), caulivotrioloxin F (compound 11), diapopyrone (compound 12), caulibysin B (compound 13), diapophthalide A (compound 14), diapophthalide B (compound 15); a compound isolated from natural origin for the first time: 3-O-desmethylphomentrioloxin (compound 3); and six known compounds: phomentrioloxin (compound 4), de-O-methyldiaporthin (compound 7), adenosine (compound 8), mellein (compound 16), palmitic acid (compound 17), and ergosterol peroxide (compound 18).

[0055] Example 4: Safety test of solid and liquid fermentation extracts and compounds of Diaporthe caulivora (DC) on human keratinocyte cell lines

[0056] 1. DC extract layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 were prepared in culture medium, and dimethyl sulfoxide reagent was used as a control group. The DC extract test concentrations were 30 and 300 μg / mL, and the compound test concentrations were 10 and 50 μM, respectively.

[0057] 2. Human keratinocyte cell line (HaCaT cells) were seeded in 96-well plates, with 1x 10 4 cells and wait for the cells to attach to the plate.

[0058] 3. Add 10 μL of 0.2 mg / mL fluorescent dye (Resazurin reagent) 2 hours before different time points (24, 48, and 72 hours), place in an incubator containing 5% CO2 and a temperature of 37°C, and detect its fluorescence (ex / em: 530nm / 590nm) and absorbance (570nm and 600nm) to determine the cell survival rate.

[0059] Example 5. Analysis of the safety results of human keratinocyte cell lines of solid and liquid fermentation extracts and compounds of Diaporthe caulivora. This experiment used a human keratinocyte line (HaCaT cell line) to test whether the DC extract and compounds 1-9 had an effect on cell growth. The results showed that compared with the 24-hour control group, the DC extracts (DC-L-EA, DC-S-EA, DC-SW) at concentrations of 30 μg / mL and 300 μg / mL had a trend of increasing cell growth over time within 48 and 72 hours, compared with the 24-hour control group. When comparing different samples at the same time, these three extracts had no significant effect on cell growth (reference Figure 5 ).

[0060] Figure 6 Compared with the 24-hour control group, the growth of cells in the control group without any treatment increased over time after 48 and 72 hours when the concentrations of 10 μM and 50 μM compounds 1-9 were compared. Compounds 1-9 also showed this trend, indicating that the compounds had no significant effect on cell growth.

[0061] Example 6: Photoprotection test of solid and liquid fermentation extracts and compounds of Diaporthe caulivora

[0062] 1. DC extract layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 were prepared in culture medium, and dimethyl sulfoxide reagent was used as a control group. The test concentrations of plant endophyte extracts were 30 and 300 μg / mL, respectively, and the test concentrations of compounds were 10 and 50 μM.

[0063] 2. Human keratinocytes (HaCaT cell line) were seeded in 96-well plates, with 1x 10 4 cells and wait for the cells to attach to the well plate.

[0064] 3. After the cells attach, add the prepared test substances - DC extraction layer and compound 1-9, and culture for 6 hours.

[0065] 4. After the time is up, the culture medium of the DC extraction layer and compound 1-9 was half-dry, and the culture medium remaining in the 96-well plate was washed with 1x phosphate buffered saline (PBS). After washing, 100 μL of 1x phosphate buffered saline was added to the 96-well plate, and the energy was 40 mJ / cm 2 The culture medium containing the DC extraction layer and compounds 1-9 was added to the 96-well plate and cultured for 24 hours.

[0066] 5. After the plate is treated, the irradiation energy is 40mJ / cm 2 After irradiation with ultraviolet light, the culture medium containing the DC extraction layer and compound 1-9 was added to the well plate and cultured for 24 hours.

[0067] 6. Add 10 μL of 0.2 mg / mL Resazurin reagent 2 hours before 24 hours, place in an incubator containing 5% carbon dioxide and a temperature of 37°C, and wait for the fluorescence (ex / em: 530nm / 590nm) and absorbance (570nm and 600nm) to detect the cell survival rate.

[0068] Example 7. Analysis of the photoprotective effects of solid and liquid fermentation extracts and compounds of Diaporthe caulivora This experiment used human keratinocytes to evaluate the survival rate of cells irradiated with ultraviolet light, and tested whether the DC extract and compounds 1-9 had a photoprotective effect on ultraviolet light. All data were compared with the control group at 0 hours and without any treatment. The growth of cells in the control group without any treatment after 24 hours tended to increase with time, and the survival rate of cells cultured for 24 hours after irradiation with ultraviolet light decreased due to damage from ultraviolet light. The protective effects of the DC extract and compounds 1-9 on cells were compared, and most of the different DC extracts had a protective effect at concentrations of 30 μg / mL and 300 μg / mL, among which DC-S-EA had the best effect (reference Figure 7 Among compounds 1-9 at 10 and 50 μM, except for compound 1 which had poor protective effect on cells damaged by ultraviolet rays, most of the compounds had protective effects, especially compounds 7, 8, and 9 which had the best protective effects (reference Figure 8 ).

[0069] Example 8: Effect of solid and liquid fermentation extracts and compounds of Diaporthe caulivora on inhibition of ultraviolet-induced reactive oxygen species

[0070] 1. DC extract layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 were prepared in culture medium, and dimethyl sulfoxide reagent was used as a control group. The concentrations of plant endophyte extracts were 30 and 300 μg / mL and the concentrations of the tested compounds were 10 and 50 μM.

[0071] 2. Human keratinocytes were seeded in 96-well plates, with 1x10 cells per well. 4 cells and wait for the cells to attach to the well plate.

[0072] 3. After the cells attach, add the prepared test substances - DC extraction layer and compound 1-9, and culture for 6 hours.

[0073] 4. After the time is up, the culture medium containing DC extract or its compound is half-dry, and the culture medium remaining in the 96-well plate is washed with 1x phosphate buffered saline, and then aspirated out, and then the fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is added to the 96-well plate, and placed in an incubator containing 5% carbon dioxide and a temperature of 37°C, away from light for 30 minutes.

[0074] 5. When the time is up, aspirate the 2',7'-dichlorodihydrofluorescein diacetate, wash it once with 100 μL of 1x phosphate buffered saline, aspirate it after washing, and add 100 μL of 1x phosphate buffered saline to the 96-well plate. Use energy 40 mJ / cm 2 The cells were irradiated with ultraviolet light and their fluorescence (ex / em: 485nm / 530nm) was detected to determine the content of reactive oxygen species in the cells.

[0075] Example 9 Analysis of the inhibitory effect of solid and liquid fermentation extracts and compounds of Diaporthe caulivora on reactive oxygen species This experiment used human keratinocytes to test whether DC extracts (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 would inhibit reactive oxygen species induced by ultraviolet rays in cells. All data were compared with the control group without ultraviolet irradiation. The fluorescence value of reactive oxygen species after ultraviolet irradiation was induced by ultraviolet rays, and the fluorescence value of reactive oxygen species after ultraviolet irradiation was increased by 40mJ / cm 2 The fluorescence value of was used as a reference to compare the inhibitory effects of DC extract layer and compounds 1-9 on intracellular reactive oxygen species. Among them, DC-S-EA, DC-SW and most of the compounds have inhibitory effects on intracellular reactive oxygen species (reference Fig. 9 ), among which compounds 7, 8, and 9 had the best inhibitory effects on intracellular reactive oxygen species (reference Fig.10 ).

[0076] Example 10. Inhibitory effect of Diaporthe caulivora extract (DC-S-EA) on UV-induced cyclooxygenase-2 (COX-2) protein expression: Western blot analysis

[0077] 1. Prepare DC-S-EA in dimethyl sulfoxide reagent. The DC extract test concentrations are 30 and 300 μg / mL. Human keratinocyte cell line cells were cultured at 2 x 10 6 The cells were seeded in a 10-cm culture dish, and DC extract was added after the cells attached and cultured for 6 hours.

[0078] 2. Aspirate half of the culture medium containing DC extract, wash the remaining culture medium once with 1x phosphate buffered saline, add 7mL of 1x phosphate buffered saline after aspirating, and use energy 20mJ / cm 2 After ultraviolet irradiation, add the culture medium containing DC-S-EA to the culture dish again, place it in an incubator containing 5% carbon dioxide and a temperature of 37°C and wait for 12 hours.

[0079] 3. After the culture medium was dried, 350 μL of radioimmunoprecipitation assay buffer (RIPA reagent) was added and the cells were scraped off and placed in a microcentrifuge tube (eppendrof) and centrifuged at 15,000 rpm at 4°C for 10 minutes. The supernatant was aspirated and placed in a new microcentrifuge tube and stored in a -80°C refrigerator.

[0080] 4. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-page) at 80 volts and transferred to a nitrocellulose membrane at 0.35 amperes for 3 hours.

[0081] 5. After the transfer is completed, soak the membrane in 5% skim milk and shake it until the next day.

[0082] 6. Wash with 1x tris (hydroxymethyl) aminomethane buffer (TBS buffer) for 10 minutes and repeat three times.

[0083] 7. Add the primary antibody of the target protein and keep at 4°C on a shaker until the next day.

[0084] 8. Wash with 1x Tris buffer for 10 minutes and repeat three times. Add secondary antibody and place on shaker for 1 hour. Wash with 1x Tris buffer for 10 minutes and repeat three times.

[0085] 9. Add 1 mL of developer to the nitrocellulose membrane, spread it evenly, and place it in a cold light imager to take pictures and store the experimental results for image quantification and analysis.

[0086] Example 11 Analysis of the inhibitory effect of Diaporthe caulivora extract (DC-S-EA) on ultraviolet-induced cyclooxygenase-2 (COX-2) protein expression In this experiment, Western blot analysis was used to investigate the inhibitory effect of DC solid-state fermentation ethyl acetate extract (DC-S-EA) on ultraviolet-induced cyclooxygenase-2 (COX-2) reaction. Compared with the 12-hour control group, the DC solid-state fermentation ethyl acetate extract (DC-S-EA) was induced by ultraviolet light. 2 The expression of cyclooxygenase-2 increased after UVB irradiation. Under the action of DC-S-EA at different concentrations of 30 μg / mL, the expression of cyclooxygenase-2 decreased, indicating that DC-S-EA inhibited the inflammatory response induced by UV rays (ref. Fig.11 ), and the effect of 30μg / mL was better than that of 300μg / mL, indicating that low concentrations can be effective.

[0087] Example 12 Safety test of Diaporthe caulivora solid and liquid fermentation extracts and compounds on mouse melanoma cells

[0088] 1. Prepare DC extract layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 in culture medium, use dimethyl sulfoxide reagent as control group, DC extract test concentration is 300 μg / mL, and compound test concentration is 50 μM.

[0089] 2. Mouse melanoma cells (B16-F10 cells) were seeded in 24-well plates, with 5 x 10 4 cells and wait for the cells to attach to the well plate.

[0090] 3. Add 10 μL of 0.2 mg / mL Resazurin reagent, place in an incubator containing 5% CO2 and a temperature of 37°C for 2 hours, and detect its fluorescence (ex / em: 530nm / 590nm) and absorbance (570nm and 600nm) to obtain the cell survival rate.

[0091] Example 13. Analysis of the safety results of solid and liquid fermentation extracts and compounds of Diaporthe caulivora on mouse melanoma cells This experiment used mouse melanoma cells (B16-F10 cells) to test whether DC extracts and compounds 1-9 had an effect on cell growth. Compared with the control group without any treatment, the results showed that the DC extracts (DC-L-EA, DC-S-EA, DC-SW) at a concentration of 300 μg / mL and the compounds 1-9 at a concentration of 50 μM had no significant effect on cell growth (reference Fig.12 and Fig.13 ).

[0092] Example 14: Inhibitory Effects of Solid and Liquid Fermentation Extracts and Compounds of Diaporthe caulivora on Tyrosinase

[0093] 1. Seed 5x10 cells in a 24-well plate. 4 To each mouse melanoma cell, 100 μl of α-melanocyte-stimulating hormone (α-MSH) (50 nM) was added to induce melanin production, and the cells were cultured in an incubator at 37° C. with 5% CO 2 for 24 hours to induce melanin production.

[0094] 2. Remove the culture matrix, add the prepared DC extraction layer (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9, and culture in an incubator for 48 hours. The concentration of the DC extraction layer is 30 and 300 μg / mL, and the concentration of the compound test is 50 μM.

[0095] 3. After the time is up, the cells are washed twice with 1x phosphate-buffered saline (500 μl per well), and then 100 μl trypsin and 500 μl 1x phosphate-buffered saline are added to collect the cells, and the cells are centrifuged at 10,000 rpm for five minutes at room temperature.

[0096] 4. Remove the supernatant, add cell tissue lysis buffer (containing phosphate buffered saline, triton X-100 and 1 mM phenylmethanesulfonylfluoride or phenylmethylsulfonylfluorid; PMSF) and mix well, react at 4°C for 30 minutes, then centrifuge at 14,000 rpm for 10 minutes, add 1 mg / ml levodopa (L-DOPA) prepared in phosphate buffered saline, react at 37°C for 3 hours, and aspirate 180 μL into a 96-well plate.

[0097] 5. Use a full-spectrum disk scanner to observe the changes in the absorbance value of the test object at a wavelength of 490nm to obtain the inhibition rate.

[0098] Example 15 Analysis of the tyrosinase inhibitory effects of solid and liquid fermentation extracts and compounds of Diaporthe caulivora This experiment used mouse melanoma cells (B16-F10 cell line) to test the tyrosinase inhibitory effects of DC extracts (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9. Compared with the control group induced by melanocyte stimulating hormone, DC-S-EA and DC-L-EA had good tyrosinase inhibitory effects (reference Fig.14 ), compound 1 had the best activity and exhibited tyrosinase inhibition effects comparable to those of the commercially available whitening agent kojic acid at 10, 50, and 100 μM (reference Fig.15 and Fig.16 ).

[0099] Example 16: Inhibitory Effects of Solid and Liquid Fermentation Extracts and Compounds of Diaporthe caulivora on Melanin

[0100] 1. Inoculate 1x10 cells in a 12-well plate. 5 500 μl of α-melanocyte stimulating hormone (50 nM) was added to each mouse melanoma cell and cultured in an incubator at 37° C. with 5% CO 2 for 24 hours to induce melanin production.

[0101] 2. Remove the culture matrix, add the prepared DC extraction layer (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9, and culture in an incubator for 48 hours. The concentration of the DC extraction layer is 30 and 300 μg / mL, and the concentration of the compound test is 50 μM.

[0102] 3. Add 200 μl of trypsin and 500 μl of 1x phosphate buffered saline, centrifuge at 3,000 rpm for eight minutes at room temperature, remove the supernatant, add 100 μl of 2N sodium hydroxide (NaOH) and mix well, place in a 95°C oven for 15 minutes, take out and place in a 96-well plate, and measure its absorbance at a wavelength of 405 nm.

[0103] Example 17 Analysis of the inhibitory effect of solid and liquid fermentation extracts and compounds of Diaporthe caulivora on melanin This experiment used mouse melanoma cells (B16-F10 cell line) to test the melanin inhibitory effect of DC extracts (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9. Compared with the control group induced by melanocyte stimulating hormone, DC-L-EA had a good melanin inhibitory effect (reference Fig.17), compounds 1 and 9 have the effect of inhibiting melanin, among which compound 1 has the best activity and has a melanin inhibitory activity close to the effect of the commercially available whitening agent kojic acid at a concentration of 100 μM (reference Fig.18 and Fig.19 ).

[0104] Example 18 Effect of Caulivotrioloxin A (Compound 1) on Tyrosinase and Tyrosinase-related Proteins: Western Blot Analysis

[0105] 1. Mouse melanoma cells were cultured at 2 x 10 6 The cells were plated in 10 cm culture dishes, α-melanocyte stimulating hormone (50 nM) was added, and the dishes were cultured in an incubator at 37° C. with 5% CO 2 for 24 hours to induce melanin production.

[0106] 2. Remove the culture medium, add the prepared test medium of compound 1 (concentrations of 5, 10, 50, 100 μM), place in an incubator and culture for 48 hours.

[0107] 3. After the culture medium was dried, 350 μL of radioimmunoprecipitation assay buffer (RIPA reagent) was added and the cells were scraped off and placed in a microcentrifuge tube (Eppendorf) and centrifuged at 15,000 rpm at 4°C for 10 minutes. The supernatant was aspirated and placed in a new microcentrifuge tube and stored in a -80°C refrigerator.

[0108] 4. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-page) at 80 volts and transferred to a nitrocellulose membrane at 0.35 amperes for 3 hours.

[0109] 5. After the transfer is completed, soak the membrane in 5% skim milk and shake it until the next day.

[0110] 6. Wash with 1x Tris buffer for 10 minutes and repeat three times.

[0111] 7. Add the primary antibody of the target protein and keep at 4°C on a shaker until the next day.

[0112] 8. Wash with 1x Tris buffer for 10 minutes and repeat three times. Add secondary antibody and place on shaker for 1 hour. Wash with 1x Tris buffer for 10 minutes and repeat three times.

[0113] 9. Add 1 mL of developer to the nitrocellulose membrane, spread it evenly, and place it in a cold light imager to take pictures and store the experimental results for image quantification and analysis.

[0114] Example 19, Analysis of the Effect of Caulivotrioloxin A (Compound 1) on Tyrosinase and Tyrosinase-related Proteins This experiment used Western blot analysis to explore the related pathways of Compound 1 on tyrosinase and tyrosinase-related proteins. Melanin is derived from tyrosine, and tyrosinase is an enzyme required for the melanin production pathway, which is divided into two pathways. Tyrosinase-related protein-1 (TRP-1) is related to the production of eumelanin, while tyrosinase-related protein-2 (TRP-2) is related to the production of pheomelanin. Fig. 20 A It can be observed that compound 1 has the effect of inhibiting the expression of tyrosinase protein at concentrations of 10, 50, and 100 μM. Fig. 20 B shows that compound 1 at concentrations of 10 and 50 μM can inhibit the expression of tyrosinase-related protein-1 (TRP-1) protein, while compound 1 at concentrations of 50 and 100 μM can slightly inhibit the expression of tyrosinase-related protein-2 (TRP-2) protein (reference Fig. 20 C), the experimental results show that the inhibitory effect of compound 1 on melanin may be due to its inhibition of tyrosinase and tyrosinase-related protein-1 protein expression.

[0115] Example 20. UVB Phototoxicity Effect of Caulivotrioloxin A (Compound 1)

[0116] 1. Compound 1 was prepared in dimethyl sulfoxide reagent. The low and high concentrations of compound 1 tested were 10 and 50 μM, respectively.

[0117] 2. Human keratinocyte cell line (HaCaT cells) were seeded in 96-well plates, with 1x 10 4 cells and wait for the cells to attach to the well plate.

[0118] 3. After the cells attach, add the prepared test compound 1 and culture for 6 hours.

[0119] 4. After the time is up, the culture medium containing compound 1 was half-dry, and the culture medium remaining in the 96-well plate was washed once with 1X PBS, and then aspirated out, and 100 μL of 1x phosphate buffered saline was added to the 96-well plate, and the energy was 40 mJ / cm 2 of UVB radiation.

[0120] 5. After irradiation, the culture medium without compound 1 was added to the 96-well plate and cultured for 24 hours. Two hours before the 24th hour, 10 μL of 0.2 mg / mL Resazurin reagent was added and placed in an incubator containing 5% CO2 and a temperature of 37°C to detect its fluorescence (ex / em: 530nm / 590nm) and absorbance (570nm and 600nm) to determine the cell survival rate.

[0121] Example 21 Analysis of the Phototoxicity Effect of Caulivotrioloxin A (Compound 1) on UVB From the above Examples 12-19, it can be confirmed that Compound 1 has the effect of inhibiting melanin and tyrosinase. In order to evaluate whether the use of Compound 1 and then exposure to light will produce phototoxicity, this experiment was conducted. This experiment used human keratinocytes (HaCaT cells) to test whether Compound 1 adds UVB to the cell damage effect. Fig.21 The results showed that compound 1 (10 μM) had no additive effect with UVB and did not cause aggravated cell damage, indicating its photosafety. Although 50 μM had an effect, it was not significant. In the future, it is recommended to use it with sunscreen or at night when manufacturing products.

[0122] Those skilled in the art will quickly appreciate that the present invention can easily achieve the objectives and obtain the results and advantages mentioned, as well as those that exist therein. The compounds, compositions, extracts and mixtures of the present invention, their manufacturing processes and methods and their uses are representatives of preferred embodiments, which are exemplary and not limited to the field of the present invention. Those skilled in the art will think of modifications and other uses therein. These modifications are all contained in the spirit of the present invention and defined in the claims. The content description and embodiments of the present invention are disclosed in detail so that those skilled in the art can make and use the present invention, even if there are various changes, modifications, and improvements therein, it should still be considered that they do not depart from the spirit and scope of the present invention.

[0123] All patents and publications mentioned in this specification are prior art in the field relevant to the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0124] The inventions properly exemplified herein may be practiced in the absence of any element, or any number of elements, limitations, or limitations not specifically disclosed herein. The terms and expressions used are descriptive and not limiting, and there is no intention to use such terms and expressions to exclude any equivalent features or portions thereof shown and described, but it should be recognized that various changes may occur within the claims of the present invention. Therefore, it should be understood that although the present invention has been specifically disclosed according to the preferred embodiments and any features, those skilled in the art will still modify and change the contents disclosed therein, and such modifications and changes are still within the scope of the patent application of the present invention.

Claims

1. A use of a Diaporthe caulivora extract for preparing a sunscreen and whitening composition, wherein the Diaporthe caulivora has a deposit number of DSM33674 in DSMZ, and wherein the Diaporthe caulivora extract comprises the compound caulibysin A.

2. The use according to claim 1, wherein the sunscreen and whitening composition is a composition having an anti-ultraviolet damage effect.

3. The use according to claim 1, wherein the sunscreen and whitening composition is a composition having a pigmentation reducing effect.

4. The use according to claim 1, wherein the sunscreen and whitening composition is used to prevent or treat discomfort or disease or photodamage caused by excessive exposure to ultraviolet rays.

5. The use according to claim 4, wherein the disorder or disease is hyperpigmentation or skin aging.

6. The use according to claim 4, wherein the photodamage state is erythema, scaling, edema, thickness change, or any combination thereof.

7. The use according to claim 1, wherein the sunscreen and whitening composition is used to prevent or treat discomfort or diseases caused by excessive pigmentation, including freckles, chloasma, stretch marks, and age spots.

8. A compound caulibysin A isolated from an extract of Diaporthe caulivora, wherein the deposit number of Diaporthe caulivora in DSMZ is DSM33674.

9. A composition comprising the compound according to claim 8.

10. The composition according to claim 9, which has an effect of preventing damage by ultraviolet rays.

11. The composition of claim 9, which has a pigmentation reducing effect.

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