TW202220683A - Use of diaporthe caulivora extract for anti-uv damage and reducing pigmentation - Google Patents

Abstract

The present invention provides the use of Diaporthe caulivora extract for preparing a composition for anti-ultraviolet damage and reducing hyperpigmentation. The present invention also provides a novel compound isolated from the extract of Diaporthe caulivora, and a composition comprising the compound.

Description

Diaporthe caulivora extract for UV protection and pigmentation reduction Uses of Lake

The present invention relates to the use of an extract of Diaporthe caulivora in preparing a composition for anti-ultraviolet damage and reducing pigmentation.

Ultraviolet (UV) in solar radiation has a great impact on the skin of living organisms. Excessive UV exposure will not only cause the skin to darken, but also make the skin surface absorb ultraviolet rays to generate free radicals; and if there are too many free radicals accumulated in the body, it will produce free radicals. Oxidative stress, unbalanced regulation of the defense system, leads to cellular damage. Although some natural substances have been proved to have the effect of ultraviolet protection and melanin inhibition by research, they are almost plant-derived extracts or compositions; the developers of microorganisms used in skin care products are currently mostly ectopic bacteria (such as Ganoderma lucidum, fungus). application, little research has been done on endophytes.

Diaporthe caulivora is a species of Diaporthe caulivora, which is a plant pathogen and often infects soybean. According to the review of past literature, it is found that its chemical composition and biological activity have not been thoroughly studied.

Although the previous literature has confirmed 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 Shell fungus ( Diaporthe caulivora ) extract can be used to inhibit melanin production or reduce pigmentation.

In view of the imperfections of the known technology, the inventor has finally conceived the case "The use of extracts from Diaporthe caulivora for anti-ultraviolet damage and pigment reduction after careful experimentation and research with perseverance The use of precipitation” can overcome the shortcomings of the prior art, the following is a brief description of the case.

Unless specifically defined otherwise, terms used in this description shall have their ordinary and common meanings understood by those skilled in the art.

The invention isolates the endophyte: Diaporthe caulivora from the leaves of Neolitsea daibuensis , and develops it into a composition with anti-ultraviolet damage and reducing pigmentation. The composition can prevent the skin from being damaged by ultraviolet rays, has the effect of whitening the skin, and can be used alone or as an active additive for medicines, cosmetics, or skin care products. The Diaporthe caulivora of the present invention has been deposited with the Food Industry Development Research Institute of Taiwan on October 21, 2020, and the deposit number is BCRC 930224. In the present invention, the solid-state fermentation product and the liquid-state fermentation product obtained by the solid-state and liquid-state fermentation of Diaporthe caulivora respectively are used as experimental objects, and the solid-state fermentation product is soaked in 95% ethanol and concentrated under reduced pressure to obtain an ethanol extract, Compound 1-18 was obtained after separation and purification by column chromatography. Among them, compounds 1, 2, 5, 6, 9, 10, 11, 12, 13, 14 and 15 are novel compounds that have not been disclosed in the previous case, and compound 3 is a compound isolated from natural sources for the first time. Cell safety test was carried out on the extract of soybean Diaporthe caulivora and the 18 compounds, and it was confirmed that it was safe to cells. The photoprotection test showed that the extract of soybean Diaporthe caulivora had ultraviolet rays Protective effect, and can suppress the rise of active oxygen species caused by ultraviolet radiation. In addition, in the melanin inhibition test, it was found that the extract of Diaporthe caulivora can inhibit tyrosinase and inhibit melanin, so that it has a good effect of reducing pigmentation. The activity test was carried out on the compounds isolated from the extract of Diaporthe caulivora , and it was found that these compounds have anti-ultraviolet light damage effect, and also have the effect of inhibiting tyrosinase and melanin. The present invention confirms that the extract of Diaporthe caulivora and its secondary metabolites have anti-ultraviolet damage and pigmentation reducing activities, and the experimental results can be used for whitening, sun protection, and anti-aging in cosmetics or skin care products and other related industries Product development and development and promotion, or used in pharmaceuticals to prevent or treat hyperpigmentation, skin aging and other discomforts or diseases caused by excessive exposure to ultraviolet rays.

Therefore, the present invention provides the use of an extract of Diaporthe caulivora for preparing a sunscreen and whitening composition. The term "sunscreen and whitening composition" used in the present invention refers to a composition with anti-ultraviolet damage and pigmentation reducing effects. In one embodiment, the sunscreen and whitening composition can protect cells to reduce UV damage, inhibit the rise of reactive oxygen species caused by UV rays, and inhibit the production of tyrosinase and melanin. The aforementioned sunscreen and whitening compositions can be used alone as pharmaceuticals, cosmetics, or skincare products, or as active additives for medicines, cosmetics, or skincare products (such as lotions, essences, lotions, and sunscreens, etc.). The aforementioned sunscreen and whitening compositions can be used to prevent or treat discomfort or disease (such as hyperpigmentation or skin aging) or photodamage conditions (such as erythema, scaling, edema, thickness changes, sunburn, suppression of immune responses, induction of tumorigenesis, or any combination thereof), and for the prevention or treatment of disorders or diseases caused by hyperpigmentation, including but not limited to freckles, chloasma, Striae of pregnancy, senile plaque, and melanoma. It can be seen from the above that the sunscreen and whitening composition of the present invention not only has the effect of sunscreen and whitening, but also has the effect of resisting skin aging, and can be used as an active additive in sunscreen, whitening, anti-skin aging products or sunscreen, whitening, and anti-skin aging products. In one embodiment, the sunscreen and whitening composition is used for preparing external cosmetics or skin preparations. In one embodiment, the extract of Diaporthe caulivora is an extract obtained by extracting the solid state fermentation of Diaporthe caulivora with a solvent, and the solvent can be an organic solvent, including but not limited to Ethanol. In another embodiment, the Diaporthe caulivora extract is an extraction obtained by performing liquid-liquid partition of a solvent and a liquid-liquid fermentation of Diaporthe caulivora The solvent can 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, diapophthalide B.

The present invention also provides a compound isolated from the extract of Diaporthe caulivora 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, diapophthalide B.

The present invention further provides a composition comprising at least one of the aforementioned compounds. In one embodiment, the composition has anti-ultraviolet damage effect. In another embodiment, the composition has a pigmentation reducing effect.

The composition of the present invention can be formed into cream, ointment, lotion, lotion, serum, Appearance such as paste or mousse. If desired, it may be applied to the skin via an aerosol form, or in a solid form such as a stick. The composition of the present invention can also be used in any form of topical formulation of pharmaceuticals, skin care products, or cosmetics, especially aqueous, hydro-alcoholic or oil-containing solutions, oil-soluble or water-soluble oil or multiple emulsions , Aqueous or oily gel, liquid, paste or solid anhydrous product. Those skilled in the art will choose adjuvants carefully. The types and amounts of adjuvants are considered according to the properties of the compounds or extracts of the present invention, and will not or substantially not be adversely affected by the contemplated additions.

The composition of the present invention may also contain adjuvants commonly used in skin care, cosmetic 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, etc. The amounts of these various adjuvants are those conventional in the field under consideration, eg, 0.01 to 20% by weight of the total composition. Depending on their nature, these adjuvants can be delivered into the fat phase, the aqueous phase, the lipid vesicles.

When the composition of the present invention is an emulsion, the proportion of the fatty phase may range from 5 to 80% by weight, eg, from 5 to 50% by weight, relative to the total weight of the composition. The oils, emulsifiers and co-emulsifiers, etc. used in the present compositions in emulsion form are selected from those conventionally used in the field under consideration. The ratio of emulsifier and co-emulsifier present in the composition ranges from 0.3 to 30% by weight, eg, from 0.5 to 20% by weight, relative to the total weight of the composition.

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

The raw materials of emulsifier and co-emulsifier that can be used in the present invention can be, 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 the hydrophilic gelling agent that can be used in the present invention can be, for example, carboxyvinyl polymers, carbomers, acrylic copolymers of acrylate/alkyl acrylate copolymers, polypropylene amide, polyamide Sugar, natural gums, and clay. The raw materials of the lipophilic gelling agent that can be used in the present invention can be, for example, modified clay of bentonite, metal salts of fatty acids, hydrophobic silica, and polyethylene.

Active agents that can be used in the present invention can be, for example, polyols, vitamins, keratolytic and/or exfoliating tablets, anti-inflammatory agents, calmants and mixtures thereof, depigmenting agents (such as koji acid), and the like derivative. Lipophilic or hydrophilic UV-screening agents, such as benzene-1,4-bis(3-methylene-10-camphorsulfonic acid), 2-ethylhexyl α -cyano, can also be used in the compositions of the present invention - β,β-diphenylacrylic acid or octocrylen, butylmethoxydibenzoylmethane, octyl methoxycinnamate and/or titanium oxide and zinc oxide.

Cosmetic powders useful in the present invention typically 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 such as Orgasol™ ( Nylon) powder, polyethylene powder, Teflon™, starch, boron nitride, lauryl lysine, copolymer microspheres, cross-linked polymethacrylate copolymer, and polysiloxane microspheres, and their analog.

[Fig. 1] The preparation process of the solid-state fermented extract 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 state fermentation of Diaporthe caulivora ; DC-S-EA is ethyl acetate dissolved layer; DC-SW is water dissolved layer

[Fig. 2] The preparation process of the liquid fermented extract of Diaporthe caulivora and its active extract layer. Solvent A is water; solvent B is ethyl acetate. Liquid-liquid partition was performed between the liquid fermentation product and ethyl acetate (v/v, 1:1) to obtain DC-L-EA as an ethyl acetate dissolved layer; DC-LW as a water dissolved layer.

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

[Fig. 4] The compound structure diagram of Diaporthe caulivora .

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

[Fig. 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.

[Fig. 7] Photoprotective effects of solid and liquid fermentation products of Diaporthe caulivora and their active extract layers on cells damaged by ultraviolet rays (concentrations of 30 and 300 μg/mL). ##: P<0.01 means there is a significant difference with the control group not irradiated with ultraviolet light; **: P<0.01 means there is a significant difference with the control group irradiated with ultraviolet light; Qu (quercetin) and atRA (all-trans retinoic acid) for the positive control group.

[Fig. 8] Photoprotective effect of solid-state fermentation products of Diaporthe caulivora ( Diaporthe caulivora ) on cells damaged by ultraviolet rays (concentrations of 10 and 50 μM). ##: P<0.01 means there is a significant difference with the control group not irradiated with ultraviolet light; **P<0.01 means there is a significant difference with the control group irradiated with ultraviolet light; Qu(quercetin) and atRA(all-trans retinoic acid) are positive control group.

[Fig. 9] Inhibitory effects of the solid and liquid fermentation products of Diaporthe caulivora and their active extract layers on intracellular reactive oxygen species induced by ultraviolet rays (concentrations of 30 and 300 μg/mL). ##: P<0.01 means there is a significant difference with the control group not irradiated with ultraviolet light; **: P<0.01 means there is a significant difference with the control group irradiated with ultraviolet light; Qu (quercetin) and atRA (all-trans retinoic acid) for the positive control group.

[Fig. 10] Inhibitory effect of compounds 1-9 isolated from solid-state fermentation of Diaporthe caulivora on UV-induced intracellular reactive oxygen species (concentrations of 10 and 50 μM). ##: P<0.01 means there is a significant difference with the control group not irradiated with ultraviolet rays; **: P<0.01 means there is a significant difference with the control group irradiated with ultraviolet rays. Qu (quercetin) and atRA (all-trans retinoic acid) were positive control groups.

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

[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 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups. Cpd: Compound.

[Fig. 13] Safety analysis of solid and liquid fermentation products of Diaporthe caulivora and its active extract layer 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 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups. Cpd: Compound.

[Fig. 14] Tyrosinase inhibitory effect of solid and liquid fermentation products of Diaporthe caulivora and its active extract layer. *: P<0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P<0.01 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups.

[Fig. 15] Tyrosinase inhibitory 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 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups. Cpd: Compound.

[Fig. 16] Tyrosinase inhibitory effect of Diaporthe caulivora solid and liquid fermentation products and their active extract layers and compound 1. [Fig. *: P<0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P<0.01 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups. Cpd: Compound.

[Fig. 17] Melanin inhibitory effect of solid and liquid fermentation products of Diaporthe caulivora and its active extract layer. *: P<0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P<0.01 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups.

[Fig. 18] The melanin inhibitory effect of compounds 1-9 isolated from the 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 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups. Cpd: Compound.

[Fig. 19] The melanin inhibitory effect of the solid and liquid fermentation products of Diaporthe caulivora and its active extract layer and compound 1. [Fig. *: P<0.05 indicates a difference from the control group containing α-melanocyte stimulating hormone inducer. **: P<0.01 means there is a significant difference with the control group containing α-melanocyte stimulating hormone inducer. Arbutin and kojic acid were the positive control groups.

[Fig. 20] The effect of compound 1 on tyrosinase and tyrosinase-related proteins. Arbutin (1 mM) and kojic acid (1 mM) were positive controls.

[FIG. 21] Evaluation of UVB phototoxicity of compound 1 . ##P<0.01 indicates a significant difference from the control group not irradiated with UVB.

The present invention may be embodied in different forms and is not limited to the examples mentioned in the following text. The following examples are merely representative of various aspects and features of the present invention.

Example 1. Process for preparing extracts of solid and liquid fermented products of Diaporthe caulivora and their active extract layers

Use a scalpel to cut the leaves of Dawu Xinmu Jiangzi into 15 pieces of about 7 × 7 mm ² in total, put them in a centrifuge tube and sterilize them with 95% alcohol for 30 seconds, then sterilize the surface with 3.5% NaClO for 2 minutes, and then sterilize them with 95% alcohol. % alcohol disinfection for 30 seconds, drained on filter paper, directly placed on MEA (malt extract agar) medium (2% malt extract medium), cultured at room temperature, and separated after the mycelium grew from the tissue After purification, the hyphae tips were cut and transplanted on MEA plates, and then stored at 25°C for 14 days. All the isolated strains were cryopreserved. The mycelium and agaric were cut into 0.5cm ² pieces, transferred to a cryovial containing 0.5mL anti-freeze protectant, and stored at -80°C until needed for experiments. Remove to reactivate. Strain identification: After the strain was left to stand at room temperature for 7 to 14 days, the DNA of the fungal hyphae was extracted with NucleoSpin ^® Plant II. The ITS1/ITS4 primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) was selected for PCR reaction, and the ITS1-5.8S-ITS2 sequence (SEQ ID NO: 3) was amplified, which is the closest to the sequence analysis results on NCBI GenBank Diaporthe caulivora .

After washing the rice, soak it with 0.2% Haibao (alkalizing agent) and 0.2% tartaric acid overnight, filter out excess water, divide into 100g cans, sterilize at high temperature and high pressure, add 15ml of nutrient solution (0.2% Haibao, 0.2% NH ₄ Cl, 0.2% yeast extract) as a solid medium for Diaporthe caulivora . When inoculating Diaporthe caulivora in solid medium, place a 9 cm plate of bacteria in 100 ml of sterile water and smash it, inoculate 10 ml per tank and stir evenly, and then incubate at 25°C in the dark. 14 days. The liquid medium was prepared by adding 30 g of corn starch (after adding corn starch, adding 2.5% α-amylase, microwave heating to gelatinize the starch), corn dipping solution (corn dipping solution) into 1.0 L of deionized water (pH=6). Steep liquor) 10g, yeast extract 5g, hipo (hipo) 2g, it was divided into 150mL/500mL conical flask. When inoculating Diaporthe caulivora in the liquid medium, place a 9 cm plate of the bacteria in 100ml of sterile water and crush it, then inoculate 10mL into the liquid medium in a sterile operating room, and place it on the Incubate for 14 days on a shaker (100 rpm) in an incubation chamber at 25°C. Refer to Figure 1 for the extraction process of Diaporthe caulivora solid fermented product (DC-S) and Figure 2 for the extraction process of Diaporthe caulivora liquid fermented product (DC-L).

The solid-state fermentation product part is shown in Figure 1. DC-S was soaked in ethanol for three days for a total of two times. After soaking, the extract was concentrated under reduced pressure to obtain an ethanol extract (DC-S-EtOH, 45.0 g). Water and ethyl acetate (v/v, 1:1) were subjected to liquid-liquid partition to obtain extraction layers of different polarities. The liquid fermented product is shown in Figure 2. DC-L and ethyl acetate (v/v, 1:1) were subjected to liquid-liquid partition to obtain extraction layers of different polarities.

Example 2. Analysis of the extraction layer results of the extracts of the solid and liquid fermentation products of Diaporthe caulivora and their activity

Solid state fermentation product: DC-S (1.4kg) was soaked in 2000ml of ethanol at room temperature for two times for three days each time, and the extract after soaking was concentrated under reduced pressure to obtain 45.0g of ethanol extract (DC-S -EtOH), water and ethyl acetate (v/v, 1:1) were used for liquid-liquid partition to obtain a water-soluble layer (DC-S-W, 11.0 g) and an ethyl acetate-dissolved layer (DC-S-W, 11.0 g) -S-EA, 1.1 g).

Liquid fermentation part: DC-L (900ml) and ethyl acetate (v/v, 1:1) were subjected to liquid-liquid partition to obtain a water-soluble layer (DC-L-W, 6.1g) and acetic acid Ethyl ester dissolved layer (DC-L-EA, 147.3 mg).

Example 3. Analysis of compounds contained in solid-state fermentation of Diaporthe caulivora

Referring to Figure 3 , the solid-state fermentation product of Diaporthe caulivora was soaked in 95% ethanol for three days for three times. The extract after soaking was concentrated under reduced pressure to obtain an ethanol extract, which was subjected to column chromatography. The components were separated and purified, and a total of 18 compounds were obtained (refer to 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 natural Compounds isolated for the first time: 3- O -desmethyl phomentrioloxin ( 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 ).

Example 4. Cell safety test of human keratinocyte strain of Diaporthe caulivora solid and liquid fermented extract layers and compounds

1. Configure DC extraction layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 in the culture medium, with dimethyl sulfite reagent as the control group, the test concentrations of DC extracts are respectively were 30 and 300 μg/mL, and compounds were tested at concentrations of 10 and 50 μM, respectively.

2. Seed the human keratinocyte cell line (HaCaT cells) in a 96-well plate, each well is 1 x 10 ⁴ cells, and wait for the cells to adhere to the well plate.

3. After 2 hours at different time points (24, 48, and 72 hours), add 10 μL of 0.2 mg/mL fluorescent dye (Resazurin reagent), and place in a culture containing 5% CO ₂ at a temperature of 37 °C. Wait in the box, and detect its fluorescence (ex/em: 530nm/590nm) and absorbance (570nm and 600nm) to obtain the cell viability.

Example 5. Analysis of the safety results of human keratinocyte strain cells of the solid and liquid fermented extract layers and compounds of Diaporthe caulivora

In this experiment, a human keratinocyte line (HaCaT cell line) was used to test whether the DC extract layer and compounds 1-9 had an effect on cell growth. The results showed that the DC extract layers (DC-L-EA, DC-S-EA, DC-SW) with a concentration of 30 μg/mL and 300 μg/mL did not have any changes in 48 and 72 hours compared with the 24-hour control group. The growth of cells in the treated control group tended to increase with time. When comparing different samples at the same time, the three extraction layers had no significant effect on cell growth (refer to Figure 5 ).

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

Example 6. Photoprotection test of solid and liquid fermented extract layers and compounds of Diaporthe caulivora

1. Configure DC extraction layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 in the medium, and use dimethyl sulfite reagent as the control group. Extracts were tested at 30 and 300 μg/mL, while compounds were tested at 10 and 50 μM, respectively.

2. Seed the human keratinocyte line (HaCaT cell line) in a 96-well plate with 1 x 10 ⁴ cells in each well, and wait for the cells to adhere to the well plate.

3. After the cells are attached, add the prepared analyte-DC extraction layer and compound 1-9 , and incubate for 6 hours.

4. After the time is up, the DC extraction layer and the culture solution of compounds 1-9 are half-dried, and then the culture solution remaining in the 96-well plate is washed with 1 times phosphate buffered saline (PBS), and then washed. After aspirating, add 100 μL of 1-fold phosphate-buffered saline into a 96-well plate, and irradiate with ultraviolet rays with an energy of 40 mJ/cm ² . The culture medium containing DC extraction layer and compound 1-9 was added again to the 96-well plate, and cultured for 24 hours.

5. After the well plate is treated, irradiate with ultraviolet rays with an energy of 40 mJ/cm ² , and after irradiation, add the culture medium containing the DC extraction layer and compounds 1-9 into the well plate, and cultivate for 24 hours.

6. Add 10 μL of 0.2 mg/mL Resazurin reagent 2 hours before 24 hours, and place it in an incubator containing 5% carbon dioxide at a temperature of 37°C to wait for the detection of its fluorescence (ex/em: 530nm/590nm). ) and absorbance values (570 nm and 600 nm) to obtain cell viability.

Example 7. Analysis of photoprotection results of solid and liquid fermented extract layers and compounds of Diaporthe caulivora

In this experiment, human keratinocyte cell lines were used to evaluate the viability of UV-irradiated cells and to test whether the DC extract layer and compounds 1-9 had UV photoprotective effects. Compared with the control group at 0 hours without any treatment, the growth of cells in the control group without any treatment after 24 hours has a trend of increasing with time, and the cells cultured for 24 hours after exposure to UV light Survival was reduced by UV damage. Comparing the protective effects of DC extraction layer and compounds 1-9 on cells, and different DC extraction layers at the concentration of 30μg/mL and 300μg/mL, most of them have protective effects, among which DC-S-EA has the most effect. good (refer to Figure 7 ). Among compounds 1-9 at 10 and 50 μM, except compound 1, which has a poor protective effect on cells damaged by ultraviolet rays, most of the compounds have protective effects, especially compounds 7, 8, and 9 have the most protective effect. good (refer to Figure 8 ).

Example 8. Inhibitory effect of the solid and liquid fermented extract layers and compounds of Diaporthe caulivora on UV-induced reactive oxygen species

1. Configure DC extraction layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 in the medium, and use dimethyl sulfite reagent as the control group. Extract concentrations were 30 and 300 μg/mL and compounds were tested at concentrations of 10 and 50 μM.

2. Seed the human keratinocyte strain in a 96-well plate, each well containing 1×10 ⁴ cells, and wait for the cells to adhere to the well plate.

3. After the cells are attached, add the prepared surrogate-DC extraction layer and compound 1-9 , and incubate for 6 hours.

4. After the time is up, the culture medium containing DC extract or its compounds is half-dried, and the culture medium remaining in the 96-well plate is washed with 1x phosphate buffered saline, washed and sucked out, and then the fluorescent dye is added. 2',7'-dichlorodihydroluciferin diacetic acid (H ₂ DCFDA) was placed in a 96-well dish and placed in an incubator containing 5% carbon dioxide at a temperature of 37° C. for 30 minutes in the dark.

5. When the time is up, aspirate 2',7'-dichlorodihydrofluorescein diacetic acid, wash once with 100 μL of 1-fold phosphate buffered saline, and aspirate after washing, and then add 100 μL of 1-fold phosphoric acid. Salt-buffered saline was placed in a 96-well plate, irradiated with ultraviolet light with an energy of 40 mJ/cm ² , and its fluorescence (ex/em: 485 nm/530 nm) was detected to obtain the content of reactive oxygen species in cells.

Example 9. Analysis of the Inhibitory Effect of Diaporthe caulivora Solid and Liquid Fermentation Extracts and Compounds on Reactive Oxygen Species

In this experiment, human keratinocyte cell lines were used to test whether DC extract layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 could inhibit the intracellular reactive oxygen species induced by ultraviolet light. All data are compared with the control group without UV irradiation. The fluorescence value of reactive oxygen species after UV irradiation is induced by UV light, and the fluorescence value of UV irradiated 40mJ/cm ² is used as the benchmark to compare DC. Inhibitory effect of 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 (refer to Figure 9 ), among which compounds 7, 8, and 9 have the best inhibitory effect on intracellular reactive oxygen species ( Refer to Figure 10 ).

Example 10. Inhibitory effect of Diaporthe caulivora extract (DC-S-EA) on UV-induced cyclooxygenase-2 (COX-2) protein expression: Western blotting method

1. Prepare DC-S-EA in dimethyl sulfite reagent, and the test concentration of DC extract is 30 and 300 μg/mL. Human keratinocyte line cells were seeded at 2 x 10 ⁶ in a 10 cm petri dish, and DC extract was added for 6 hours after the cells were attached.

2. Suck the culture medium containing DC extract half dry, wash the residual culture medium once with 1 times phosphate buffered saline, and then add 7 mL of 1 times phosphate buffered saline after aspirating, and use the energy of 20mJ/cm ² . After ultraviolet irradiation, the culture medium containing DC-S-EA was added again to the culture dish, and it was placed in an incubator containing 5% carbon dioxide at a temperature of 37 °C for 12 hours.

3. Add 350 μL of radioimmunoprecipitation assay buffer (RIPA reagent) and scrape off the cells, place them in a microcentrifuge tube (eppendrof) and centrifuge at 15,000 rpm for 10 minutes at 4°C. , the supernatant was aspirated and placed in a new microcentrifuge tube and stored in a -80°C refrigerator.

4. Use sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-page) to perform protein separation electrophoresis at 80 volts, and transfer to nitrocellulose membrane at 0.35 ampere for 3 hours ( Blotting membrane).

5. After the transfer, soak the membrane in 5% skim milk and shake it until the next day.

6. Washing with 1-fold tris(hydroxymethyl)aminomethane buffer (tris(hydroxymethyl)aminomethane; TBS buffer) for 10 minutes was repeated three times.

7. Add primary antibody to target protein on shaker and keep at 4°C until the next day.

8. Wash with 1x Tris buffer for 10 minutes and repeat three times, add secondary antibody and place on a shaker for 1 hour, and wash with 1x Tris buffer for 10 minutes and repeat three times.

9. Add 1 mL of developer on the nitrocellulose membrane, disperse it evenly, and put it into a luminescent imager to take pictures and store the experimental results for image quantification and analysis.

Example 11. Analysis of inhibitory effect of Diaporthe caulivora extract (DC-S-EA) on UV-induced cyclooxygenase-2 (COX-2) protein expression

In this experiment, the western blot method was used to investigate the inhibitory effect of DC solid-state fermentation ethyl acetate extraction layer (DC-S-EA) on UV-induced cyclooxygenase-2 (COX-2) reaction. Compared with the control group for 12 hours, the expression of cyclooxygenase-2 increased after being irradiated by UVB with an energy of 20 mJ/cm ² . Under the action of DC-S-EA at different concentrations of 30 μg/mL, the performance of cyclooxygenase-2 The decrease indicates that DC-S-EA can inhibit the inflammatory response induced by ultraviolet light (refer to Figure 11 ), and the effect of 30 μg/mL is better than that of 300 μg/mL, indicating that low concentrations can be effective.

Example 12. Safety test on mouse melanoma cells of Diaporthe caulivora solid and liquid fermented extract layers and compounds

1. Configure DC extraction layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 in the culture medium, take dimethyl sulfite reagent as the control group, and the test concentration of DC extract is 300 μg/mL, while compounds were tested at a concentration of 50 μM.

2. The mouse melanoma cells (B16-F10 cells) were seeded in a 24-well plate, each well was 5 x 10 ⁴ cells, and the cells were allowed to adhere to the well plate.

3. Add 10 μL of 0.2 mg/mL Resazurin reagent, place it in an incubator with 5% CO ₂ and a temperature of 37°C for 2 hours, and detect its fluorescence (ex/em: 530nm/590nm) and absorbance values (570nm and 600nm) to obtain cell viability.

Example 13. Analysis of the safety results of the solid and liquid fermentation products of Diaporthe caulivora and the mouse melanoma cell safety results of the compounds

Mouse melanoma cells (B16-F10 cells) were used in this experiment to test whether DC extract layers and compounds 1-9 had an effect on cell growth. Compared with the control group without any treatment, the results showed that the DC extract layer (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 effect on the cells. Growth was not significantly affected (see Figures 12 and 13 ).

Example 14. Inhibitory effect of tyrosinase by solid and liquid fermented extract layers and compounds of Diaporthe caulivora

1. Inoculate 5× ^{10 4} mouse melanoma cells in a 24-well plate, add 100 μl of α-melanocyte-stimulating hormone (α-MSH) (50nM) to induce melanin production, and place at 37 Incubate for 24 hours at °C in an incubator containing 5% CO2 to induce melanin production.

2. Remove the culture medium, add the prepared DC extraction layer (DC-L-EA, DC-S-EA, DC-SW) and compound 1-9 , and place it in an incubator for 48 hours. The concentrations were 30 and 300 μg/mL, while the concentration of the compounds tested was 50 μM.

3. After the time is up, wash the cells twice with 1x phosphate-buffered saline (500 μl per well), then add 100 μl of trypsin and 500 μl of 1x phosphate-buffered saline to collect cells and store at room temperature. Centrifuge at 10,000 rpm for five minutes.

4. Remove the supernatant and add a cell tissue lysis buffer (including phosphate buffered saline, polyethylene glycol octyl phenyl ether (triton X-100) and 1 mM benzyl) Sulfonyl fluoride (phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluorid; PMSF)) was mixed evenly, reacted at 4°C for 30 minutes, then centrifuged at 14,000 rpm for 10 minutes, and added Levodopa at 1 mg/ml in phosphate-buffered saline; L-DOPA), react at 37°C for 3 hours, and aspirate 180 μL into a 96-well plate.

5. Use a full-spectrum disc scanner to observe the change of the absorbance value of the analyte at a wavelength of 490 nm to obtain the inhibition rate.

Example 15. Analysis of tyrosinase inhibitory effect of solid and liquid fermented extract layers and compounds of Diaporthe caulivora

In this experiment, mouse melanoma cells (B16-F10 cell line) were used to test the tyrosinase inhibitory effect of DC extract layers (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 have good tyrosinase inhibitory effect (refer to Figure 14 ), and compound 1 has the best activity, and it has the best activity at 10, 50, and 100 μM exhibited a tyrosinase inhibitory effect comparable to that of a commercial whitening agent, kojic acid (refer to FIG. 15 and FIG. 16 ).

Example 16. Inhibitory effect of the solid and liquid fermented extract layers and compounds of Diaporthe caulivora on melanin

1. Seed 1x10 ⁵ mouse melanoma cells in a 12-well dish, add 500 μl of α-melanocyte stimulating hormone (50 nM), and place in a 37°C, 5% CO ₂ incubator for 24 hours to induce Melanin production.

2. Remove the culture medium, add the prepared DC extraction layer (DC-L-EA, DC-S-EA, DC-SW) and compound 1-9 , and place it in an incubator for 48 hours. Concentrations were 30 and 300 μg/mL, while compounds were tested at a concentration of 50 μM.

3. Add 200 μl trypsin and 500 μl 1x phosphate buffered saline, centrifuge at 3,000 rpm for 8 minutes at room temperature, remove the supernatant, add 100 μl 2N sodium hydroxide (NaOH), mix well, and place at 95°C The oven was heated for 15 minutes, and the 96-well plate was taken out, and its absorbance value was measured at a wavelength of 405 nm.

Example 17. Analysis of the inhibitory effect of Diaporthe caulivora solid and liquid fermentation extract layers and compounds on melanin

In this experiment, mouse melanoma cells (B16-F10 cell line) were used to test the melanin inhibitory effect of DC extract layers (DC-L-EA, DC-S-EA, DC-SW) and compounds 1-9 . Compared with the control group induced by melanocyte stimulator, DC-L-EA has a good melanin inhibitory effect (refer to Figure 17 ), compound 1 and compound 9 have melanin inhibitory effect, and compound 1 has the best activity at a concentration of 100 μM , has a melanin inhibitory activity close to the effect of the commercially available whitening agent kojic acid (refer to Figure 18 and Figure 19 ).

Example 18. The effect of compound 1 (Caulivotrioloxin A) on tyrosinase and tyrosinase-related proteins: Western blotting method

1. Murine melanoma cells were seeded at 2 x 10 ⁶ in a 10 cm petri dish, added with α -melanocyte stimulating hormone (50 nM), and placed at 37°C in an incubator containing 5% CO ₂ for 24 hours. Induce melanin production.

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

3. Add 350 μL of radioimmunoprecipitation assay buffer (RIPA reagent) and scrape off the cells, place them in a microcentrifuge tube (Eppendorf) and centrifuge at 15,000 rpm for 10 minutes at 4°C. , the supernatant was aspirated Store in a -80°C freezer in a new microcentrifuge tube.

4. Use sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-page) to perform protein separation electrophoresis at 80 volts, and transfer to nitrocellulose membrane at 0.35 ampere for 3 hours ( Blotting membrane).

5. After the transfer, soak the membrane in 5% skim milk and shake it until the next day.

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

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

8. Wash with 1x Tris buffer for 10 minutes and repeat three times, add secondary antibody and place on a shaker for 1 hour, and wash with 1x Tris buffer for 10 minutes and repeat three times.

9. Add 1 mL of developer on the nitrocellulose membrane, disperse it evenly, and put it into a luminescent imager to take pictures and store the experimental results for image quantification and analysis.

Example 19. Analysis of the effect of compound 1 (Caulivotrioloxin A) on tyrosinase and tyrosinase-related proteins

In this experiment, the Western blotting method was used to explore the related pathways of compound 1 to tyrosinase and tyrosinase-related proteins. Melanin is derived from tyrosine, and tyrosinase is the pathway of melanin production. The enzymes required for the production of t tyrosinase related protein; TRP-2) is related to the production of pheomelanin. While Figure 20A shows that Compound 1 has the effect of inhibiting the expression of tyrosinase protein at concentrations of 10, 50 and 100 μM, and Figure 20B shows that Compound 1 can inhibit tyrosinase-related protein- 1 (TRP-1) protein expression, and compound 1 at concentrations of 50 and 100 μM can slightly inhibit the expression of tyrosinase-related protein-2 (TRP-2) protein (refer to Figure 20C ), the compound 1 can be known from the experimental results. 1 The inhibitory effect on melanin may be due to its inhibition of tyrosinase and tyrosinase-related protein-1 protein expression.

Example 20. UVB phototoxicity of compound 1 (Caulivotrioloxin A)

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

2. Seed the human keratinocyte cell line (HaCaT cells) in a 96-well plate, each well is 1 x 10 ⁴ cells, and wait for the cells to adhere to the well plate.

3. After the cells are attached, add the prepared analyte compound 1 and culture for 6 hours.

4. After the time is up, half-dry the culture medium containing compound 1 , wash the culture medium remaining in the 96-well plate once with 1X PBS, aspirate after washing, and then add 100 μL of 1X phosphate-buffered saline to 96 In the well plate, UVB irradiation with an energy of 40 mJ/cm ² was used.

5. After irradiation, add the culture medium without compound 1 to the 96-well plate, incubate for 24 hours, and add 10 μL of 0.2 mg/mL Resazurin reagent 2 hours before the 24 hours, and place it at a temperature containing 5% CO ₂ . The cell viability was obtained by waiting to detect its fluorescence (ex/em: 530nm/590nm) and absorbance (570nm and 600nm) in an incubator at 37°C.

Example 21. Analysis of the phototoxicity effect of compound 1 (Caulivotrioloxin A) 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 will cause phototoxicity when irradiated with light, this experiment was carried out. In this experiment, human keratinocyte cell line (HaCaT cells) was used to test whether compound 1 added UVB to damage cells. Figure 21 shows that Compound 1 (10 μM) has no additive effect with UVB, and does not cause aggravating damage to cells, indicating its photosafety. 50 μM had an effect, but it was not significant. It is recommended to use with sunscreen or at night when making products in the future.

One skilled in the art will quickly appreciate that the present invention readily accomplishes its objectives and obtains the ends and advantages mentioned, as well as those contained therein. The compounds, compositions, extracts and mixtures of the present invention, procedures and methods for their manufacture, and uses thereof are representative of preferred embodiments, which are exemplary and not limited to the field of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are all contained in the spirit of the present invention and are defined in the scope of the patent application. The content description and embodiments of the present invention are disclosed in detail, so that any person skilled in the art can make and use the present invention, even if there are various changes, modifications, and advancements therein, it should still be regarded as not departing from the present invention. spirit and scope.

All patents and publications mentioned in the specification are subject to the general art in the field related 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.

Inventions suitably exemplified herein may be practiced in the absence of any element, or many elements, limitations, or limitations not specifically disclosed herein. The terms and expressions used are intended to be descriptive rather than restrictive in the description, and there is no intention to use such terms and expressions to exclude any features or parts equivalent to those shown and described, but it should be understood that in this Various changes are possible within the scope of the patent application for the invention. Therefore, it should be understood that although the present invention has been specifically disclosed in terms of preferred embodiments and any features, it is well known that 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.

<110> Kaohsiung Medical University Institute of Food Industry Development

<120> Use of Diaporthe caulivora extract for anti-ultraviolet damage and reducing pigmentation

<130> 3477-KMU-TW

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Claims (10)

Use of a Diaporthe caulivora extract for preparing a sunscreen and whitening composition. The use according to claim 1, wherein the sunscreen and whitening composition is a composition with anti-ultraviolet damage effect. The use according to claim 1, wherein the sunscreen and whitening composition is a composition having the effect of reducing pigmentation. The use of claim 1, wherein the sunscreen whitening composition is for preventing or treating a condition or disease or photodamage state caused by overexposure to ultraviolet light, wherein the condition or disease is hyperpigmentation or skin aging, and wherein The photodamage state is erythema, scaling, edema, thickness change, sunburn, suppressed immune response, tumorigenesis, or any combination thereof. The use according to claim 1, wherein the sunscreen and whitening composition is used to prevent or treat discomfort or disease caused by hyperpigmentation, including freckles, chloasma, striae of pregnancy, Senile plaque, and melanoma. The use of claim 1, wherein 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, diapophthalide B. A compound isolated from the extract of Diaporthe caulivora 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, diapophthalide B. A composition comprising at least one compound as claimed in claim 7. The composition of claim 8, which has the effect of resisting UV damage. The composition of claim 8, which has the effect of reducing pigmentation.

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