CN116509909A - Use of soybean mesotheca extract for resisting ultraviolet injury and reducing pigmentation - Google Patents
Use of soybean mesotheca extract for resisting ultraviolet injury and reducing pigmentation Download PDFInfo
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- CN116509909A CN116509909A CN202310317717.8A CN202310317717A CN116509909A CN 116509909 A CN116509909 A CN 116509909A CN 202310317717 A CN202310317717 A CN 202310317717A CN 116509909 A CN116509909 A CN 116509909A
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
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Abstract
The invention provides a use of an extract of soybean chaetomium (Diaporthe caulivora) for preparing a composition for resisting ultraviolet injury and reducing pigmentation. The invention also provides a novel compound isolated from an extract of soybean chaetomium (Diaporthe caulivora), and compositions comprising the same.
Description
The application is a divisional application of an invention patent application with the application number of 202011344831.2 and the application date of 2020, 11 and 26, and the invention name of the application of the soybean mesotheca extract for resisting ultraviolet injury and reducing pigmentation.
Technical Field
The present invention relates to the use of an extract of chaetomium sojae (Diaporthe caulivora) for the preparation of a composition for the anti-uv damage and pigmentation reduction.
Background
Ultraviolet (UV) in solar radiation has a great influence on the skin of organisms, and excessive UV radiation can cause the skin to become black and cause the skin surface layer to absorb UV to generate free radicals; however, if excessive radicals accumulate in the body, oxidative stress is generated, so that the defense system is not regulated in a balanced manner, and the cells are damaged. Although some natural substances have been studied to demonstrate the effect of protecting against ultraviolet rays and inhibiting melanin, they are almost extracts or compositions of plant origin; regarding the application of microorganisms to the application of the maintenance product developers, most of the application of the microorganisms to the exogenous bacteria (such as ganoderma lucidum and edible fungus) is performed, and the research on the endogenous bacteria is very little.
The soybean mesotheca (Diaporthe caulivora) is a strain of the family mesotheca, which is a plant pathogen and commonly infects soybeans; according to the past literature review, the chemical components and biological activities of the compounds have not been thoroughly studied.
While the prior literature has demonstrated anti-inflammatory activity of the soybean chaetomium (Diaporthe caulivora), it has not been demonstrated that the soybean chaetomium (Diaporthe caulivora) extract can be used to prevent skin damage caused by ultraviolet radiation, nor that the soybean chaetomium (Diaporthe caulivora) extract can be used to inhibit melanogenesis or reduce pigmentation.
The inventor aims at the defects of the prior art, through careful experiments and researches, and finally designs the application of the soybean chaetomium (Diaporthe caulivora) extract in resisting ultraviolet injury and reducing pigmentation with the help of the mind of being inexhaustible, and can overcome the defects of the prior art, and the following is a brief description of the application.
Disclosure of Invention
Unless specifically defined otherwise, the terms used in this description will have the ordinary and customary meaning as known to those skilled in the art.
Endophytes are isolated from the leaves of the large Wu Xinmu ginger (Neolitsea daibuensis): the soybean chaetomium (Diaporthe caulivora) is developed into a composition with the effects of resisting ultraviolet injury and reducing pigmentation, can prevent skin from being damaged by ultraviolet rays, has the effect of whitening skin, and can be used singly or as an active additive of medicines, cosmetics or maintenance products. The soybean alternaria alternata of the invention, which is classified and named as soybean alternaria alternata (Diaporthe caulivora), is deposited in German biological Depositary (DSMZ) on the 10 th month 23 th year 2020, and has the deposit number: DSM33674.
The invention takes solid state fermentation products and liquid state fermentation products obtained by respectively solid state and liquid state fermentation of soybean backset fungus (Diaporthe caulivora) as experimental targets, the solid state fermentation products are soaked in 95% ethanol, the ethanol extract is obtained after decompression concentration, and the compounds 1-18 are obtained after separation and purification by column chromatography. Wherein compounds 1, 2, 5, 6, 9, 10, 11, 12, 13, 14 and 15 are novel compounds, and compound 3 is a compound isolated from a natural material for the first time. The extract of soybean alternaria alternata (Diaporthe caulivora) and the 18 compounds were subjected to a cell safety test, and the cell safety was confirmed, and the extract of soybean alternaria alternata (Diaporthe caulivora) was shown to have an ultraviolet protective effect in the photo-protective test, and to suppress the increase of active oxygen species due to ultraviolet irradiation. In addition, in a melanin inhibiting test, the extract of the soybean chaetomium (Diaporthe caulivora) has the effects of inhibiting tyrosinase and inhibiting melanin, so that the extract has good effect of reducing pigmentation. The compounds separated from the soybean mesotheca (Diaporthe caulivora) extract are subjected to an activity test, and the compounds are found to have an ultraviolet injury resistance effect and also have the effect of inhibiting tyrosinase and melanin. The invention proves that the soybean-based seashell (Diaporthe caulivora) extract and the secondary metabolite thereof have the activities of resisting ultraviolet injury and reducing pigmentation, and the experimental result can be used for the development and popularization of whitening, sun-screening and anti-aging products in cosmetics, maintenance products and other related industries, or used for medicines for preventing or treating discomfort or diseases such as hyperpigmentation, skin aging and the like caused by overexposure to ultraviolet rays.
Accordingly, the present invention provides the use of an extract of chaetomium sojae (Diaporthe caulivora) for the preparation of a sunscreen whitening composition. The term "sunscreen whitening composition" as used herein refers to a composition having an anti-uv effect and a pigmentation reducing effect. In one embodiment, the sunscreen and whitening composition protects cells from uv damage, inhibits uv-induced increases in reactive oxygen species, and inhibits tyrosinase and melanin production. The sunscreen and whitening composition can be used as a medicine, a cosmetic or a care product alone or as an active additive of a medicine, a cosmetic or a care product (such as toning lotion, essence, emulsion, sun cream and the like). The aforementioned sunscreen and whitening composition may be used for preventing or treating discomfort or disease caused by excessive exposure to ultraviolet rays (such as hyper-pigmentation, skin aging, etc.) or photodamage state (such as erythema, scaling, edema, thickness change, sunburn, immune response inhibition, tumorigenesis (tumourigenesis), or any combination thereof), and for preventing or treating discomfort or disease caused by hyper-pigmentation, including but not limited to freckle (freckle), chloasma (chloasma), striae gravidarum (striae of pregnancy), senile plaque (senile), and melanoma (melanoma). As can be seen from the above, the sun-screening and whitening composition of the present invention has the effect of resisting skin aging in addition to sun-screening and whitening, and can be used as an active additive in sun-screening, whitening and anti-skin aging products or sun-screening, whitening and anti-skin aging products. In one embodiment, the sunscreen whitening composition is used to prepare a cosmetic or skin formulation for external use. In one embodiment, the extract of the soybean chaetomium (Diaporthe caulivora) is obtained by extracting a solid state fermentation product of the soybean chaetomium (Diaporthe caulivora) with a solvent, which may be an organic solvent including but not limited to ethanol. In another embodiment, the extract of the soybean chaetomium (Diaporthe caulivora) is obtained by performing an equal volume liquid-liquid partition (liquid-liquid partition) of a liquid fermentation product of the soybean chaetomium (Diaporthe caulivora) with a solvent, wherein the solvent may be an organic solvent, including but not limited to ethanol. In one embodiment, the extract of chaetomium sojae (Diaporthe caulivora) comprises at least one compound selected from the group consisting of: caulivotrioloxin A, caulivotrioloxin B, caulivotrioloxin C, caulivotrioloxin D, caulivotrioloxin E, caulivotrioloxin F, calibysin A, calibysin B, diapopyrne, diapophalide A, diapophalide B.
The present invention also provides a compound isolated from an extract of chaetomium sojae (Diaporthe caulivora) selected from the group consisting of: caulivotrioloxin A, caulivotrioloxin B, caulivotrioloxin C, caulivotrioloxin D, caulivotrioloxin E, caulivotrioloxin F, calibysin A, calibysin B, diapopyrne, diapophalide A, diapophalide B.
The invention further provides a composition comprising at least one of the foregoing compounds. In one embodiment, the composition has an anti-UV effect. In another embodiment, the composition has the effect of reducing pigmentation.
The composition of the present invention may form the appearance of a cream, ointment, lotion, pack, slurry, paste or mousse. It may be applied to the skin via aerosol form if desired, or may be in solid form, such as in stick form. The compositions of the invention may also be used in any pharmaceutical, cosmetic, care or oil-containing solution, in particular in the form of a topical formulation, in aqueous, aqueous-alcoholic or oily solution, in water or in oil or multiple emulsions, in aqueous or oily gels, in liquid, pasty or solid anhydrous products. The person skilled in the art will be careful to choose the adjuvants, the kind of adjuvants and the amounts thereof being such that the nature of the compounds or extracts according to the invention is considered, without or substantially without adverse effects due to the envisaged addition.
The compositions of the present invention may also contain adjuvants commonly found in cosmetic, dermatological or dermatological preparations, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, sunscreens, pigments, odor absorbers, dyes and the like. The amounts of these different adjuvants are known in the field under consideration, for example: 0.01 to 20% by weight of the total composition. Depending on their nature, these adjuvants can be fed into the fatty phase, the aqueous phase, the fat vesicles.
When the composition of the invention is an emulsion, the proportion of fatty phase may range from 5 to 80% by weight, relative to the total weight of the composition, for example: from 5 to 50% by weight. The oils, emulsifiers, co-emulsifiers and the like used in the present compositions in the form of emulsions are selected from agents known to be used in the field under consideration. The ratio of emulsifier to co-emulsifier present in the composition ranges from 0.3 to 30 wt%, relative to the total weight of the composition, for example: from 0.5 to 20% by weight.
The oil raw material which can be used in the present invention may be, for example, a mineral oil such as liquid petroleum, avocado oil, vegetable oil such as soybean oil, animal oil such as lanolin, synthetic oil, perhydrosqualene (perhydrosqualene), silicone oil, cyclomethicone (cyclomethicone), fluorinated oil, or perfluoropolyether. As the fatty substance, cetyl alcohol, fatty acid, carnauba wax, ceresin wax, etc. can also be used.
The emulsifier and co-emulsifier materials useful in the present invention may be, for example: fatty acid esters of polyethylene glycol (e.g., PEG-20 stearate), and fatty acid esters of glycerol (e.g., glyceryl stearate). Hydrophilic gellant materials useful in the present invention may be, for example: carboxyvinyl polymers, carbomers, acrylic copolymers of acrylate/alkyl acrylate copolymers, polyacrylamides, polysaccharides, natural gums, and clays. The lipophilic gelling agent materials useful in the present invention may be, for example: bentonite modified clay, fatty acid metal salts, hydrophobic silica, and polyethylene.
Active agents useful in the present invention may be, for example: polyols, vitamins, keratolytic and/or descaling tablets, anti-inflammatory agents, sedatives (calmant) and mixtures thereof, depigmenting agents (e.g., kojic acid), and derivatives thereof. Lipophilic or hydrophilic UV screening agents, such as benzene-1, 4-bis (3-methylene-10-camphorsulfonic acid), 2-ethylhexyl alpha-cyano-beta, beta-diphenylacrylic acid or octocrylene (octocrylen), butyl methoxydibenzoylmethane, octyl methoxycinnamate and/or titanium oxide and zinc oxide, may also be used in the compositions of the invention.
Cosmetic powders useful in the present invention typically comprise 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 (Nylon) powder such as Orgasol (TM), polyethylene powder, teflon (TM), starch, boron nitride, lauroyl-lysine, copolymer microspheres, crosslinked polymethacrylate copolymers, silicone beads, and the like.
Description of biological Material preservation
Classification naming: soy backset fungus (Diaporthe caulivora)
Preservation mechanism: german collection of organisms (Leibnitz-institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH)
The preservation organization is abbreviated as: DSMZ
Address: brinz holven street 7B38124 (Inhoffenstra βe7b,38124braunschweig, germany) date of deposit: 2020, 10 and 23 days
Preservation number: DSM33674.
Drawings
FIG. 1. Preparation flow of extract of solid state fermentation product of Soy-bean Chaetomium (Diaporthe caulivora) and active extract layer thereof. Solvent A is water; solvent B is ethyl acetate. DC-S-EtOH is ethanol extract of solid fermentation product of soybean chaetomium (Diaporthe caulivora); DC-S-EA is ethyl acetate dissolved layer; DC-S-W is a water-soluble layer.
FIG. 2. Preparation of extract of liquid fermentation product of Gekko sojae (Diaporthe caulivora) and active extract layer thereof. Solvent A is water; solvent B is ethyl acetate. Liquid-liquid partitioning (liquid-liquid partition) is carried out on the liquid fermentation product and ethyl acetate (v/v, 1:1), and DC-L-EA is obtained as an ethyl acetate dissolved layer; DC-L-W is a water-soluble layer.
FIG. 3A process for the isolation of an ethanol extract of a solid state fermentation of Gekko sojae (Diaporthe caulivora). D is dichloromethane; m is methanol; * Is a new compound; * Are compounds isolated for the first time from natural sources.
FIG. 4 shows the structure of the compound of Gekko sojae (Diaporthe caulivora).
FIG. 5 safety analysis of solid and liquid fermentation products of Aschersonia sojae (Diaporthe caulivora) and their active extract layers at a concentration of 30. Mu.g/mL. (B) Safety analysis of solid and liquid fermentation products of soybean chaetomium (Diaporthe caulivora) and active extract layers thereof at 300 μg/mL.
FIG. 6 safety analysis of (A) Compounds 1-9 isolated from solid state fermentation of Gekko sojae at a concentration of 10. Mu.M (Diaporthe caulivora). (B) Safety analysis of compounds 1-9 isolated from solid state fermentation of chaetomium sojae (Diaporthe caulivora) at a concentration of 50. Mu.M. * P <0.01 indicates a significant difference from the control group irradiated with ultraviolet rays.
FIG. 7 photo-protecting effect of solid and liquid fermentation products of Gekko sojae (Diaporthe caulivora) and its active extract layer on cells injured by ultraviolet rays (concentration of 30 and 300. Mu.g/mL). P <0.01 indicates a significant difference from the control group without uv irradiation; * P <0.01 represents a significant difference from the control group irradiated with ultraviolet rays; qu (quercetin ) and attA (all-trans retinoic acid, all-trans retinoic acid) were positive controls.
FIG. 8 photo-protecting effect of solid state fermentation product compounds 1-9 of Gekko sojae (Diaporthe caulivora) on cells injured by ultraviolet rays (concentrations 10 and 50. Mu.M). P <0.01 indicates a significant difference from the control group without uv irradiation; * P <0.01 represents a significant difference from the control group irradiated with ultraviolet rays; qu (quercetin) and attA (all-trans retinoic acid) were positive controls.
FIG. 9 shows the inhibitory effect of solid and liquid fermentation products of soybean hypocrea (Diaporthe caulivora) and active extract layers thereof on intracellular active oxygen substances induced by ultraviolet rays (concentration: 30 and 300. Mu.g/mL). P <0.01 indicates a significant difference from the control group without uv irradiation; * P <0.01 represents a significant difference from the control group irradiated with ultraviolet rays; qu (quercetin) and attA (all-trans retinoic acid) were positive controls.
FIG. 10 shows the inhibitory effect of compounds 1 to 9 isolated from solid state fermentation of soybean hypocrea (Diaporthe caulivora) on intracellular active oxygen species induced by ultraviolet rays (concentrations of 10 and 50. Mu.M). P <0.01 indicates a significant difference from the control group without uv irradiation; * P <0.01 indicates a significant difference from the control group irradiated with ultraviolet rays. Qu (quercetin) and attA (all-trans retinoic acid) were positive controls.
FIG. 11 inhibition of ultraviolet-induced COX-2 protein expression by ethyl acetate extract layer (DC-S-EA) of solid state fermentation product of Asclepiadaceae (Diaporthe caulivora).
FIG. 12 safety analysis of solid and liquid fermentation products of Gekko sojae (Diaporthe caulivora) and active extract layers (300. Mu.g/mL) and isolated compounds 1-9 (50. Mu.M) in murine melanoma cells. * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups. Cpd, compound.
FIG. 13 safety analysis of solid and liquid fermentation products of Gekko sojae (Diaporthe caulivora), active extract layers thereof and compound 1 in mouse melanoma cells. * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups. Cpd, compound.
FIG. 14 tyrosinase inhibiting effect of solid and liquid fermentation products of Gekko sojae (Diaporthe caulivora) and active extract layers thereof. * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups.
FIG. 15 tyrosinase inhibitory effect of compounds 1-9 isolated from solid state fermentation of Gekko sojae (Diaporthe caulivora). * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups. Cpd: a compound.
FIG. 16 tyrosinase inhibitory effect of solid and liquid fermentation products of Gekko sojae (Diaporthe caulivora), active extraction layers thereof and compound 1. * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups. Cpd, compound.
FIG. 17 melanin inhibiting effects of solid and liquid ferments of Soy-base fungus (Diaporthe caulivora) and active extract layers thereof. * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups.
FIG. 18 melanin inhibiting effects of compounds 1-9 isolated from solid state fermentation of Gekko sojae (Diaporthe caulivora). * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups. Cpd, compound.
FIG. 19 shows the melanin inhibiting effects of solid and liquid fermentation products of Gekko sojae (Diaporthe caulivora), active extract layers thereof, and Compound 1. * P <0.05 indicates a difference from the control group containing the alpha melanocyte stimulating agent. * P <0.01 indicates a significant difference from the control group containing the alpha melanocyte stimulating agent. Arbutin (arbutin) and kojic acid (kojic acid) are positive control groups.
FIG. 20 effects of Compound 1 on tyrosinase and tyrosinase-related proteins. Arbutin (arbutin) (1 mM) and kojic acid (1 mM) were used as positive control groups.
Figure 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 UVB-irradiated control group.
Detailed Description
The invention may be embodied in different forms and is not limited to the examples set forth below. The following examples are merely representative of various aspects and features of the present invention.
EXAMPLE 1 preparation procedure of extract of solid and liquid fermentation products of Phyllostachys sojae (Diaporthe caulivora) and active extract layer thereof the Wu Xinmu ginger leaves were cut to about 7X 7mm using a scalpel 2 Putting 15 pieces in a centrifuge tube, sterilizing with 95% alcohol for 30 seconds, sterilizing with 3.5% NaClO for 2 minutes, sterilizing with 95% alcohol for 30 seconds, draining on filter paper, directly placing on MEA (malt extract agar) culture medium (2% malt extract culture medium), culturing at room temperature, separating and purifying after mycelia grow out of tissue, cutting mycelia tip, transplanting on MEA flat plate, culturing at 25deg.C for 14 days, and preserving. All isolated strains were cryopreserved and mycelia together with the watercress were cut to 0.5cm 2 The large and small blocks are transferred into a freezing tube containing 0.5mL of antifreeze protective agent, stored at-80 ℃, and taken out for reactivation when the experiment needs. And (3) strain identification: the strain was allowed to stand at room temperature for 7 to 14 days toPlant II extracts DNA of fungal hyphae. And (3) selecting an ITS1/ITS4 primer pair (SEQ ID NO:1 and SEQ ID NO: 2) for PCR reaction, amplifying an ITS1-5.8S-ITS2 sequence (SEQ ID NO: 3), wherein the sequence is closest to the sequence analysis result on NCBI GenBank to obtain the soybean chaetomium (Diaporthe caulivora).
Soaking rice in 0.2% sea treasure (alkaline agent) and 0.2% tartaric acid overnight, filtering to remove excessive water, packaging into 100g each tank, sterilizing at high temperature under high pressure, and adding 15ml of nutrient solution (0.2% sea treasure and 0.2% NH) before inoculation 4 Cl, 0.2% yeast extract) as a solid medium for soybean chaetomium (Diaporthe caulivora). Inoculating soybean mesotheca (Diap)The calanivora) was placed in a solid medium, a strain which had grown over a 9 cm plate was broken up in 100ml of sterile water, inoculated with 10ml of each pot, stirred uniformly, and then subjected to stationary culture at 25℃for 14 days under dark conditions. The liquid medium was prepared by adding 30g of corn starch (after adding corn starch, adding 2.5% alpha-amylase, and heating with microwave to gelatinize the starch), 10g of corn steep liquor (corn steep liquor), 5g of yeast extract, and 2g of sea cucumber (hipo) to 1.0L of deionized water (ph=6), and packaging in 150mL/500mL triangular flask. When soybean chaetomium (Diaporthe caulivora) was inoculated to a liquid medium, a strain full of 9 cm plates was broken up by placing it in 100mL of sterile water, then 10mL of the liquid medium was inoculated to a sterile operation chamber, and the culture was carried out on a shaker (100 rpm) of a culture chamber at 25℃for 14 days after inoculation. The extraction process of solid state fermentation product (DC-S) of soybean chaetomium (Diaporthe caulivora) can be referred to in FIG. 1; the extraction process of the liquid fermentation product (DC-L) of the soybean chaetomium (Diaporthe caulivora) can be referred to as figure 2.
The solid state fermentation product is shown in FIG. 1, DC-S is soaked in ethanol for three days, the total of two times, the soaked extract is decompressed and concentrated to obtain ethanol extract (DC-S-EtOH, 45.0 g), and liquid-liquid distribution (liquid-liquid partition) is carried out by using water and ethyl acetate (v/v, 1:1) to obtain extraction layers with different polarities. Liquid fermentation fraction as shown in FIG. 2, DC-L and ethyl acetate (v/v, 1:1) were subjected to liquid-liquid partition (liquid-liquid partition) to obtain extraction layers of different polarities.
EXAMPLE 2 analysis of results of extract layer of solid and liquid fermentation products of Phyllostachys sojae (Diaporthe caulivora) and their Activity
Solid state ferment part: DC-S (1.4 kg) was soaked with 2000ml of ethanol at room temperature twice a three-day period, the soaked extract was concentrated under reduced pressure to give 45.0g of ethanol extract (DC-S-EtOH), and liquid-liquid partition (liquid-liquid partition) was performed using water and ethyl acetate (v/v, 1:1) to give a water-soluble layer (DC-S-W, 11.0 g) and an ethyl acetate-soluble layer (DC-S-EA, 1.1 g).
Liquid ferment part: liquid-liquid partition (liquid-liquid partition) was performed on DC-L (900 ml) and ethyl acetate (v/v, 1:1) to obtain a water-soluble layer (DC-L-W, 6.1 g) and an ethyl acetate-soluble layer (DC-L-EA, 147.3 mg).
Example 3 analysis of Compounds contained in solid state fermentation of Phyllostachys sojae (Diaporthe caulivora) referring to FIG. 3, solid state fermentation of Phyllostachys sojae (Diaporthe caulivora) was soaked with 95% ethanol three times a day, and the soaked extract was concentrated under reduced pressure to obtain ethanol extract, and the ethanol extract was subjected to component separation and purification by column chromatography to obtain 18 compounds (referring to FIG. 4) containing 11 novel compounds: caulivotrioloxin A (compound 1), caulivotrioloxin B (compound 2), caulivotrioloxin E (compound 5), caulivotrioloxin D (compound 6), calibysin a (compound 9), caulivotrioloxin C (compound 10), caulivotrioloxin F (compound 11), diapopyrone (compound 12), calibysin B (compound 13), diapophalide a (compound 14), diapophalide B (compound 15); a compound of first isolation of a natural substance: 3-O-desmethyl phomentrioloxin (compound 3); six known compounds: phomentrioxolin (compound 4), de-O-methylldiaporthin (compound 7), adenosine (compound 8), mellin (compound 16), palmitic acid (compound 17), and ergosterol peroxide (compound 18).
EXAMPLE 4 human keratinocyte cell line cell safety test of solid and liquid fermentation extract layer and Compound of Phyllostachys sojae (Diaporthe caulivora, DC)
1. DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and compounds 1-9 were prepared in a medium, and a dimethylsulfoxide reagent was used as a control group, with DC extract test concentrations of 30 and 300. Mu.g/mL, respectively, and compound test concentrations of 10 and 50. Mu.M, respectively.
2. Human keratinocyte cell lines (HaCaT cells) were seeded in 96-well plates, each well 1x 10 4 Individual cells and to be attached to the plate.
3. 10. Mu.L of 0.2mg/mL fluorescent dye (Resazurin reagent) was added 2 hours before the various time points (24, 48, and 72 hours) and placed in a solution containing 5% CO 2 The cell viability was determined by waiting in an incubator at 37℃and measuring the fluorescence (ex/em: 530nm/590 nm) and absorbance (570 nm and 600 nm) thereof.
Example 5, solid and liquid fermentation extract layer of chaetomium sojae (Diaporthe caulivora) and results of compound analysis of human keratinocyte strain cell safety this experiment uses human keratinocyte strain (HaCaT cell line) to test whether DC extract layer and compounds 1-9 have an effect on cell growth. The results showed that the growth of cells in the control group without any treatment tended to increase with time over 48 and 72 hours compared to the 24-hour control group with DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) at concentrations of 30. Mu.g/mL and 300. Mu.g/mL. These three extracts had no significant effect on cell growth when compared to the time of the different samples (see figure 5).
FIG. 6 shows that compounds 1-9 at concentrations of 10. Mu.M and 50. Mu.M showed a trend of increasing growth over time for the control group without any treatment over 48 and 72 hours as compared to the 24 hour control group. This trend is also true for compounds 1-9, indicating that compounds have no significant effect on cell growth.
EXAMPLE 6 light protection test of solid and liquid fermentation extract layer and Compound of Gekko Swinhonis (Diaporthe caulivora)
1. DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and compounds 1-9 were prepared in a medium, and a dimethylsulfoxide reagent was used as a control group, wherein the test concentrations of endophyte extracts in the plants were 30 and 300. Mu.g/mL, respectively, and the test concentrations of the compounds were 10 and 50. Mu.M, respectively.
2. Human keratinocyte cell lines (HaCaT cell lines) were seeded in 96-well plates, each well being 1x 10 4 Individual cells and to be attached to the well plate.
3. After the cells are attached, the prepared test substance-DC extraction layer and the compound 1-9 are added for culturing for 6 hours.
4. After the time is up, the culture solution of the DC extraction layer and the compounds 1-9 is sucked and semidried, the culture solution remained in the 96-well plate is washed by using 1-fold phosphate buffer physiological saline (phosphate buffered saline; PBS), 100 mu L of 1-fold phosphate buffer physiological saline is added into the 96-well plate after washing, and the energy is utilized to 40mJ/cm 2 Ultraviolet radiation of (a) is provided. Adding DC extract layer and compound again1-9 in 96-well plate for 24 hr.
5. After the treatment of the pore plate, the energy of irradiation is 40mJ/cm 2 After the irradiation, the culture solution containing the DC extraction layer and the compounds 1 to 9 was added to the well plate and cultured for 24 hours.
6. And 10. Mu.L of 0.2mg/mL of Resazurin reagent was added before 2 hours for 24 hours, and the mixture was placed in an incubator containing 5% carbon dioxide at 37℃and kept waiting for detection of fluorescence (ex/em: 530nm/590 nm) and absorbance (570 nm and 600 nm) to determine the cell viability.
Example 7, light protection results of solid and liquid fermentation extract layer of chaetomium sojae (Diaporthe caulivora) and compounds this experiment uses human keratinocyte strain to evaluate the viability of cells irradiated with ultraviolet rays and test whether the DC extract layer and compounds 1-9 have an ultraviolet light protection effect. All data showed a trend of increasing growth of cells of the control group which had not been treated with any treatment for 24 hours as compared with the control group which had not been treated with any treatment for 0 hour, and cell viability after 24 hours of incubation with ultraviolet rays was impaired by ultraviolet rays. Comparing the protective effect of the DC extract layer and compounds 1-9 on cells, and the different DC extract layers were mostly protective at concentrations of 30 μg/mL and 300 μg/mL, with the best effect being DC-S-EA (see FIG. 7). On the other hand, of the compounds 1 to 9 having a concentration of 10 and 50. Mu.M, most of the compounds have the best protective effect, except that the compound 1 has the poor protective effect on cells damaged by ultraviolet rays, and in particular, the compounds 7, 8 and 9 have the best protective effect (see FIG. 8).
EXAMPLE 8 solid and liquid fermentation product extract layer of Soy-Equisetum (Diaporthe caulivora) and Compound inhibitory Effect on ultraviolet-induced active oxygen substances
1. DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and compounds 1-9 were prepared in a medium, and a dimethylsulfoxide reagent was used as a control group, and the concentration of endophyte extract in the plant was 30 and 300. Mu.g/mL and the concentration of the compound was 10 and 50. Mu.M.
2. Human keratinocyte cell lines were seeded in 96-well plates, each well being 1x10 4 The number of cells in a cell is one,and the cells are allowed to adhere to the well plate.
3. After the cells are attached, the prepared test substance-DC extraction layer and the compound 1-9 are added for culturing for 6 hours.
4. After the lapse of time, the culture solution containing the DC extract or the compound thereof is sucked semi-dry, the culture solution remained in the 96-well plate is washed with 1-fold phosphate buffer physiological saline, and after washing, sucked out, and then fluorescent dye 2',7' -dichlorofluorescein diacetate (H) is added 2 DCFDA) was placed in 96-well plates and placed in an incubator containing 5% carbon dioxide at 37 ℃ for 30 minutes in the dark.
5. When the time is up, sucking out 2',7' -dichlorofluorescein diacetate, washing with 100. Mu.L of 1-fold phosphate buffer physiological saline for 1 time, sucking out after washing, adding 100. Mu.L of 1-fold phosphate buffer physiological saline into a 96-well plate, and using energy of 40mJ/cm 2 The content of intracellular active oxygen species was determined by measuring the fluorescence (ex/em: 485nm/530 nm) of the ultraviolet radiation of (C).
Example 9 analysis of the effect of solid and liquid fermentation extract layers and Compounds of Equipped soybean (Diaporthe caulivora) on active oxygen substance inhibition this experiment used human keratinocyte cell lines to test whether DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and compounds 1-9 inhibited intracellular UV-induced active oxygen substances. All the data showed that the fluorescence value of the active oxygen substance after irradiation with ultraviolet rays was increased by induction of ultraviolet rays and was 40mJ/cm after irradiation with ultraviolet rays, as compared with the control group without irradiation with ultraviolet rays 2 Based on the fluorescence value of (2), the inhibitory effect of the DC extraction layer and the compounds 1 to 9 on intracellular active oxygen species was compared. Among them, DC-S-EA, DC-S-W and most of the compounds had inhibitory effects on intracellular active oxygen substances (see FIG. 9), and among them, the inhibitory effects on intracellular active oxygen substances were optimal by compounds 7, 8 and 9 (see FIG. 10).
Example 10 inhibition of ultraviolet-induced cyclooxygenase-2 (COX-2) protein expression by extract of Asclepiadaceae (Diaporthe caulivora) (DC-S-EA): western blot analysis
1. Preparing DC-S-EA in dimethyl sulfoxide reagent, wherein the test concentration of DC extract is 30 and 300. Mu.g/mL. Human keratinocyte cell line cell at 2x 10 6 The cells were plated in 10 cm dishes and cultured for 6 hours with the addition of DC extract after cell attachment.
2. The culture solution containing DC extract is sucked semi-dry, the residual culture solution is washed for 1 time by using phosphate buffer physiological saline, 7mL of 1 time by using phosphate buffer physiological saline is added after sucking out, and the energy is utilized for 20mJ/cm 2 The culture medium containing DC-S-EA was added again to the dish and left to stand in an incubator containing 5% carbon dioxide at 37℃for 12 hours.
3. After the culture broth was blotted dry, 350. Mu.L of radioimmunoprecipitation buffer (radioimmunoprecipitation assay buffer; RIPA reagent) was added and the cells scraped off, placed in a microcentrifuge tube (eppendof) and centrifuged at 15,000rpm at 4℃for 10 minutes, and the supernatant was aspirated into a fresh microcentrifuge tube and stored in a-80℃refrigerator.
4. Protein separation electrophoresis was performed at 80 volts using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-page) and transferred onto nitrocellulose membrane (Blotting membrane) at 0.35 amp for 3 hours.
5. After the transfer of the film was completed, the film was immersed in 5% skim milk and shaken until every other day.
6. Washing with 1-fold tris buffer (tris (hydroxymethyl) aminomethane; TBS buffer) for 10 minutes was repeated three times.
7. Primary antibodies to the protein of interest were added and kept on a shaker at 4 ℃ until every other day.
8. The washing with 1-fold tris buffer for 10 min was repeated three times, the secondary antibody was added and placed on a shaker for 1 hour, and the washing with 1-fold tris buffer for 10 min was repeated three times.
9. 1mL of developer is added on the nitrocellulose membrane, and after being evenly dispersed, the nitrocellulose membrane is put into a luminescence imager to take pictures and store experimental results for image quantification and analysis.
EXAMPLE 11 ultraviolet-induced epoxy Synthesis of extract (DC-S-EA) of Phytolacca sojae (Diaporthe caulivora)Inhibition effect analysis of enzyme-2 (COX-2) protein expression level the present experiment used western blot analysis to perform inhibition of the ultraviolet-induced cyclooxygenase-2 (COX-2) reaction by the ethyl acetate extract layer (DC-S-EA) of the DC solid state fermentation. Compared with the 12-hour control group, the energy is 20mJ/cm 2 The increase in cyclooxygenase-2 expression after UVB irradiation, and the decrease in cyclooxygenase-2 expression under the action of DC-S-EA at different concentrations of 30. Mu.g/mL, indicated that DC-S-EA inhibited the inflammatory reaction induced by UV (see FIG. 11), and that 30. Mu.g/mL was better than 300. Mu.g/mL, indicating that low concentrations were effective.
Example 12, solid and liquid fermentation extract layer of Soy-Etsche (Diaporthe caulivora) and test for safety of Compound in murine melanoma cells
1. DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and compounds 1-9 were prepared in a medium, and a concentration of 300. Mu.g/mL was measured for DC extract and 50. Mu.M was measured for compound using a dimethylsulfoxide reagent as a control group.
2. Mouse melanoma cells (B16-F10 cells) were seeded in 24-well plates, each 5X 10 4 Individual cells and to be attached to the well plate.
3. mu.L of 0.2mg/mL Resazurin reagent was added and the mixture was placed in a reaction vessel containing 5% CO 2 The cell viability was determined by waiting 2 hours in an incubator at 37℃and measuring the fluorescence (ex/em: 530nm/590 nm) and absorbance (570 nm and 600 nm).
Example 13, solid and liquid fermentation extract layer of chaetomium sojae (Diaporthe caulivora) and compound safety results analysis of mouse melanoma cells this experiment uses mouse melanoma cells (B16-F10 cells) to test whether DC extract layer and compounds 1-9 have an effect on cell growth. The results showed that the DC extract layer (DC-L-EA, DC-S-EA, DC-S-W) at a concentration of 300. Mu.g/mL and compounds 1 to 9 at a concentration of 50. Mu.M had no significant effect on cell growth compared to the control group without any treatment (see FIGS. 12 and 13).
EXAMPLE 14 inhibition of tyrosinase by solid and liquid fermentation extract layer and Compounds of Phytocheca sojae (Diaporthe caulivora)
1. Seeding in 24-well plates 5X10 4 In each mouse melanoma cell, 100 μl of alpha melanocyte-stimulating hormone (alpha-melanocyte-stimulating hormone; alpha-MSH) (50 nM) was added to induce melanin production (melanin), and the mixture was placed at 37deg.C with 5% CO 2 Is cultured for 24 hours in an incubator of (2) to induce melanogenesis.
2. The culture medium was removed, and after adding the prepared DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and the compounds 1 to 9, the mixture was placed in an incubator and cultured for 48 hours, the concentration of the DC extract layer was 30 and 300. Mu.g/mL, and the concentration of the compound test was 50. Mu.M.
3. After the time had elapsed, the cells were washed twice with 1-fold phosphate-buffered saline (500. Mu.l per well), followed by the addition of 100. Mu.l of tyrosine (trypsin) and 500. Mu.l of 1-fold phosphate-buffered saline, and collected, and centrifuged at 10,000 rpm for five minutes at room temperature.
4. The supernatant was removed, a tissue lysis buffer (lysia buffer) containing phosphate buffered saline, polyethylene glycol octylphenyl ether (triton X-100) and 1mM phenylmethylsulfonyl fluoride (phenylmethanesulfonylfluoride or phenylmethylsulfonylfluorid; PMSF) was added, and the mixture was mixed well, reacted at 4℃for 30 minutes, centrifuged at 14,000 rpm for 10 minutes, and Levodopa (Levodopa; L-DOPA) 1mg/ml in phosphate buffered saline was added, reacted at 37℃for 3 hours, and 180. Mu.L to 96 well plates were aspirated.
5. And observing the change of the absorbance value of the object to be detected by using a full-spectrum disk scanner at the wavelength of 490nm to obtain the inhibition rate.
Example 15 analysis of tyrosinase inhibitory effects of solid and liquid fermentation extract layers and Compounds of Sonchi (Diaporthe caulivora) the tyrosinase inhibitory effects of DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and compounds 1-9 were tested using mouse melanoma cells (B16-F10 cell line) in this experiment. Compared with the control group induced by melanocyte stimulating hormone, DC-S-EA and DC-L-EA have good tyrosinase inhibitory effect (refer to FIG. 14), and compound 1 has optimal activity and exhibits tyrosinase inhibitory effect equivalent to that of commercially available whitening agent kojic acid (kojic acid) at 10, 50 and 100. Mu.M (refer to FIGS. 15 and 16).
EXAMPLE 16 inhibition of melanin by solid and liquid fermentation extract layers and Compounds of Phytolacca sojae (Diaporthe caulivora)
1. Seeding in 12-well plates 1X10 5 Mu.l of alpha melanocyte stimulating hormone (50 nM) was added to each mouse melanoma cell, and the mixture was placed at 37℃with 5% CO 2 Is cultured for 24 hours in an incubator of (2) to induce melanogenesis.
2. The culture medium was removed, and after adding the prepared DC extract layers (DC-L-EA, DC-S-EA, DC-S-W) and the compounds 1 to 9, the mixture was placed in an incubator and cultured for 48 hours, the concentration of the DC extract layer was 30 and 300. Mu.g/mL, and the concentration of the compound test was 50. Mu.M.
3. 200. Mu.l of trypsin and 500. Mu.l of 1-fold phosphate buffer physiological saline were added, centrifuged at room temperature for 3,000 rpm for eight minutes, the supernatant was removed, 100. Mu.l of 2N sodium hydroxide (NaOH) was added and mixed well, and the mixture was placed in a 95℃oven and heated for 15 minutes, and the mixture was taken out and placed in a 96-well plate, and the absorbance was measured at a wavelength of 405 nm.
Example 17 inhibition of solid and liquid fermentation extract layers of chaetomium sojae (Diaporthe caulivora) and compounds on melanin analysis this experiment uses mouse melanoma cells (B16-F10 cell lines) to test the melanin (melanin) inhibiting effect of the DC extract layers (DC-L-EA, DC-S-W) and compounds 1-9. Compared with the control group induced by melanocyte stimulating hormone, DC-L-EA has good melanin inhibiting effect (see FIG. 17), and compound 1 and compound 9 have melanin inhibiting effect, wherein compound 1 has optimal activity and melanin inhibiting activity close to that of commercially available whitening agent kojic acid (kojic acid) at concentration of 100 μm (see FIGS. 18 and 19).
Examples 18 and Caulivotrioloxin A (Compound 1) action on tyrosinase and tyrosinase-related proteins: western blot analysis
1. Mouse melanoma cells at 2x 10 6 Adding alpha melanocyte stimulating hormone (50 nM) into 10 cm culture dish, standing at 37deg.C, and adding 5% CO 2 Is cultured for 24 hours in an incubator of (2) to induce melanogenesis.
2. The culture medium was removed, and the prepared test medium of Compound 1 (at concentrations of 5, 10, 50, 100. Mu.M) was added and incubated in an incubator for 48 hours.
3. After the culture broth was blotted, 350. Mu.L of a radioimmunoprecipitation buffer (radioimmunoprecipitation assay buffer; RIPA reagent) was added and the cells scraped off, and the mixture was placed in a microcentrifuge tube (Eppendorf) and centrifuged at 15,000rpm at 4℃for 10 minutes, and the supernatant was aspirated and stored in a fresh microcentrifuge tube in a-80℃refrigerator.
4. Protein separation electrophoresis was performed at 80 volts using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-page) and transferred onto nitrocellulose membrane (Blotting membrane) at 0.35 amp for 3 hours.
5. After the transfer of the film was completed, the film was immersed in 5% skim milk and shaken until every other day.
6. The washing with 1-fold tris buffer for 10 min was repeated three times.
7. Primary antibodies to the protein of interest were added and kept on a shaker at 4 ℃ until every other day.
8. The washing with 1-fold tris buffer for 10 min was repeated three times, the secondary antibody was added and placed on a shaker for 1 hour, and the washing with 1-fold tris buffer for 10 min was repeated three times.
9. 1mL of developer is added on the nitrocellulose membrane, and after being evenly dispersed, the nitrocellulose membrane is put into a luminescence imager to take pictures and store experimental results for image quantification and analysis.
Examples 19, caulivotrioloxin A (Compound 1) analysis of tyrosinase and tyrosinase-related proteins the present experiment was conducted using Western blot analysis to investigate the pathways of Compound 1 for tyrosinase and tyrosinase-related proteins, melanin being an enzyme required for the formation of melanin by tyrosine (tyrosinase), which is divided into two pathways, tyrosinase-related protein-1 (tyrosinase related protein; TRP-1) was associated with the formation of eumelanin (eumelanin), whereas tyrosinase-related protein-2 (tyrosinase related protein; TRP-2) was associated with the production of pheomelanin. While FIG. 20A shows that compound 1 has an effect of inhibiting the expression level of tyrosinase protein at concentrations of 10, 50 and 100. Mu.M, FIG. 20B shows that compound 1 can inhibit the expression level of tyrosinase-related protein-1 (TRP-1) protein at concentrations of 10 and 50. Mu.M, and that compound 1 can slightly inhibit the expression level of tyrosinase-related protein-2 (TRP-2) protein at concentrations of 50 and 100. Mu.M (see FIG. 20C), the effect of compound 1 on melanin can be obtained from the experimental results, probably due to the inhibition of tyrosinase and tyrosinase-related protein-1 protein expression levels.
UVB phototoxic Effect of examples 20, caulivotrioloxin A (Compound 1)
1. Compound 1 was placed in dimethylsulfoxide reagent and tested at low and high concentrations of 10 and 50 μm, respectively.
2. Human keratinocyte cell lines (HaCaT cells) were seeded in 96-well plates, each well 1x 10 4 Individual cells and to be attached to the well plate.
3. After cell attachment, the prepared test compound 1 was added and cultured for 6 hours.
4. After the lapse of time, the culture solution containing Compound 1 was half-dried by pipetting, washing 1 XPBS for 1 time, and then pipetting out, and then adding 100. Mu.L of 1 Xphosphate buffered saline in 96-well plates, using energy of 40mJ/cm 2 Is irradiated with UVB.
5. After irradiation, the culture medium without compound 1 was added to a 96-well plate, incubated for 24 hours, and 10. Mu.L of 0.2mg/mL Resazurin reagent was added 2 hours before 24 hours, and placed in a medium containing 5% CO 2 The cell viability was determined by waiting for fluorescence (ex/em: 530nm/590 nm) and absorbance (570 nm and 600 nm) in an incubator at 37 ℃.
Analysis of phototoxicity effects of UVB in examples 21 and Caulivotrioloxin A (Compound 1) from examples 12 to 19 above, it was confirmed that Compound 1 has melanin and tyrosinase inhibiting effects, and this experiment was conducted in order to evaluate whether phototoxicity was generated by further irradiation with Compound 1. The experiment uses human keratinocyte cell lines (HaCaT cells) to test whether compound 1 was added with UVB for cell damage. FIG. 21 shows that compound 1 (10. Mu.M) has no additive effect with UVB, does not cause the aggravated damaging effect of cells, and shows its photoprotection. Although 50 μm had an effect, it was not significant. It is recommended to use the product with sunscreen agent or at night.
Those skilled in the art will readily appreciate that the present invention may be readily utilized as a basis for the obtaining the objects and obtaining the ends and advantages mentioned, as well as those inherent therein. The compounds, compositions, extracts and mixtures of the invention, processes and methods for their manufacture and use are representative of the preferred embodiments, which are exemplary and not limiting in scope. Those skilled in the art will recognize where modifications and other uses may occur. Such modifications are intended to fall within the spirit of the invention and are defined in the claims. The present disclosure and examples are described in such detail as to enable those skilled in the art to make and use the invention, and it is intended that various changes, modifications and improvements be made therein without departing from the spirit and scope of the invention.
All patents and publications mentioned in the specification are directed to the state of the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The invention suitably illustrated herein may be practiced in the absence of any element, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that while the invention has been specifically disclosed in terms of preferred embodiments and optional features, modification and variation of the inventions herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the claims.
Claims (11)
1. Use of an extract of alternaria sojae (Diaporthe caulivora) having the accession number DSM33674 in the DSMZ for the preparation of a sun protection and whitening composition, wherein the extract of alternaria sojae comprises the compound calibysin a.
2. The use according to claim 1, wherein the sun protection whitening composition is a composition having an anti-uv effect.
3. The use according to claim 1, wherein the sunscreen whitening composition is a composition having a pigmentation reducing effect.
4. The use according to claim 1, wherein the sunscreen whitening composition is for preventing or treating discomfort or disease or photo-injury conditions caused by overexposure to ultraviolet light.
5. The use according to claim 4, wherein the discomfort or disease is hyper-pigmentation or skin aging.
6. The use of claim 4, wherein the photodamaged condition is erythema, scaling, edema, thickness changes, sunburn, immune response suppression, tumorigenesis, or any combination thereof.
7. The use according to claim 1, wherein the sun protection and whitening composition is used for preventing or treating discomfort or disease caused by hyper-pigmentation, including freckles, chloasma, striae of pregnancy, age spots, and melanoma.
8. A compound calibysin a isolated from an extract of alternaria sojae (Diaporthe caulivora) deposited under the DSMZ accession No. DSM33674.
9. A composition comprising the compound of claim 8.
10. The composition of claim 9 having an anti-uv effect.
11. The composition of claim 9, having the effect of reducing pigmentation.
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