TW202317165A - Fermentation products, process for preparing and uses of the same - Google Patents

Abstract

Disclosed herein is a process for preparing a fermentation product of a traditional Chinese medicine compound. Also disclosed herein is that the fermentation product of the traditional Chinese medicine compound can be used in reducing ultraviolet damage and inhibiting melanogenesis.

Description

Fermentation product of traditional Chinese medicine compound, its preparation method and use

The invention relates to a method for preparing a fermented product of a traditional Chinese medicine compound and the fermented product. The present invention also relates to the application of the fermentation product in slowing down ultraviolet damage and inhibiting melanin production.

Ultraviolet damage is caused by exposure of any part of a human individual to ultraviolet radiation for a long time, and may cause a series of acute and chronic symptoms, among which acute symptoms usually include photodamage, erythema, etc. , inflammation (inflammation), tanning (tanning), and chronic symptoms usually include skin aging (skin aging) [also known as photoaging (photoaging)], severe cases may even lead to tumors or cancer [eg, skin cancer (skin cancer) cancer)].

Melanogenesis (that is, melanin synthesis) refers to when skin melanocytes (dermal melanocytes) are subjected to environmental factors (such as ultraviolet (ultraviolet, UV)) or physiological factors (such as fatigue (fatigue), stress ( After the induction of stress), chronic inflammation (chronic inflammation) and abnormal release of α-melanocyte stimulating hormone (α-MSH) in the body], the tyrosine (tyrosine) in melanocytes The process of being converted into melanin through the catalysis of tyrosinase (it is the rate-limiting step of melanin production) and a series of redox reactions. Although melanin can protect the skin from the above-mentioned ultraviolet rays, when melanin is accumulated in large amounts on the skin or distributed abnormally, it may cause skin disorders such as lentigines, freckles, black skin disease (melasma), age spots (age spots) and hyperpigmentation (hyperpigmentation) and so on.

Currently, topical steroids, non-steroidal anti-inflammatory drugs (NSAIDs), antihistamines, and sunscreens are often used clinically to treat and/or prevent UV damage; And use melanogenesis inhibitors such as hydroquinone, arbutin, and kojic acid to lighten or remove melanin accumulated on the skin.

However, the therapeutic effects of these drugs are still not satisfactory, and may cause serious side effects to patients. Therefore, relevant researchers in this field try to find active components from natural sources (including Chinese herbal medicines and probiotics) that can be used to alleviate ultraviolet damage and inhibit melanin production. For example, TW I393568 B discloses a Chinese herbal compound [it contains Angelica dahurica , Ampelopsis japonica , Atractylodes macrocephala , Typhonium giganteum , Poria cocos , Bletilla striata striata ) and Asarum heterotropoides ], the ethanol extract was found through in vitro experiments to inhibit the tyrosinase activity in human metastatic melanoma cell line A2058 and reduce its The amount of melanin is produced, and it has been confirmed to have whitening effect through human experiments.

In addition, in CC Tsai et al . (2013), Molecules , 18:14161-14171, Tsai et al. used the culture supernatant of Lactobacillus rhamnosus strain LRH113 for α,α-diphenyl-β-bitter The analysis of hydrazyl radical scavenging ability [α,α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging ability] and the test of tyrosinase inhibitory activity, the experimental results showed that the culture supernatant of Lactobacillus rhamnosus LRH113 The solution has good antioxidant activity and can effectively inhibit tyrosinase activity.

Although there are the above-mentioned literature reports, there is still a need in the art for drugs that can effectively slow down the damage of ultraviolet rays and inhibit the production of melanin.

Summary of the invention

In the present invention, the applicant uses a medium containing 8 kinds of Chinese herbal medicines: Atractylodes macrocephala, Bletilla striata, Poria cocos, Poria cocos, Baixian, peony, Tribulus and pearl powder to ferment and cultivate Lactobacillus rhamnosus , The obtained fermentation product was unexpectedly found through experiments, not only effectively inhibiting tyrosinase and melanin production, but also effectively improving ultraviolet-induced skin damage.

Therefore, in a first aspect, the present invention provides a method for preparing a fermentation product of a Chinese medicine compound, which includes: using a medium containing a Chinese medicine compound to ferment and cultivate Lactobacillus rhamnosus, and collecting the obtained fermentation culture Wherein, the traditional Chinese medicine compound contains Atractylodes macrocephala, Bletilla striata, Poria cocos, Poria cocos, Baixian, peony, Tribulus terrestris, and pearl powder.

In a second aspect, the present invention provides a fermented product of a traditional Chinese medicine compound prepared by using a method as described above.

In a third aspect, the present invention provides a use of the fermentation product of the above-mentioned traditional Chinese medicine compound for the preparation of a medicine or cosmetic for alleviating ultraviolet damage.

In a fourth aspect, the present invention provides a use of the above-mentioned traditional Chinese medicine compound fermentation product for preparing a medicine or cosmetic for inhibiting melanin production.

In a fifth aspect, the present invention provides a method for mitigating ultraviolet damage, which comprises administering a fermented product of the traditional Chinese medicine compound as described above to an individual in need thereof.

In a sixth aspect, the present invention provides a method for inhibiting melanin production, which comprises administering a fermentation product of the above-mentioned traditional Chinese medicine compound to an individual in need thereof.

Detailed Description of the Invention

It is to be understood that if any prior publication is cited herein, that prior publication does not constitute an acknowledgment that, in Taiwan or any other country, that prior publication forms a common practice in the art part of knowledge.

For the purposes of this specification, it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Of course, the invention is in no way limited by the methods and materials described.

The invention provides a method for preparing a fermentation product of a Chinese medicine compound, which comprises: using a culture medium containing a Chinese medicine compound to ferment and cultivate Lactobacillus rhamnosus, and collecting the obtained fermentation culture, wherein the Chinese medicine compound contains Atractylodes macrocephala , Bletilla striata , Ampelopsis japonica , Poria cocos , Dictamnus dasycarpus , Paeonia lactiflora , Tribulus terrestris , and pearl powder. The present invention also provides a fermented product of a traditional Chinese medicine compound prepared by using the above-mentioned method.

According to the present invention, the Atractylodes Rhizome used in the Chinese medicine compound can come from the rhizome (rhizome) of Atractylodes Rhizome; the Bletilla striata used in the Chinese medicine compound can come from the corm (corm) part of Bletilla striata; from the tuberous root part of Poria cocos; the Poria cocos used in the Chinese medicine compound can come from the peeled sclerotium part of Poria cocos; Radix Paeoniae Alba) root bark part; the peony used in the Chinese medicine compound can come from the root of Radix Paeoniae Alba (also known as Radix Paeoniae Alba, Hangbai or Golden Paeoniae Alba); Tribulus terrestris used in the Chinese medicine compound can come from fruit; and the pearl powder used in the Chinese medicine compound can come from the outer layer of pearl and the inner layer of the shell (nacre) present in most molluscs. In a preferred embodiment of the present invention, the Chinese medicine compound contains dried powders of 8 kinds of Chinese herbal medicines, namely Atractylodes Rhizome, Bletilla striata corm, Bletilla striata root tuber, Poria cocos sclerotia, Baixian root bark, Paeoniae officinalis root, and nacre.

According to the present invention, based on the total weight of the culture medium, the Chinese medicine compound has a content of 1 g/L.

In a preferred embodiment of the present invention, the Chinese medicine compound contains 12.5% (w/w) Atractylodes Rhizome; 12.5% (w/w) Bletilla striata; 12.5% (w/w) Bletilla striata; Poria cocos /w); Baixian 12.5% (w/w); Peony 12.5% (w/w); Tribulus terrestris 12.5% (w/w); and Pearl powder 12.5% (w/w).

As used herein, the term "Lactobacillus rhamnosus" is intended to cover those strains of Lactobacillus rhamnosus that are readily available to those skilled in the art (for example, those that can be purchased from domestic or foreign depositories), Or Lactobacillus rhamnosus strains isolated and purified from natural sources by using the customary microorganism isolation method in this art. Preferably, the Lactobacillus rhamnosus strain is Lactobacillus rhamnosus GG (BCRC 16000).

As used herein, the terms "culturing", "fermentation" and "cultivation" are used interchangeably.

According to the present invention, the culture medium suitable for culturing the Lactobacillus rhamnosus is well known to those skilled in the art, including, but not limited to: MRS broth medium (MRS broth).

Preferably, the fermentation product of the traditional Chinese medicine compound is a fluid substantially free of bacteria.

As used herein, the term "substantially free of" means lacking a meaningful amount of a specified component. Preferably, the content of the component has no measurable effect on the properties of the fermentation product of the traditional Chinese medicine compound. More preferably, the fermentation product of the traditional Chinese medicine compound does not contain this component at all.

According to the present invention, the fluid substantially free of bacterial cells is obtained by performing a separation process on the fermentation culture.

According to the present invention, the separation treatment is selected from the group consisting of filtration, centrifugation treatment, concentration treatment, and combinations thereof.

In a preferred embodiment of the present invention, the fluid without bacterial cells is obtained by filtering the fermentation culture using a filter paper with a pore size of 0.45 μm.

Based on the above-mentioned beneficial biological activity, the applicant believes that the fermentation product of the traditional Chinese medicine compound of the present invention has a high potential to be developed into a drug or cosmetic for slowing down the damage caused by ultraviolet rays. Therefore, the present invention provides a use of the fermentation product of the above-mentioned traditional Chinese medicine compound for preparing a medicine or cosmetic for alleviating ultraviolet damage.

The present invention also provides a method for mitigating ultraviolet damage, which comprises administering a fermented product of the traditional Chinese medicine compound to an individual in need of mitigating ultraviolet damage.

As used herein, "mitigating" UV damage means preventing, reducing, alleviating, ameliorating, relieving, or controlling one or more of UV damage. A clinical sign, and progress in lowering, stopping, or reversing the severity of a condition or symptom of UV damage being treated ( progress).

As used herein, the term "ultraviolet damage" means symptoms caused by any part of a human individual after ultraviolet overexposure (ultraviolet overexposure), including, but not limited to: skin inflammation (skin inflammation), delayed delayed tanning reaction, skin aging (also known as photoaging), skin cancer, severe pains in the joints and muscles, and around the eyes joints and muscles and around the eyes), shock, fever, nausea, vomiting, and generalized weakness.

In a preferred embodiment of the present invention, the ultraviolet damage is photoaging.

As used herein, the term "photoaging" means extrinsic skin aging caused by exposure to ultraviolet rays. Conditions of photoaging include, but are not limited to: telangiectasia, epidermis thickening, skin atrophy, degradation of collagen and elastin, elastosis (elastosis), poor skin elasticity, rough skin texture, dryness, wrinkle formation, and pigmentary change (eg, freckles, spots ( freckles), hypopigmentation or hyperpigmentation], etc.

According to the present invention, the pharmaceutical product can be manufactured into a dosage form suitable for parenteral administration, oral administration or topical administration using techniques well known to those skilled in the art ( dosage form).

According to the present invention, the medicine may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may contain one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes and the like. The selection and quantities of these reagents are within the professionalism and routine skill of those skilled in the art.

According to the present invention, the medicinal product may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, intrapleural injection, intramuscular injection injection), intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection injection), intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration.

According to the present invention, the medicinal product can be manufactured into a dosage form suitable for oral administration by techniques well known to those skilled in the art, including, but not limited to, sterile powder, tablet, troche ), lozenge, pellet, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup , elixirs, syrups, and the like.

In accordance with the present invention, the medicinal product may also be formulated as an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to: emulsions , gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion , serum, paste, foam, drop, suspension, salve, and bandage.

According to the present invention, the external preparation is prepared by mixing the medicinal product of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives (additives) selected from the group consisting of water, alcohols, glycols, hydrocarbons (such as petroleum jelly and white Vaseline (white petrolatum)], wax (wax) (such as paraffin (paraffin) and yellow wax (yellow wax)], preservatives (preserving agents), antioxidants (antioxidants), surfactants (surfactants), absorption enhancers (absorption enhancers), stabilizing agents, gelling agents (such as carbopol ^® 941 (carbopol ^® 941), microcrystalline cellulose, and carboxymethylcellulose (carboxymethylcellulose)], active Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents ( occlusive agents), emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants ( propellants) etc. The selection and amounts of these additives are within the professionalism and routine skill of those skilled in the art.

According to the present invention, the dosage and frequency of administration of the fermentation product of the Chinese herbal compound will vary depending on the following factors: the severity of the ultraviolet damage to be slowed down, the route of administration, and the age, physical condition and age of the individual to be slowed down by the ultraviolet damage. reaction.

According to the present invention, the cosmetic may further comprise a cosmetically acceptable adjuvant which is widely used in cosmetic manufacturing techniques. For example, the cosmetically acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants , coloring agent, thickening agent, filler, fragrance and odor absorber. The selection and quantities of these reagents are within the professionalism and routine skill of those skilled in the art.

According to the present invention, the cosmetic product can be manufactured into a form suitable for skin care or makeup using techniques well known to those skilled in the art, including, but not limited to: aqueous solution, water - alcohol solution (aqueous-alcohol solution) or oily solution (oily solution), in the form of oil-in-water type (oil-in-water type), oil-in-water type (water-in-oil type) or composite type emulsion, gel Gels, ointments, creams, masks, patches, packs, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, drops, mousses ( mousse), sunblock, tonic water, foundation, eyeshadow, makeup remover products, soap and other body cleansing products, etc.

According to the present invention, the cosmetic product may also be used in combination with one or more known active external use agents selected from the group consisting of: whitening agents (such as tretinoin, catechin (catechin, kojic acid, arbutin and vitamin C)], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers absorbers), plant extracts (such as aloe extract), skin nutrients, anesthetics, anti-acne agents, antipruritics , analgesic, antiperspirant, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antiaging agents ), antiwrinkle agents, antiseborrheic agents, wound-healing agents, corticosteroids, hormones, free radical scavengers (such as catalase), anti Oxidants (such as vitamin E), cytokines (such as interleukin-1), thiols (such as amifostine), and steroids (such as 5-androstenediol). The selection and amount of these topical agents are within the professionalism and routine skill of those skilled in the art.

According to the invention, the cosmetic product is in a form for topical application.

The present invention also provides a use of the above-mentioned traditional Chinese medicine compound fermentation product for preparing a medicine or cosmetic for inhibiting melanin production.

The present invention also provides a method for inhibiting melanin production, which includes administering a fermentation product of the above-mentioned traditional Chinese medicine compound to an individual who needs to inhibit melanin production.

As used herein, the terms "inhibition of melanogenesis" and "inhibition of melanin synthesis", "depigmenting", "lightening the melanin", " The terms "whitening", "skin color lightening", "bleaching", "whitening", "brightening", "sunburn" and "sunburn" are used interchangeably .

According to the present invention, the administration route, dosage form and available pharmaceutically acceptable carrier of the drug are as described above; and the application form of the cosmetic, the available cosmetically acceptable adjuvant, And the external preparations that can be used in combination are as described above. Detailed Description of the Preferred Embodiment

The present invention will be further described in terms of the following examples, but it should be understood that these examples are for illustration purposes only, and should not be construed as limitations on the implementation of the present invention. Examples General experimental materials: 1. Source and cultivation of Lactobacillus rhamnosus GG:

Lactobacillus rhamnosus GG (BCRC 16000) (corresponding to ^ATCC® 53103™) used in the following examples was purchased from the biological resources of the Food Industry Research and Development Institute (FIRDI) in Taiwan Preservation and Research Center (Biosource Collection and Research Center, BCRC). 2. Source and culture of mouse skin melanoma cell line B16F10:

The mouse skin melanoma cell line B16F0 (BCRC 60029) (corresponding to ^ATCC® CRL6322™) used in the following examples was purchased from BCRC.

B16F0 cells were cultured in a petri dish containing RPMI 1640 medium) [supplemented with 10% FBS (Hyclone), 100 μg/mL penicillin and 100 μg/mL streptomycin], and Cultures were carried out in an incubator whose culture conditions were set at 37°C and 5% CO ₂ . Thereafter, replace with fresh medium approximately every 2-3 days. When the cell density reaches about 80-90% confluence, remove the medium and wash the cells with phosphate buffered saline (Phosphate Buffered Saline, PBS) for a total of 2 times, then add trypsin-EDTA (trypsin-EDTA) to detach the cells from the bottom of the dish. Afterwards, add fresh medium to neutralize the activity of trypsin and repeatedly aspirate and wash the medium with a pipette to fully break up the cells, then distribute the resulting cell suspension into a new Petri dish, and incubate The conditions were set to be cultured in an incubator at 37°C with 5% CO ₂ . General experimental methods: 1. Statistical analysis:

In the following examples, the experiments of each group were repeated, and the experimental data obtained were statistically analyzed using GraphPad Prism 5.01 software (San Diego, CA, USA), and expressed as "average value (mean) ± standard deviation ( standard deviation, SD)" to indicate. Differences among the experimental data of each group were evaluated by one-way analysis of variance (ANOVA) combined with Tukey's post hoc test or Dunnett's test. If the obtained statistical comparison result is p <0.05, it means there is statistical significance. Embodiment 1. prepare the fermented product of compound traditional Chinese medicines (TCM) of the present invention

The types and parts of the 8 Chinese medicines used to prepare the fermentation products of the Chinese medicine compound of the present invention in this example are shown in Table 1 below. Table 1. Chinese herbal medicines used in the preparation of traditional Chinese medicine compound type Application area Atractylodes macrocephala (Atractylodes macrocephala ) rhizome Baiji ( Bletilla striata ) bulb White scorpion ( Ampelopsis japonica ) root Poria cocos ( Poria cocos ) Sclerotium (sclerotium) White Fresh ( Dictamnus dasycarpus ) root bark Peony ( Paeonia lactiflora ) root Tribulus terrestris fruit pearl nacre

First, the dry powder of the above 8 kinds of Chinese herbal medicines (all purchased from Keda Pharmaceutical) was 1:1:1:1:1:1:1:1 (w/w/w/w/w/w /w/w) ratio is mixed to obtain a Chinese medicine compound.

Then, the traditional Chinese medicine compound was prepared as a medium with the formula shown in Table 2 below, and Lactobacillus rhamnosus GG was inoculated into 200 mL of the medium at a concentration of 2.2×10 ⁷ CFU/mL , followed by fermentation at 37°C under anaerobic conditions for 48 hours. Table 2. Formulas of medium containing Chinese herbal compound Element content Chinese herbal compound 1g/L Tryptone 15g/L beef extract 2.5g/L Yeast extract 7.5g/L Cysteine HCl 0.05 g/L lactose 2.5g/L Glucose 7.5g/L Dipotassium hydrogen phosphate (K ₂ HPO ₄ ) 4.5g/L Tween-80 80 mL/L The balance is distilled water, and the pH value is adjusted to 6.5±0.1

Afterwards, the obtained fermentation culture was centrifuged at 8,000 × g for 25 minutes, and the supernatant was filtered through a filter paper with a pore size of 0.45 μm, and the filtrate thus obtained was vacuum evaporated at 50°C treatment (vacuum evaporation treatment) and freeze-drying treatment, so as to obtain the traditional Chinese medicine compound fermentation product in the form of dry powder, and store it at 4°C for future use. Embodiment 2. The cell viability assay (cell viability assay) experimental method of the fermentation product of Chinese medicine compound prescription of the present invention :

First, the B16F0 cells that were subcultured according to item 2 of the above "General Experimental Materials""Source and Culture of Mouse Skin Melanoma Cell Line B16F10" were divided into 6 groups, including 1 control group and 5 experimental groups (ie experimental groups 1 to 5). Cells in each group were cultured in 96-well plates (96-well plate) containing RPMI 1640 medium (supplemented with 10% FBS, 100 μg/mL penicillin and 100 μg/mL streptomycin), and placed in an incubator. (37°C, 5% CO ₂ ) for cultivation. Then, an appropriate amount of the Chinese medicine compound fermentation product obtained in Example 1 was added to the cell cultures of the experimental groups 1 to 5, so that the experimental groups 1 to 5 had a final concentration of 0.05, 0.1, 0.2, 0.25 and 0.5 wt% of traditional Chinese medicine compound fermentation products. In addition, the cell culture of the control group did not contain the traditional Chinese medicine compound fermentation product. After each group of cell cultures was cultured in an incubator (37°C, 5% CO ₂ ) for 48 hours, the liquid in each well was removed, and 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide {3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide} (MTT, 0.5 mg/mL, 100 µL) was added to each well and Incubation was carried out at 37°C for 4 hours. Afterwards, remove the liquid in each well, then add 100 µL of dimethyl sulfoxide (DMSO) and mix well, and then read each well with an ELISA reader (ELISA reader) at a wavelength of 570 nm. Absorbance (OD ₅₇₀ ) of the well.

The cell viability percentage (%) is calculated by substituting the obtained cell number into the following formula (1): Formula (1) : A = (B/C) × 100 where: A = cell viability percentage ( %) B = the OD ₅₇₀ absorbance value measured by each group C = the OD ₅₇₀ absorbance value measured by the control group

Afterwards, the obtained experimental data were analyzed according to the method described in item 1 "Statistical Analysis" of the above "General Experimental Methods". result:

Figure 1 shows the measured cell viability percentage of B16F0 cells after being treated with different concentrations of fermentation products of traditional Chinese medicine compound. It can be seen from FIG. 1 that there is no significant difference in the viability percentage of cells in each experimental group compared with the control group. The result of this experiment shows that the fermentation product of the traditional Chinese medicine compound according to the present invention has no cytotoxicity and will not cause damage to the skin. Example 3. Efficacy evaluation of the fermentation product of the traditional Chinese medicine compound of the present invention in inhibiting melanogenesis in vitro

In this example, the applicant used α-melanocyte stimulating hormone (α-MSH) to induce melanin production in B16F0 cells, and measured melanin content and tyrosinase activity (tyrosinase activity) to evaluate the effectiveness of the fermentation product of the Chinese medicine compound of the present invention in inhibiting melanin production. Experimental method: A , treatment of α -MSH and the fermentation product of the traditional Chinese medicine compound of the present invention:

First, the B16F0 cells that were subcultured according to item 2 of the above "General Experimental Materials""Source and Culture of Mouse Skin Melanoma Cell Line B16F0" were divided into 5 groups, including 1 normal control group , 1 pathological control group (pathological control) and 3 experimental groups (ie, experimental groups 1 to 3). Each group of cells was cultured in a 6-well culture plate containing RPMI 1640 medium (supplemented with 10% FBS, 100 μg/mL penicillin and 100 μg/mL streptomycin) at a quantity of 2×10 ⁵ cells/well and cultured overnight in an incubator (37°C, 5% CO ₂ ).

Next, the cell cultures of the pathological control group and experimental groups 1 to 3 were replaced with RPMI medium supplemented with 100 nM α-MSH to induce melanin production, and the experimental groups 1 to 3 were additionally added with an appropriate amount of The traditional Chinese medicine compound fermentation product obtained in Example 1, so that the experimental groups 1 to 3 respectively have a final concentration of 0.05, 0.2 and 0.5% (w/w) of the traditional Chinese medicine compound fermentation product. In addition, the cell culture of the normal control group was replaced with fresh RPMI medium not containing α-MSH. After each group was cultured in an incubator (37° C., 5% CO ₂ ) for 48 hours, the cell cultures of each group were harvested. B. Determination of melanin content :

The determination of melanin content is carried out with reference to the method described in SY Park et al. (2011), Arch. Dermatol. Res. , 303(10):737-744. Briefly, the cell culture obtained in item A above was washed twice with PBS, followed by the addition of 2N NaOH (in 10% DMSO) and mixing well, followed by heat treatment at 80°C It took 1 hour. Afterwards, the resulting mixture was centrifuged at 3,000 rpm for 3 minutes, and then 100 μL of supernatant was collected, and the absorbance (OD ₄₀₅ ) was measured with an ELISA reader at a wavelength of 405 nm.

The melanin content (%) is calculated by substituting the measured absorbance value (OD ₄₀₅ ) into the following formula (2): Formula (2) : D=(E/F)×100 where: D=melanin content (%) E=the OD ₄₀₅ absorbance value measured by each group F=the OD ₄₀₅ absorbance value measured by the normal control group

Afterwards, the obtained experimental data were analyzed according to the method described in item 1 "Statistical Analysis" of the above "General Experimental Methods". C , the mensuration of tyrosinase activity (tyrosinase activity) :

The determination of cell tyrosinase activity is generally carried out with reference to the method described in Y. Ye et al. (2010), J. Ethnopharmacol. , 129(3):387-90 with slight modifications. Briefly, each group of cell cultures obtained in item A above was washed with ice-cold PBS twice in total, and then centrifuged. Afterwards, the supernatant was removed and a dissolution buffer (lysis buffer) [PBS containing 1% (v/v) Triton X-100, pH 6.8] was added respectively and mixed well. Afterwards, the resulting cell mixture was frozen for 15 minutes, and then moved to room temperature for thawing for 2 minutes. Next, the obtained cell lysate (cell lysate) was processed, and then the supernatant was collected and 100 μL of 2.5 mM 3,4-dihydroxyphenylalanine (3,4-dihydroxyphenylalanine, DOPA) solution (mixed in 0.1 M in PBS, pH 7.0) and the reaction was carried out at 37°C for 1 hour. Afterwards, the absorbance (OD ₄₉₀ ) of the formed mixture was measured with an ELISA reader at a wavelength of 490 nm.

Tyrosinase activity (%) was calculated by substituting the measured absorbance (OD ₄₉₀ ) into the following formula (3): Formula (3) : G=(H/I)×100 where: G =tyrosinase activity (%) H=the OD ₄₉₀ absorbance value measured by each group I=the OD ₄₉₀ absorbance value measured by the normal control group

Afterwards, the obtained experimental data were analyzed according to the method described in item 1 "Statistical Analysis" of the above "General Experimental Methods". Results: A. Determination of melanin content:

Figure 2 shows the measured melanin content of B16F0 cells induced to produce melanin in each group after being treated with different concentrations of fermentation products of Chinese herbal medicine. It can be seen from FIG. 2 that the melanin content of the pathological control group is significantly higher than that of the normal control group, which indicates that α-MSH can successfully induce melanin production in B16F0 cells. Compared with the pathological control group, the melanin content of experimental groups 1 to 3 has a downward trend, and it will become more obvious with the increase of the concentration of the fermentation product of the traditional Chinese medicine compound. Among them, the melanin content of experimental group 3 It was significantly lower than that of the pathological control group. In particular, the tyrosinase activity of the experimental group 3 was even slightly lower than that of the normal control group. The experimental results show that the fermentation product of the traditional Chinese medicine compound of the present invention has an excellent inhibitory effect on the induced melanin production. B , the determination of tyrosinase activity:

Figure 3 shows the tyrosinase activity measured after the B16F0 cells induced to produce melanin in each group were treated with different concentrations of fermentation products of traditional Chinese medicine compound. It can be seen from Figure 3 that the tyrosinase activity of the pathological control group was significantly higher than that of the pathological control group, which indicated that α-MSH could effectively promote the activity of tyrosinase, thereby inducing the melanin production of B16F0 cells. Compared with the pathological control group, the tyrosinase activity of the experimental groups 1 to 3 all had a downward trend, and it became more obvious with the increase of the concentration of the fermentation product of the traditional Chinese medicine compound. Among them, the experimental group 3 The tyrosinase activity was significantly lower than that of the pathological control group. In particular, the tyrosinase activity of the experimental group 3 was even slightly lower than that of the normal control group. The experimental results show that the fermentation product of the traditional Chinese medicine compound of the present invention can effectively inhibit the tyrosinase activity of melanocytes induced to produce melanin. Example 4. Efficacy evaluation of the fermentation product of the traditional Chinese medicine compound of the present invention in the in vivo ( in vivo ) anti-ultraviolet - induced skin damage (UV-induced skin damage)

In this example, the applicant used ultraviolet light to induce skin damage in mice, and evaluated the anti-ultraviolet-induced skin damage effect of the fermentation product of the traditional Chinese medicine compound of the present invention in vivo by histopathological analysis. Experimental materials: 1. Experimental animals:

The male C57BL/6J mice used in this example were purchased from the National Laboratory Animal Center, R.O.C. All experimental animals were kept in an individually air-conditioned animal room with 12 hours of light and 12 hours of darkness, room temperature maintained at 22±2°C and relative humidity maintained at 50±5%, and water and feed were adequately supplied. The handling of experimental animals and all experimental procedures are approved by the Institutional Animal Care and Use Committee of China Medical University, and according to the National Institutes of Health (National Institutes of Health, NIH) in accordance with the Guidelines for the Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals). 2. Preparation of test solutions of different concentrations of traditional Chinese medicine compound fermentation products:

An appropriate amount of the traditional Chinese medicine compound fermentation product obtained in Example 1 above is dissolved in propylene glycol-ethanol aqueous solution [wherein propylene glycol: ethanol: water = 1: 1: 8 (v/v/v)], and is formulated into A test solution of 12% (w/w) Chinese medicine compound fermentation product is ready for use. Experimental method: A , the application of the fermentation product of the Chinese medicine compound of the present invention and the irradiation of ultraviolet rays:

First, the backs of all the mice were shaving, and randomly divided into a normal control group, an ultraviolet control group and an experimental group. Then, smear the test solution on the back of the mice in the experimental group (the dosage is: apply 100 μL of a 6% (w/w) Chinese medicine compound fermentation product solution to a fixed skin area), and the normal control group And the back of the mice in the ultraviolet control group were smeared with 100 μL of propylene glycol-ethanol aqueous solution.

After the back skin of the mice is completely dry (about 2 hours), use an ultraviolet irradiation instrument [equipped with a UV-B lamp] to irradiate the back skin of the mice in the ultraviolet control group and the experimental group with ultraviolet light, each time a single The ultraviolet dose (single UV-B dosage) was 150 mJ/cm ² . As for the mice in the normal control group, they did not receive any ultraviolet radiation treatment.

The application of the fermentation product of the above-mentioned traditional Chinese medicine compound-ultraviolet irradiation treatment was carried out 3 times a week for a total of 8 weeks. Afterwards, the mice of each group were sacrificed, and the dorsal skin tissue of the mice of each group was collected using surgical scissors. B. Preparation of tissue slices:

Soak the back skin tissue of each group of mice obtained in item A above in fixative solution [which contains formaldehyde: acetic acid: 70% (v/v) ethanol = 1:1:20 (v/v/v) ] for 24 hours, followed by dehydration with 70%, 90% and 100% (v/v) ethanol in sequence, and then replacing ethanol with xylene and soaking for 48 hours. Afterwards, the fixed and dehydrated tissue samples are embedded in paraffin, and then sliced to obtain tissue sections. After that, they were used for the staining of items C to E below. C. Collagen staining : _

Collagen was stained by Masson's staining using a Trichrome Stain (Masson) Kit (purchased from Sigma-Aldrich), and then observed and photographed using an optical microscope. D , epidermis staining :

The staining of the epidermis was performed using hematoxylin-eosin according to well-known and routine techniques for those skilled in the art, followed by observation and photographing using an optical microscope. E. Melanin staining:

Melanin was stained by Fontana Masson staining using a NovaUltra™ Fontana Masson staining kit, followed by observation and photographing using an optical microscope. Results: A. Collagen staining:

Figure 4 shows the results observed by Masson's staining of the back skin tissues of the mice in each group. It can be seen from Figure 4 that, compared with the mice in the normal control group, the collagen layer of the mice in the ultraviolet control group was significantly thinner, which means that ultraviolet irradiation can induce collagen degradation in mouse skin. Compared with mice in the UV control group, mice in the experimental group had significantly thicker collagen layers. The results of this experiment show that the fermentation product of the traditional Chinese medicine compound of the present invention can effectively prevent and/or improve the thinning of the collagen layer induced by ultraviolet radiation. B. Epidermis staining :

FIG. 5 shows the results observed by hematoxylin-eosin staining of the back skin tissues of the mice in each group. It can be seen from Figure 5 that compared with the mice in the normal control group, the epidermis (marked by the red line) of the mice in the ultraviolet control group was obviously thicker, and the surface was less smooth, which indicated that ultraviolet radiation could induce skin damage in mice. The epidermis thickens and produces fine lines. Compared with the mice in the ultraviolet control group, the epidermis of the mice in the experimental group was significantly thinner, and even thinner and smoother than that of the normal control group. The experimental results show that the fermentation product of the traditional Chinese medicine compound of the present invention can effectively prevent and/or improve the thickening of the epidermis induced by ultraviolet radiation. C. Melanin staining:

Fig. 6 shows the results observed by Fontana-Masson staining of the back skin tissues of mice in each group. It can be seen from FIG. 6 that, compared with the mice in the normal control group, more melanin granules (melanin granules) were obviously formed in the skin tissue of the mice in the ultraviolet control group. Compared with the mice in the ultraviolet control group, the melanin granules in the skin tissue of the mice in the experimental group were greatly reduced. The experimental results show that the fermentation product of the traditional Chinese medicine compound of the present invention can effectively prevent and/or improve the pigmentation induced by ultraviolet radiation.

Based on the above experimental results, it can be seen that the fermentation product of the traditional Chinese medicine compound of the present invention can effectively improve the damage caused by ultraviolet rays to the skin (including thickening of the epidermis, melanin production and collagen reduction), and is expected to prevent and/or delay photoaging. The effect.

While the invention has been described with reference to specific examples thereof, obviously many modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the claims attached hereto.

The above-mentioned and other objects, features and advantages of the present invention will become apparent after referring to the following detailed description, preferred embodiments and accompanying drawings, wherein: Fig. 1 is B16F0 cells treated with different The measured cell viability percentage after the fermented product of compound Chinese herbal medicine at different concentrations; Figure 2 shows the melanin content measured after being treated with fermented product of compounded Chinese herbal medicine at different concentrations in B16F0 cells induced by each group, wherein "*" means: when compared with the normal control group, p <0.05; and "##" means: when compared with the pathological control group, p <0.01; The tyrosinase activity measured after treating the fermentation products of Chinese herbal compound with different concentrations, where "***" means: when compared with the normal control group, p <0.001; and "###" means: when Compared with the pathological control group, p <0.001; Figure 4 shows the results observed by Masson's staining of the back skin tissues of mice in each group after 8 weeks of ultraviolet radiation; Figure 5 shows the results of 8 weeks of ultraviolet radiation After the irradiation, the back skin tissues of mice in each group were observed by hematoxylin-eosin staining; and Figure 6 shows that after 8 weeks of ultraviolet irradiation, the back skin tissues of mice in each group were stained by Fontana - Observed by Masson staining.

Claims (9)

A method for preparing a fermented product of a compound Chinese medicine, comprising: Using a medium containing a traditional Chinese medicine compound to ferment and cultivate Lactobacillus rhamnosus, and collecting the obtained fermentation culture, Wherein, the traditional Chinese medicine compound contains Atractylodes macrocephala, Bletilla striata, Poria cocos, Poria cocos, Baixian, peony, Tribulus terrestris, and pearl powder. The method of claim 1, wherein the Chinese medicine compound has a content of 1 g/L based on the total weight of the culture medium. The method of claim item 1, wherein the calculation is based on the total weight of the Chinese medicine compound, and the Chinese medicine compound contains: 12.5% (w/w) Atractylodes macrocephala; 12.5% (w/w) bletilla striata; 12.5% (w/w) white pomegranate; 12.5% (w/w) Poria cocos; 12.5% (w/w) white fresh; 12.5% (w/w) peony; 12.5% (w/w) Tribulus terrestris; and 12.5% (w/w) pearl powder. The method according to claim 1, wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus GG (BCRC 16000). The method according to claim 1, further comprising performing a separation process on the fermentation culture to obtain a fluid free of bacterial cells. A fermented product of a compound traditional Chinese medicine, which is prepared by using a method as described in any one of claims 1 to 5. A fermented product of a traditional Chinese medicine compound according to claim 6 is used for preparing a medicine or cosmetic for slowing down ultraviolet rays. The use according to claim 7, wherein the ultraviolet damage includes photoaging. A fermented product of a traditional Chinese medicine compound according to claim 6 is used for preparing a medicine or cosmetic for inhibiting melanin production.

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