A fermented dendrobium extract, a preparation method thereof, and use thereof in a whitening cosmetic

Abstract

The present application provides a kind of fermented dendrobium extract and preparation method, by the unique new generation metabolite, still directional conversion the active ingredient of original dendrobium, generates the rare compound of more active;The present application further provides the use of fermented dendrobium extract in whitening cosmetics, belongs to the field of cosmetics and biotechnology, the product of fermented dendrobium prepared by lactobacillus plantarum fermentation process, not only directional conversion the active ingredient of original dendrobium, also generates a variety of active new generation metabolite and rare compound, the tyrosinase enzyme inhibitory activity and melanin production inhibition effect are significantly enhanced, to overcome the deficiency of the low whitening activity of existing dendrobium extract, lack of characteristic active ingredient spectrum.The present application fermentation process sucrose etc. component is completely consumed, macromolecular substance is degraded, reduces the potential irritability to skin.

Description

Technical Field

[0001] This invention provides a fermented Dendrobium extract and its preparation method. Through unique new metabolites, the original active ingredients in Dendrobium are also directionally transformed to generate rare compounds with stronger activity. This invention further provides the use of fermented Dendrobium extract in whitening cosmetics, belonging to the field of bio-extraction and cosmetic technology. Background Technology

[0002] Excessive melanin deposition is the direct cause of dull skin tone and age spots. Tyrosinase is a key rate-limiting enzyme in the melanin biosynthesis pathway, catalyzing the hydroxylation of L-tyrosine to L-DOPA, which is further oxidized to dopaquinone, ultimately forming melanin through a series of reactions. Therefore, inhibiting tyrosinase activity is one of the core strategies for screening and evaluating skin-whitening active ingredients.

[0003] Arbutin, a natural glycoside tyrosinase inhibitor extracted from bearberry leaves, is a popular whitening agent in cosmetics. Kojic acid, a fungal metabolite, is a competitive inhibitor of tyrosinase, but it can irritate the skin. Vitamin C and its derivatives work by reducing melanin intermediates, but their stability is poor. Each of these whitening agents has its limitations: arbutin and kojic acid only achieve satisfactory inhibitory effects at medium to high concentrations, while vitamin C has poor stability. Therefore, developing novel, highly effective, safe, and naturally derived whitening active ingredients has significant industrial value and market demand.

[0004] Dendrobium spp., a perennial herb belonging to the genus Dendrobium in the Orchidaceae family, is a traditional and precious Chinese medicinal herb. Modern research shows that Dendrobium contains various active ingredients such as bibenzyls, stilbenoids, flavonoids, and polysaccharides, exhibiting antioxidant, anti-inflammatory, and immunomodulatory biological activities. Existing literature reports that Dendrobium extracts possess certain tyrosinase inhibitory activity; for example, Dendrobium officinale extract has been used to prepare skin-whitening compositions (CN201611152415, CN202410620467, etc.). However, the tyrosinase inhibitory activity of traditional Dendrobium extracts is relatively weak, usually requiring higher concentrations to achieve satisfactory results. Furthermore, its active ingredients are mainly native metabolites, lacking rare, highly active metabolites produced through microbial transformation. Summary of the Invention

[0005] This invention provides a fermented Dendrobium extract and its preparation method. The fermentation process using Lactobacillus plantarum produces a large amount of fermented and directionally transforms the original active ingredients in Dendrobium, generating new metabolites and rare compounds with stronger activity.

[0006] The fermented Dendrobium product generated by the preparation method described in this invention has significantly enhanced tyrosinase inhibition activity and melanin production inhibition effect, thus overcoming the shortcomings of existing Dendrobium extracts, such as low whitening activity and lack of characteristic active ingredient spectrum.

[0007] The present invention discloses a method for preparing fermented Dendrobium extract, comprising two steps: extraction and fermentation. Step 1: Preparation of extract: Take dried Dendrobium officinale powder, add 20-30 times the amount of softened water, and add the following nutrient fortifiers: yeast extract 5-10 g / L, K2HPO4 1-2 g / L, sodium acetate 3-5 g / L, MgSO4·7H2O 0.2-0.5 g / L, MnSO4 0.05-0.08 g / L, Tween 80 1-2 mL / L, and autoclave for extraction and sterilization. Step 2: Anaerobic fermentation. After the sterilized extract is cooled, 5%~10% (v / v) of Lactobacillus plantarum activated on MRS medium is inoculated and cultured anaerobically for 24~48 hours. After fermentation, the extract is centrifuged and filtered to obtain the fermented Dendrobium extract.

[0008] Preferably, the extract in step one is prepared according to the following formula: the ratio of Dendrobium powder to softened water is 1:20 (w / v), yeast extract 8 g / L, K2HPO4 2 g / L, sodium acetate 3 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.05 g / L, and Tween 80 1 mL / L.

[0009] The fermented Dendrobium extract prepared by the above method has a characteristic component spectrum that distinguishes it from the unfermented Dendrobium extract. Full-spectrum identification by UHPLC-Q-Orbitrap MS revealed that it contains one or more of the following markers: The first category consists of fermentation biomarkers, which were not detected in unfermented Dendrobium extract. These include: alpha-ketoglutarate (≥20,000 μg / g), N1,N4-bis[N-(3,4,5-trimethoxyphenyl)carbamoimido]-1,4-piperazine dicarboxamide (≥18,000 μg / g), oleoyl-L-α-lysophosphatidylcholine (≥6,000 μg / g), and procholinesterone (≥1,500 μg / g). μg / g), 4-[(butylcarbamoyl)amino]-1-ethyl-1H-pyrazole-5-carboxamide (content ≥700 μg / g), (2S)-3-(4-hydroxyphenyl)-2-({[(3S,4S,5R)-2,3,4-trihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]methyl}amino)propionic acid (content ≥700 μg / g), 3-isopropylmalic acid (content ≥150 μg / g), norsine (content ≥120 μg / g), glycocholic acid (content ≥100 μg / g), and leucopicrin (content ≥35 μg / g).

[0010] The second category consists of biomarkers that show a significant increase in fermentation compared to unfermented Dendrobium extract, including: N-[2-(3,4-dimethoxyphenyl)ethyl][1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (≥200-fold increase), 4-pyridoxine (≥1,000-fold increase), 4-[5-hydroxy-6-(2-hydroxy-3-methyl-4-methylenetetrahydro-2-furan)- 1,3-Dioxane-4-yl]-2,6-piperidinedione (≥100-fold increase), 1-[(1-cyanocyclohexyl)amino]-1-oxo-2-propyl(1,3-dimethyl-2,6-dione-1,2,3,6-tetrahydro-7H-purine-7-yl)acetate (≥1,000-fold increase), DL-tyrosine (≥40-fold increase), isochlorogenic acid C (≥10-fold increase and content ≥600 μg / g), nicotinamide (≥25-fold increase), (15Z)-9,12,13-trihydroxy-15-octadecenoic acid (≥20-fold increase), methyl-D-erythritol phosphate (≥20-fold increase).

[0011] The present invention also provides the application of the above-mentioned fermented Dendrobium extract in the preparation of whitening, spot-removing and skin-brightening cosmetic compositions.

[0012] The fermented Dendrobium extract of the present invention exhibits superior tyrosinase inhibitory activity at a concentration of 100 μg / mL compared to 0.5 mM (approximately 136 μg / mL) arbutin, and its whitening activity is significantly enhanced and significantly higher than that of the unfermented Dendrobium extract at the same concentration.

[0013] Targeted generation of rare active substances: During fermentation, resveratrol is directionally converted into paclitaxel, flavonoid glycosides hydrolyze to release luteolin, and monocaffeoylquinic acid is converted into isochlorogenic acid C, producing more active rare compounds. Fourth, the process is simple and cost-controllable: The extraction and fermentation are integrated into one design, eliminating the need for separate fermentation culture medium preparation; it uses commonly used safe strains and conventional equipment in the cosmetics industry.

[0014] The positive effects of this invention are as follows:

[0015] The fermented Dendrobium officinale product prepared through the fermentation process of Lactobacillus plantarum not only directionally transforms the original active ingredients in Dendrobium officinale, but also generates more than 20 new metabolites and rare compounds with stronger activity. This significantly enhances the inhibitory activity on tyrosinase and the inhibition of melanin production, overcoming the shortcomings of existing Dendrobium officinale extracts, such as low whitening activity and a lack of characteristic active ingredient profiles. In this invention's fermentation process, components such as sucrose are completely consumed, and large molecules are degraded, reducing potential skin irritation. Attached Figure Description

[0016] Figure 1 This is the positive ion mode of the total ion current (TIC) spectrum of fermented Dendrobium in this invention; Figure 2 This is the negative ion mode of the total ion current (TIC) diagram of fermented Dendrobium in this invention; Figure 3 This is the positive ion mode of the total ion current (TIC) spectrum of the Dendrobium extract of the present invention; Figure 4 This is the negative ion mode of the total ion current (TIC) diagram of the Dendrobium extract of the present invention; Figure 5 The results of the fermented Dendrobium of this invention are compared with those of Dendrobium and arbutin tyrosinase inhibition experiments. Figure 6 This is a comparison of the melanin inhibition test results of fermented Dendrobium and Dendrobium and arbutin in this invention. Detailed Implementation

[0017] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention. Example 1

[0018] Step 1: Take dried Dendrobium officinale (iron-skin Dendrobium), pulverize it to 3060 mesh, add softened water at a material-to-liquid ratio of 1:20 (w / v), and then add nutrient fortifiers according to the following formula: yeast extract 8 g / L, K2HPO4 2 g / L, sodium acetate 3 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.05 g / L, and Tween 80 1 mL / L. Place the mixture in an autoclave and treat it at 115°C for 30 minutes to achieve simultaneous extraction of Dendrobium officinale active ingredients and sterilization of the culture medium.

[0019] Step 2: After sterilization, cool the extract to 35-40°C and inoculate it with Lactobacillus plantarum culture that has been activated and cultured to the logarithmic phase in MRS medium under aseptic conditions. The inoculation amount is 10% (v / v). Transfer it to an anaerobic fermenter and incubate it in aerobic culture at 37°C for 36 hours.

[0020] Step 3: After fermentation, centrifuge the fermentation broth at 4°C and 8000 rpm for 10 minutes, take the supernatant, filter it through a 0.22 μm microporous membrane for sterilization, and collect the filtrate to obtain the fermented Dendrobium extract. Example 2

[0021] Step 1: Grind dried Dendrobium officinale into powder to 3060 mesh. Add softened water at a material-to-liquid ratio of 1:20 (w / v), and then add nutrient fortifiers according to the following formula: yeast extract 5 g / L, K2HPO4 1 g / L, sodium acetate 3 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.05 g / L, and Tween 80 1 mL / L. Place the mixture in an autoclave and treat at 115°C for 30 minutes.

[0022] Steps two and three are the same as in Example 1. Example 3

[0023] Step 1: Grind dried Dendrobium officinale into powder to 3060 mesh. Add softened water at a material-to-liquid ratio of 1:30 (w / v), and then add nutrient fortifiers according to the following formula: yeast extract 10 g / L, K2HPO4 2 g / L, sodium acetate 5 g / L, MgSO4·7H2O 0.5 g / L, MnSO4 0.08 g / L, and Tween 80 2 mL / L. Place the mixture in an autoclave and treat at 115°C for 30 minutes.

[0024] Steps two and three are the same as in Example 1. Example 4

[0025] Full-spectrum component analysis of Dendrobium officinale extract The fermented Dendrobium officinale extract (ZLFJ-01) prepared in Example 1 and the control unfermented Dendrobium officinale extract (ZLTQ-01, prepared in the same way as in Example 1 but omitting the inoculation and fermentation steps) were identified by ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap mass spectrometry (UHPLC-Q-Orbitrap MS) in positive ion mode and negative ion mode, respectively.

[0026] Chromatographic conditions: The column was an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm); mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution program: 0-2 min, 5% B; 2-15 min, 5%-95% B; 15-17 min, 95% B; 17-17.5 min, 95%-5% B; 17.5-20 min, 5% B; flow rate 0.3 mL / min; column temperature 40°C; injection volume 2 μL.

[0027] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion modes were collected separately; scan range m / z 100~1500; resolution 70,000 (MS full scan); capillary temperature 350°C; spray voltage 3.5 kV (positive ion) / 2.8 kV (negative ion).

[0028] 729 compounds were identified using the positive ion mode and 322 compounds were identified using the negative ion mode, such as... Figures 1-4 As shown.

[0029] The following experimental examples demonstrate the positive effects of the present invention.

[0030] Experimental Example 1:

[0031] (1) Fermentation biomarkers: The following compounds were not detected in the unfermented Dendrobium extract but were present in significant amounts in the fermented Dendrobium extract, including: alpha-ketoglutarate (22,156 μg / g), N1,N4-bis[N-(3,4,5-trimethoxyphenyl)carbamoylimino]-1,4-piperazine dicarboxamide (20,037 μg / g), oleoyl-L-α-lysophosphatidic acid (6,664 μg / g), procholinesterase (1,693 μg / g), and 4-[(butylcarbamoyl)amino]-1-ethyl-1H-pyrazole-5-carboxamide (758 μg / g). μg / g), (2S)-3-(4-hydroxyphenyl)-2-({[(3S,4S,5R)-2,3,4-trihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]methyl}amino)propionic acid (754 μg / g), 3-isopropylmalic acid (173 μg / g), norsine (134 μg / g), glycocholic acid (115 μg / g), leucine (41 μg / g), 4-[(2-cyclohexenylethyl)amino]-4-oxobut-2-enoic acid (79 μg / g), taurine (55 μg / g), N-(3-hydroxyphenyl)acetamide (137 μg / g).

[0032] (2) Significantly increased markers of fermentation: Compared with unfermented Dendrobium extract, the following compounds were significantly increased in fermented Dendrobium extract: 4-pyridoxine (4,816 μg / g, an increase of 1,152 times), N-[2-(3,4-dimethoxyphenyl)ethyl][1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (27,549 μg / g, an increase of 215 times), 1,2-di-O-methyl-4-[(2R)-2,4-dihydrobutamido]-4,6-dideoxy-α-D-mannopyranoside (20,825 μg / g). μg / g, an increase of 119 times), 4-[5-hydroxy-6-(2-hydroxy-3-methyl-4-methylenetetrahydro-2-furan)-1,3-dioxane-4-yl]-2,6-piperidinedione (5,666 μg / g, an increase of 120 times), 1-[(1-cyanocyclohexyl)amino]-1-oxo-2-propyl(1,3-dimethyl-2,6-dione-1,2,3,6-tetrahydro-7H-purine-7-yl)acetate (1,155 μg / g, an increase of 1,117 times), DL-tyrosine (1,050 μg / g, an increase of 49 times), isochlorogenic acid C (678 μg / g, an increase of 11 times), nicotinamide (493 μg / g, an increase of 29 times), (15Z)-9,12,13-trihydroxy-15-octadecenoic acid (2,221 μg / g, an increase of 119 times), 4-[5-hydroxy-6-(2-hydroxy-3-methyl-4-methylenetetrahydro-2-furan)-1,3-dioxane-4-yl]-2,6-piperidinedione (5,666 μg / g, an increase of 120 times), 1-[(1-cyanocyclohexyl)amino]-1-oxo-2-propyl(1,3-dimethyl-2,6-dione-1,2,3,6-tetrahydro-7H-purine-7-yl)acetate (1,155 μg / g, an increase of 1,117 times), DL-tyrosine (1,050 μg / g, an increase of 49 times), isochlorogenic acid C (678 μg / g, an increase of 11 times), nicotinamide μg / g, an increase of 24 times), methyl-D-erythritol phosphate (17,808 μg / g, an increase of 23 times).

[0033] (3) Evidence of fermentation transformation: Resveratrol (1.30 μg / g, positive ion mode; 3.96 μg / g, negative ion mode) and dihydroresveratrol (8.25 μg / g, negative ion mode) detected in the unfermented Dendrobium extract completely disappeared after fermentation, while its hydroxylation transformation product, paclitaxel (40.83 μg / g), was newly formed in the fermented Dendrobium extract. Rutin (2.02 μg / g), isoquercitrin (18.22 μg / g, negative ion mode), vitexin glucoside (7.96 μg / g), and other flavonoid glycosides detected in the unfermented Dendrobium extract completely disappeared after fermentation, while luteolin (29.03 μg / g) was newly formed. Phenolic acids such as caffeic acid (9.89 μg / g, negative ion mode), chlorogenic acid (0.69~42.05 μg / g), and cryptochlorogenic acid (7.67 μg / g) detected in unfermented Dendrobium extract completely disappeared after fermentation. At the same time, the content of isochlorogenic acid C increased significantly from 61.24 μg / g to 678.45 μg / g (an 11-fold increase), and the content of isochlorogenic acid B increased from 5.60 μg / g to 37.06 μg / g (a 7-fold increase).

[0034] The above results indicate that fermentation treatment with Lactobacillus plantarum significantly altered the composition profile of Dendrobium extract, not only producing a large number of fermentation-specific new metabolites, but also directionally transforming the original active ingredients in Dendrobium, generating rare compounds with stronger activity.

[0035] Experimental Example 2:

[0036] Samples to be tested: Fermented Dendrobium officinale extract (ZLFJ-01), unfermented Dendrobium officinale extract (ZLTQ-01), and arbutin (Sigma-Aldrich) prepared in Example 1. Cell line: B16-F10 mouse melanoma cells (ATCC).

[0037] B16-F10 cells were seeded in 96-well plates (1×10⁻⁶ cells per well). 4 Cells / well were cultured for 24 hours. Different concentrations of fermented Dendrobium officinale extract (25, 50, 100, 200 μg / mL), unfermented Dendrobium officinale extract (25, 50, 100, 200 μg / mL), and arbutin (0.5 mM, i.e., 136 μg / mL) were added. Simultaneously, α-MSH (100 nM) was added to induce melanin production. After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with PBS. A 1% Triton X-100 solution in PBS (containing 1 mg / mL L-DOPA) was added to each well, and the cells were incubated at 37°C for 1 hour. The absorbance was measured at 475 nm, and the relative tyrosinase activity was calculated.

[0038] The results showed that the fermented Dendrobium officinale extract at a concentration of 100 μg / mL significantly inhibited tyrosinase activity in B16-F10 cells compared to the same concentration of unfermented Dendrobium officinale extract, and was also superior to 0.5 mM (136 μg / mL) arbutin. Within the tested concentration range, the inhibition of tyrosinase activity by the fermented Dendrobium officinale extract was concentration-dependent. Specifically, the inhibition rate of tyrosinase activity by the 100 μg / mL fermented Dendrobium officinale extract was more than 2 times higher than that by the same concentration of unfermented Dendrobium officinale extract. The inhibitory effect of the 200 μg / mL fermented Dendrobium officinale extract on tyrosinase activity was further enhanced (see [link to relevant documentation]). Figure 5 ).

[0039] The above results indicate that the tyrosinase inhibitory activity of Dendrobium extract was significantly enhanced by fermentation treatment with Lactobacillus plantarum, and it achieved a better effect than the classic whitening agent arbutin (0.5 mM) at a low concentration (100 μg / mL).

[0040] Experimental Example 3:

[0041] B16-F10 cells were seeded in 6-well plates (2 × 10⁻⁶ cells per well). 5 Cells / well were cultured for 24 hours. Different concentrations of fermented Dendrobium officinale extract (25, 50, 100, 200 μg / mL), unfermented Dendrobium officinale extract (25, 50, 100, 200 μg / mL), and arbutin (0.5 mM, i.e., 136 μg / mL) were added, along with α-MSH (100 nM) to induce melanin production. After 72 hours of culture, the culture medium was discarded, and the cells were washed twice with PBS. 1 mol / L NaOH (containing 10% DMSO) was added to each well, and the mixture was heated at 80°C for 1 hour to dissolve the melanin. The absorbance was measured at 405 nm, and the relative melanin content was calculated (see [reference]). Figure 6 ).

[0042] Results showed that fermented Dendrobium officinale extract at a concentration of 100 μg / mL significantly inhibited melanin production in B16-F10 cells compared to unfermented Dendrobium officinale extract at the same concentration, and was also superior to 0.5 mM (136 μg / mL) arbutin. Within the concentration range of 25–200 μg / mL, the inhibition of melanin production by fermented Dendrobium officinale extract was concentration-dependent.

[0043] The above results indicate that fermented Dendrobium extract can effectively inhibit melanin production in B16 melanoma cells induced by α-MSH within a safe concentration range. Its inhibitory effect is superior to that of unfermented Dendrobium extract at the same concentration and 0.5 mM arbutin, demonstrating good potential for skin whitening applications.

Claims

1. A fermented Dendrobium extract, characterized in that... It mainly includes the following substances: Alpha-ketoglutaric acid; N1,N4-bis[N-(3,4,5-trimethoxyphenyl)carbamoimidin]-1,4-piperazine dicarboxamide; oleoyl-L-α-lysophosphatidic acid; procholinesterone; 4-[(butylcarbamoyl)amino]-1-ethyl-1H-pyrazole-5-carboxamide; (2S)-3-(4-hydroxyphenyl)-2-({[(3S,4S,5R)-2,3,4-trihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]methyl}amino)propionic acid; 3-isopropylmalic acid; norsine; glycinecholic acid; leucovorin; It also includes: N-[2-(3,4-dimethoxyphenyl)ethyl][1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide; 4-pyridoxic acid; 4-[5-hydroxy-6-(2-hydroxy-3-methyl-4-methylenetetrahydro-2-furan)-1,3-dioxane-4-yl]-2,6-piperidinedione; 1-[(1-cyanocyclohexyl)amino]-1-oxo-2-propyl(1,3-dimethyl-2,6-dione-1,2,3,6-tetrahydro-7H-purine-7-yl)acetate; DL-tyrosine; isochlorogenic acid C; nicotinamide; (15Z)-9,12,13-trihydroxy-15-octadecenoic acid; methyl-D-erythritol phosphate.

2. The method for preparing fermented Dendrobium extract as described in claim 1, comprising two steps: extraction and fermentation: Step 1 is the preparation of the extract: Take dried Dendrobium officinale powder, add 20-30 times the amount of softened water, and add the following nutrient fortifiers: yeast extract 5-10 g / L, K2HPO4 1-2 g / L, sodium acetate 3-5 g / L, MgSO4·7H2O 0.2-0.5 g / L, MnSO4 0.05-0.08 g / L, Tween 80 1-2 mL / L, and autoclave for extraction and sterilization; Step two is anaerobic fermentation: After the sterilized extract is cooled, 5%~10% (v / v) of Lactobacillus plantarum activated on MRS medium is inoculated and anaerobic cultured for 24~48 hours. After fermentation, centrifuge and filter to obtain fermented Dendrobium extract.

3. A method for preparing fermented Dendrobium extract as described in claim 1 or 2, characterized in that: In step one, the extract is prepared according to the following formula: the ratio of Dendrobium powder to softened water is 1:20 (w / v), yeast extract 8 g / L, K2HPO4 2 g / L, sodium acetate 3 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.05 g / L, and Tween 80 1 mL / L.

4. The use of the fermented Dendrobium extract as described in claim 1 in the preparation of whitening cosmetic compositions.

5. The use of the fermented Dendrobium extract as described in claim 1 in the preparation of a skin-brightening and freckle-removing cosmetic composition.

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