Dendrobium candidum sprout extract and use thereof

Abstract

The present application relates to Dendrobium candidum tender bud extract and its use, specifically to the application of Dendrobium candidum tender bud extract obtained by differentiation induction of Dendrobium candidum stem cells in the preparation of products with whitening and / or freckle-removing effects; wherein the differentiation induction is carried out in MS medium containing 0.1-1 g / L NAA, 0.5-2 mg / L KT, 15-30 g / L glucose and 10-100 g / L maltose. The Dendrobium candidum tender bud extract obtained by differentiation of Dendrobium candidum stem cells provided by the present application can significantly inhibit the activity of tyrosinase, thereby achieving the effects of whitening and freckle removal.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of biological skin care products, and in particular to a Dendrobium officinale young shoot extract and application thereof. Background Art

[0002] Dendrobium officinale ( Dendrobium officinale Dendrobium officinale is a perennial herbaceous plant in the genus Dendrobium of the Orchidaceae family. Its stem is upright, cylindrical, unbranched, multi-noded, and has bi-row leaves. Currently, Dendrobium officinale is still obtained through traditional cultivation, resulting in numerous raw material bottlenecks, including wide variations in active ingredients, difficulty quantifying active ingredients, and a shortage of authentic medicinal materials. Plant stem cells reside within specialized structures within the plant's meristem and possess remarkable regenerative abilities. Plant root and shoot stem cells have very similar regulatory mechanisms and effects to animal stem cells, even sharing identical regulatory molecules. Research indicates that young shoots cannot be obtained in large quantities through cultivation.

[0003] At the same time, the efficacy of traditional Dendrobium officinale mainly focuses on strengthening the spleen and stomach, promoting gastric juice secretion, and helping to improve indigestion caused by spleen and stomach disharmony. As for other uses, they have not yet been developed, especially the use of Dendrobium officinale sprout extract in the field of skin care, especially the application related to whitening and freckle removal, which has not been reported.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] In view of the above-mentioned technical defects, the present invention provides an extract of Dendrobium officinale stems (protocorms) differentiated from Dendrobium officinale stem cells and its new use in inhibiting melanin production, thereby achieving whitening and freckle removal.

[0006] To achieve the above purpose, the technical solution of the present invention is:

[0007] The present invention provides a Dendrobium officinale tender shoot extract obtained by inducing differentiation of Dendrobium officinale stem cells, wherein the differentiation induction is carried out in an MS culture medium containing 0.1-1 g / L NAA, 0.5-2 mg / L KT, 15-30 g / L glucose and 10-100 g / L maltose.

[0008] Preferably, the differentiation induction is carried out in MS medium containing 0.3-0.6 g / L NAA, 0.5-1.5 mg / L KT, 15-25 g / L glucose and 40-60 g / L maltose.

[0009] Preferably, the differentiation induction is carried out in MS medium containing 0.5 g / L NAA, 1 mg / L KT, 20 g / L glucose and 50 g / L maltose.

[0010] Preferably, the differentiation induction condition parameters are: light intensity of 1800-2500 Lux, red to blue light ratio of 3-5:1, light exposure time of 6-12 hours per day, and culture for 30-45 days.

[0011] Preferably, the method for preparing the Dendrobium officinale stem cells comprises:

[0012] (a) Stem cell cultivation: Multiple fragments of primary Dendrobium officinale stem cells derived from the tips of young stems of Dendrobium officinale are spaced apart and distributed on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are cultured in the dark. The multiple fragments are spaced apart so that the cultivated Dendrobium officinale stem cell clumps do not contact each other;

[0013] (b) Stem cell screening: Select the Dendrobium officinale stem cell clusters that are obviously growing fast, with uniform color and morphology during cultivation, and cut them into multiple fragments;

[0014] (c) Stem cell subculture: The cut Dendrobium officinale stem cell fragments are distributed at intervals on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are subcultured in the dark; after each generation of culture and before the next subculture, the stem cell screening step (b) is performed to obtain a Dendrobium officinale stem cell cluster from a clone;

[0015] (d) Stem cell screening: Select the D. officinale stem cell clusters that grow significantly faster during cultivation;

[0016] (e) Establishing stem cell lines: The selected Dendrobium officinale stem cell clusters are subcultured on a solid plant stem cell culture medium in the dark; after each subculture, the above-mentioned stem cell screening step (d) is performed before the next subculture until one or more plant stem cell lines are obtained;

[0017] (f) Stem cell line acclimation: The debris of one or more established Dendrobium officinale stem cell lines are distributed at intervals on a solid-state conditioned plant stem cell culture medium, and the Dendrobium officinale stem cells are subcultured under dark conditions; after each generation of cultivation and before the next generation of cultivation, the clumps that are brown or have changed color or have a slow division rate compared with the previous generation are eliminated. After multiple generations of cultivation, one or more Dendrobium officinale stem cells with stable traits, stable division, and fast division are obtained.

[0018] Preferably, the method for preparing Dendrobium officinale stem cells further includes the following steps before step (a): chopping the static center of Dendrobium officinale into fragments, amplifying and cultivating callus tissue under dark conditions, then chopping the callus tissue into fragments, distributing them at intervals in a solid callus induction medium for amplification, and then subculturing the amplified callus tissue; after the end of each generation of cultivation and before the cultivation of the next generation, selecting the amplified callus tissue with consistent color and morphology during cultivation, removing the browned part and dividing it into small pieces, and then distributing them at intervals in a solid callus induction medium, wherein the interval distribution ensures that the callus tissue clumps grown after cultivation do not contact each other, and after multiple generations of cultivation, the primary Dendrobium officinale stem cells are obtained; the obtained primary Dendrobium officinale stem cells are then cultured in step (a).

[0019] Preferably, in the Dendrobium officinale young shoot extract, the polysaccharide content is less than 1 g / L; and / or the alcohol extract content is greater than 1 g / L; and / or the polyphenol content is greater than 100 mg / L; and / or the alkaloid content is greater than 200 mg / L.

[0020] Preferably, the Dendrobium officinale sprout extract is obtained by water extraction or alcohol extraction.

[0021] Preferably, the Dendrobium officinale sprout extract is obtained by water extraction, which comprises the following steps:

[0022] (1) Take fresh Dendrobium officinale shoots and dry them;

[0023] (2) Powdering the dried young shoots of Dendrobium officinale, soaking them, and filtering them to obtain a primary stock solution;

[0024] (3) filtering the primary stock solution step by step to obtain a secondary stock solution;

[0025] (4) Filtering the secondary stock solution again to obtain a Dendrobium officinale extract, namely, a Dendrobium officinale sprout extract.

[0026] Preferably, in step (3), the step of stepwise filtration is as follows: the primary stock solution is filtered sequentially through filters with precisions of 20 μm, 10 μm, 5 μm, 1 μm, and 0.45 μm.

[0027] Preferably, the pressure during the step-by-step filtration is less than 0.2 MPa.

[0028] Preferably, in step (2), the mass ratio of the powder to water is 1:20-1:50, and the soaking time is 2-5 hours.

[0029] Preferably, in step (4), the second filtration is performed using a filter with an accuracy of 0.22 μm.

[0030] The present invention provides use of any of the extracts described above in preparing products with whitening and / or spot-lightening effects.

[0031] Preferably, the product is a cosmetic, and its dosage form includes emulsion, water, oil or gel;

[0032] Preferably, the product is a medicine, and its dosage forms include oils, emulsions, ointments, pastes, coatings, gels, aerosols, sprays, solutions, and liniments.

[0033] The beneficial effects of the present invention are:

[0034] This invention aims to provide a method for cultivating young stems (protocorms) of Dendrobium officinale by utilizing plant stem cell technology and directed differentiation techniques, as well as the safe application of aqueous extracts in skincare products for whitening and freckle removal. This approach aims to enhance the medicinal value and safety of Dendrobium officinale and significantly increase the content and stability of whitening active ingredients. The stem-derived young stem extract provided by this invention can significantly inhibit tyrosinase activity, thereby reducing melanin synthesis and significantly improving whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the cytotoxicity test results of the stem cell-derived Dendrobium officinale extract (SDO) and the commercial Dendrobium officinale extract (DO) provided in this application.

[0036] Figure 2 Schematic diagram of the inhibitory effects of stem cell-derived Dendrobium officinale extract (SDO) and commercial Dendrobium officinale extract (DO) on melanin synthesis in melanocytes provided in this application.

[0037] Figure 3 This is a graph showing the variance analysis results of the metabolomics difference analysis between the stem cell-derived Dendrobium officinale extract (SDO) and the commercial Dendrobium officinale extract (DO) provided in this application, where red indicates downregulated components, blue indicates upregulated components, and green indicates unchanged components. The multiplication factor log2 FC(SDO / DO)≥5, p<0.01. DETAILED DESCRIPTION

[0038] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0039] At present, many whitening products on the market rely on chemical whitening agents, such as hydroquinone, arbutin, etc. Although these ingredients can inhibit melanin production in the short term, long-term use may cause adverse reactions such as skin irritation and photosensitivity, and the effect is limited. Based on this, the inventors of the present invention, after extensive and long-term research, directed the differentiation of Dendrobium officinale stem cells obtained from the Chinese herbal medicine plant stem cell platform established in the early research of the present invention into Dendrobium buds, and verified through metabolomics that the stem cell-derived Dendrobium officinale extract is specifically enriched with key whitening ingredients (such as polyphenols and alkaloids); the extract contains small molecule sugars (such as fructose and sucrose, which enhance skin moisturizing properties), rich alcohol extracts, alkaloids (such as dendrobium alkaloids, which reduce melanin synthesis), polyphenols (such as resveratrol and ferulic acid, which inhibit tyrosinase activity), etc., and its active ingredient content is 5 to 15 times higher than that of traditional Dendrobium officinale. When applied to skin care products, it can effectively inhibit tyrosinase activity, reduce melanin synthesis, and thus achieve the effect of whitening and removing spots. Compared with traditional Dendrobium, the whitening effect of the Dendrobium officinale buds of the present invention is improved by at least 50%.

[0040] The present invention provides a Dendrobium officinale tender shoot extract obtained by inducing differentiation of Dendrobium officinale stem cells, wherein the differentiation induction is carried out in an MS culture medium containing 0.1-1 g / L NAA, 0.5-2 mg / L KT, 15-30 g / L glucose and 10-100 g / L maltose.

[0041] In one embodiment, the concentration of NAA is 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc.

[0042] In one embodiment, the concentration of KT is 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, etc.

[0043] In one embodiment, the concentration of glucose is 16g / L, 17g / L, 18g / L, 19g / L, 20g / L, 21g / L, 22g / L, 23g / L, 24g / L, 25g / L, 26g / L, 27g / L, 28g / L, 29g / L, 30g / L, etc.

[0044] In one embodiment, the concentration of maltose is 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, etc.

[0045] In one embodiment, the differentiation induction is performed in MS medium containing 0.3-0.6 g / L NAA, 0.5-1.5 mg / L KT, 15-25 g / L glucose, and 40-60 g / L maltose.

[0046] In one embodiment, the differentiation induction is performed in MS medium containing 0.5 g / L NAA, 1 mg / L KT, 20 g / L glucose and 50 g / L maltose.

[0047] In one embodiment, the differentiation induction condition parameters are: light intensity of 1800-2500 Lux, red to blue light ratio of 3-5:1, light exposure time of 6-12 hours per day, and culture for 30-45 days.

[0048] In one embodiment, the light intensity is 1800 Lux, 1850 Lux, 1900 Lux, 1950 Lux, 2000 Lux, etc.

[0049] In one embodiment, the ratio of red to blue light is 3.5:1, 4:1, 4.5:1, 5:1, etc.

[0050] In one embodiment, the daily illumination time is 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, etc.

[0051] In one embodiment, the culture is for 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, etc.

[0052] In one embodiment, the differentiation induction condition parameters are: light intensity of 1800-2200 Lux, red to blue light ratio of 3.5-4.5:1, light exposure time of 7-9 hours per day, and culture for 30-45 days.

[0053] The present invention specifically induces Dendrobium officinale stem cells, and the resulting Dendrobium officinale sprouts can effectively inhibit tyrosinase activity and have excellent whitening / freckle removal effects.

[0054] In one embodiment, the polysaccharide content in the Dendrobium officinale young shoot extract is less than 1 g / L.

[0055] In one embodiment, the polysaccharide content in the Dendrobium officinale young shoot extract is less than 0.5 g / L.

[0056] In one embodiment, the polysaccharide content in the Dendrobium officinale young shoot extract is 0.48 g / L.

[0057] In one embodiment, the content of the alcohol extract in the Dendrobium officinale young shoot extract is greater than 1 g / L.

[0058] In one embodiment, the content of the alcohol extract in the Dendrobium officinale young shoot extract is greater than 2 g / L.

[0059] In one embodiment, the content of the alcohol extract in the Dendrobium officinale sprout extract is greater than 2.5 g / L.

[0060] In one embodiment, the content of the alcohol extract in the Dendrobium officinale sprout extract is 2.5-3 g / L.

[0061] In one embodiment, the content of the alcohol extract in the Dendrobium officinale sprout extract is 2.928 g / L.

[0062] In one embodiment, the polyphenol content in the Dendrobium officinale young shoot extract is greater than 100 mg / L.

[0063] In one embodiment, the polyphenol content in the Dendrobium officinale young shoot extract is greater than 250 mg / L.

[0064] In one embodiment, the polyphenol content in the Dendrobium officinale young shoot extract is 300-330 mg / L.

[0065] In one embodiment, the polyphenol content in the Dendrobium officinale young shoot extract is 319.2 mg / L.

[0066] In one embodiment, the alkaloid content in the Dendrobium officinale young shoot extract is greater than 200 mg / L.

[0067] In one embodiment, the alkaloid content in the Dendrobium officinale young shoot extract is greater than 400 mg / L.

[0068] In one embodiment, the alkaloid content in the Dendrobium officinale young shoot extract is 430-480 mg / L.

[0069] In one embodiment, the alkaloid content in the Dendrobium officinale young shoot extract is 463.6 mg / L.

[0070] In one embodiment, resveratrol and rutin are highly expressed in the Dendrobium officinale young shoot extract.

[0071] Furthermore, in a certain embodiment, in the Dendrobium officinale extract, cinnamic acid, ferulic acid, caffeic acid phenethyl ester, acetyl glycitin, hexylresorcinol, thiamine, nifedipine, (±)-equol, caffeine, isoferulic acid, isoliquiritigenin, demethoxycurcumin, vitamin A, and eriodictyol are all highly expressed.

[0072] In the present application, the Dendrobium officinale stem cells are obtained through the Chinese herbal medicine plant stem cell technology platform established in the preliminary research of the present invention. The method of the technology platform has been recorded in the patent document CN 116445393B, and the contents disclosed in the above document are incorporated herein by reference in their entirety.

[0073] In one embodiment, the method for preparing the Dendrobium officinale stem cells comprises:

[0074] (a) Stem cell cultivation: Multiple fragments of primary Dendrobium officinale stem cells derived from the tips of young stems of Dendrobium officinale are spaced apart and distributed on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are cultured in the dark. The multiple fragments are spaced apart so that the cultivated Dendrobium officinale stem cell clumps do not contact each other;

[0075] (b) Stem cell screening: Select the D. officinale stem cell clusters that are obviously growing fast, with uniform color and morphology during cultivation, and cut them into multiple fragments;

[0076] (c) Stem cell subculture: The cut Dendrobium officinale stem cell fragments are distributed at intervals on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are subcultured in the dark; after each generation of culture and before the next subculture, the stem cell screening step (b) is performed to obtain a Dendrobium officinale stem cell cluster from a clone;

[0077] (d) Stem cell screening: Select the D. officinale stem cell clusters that grow significantly faster during cultivation;

[0078] (e) Establishing stem cell lines: The selected Dendrobium officinale stem cell clusters are subcultured on a solid plant stem cell culture medium in the dark; after each subculture, the above-mentioned stem cell screening step (d) is performed before the next subculture until one or more plant stem cell lines are obtained;

[0079] (f) Stem cell line domestication: the debris of one or more established Dendrobium candidum stem cell lines are again spaced and distributed on the solid-state condition plant stem cell culture medium, and the Dendrobium candidum stem cells are subcultured in dark conditions; after each generation of cultivation, the brown or color changed or slowly divided mass is eliminated compared with the previous generation before the next generation of cultivation, and after multiple generations of cultivation, one or more Dendrobium candidum stem cells with stable traits, stable division and rapid division are obtained.

[0080] In a certain embodiment, the preparation method of the Dendrobium candidum stem cells further comprises the following steps before step (a): cutting the quiescent center of Dendrobium candidum into debris, expanding and cultivating the callus in dark conditions, then cutting the callus into debris, spacing and distributing it in the solid-state callus induction medium for expansion, and then subculturing the expanded callus; after each generation of cultivation, the expanded callus with consistent color and morphology is selected, the brown part is removed, and then cut into small pieces, which are again spaced and distributed in the solid-state callus induction medium, and the spacing and distribution is such that the callus clumps after growth do not contact each other, and after multiple generations of cultivation, the primary Dendrobium candidum stem cells are obtained; the obtained primary Dendrobium candidum stem cells are subcultured in step (a).

[0081] In a certain embodiment, the time for one generation of cultivation of the callus is 8-20 days or the callus grows more than 60% of the total area of the culture vessel.

[0082] In a certain embodiment, the subculture of the callus is carried out for 2-5 generations, and then the subculture of the callus is stopped.

[0083] In a certain embodiment, in steps (b) and (c), the subculture is not less than 5 times.

[0084] In a certain embodiment, in steps (d) and (e), the subculture is not less than 5 times.

[0085] In a certain embodiment, during the subculture of step (e), the subculture is carried out for 20-40 days, and then the subculture is expanded at a ratio of 1:4-6.

[0086] In a certain embodiment, the spacing and distribution distance between two adjacent debris is more than three times the size of the debris.

[0087] In a specific and preferred embodiment, the Dendrobium candidum stem cells are obtained by the following method:

[0088] (1) Obtain Dendrobium officinale callus: Dendrobium officinale tender stem tip is sterilized with mercury chloride, the tip tissue is chopped, the plant quiescent center tissue is exposed, the inhibition function of the quiescent center to the plant stem cell is destroyed, the reverse differentiation is strengthened, and the plant callus is obtained by solid medium culture in callus induction medium (MS basic medium + 1.0 mg / L NAA + 0.8 mg / L 6-BA + 25 g / L sucrose + 7.0 g / L agar, pH 5.8). The callus is a kind of tissue newly generated on the surface of the injured part when the plant is injured by external adverse conditions. The callus contains plant stem cells.

[0089] (2) Expand the callus: the Dendrobium officinale callus is transferred to the callus induction medium, and is expanded for multiple times under the condition of no visible light. The steps are as follows: the callus is placed on the solid callus induction medium for culture, after the callus is formed, the callus with basically consistent color and morphology is selected, the brown part is removed, the callus is divided into small pieces, and is laid on the medium for subculture expansion with interval. The subculture is performed every 20 days or when the callus grows more than 60% of the total area. The subculture is performed for 5 times, the brown part is continuously removed during the subculture, and the well-cultured tissue is retained. The culture temperature condition is 25°C, and a large amount of callus is obtained.

[0090] (3) Isolation of plant stem cells: the callus with consistent color and morphology after 5 times of expansion is selected during the expansion process, 9 uniform pieces are selected, the callus of one piece is chopped into 1 mm or so debris, and is transferred to the solid plant stem cell culture medium (MS basic medium + 1.0 mg / L 6-BA + 25 g / L sucrose + 10 g / L potato juice + 15 g / L banana juice + 7.0 g / L agar, pH 5.8) for culture. The thin distribution is performed under the condition of no visible light, that is, the distance between each debris is 5 times the diameter of the debris, so that the stem cell debris maintains a clear distance, so that the callus does not contact each other after growth, and the plant stem cells from one clone are ensured to be separated. The brown piece is removed in time during the culture. The culture temperature condition is preferably 25°C, and the growth is 20 days.

[0091] (4) Plant stem cell screening and subculture: Isolate and select the clumps that are obviously growing fast (1.5 times the growth rate or more) and have consistent color and morphology in step 3. Cut each clump into fragments of about 1 mm and place them in a new solid plant stem cell culture medium for thin distribution again. That is, the distance between each fragment is 5 times the diameter of the fragment, so that the stem cell fragments maintain a clear distance so that the clumps will not touch each other after growing up. Cultivate and subculture under conditions without visible light to ensure that plant stem cells from a clone are isolated. According to the above process, the fragments of the stem cell clusters are thinly distributed and cultured 5 times in this step. After each generation of cultivation and before the next subculture, the plant stem cell clusters that are obviously growing fast, consistent in color and morphology are selected and cut into multiple fragments for the next generation of cultivation. The temperature condition is 25℃.

[0092] (5) Plant stem cell screening and line establishment: Select multiple stem cell clusters with rapid growth and stable color and morphology from step (4) and perform five stabilization cultures. Instead of sparsely distributed cultures, each clone is expanded and cultured. After 20 days of culture, the clones are subcultured at a ratio of 1:5. Unstable clones are deleted during the culture process to establish multiple stem cell lines. The temperature is 25°C.

[0093] The Dendrobium officinale stem cells obtained by the above-mentioned method can maintain the variety's continuous and stable high expression of target substances while maintaining a stable division rate.

[0094] In one embodiment, the Dendrobium officinale stem cells are expanded and cultured before differentiation induction culture, and the expansion and culture specifically comprises: solid culture of the Dendrobium officinale stem cells for 60 to 90 days, followed by liquid culture for 30 to 45 days. The solid culture medium is MS medium containing 0.5 mg / L NAA, 0.5 mg / L KT, 20 g / L glucose, and 10 g / L agar. The liquid culture medium is MS medium containing 0.5 mg / L NAA, 0.5 mg / L KT, and 20 g / L glucose.

[0095] In one embodiment, the Dendrobium officinale young shoot extract is obtained by water extraction or alcohol extraction.

[0096] In one embodiment, the Dendrobium officinale sprout extract is obtained by water extraction, and the water extraction method comprises the following steps:

[0097] (1) Take fresh Dendrobium officinale shoots and dry them;

[0098] (2) Powdering the dried Dendrobium officinale buds, soaking them, and filtering them to obtain a primary stock solution;

[0099] (3) filtering the primary stock solution step by step to obtain a secondary stock solution;

[0100] (4) filtering the secondary stock solution again to obtain the Dendrobium candidum extract, i.e. the Dendrobium candidum tender bud extract.

[0101] In an embodiment, the drying temperature is 40-55℃, such as 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, etc.

[0102] In an embodiment, the drying duration is 18-24 hours, such as 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, etc.

[0103] In an embodiment, in step (3), the step of filtering gradually is as follows: filtering the primary stock solution through filters with precision of 20μm, 10μm, 5μm, 1μm and 0.45μm in sequence.

[0104] In an embodiment, the pressure in the step of filtering gradually is less than 0.2MPa.

[0105] In an embodiment, in step (2), the mass ratio of the powder to water is 1:20-1:50, and the soaking duration is 2-5 hours.

[0106] In an embodiment, in step (2), the particle size of the powder is less than or equal to 120 mesh.

[0107] In an embodiment, in step (2), the filtering is performed by using a filter with precision of 5µm.

[0108] In an embodiment, the soaking duration is 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, etc.

[0109] In an embodiment, in step (4), the filtering again is performed by using a filter with precision of 0.22μm.

[0110] The application provides use of the extract of any one of the above embodiments in preparation of a product with whitening and / or spot-fading efficacy.

[0111] In an embodiment, the product is a cosmetic product, and the dosage form thereof includes emulsion, water agent, oil agent or gel.

[0112] In an embodiment, the product is a pharmaceutical product, and the dosage form thereof includes oil agent, emulsion, paste, paste, film coating agent, gel, aerosol, spray, solution, liniment.

[0113] The present invention is further illustrated below by means of specific examples. Unless otherwise specified, "%" represents percentage by mass. The materials and reagents used in the following examples, unless otherwise noted, are commonly used in the art and are commercially available or synthesized by known methods. Experimental procedures in the following examples, where conditions are not specified, were generally performed according to conventional experimental conditions or those recommended by the manufacturers of the relevant reagents (kits). Example 1

[0114] In this embodiment, the Dendrobium officinale stem cells used were obtained by the method described in patent document CN 116445393B.

[0115] (1) Cultivation and proliferation of Dendrobium officinale stem cells

[0116] The indoor environment is a clean room with a cleanliness level of 100,000 or above, maintaining a room temperature of 25℃~28℃ and an air relative humidity of 40%~70%. The production environment, production water and filtered water should comply with the corresponding standards respectively.

[0117] Solid culture medium (MS medium + 0.5 mg / L NAA + 0.5 mg / L KT + 20 g / L glucose + 10 g / L agar solid culture medium) was used to culture Dendrobium officinale stem cells for 60 to 90 days, and the culture medium was replaced every 25 to 30 days.

[0118] Dendrobium officinale stem cells were cultured in a liquid medium (MS medium + 0.5mg / L NAA + 0.5mg / L KT + 20g / L glucose). Using an inoculum size of 20g / L–30g / L fresh weight, the stem cells were transferred to 2L–5L glass bottles for liquid culture using aseptic techniques. Sterile air was introduced into the culture medium at a flow rate of 0.75L / min–1L / min. The culture period was 30–45 days, with the medium replaced every 15–20 days.

[0119] (2) Induced differentiation of Dendrobium officinale stem cells

[0120] When plant stem cells have expanded to a certain number, they enter the stem cell differentiation stage. Liquid culture medium (MS medium + 0.5mg / L NAA + 1mg / L KT + 20g / L glucose + 50g / L maltose) is used for differentiation induction. The specific steps are as follows:

[0121] According to the inoculum volume of 40g / L~50g / L, the stem cells of Dendrobium officinale were transferred to a 10L glass bottle for culture. o C~28 oC, relative humidity 40%-70%, uniform soft artificial light supplemented with a 4:1 ratio of red to blue light, light intensity of 2000 Lux, and a light-dark cycle of 8h:16h for induced differentiation. Simultaneously, sterile air was introduced into the culture flask at a flow rate of 0.75L / min-1L / min during the incubation process. The incubation period was 30-45 days. Harvest the young stems when their total volume reached 2 / 3 of the total volume of the culture flask.

[0122] Example 2 Preparation of Dendrobium officinale sprout extract

[0123] The young buds of Dendrobium officinale obtained in Example 1 were washed 3 to 5 times until clear. The cleaned fresh buds were placed in a protective washing bag and placed in a special dehydrator for fresh buds for dehydration. The dehydrated fresh buds were placed flat in an oven and dried at a temperature of 45°C for 18 to 24 hours; the dried product was beaten into a 120-mesh dry powder, and soaked in pure water for 3 hours at a mass ratio of 1:40. The solids were filtered with a 50-mesh gauze, and then filtered with PP10 20μm, PP10 10μm, PP10 5μm, PP10 1μm, PP10 0.45μm, and PES10 0.45μm. The pressure during filtration could not exceed 0.2 MPa to obtain the filtered primary stock solution. The above filtration step was completed using a Kammer intelligent peristaltic pump UIP. The primary stock solution was sterilized by ultraviolet and then tested. The sterile primary stock solution was selected for secondary filtration using a PES10 0.22μm membrane to obtain the Dendrobium officinale sprout extract (SDO).

[0124] The same method as above was used to obtain a traditional medicinal Dendrobium officinale extract as a control, namely the commercial Dendrobium officinale extract (DO). Example 3

[0125] To explore the whitening effect of stem cell-derived Dendrobium officinale, this example uses a human melanocyte model to analyze the regulatory effect of the sample on melanin production through cytotoxicity testing and melanin synthesis inhibition experiments.

[0126] (1) Cytotoxicity test

[0127] Human melanocytes were cultured at a rate of 1×10 4The cells were seeded into 96-well plates at a density of 1000 cells / well and incubated overnight in a 37°C, 5% CO2 incubator. The Dendrobium officinale extract product (SDO) and the commercial Dendrobium officinale extract (DO) of the present application were set with 8 concentration gradients (%, V / V), 10.00%, 5.00%, 2.50%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08%, with 3 replicate wells in each group, and the positive control was a culture medium containing 10% DMSO. After 24 hours of administration, MTT working solution (0.5 mg / mL) was added and incubated for 2 hours. After dissolution, the OD value at 490 nm was measured to calculate the relative cell viability. The cytotoxicity test results of each group were 1 and Figure 1 .

[0128] Table 1

[0129]

[0130] From Table 1 and Figure 1 It can be seen that the overall difference between the Dendrobium officinale extract of the present application and the commercial Dendrobium officinale extract is not much. The cell viability of both samples is greater than 90% when the concentration is ≤3.00%, and there is no significant effect on cell viability.

[0131] (2) Inhibit melanin synthesis

[0132] The Dendrobium officinale extract (SDO) and the commercial Dendrobium officinale extract (DO) of this application were set at four concentrations (%, V / V): 0.05%, 0.50%, 3.00%, and 10.00%. The cells were set up as a blank control (BC), a positive control (kojic acid, 0.3% m / V), and a sample group (0.05%~10.00% V / V). After 24 hours of drug addition, the melanin was dissolved in 1M NaOH (containing 10% DMSO), incubated at 80℃ for 1 hour, and the absorbance at 405 nm was measured to calculate the inhibition rate. The results are shown in Table 2 and Figure 2 shown.

[0133] Table 2

[0134]

[0135] From Table 2 and Figure 2It can be seen that the higher the concentration of the stem cell-derived Dendrobium officinale sample in this application, the more significant the inhibition rate of cellular melanin content, while commercial Dendrobium officinale has almost no inhibition rate. The inhibition rate of stem cell-derived Dendrobium officinale at a concentration of 10.00% reached 40.08%, close to the positive control kojic acid (74.44%). Compared with the blank control, there was no significant difference in the inhibition rate of commercial Dendrobium officinale in the 0.05%, 0.50%, 3.00%, and 10.00% concentration groups, while the inhibition rate of stem cell-derived Dendrobium officinale was significantly different (p < 0.05), with p < 0.01 for the 3.00% and 10.00% groups. Combined with cellular safety and inhibitory effect, stem cell-derived Dendrobium officinale at a concentration of 3.00% to 10.00% is recommended for use in whitening skin care product formulations. Example 4

[0136] The stem cell-derived Dendrobium officinale extract obtained in this application was prepared into a hydrogel for human freckle removal testing.

[0137] The formula of the hydrogel is shown in Table 3. A hydrogel without the stem cell-derived Dendrobium officinale extract of the present application was used as a control.

[0138] Table 3

[0139]

[0140] This study enrolled 60 subjects with dark spots and dark skin, aged 35-56 years (mean age 39.27 years). Subjects were randomly divided into two groups of 30 based on their skin spot count. One group received a gel containing stem-derived Dendrobium officinale extract, while the other group received a blank control gel without stem-derived Dendrobium officinale extract. Instructions for use: After cleansing and toning morning and evening, apply an appropriate amount of this product to the facial skin and gently massage until absorbed. Use for 60 days. VISIA images were collected and subjects' skin condition was self-assessed on days 0, 15, 30, and 60.

[0141] After 60 days of using the hydrogel containing the stem-derived Dendrobium officinale extract of the present application, none of the 30 subjects experienced any adverse reactions. The results of the dark spot test on the 30 subjects are shown in Table 4. The improvement rate of dark spots is calculated as follows: Improvement rate (%) = (baseline value - test value after N days of use) / baseline value * 100%.

[0142] Table 4

[0143]

[0144] As can be seen from Table 4, after 30 days of using the sample, the improvement rate of dark spots on the skin in the extract group was 35.76%, which was significantly better than the baseline value, and there was no significant difference in the blank control group before and after use; after 60 days of using the sample, the improvement rate of dark spots on the skin in the extract group was 59.30%, which was significantly better than the baseline value, and there was no significant difference in the blank control group before and after use. Example 5

[0145] The stem cellized Dendrobium officinale extract obtained according to the preparation method of Example 2 and the commercial Dendrobium officinale extract were dried to obtain extract powders, and the chemical component content was analyzed.

[0146] (1) Take 12.5 L of the extract prepared in Example 2 to prepare 50 g of extract powder. Add distilled water at a solid-liquid ratio of 1:10 (g / mL). Extract under reflux in a water bath for 1.5-2 hours each time, 2-3 times. Combine the extracts and concentrate under reduced pressure.

[0147] The concentrate was subjected to polyamide adsorption method to remove protein (120 g / L, adsorption for 12 h, elution with 5 times distilled water), and then precipitated with 80% anhydrous ethanol (3 times anhydrous ethanol, added with stirring, sealed and kept at low temperature for 12 h, centrifuged. Repeated 3 times). The precipitate was the stem cellized Dendrobium officinale polysaccharide. The yield of polysaccharides is shown in Table 5.

[0148] Analysis of the polysaccharide composition revealed that the sugars in the stem cell-derived Dendrobium officinale extract powder primarily consisted of fructose, sucrose, and glucose. The polysaccharide content was relatively low, resulting in unremarkable results from liquid chromatography-mass spectrometry. Commercial Dendrobium officinale extract powder contained a higher content of polysaccharides, primarily fructose, sucrose, glucose, and maltose, as well as some other oligosaccharides.

[0149] (2) Take 50 g of the extract powder and extract it twice with 1000 mL of 95% ethanol under reflux for 1.5 h. Combine the extracts and concentrate to dryness under reduced pressure. Calculate the yield of the alcohol extract (see Table 5).

[0150] Table 5

[0151]

[0152] (3) Take 10g of the extract powder and place it in a round-bottom flask. Add 70% ethanol solution at a solid-liquid ratio of 1:10. Extract under reflux in a 75℃ water bath for 1-1.5 hours, and extract 2-3 times. Combine the extracts and concentrate under reduced pressure. Further purify with macroporous resin D101, eluting with 20%, 40%, 60%, and 80% ethanol, respectively. Collect the eluates, dry them, and test the polyphenol content. The results are as follows: The 40% elution fraction has the highest polyphenol content and the best purity. The polyphenol content and yield are shown in Table 6.

[0153] (4) Take 10 g of the extract powder and soak it in 10 times the amount of 90% ethanol for 12 h, then reflux extract for 2 h, three times in a row. After the alcohol extract is concentrated under reduced pressure until there is no alcohol smell, it is dissolved in 5% hydrochloric acid, filtered, and the acid solution is extracted with petroleum three times, defatted, and alkalized with concentrated ammonia water to pH = 10. The alkaline solution is extracted with chloroform twice, and the solvent is recovered to obtain the crude alkaloid extract. The alkaloid content and yield are shown in Table 6.

[0154] Table 6

[0155] Example 6

[0156] The stem cellized Dendrobium officinale extract obtained according to the preparation method of Example 2 and the commercial Dendrobium officinale extract were dried to obtain extract powders, and the chemical component content was analyzed.

[0157] Extracts from stem-derived Dendrobium officinale extract (SDO) and commercial Dendrobium officinale extract (DO) were obtained according to the method in point (3) of Example 5. The metabolites of DO and SDO were analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) in positive and negative ion modes, respectively, with a scan range of 80–800 m / z. Differential metabolites (FC ≥ 5, p < 0.05, VIP ≥ 1) were screened by ANOVA and OPLS-DA, and key molecules were verified by combining literature.

[0158] The results of differential metabolite identification showed that 173 metabolites were significantly down-regulated in SDO and 9 were up-regulated in DO under positive ion mode ( Figure 3 In negative ion mode, 189 metabolites were down-regulated in SDO and 45 were up-regulated in DO ( Figure 3 ANOVA and OPLS-DA analysis showed that the expression of 16 metabolites in SDO was significantly higher than that in DO, among which resveratrol and harmine were highly expressed (Table 7).

[0159] Table 7

[0160]

[0161] The QTOF-MS verification results showed that the resveratrol concentration in SDO was 5.8 times that of DO, and the rutin concentration in SDO was 51 times that of DO, indicating that SDO was significantly enriched in these two components.

[0162] Potential targets of resveratrol and harmine were obtained through databases such as PubChem and TCMSP, and the intersection with whitening and freckle-removing targets (from GeneCards) was taken to obtain 280 core targets. In addition, the protein interaction network was constructed using the STRING database, and the PPI network TSV file constructed by Cytoscape was used to perform network topology structure screening for core targets. The score of each node was calculated using the MCC algorithm, and 125 core targets were obtained. The 10 nodes with the highest scores were further screened as potential core therapeutic targets. Sorted from high to low by score, the top 10 targets were AKT1, TNF, SRC, STAT3, CTNNB1, EGFR, ALB, HSP90AA1, BCL2, and ESR1. AutoDock Vina was then used to verify the binding activity of resveratrol and harmine to the core targets (binding energy ≤-5.0 kcal·mol -1 for effective combination).

[0163] The results are as follows: Resveratrol binds to HARS (binding energy = -8 kcal·mol -1 ), resveratrol and JAK2 (binding energy = -7.9 kcal·mol -1 ) and carnosine bind to JAK2 (binding energy = -7.9 kcal·mol -1 ) showed strong binding activity, confirming its ability to regulate whitening-related pathways. The binding ability is shown in Table 8, where the unit is kcal·mol -1 . Core targets such as HARS and JAK2 are closely related to the NF-κB, JAK-STAT, PI3K-Akt, MAPK, and mTOR signaling pathways. Studies have shown that resveratrol inhibits NF-κB phosphorylation, reduces the release of pro-inflammatory factors (IL-6, TNF-α), and activates the Nrf2 pathway to enhance antioxidant capacity, thereby reducing oxidative stress-induced pigmentation. Carnosine directly inhibits tyrosinase expression and reduces melanin synthesis by inhibiting DYRK1A kinase and blocking NFATC3 nuclear translocation. Resveratrol and carnosine are the core mechanisms of SDO's whitening and spot-removing functions, providing a scientific basis for its development in natural skin care products.

[0164] Table 8

[0165]

[0166] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A Dendrobium officinale sprout extract obtained by inducing differentiation of Dendrobium officinale stem cells, characterized in that: The differentiation induction was carried out in MS medium containing 0.5 mg / L NAA, 1 mg / L KT, 20 g / L glucose and 50 g / L maltose; The differentiation induction condition parameters are: light intensity of 1800-2500 Lux, red to blue light ratio of 3-5:1, and light exposure time of 6-12 hours per day; The preparation method of the Dendrobium officinale stem cells comprises: (a) Stem cell cultivation: Multiple fragments of primary Dendrobium officinale stem cells derived from the tips of young stems of Dendrobium officinale are spaced apart and distributed on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are cultured in the dark. The multiple fragments are spaced apart so that the cultivated Dendrobium officinale stem cell clumps do not contact each other; (b) Stem cell screening: Select the Dendrobium officinale stem cell clusters that are obviously growing fast, with uniform color and morphology during cultivation, and cut them into multiple fragments; (c) Stem cell subculture: The cut Dendrobium officinale stem cell fragments are distributed at intervals on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are subcultured in the dark; after each generation of culture and before the next subculture, the stem cell screening step (b) is performed to obtain a Dendrobium officinale stem cell cluster from a clone; (d) Stem cell screening: Select the D. officinale stem cell clusters that grow significantly faster during cultivation; (e) Establishing stem cell lines: The selected Dendrobium officinale stem cell clusters are subcultured on a solid plant stem cell culture medium in the dark; after each subculture, the above-mentioned stem cell screening step (d) is performed before the next subculture until one or more plant stem cell lines are obtained; (f) Stem cell line acclimation: The debris of one or more established Dendrobium officinale stem cell lines are distributed at intervals on a solid-state conditioned plant stem cell culture medium, and the Dendrobium officinale stem cells are subcultured under dark conditions; after each generation of cultivation and before the next generation of cultivation, the clumps that are brown or have changed color or have a slow division rate compared with the previous generation are eliminated. After multiple generations of cultivation, one or more Dendrobium officinale stem cells with stable traits, stable division, and fast division are obtained.

2. The extract according to claim 1, characterized in that The differentiation induction culture is carried out for 30 to 45 days.

3. The extract according to claim 1, characterized in that: The preparation method of the Dendrobium officinale stem cells further includes the following steps before step (a): chopping the static center of the Dendrobium officinale into fragments, amplifying and cultivating callus tissue under dark conditions, then chopping the callus tissue into fragments, distributing them at intervals in a solid callus induction culture medium for amplification, and then subculturing the amplified callus tissue; after each generation of cultivation and before the next generation of cultivation, selecting amplified callus tissue with consistent color and morphology during cultivation, removing the browned part and dividing it into small pieces, and then distributing them at intervals in a solid callus induction culture medium, wherein the spacing distribution prevents the callus tissue clumps from contacting each other after cultivation, and after multiple generations of cultivation, obtaining the primary Dendrobium officinale stem cells; and the obtained primary Dendrobium officinale stem cells are then subjected to the cultivation of step (a).

4. The extract according to claim 1, characterized in that In the Dendrobium officinale young shoot extract, the polysaccharide content is less than 1 g / L; and / or the alcohol extract content is greater than 1 g / L; and / or the polyphenol content is greater than 100 mg / L; and / or the alkaloid content is greater than 200 mg / L.

5. The extract according to claim 1, characterized in that The Dendrobium officinale tender shoot extract is obtained by water extraction or alcohol extraction.

6. The extract according to claim 5, characterized in that The Dendrobium officinale sprout extract is obtained by water extraction, which comprises the following steps: (1) Take fresh Dendrobium officinale shoots and dry them; (2) Powdering the dried young shoots of Dendrobium officinale, soaking them, and filtering them to obtain a primary stock solution; (3) filtering the primary stock solution step by step to obtain a secondary stock solution; (4) Filtering the secondary stock solution again to obtain a Dendrobium officinale extract, namely, a Dendrobium officinale sprout extract.

7. The extract according to claim 6, characterized in that The step-by-step filtration steps are as follows: filtering the primary stock solution through filters with precisions of 20 μm, 10 μm, 5 μm, 1 μm, and 0.45 μm in sequence; And / or, the pressure during the step-by-step filtration is less than 0.2 MPa; And / or, in step (2), the mass ratio of the powder to water is 1:20-1:50, and the soaking time is 2-5 hours; And / or, in step (4), the second filtration is performed using a filter with an accuracy of 0.22 μm.

8. Use of the extract according to any one of claims 1 to 7 in the preparation of a product having whitening and / or spot-lightening effects.

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