New application of dendrobium officinale tender shoot extract

Abstract

The invention relates to a new application of a dendrobium officinale tender shoot extract, in particular to an application of the dendrobium officinale tender shoot extract obtained by differentiation induction of dendrobium officinale stem cells in preparation of a medicine for treating and / or preventing tumors, especially an application in treating and / or preventing melanoma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a new use of a Dendrobium officinale sprout extract, and especially to an use of the Dendrobium officinale sprout in preparing a medicine for treating and / or preventing tumors. Background Art

[0002] Melanoma, one of the most aggressive types of skin cancer, originates from melanocytes in the basal layer of the epidermis. Melanomas are categorized based on their developmental pathways, including superficial spreading melanoma (low-accumulated sun-damage melanoma) and lentigo maligna melanoma (high-accumulated sun-damage melanoma). Melanoma development is influenced by both the tumor cells themselves and their surrounding immune microenvironment. Within this microenvironment, the infiltration of immune cells such as T cells, macrophages, and dendritic cells influences melanoma proliferation.

[0003] Early diagnosis is crucial for improving the clinical treatment of melanoma. Pathologically, melanoma is characterized by cellular atypia, increased mitotic activity, and irregular pigmentation. The standard treatment for early-stage melanoma is surgical resection, which generally achieves good results. However, for advanced or metastatic melanoma, drug therapy is the mainstay.

[0004] At present, immunotherapy, targeted therapy and immunotherapy have also made significant progress in the treatment of melanoma. However, the above methods have defects such as large side effects and high costs. At the same time, factors such as acquired drug resistance, low sensitivity and adverse drug reactions limit their clinical application and efficacy. In view of this, it is still necessary to further explore efficient means of treating melanoma. Summary of the Invention

[0005] In view of the above-mentioned technical deficiencies, the present invention provides a new application of an extract of Dendrobium officinale shoots (protocorms) differentiated from Dendrobium officinale stem cells in the treatment of tumors, especially in the treatment of melanoma.

[0006] To achieve the above purpose, the technical solution of the present invention is: The present invention provides use of a Dendrobium officinale sprout extract obtained by inducing differentiation of Dendrobium officinale stem cells in preparing a medicine for treating and / or preventing tumors.

[0007] Preferably, the drug can mediate tumor cell apoptosis by inducing tumor cell autophagy.

[0008] Preferably, the drug is capable of inhibiting the activation of the mTOR pathway.

[0009] Preferably, the drug can regulate the expression of the autophagy-related protein Beclin-1.

[0010] Preferably, the tumor is melanoma.

[0011] Preferably, the method for preparing 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 on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are cultured in the dark. The spacing between the multiple fragments is sufficiently large so that the cultivated Dendrobium officinale stem cell clumps do not contact each other; (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; (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.

[0012] 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 basically 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 spacing distance is large enough so 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).

[0013] Preferably, 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.

[0014] 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.

[0015] Preferably, the Dendrobium officinale sprout extract is obtained by water extraction or alcohol extraction.

[0016] Preferably, 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 Dendrobium officinale buds, 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.

[0017] 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.

[0018] Preferably, the pressure during the step-by-step filtration is less than 0.2 MPa.

[0019] 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.

[0020] Preferably, in step (4), the second filtration is performed using a filter with an accuracy of 0.22 μm.

[0021] The beneficial effects of the present invention are: The present invention aims to utilize plant stem cell technology and directed differentiation techniques to provide a novel application of an aqueous extract of the young stems (protocorms) of Dendrobium officinale for the treatment and / or prevention of tumors, particularly for inhibiting the growth of melanoma. The stem-derived Dendrobium officinale extract provided herein can effectively induce autophagy, particularly mitochondrial autophagy, thereby causing tumor cell apoptosis. The stem-derived Dendrobium officinale extract provided herein is safe, non-toxic, and low-cost for the treatment of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 2 Schematic diagram of the cell apoptosis status detected by flow cytometry in each group in Example 4 of the present application.

[0024] Figure 3 Schematic diagram of the cell apoptosis rate of each group detected by flow cytometry in Example 4 of the present application.

[0025] Figure 4 This is the SDS-PAGE electrophoresis result of mTOR phosphorylation in each group in Example 5 of the present application.

[0026] Figure 5 Schematic diagram of the mTOR phosphorylation level in each group in Example 5 of the present application.

[0027] Figure 6 This is a diagram showing the SDS-PAGE electrophoresis results of LC3-II of each group in Example 6 of the present application.

[0028] Figure 7 These are the SDS-PAGE electrophoresis results of Beclin-1 and p62 proteins in each group in Example 6 of the present application.

[0029] Figure 8 Schematic diagram of the LC3-II / LC3-I expression ratios of each group in Example 6 of the present application.

[0030] Figure 9 Schematic diagram of the expression levels of Beclin-1 and p62 in each group in Example 6 of the present application.

[0031] Figure 10 Schematic diagram of the in situ staining results of LC3 protein in A375 cells of each group in Example 7 of the present application.

[0032] Figure 11 Schematic diagram of the tumor weight of the mouse melanoma model in each group in Example 8 of the present application. DETAILED DESCRIPTION

[0033] 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.

[0034] Currently, melanoma is a skin cancer that originates from melanocytes. Melanoma usually begins on skin that is frequently exposed to sunlight, including the skin of the arms, back, face, and legs. Melanoma is the most dangerous type of skin cancer, with a high degree of malignancy, and is one of the main causes of death from skin cancer. Current treatments mostly rely on small molecule drugs or molecular immunotherapy, which have the disadvantages of high cost and large side effects. Based on this, the inventors of the present invention, after a lot of long-term research, directed the differentiation of Dendrobium officinale stem cells obtained from the Chinese herbal plant stem cell platform established in the early research of the present invention into Dendrobium buds, and found that the Dendrobium officinale bud extract can inhibit melanin synthesis and has the effect of lightening spots and whitening. It was further found that the extract obtained in this way can effectively inhibit the growth of melanoma, providing a new approach to the treatment of melanoma.

[0035] The present invention provides use of a Dendrobium officinale sprout extract obtained by inducing differentiation of Dendrobium officinale stem cells in preparing a medicine for treating and / or preventing tumors.

[0036] In one embodiment, the drug can mediate tumor cell apoptosis by inducing tumor cell autophagy.

[0037] In one embodiment, the drug can mediate tumor cell apoptosis by inducing mitochondrial autophagy in tumor cells.

[0038] In one embodiment, the drug is capable of inhibiting mTOR pathway activation.

[0039] In one embodiment, the drug can regulate the expression of the autophagy-related protein Beclin-1.

[0040] In one embodiment, the drug can upregulate the expression of the autophagy-related protein Beclin-1.

[0041] In one embodiment, the tumor is melanoma.

[0042] The present invention specifically induces Dendrobium officinale stem cells, and the resulting Dendrobium officinale shoots can effectively inhibit the growth of melanoma.

[0043] 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.

[0044] In one embodiment, the method for preparing 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 on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are cultured in the dark. The spacing between the multiple fragments is sufficiently large so that the cultivated Dendrobium officinale stem cell clumps do not contact each other; (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; (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.

[0045] 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 basically 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 spacing distance is large enough so 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).

[0046] 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.

[0047] In one embodiment, the method for preparing 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 on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are cultured in the dark. The spacing between the multiple fragments is sufficiently large so that the cultivated Dendrobium officinale stem cell clumps do not contact each other; (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; (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.

[0048] In a certain embodiment, 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 medium for amplification, and then subculturing the amplified callus tissue; after each generation of cultivation and before the next generation of cultivation, selecting the amplified callus tissue with basically 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 spacing distance is large enough so 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).

[0049] In one embodiment, when the callus tissue is cultured, the time for one generation of culture is 8 to 20 days or the time when the callus tissue grows to more than 60% of the total area of the culture container.

[0050] In one embodiment, the callus tissue is subcultured for 2 to 5 generations, and then the subculture of the callus tissue is terminated.

[0051] In one embodiment, in steps (b) and (c), subculturing is performed no less than 5 times.

[0052] In one embodiment, in steps (d) and (e), subculturing is performed no less than 5 times.

[0053] In one embodiment, during the subculture process of step (e), after culturing for 20 to 40 days, the cells are subcultured at a ratio of 1:4 to 6.

[0054] In one embodiment, the spacing distance between two adjacent chips is more than three times the size of the chips.

[0055] In a specific and preferred embodiment, the Dendrobium officinale stem cells are obtained by the following method: (1) Obtaining Dendrobium officinale callus: The tip of the tender stem of Dendrobium officinale was disinfected with mercuric chloride, and the tip tissue was minced to expose the plant's quiescent center tissue, thereby destroying the inhibitory function of the quiescent center regulatory cells on plant stem cells and enhancing reverse differentiation. The plant callus was cultured in a solid medium in a callus induction medium (MS basal medium + 1.0 mg / L NAA + 0.8 mg / L 6-BA + 25 g / L sucrose + 7.0 g / L agar, pH 5.8) to obtain plant callus tissue. Callus tissue is a new tissue that grows on the surface of the wound when the plant is damaged by adverse external conditions. Callus tissue contains plant stem cells.

[0056] (2) Callus expansion: The callus of Dendrobium officinale was transferred to a callus induction medium and amplified multiple times in the absence of visible light. The steps are as follows: the callus was cultured on a solid callus induction medium. After the callus was formed, the callus with basically the same color and morphology was selected, and the browned part was removed. The callus was divided into small pieces and spread on the medium at intervals for subculture. One generation was performed every 20 days or when the callus grew to more than 60% of the total area. Five subcultures were performed. During the subculture process, the browned part was continuously removed and the tissue with better culture was retained. The culture temperature was 25°C, and a large amount of callus was obtained.

[0057] (3) Plant stem cell isolation: During the amplification process, callus tissues that remain consistent in color and morphology after 5 amplifications are selected. The above 9 uniform clumps are selected, and the callus tissue of one clump is cut into fragments of about 1 mm. The fragments are transferred to a solid plant stem cell culture medium (MS basal 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 callus tissues are thinly distributed under the condition of no visible light, 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 callus tissues will not contact each other after growing, ensuring that plant stem cells from one clone are isolated. Browning clumps are removed in time during the culture process. The culture temperature is preferably 25°C and the growth period is 20 days.

[0058] (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℃.

[0059] (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 more stabilization cultures. This means that each clone is expanded and cultured for 20 days at a 1:5 subculture ratio. Unstable clones are removed during the culture process to establish multiple stem cell lines. The temperature is 25°C.

[0060] The plant stem cells obtained by the above-mentioned method can maintain the variety's continuous and stable high expression of the target substance while maintaining a stable division rate.

[0061] In one embodiment, the differentiation induction is performed 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] In one embodiment, the light intensity is 1800 Lux, 1850 Lux, 1900 Lux, 1950 Lux, 2000 Lux, etc.

[0070] In one embodiment, the ratio of red to blue light is 3.5:1, 4:1, 4.5:1, 5:1, etc.

[0071] In one embodiment, the daily illumination time is 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, etc.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] In one embodiment, the Dendrobium officinale young shoot extract is obtained by water extraction or alcohol extraction.

[0076] In one embodiment, the Dendrobium officinale sprout extract is obtained by water extraction, and the water extraction method comprises the following steps: (1) Take fresh Dendrobium officinale shoots and dry them; (2) Powdering the dried Dendrobium officinale buds, 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.

[0077] In one embodiment, the drying temperature is 40-55°C, for example, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc.

[0078] In one embodiment, the drying time is 18 to 24 hours, for example, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, etc.

[0079] In one embodiment, in step (3), the step of stepwise filtration is as follows: the primary stock solution is filtered sequentially through filters with accuracies of 20 μm, 10 μm, 5 μm, 1 μm, and 0.45 μm.

[0080] In one embodiment, the pressure during the step-by-step filtration is less than 0.2 MPa.

[0081] In one embodiment, in step (2), the mass ratio of the powder to water is 1:20 to 1:50, and the soaking time is 2 to 5 hours.

[0082] In one embodiment, in step (2), the particle size of the powder is less than or equal to 120 mesh.

[0083] In one embodiment, in step (2), the filtration is performed using a filter with a particle size of 5 μm.

[0084] In one embodiment, the soaking time is 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, etc.

[0085] In one embodiment, in step (4), the second filtration is performed using a filter with an accuracy of 0.22 μm.

[0086] Analysis revealed that the extract of Dendrobium officinale young shoots obtained in the present application contained 0.48 g / L of polysaccharides, 2.928 g / L of alcohol extract, 319.2 mg / L of polyphenols, and 463.6 mg / L of alkaloids.

[0087] Furthermore, the Dendrobium officinale extract obtained in this application contains high levels of cinnamic acid, ferulic acid, caffeic acid phenethyl ester, acetyl glycitin, hexylresorcinol, thiamine, nifedipine, (±)-equol, caffeine, isoferulic acid, isoliquiritigenin, demethoxycurcumin, vitamin A, and eriodictyol. In particular, the resveratrol concentration in SDO (the Dendrobium officinale extract obtained in this application) is 5.8 times that of DO (the commercial Dendrobium officinale extract), and the rutin concentration in SDO is 51 times that of DO.

[0088] 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

[0089] In this embodiment, the Dendrobium officinale stem cells used were obtained by the method described in patent document CN 116445393B.

[0090] (1) Cultivation and proliferation of Dendrobium officinale stem cells 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.

[0091] 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.

[0092] 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.

[0093] (2) Induced differentiation of Dendrobium officinale stem cells 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: 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 o C, 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. Example 2 Preparation of Dendrobium officinale sprout extract

[0094] 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 45 ° C for 18 to 24 hours; the dried product was beaten into a 120-mesh dry powder, soaked in pure water at a mass ratio of 1:40 for 3 hours, and then the solids were filtered with a 50-mesh gauze. Then, the solids were filtered with PP10 20μm, PP10 10μm, PP10 5μm, PP10 1μm, PP10 0.45μm, and PES10 0.45μm in sequence. 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 0.22 μm filter membrane to obtain the Dendrobium officinale sprout extract.

[0095] 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

[0096] 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 are shown in Tables 1 and Figure 1 .

[0097] Table 1

[0098] 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. Example 4

[0099] To explore the effect of stem cell-derived Dendrobium officinale extract on melanoma cell apoptosis, this example used flow cytometry to stain melanoma cells and observe cell survival and death.

[0100] Human melanoma A375 cells in logarithmic growth phase were collected and 5×10 4 / L was placed in a cell culture flask, 2 mL of cell suspension was added to each flask, and cultured for 24 h. The experiment was divided into the following groups containing the extract of this application (SDOP): 0 (control group), 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / L group and 1.6 mg / L group containing 0.01 mg / L chloroquine (CQ, a specific autophagy inhibitor that mainly inhibits the fusion of autophagosomes and lysosomes in cells). After incubation for 48 h, the cells were collected, centrifuged at 1000 r / min, and the supernatant was discarded. The cells were washed with PBS, collected, and the following operations were performed in an ice bath in the dark: the cells were suspended with the binding buffer of the Biyuntian Annexin V-FITC Cell Apoptosis Detection Kit, and the cell concentration was adjusted to 5×10 4 / L, added Annexin-FITC and PI staining solution, mixed and incubated for 15 min, and cell apoptosis was detected by flow cytometry within 1 h.

[0101] The results are as follows Figure 2 and 3As shown, the proportion of apoptotic cells in the drug-treated cells was significantly increased compared with the blank and normal control groups (P < 0.001, n = 3). Annexin V and PI double staining revealed that the apoptotic rates (the sum of early apoptotic Q4 and late apoptotic Q2) were 0%, 25.9%, 32%, 42%, and 56.6% at drug concentrations of 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL, respectively. The apoptotic rate increased with increasing drug concentration. When cells were treated with 1.6 mg / mL of the drug and 0.01 mg / mL of CQ chloroquine (an autophagy inhibitor), the apoptotic rate was 30.9%, a 25.7% decrease compared to cells treated without the inhibitor at the same drug concentration, and the survival rate of cancer cells was increased. This indicates that drug treatment induces autophagy in cells and that the drug promotes apoptosis by inducing autophagy. This result was statistically significant. Example 5

[0102] Six groups of A375 cells were treated with the following extract concentrations: 0, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL, a 1.6 mg / L group containing 0.01 mg / L chloroquine (CQ), and a 0.01 mg / mL chloroquine (CQ) group. A375 cells were treated with different doses of stem-derived Dendrobium officinale extract for 48 hours before immunoblotting analysis. After washing twice with PBS, cells were lysed on ice for 30 minutes using pre-chilled protein lysis buffer and centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was collected and quantified using the BCA assay. Proteins were separated by SDS-PAGE and electrophoretically transferred to a 0.22 μm PVDF membrane. The membrane was then blocked and washed. Primary antibodies were added overnight at 4°C, followed by incubation with fluorescent secondary antibodies for 1 hour at room temperature. The experiment was repeated three times.

[0103] SDS-PAGE electrophoresis results are shown in Figure 4 , the phosphorylation level of mTOR is as Figure 5 The results showed that the drug group inhibited the phosphorylation of mTOR in A375 cells, inhibited the activation of the mTOR pathway, and promoted autophagy and apoptosis in human melanoma cells. The phosphorylation level of mTOR was significantly inhibited, and the inhibitory effect was dose-dependent. Example 6

[0104] LC3 is a universal marker protein for autophagy, consisting of two isoforms: LC3-I (LC3BI) and LC3-II (LC3BII). LC3-I is a soluble cytoplasmic protein expressed under normal conditions. When cells initiate autophagy, LC3-I interacts with the autophagy protein PE through a ubiquitin-like modification process, converting it into the membrane protein LC3-II. LC3-II expression reflects the maturation stage of autophagy. LC3-II levels are associated with intracellular autophagosomes and are a common marker of autophagic activity. Beclin-1 promotes the formation of autophagic vacuoles during autophagy in melanoma cells, and p62 is a selective autophagy substrate. Therefore, the expression of LC3-II, LC3-I, p62, and Beclin-1 is monitored simultaneously to assess their relationship with autophagy.

[0105] Six groups of A375 cells were treated with the following extract concentrations: 0, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL, a 1.6 mg / L group containing 0.01 mg / L chloroquine (CQ), and a 0.01 mg / mL chloroquine (CQ) group. A375 cells were treated with different doses of stem-derived Dendrobium officinale extract for 48 hours before immunoblotting analysis. After washing twice with PBS, cells were lysed on ice for 30 minutes using pre-chilled protein lysis buffer and centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was collected and quantified using the BCA assay. Proteins were separated by SDS-PAGE and electrophoretically transferred to a 0.22 μm PVDF membrane. The membrane was then blocked and washed. Primary antibodies were added overnight at 4°C, followed by incubation with fluorescent secondary antibodies for 1 hour at room temperature. The experiment was repeated three times.

[0106] The SDS-PAGE electrophoresis results of LC3-II are shown in Figure 6 The results of SDS-PAGE electrophoresis of Beclin-1 and p62 proteins are shown in Figure 7 , the expression levels of LC3-II / LC3-I, Beclin-1, and p62 are as follows Figures 8 to 9 The results showed that SDO upregulated the expression of the autophagy-related protein Beclin-1, increased the LC3-II / LC3-I ratio, and decreased p62 protein expression. This indicates that autophagy is ongoing, whereas autophagy is inhibited. Example 7

[0107] The experiment set up 3 groups to treat A375 cells, which were the following groups containing the following extract concentrations: 0, 1.6 mg / mL, and a 1.6 mg / L group containing 0.01 mg / L chloroquine (CQ). After treatment, each group of cells were washed with PBS for 10 minutes for a total of 3 times, fixed with paraformaldehyde at room temperature for 30 minutes, washed twice with PBS, added with 0.25% Triton X-100 and placed on ice for 5 minutes, and the cells were blocked with blocking solution at room temperature for 2 hours. Then, LC3 antibody was added and incubated at 4°C overnight. On the second day, secondary antibody labeled with Alexa Fluor 488 was added, washed with PBS for 10 minutes for a total of 3 times, and DAPI nuclear staining (blue) was performed and observed under a confocal microscope. The results are shown in Figure 2. Figure 10 As shown, bright green spots (equivalent to autophagosomes) were formed under the microscope.

[0108] The results showed that LC3-II autophagy protein (LC3B) was hardly expressed in melanoma cells, but the expression level increased after adding 1.6 mg / mL of the drug. The fluorescence intensity and expression level were higher after adding the CQ autophagy inhibitor. This was because CQ inhibited the fusion of autophagosomes and lysosomes, resulting in the inability to degrade the protein and causing aggregation, which was consistent with the WB results. Example 8

[0109] A mouse model was established using B16 melanoma to evaluate the pharmacodynamics of the stem cell-derived Dendrobium officinale obtained in this application and traditional Dendrobium extract on the growth of melanoma.

[0110] The specific steps are as follows: 1. Cell preparation: Culture B16-F10 cells in DMEM medium until the logarithmic growth phase. Collect the cells and resuspend them in PBS to adjust the cell concentration to 1×10 6 / mL.

[0111] 2. Mouse anesthesia: Anesthetize C57BL / 6J mice.

[0112] 3. Injection: Inject 0.1 mL of cell suspension (about 1×10 5 cells).

[0113] 4. Observation: Regularly sacrifice mice, remove tumors, and measure tumor volume using the formula: Tumor volume (mm³) = (length × width²) / 2. Observe tumor growth and the survival status of the mice.

[0114] 5. The experiment was divided into 6 groups, with 15 C57BL / 6J mice in each group, and they received the following treatments: high-dose of stem cell-derived Dendrobium officinale (SDO) extract group (2 mg / kg), low-dose of SDO group (1 mg / kg), high-dose of commercial Dendrobium (DO) group (2 mg / kg), low-dose of commercial Dendrobium (DO) group (1 mg / kg), control drug (5-fluorouracil group, the dosage was based on the standard dose), and blank control group (PBS).

[0115] The survival status and tumor growth of the mice were observed. The mice were killed 4 weeks after the above treatment and the tumor weight of each group was weighed. The tumor weight of each group is shown in Table 2 and Figure 11 shown.

[0116] Table 2

[0117] The results showed that the high-dose SDO group had the smallest average tumor weight (0.213 g), which was significantly different from the blank control group (1.000 g) (P < 0.01), indicating that the high-dose SDO group had the greatest inhibitory effect on tumor growth. The low-dose SDO group had the second-largest average tumor weight (0.350 g), which was significantly different from the blank control group (P < 0.01), but the effect was not as strong as that of the high-dose group. The average tumor weights of the high-dose DO group and the low-dose DO group were 0.850 g and 0.900 g, respectively, which were not significantly different from the blank control group (P > 0.05), indicating that traditional Dendrobium candidum had little inhibitory effect on tumor growth. The average tumor weight of the 5-fluorouracil group was 0.410 g, which was significantly different from the blank control group (P < 0.01) and the high-dose SDO group (P < 0.05), indicating that 5-fluorouracil had some inhibitory effect on tumor growth, but the effect was not as strong as that of the high-dose SDO group. Based on this, the stem cell-derived Dendrobium officinale (SDO) extract provided in this application has the most significant inhibitory effect on B16 melanoma at high doses, and also has a certain inhibitory effect at low doses, while the effect of traditional Dendrobium (DO) is not obvious.

[0118] 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. Use of a Dendrobium officinale shoot extract obtained by inducing differentiation of Dendrobium officinale stem cells in the preparation of a drug for treating and / or preventing tumors.

2. The use according to claim 1, characterized in that The drug can mediate tumor cell apoptosis by inducing tumor cell autophagy.

3. The use according to claim 1, characterized in that The tumor is melanoma.

4. The use according to claim 1, characterized in that 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 on a solid plant stem cell culture medium, and the Dendrobium officinale stem cells are cultured in the dark. The spacing between the multiple fragments is sufficiently large 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.

5. The use according to claim 4, 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 the end of each generation of cultivation and before the cultivation of the next generation, selecting amplified callus tissue with basically 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 distance is large enough so 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).

6. The use according to claim 1, characterized in that 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; And / or, the differentiation induction condition parameters are: light intensity of 1800-2500 Lux, red light to blue light ratio of 3-5:1, light exposure time of 6-12 hours per day, and culture for 30-45 days.

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

8. The use according to claim 7, 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 Dendrobium officinale buds, 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.

9. The use according to claim 8, characterized in that In step (3), the step of filtering step by step is as follows: filtering the primary stock solution through filters with a precision 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.

10. The use according to claim 8, characterized in that In step (2), the mass ratio of the powder to water is 1:20 to 1:50, and the soaking time is 2 to 5 hours; and / or, In step (4), the second filtration is performed using a filter with an accuracy of 0.22 μm.

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