Method for increasing content of effective components of dendrobium officinale and application

Abstract

The invention relates to a method for increasing the content of effective components of dendrobium officinale and application of the method. Dendrobium officinale explants are cultured through a subculture medium containing cactaceae plant components, and dendrobium officinale tissue culture seedlings with the content of the effective components increased are obtained; wherein the concentration of the cactaceae plant component in the subculture medium is 0.5 g / L to 3 g / L. In the subculture process of the dendrobium officinale, the stress effect of cactaceae plant components is utilized to ensure that the multiplication coefficient of subculture of the dendrobium officinale is high, so that more dendrobium officinale single plants can be obtained, and meanwhile, the content of effective components in the dendrobium officinale is doubled and even dozens of times increased; and sufficient resources are provided for new drug development and medicine.

Description

Technical Field

[0001] The present invention belongs to the field of plant tissue culture, and particularly relates to a method and application for increasing the content of active ingredients in Dendrobium officinale Background Art

[0002] Dendrobium officinale Kimura & Migo is a plant of the genus Dendrobium in the Orchidaceae family, mainly distributed on semi-shady and humid rock surfaces in mountainous areas of Anhui, Zhejiang, Fujian and other places in China. Its stem is used as medicine, which has the effects of promoting gastric juice secretion, enhancing appetite, promoting digestion, etc., and has good curative effects on gastric diseases such as disharmony between the spleen and stomach and indigestion. At the same time, it is rich in nutrients such as polysaccharides, flavonoids, phenols, and amino acids, which have the functions of moisturizing, delaying aging, promoting liver metabolism, detoxifying and bile secretion, and can also enhance immune function and improve the body's resistance. However, the germination rate of Dendrobium officinale seeds under natural conditions is very low, usually less than 5%, and even if germinated, the survival rate is extremely low, making it difficult to meet the social demand for Dendrobium officinale through conventional propagation means. Currently, the propagation of Dendrobium officinale through plant tissue culture technology not only shortens the seedling raising cycle but also realizes large-scale production, providing high-quality tissue culture seedlings for the market.

[0003] However, the content of active ingredients such as alkaloids, dendrobioside, and flavonoids in Dendrobium officinale is relatively low, restricting its application in new drug development and the pharmaceutical field. Therefore, developing a method that can increase the content of active ingredients in Dendrobium officinale is of great significance for making full use of Dendrobium officinale resources and promoting the development of related industries. Summary of the Invention

[0004] The purpose of the present invention is to provide a method and application for increasing the content of active ingredients in Dendrobium officinale.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a method for increasing the content of active ingredients in Dendrobium officinale, wherein the explants of Dendrobium officinale are cultured in a subculture medium containing components of the Cactaceae family to obtain tissue culture seedlings of Dendrobium officinale with increased content of active ingredients; wherein, the concentration of the components of the Cactaceae family in the subculture medium is 0.5 - 3 g / L.

[0007] The present invention utilizes the stress of cactus plant components on Dendrobium officinale, so as to increase the content of its active ingredients, which helps in the extraction, processing of pharmaceutical compounds and the development of new drugs. The stress mechanism of plants refers to a series of physiological and molecular response mechanisms of plants when facing environmental stress. These mechanisms help plants adapt to and resist various adverse environmental conditions, such as drought, salinity, low temperature, pests and diseases, etc. Specifically, plants will adjust their own growth and development to adapt to the changes in the external environment under adversity. Usually, plants will initiate secondary metabolic pathways under stress conditions and produce some compounds with defensive functions, such as alkaloids, flavonoids, etc. These substances help plants resist pests and diseases and adverse environments.

[0008] According to some specific embodiments, the cactus plant components are derived from one or more of cactus, Nopalea cochenillifera, Opuntia monacantha, Opuntia ficus-indica, Opuntia microdasys, Echinopsis tubiflora.

[0009] According to some specific embodiments, the cactus plant components come from the fleshy part of cactus plants.

[0010] According to some specific embodiments, the cactus plant components are added in the form of a slurry obtained by grinding fresh cactus plants with or without water, or in the form of a powder after drying the cactus plants.

[0011] According to some specific embodiments, the concentration of the cactus plant components in the subculture medium is 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L or 3 g / L.

[0012] According to some specific embodiments, the concentration of the cactus plant components in the subculture medium is 0.9 - 2.8 g / L. Further, the concentration of the cactus plant components in the subculture medium is 1.2 - 2.5 g / L.

[0013] According to some specific embodiments, the subculture medium is based on an incremented MS medium, and the incremented MS medium is one in which the concentrations of KNO3, NH4NO3, MgSO4·7H2O, and KH2PO4 in the MS medium are each independently increased by 25% - 75%. The subculture medium also contains CPPU with a final concentration of 2 - 3 mg / L and NAA with a final concentration of 0.2 - 0.6 mg / L.

[0014] Furthermore, the subculture medium also contains agar and sucrose. Further, the final concentration of the agar is 6 - 8 g / L, and the final concentration of the sucrose is 25 - 35 g / L.

[0015] Furthermore, the increased MS medium is such that the concentrations of KNO3, NH4NO3, MgSO4·7H2O, and KH2PO4 in the MS medium are each independently increased by 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75%.

[0016] Furthermore, the increased MS medium is such that the concentrations of KNO3, NH4NO3, MgSO4·7H2O, and KH2PO4 in the MS medium are each independently increased by 40% - 60%.

[0017] Furthermore, the increase amounts of the concentrations of KNO3, NH4NO3, MgSO4·7H2O, and KH2PO4 in the MS medium are the same.

[0018] Furthermore, the final concentration of CPPU in the subculture medium is 2 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L or 3 mg / L.

[0019] Furthermore, the final concentration of NAA in the subculture medium is 0.2 mg / L, 0.21 mg / L, 0.22 mg / L, 0.23 mg / L, 0.24 mg / L, 0.25 mg / L, 0.26 mg / L, 0.27 mg / L, 0.28 mg / L, 0.29 mg / L, 0.3 mg / L, 0.31 mg / L, 0.32 mg / L, 0.33 mg / L, 0.34 mg / L, 0.35 mg / L, 0.36 mg / L, 0.37 mg / L, 0.38 mg / L, 0.39 mg / L, 0.4 mg / L, 0.45 mg / L, 0.5 mg / L, 0.55 mg / L or 0.6 mg / L.

[0020] According to some specific embodiments, the Dendrobium officinale explant is the stem of a Dendrobium officinale plant, the stem of a Dendrobium officinale tissue culture seedling obtained by culturing without components of the Cactaceae family, or the stem of a Dendrobium officinale tissue culture seedling obtained by culturing with components of the Cactaceae family.

[0021] According to some specific embodiments, the culture conditions are as follows: temperature 25 - 30 °C, humidity 50 - 70%, light intensity 3500 - 5000 lux, 8 - 12 hours of light per day, and 25 - 35 days of culture as one cycle.

[0022] According to some specific embodiments, the active ingredients include one or more of stilbenes, sesquiterpenes, flavonoids, alkaloids, terpenoids, phenolic acids, tannins, and polysaccharides.

[0023] According to some specific embodiments, the active ingredients include, but are not limited to, one or more of Dendrobine A, Dendrobine C, Dendroside F, Eriodictyol, Isofraxidin, Hordenine, Crocinamide, Oleuropein, 1-(4-Hydroxybenzoyl)glucose, 2,4-Dihydroxyquinoline, 2-Piperidone, 3-Hydroxypterostilbene, 3-O-Methyl ellagic acid, Vitexin glucoside, 4,6-Dihydroxyquinoline.

[0024] The second aspect of the present invention provides the application of components of the Cactaceae family in culturing Dendrobium officinale tissue culture seedlings to increase the content of active ingredients in Dendrobium officinale tissue culture seedlings.

[0025] The third aspect of the present invention provides a method for preparing a Dendrobium officinale extract, which uses the Dendrobium officinale tissue culture seedlings obtained by culturing by the method described above as raw materials to extract the Dendrobium officinale extract.

[0026] Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0027] During the subculture process of Dendrobium officinale in the present invention, by utilizing the stress effect of components of the Cactaceae family, while ensuring a relatively high multiplication coefficient in the subculture of Dendrobium officinale, so that more individual Dendrobium officinale plants can be obtained, the content of active ingredients in Dendrobium officinale doubles or even increases by dozens of times, providing sufficient resources for new drug development and medicine. Description of the Drawings

[0028] Figure 1 A photograph of the Dendrobium officinale tissue culture seedlings for Comparative Example 1;

[0029] Figure 2 A photograph of the Dendrobium officinale tissue culture seedlings for Example 2. Detailed Embodiments

[0030] In the following text, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive.

[0031] All features disclosed in this invention, or steps in all methods or processes disclosed, except for mutually exclusive features or steps, can be combined in any manner.

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Unless otherwise stated, they can all be replaced by other equivalent or similar-purpose alternative features. Unless otherwise stated, each feature is just an example among a series of equivalent or similar features. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention.

[0033] The terms used in this invention generally have the meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements for specific use, and the implementation conditions not specified are conventional conditions in this industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0034] In this invention, operations without special instructions are carried out at room temperature. The raw materials in this application can be obtained commercially or prepared by conventional methods in the prior art.

[0035] The MS medium in the present invention can be obtained commercially or prepared by oneself. All components of the MS medium refer to: KNO3 1900 mg / L, NH4NO3 1650 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, CaCl2·2H2O 440 mg / L, Na2EDTA·2H2O 37.25 mg / L, FeSO4·7H2O 27.8 mg / L, MnSO4·4H2O 16.9 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, NaMoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, VB1 0.1 mg / L, VB6 0.5 mg / L, and glycine 2 mg / L.

[0036] Determination of the solid content of cactus: Clean the stem of the cactus, remove the thorns on the surface, and dry the surface moisture for later use. Remove the cactus epidermis, take the fleshy part of the cactus, weigh 30 grams of peeled flesh, cut it into pieces, and dry it in an oven at 60 °C for about 13 hours until constant weight. The weight of the dried peeled flesh is 1.82 grams.

[0037] Determination of the solid content of prickly pear: Clean the stem of the prickly pear, remove the thorns on the surface, and dry the surface moisture for later use. Remove the prickly pear epidermis, take the fleshy part of the prickly pear, weigh 30 grams of peeled flesh, cut it into pieces, and dry it in an oven at 60 °C for about 13 hours until constant weight. The weight of the dried peeled flesh is 2.18 grams.

[0038] In the present invention, the cactus family plant component added to the medium can be added in the form of powder ground from the dried peeled flesh, or directly ground into a slurry in the form of fresh (wet) peeled flesh, or added in the form of a slurry ground from fresh (wet) peeled flesh added with a certain amount of water. Grinding with water is beneficial to the grinding operation. However, since the fleshy part of the cactus family plant itself has a high water content, it can also be directly ground.

[0039] Among them, the content of the cactus family plant component in the medium is calculated based on the solid content of the cactus family plant component. When preparing, add the powder or slurry of the cactus family plant component to the medium and make up the volume to 1 L with water.

[0040] Example 1

[0041] (1) Preparation of cactus grinding liquid

[0042] Wash the stem of the cactus clean, remove the spines on the surface, and let the surface moisture dry for later use. Remove the epidermis of the cactus, take the fleshy part of the cactus, weigh 15 grams of peeled flesh, cut it into pieces, and process it with a grinder to obtain cactus grinding liquid, where the content of the cactus component is about 0.91 grams.

[0043] (2) Cultivate tissue-cultured seedlings of Dendrobium officinale

[0044] Cut a section of the stem of the tissue-cultured seedlings of Dendrobium officinale and culture it in the subculture medium for 30 days. The culture conditions are: control the culture temperature at 27 + 2 °C, light 10 h / d, light intensity 4000 lux, relative humidity 60%;

[0045] The subculture medium is: 3 / 2 MS medium, 2.5 mg / L of CPPU (forchlorfenuron, C 12 H 10 ClN3O), 0.4 mg / L of NAA (1-naphthaleneacetic acid), 0.91 g / L of cactus component, 7 g / L of agar, and 30 g / L of sucrose.

[0046] Among them, 3 / 2 MS medium means that the contents of four components, namely KNO3, NH4NO3, MgSO4·7H2O, and KH2PO4 in the MS medium, are increased by half, and the contents of other components remain unchanged. That is, the concentration of KNO3 in the 3 / 2 MS medium is 2850 mg / L, the concentration of NH4NO3 is 2475 mg / L, the concentration of KH2PO4 is 255 mg / L, and the concentration of MgSO4·7H2O is 555 mg / L.

[0047] Example 2

[0048] It is basically the same as Example 1, except for the concentration of the cactus component in the subculture medium. The concentration of the cactus component in Example 2 is 1.82 g / L.

[0049] Example 3

[0050] It is basically the same as Example 1, except for the concentration of the cactus component in the subculture medium. The concentration of the cactus component in Example 3 is 2.73 g / L.

[0051] Example 4

[0052] It is basically the same as Example 1, except that the added component in the subculture medium is the cactus component, and the concentration of the cactus component is 2.18 g / L. That is, the cactus (a plant of the Cactaceae family) is replaced with the stress material. By comparing Example 2 and Example 4, it can be seen that the proliferation coefficients of the subcultured seedlings of Dendrobium officinale under the stress of the cactus component and the stress of the cactus component are very little different. It can be seen that using different plants of the Cactaceae family as stress materials can achieve the same effect.

[0053] Comparative Example 1

[0054] It is basically the same as Example 1, except that the cactus component is not added to the subculture medium, that is, the concentration of the cactus component in this comparative example is 0 g / L.

[0055] Comparative Example 2

[0056] It is basically the same as Example 1, except for the concentration of the cactus component in the subculture medium, that is, the concentration of the cactus component in this comparative example is 3.64 g / L.

[0057] The growth conditions and proliferation coefficients of the tissue-cultured seedlings of Dendrobium officinale in each example and comparative example were observed, and the results are shown in Table 1 below. Proliferation coefficient (K): The multiple by which the number of the culture in the later culture increases compared to the previous culture is called the proliferation coefficient.

[0058] Table 1

[0059]

[0060] It can be seen from Table 1 above that when the cactus component is added, with the increase of the added concentration, under the stress of the cactus component, the proliferation coefficient of the tissue-cultured seedlings of Dendrobium officinale gradually decreases, and the leaves of Dendrobium officinale gradually change from dark green to light green. When the addition reaches 1.82 g / L, the whole leaves of the tissue-cultured seedlings of Dendrobium officinale turn white, and they grow well, and the proliferation coefficient decreases, but the decrease amplitude is relatively not obvious. Under the stress when the concentration of the cactus component reaches 3.64 g / L, the proliferation coefficient of Dendrobium officinale drops from 8.1 in Comparative Example 1 (without adding the cactus component) to 2.6, and the tissue-cultured seedlings of Dendrobium officinale grow slowly, the leaves are narrow and yellowish, the plants are short, and even some tissue-cultured seedlings of Dendrobium officinale die. Finally, it is screened that when the concentration of the cactus component is 1.82 g / L, the stress effect is relatively obvious and the proliferation coefficient is high.

[0061] Figure 1 The photo shows the tissue-cultured seedlings of Dendrobium officinale in Comparative Example 1. It can be seen that when the cactus component is not added, the tissue-cultured seedlings of Dendrobium officinale grow well, have good differentiation, and the leaves are green. Figure 2The photograph of the tissue culture seedlings of Dendrobium officinale in Example 2 can be seen. Under the stress of the cactus component, the tissue culture seedlings of Dendrobium officinale grow and differentiate normally, but the leaves turn milky white or even white.

[0062] In this application, chromatography is combined with mass spectrometry to identify the component analysis of the tissue culture seedlings of Dendrobium officinale under the stress of the cactus component. Chromatography is used for substance separation, and mass spectrometry is used for substance identification. Among them, high performance liquid chromatography tandem mass spectrometry can accurately determine the quantity and quality. The research on metabolic components plays a very important role in improving crop yield and enhancing the medicinal value of plants.

[0063] The detection and analysis of metabolic components are divided into two groups. One is the tissue culture seedlings of Dendrobium officinale in subculture without adding cactus components as the control group; the other is the tissue culture seedlings of Dendrobium officinale in subculture with 1.82 g / L cactus components added as the experimental group. Both groups use the leaves of the tissue culture seedlings of Dendrobium officinale in subculture as materials, and through the dry sample extraction method, qualitative and quantitative analysis of chromatography and mass spectrometry are carried out.

[0064] Through qualitative and quantitative analysis of chromatography and mass spectrometry, more than 200 kinds of alkaloids, more than 150 kinds of flavonoids, more than 170 kinds of terpenoids and other substances are detected. Only several substances are listed in Table 2 below as examples, and these substances are all valuable in the pharmaceutical and pharmaceutical industries.

[0065] Table 2

[0066]

[0067] It can be seen from Table 2 that for substances such as dendrobioside F, hordenine, and croamide, the relative content in the experimental group is much higher than that in the control group, and some have increased several times or even ten times. Thus, it can be seen that by applying the stress of the cactus component to the tissue culture seedlings of Dendrobium officinale, the effective components can be increased, which is helpful for the extraction and processing of pharmaceutical compounds and the development of new drugs.

[0068] Among them, alkaloids are widely used in the pharmaceutical field. The effect of hordenine is similar to that of ephedrine or ephedrine. It has the effects of relaxing bronchial smooth muscle, contracting blood vessels, vasopressin, raising blood pressure and exciting the central nervous system. It can be used to relieve bronchitis and bronchial asthma, can enhance the tension and movement of the uterus, and there is a dose-effect relationship. For croamide, it is mainly used in the medical field to treat diseases such as rheumatoid arthritis. And dendrobioside in Dendrobium officinale can significantly enhance the immune function of the body, activate the immune system, enhance the body's natural resistance, and fight various diseases.

[0069] In this application, stress is applied during the subculture process of Dendrobium officinale. Since the multiplication coefficient of the subculture of Dendrobium officinale is relatively high, more individual plants of Dendrobium officinale can be obtained, which can be used for processing, extraction and separation. Moreover, after stress, the amounts of many alkaloids, polysaccharides and flavonoids increase by several times or even dozens of times, providing sufficient resources for new drug development and medicine. Therefore, this application is of great significance.

[0070] The above has described the present invention in detail. The purpose is to enable those skilled in this field to understand the content of the present invention and implement it. However, this cannot limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for increasing the content of active ingredients in Dendrobium officinale, characterized in that: By culturing Dendrobium officinale explants in a subculture medium containing components of the Cactaceae family, Dendrobium officinale tissue culture seedlings with increased active ingredient content are obtained; wherein the concentration of the Cactaceae plant components in the subculture medium is 0.5 - 3 g / L.

2. The method for increasing the content of active ingredients of Dendrobium officinale according to claim 1, wherein: The Cactaceae plant components are derived from one or more of Opuntia, Nopalea cochenillifera, Opuntia monacantha, Opuntia ficus-indica, Opuntia microdasys, or Echinopsis tubiflora.

3. The method for increasing the content of effective components of Dendrobium officinale according to claim 1, characterized in that: The Cactaceae plant components are from the fleshy part of the Cactaceae plant.

4. The method for increasing the content of active ingredients of Dendrobium officinale according to claim 1, wherein: The Cactaceae plant components are added in the form of a slurry obtained by grinding fresh Cactaceae plants with or without water, or in the form of a powder obtained after drying the Cactaceae plants.

5. The method for increasing the content of active ingredients of Dendrobium officinale according to claim 1, characterized in that: The concentration of the Cactaceae plant components in the subculture medium is 0.9 - 2.8 g / L.

6. The method for increasing the content of effective components of Dendrobium officinale according to claim 5, characterized in that: The concentration of the Cactaceae plant components in the subculture medium is 1.2 - 2.5 g / L.

7. The method for increasing the content of effective components of Dendrobium officinale according to claim 1, characterized in that: The subculture medium is based on an incremented MS medium, and the incremented MS medium is one in which the concentrations of KNO3, NH4NO3, MgSO4·7H2O, and KH2PO4 in the MS medium are each independently increased by 25% - 75%. The subculture medium also contains CPPU with a final concentration of 2 - 3 mg / L and NAA with a final concentration of 0.2 - 0.6 mg / L.

8. The method for increasing the content of active ingredients of Dendrobium officinale according to claim 1, characterized in that: The Dendrobium officinale explants are the stems of Dendrobium officinale plants, the stems of Dendrobium officinale tissue culture seedlings obtained by culturing without Cactaceae plant components, or the stems of Dendrobium officinale tissue culture seedlings obtained by culturing with Cactaceae plant components.

9. Application of Cactaceae plant components in culturing Dendrobium officinale tissue culture seedlings to increase the active ingredient content of Dendrobium officinale tissue culture seedlings.

10. A method for preparing a Dendrobium officinale extract, characterized in that: Using the Dendrobium officinale tissue culture seedlings obtained by culturing according to the method described in any one of claims 1 to 8 as raw materials, the extraction of Dendrobium officinale extracts is carried out.

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