A preparation method of dendrobium root tip plant stem cells and its application in lowering blood sugar

Abstract

The invention discloses a preparation method of dendrobium root tip plant stem cells and their application in reducing blood sugar. The dendrobium root tip plant stem cell powder is obtained mainly through several different steps such as specimen pretreatment, specimen treatment, induction shaking table culture, liquid replacement, subculture, cryopreservation and harvesting, drying and crushing. Then, the obtained dendrobium plant stem cell powder has a significant blood sugar-lowering effect at a dose of 100 mg / kg / d, and can be used to prevent or treat diabetes.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for preparing dendrobium root tip plant stem cells and an application thereof in reducing blood sugar. Background Art

[0002] Plants have always played an important role in traditional and modern medicine. Nearly 80% of the world's population relies on plant extracts to maintain their vital health. Medicinal plants refer to plants that can be used as medicine in whole, in part, or in their secretions. For example, motherwort and selfheal, which are all used as medicine, ginseng, datura, belamcanda, platycodon, rhodiola, which are partially used as medicine, and cinchona, which is used as medicine after extraction.

[0003] In recent years, the research on Dendrobium has attracted more and more attention. Its rich alkaloids, polysaccharides and other biologically active ingredients have significant blood sugar lowering, anti-tumor, immunity enhancement, cardiovascular protection, and antioxidant capabilities, which are the focus of people's attention. In the latest version of the 2020 edition of the Chinese Pharmacopoeia, Dendrobium is defined as the cultivated products of the orchid plants Dendrobium nobile Lindl., Dendrobium huoshanense CZTang et SJCheng, Dendrobium chrysotoxum Lindl. or Dendrobium fimbriatum Hook. and their fresh or dried stems of similar species of plants of the same genus, while Dendrobium officinale appears as a single product.

[0004] At present, more than 50% of the world's medicines require these endangered medicinal plants as raw materials. In the past 10 years, herbal treatments in North America and Europe have increased by about 10% each year. Today, the market value of herbal treatments in North America and Europe is estimated to be 20 billion US dollars. The well-known environmental protection organization, the World Wildlife Fund (WWF), revealed that there are 4,000 to 10,000 species of plants in the world that are facing survival threats.

[0005] At the same time, due to the slow growth of medicinal plants, limited natural sources, raw materials susceptible to environmental and toxin pollution, active ingredient reduction and other factors, it is difficult to adapt to the needs of industrial production, further exacerbating the threat to the survival of medicinal plants. Therefore, it is urgent to develop a method to stably obtain plant stem cells with multiple active ingredients. Summary of the invention

[0006] The purpose of the present invention is to provide a method for obtaining Dendrobium plant stem cells.

[0007] According to a first aspect of the present invention, a method for preparing Dendrobium root tip plant stem cells is provided, comprising the following steps:

[0008] 1) Specimen pretreatment: remove all debris attached to the picked Dendrobium, clean and disinfect it, and then transfer the root tip of Dendrobium to a glass dish in a biosafety cabinet for later use;

[0009] 2) Specimen processing: After everything is ready, first use a surgical blade to remove the outermost epidermis of the root tip of the Dendrobium, then slowly further ring the Dendrobium, find the meristem, cut it into small pieces or filaments, place it in antioxidant solution, and after it is completely removed, rinse it with culture medium, and then transfer it to culture medium;

[0010] 3) Induced shaking culture: adding biological inducer to the culture medium into which the Dendrobium root tip plant stem cells have been transferred; placing the culture bottle in a shaking incubator for culture;

[0011] 4) Changing the medium, subculturing, freezing and harvesting: After 24-48 hours, the culture medium containing the root tip tissue of Dendrobium officinale is filtered, the filter residue is removed, and the culture medium and biological inducer are added again to further expand the culture; after the number of plant stem cells of the root tip of Dendrobium officinale to be cultured is sufficient, centrifugation, freezing or harvesting are performed;

[0012] 5) Drying and pulverizing: The harvested Dendrobium root tip plant stem cells are freeze-dried or microwave-dried. After being completely dried, they are pulverized and sieved using a small pulverizer to obtain Dendrobium plant stem cell powder.

[0013] Preferably, the cleaning and disinfection steps in step 1) are specifically as follows: rinse with tap water; place the rinsed root tip of Dendrobium officinale in a sterilized basin, disinfect with 75% ethanol for 1-5 minutes, then rinse with sterile water 2-4 times, then disinfect with 0.1-1% sodium hypochlorite disinfectant for 1-5 minutes, and then continue to rinse with sterile water for 2-4 times.

[0014] Preferably, the biological inducer in steps 3) and 4) is composed of 6-BA, NAA, amphotericin B and dual-antibody. Further, the concentration of 6-BA added to the culture medium is 0.1-2 mg / L, the concentration of NAA added to the culture medium is 0.1-2 mg / L, the concentration of amphotericin B added to the culture medium is 0.1-2 mg / L, and the concentration of dual-antibody added to the culture medium is (volume percentage) 0.5-2%.

[0015] Preferably, in step 3), the rotation speed of the shaking table is controlled to be 120-150 r / min and the temperature is controlled to be 20-25°C.

[0016] Preferably, the drying step in step 5) is performed by freeze drying or microwave drying.

[0017] Preferably, the culture medium is MS medium or B5 medium.

[0018] In the present invention, an oscillator shaker culture is used, which is of great significance. During the shaking process, a mild rheological force can be applied to the cell clusters in the culture medium to break them into smaller cell clusters or single cells; it can also facilitate the exchange of substances between the culture medium and the cells; it can also enable gas exchange between air through the gas-liquid interface, ensuring the oxygen required for cell growth and breathing, and at the same time facilitating the release of carbon dioxide.

[0019] At the same time, it should be noted that the present invention uses a combination of 6-BA, NAA, amphotericin B and double antibiotics as an inducer to achieve the effect of promoting the growth of plant stem cells and inhibiting bacteria.

[0020] 6-BA, or 6-benzylaminopurine, is the first artificially synthesized cytokinin. It has many physiological effects, such as promoting cell division, promoting differentiation of non-differentiated tissues, promoting cell enlargement and fattening, promoting seed germination, inducing the growth of dormant buds, and inhibiting or promoting the elongation growth of stems and leaves.

[0021] NAA, i.e., naphthaleneacetic acid, is a plant growth regulator of the auxin class, which has the effect of promoting cell division and expansion. In addition to the basic functions of general auxins, it can also promote the formation of plant adventitious roots and roots. In the present invention, plant stem cells from the root tip of Dendrobium officinale are used, and the addition of NAA has a unique and important significance.

[0022] Amphotericin B is a polyene antifungal antibiotic that can inhibit fungal growth by affecting cell membrane permeability. Double antibiotics, a mixture of penicillin and streptomycin, can inhibit bacterial growth and avoid cell contamination. It is of great significance in the early stages of the experiment and can protect the growth of the obtained plant stem cells.

[0023] In addition, the specific proportion of the MS culture medium in the present invention is: 4.23 g of macroelements, 85.3 mg of trace elements, 103.1 mg of vitamins, and 15.0 g of sucrose.

[0024] Furthermore, the specific ratio of the B5 culture medium is: 3.1 g of inorganic salts and vitamins, 0.1 g of inositol, and 20.0 g of sucrose.

[0025] The Dendrobium root tip plant stem cells prepared by the above method can be used in medicines for preventing and treating diabetes. Therefore, according to another aspect of the present invention, a medicine or health food with blood sugar lowering effect is provided, the components of which include the Dendrobium root tip plant stem cells prepared by the present invention.

[0026] In the present invention, the most groundbreaking technology is to use the root tip of Dendrobium to induce the production of Dendrobium root tip plant stem cells through biological methods, and at the same time use the powder of Dendrobium root tip plant stem cells as the main raw material to study its application in lowering blood sugar and develop medicines or compound health products with significant blood sugar lowering effects. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the following embodiments are specifically cited, and their role is understood to be an explanation of the present invention rather than any form of limitation of the present invention.

[0028] Example 1 Cultivation method of Dendrobium root tip plant stem cells

[0029] Remove all kinds of debris attached to the picked Dendrobium and rinse it with tap water for more than 2 hours. Place the rinsed root tip of Dendrobium in a sterilized basin, disinfect it with 75% ethanol for 2 minutes, and then rinse the root tip of Dendrobium with sterilized water 4 times. Use 1% sodium hypochlorite disinfectant for secondary disinfection for 2 minutes, and then continue to rinse with sterilized water for 4 times. After rinsing, transfer the root tip of Dendrobium to the glass dish of the biological safety cabinet. Use a surgical blade to remove the outermost epidermis of the root tip of Dendrobium, and then slowly ring the Dendrobium further, find the meristem, and cut it into small pieces or filaments. Place it in a 1mol / L sodium thiosulfate antioxidant solution until it is completely removed. Rinse it twice with MS culture medium and then transfer it to MS culture medium. Add 1mg / L 6-BA, 1mg / L NAA, 2mg / L amphotericin B, and 1% double-antibiotic inducer. The culture bottle was placed in a shaker, and the speed of the shaker was controlled to be 120 r / min and the temperature was controlled to be 22° C. After 24 hours, the culture medium containing the root tip tissue of Dendrobium was filtered, the filter residue was removed, and the culture medium and inducer were added to further expand the culture. After the culture was completed, the process of centrifugation, freezing, drying, and crushing was performed to obtain the Dendrobium plant stem cell powder.

[0030] Example 2 Investigation of the yield of Dendrobium root tip plant stem cells under different culture medium conditions

[0031] Using the method in Example 1, an equal amount of root tips of Dendrobium officinale were taken to culture stem cells, and different culture media or inducers were used for culture, as shown in Table 1.

[0032] Table 1 Grouping methods of different culture media or inducers

[0033]

[0034] Through the above experiments, the obtained Dendrobium root tip plant stem cells were freeze-dried and then crushed. The amount of dry powder obtained each time was weighed. Each group conducted 5 parallel experiments and took the average value. The final experimental results are shown in Table 2.

[0035] Table 2 Effects of different culture media or inducer groups on the yield of plant stem cells from the root tips of Dendrobium officinale

[0036]

[0037]

[0038] Example 3 Effect of Dendrobium root tip plant stem cells on promoting the growth and differentiation of rat insulin cells in vitro

[0039] The rat insulin (INS-1) cell line was selected, and the MTT method was used to determine the activity of Dendrobium root tip plant stem cells in promoting cell proliferation in vitro. The concentration of the determination was 0-100μg / ml, and the treatment time was 72 hours. The results showed that Dendrobium root tip plant stem cells had a significant effect on promoting the growth and differentiation of rat insulin cells. T-test was used for statistical analysis, and p<0.05 was significantly different. The specific results are shown in Table 3.

[0040] Table 3 Growth promotion effect of Dendrobium root tip plant stem cells on rat insulin cells (%) (M±SE)

[0041]

[0042] The results showed that the plant stem cells from the root tip of Dendrobium officinale significantly promoted the proliferation of rat insulin cells, **p<0.01 compared with the blank group.

[0043] Example 4 Animal Function Experiment

[0044] The powder of Dendrobium root tip plant stem cells was prepared by the method in Example 1, and then animal function tests were carried out according to the Technical Specifications for Inspection and Evaluation of Health Food (2003 Edition) of the Ministry of Health.

[0045] 80 male Kunming mice aged 4 weeks were purchased. After being fed normally in the animal breeding room for 1 week to adapt to the environment, the weight and blood sugar level of the mice were measured. 10 mice were used as the blank control group, fed normally, and always given ordinary feed. The remaining 70 mice were intraperitoneally injected with streptozotocin (STZ) at a dose of 70 mg / kg. The blood sugar level was measured 72 hours later. The mice with a blood sugar level of 10-25 mmol / l were used as model animals. The model animals were always given high-sugar feed. 50 model mice were selected and divided into 5 groups according to the blood sugar level, and the average blood sugar level of each group was close, with 10 mice in each group, plus mice as the blank control group, a total of 6 groups, 6 groups are: blank control group, model group: gavage of diabetic mice with normal saline 0.3 ml / d; positive control group (metformin group 150 mg / kg / d): low dose of Dendrobium root tip plant stem cells 25 mg / kg / d; medium dose group: 50 mg / kg / d; high dose group: 100 mg / kg / d. The drug was administered by gavage once a day for 14 consecutive days, and the blood glucose level was measured after fasting. The specific results are shown in Table 4:

[0046] Table 4 Effects of Dendrobium root tip plant stem cells on STZ-induced diabetic mouse model

[0047]

[0048]

[0049] Compared with the blank control group, ▲ p<0.05; compared with the model group, *p<0.05, **p<0.01

[0050] Table 4 shows that after the successful modeling of diabetic mice, the blood sugar level of the model group increased after eating high-sugar feed, the blood sugar level of the positive control group decreased after being given the positive drug metformin, and the blood sugar level of the diabetic mice given Dendrobium root tip plant stem cells decreased significantly, which is equivalent to the clinical first-line drug metformin, indicating that it has a significant effect on reducing the increase in blood sugar caused by streptozotocin (STZ), and there is a certain dose-effect relationship, and the effect is good.

Claims

1. A method for preparing Dendrobium root tip plant stem cells, characterized in that: The following steps are involved: 1) Specimen pretreatment: remove all debris attached to the picked Dendrobium, clean and disinfect it, and then transfer the root tip of Dendrobium to a glass dish in the biosafety cabinet for later use; 2) Specimen processing: After everything is ready, first use a surgical blade to remove the outermost epidermis of the root tip of the Dendrobium, then slowly further ring the Dendrobium, find the meristem, cut it into small pieces or filaments, place it in antioxidant solution, and after it is completely removed, rinse it with culture medium and then transfer it to culture medium; 3) Induced shaking culture: add biological inducer to the culture medium with the Dendrobium root tip plant stem cells transferred; place the culture bottle in a shaking table for culture; control the speed of the shaking table to 120-150r / min and the temperature to 20-25℃; 4) Medium replacement, subculturing, freezing and harvesting: After 24-48 hours, filter the culture medium containing the root tip tissue of Dendrobium officinale, remove the filter residue, re-add the culture medium and biological inducer, and further expand the culture; centrifuge, freeze or harvest after the number of plant stem cells in the root tip of Dendrobium officinale is sufficient; 5) Drying and pulverizing: freeze-drying or microwave-drying the harvested Dendrobium root tip plant stem cells. After they are completely dried, they are pulverized and sieved using a small pulverizer to obtain Dendrobium plant stem cell powder; The culture medium is MS culture medium, and the biological inducer is composed of 1 mg / L 6-BA, 1 mg / L NAA, 2 mg / L amphotericin B and 1% double antibiotic; The antioxidant liquid is 1 mol / L sodium thiosulfate.

2. The preparation method according to claim 1, characterized in that: The cleaning and disinfection steps described in step 1) are specifically as follows: rinse with tap water; place the rinsed root tip of Dendrobium officinale in a sterilized basin, disinfect with 75% ethanol for 1-5 minutes, then rinse with sterilized water 2-4 times, then disinfect with 0.1-1% sodium hypochlorite disinfectant for 1-5 minutes, and then continue to rinse with sterilized water for 2-4 times.

3. Use of the preparation method according to any one of claims 1-2 in the preparation of medicines or health foods with hypoglycemic effects.

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