A culture method for improving synthesis of physiological active substances by ginseng and kudzu root

Abstract

The application belongs to the technical field of biology, and discloses a culture method for improving synthesis of physiological active substances of ginseng and kudzu root, wherein the culture method is to culture adventitious roots of ginseng and kudzu root together, and the culture medium used in the culture is as follows: taking MS culture medium as a basic culture medium, 30-50 g / L sucrose, 1.0-6.0 mg / L IBA, 0.2-0.6 mg / L NAA, and 0.1-0.3 mg / L KT, and pH is 5.6-6.0. The method has the characteristics of short production cycle, high efficiency and factory production for producing adventitious roots of ginseng and kudzu root, and provides technical support for developing ginseng and kudzu root biological medicines and series of health care products by using a fermentation engineering technology platform.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a cultivation method for improving the synthesis of physiologically active substances by ginseng and kudzu root. Background Art

[0002] Pueraria lobata (Kueraria lobata) is a perennial deciduous vine in the genus Pueraria, subfamily Faboideae, of the Fabaceae family. There are approximately 30 species of this genus worldwide. Pueraria lobata can improve blood circulation and blood flow, and has benefits such as reducing fever, clearing rashes, promoting yang and stopping diarrhea, and relieving hangovers. Currently, over 70 phytochemicals have been identified from Pueraria lobata, classified according to their chemical composition into several major categories, including isoflavones, triterpenoid saponins, coumarins, and alkaloids. Isoflavones and triterpenoid saponins are generally considered to be the primary active components of Pueraria lobata. Among these isoflavones, puerarin, genistein, and formononetin are the main active components. Puerarin has been extensively studied for its various pharmacological activities, including cardioprotection, vasodilation, cardiovascular protection, anti-inflammatory, antioxidant, neuroprotective, pain relief, bone formation promotion, alcohol intake suppression, anticancer, hepatoprotective, and estrogenic activity. Experimental and clinical studies have reported that puerarin is also widely used in the treatment of cardiovascular disease, diabetes and its complications, osteonecrosis, and cancer. In addition, puerarin can delay gastric emptying, slow the absorption rate of alcohol, reduce intestinal permeability, and protect the gastrointestinal mucosa. Puerarin can also regulate the activity of enzymes involved in ethanol metabolism, such as alcohol dehydrogenase and aldehyde dehydrogenase, and selectively inactivate cytochromes, thereby reducing the production of free radicals and alleviating liver cell damage. Puerarin can also increase the activity of antioxidant enzymes in the body and reduce oxidative stress in tissues caused by liver damage. Pueraria starch is the main edible ingredient in kudzu root. The amylose content in kudzu root starch is approximately 19.8-60.5%, and the fat, protein, and ash contents are all less than 1%. It also contains flavonoids such as total flavonoids and puerarin, and is rich in many mineral elements essential to the human body (iron, phosphorus, calcium, zinc, potassium, etc.).

[0003] The chemical composition of ginseng (Panax ginseng CA Meyer) primarily includes saponins, sugars, volatile components, organic acids and their esters, proteins, enzymes, sterols and their glycosides, polypeptides, lignin, and nitrogen-containing compounds. Ginsenosides and ginseng polysaccharides are the primary active ingredients and are widely used clinically. Ginsenosides have certain activities in regulating the immune and endocrine systems. Under certain conditions, they can increase or decrease blood pressure, enhance cardiac function, and also have a strong cardioprotective effect. Ginseng has both calming and soothing effects and refreshing and stimulating effects for those with weak constitutions. Ginseng extracts can antagonize many stimulant drugs, effectively counteracting the effects of central nervous system depressants such as chloral hydrate. Ginseng is also commonly used in modern clinical practice to improve abnormal immune responses in a variety of diseases, including ulcerative colitis. In summary, as a component of health foods, ginseng exerts significant pharmacological effects in regulating immune function, providing sedation, and protecting the heart.

[0004] The unscientific exploitation and utilization of kudzu and ginseng resources has severely damaged kudzu resources, leading to the decline and even disappearance of some high-quality resources. Furthermore, the limited number of cultivated varieties and the lag in resource conservation and variety breeding research have led to an increase in pests and diseases, degradation of plant characteristics, and a continuous decline in quality, resistance, and yield. In vitro culture of plant cells, tissues, and organs is a key tool for germplasm creation. Unrestricted by time and space, it can be used for the efficient production of bioactive substances from medicinal plants.

[0005] To this end, by co-culturing the adventitious roots of Pueraria lobata and Panax ginseng, the biosynthesis and accumulation of rare ginsenosides in Panax ginseng can be improved and enhanced by utilizing the induction and activation effects of puerarin in Pueraria lobata tissue cells on enzymes involved in rare saponin metabolism in Panax ginseng adventitious roots. Furthermore, the saponins secreted by Panax ginseng can inhibit the browning of Pueraria lobata adventitious roots and promote their growth. Therefore, the present invention provides a cultivation method that increases the synthesis of physiologically active substances in Panax ginseng and Pueraria lobata, has a short production cycle, and is highly efficient. Furthermore, the co-cultured Pueraria lobata and Panax ginseng is also a high-quality health food ingredient. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a cultivation method for improving the synthesis of physiologically active substances by ginseng and kudzu root. The present invention utilizes the co-cultivation of the adventitious roots of ginseng and kudzu root to significantly increase the content of ginsenosides and puerarin, which is beneficial to the accumulation of active substances. In addition, the combination of the two adventitious roots of ginseng and kudzu root can be directly used as a raw material for health products and used in the food, medicine and other industries to meet people's needs.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A cultivation method for improving the synthesis of physiologically active substances by ginseng and kudzu root comprises co-cultivating ginseng and kudzu root through adventitious rooting.

[0009] When different varieties are co-cultured, they inhibit each other's growth, which ultimately leads to a decrease in the yield of harvested cells. However, the present invention screened the optimal combination and found that the adventitious root biomass was higher when ginseng and kudzu root were co-cultured. Co-cultivation can significantly increase the content of ginsenosides and puerarin, which is beneficial to the accumulation of active substances.

[0010] Preferably, the culture medium used for the co-cultivation is: MS medium as the basal medium, 30-50 g / L sucrose, and pH 5.8-6.5.

[0011] The improved MS liquid basic culture medium adopted by the present invention can realize the co-cultivation of ginseng and kudzu root adventitious roots, and simultaneously obtain a large amount of pharmacologically active substances.

[0012] Preferably, the culture medium used for the co-cultivation is: MS medium as the basal medium, 30-50 g / L sucrose, 1.0-6.0 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, and pH 5.6-6.0.

[0013] The present invention uses a modified MS culture medium as a basic culture medium, uses sucrose and starch produced by kudzu root as carbon sources, and uses hormones to regulate the growth of adventitious roots. IBA has a promoting effect on the growth of adventitious roots. Root hairs grow in the root tip elongation zone of the adventitious roots in 5-7 days, the root hairs increase significantly in 8-14 days, the new adventitious roots grow rapidly in 15-30 days, the growth rate slows down in 31-40 days, and the root diameter becomes thicker. IBA has a function of promoting the growth of adventitious roots and is beneficial to the accumulation and synthesis of active substances. IBA and NAA both play a role in the growth and development of adventitious roots. IBA has a promoting effect on the elongation of adventitious root branches, and NAA is beneficial to the lateral thickening growth of adventitious roots. It can also cause cells to re-heal, and the combination is supplemented with KT. The hormone combination can achieve rapid growth of the adventitious roots of ginseng and kudzu root when co-cultured, solving the problem of high phenolic content in kudzu root inhibiting adventitious root growth. At the same time, starch, a secondary metabolite of kudzu root, can provide a carbon source for the adventitious roots during their growth, significantly increasing the content of ginsenosides and puerarin. Puerarin can also induce the expression of phenylalanine ammonia lyase (PAL), serine carboxylase (SCL), squalene cyclase (SQS), and ginsenoside synthase (GTS) during the synthesis of ginsenosides, promoting the accumulation of ginsenosides. The catalytic product of SQS is an important substrate for the synthesis of the rare ginsenoside Rg3. Therefore, the method of the present invention can significantly increase the content of ginsenosides and puerarin.

[0014] Preferably, the culture medium used for the co-cultivation is: MS medium as the basal medium, 30 g / L sucrose, 2.0 mg / L IBA, 0.2 mg / L NAA, 0.1 mg / L KT, and pH 5.8.

[0015] The present invention takes the co-cultivated adventitious roots of Pueraria lobata and ginseng as the research object, performs liquid suspension culture in a triangular flask, and determines the changes in the content of total ginsenosides and puerarin in the culture medium and the dry weight of the adventitious roots to explore the dynamic changes in adventitious root growth and the accumulation of physiologically active substances. A growth kinetic model of the co-cultivated adventitious roots is established, and the accumulation of target substances in the adventitious roots is promoted by regulating the hormones IBA, NAA, and KT.

[0016] Preferably, the co-cultivation conditions are: culturing in the dark on a shaking platform at 24-26°C for 35-40 days.

[0017] Preferably, the rotation speed of the shaking table is 80-120 rpm.

[0018] Preferably, the mass ratio of the ginseng adventitious roots to the kudzu root adventitious roots is 1:1-1.5, and the total inoculation amount of the ginseng adventitious roots and the kudzu root adventitious roots is: 5-10 g of adventitious roots per 100 mL of the culture medium.

[0019] Preferably, the preparation method of the ginseng adventitious root is as follows: cutting a 3-5 year old fresh ginseng root into segments, disinfecting and rinsing it, placing it in a ginseng adventitious root culture medium and dark culturing it at 20-26°C for 30-35 days to produce ginseng adventitious roots, wherein the inoculum amount is 5-10 g per 100 mL of the ginseng adventitious root culture medium, wherein the ginseng adventitious root culture medium is: 1 / 2-1 / 4 MS, 30-50 g / L sucrose, 1-5 g / L glucomannan, 0.3-0.5% agar, 1-5 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, pH 5.8-6.5;

[0020] The preparation method of the kudzu root adventitious roots comprises the following steps: cutting wild fresh kudzu roots into segments, disinfecting and rinsing the segments, placing the segments in a culture medium, and dark culturing the segments in the kudzu root adventitious root culture medium at 20-26°C for 30-35 days to produce kudzu root adventitious roots, wherein the inoculum amount is 5-10 g per 100 mL of the kudzu root adventitious root culture medium, wherein the kudzu root adventitious root culture medium is based on B5 culture medium, supplemented with 30-50 g / L sucrose, 0.5-1% kudzu root powder, 0.3-0.5% agar, 1-5 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, and 0.5-1% activated carbon, and has a pH value of 5.8-6.5.

[0021] The present invention uses kudzu root powder to prepare a natural culture medium, which provides macroelements, trace elements and carbon sources for the adventitious roots to be cultivated; the substances contained in the kudzu root powder provide a substrate for puerarin in the kudzu root adventitious roots; in addition, hormones are used to regulate the growth of the adventitious roots, so that the target product cultivated is safer and more reliable.

[0022] Preferably, the specific steps of segmenting, disinfecting and rinsing are: cutting into 1-2 cm segments, disinfecting with 70-75% alcohol for 10-20 seconds, then disinfecting with 0.1-0.5% mercurous chloride solution for 20-30 minutes, and then rinsing with sterile water for 3-5 times.

[0023] The adventitious root product of Pueraria ginseng obtained by the cultivation method described above.

[0024] As described above, the products of Pueraria lobata and Panax ginseng adventitious roots are used in the fields of food, health care products and preparation of medicines.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention adopts liquid suspension culture to co-cultivate ginseng and kudzu adventitious roots, and the dry weight of the co-cultivated adventitious roots harvested reaches 5.34 g / L. The adventitious roots of the two plants can grow and proliferate normally when co-cultivated. When different varieties are co-cultivated, the varieties inhibit each other's growth, which ultimately leads to a decrease in the harvested cell yield. However, by screening the best combination, the present invention can harvest the two co-cultivated adventitious roots with a higher biomass.

[0027] (2) The present invention obtains ginsenosides and puerarin by co-culturing ginseng and kudzu root adventitious roots. The total ginseng content is 0.64%, which is 1.42 times the value of 0.45% for total ginsenosides specified in the Chinese Pharmacopoeia; the puerarin content is 3.6%, which is 1.5 times the value of 2.4% for puerarin specified in the Chinese Pharmacopoeia. At the same time, the invention can stimulate the production of active substances that are not detected in single culture.

[0028] (3) The present invention can achieve rapid growth of the adventitious roots of ginseng and kudzu root when co-cultivated through hormone combination, thus solving the problem that the high content of phenolic substances in kudzu root inhibits the growth of adventitious roots; starch, a secondary metabolite of kudzu root, can provide a carbon source for the adventitious roots during their growth; at the same time, co-cultivation can greatly increase the content of ginsenosides and puerarin, which is beneficial to the accumulation of active substances; in addition, the combination of the two adventitious roots of ginseng and kudzu root can be directly used as a raw material for health products.

[0029] (4) The active substance obtained by the present invention is a natural effective ingredient synthesized by ginseng and kudzu root. The combination of kudzu root and ginseng is a commonly used Chinese medicine pair in clinical practice. The combination of the two can be used to treat diseases such as spleen and stomach qi deficiency, exogenous diseases, fever, diabetes, measles, stroke and hemiplegia. According to traditional Chinese medicine theory, kudzu root is sweet and cool in nature, which can restrain the warm and dry ginseng. The dispersing power of kudzu root can be suppressed by the astringent effect of ginseng. The two complement each other and can also complement each other. It is an example of Chinese medicine compatibility to increase efficacy and reduce toxicity. It can be used in food, medicine and other industries to meet people's needs.

[0030] (5) The cultivation method of the present invention has a short production cycle, high efficiency, and can be produced in a factory. It provides a new method for producing physiologically active substances such as saponins and puerarin, and provides new ideas for the rational development and utilization of kudzu root and ginseng. It can effectively protect wild Chinese herbal medicine resources and provide opportunities for the modernization of traditional Chinese herbal medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0032] Figure 1 This is a diagram showing the adventitious root growth status of the combination of culture medium MS + 4.0 mg / L IBA + 0.2 mg / L NAA + 0.3 mg / L KT in Example 1. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0034] MS medium is a commonly used basic culture medium. It was designed by Murashige and Skoog for tobacco cell culture in 1962. It is characterized by high concentrations of inorganic salts and ions, and is a relatively stable ion balance solution. It has a high nitrate content, and the quantity and proportion of its nutrients are appropriate, which can meet the nutritional and physiological needs of plant cells. Therefore, it has a wide range of applications. Most plant tissue cultures use it as the basic culture medium for rapid propagation.

[0035] Example 1

[0036] A method for increasing the production of physiologically active substances from ginseng and kudzu root comprises the following specific steps:

[0037] (1) Preparation of modified MS medium: Weigh the components of MS medium (Beijing Kangbaisi Technology Co., Ltd.) in proportion, add 30 g / L sucrose, dissolve in water, adjust the pH to 5.8, and stir evenly to obtain the modified MS liquid basal medium;

[0038] After adding hormones IBA, NAA and KT to the obtained modified MS liquid basal medium, adjusting the pH to 5.8, stirring evenly and then sterilizing to obtain a co-culture medium;

[0039] (2) Transfer of adventitious roots: The adventitious roots of Pueraria lobata cultured for 30 days and the sterile adventitious roots of ginseng with a growth period of 30 days were used as explants. Ginseng and Pueraria lobata adventitious roots were cut into small pieces on a clean bench and transferred into the adventitious root co-culture medium at a ratio of 1:1. The inoculation amount was 8% (8 g of adventitious roots were inoculated per 100 mL of culture medium). The adventitious roots were placed in a shaker for dark culture under the following conditions: culture temperature of 26°C and shaker speed of 100 rpm. After 40 days of culture, the adventitious roots of Pueraria lobata and ginseng were collected.

[0040] The preparation method of ginseng adventitious roots is as follows: 3-year-old fresh ginseng roots are cut into 1-2 cm segments, disinfected with 70% alcohol for 10 seconds, then disinfected with 0.5% mercurous chloride solution for 30 minutes, and then rinsed with sterile water three times, and placed in ginseng adventitious root culture medium. After incubation in the dark at 20-26°C for 30 days, a large number of new sterile ginseng adventitious roots are generated. The inoculum size is 10 g per 100 mL of ginseng adventitious root culture medium. The ginseng adventitious root culture medium consists of 1 / 2 MS + 30 g / L sucrose + 1 g / L glucomannan + 0.3% agar + 1 mg / LIBA + 0.2 mg / L NAA + 0.1 mg / L KT, pH 5.8.

[0041] The method for preparing kudzu root adventitious roots comprises: taking wild fresh kudzu root, cutting its root tip into 1-2 cm segments, disinfecting with 70% alcohol for 10 seconds, then disinfecting with 0.1% mercurous chloride solution for 20 minutes, rinsing with sterile water three times, placing in culture medium and culturing in the dark at 20-26°C, and after 30 days, a large number of kudzu root adventitious roots are newly generated; the inoculum size is 10 g per 100 mL of kudzu root adventitious root culture medium, wherein the kudzu root adventitious root culture medium is: B5 medium as a base medium supplemented with 30 g / L sucrose, 0.5% kudzu root powder, 0.3% agar, 1 mg / L IBA, 0.2 mg / L NAA, 0.1 mg / L KT, 0.5% activated carbon, and pH 5.8;

[0042] The obtained adventitious root product of Pueraria ginseng was used to determine the content of ginsenosides and puerarin by high performance liquid chromatography (referring to General Chapter 0512 of the Chinese Pharmacopoeia). The amount of hormone added and the test results are shown in Table 1.

[0043] Comparative Example 2 (Ginseng)

[0044] The difference from Example 1 is that only ginseng adventitious roots were inoculated, and the other parameters and steps were the same as in Example 1;

[0045] The obtained ginsenoside content of the adventitious root product of Panax ginseng was determined by high performance liquid chromatography (referring to General Chapter 0512 of the Chinese Pharmacopoeia). The test results are shown in Table 1.

[0046] Comparative Example 2 (Pueraria lobata)

[0047] The difference from Example 1 is that only adventitious roots of Pueraria lobata were inoculated, and the other parameters and steps were the same as those in Example 1;

[0048] The puerarin content of the obtained adventitious root product of Pueraria lobata was determined by high performance liquid chromatography (referring to General Chapter 0512 of the Chinese Pharmacopoeia). The test results are shown in Table 1.

[0049] Table 1 Results of the content of total saponins and puerarin in the products of Example 1 and Comparative Examples 1-2

[0050]

[0051]

[0052] From the data in Table 1, we can see that in the co-culture of ginseng and kudzu root adventitious roots, the adventitious root biomass of the combination of medium MS+4.0mg / LIBA+0.2mg / LNAA+0.3mg / L KT was higher (see Figure 1 ), the total saponins and puerarin content are the highest, the total ginseng content is 0.64%, which is higher than the value specified in the "Chinese Pharmacopoeia" for total ginsenosides of ginseng, and the puerarin content is 3.6%, which is higher than the value specified in the "Chinese Pharmacopoeia" for the puerarin content in Pueraria.

[0053] The various embodiments are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to in detail.

[0054] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for increasing the synthesis of physiologically active substances in ginseng and kudzu root, characterized in that: The culture method comprises co-culturing ginseng and kudzu root for adventitious roots; The co-culture medium is: MS medium as the base medium, 30-50 g / L sucrose, 2-4 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, pH 5.6-6.0; The mass ratio of the ginseng adventitious roots to the kudzu root adventitious roots is 1:1-1.5, and the total inoculation amount of the ginseng adventitious roots and the kudzu root adventitious roots is: 5-10 g of adventitious roots are inoculated per 100 mL of the culture medium; The preparation method of ginseng adventitious roots is as follows: cutting 3-5 year old fresh ginseng roots, disinfecting and rinsing them, placing them in a ginseng adventitious root culture medium and dark culturing them at 20-26° C. for 30-35 days to produce ginseng adventitious roots, wherein the inoculum amount is 5-10 g per 100 mL of the ginseng adventitious root culture medium, wherein the ginseng adventitious root culture medium comprises: 1 / 2-1 / 4 MS, 30-50 g / L sucrose, 1-5 g / L glucomannan, 0.3-0.5% agar, 1-5 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, and a pH of 5.8-6.5; The preparation method of the kudzu root adventitious roots comprises the following steps: cutting wild fresh kudzu roots into segments, disinfecting and rinsing the segments, placing the segments in a culture medium, and dark culturing the segments in the kudzu root adventitious root culture medium at 20-26°C for 30-35 days to produce kudzu root adventitious roots, wherein the inoculation amount is 5-10 g per 100 mL of the kudzu root adventitious root culture medium, wherein the kudzu root adventitious root culture medium is based on B5 culture medium, supplemented with 30-50 g / L sucrose, 0.5-1% kudzu root powder, 0.3-0.5% agar, 1-5 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, and 0.5-1% activated carbon, and has a pH value of 5.8-6.

5.

2. The method for increasing the synthesis of physiologically active substances by ginseng and kudzu root according to claim 1, characterized in that: The co-cultivation conditions are: culturing in the dark at 24-26° C. on a shaking platform for 35-40 days.

3. The method for increasing the ability of ginseng and kudzu root to synthesize physiologically active substances according to claim 2, wherein: The rotation speed of the shaking table is 80-120 rpm.

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