A co-culture method and medium for improving the content of physiological active substances in panax notoginseng and pueraria lobata adventitious roots

Abstract

The present application provides a method for co-culturing adventitious roots of Panax notoginseng and Pueraria lobata to improve the content of physiological active substances, and a culture medium. The present application belongs to the field of biotechnology, and particularly relates to a method for co-culturing adventitious roots of medicinal Panax notoginseng and Pueraria lobata. The adventitious roots of the two combined species are suspended in a MS medium containing 2.0-6.0 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, 30 g / L sucrose, and pH 5.8 for culturing, so that the co-culturing of adventitious roots of Panax notoginseng and Pueraria lobata can be realized, and a large amount of physiological active substances can be obtained. The adventitious roots of Panax notoginseng and Pueraria lobata produced by the method have the characteristics of short production cycle, high efficiency, and factory production, and lay a theoretical and practical foundation for developing Panax notoginseng and Pueraria lobata biological medicines and a series of health care products by using fermentation technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a method for improving the content of physiological active substances in Panax notoginseng and Pueraria lobata adventitious root co-culture and a culture medium. BACKGROUND

[0002] Pueraria lobata is a perennial deciduous vine of the legume family, Fabaceae, in the subfamily Papilionoideae. It is one of the medicinal plants in traditional Chinese medicine. There are about 30 species of Pueraria worldwide. Pueraria lobata can accelerate blood circulation and blood flow, and has the effects of fever reduction, rash, yang rising, diarrhea stopping, and alcohol resolving. At present, more than 70 kinds of phytochemicals have been identified in Pueraria lobata, which can be divided into isoflavones, triterpene saponins, coumarins, and alkaloids according to their chemical composition. Isoflavones and triterpene saponins are generally considered to be the main active ingredients of Pueraria lobata. Among them, isoflavones mainly include puerarin, genistein, and formononetin. Puerarin has been widely studied due to its various pharmacological activities, including heart protection, vasodilation, cardiovascular protection, anti-inflammatory, antioxidant, neuroprotection, pain relief, promotion of bone formation, inhibition of alcohol intake, anticancer, liver protection, and estrogen activity. Experimental and clinical studies have reported that puerarin is also widely used in the treatment of cardiovascular diseases, diabetes and its complications, bone necrosis, and cancer. In addition, puerarin can delay gastric emptying, slow down the absorption rate of alcohol, reduce intestinal wall permeability, and protect the gastrointestinal mucosa. At the same time, puerarin can also regulate the activity of enzymes related to ethanol metabolism, such as alcohol dehydrogenase and aldehyde dehydrogenase, selectively inactivate cytochrome, thereby reducing the production of free radicals and reducing liver cell damage. Puerarin can increase the activity of antioxidant enzymes in the body and reduce oxidative stress in tissues caused by liver damage.

[0003] Panax notoginseng (Burk.) F.H.Chen, also known as Panax notoginseng, is a perennial herb of the Araliaceae family. Panax notoginseng is called Panax notoginseng because it can be harvested after three to seven years of cultivation and each plant has three leaf stalks with seven leaves on each stalk. Panax notoginseng is warm in nature and slightly bitter in taste, and the whole plant can be used as medicine. It has the effects of promoting blood circulation, removing blood stasis, and relieving swelling and pain. The famous Chinese patent medicine "Yunnan Baiyao" and "Pianzaihuang" are made from Panax notoginseng. Due to its unique and obvious effects, Panax notoginseng has been increasingly used in various industries. In recent years, there have been numerous products related to cardiovascular and cerebrovascular diseases, and the use of Panax notoginseng has continued to rise in various industries. With continuous research, scholars have discovered compounds such as total saponins, vitamins, and alcohols in Panax notoginseng, as well as inorganic elements such as potassium, magnesium, and calcium. These active ingredients are the basis for Panax notoginseng's unique properties, such as hemostasis, anti-inflammatory, and antioxidant effects. Currently, saponins are one of the main active ingredients of Panax notoginseng, and research on flavonoids, amino acids, and polyacetylenic alcohols in Panax notoginseng has also been reported.

[0004] Total saponins are the main effective components of Panax notoginseng, which has the effect of activating blood and removing blood stasis, and can reduce lipid and prevent and treat thrombosis. Total flavonoids of Pueraria lobata are the main effective components of Pueraria lobata, including puerarin, daidzein and other components, which have the effects of improving blood circulation, reducing blood sugar and reducing blood lipid. Puerarin and Panax notoginseng mainly have the effects of activating blood and removing blood stasis in traditional Chinese medicine, which can improve blood circulation, especially microcirculation. They are mainly used for treating cardiovascular and cerebrovascular diseases, hypertension and cerebral thrombosis. Pueraria lobata and Panax notoginseng are used as functional food raw materials, and simultaneous administration can have the effects of regulating the spleen and stomach, activating blood and removing blood stasis, and can also enhance the digestion and absorption of nutrients in the body, improve the body's immunity and enhance the effect of gastrointestinal function.

[0005] With the unscientific exploitation and utilization of Pueraria lobata and Panax notoginseng resources, the resources of Pueraria lobata are seriously damaged, and some excellent resources are decreasing and disappearing. In addition, due to the single planting variety, the research on resource protection and variety breeding is lagging behind, which leads to the increase of diseases and pests, the degeneration of species, the continuous decline of quality, resistance and yield. It is found that the in vitro culture of plant root organs is one of the important means for creating germplasm, and it is not limited by time and space environment, and it has no "heavy toxic side effects" and pesticide residues, which is a high-quality functional food, cosmetics and pharmaceutical raw materials.

[0006] Therefore, it is necessary to provide a culture medium and a culture method to solve the above problems. SUMMARY

[0007] According to the above technical problems, a Panax notoginseng and Pueraria lobata adventitious root co-culture method and culture medium for improving the synthesis content of physiological active substances are provided. The present application uses MS as the basic medium, adds sucrose, and uses plant hormones IBA, NAA and KT to regulate the growth of adventitious roots.

[0008] The technical means adopted by the present application are as follows:

[0009] A Panax notoginseng and Pueraria lobata adventitious root co-culture method for improving the synthesis content of physiological active substances, which suspends the adventitious roots of the combination of Panax notoginseng and Pueraria lobata in MS medium containing 2.0-6.0 mg / L IBA, 0.2-0.6 mg / L NAA, 0.1-0.3 mg / L KT, 30 g / L sucrose and pH 5.8 to culture, realizing the co-culture of Panax notoginseng and Pueraria lobata adventitious roots, and obtaining a large amount of physiological active substances.

[0010] Further, it specifically includes the following steps:

[0011] S1, preparation of the medium: 20g of MS medium powder and 20g of sucrose are weighed for each 1 liter of medium, the above ingredients are added to 1L of water, the pH is adjusted to 5.8, and the MS liquid basic medium is obtained after uniform stirring;

[0012] S2, hormone adjustment of adventitious root co-culture medium: 1.0-6.0mg / L IBA, 0.2-0.6mg / L NAA, and 0.1-0.3mg / L KT are added to the medium obtained in step S1, the pH is adjusted to 5.8, and the adventitious root co-culture medium is obtained after sterilization after uniform stirring;

[0013] S3, transfer of adventitious roots: the sterile adventitious roots of Gegen with a growth cycle of 25-30d and the sterile adventitious roots of Sanqi with a growth cycle of 30-45d are used as explants;

[0014] In the clean bench, the sterile adventitious roots of Sanqi and Gegen are cut into small pieces, which are transferred into the adventitious root co-culture medium obtained in step S2 at a ratio of 1:1, and are placed in a shaking bed for dark culture.

[0015] Further, in step S2, 4.0mg / L IBA, 0.2mg / L NAA, and 0.3mg / L KT are added to the medium obtained in step S1, the pH is adjusted to 5.8, and the adventitious root co-culture medium is obtained after sterilization after uniform stirring.

[0016] Further, the culture conditions in step S3 are as follows: under the condition of 20-30℃, the culture is carried out in the dark for 35-40d, and the shaking bed rotation speed is 100rpm, so that a large amount of physiological active substances can be obtained.

[0017] The application also provides an adventitious root co-culture medium for improving the content of physiological active substances of Sanqi and Gegen, which is MS medium containing 4.0mg / L IBA, 0.2mg / L NAA, 0.3mg / L KT, and 30g / L sucrose, and the pH is 5.8; and the culture is carried out at 26℃ and in the dark, and the shaking bed rotation speed is 100rpm.

[0018] Compared with the prior art, the application has the following advantages:

[0019] 1. The dry weight of the co-cultured adventitious roots harvested from the co-cultured adventitious roots of Sanqi and Gegen reaches 8.65g / L, and the adventitious roots of the two plants can grow and proliferate normally when co-cultured. The defect that the growth of different varieties is inhibited and the yield of harvested cells is reduced is overcome. The biomass of the two co-cultured adventitious roots harvested by culture in the optimal medium is high.

[0020] 2. In this invention, when adventitious roots of Panax notoginseng and Pueraria lobata are co-cultured, Panax notoginseng saponins and Puerarin are obtained. The total Panax notoginseng saponin content reaches 8.64% dry weight, which is 1.29 times the total content of 6.72% specified in the Chinese Pharmacopoeia; the Puerarin content is 3.1% dry weight, which is 1.30 times the total content of 2.4% specified in the Chinese Pharmacopoeia.

[0021] 3. This invention takes the co-cultured adventitious roots of kudzu root and Panax notoginseng as the research object. By measuring the changes in the content of total Panax notoginseng saponins and puerarin in the culture medium and the dry weight of adventitious roots, the dynamic changes in the growth of adventitious roots and the accumulation of physiologically active substances are explored, and a kinetic model of co-cultured adventitious root growth is established. The accumulation of target substances in adventitious roots is promoted by regulating the hormones IBA, NAA, and KT.

[0022] In summary, the method of this invention for producing adventitious roots of Panax notoginseng and kudzu has the advantages of short production cycle, high efficiency, and industrial-scale production. It also lays a theoretical and practical foundation for the application of fermentation technology to develop Panax notoginseng and kudzu biopharmaceuticals and a series of health products.

[0023] The active substances obtained by this invention are natural effective components biosynthesized from Panax notoginseng and Pueraria lobata. When used in combination in traditional Chinese medicine, they mainly have the effects of promoting blood circulation and removing blood stasis, improving blood circulation, especially microcirculation. Clinically, they are mainly used to treat cardiovascular and cerebrovascular diseases, hypertension, and cerebral thrombosis. They can also be used in the food and pharmaceutical industries, meeting people's needs, effectively protecting wild Chinese herbal medicine resources, and providing opportunities for the modernization of traditional Chinese herbal medicine.

[0024] Based on the above reasons, this invention can be widely promoted in the field of co-cultivation technology of adventitious roots of Panax notoginseng and Pueraria lobata. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows actual photos of adventitious roots of Panax notoginseng and Pueraria lobata cultured using the adventitious root co-culture medium provided by this invention. Detailed Implementation

[0027] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of the present application.

[0028] The present application provides a Panax notoginseng and Pueraria lobata adventitious root co-culture medium for improving the content of physiological active substances and a culture method thereof, and illustrates the application of the medium in the co-culture of Panax notoginseng and Pueraria lobata adventitious roots, so that the co-culture of Panax notoginseng and Pueraria lobata adventitious roots is realized, and a large amount of physiological active substances are obtained.

[0029] First, the adventitious root co-culture medium is prepared, specifically including the following steps:

[0030] S1, preparation of the medium: 20 g of MS medium powder and 20 g of sucrose are weighed for each 1 liter of the medium, the above ingredients are added into 1 L of water, the pH is adjusted to 5.8, and the MS liquid basic medium is obtained after uniform stirring;

[0031] S2, hormone adjustment of the adventitious root co-culture medium: 1.0-6.0 mg / L of IBA, 0.2-0.6 mg / L of NAA and 0.1-0.3 mg / L of KT are added to the medium obtained in step S1, the pH is adjusted to 5.8, and the adventitious root co-culture medium is obtained after sterilization after uniform stirring; preferably, 4.0 mg / L of IBA, 0.2 mg / L of NAA and 0.3 mg / L of KT are added, the pH is adjusted to 5.8, and the adventitious root co-culture medium is obtained after sterilization after uniform stirring.

[0032] Then, the adventitious roots are transferred, and the sterile adventitious roots of Pueraria lobata with a growth cycle of 25-30 days and the sterile adventitious roots of Panax notoginseng with a growth cycle of 30-45 days are used as explants.

[0033] In the clean bench, the sterile adventitious roots of Panax notoginseng and Pueraria lobata are cut into small pieces, which are transferred into the adventitious root co-culture medium obtained in step S2 at a ratio of 1:1, and liquid suspension culture is carried out in a 250 ml triangular flask, and dark culture is carried out in a shaking bed, and the culture conditions are as follows: 20-30℃, dark culture for 35-40 days, and the shaking bed rotation speed is 100 rpm, so that a large amount of physiological active substances can be obtained.

[0034] During cultivation, root hairs emerged from the root tip elongation zone of adventitious roots at 5-7 days, with a significant increase in root hairs at 8-14 days. New adventitious roots grew rapidly at 15-30 days, and the growth rate slowed at 31-40 days, with the root diameter increasing. Both IBA and NAA played a role in the growth and development of adventitious roots. IBA promoted branching and elongation of adventitious roots, which was beneficial for the accumulation and synthesis of active substances. NAA promoted lateral thickening of adventitious roots and could also induce cell re-calling. Combined with KT, this promoted the growth of both Panax notoginseng and Pueraria lobata adventitious roots, leading to the accumulation of notoginsenosides and puerarin, respectively.

[0035] The adventitious roots cultivated according to this invention were subjected to high performance liquid chromatography (referring to General Chapter 0512 of the Chinese Pharmacopoeia) to determine the contents of total saponins and puerarin in Panax notoginseng. The results are shown in Table 1.

[0036] Table 1. Effects of different hormone ratios on the growth of co-cultured adventitious roots and the accumulation of total saponins and puerarin.

[0037]

[0038] The results showed that in the co-culture of adventitious roots of Panax notoginseng and Pueraria lobata, the combination of MS medium + 4.0 mg / L IBA + 0.2 mg / L NAA + 0.3 mg / L KT medium resulted in higher adventitious root biomass (e.g., ...). Figure 1 The sample shown had the highest total saponin and puerarin content, with a total saponin content of 8.64% dry weight. The Chinese Pharmacopoeia specifies a total saponin content of 6.72%. The puerarin content was ≥3.1% dry weight, while the Chinese Pharmacopoeia specifies a puerarin content of not less than 2.4%.

[0039] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for increasing the content of physiologically active substances in the adventitious roots of Panax notoginseng and Pueraria lobata co-cultured, characterized in that, Specifically comprising the following steps: S1, preparation of the medium: weigh 20 g of MS medium powder and 30 g of sucrose per 1 liter of medium, add the above ingredients to 1 L of water, adjust the pH to 5.8, and stir uniformly to obtain the MS liquid basic medium; S2, hormone adjustment of the adventitious root co-culture medium: add 2.0-6.0 mg / L IBA, 0.2-0.6 mg / L NAA, and 0.1-0.3 mg / L KT to the medium obtained in step S1, adjust the pH to 5.8, stir uniformly, sterilize, and obtain the adventitious root co-culture medium; S3, transfer of the adventitious roots: use the cultured aseptic adventitious roots of Gegen with a growth cycle of 25-30 days and the aseptic adventitious roots of Sanqi with a growth cycle of 30-45 days as explants; On the clean bench, cut the aseptic adventitious roots of Gegen and Sanqi into small pieces, transfer them into the adventitious root co-culture medium obtained in step S2 at a ratio of 1:1, and place them in the dark for culture on a shaker.

2. The method of claim 1, wherein the method is characterized by, In step S2, add 4.0 mg / L IBA, 0.2 mg / L NAA, and 0.3 mg / L KT to the medium obtained in step S1, adjust the pH to 5.8, stir uniformly, sterilize, and obtain the adventitious root co-culture medium.

3. The method of claim 1, wherein the method is characterized by, The culture conditions in step S3 are as follows: cultivate in the dark at 20-30°C for 35-40 days, and the shaker speed is 100 rpm, so that a large amount of physiologically active substances can be obtained.

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