CN117821364A - Puerarin adventitious root culture medium for improving puerarin content and application thereof - Google Patents

Abstract

The invention belongs to the field of plant cells, and discloses a pueraria adventitious root culture medium for improving puerarin content and application thereof, wherein the culture medium is based on an MS culture medium, IBA is 1.0-3.0mg/L, sucrose is 25-30g/L, kong Qumei elicitor is 50-200mg/L, and pH is 5.6-6.0. The method for producing the adventitious roots of the kudzuvine root has the characteristics of short production period, high efficiency, industrialized production and the like, and provides technical support for developing kudzuvine root biological medicaments and series of health-care products by applying a fermentation engineering technical platform.

Description

Puerarin adventitious root culture medium for improving puerarin content and application thereof

Technical Field

The invention belongs to the technical field of plant cells, and particularly relates to a kudzuvine root adventitious root culture medium for improving puerarin content and application thereof.

Background

The radix Puerariae (Pueraria lobata) is perennial deciduous vine of Pueraria of Papilionaceae of Leguminosae, and the total number of Pueraria plants is about 30 worldwide; pueraria lobata has effects of accelerating blood circulation and blood flow, and has effects of relieving fever, promoting eruption, invigorating yang, relieving diarrhea, and relieving alcoholic intoxication. At present, more than 70 phytochemicals have been identified from kudzuvine root, and the phytochemicals can be classified into isoflavone, triterpenoid saponin, coumarin, alkaloid and the like according to the chemical components, and the isoflavone and the triterpenoid saponin are generally considered to be main active components of the kudzuvine root. Wherein the isoflavone is mainly puerarin, genistein, formononetin, etc. Puerarin has been studied extensively for its potent various pharmacological activities including cardioprotection, vasodilation, cardiovascular protection, anti-inflammatory, antioxidant, neuroprotection, pain relief, bone formation promotion, alcohol intake inhibition, anti-cancer, liver protection, estrogenic activity. Experimental and clinical studies have reported that puerarin is also widely used in cardiovascular disease; diabetes and its complications; osteonecrosis and cancer treatment. In addition, puerarin can delay gastric emptying, slow down the absorption rate of alcohol, reduce intestinal wall permeability and protect gastrointestinal mucosa. Meanwhile, puerarin can also regulate the activity of enzymes related to ethanol metabolism, such as ethanol dehydrogenase and aldehyde dehydrogenase, and selectively inactivate cytochromes, so that the generation of free radicals is reduced, and the damage to liver cells is reduced; puerarin can improve in vivo antioxidant enzyme activity, and reduce oxidative stress in tissue caused by liver injury. In addition, the radix Puerariae starch is main edible component, and has amylose content of 19.8-60.5% in radix Puerariae starch, fat, protein, ash content of less than 1%, and flavonoid such as total flavone, puerarin, etc., and is rich in mineral elements (iron, phosphorus, calcium, zinc, potassium, etc.) necessary for human body.

With the development and utilization of radix Puerariae and Ginseng radix resources, the radix Puerariae resources are seriously damaged, and some excellent resources are being reduced and disappeared. In addition, due to single planting variety, resource protection and lag in variety breeding research, diseases and insect pests are increased, the variety is degraded, and the quality, the resistance and the yield are continuously reduced. In vitro culture of plant cells, tissues and organs is one of the important means for germplasm creation, and can be used for efficient production of bioactive substances in medicinal plants without environmental restrictions of time and space.

Therefore, the method for culturing the kudzuvine root, which can improve the content of puerarin, has short production period and high efficiency, is a problem to be solved by the technicians in the field.

Disclosure of Invention

In order to solve the technical problems, the invention provides a pueraria adventitious root culture medium for improving puerarin content and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a pueraria adventitious root culture medium for improving puerarin content uses MS culture medium as basic culture medium, IBA 1.0-3.0mg/L, sucrose 25-30g/L, kong Qumei inducer 50-200mg/L (total sugar/culture medium) and pH 5.6-6.0.

According to the invention, MS is used as a basic culture medium, and the growth of adventitious roots and puerarin accumulation are regulated and controlled by changing carbon sources, hormones and adding fungus inducers; the IBA has a promoting effect on the growth of adventitious roots, root hairs grow in a root tip elongation area of 3-5d adventitious roots, 6-14d root hairs are obviously increased, 15-23d new adventitious roots grow rapidly, the growth speed of 23-30d is slow, the root diameter is thickened, the IBA has the effect of promoting the growth of adventitious roots, the IBA is beneficial to the accumulation and synthesis of active substances, the IBA has a promoting effect on the elongation of branches of adventitious roots, and the accumulation of puerarin has a certain influence; kong Qumei elicitors (fungal elicitors) can regulate secondary metabolite accumulation in adventitious roots through signal transduction; therefore, the culture medium formula which is most favorable for the accumulation of the adventitious root growth active substances is obtained by adjusting the components of the culture medium of the adventitious root of the kudzuvine root.

Preferably, the IBA is 2.0mg/L and the Kong Qumei inducer is 100mg/L (total sugar/medium).

Preferably, the preparation method of the Kong Qumei inducer comprises the following steps: inoculating Aspergillus nidulans to PDA culture medium, dark culturing at 26-28deg.C for 6-9d, collecting mycelium after velvet, thinning or microcreping, sterilizing, adding water, grinding, filtering to obtain Kong Qumei inducer solution, and measuring total sugar content by anthrone colorimetric method.

Preferably, the PDA culture medium is a solid culture medium of 8-12g/L of potato extract powder, 10-15g/L of glucose and 8-10g/L of agar.

The application of the culture medium in the culture of the adventitious roots of the kudzuvine root is described above.

The adventitious roots of the kudzuvine root can be rapidly propagated in a large amount in a short time through liquid suspension culture, and a large amount of physiologically active substances are obtained at the same time, so that the good properties of the parent are effectively maintained, the variety is prevented from being mixed and viruses are prevented from accumulating in the plant body, and the method is free from season limitation.

The invention is characterized in that the puerarin is obtained by culturing the adventitious roots of the pueraria, the puerarin is mainly obtained from the cultivated pueraria, and various effective pharmacological activities of the puerarin are widely studied, wherein the pharmacological activities include heart protection, vasodilation, cardiovascular protection, anti-inflammatory, antioxidation, neuroprotection, pain relief, bone formation promotion, alcohol intake inhibition, anticancer, liver protection, estrogen activity and the like,

preferably, the culturing conditions are as follows: culturing at 25-27deg.C for 30-35d in dark.

Preferably, the cultivation is on a shaker at a rotation speed of 90-120 rpm.

Preferably, the inoculation amount of the adventitious roots of the kudzuvine root is 6-8g/L.

Preferably, the preparation method of the adventitious roots of the kudzuvine root comprises the following steps: taking field-planted kudzuvine root, taking 0.5-1cm stem as an explant, cutting 3-4 pieces of the explant, placing the cut explant on a sterile adventitious root culture medium, culturing for 25-35d in dark at 25 ℃, and selecting adventitious roots of the kudzuvine root which grow vigorously on a solid culture medium and have no browning and strong meristematic ability.

Preferably, the adventitious root medium is: taking an MS culture medium as a basic culture medium, adding 1-3mg/L IBA, 25-30g/L sucrose and 4-6g/L agar powder, uniformly mixing, and sterilizing.

The wild pueraria adventitious roots are prepared by the culture method, and the wild pueraria adventitious roots are subjected to scale culture, so that the culture can be used as a raw material for producing puerarin to replace wild pueraria resources, and the purpose of protecting the pueraria resources is achieved.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention adopts liquid suspension culture to culture the adventitious roots of the kudzuvine root, the wet weight of the harvested adventitious roots is 73g/L, the dry weight is 6.6g/L, the growth of the adventitious roots is 10 times in 30 days, the puerarin content is 22.1% of the dry weight, and the total content is 9.2 times of the puerarin specified value of 2.4% in Chinese pharmacopoeia.

(2) In the invention, the dynamic change rule of the growth of the adventitious roots and the accumulation of physiologically active substances is ascertained by measuring the content of puerarin in the adventitious roots, the wet weight and dry weight of the adventitious roots and other change conditions in liquid suspension culture, and a co-culture adventitious root growth dynamics model is established; by means of hormonal regulation, the accumulation of target substances in adventitious roots is promoted.

(3) The culture medium for culturing the kudzuvine root has the advantages of short production period, high efficiency and industrialized production, provides a new method for producing physiologically active substances such as puerarin, provides a new idea for reasonable development and utilization of the kudzuvine root, can effectively protect wild Chinese herbal medicine resources, and provides a opportunity for modern development of traditional Chinese herbal medicines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the growth state of adventitious roots in examples 1 and 2;

FIG. 2 is a chromatogram of the adventitious root puerarin of examples 1 and 2.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The MS culture medium is a common basic culture medium, is designed for tobacco cell culture in 1962 by Murashige and Skoog, and is characterized in that the concentration of inorganic salt and ions is higher, the MS culture medium is a stable ion balance solution, the nitrate content is high, the quantity and the proportion of nutrients are proper, and the nutrient and physiological requirements of plant cells can be met, so that the MS culture medium has a wider application range, and most plant tissue cultures are quickly propagated by using the MS culture medium as the basic culture medium of the culture medium.

Example 1

A method for culturing adventitious roots of radix Puerariae for increasing puerarin content comprises the following steps:

(1) Preparation of modified MS medium: weighing 20g of MS culture medium powder (Beijing Kang Beisi technology Co., ltd.) and 30g of sucrose per 1 liter of culture medium, adding the MS culture medium powder and sucrose into 1L of water, and uniformly stirring to obtain an MS liquid basic culture medium with sucrose concentration of 30 g/L;

adding IBA with three gradients of 1.0, 2.0 and 3.0mg/L into the obtained MS liquid basic culture medium, regulating the pH value to 5.8, uniformly stirring, and sterilizing to obtain an improved MS culture medium;

(2) Transfer of adventitious roots: taking field planted kudzuvine root as an explant experimental material after detoxification, selecting 0.5-1cm stem as a basic culture medium, sequentially adding 2mg/L IBA, 30g/L sucrose and 6g/L agar powder into the basic culture medium, uniformly mixing, pouring the mixture into a 30mL glass bottle for sterilization for standby, placing the prepared 4 explant sections on a sterile adventitious root culture medium, culturing for 30d under the dark condition at 25 ℃, selecting adventitious roots with vigorous growth and no browning, taking the adventitious roots with strong labor-saving capability as explants, cutting the adventitious roots into small sections on an ultra-clean workbench, transferring the small sections into an improved MS culture medium, and culturing in a shaking table in a dark manner, wherein the suspension culture condition of the adventitious roots is as follows: the culture temperature is 26 ℃, the rotation speed of a shaking table is 110rpm, after 30d culture, the growth condition is observed, the result is shown as a figure 1, wherein 1-3 in the figure 1 are growth state diagrams of IBA 1, 2 and 3mg/L in sequence, and the figure shows that when the concentration of IBA is 2mg/L, the growth form of adventitious roots is best, the occurrence density of the adventitious roots is obviously increased, and the growth is prolonged when the root length is relatively low before; when the concentration is 1mg/L, the number of the induced callus on the explant is relatively large, the number of adventitious roots on the surface of the callus is relatively small, and the length of the adventitious roots is relatively long; when the addition concentration of IBA is 3mg/L, the adventitious roots are seriously brown, so that 2mg/LIBA is comprehensively considered to be selected as the optimal addition concentration of adventitious root induction;

meanwhile, collecting the product of the adventitious roots of the radix puerariae, measuring the puerarin content of the obtained product of the adventitious roots of the radix puerariae by adopting a high performance liquid chromatography (refer to general rule 0512 of Chinese pharmacopoeia), wherein the result is shown in figure 2, and 1-3 in figure 2 are chromatograms of IBA 1, 2 and 3mg/L in sequence;

the results of the hormone addition and the wet weight and dry weight of the adventitious roots are shown in Table 1;

comparative example 1 was carried out without adding IBA, the other parameters and steps were the same as in example 1, the relevant results are shown in Table 1, the influence of different hormone ratios in Table 1 on the growth of adventitious roots of Pueraria lobata and the accumulation of puerarin

As shown in Table 1, the concentration of IBA in the culture medium for culturing adventitious roots of radix Puerariae is optimal when the concentration is 2.0mg/L, the dry weight of the adventitious roots is 6.2g/L, the wet weight is 57g/L, the puerarin content of the adventitious roots of radix Puerariae is 6% of the dry weight, and the total content is 2.5 times of the specified value of puerarin in Chinese pharmacopoeia; compared with the examples, when IBA is not added, the biomass of the adventitious roots of the kudzuvine root and the puerarin content are lower.

Example 2

A method for culturing adventitious roots of radix Puerariae for increasing puerarin content comprises the following steps:

(1) Preparation of modified MS medium: weighing 20g of MS culture medium powder, 30g of sucrose and 2.0mg of IBA per 1 liter of culture medium, adding the MS culture medium powder, the IBA and the sucrose into 1L of water, and uniformly stirring to obtain an MS liquid basic culture medium with the sucrose concentration of 30g/L, IBA and the concentration of 2.0 mg/L;

adding 50, 100 and 200mg/L (total sugar/culture medium) of three gradient Kong Qumei inducers into the obtained MS liquid basic culture medium, regulating the pH value to 5.8, uniformly stirring, and sterilizing to obtain an improved MS culture medium; the preparation method of the Kong Qumei inducer comprises the following steps: 2 pieces of aspergillus porus are selected and inoculated on 25mL of PDA culture medium for activation, the culture medium is cultivated for 7d in the dark at 26 ℃ and has velvet-like texture, the culture medium is flat, thin or microcrepe, (the PDA culture medium is a solid culture medium added with 8g/L of potato extract powder, 10g/L of glucose and 8g/L of agar), the cultivated Kong Qumei is sterilized for 20min at 101kPa and 121 ℃, mycelia are centrifugally collected, washed by sterile water, fully ground and filtered, distilled water is used for constant volume to 50mL, and the total sugar content is measured to be 10.03mg/mL by an anthrone colorimetric method;

(2) Transfer of adventitious roots: taking field planted kudzuvine root as an explant experimental material after detoxification, selecting 0.5-1cm stem as a basic culture medium, sequentially adding 2mg/L IBA, 30g/L sucrose and 6g/L agar powder into the basic culture medium, uniformly mixing, pouring the mixture into a 30mL glass bottle for sterilization for standby, placing the prepared 4 explant sections on a sterile adventitious root culture medium, culturing for 30d under the dark condition at 25 ℃, selecting adventitious roots with vigorous growth and no browning, taking the adventitious roots with strong labor-saving capability as explants, cutting the adventitious roots into small sections on an ultra-clean workbench, transferring the small sections into an improved MS culture medium, and culturing in a shaking table in a dark manner, wherein the suspension culture condition of the adventitious roots is as follows: the culture temperature is 26 ℃, the rotation speed of a shaking table is 110rpm, after 30 days of culture, the growth condition is observed, and the result is shown as a figure 1, wherein 4-6 in the figure 1 are sequentially shown as a Kong Qumei inducer growth state diagram of 50mg/L, 100mg/L and 200mg/L, and the figure shows that most of adventitious roots added with 50mg/L of aspergillus nidulans are aggregated; the adventitious roots added with 100mg/L aspergillus nidulans grow more dispersedly, the roots are thick and long, lateral roots grow and the main roots which are elongated are less, and the color is dark brown; adventitious root side roots treated with high concentration (200 mg/L) Kong Qumei are thickened and reduced in number, and a small amount of scattered callus exists, so that the whole body is light brown;

meanwhile, collecting the product of the adventitious roots of the radix puerariae, measuring the puerarin content of the obtained product of the adventitious roots of the radix puerariae by adopting a high performance liquid chromatography (refer to general rule 0512 of Chinese pharmacopoeia), wherein the result is shown in figure 2, and 4-6 in figure 2 are chromatograms of 50, 100 and 200mg/L of the inducer of Kong Qumei in sequence;

the results of the hormone addition and the correlation of the wet weight and dry weight of the adventitious roots are shown in Table 2,

TABLE 2 influence of different Kong Qumei inducer ratios on growth of adventitious roots of Pueraria lobata and Puerarin accumulation

Kong Qumei elicitor (mg/L)	Wet weight (g/L)	Dry weight (g/L)	Puerarin content (%)
				50	49	4.8	8.9
100	73	6.6	22.1
				200	79	6.9	9.8

As is clear from Table 2, the culture medium Aspergillus porus inducer concentration is optimal when the concentration of the culture medium Aspergillus porus inducer for culturing the adventitious roots of the kudzuvine root is 100mg/L, the dry weight of the adventitious roots is 6.6g/L, the wet weight is 73g/L, the puerarin content of the adventitious roots of the kudzuvine root is 22.1% of the dry weight, and the total content is 9.2 times of the specified value of puerarin of Chinese pharmacopoeia.

The various embodiments are described in a progressive manner, each embodiment focusing on differences from the other embodiments, and identical and similar parts between the various embodiments are sufficient to be seen with each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. A kudzuvine root adventitious root culture medium for improving puerarin content is characterized in that the culture medium takes an MS culture medium as a basic culture medium, IBA is 1.0-3.0mg/L, sucrose is 25-30g/L, kong Qumei inducer is 50-200mg/L, and pH is 5.6-6.0.

2. The pueraria adventitious root culture medium with increased puerarin content according to claim 1, wherein said IBA is 2.0mg/L and said Kong Qumei inducer is 100mg/L.

3. The culture medium for adventitious roots of kudzuvine root for increasing puerarin content according to claim 1, wherein the preparation method of the Kong Qumei inducer is as follows: inoculating Aspergillus nidulans to PDA culture medium, dark culturing at 26-28deg.C for 6-9d, collecting mycelia after velvet, thinning or microcreping, sterilizing, grinding with water, and filtering to obtain Kong Qumei inducer solution.

4. The pueraria adventitious root culture medium for increasing puerarin content according to claim 3, wherein the PDA culture medium is a solid culture medium of 8-12g/L of potato extract powder, 10-15g/L of glucose and 8-10g/L of agar.

5. Use of the culture medium according to any one of claims 1-4 in the cultivation of adventitious roots of kudzuvine root.

6. The use according to claim 5, wherein the conditions of the culture are: culturing at 26+ -1deg.C for 30-35d in dark place.

7. The use according to claim 5, wherein the cultivation is on a shaker at a speed of 90-120 rpm.

8. The use according to claim 5, wherein the inoculum size of adventitious roots of kudzuvine root is 6-8g/L.

9. The application of claim 8, wherein the preparation method of the adventitious roots of the kudzuvine root comprises the following steps: after the kudzuvine root is detoxified, 0.5-1cm stem is selected as an explant, 3-4 pieces of the explant are cut and placed on a sterile adventitious root culture medium, and the culture is carried out for 25-35d under the dark condition at 25 ℃.

10. The use according to claim 9, wherein the adventitious root culture medium is: taking an MS culture medium as a basic culture medium, adding 1-3mg/L IBA, 25-30g/L sucrose and 4-6g/L agar powder, uniformly mixing, and sterilizing.