CN110100739A - A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation - Google Patents
A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation Download PDFInfo
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- CN110100739A CN110100739A CN201910547865.2A CN201910547865A CN110100739A CN 110100739 A CN110100739 A CN 110100739A CN 201910547865 A CN201910547865 A CN 201910547865A CN 110100739 A CN110100739 A CN 110100739A
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- radix astragali
- astragalus root
- cepha
- ethephon
- chloroethyl
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The present invention provides a kind of methods for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, comprising the following steps: S1, astragalus root of Radix Astragali is inoculated into improvement B5 fluid nutrient medium, is cultivated 27~33 days；Improve B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+sucrose 25~35g/L+, 1.5~4.0mg/L of indolebutyric acid, pH5.8~6.0；S2, the ethanol solution that ethephon (CEPHA),2-(chloroethyl) phosphonic acid is added into S1 culture medium, make 5~50 μM/L of concentration of ethephon (CEPHA),2-(chloroethyl) phosphonic acid in culture medium, cultivate 2~7 days；S3, astragalus root of Radix Astragali is collected, it is dry.The present invention in improvement B5 medium by cultivating astragalus root of Radix Astragali, and ethephon (CEPHA),2-(chloroethyl) phosphonic acid is added as elicitor, the raising of biomass and isoflavone content in astragalus root of Radix Astragali is effectively facilitated, and incubation is simple, mild condition, suitable for the large-scale production of Radix Astragali and the industrialized production of gathering isoflavone, realistic meaning with higher.
Description
Technical field
The present invention relates to field of biotechnology, specially a kind of method for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation.
Background technique
Radix Astragali is the dry root of astragalus mongholicus or astragalus membranaceus of leguminous plant, has tonifying Qi and lifting yang, strengthening exterior and reducing sweat, detoxification row
Purulence, inducing diuresis for removing edema and myogenic and other effects are usually used in the industries such as drug, health care product, food.Isoflavones be Radix Astragali chief active at
Point, there is anti-oxidant, antiviral, hypoglycemic, protection nerve, inhibit melanin to be formed and improve the pharmacological actions such as fatigue, still
Gathering isoflavone low output.Gathering isoflavone mainly includes calycosin glucoside, ononin, calycosin and awns
Handle florigen etc., wherein calycosin glucoside is one of evaluation Milkvetch Root quality " labeled compound ".
Currently, Radix Astragali relies primarily on artificial cultivation, however cultivation Radix Astragali growth cycle is long, is often stranded by disease, worm, crop smothering
It disturbs, and isoflavone content is also unstable, is often subject to the influence of environmental factor.In order to solve this problem, meet people couple
The needs of gathering isoflavone, people attempt to produce using the method for tissue cultures such as callus, hairy and adventitious root culture
Gathering isoflavone.But it has not yet to see and promotes astragalus root of Radix Astragali in conjunction with addition ethephon (CEPHA),2-(chloroethyl) phosphonic acid using 3/4 improvement B5 medium
The report of Its Isoflavonoid Accumulation.
Summary of the invention
The purpose of the present invention is to solve the deficiencies of above-mentioned technology, provide a kind of promotion astragalus root of Radix Astragali Its Isoflavonoid Accumulation
Method, by 3/4 improvement B5 Liquid Culture in add inducible factor ethephon (CEPHA),2-(chloroethyl) phosphonic acid, effectively facilitated astragalus root of Radix Astragali biomass
With the raising of isoflavone content.
To achieve the above object, the technical solution adopted by the present invention is that:
A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, comprising the following steps:
S1, astragalus root of Radix Astragali is inoculated into improvement B5 fluid nutrient medium, is cultivated 27~33 days；
The improvement B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+25~35g/L+ of sucrose indolebutyric acid 1.5
~4.0mg/L, pH 5.0~6.0；
S2, the ethanol solution that ethephon (CEPHA),2-(chloroethyl) phosphonic acid is added into the improvement B5 fluid nutrient medium of S1, the improvement B5 fluid nutrient medium
The concentration of middle ethephon (CEPHA),2-(chloroethyl) phosphonic acid is 5~50 μM/L, is cultivated 2~7 days；
S3, the astragalus root of Radix Astragali after S2 culture is collected, be cleaned and dried.
Preferably, the condition of culture of the S1 and S2 are as follows: 25 ± 2 DEG C of temperature, no light is sterile.
Preferably, the concentration of the ethanol solution of ethephon (CEPHA),2-(chloroethyl) phosphonic acid is 100mM/mL, the ethanol solution of the ethephon (CEPHA),2-(chloroethyl) phosphonic acid in the S2
It sterilizes by 0.45 μm of membrane filtration.
Preferably, drying temperature is 55~60 DEG C in the S3, and drying time is 2~3 days.
Compared with prior art, beneficial effects of the present invention are as follows:
Ethephon (CEPHA),2-(chloroethyl) phosphonic acid is added as elicitor by cultivating astragalus root of Radix Astragali in improvement B5 nutrient solution in the present invention, has
Effect promotes the raising of biomass and isoflavone content in astragalus root of Radix Astragali, and incubation is simple, mild condition, is suitable for Huang
The large-scale production of stilbene and the industrialized production of gathering isoflavone, meet the needs of people are to gathering isoflavone.
Specific embodiment
Below by specific embodiment example, the present invention will be described in detail.The scope of the present invention is not limited to the tool
Body embodiment.
The 3/4B5 fluid nutrient medium that the present invention uses refers to that the total volume of fluid nutrient medium is constant, and nutritional ingredient is reduced into
The formula of 3/4,3/4B5 fluid nutrient medium originally such as table 1.
The formula of 1 3/4B5 fluid nutrient medium of table
Embodiment 1
The present embodiment provides a kind of methods for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, comprising the following steps:
S1, adventitious roots delicate, that differentiation is more will be grown and namely grow to carry out good astragalus root of Radix Astragali and be inoculated into improve B5
It in fluid nutrient medium, cultivates 30 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
Improve B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+sucrose 25g/L+ indolebutyric acid 1.5mg/L, pH=
5.8；
The ethanol solution of S2, the ethephon (CEPHA),2-(chloroethyl) phosphonic acid that compound concentration is 100mM/mL, and by 0.45 μm of membrane filtration sterilizing, to S1
Improvement B5 fluid nutrient medium in be added ethephon (CEPHA),2-(chloroethyl) phosphonic acid ethanol solution, until culture medium in ethephon (CEPHA),2-(chloroethyl) phosphonic acid concentration be 10 μM/L, training
It supports 5 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
S3, the astragalus root of Radix Astragali after S2 culture to be collected, that is, takes out adventitious root from culture medium, tap water rinses,
And 2 days are dried at 60 DEG C until constant weight.
Embodiment 2
The present embodiment provides a kind of methods for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, comprising the following steps:
S1, adventitious roots delicate, that differentiation is more will be grown and namely grow to carry out good astragalus root of Radix Astragali and be inoculated into improve B5
It in fluid nutrient medium, cultivates 30 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
Improve B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+sucrose 28g/L+ indolebutyric acid 2.0mg/L, pH=
5.8；
The ethanol solution of S2, the ethephon (CEPHA),2-(chloroethyl) phosphonic acid that compound concentration is 95mM/mL, and by 0.45 μm of membrane filtration sterilizing, to S1
Improvement B5 fluid nutrient medium in be added ethephon (CEPHA),2-(chloroethyl) phosphonic acid ethanol solution, until culture medium in ethephon (CEPHA),2-(chloroethyl) phosphonic acid concentration be 8 μM/L, culture
7 days, condition of culture was 25 ± 2 DEG C of temperature, and no light is sterile；
S3, the astragalus root of Radix Astragali after S2 culture to be collected, that is, takes out adventitious root from culture medium, tap water rinses,
And 3 days are dried at 58 DEG C until constant weight.
Embodiment 3
A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, comprising the following steps:
S1, namely growths delicate, that differentiation is more will be grown carry out good astragalus root of Radix Astragali and be inoculated into improvement B5 liquid training
It supports in base, cultivates 27 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
Improve B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+sucrose 30g/L+ indolebutyric acid 2.5mg/L, pH=
5.8；
The ethanol solution of S2, the ethephon (CEPHA),2-(chloroethyl) phosphonic acid that compound concentration is 100mM/mL, and by 0.45 μm of membrane filtration sterilizing, to S1
Improvement B5 fluid nutrient medium in be added ethephon (CEPHA),2-(chloroethyl) phosphonic acid ethanol solution, until culture medium in ethephon (CEPHA),2-(chloroethyl) phosphonic acid concentration be 10 μM/L, training
It supports 5 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
S3, the astragalus root of Radix Astragali after S2 culture to be collected, that is, takes out adventitious root from culture medium, tap water rinses,
And 3 days are dried at 55 DEG C until constant weight.
Embodiment 4
A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, comprising the following steps:
S1, namely growths delicate, that differentiation is more will be grown carry out good astragalus root of Radix Astragali and be inoculated into improvement B5 liquid training
It supports in base, cultivates 33 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
Improve B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+sucrose 32g/L+ indolebutyric acid 3.0mg/L, pH=
5.8；
The ethanol solution of S2, the ethephon (CEPHA),2-(chloroethyl) phosphonic acid that compound concentration is 100mM/mL, and by 0.45 μm of membrane filtration sterilizing, to S1
Improvement B5 fluid nutrient medium in be added ethephon (CEPHA),2-(chloroethyl) phosphonic acid ethanol solution, until culture medium in ethephon (CEPHA),2-(chloroethyl) phosphonic acid concentration be 5 μM/L, culture
5 days, condition of culture was 25 ± 2 DEG C of temperature, and no light is sterile；
S3, the astragalus root of Radix Astragali after S2 culture to be collected, that is, takes out adventitious root from culture medium, tap water rinses,
And 2 days are dried at 60 DEG C until constant weight.
Embodiment 5
A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, comprising the following steps:
S1, namely growths delicate, that differentiation is more will be grown carry out good astragalus root of Radix Astragali and be inoculated into improvement B5 liquid training
It supports in base, cultivates 30 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
Improve B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+sucrose 35g/L+ indolebutyric acid 4.0mg/L, pH=
5.8；
The ethanol solution of S2, the ethephon (CEPHA),2-(chloroethyl) phosphonic acid that compound concentration is 100mM/mL, and by 0.45 μm of membrane filtration sterilizing, to S1
Improvement B5 fluid nutrient medium in be added ethephon (CEPHA),2-(chloroethyl) phosphonic acid ethanol solution, until culture medium in ethephon (CEPHA),2-(chloroethyl) phosphonic acid concentration be 50 μM/L, training
It supports 2 days, condition of culture is 25 ± 2 DEG C of temperature, and no light is sterile；
S3, the astragalus root of Radix Astragali after S2 culture to be collected, that is, takes out adventitious root from culture medium, tap water rinses,
And 3 days are dried at 55 DEG C until constant weight.
Comparative example 1
Commercially available gathering isoflavone standard items, calycosin standard items, calycosin glucoside standard items, awns handle
Florigen standard items, ononin standard items.
Comparative example 2
It is identical as the process of embodiment 1, the difference is that being added without the ethanol solution of ethephon (CEPHA),2-(chloroethyl) phosphonic acid, that is, without S2.
We have carried out gathering isoflavone content to the sample of embodiment 1- embodiment 5 and comparative example 1 and comparative example 2
HPLC analysis, detailed process is as follows:
1, laboratory apparatus and reagent
Laboratory apparatus: Shimadzu highly effective liquid phase chromatographic system (LC-10ATVP Plus), binary geopressure gradient pump (LC-
10ADVP), UV, visible light dual wavelength detector (SPD-10AVP Plus)；
Reagent: calycosin glucoside, ononin, calycosin, onocerin reference substance are purchased from Shanghai
Rong He Pharmaceutical Technology Co., Ltd；Hplc grade methanol, chromatographic grade acetonitrile, ultrapure water are other pure to analyze.
2, experimental method
2.1 chromatographic condition
It is tested using China's spectrum C18 reverse chromatograms column (5 μm, 4.6 × 250mm), mobile phase A is water, and Mobile phase B is second
Nitrile；Flow velocity is 0.8mL/min；Detection wavelength: 230nm, column temperature: 30 DEG C, sampling volume is 20 μ L, binary gradient elutes condition: B
Pump concentration 0~30min be 15 → 55%, 30~35min be 55 → 100%, 35~40min be 100 → 15%, 40~
45min is 15%.
The preparation of 2.2 test solutions
It weighs 0.5g astragalus root of Radix Astragali powder in a round bottom flask, analysis level methanol 50mL is added, is returned in 80 DEG C of water-baths
Stream 4 hours, withdrawing fluid is filtered, is concentrated to dryness in 60 DEG C of rotary evaporations, is then dissolved with 5mL hplc grade methanol, is passed through
0.22 μm of miillpore filter impurity elimination, is filled into 10mL centrifuge tube, as test solution, that is, 5 He of embodiment 1- embodiment
The test solution of the sample of comparative example 2.
We distinguish the commercially available calycosin of accurate weighing 1mg, calycosin glucoside, ononin, awns
Handle florigen standard items are added 0.1mL hplc grade methanol and sufficiently dissolve, and are prepared into the mixing for being 0.1mg/mL containing single mark concentration
Standard solution, that is, comparative example 1 sample test solution.
1 isoflavone content of table
Table 1 is the isoflavone content of embodiment and comparative example sample, as it can be seen from table 1 the Huang of the method for the present invention culture
Stilbene adventitious root, cultivation cycle is short, and isoflavone content is high, and the content of calycosin glucoside meets the People's Republic of China (PRC)
(>=0.020%, dry weight meter) is required as defined in pharmacopeia (2015) version.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (4)
1. a kind of method for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation, which comprises the following steps:
S1, astragalus root of Radix Astragali is inoculated into improvement B5 fluid nutrient medium, is cultivated 27~33 days；
The improvement B5 Liquid Culture based formulas are as follows: 3/4B5 fluid nutrient medium+25~35g/L+ of sucrose indolebutyric acid 1.5~
4.0mg/L, pH 5.0~6.0；
S2, the ethanol solution that ethephon (CEPHA),2-(chloroethyl) phosphonic acid is added into the improvement B5 fluid nutrient medium of S1, second in the improvement B5 fluid nutrient medium
The concentration of alkene benefit is 5~50 μM/L, is cultivated 2~7 days；
S3, the astragalus root of Radix Astragali after S2 culture is collected, be cleaned and dried.
2. a kind of method for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation according to claim 1, which is characterized in that the S1
With the condition of culture of S2 are as follows: 25+2 DEG C of temperature, no light is sterile.
3. a kind of method for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation according to claim 1, which is characterized in that the S2
The concentration of the ethanol solution of middle ethephon (CEPHA),2-(chloroethyl) phosphonic acid is 95~100mM/mL, and the ethanol solution of the ethephon (CEPHA),2-(chloroethyl) phosphonic acid passes through 0.45 μm of membrane filtration
Sterilizing.
4. a kind of method for promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation according to claim 1, which is characterized in that the S3
Middle drying temperature is 55~60 DEG C, and drying time is 2~3 days.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114982638A (en) * | 2022-05-19 | 2022-09-02 | 山西大学 | Chemical inducer for astragalus adventitious root tissue culture and application of chemical inducer in astragalus adventitious root culture |
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