CN103651123B - A kind of method of producing the two glycosides of Chinese herbaceous peony - Google Patents
A kind of method of producing the two glycosides of Chinese herbaceous peony Download PDFInfo
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Abstract
The invention discloses a kind of method of producing the two glycosides of Chinese herbaceous peony, comprising: induction in access inducing culture after explant sterilization is produced callus; By described callus access proliferated culture medium, after carrying out squamous subculture under illumination condition, from callus, after squamous subculture completes, extract the two glycosides of Chinese herbaceous peony.Compared with the two glycosides content of the Chinese herbaceous peony of cultivation Chinese herbaceous peony, the content of the two glycosides of Chinese herbaceous peony and it is quite even higher in the callus that the present invention cultivates, but the integration time of the two glycosides of Chinese herbaceous peony shortened to 30 ~ 60 days from 2 ~ 4 years, and can avoid the pollution of the harmful substances such as the Environmental Pesticide such as soil, water remains, heavy metal in whole process.Therefore, utilize this method to produce the two glycosides of Chinese herbaceous peony, not only can significantly the production time, reduce production cost, and the Chinese herbaceous peony produced pair glycosides has higher safety.
Description
Technical field
The present invention relates to natural drug extractive technique field, particularly relate to a kind of method of producing the two glycosides of Chinese herbaceous peony.
Background technology
Chinese herbaceous peony (Paeonia lactiflora Pall.) is Paeoniaceae Paeonia (Paeonia) perennial root herbaceous plant, has long cultivation history, is loved by the people since ancient times, is described as " flower phase ".
Chinese herbaceous peony is not only a kind of ornamental plants, or a kind of medicinal plant.The root of Chinese herbaceous peony can be used as medicine, and does not directly dry person for the radix paeoniae rubrathe after gathering, and boils the person of drying again for the root of herbaceous peony in rearmounted boiling water of gathering after boiling rear removing crust or peeling.The radix paeoniae rubrathe has clearing heat and cooling blood, blood stasis removing analgesic function, is used for the treatment of heat and enters ying blood, and febrile virulent maculae, hematemesis and epistaxis, red eye, swell pain, liver-depressed hypochondrium pain, through closing dysmenorrhoea, Disorder lump in the abdomen stomachache, injury from falling down, carbuncle swell sore; The root of herbaceous peony has nourishing blood for regulating menstruation, astringing YIN to stop sweating, easing the affected liver to relieve pain, suppressing liver-YANG function, be used for the treatment of that the deficiency of blood is sallow, irregular menstruation, spontaneous perspiration, night sweat, hypochondriac pain, stomachache, four limbs contraction pain, have a headache dizzy.The Chinese herbaceous peony comprising the radix paeoniae rubrathe and the root of herbaceous peony is a kind of parts of generic medicinal plants, and its principle active component is all Paeoniflorin and albiflorin, and these two kinds of compositions are collectively referred to as the two glycosides of Chinese herbaceous peony.Modern pharmacology experiment shows, the two glycosides of Chinese herbaceous peony suppresses the autoimmunity reaction of human body by multiple approach, therefore has anti-inflammatory, pain relieving, protects the liver and the multiple pharmacological effect such as anti-autoimmunity.At present, the two glycosides capsule of Chinese herbaceous peony being active ingredient with the two glycosides of Chinese herbaceous peony for the clinical treatment of rheumatoid arthritis, and achieves good result for the treatment of.
Along with the medical value of the two glycosides of Chinese herbaceous peony day by day come into one's own, range of application day by day widens, the production of the two glycosides of Chinese herbaceous peony has become the key factor that the two glycosides of restriction Chinese herbaceous peony is generally applied.
The traditional preparation methods of the two glycosides of Chinese herbaceous peony first plants Chinese herbaceous peony, taking Peony Root, obtaining the radix paeoniae rubrathe or the root of herbaceous peony through drying the process of preparing Chinese medicine until Chinese herbaceous peony growth after 2 ~ 4 years.Pharmacy corporation therefrom two glycosides of separation and Extraction Chinese herbaceous peony again after the root of herbaceous peony place of production purchase radix paeoniae rubrathe or the root of herbaceous peony.Because Chinese herbaceous peony growth cycle is long, so the whole production process cycle is longer.In addition, in the radix paeoniae rubrathe and the root of herbaceous peony, the content of the two glycosides of Chinese herbaceous peony is lower, be generally about 2% of dry weight, and the content of the two glycosides of Chinese herbaceous peony is also subject to the Chinese herbaceous peony growth place of production, season and weather, and the impact of the many factors such as cultivation management level and usually cause existing larger fluctuation, the cost making to extract the two glycosides of Chinese herbaceous peony from the radix paeoniae rubrathe or the root of herbaceous peony is high, efficiency is low, not only limits throughput, and the price of final products also remains high.These deficiencies are also that traditional natural drug extracts and produces the Universal Problems faced.
Based on above-mentioned deficiency, people attempt adopting plant cell engineering means, carry out the large-scale culture of plant cell in the factory, and by the control of condition of culture and preferably, improve the content of active ingredient in plant cell, shorten the time of active ingredient accumulation, ensure the stable of effective constituent concentration, thus shorten the production time, reduce production cost, increase production scale.The method utilizing plant tissue culture technique to produce secondary metabolites at present achieves successfully on multiple medicinal plant, and the pilot scale culture all having begun through plant cell as the saponin(e material in the medicinal plants such as ginseng, pseudo-ginseng, digitalis is produced.
In order to meet the demand of extensive patients to the two glycosides medicine of Chinese herbaceous peony, the financial burden of reduction patient medication, being also necessary to adopt the production method of the pilot scale culture technology of plant cell to the two glycosides of Chinese herbaceous peony to upgrade, reducing production cost, shorten the production time.But up to now, any patent or Research Literature is not yet had to report this method, and the technology at present about Chinese herbaceous peony callus tissue culture also mainly concentrates in callus of induce, plant regeneration, there are not the content about the two glycosides of Chinese herbaceous peony in callus and product quantifier elimination.
Summary of the invention
The invention provides a kind of method of producing the two glycosides of Chinese herbaceous peony, by evoked callus under specified conditions, substantially reduce the production time, reduce cost, and ensure that the two glycosides of Chinese herbaceous peony has higher content in callus.
Produce a method for the two glycosides of Chinese herbaceous peony, comprising:
(1) induction in access inducing culture after the sterilization of Chinese herbaceous peony explant is produced callus;
(2) by described callus access proliferated culture medium, under illumination condition, carry out squamous subculture, from callus, after squamous subculture completes, extract the two glycosides of Chinese herbaceous peony;
Wherein, described proliferated culture medium is:
1/2MS or WPM medium containing 0.5 ~ 0.75mg/L6-benayl aminopurine, 0.5 ~ 1mg/L methyl α-naphthyl acetate and 0.025 ~ 0.1mg/L disleave spirit, wherein, 1/2MS or WPM medium contains 25 ~ 35g/L sucrose and 6 ~ 8g/L agar.
Proliferated culture medium not only affects the proliferation times of callus, also affect the content of the two glycosides of Chinese herbaceous peony in callus simultaneously, the hormone combinations of 6-benzyl aminopurine, methyl α-naphthyl acetate and disleave spirit is selected in the proliferated culture medium of the application, and control the reasonable volume of often kind of hormone, callus not only can be made to grow faster, the content of the two glycosides of Chinese herbaceous peony in callus can also be improved simultaneously.
Preferred further, described proliferated culture medium is:
1/2MS or WPM medium containing 0.5mg/L6-benayl aminopurine, 1mg/L methyl α-naphthyl acetate and 0.1mg/L disleave spirit, wherein, 1/2MS or WPM medium contains 30g/L sucrose and 8g/L agar.
Generally, the pH of described proliferated culture medium is 4.5 ~ 7.0.
Described Chinese herbaceous peony can be the various Chinese herbaceous peony varieties of plant of the ground such as Hui nationality, Jinhua, Zhejiang cultivation, and concrete kind can be " cattail's spike ", " lines ", " numb stubble " or " shank ".
Described explant can be stem, petiole, blade or root.
When sterilizing to explant, disinfectant used can be alcohol, mercuric chloride, clorox, hydrogen peroxide etc.In order to reach better Disinfection Effect, avoid polluting, usually by multiple disinfectant with the use of, and strict time controlling sterilization, prevent from killing explant somatocyte, affect inductivity.
The method of described sterilization is: after being cleaned up by the explant of Chinese herbaceous peony, after 70 ~ 75% alcohol disinfecting 10 ~ 60s, after soaking 5 ~ 30min, cleans up with 2 ~ 10%NaClO.
Inducing culture is the key factor of explant dedifferentiation, adds exogenous hormone in the medium and explant dedifferentiation can be induced to form callus, affect the quality of callus, thus affect the content of the two glycosides of Chinese herbaceous peony in final callus.Because callus is exogenous hormone and the interactional result of plant explants endogenous hormones, and the not intricate and endogenous hormones of kindred plant explant endogenous hormones and the different of the exogenous hormone mechanism of action, result in the kind of the required hormone added of explant of not kindred plant, the content of hormone, the proportioning of hormone have very big difference.
Described inducing culture is:
Containing MS, 1/2MS or WPM medium of 0.5 ~ 2mg/L6-benayl aminopurine, 0.2 ~ 1mg/L methyl α-naphthyl acetate and 0.2 ~ 0.5mg/L2,4-dichlorophenoxyacetic acid, wherein, MS, 1/2MS or WPM medium contains 25 ~ 35g/L sucrose and 6 ~ 8g/L agar.
Preferred further, described inducing culture is:
Containing MS, 1/2MS or WPM medium of 2mg/L6-benayl aminopurine, 0.2mg/L methyl α-naphthyl acetate and 0.2mg/L2,4-dichlorophenoxyacetic acid, wherein, MS, 1/2MS or WPM medium contains 30g/L sucrose and 8g/L agar.
If when selecting medium based on 1/2MS, Ca in this 1/2MS medium
2+concentration is generally 0.003mol/L.
Generally, the pH of described inducing culture is 4.5 ~ 7.0.
Except inducing culture, inducing culturing condition also affects the inductivity of callus, general, and the temperature of described induction is 18 ~ 30 DEG C, and the time is 18 ~ 60 days, during induction, and light application time is 8 ~ 20 hours/day, and intensity of illumination is 800 ~ 2500lx; Preferably, the temperature of described induction is 22 ~ 25 DEG C, and the time is 20 ~ 30 days, and light application time is 12 ~ 16 hours/day, and intensity of illumination is 1200 ~ 1600lx; Preferred, the temperature of described induction is 24 DEG C, and the time is 25 days, and light application time is 14 hours/day, and intensity of illumination is 1500lx.
In addition, the condition of squamous subculture also affects the content of the two glycosides of Chinese herbaceous peony in callus, and the temperature of described squamous subculture is 18 ~ 30 DEG C, and light application time is 8 ~ 20 hours/day, and intensity of illumination is 800 ~ 2500lx; Preferably, the temperature of described squamous subculture is 22 ~ 25 DEG C, and light application time is 12 ~ 16 hours/day, and intensity of illumination is 1200 ~ 1600lx; Preferred, the temperature of described squamous subculture is 24 DEG C, and light application time is 14 hours/day, and intensity of illumination is 1500lx.
The algebraically of described squamous subculture was generally for 2 ~ 20 generations, and each squamous subculture cycle is 18 ~ 60 days, and preferably, the algebraically of described squamous subculture was generally for 4 ~ 6 generations, and each squamous subculture cycle is 30 ~ 35 days.
The method of the two adoptable routine of glycosides of the Chinese herbaceous peony in callus is extracted, and comprising: get callus, and oven dry, pulverizing are placed on lixiviate in solvent, and supernatant is the crude extract of the two glycosides of Chinese herbaceous peony.
As required, can to the further separation and purification of crude extract.
Described solvent can be methyl alcohol, ethanol, n-butanol etc., is specifically as follows 80% methanol aqueous solution.
Compared with prior art, beneficial effect of the present invention is:
Plant tissue culture technique is more conventional technology, be mainly used in the several field of production of Fast-propagation, the production of virus-free seedling, seed selection new varieties, Germ-plasma resources protection and secondary metabolites, patent " a kind of method for tissue culture of paeonia lactiflora " (application number 201010102618.0) describes a kind of is the numerous soon method of Chinese herbaceous peony, but it only provides the method making the stable growth fast in Chinese herbaceous peony indoor; Document " induction and differentiations of 4 kind Chinese herbaceous peony callus " (Agricultural University Of Hunan's journal the 37th volume the 2nd phase in 2011) also only have studied the principal element affecting callus induction and differentiation, and does not meet relevant report for the research of the principal element etc. utilizing callus tissue culture technology to produce the two glycosides of Chinese herbaceous peony and affect Chinese herbaceous peony pair glycosides output.
The two glycosides of Chinese herbaceous peony is produced in the cultivation that the present invention also carries out callus under given conditions by induction Chinese herbaceous peony explant formation callus; not only two for Chinese herbaceous peony glycosides can be shortened to 1 ~ 2 month from 2 ~ 4 years the integration time in plant; and compared with the two glycosides content of the Chinese herbaceous peony in Peony Root; in callus, the content of the two glycosides of Chinese herbaceous peony is quite even higher with it; also more stable, be more conducive to large-scale production.These advantages can solve current wild herbaceous peony preferably cannot meet the needs of problems of market to the two glycosides of Chinese herbaceous peony, can realize Chinese herbaceous peony two glycosides short time, produce low-costly and in high volume, have the society and economic worth that are highly profitable.
In addition, the whole process of the two glycosides accumulation of callus induction, growth and Chinese herbaceous peony is all in indoor environment, not only not by region, season and climatic influences, therefore, method of the present invention can also effectively be avoided occurring heavy metal pollution and residue of pesticide in the two glucoside extract of Chinese herbaceous peony, and the two glycosides of Chinese herbaceous peony of extraction has higher safety.
Accompanying drawing explanation
Fig. 1 is the two glycosides assay chromatogram of Chinese herbaceous peony in embodiment 6 Chinese herbaceous peony " lines " Callus of Leaf;
Fig. 2 is the two glycosides assay chromatogram of Chinese herbaceous peony in embodiment 6 Chinese herbaceous peony " lines " callus from stem segment;
Fig. 3 is the two glycosides assay chromatogram of Chinese herbaceous peony in embodiment 6 Chinese herbaceous peony " lines " petiole callus;
Fig. 4 is the two glycosides assay chromatogram of Chinese herbaceous peony in embodiment 6 Chinese herbaceous peony " lines " root-derived callus;
Fig. 5 changes the two glycosides assay chromatogram of Chinese herbaceous peony in Multiplying culture based component (plant hormone) rear blade callus in comparative example 1;
Fig. 6 changes the two glycosides assay chromatogram of Chinese herbaceous peony in condition of culture (temperature) rear blade callus in comparative example 2.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained.
Test medium
Inducing culture: WPM medium+sucrose 30g/L+ agar 8g/L+6-benayl aminopurine 2mg/L+ methyl α-naphthyl acetate 0.2mg/L+2,4-dichlorophenoxyacetic acid 0.2mg/L, the pH value of medium is about 5.2.
Proliferated culture medium: WPM medium+sucrose 30g/L+ agar 8g/L+6-benayl aminopurine 0.5mg/L+ methyl α-naphthyl acetate 1mg/L+ disleave spirit 0.1mg/L, the pH value of medium is about 5.2.
The formula of WPM medium is in table 1.
Table 1
The cultivation of embodiment 1 Chinese herbaceous peony Callus of Leaf
(1) sterilization of explant
The blade of the Chinese herbaceous peony (comprising " cattail's spike " and " lines " two kinds, original producton location Hui nationality) of clip laboratory cultures is after liquid detergent dilution soaks rinsing 20min, clean with tap water.Blot the moisture on blade with blotting paper, with 75% alcohol disinfecting 30s in superclean bench, then soak 10min with 10%NaClO, aseptic water washing 5 times, finally blot moisture on blade with the filter paper of sterilizing.
(2) Fiber differentiation of callus
Blade after sterilization being cleaned is cut into about 1cm
2, insert in inducing culture and carry out Fiber differentiation, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx, cultivation cycle 25 days.The callus induction rate of two kind Chinese herbaceous peony blades used is all greater than 90%.
(3) Multiplying culture of callus
To choose in above-mentioned steps the callus that not brownization, growth conditions are stable, cultivate in proliferated culture medium of transferring after it is cut into fritter in super-clean bench, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx.
Be a squamous subculture cycle with 32d, when squamous subculture is to forth generation, the proliferation times of two kinds of Chinese herbaceous peony callus is respectively 5.56(" lines " and plants) and 4.90(" cattail's spike " kind).
The cultivation of embodiment 2 Chinese herbaceous peony stem callus
(1) sterilization of explant
The stem of the Chinese herbaceous peony (comprising " cattail's spike " and " lines " two kinds, original producton location Hui nationality) of clip laboratory cultures is soak rinsing 20min in liquid detergent dilution after, clean with tap water; Blot the moisture on stem with blotting paper, with 75% alcohol disinfecting 30s in superclean bench, then soak 10min with 10%NaClO, aseptic water washing 5 times, finally blot the moisture on stem with the filter paper of sterilizing.
(2) Fiber differentiation of callus
Stem after sterilization being cleaned is cut into the stem section of 0.3 ~ 0.5cm, and insert in inducing culture and carry out Fiber differentiation, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx, cultivation cycle 25 days.The inductivity of Cultivars of Chinese Herbaceous Peony " lines " stem is 23.1%, and " cattail's spike " is 20.0%.
(3) Multiplying culture of callus
To choose in above-mentioned steps the callus that not brownization, growth conditions are stable, in super-clean bench, cultivate in proliferated culture medium of transferring after being cut to fritter, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx.
Be a squamous subculture cycle with 32d, when squamous subculture is to forth generation, the proliferation times of two kinds of callus is respectively 3.9(" lines " and plants) and 3.2(" cattail's spike " kind).
The cultivation of embodiment 3 Chinese herbaceous peony petiole callus
(1) sterilization of explant
The petiole of the Chinese herbaceous peony (comprising " cattail's spike " and " lines " two kinds, original producton location Hui nationality) of clip laboratory cultures is after liquid detergent dilution soaks rinsing 20min, clean with tap water.Blot petiolar moisture with blotting paper, with 75% alcohol disinfecting 30s in superclean bench, then soak 10min with 10%NaClO, aseptic water washing 5 times, then blot moisture on petiole with the filter paper of sterilizing.
(2) Fiber differentiation of callus
Petiole after sterilization being cleaned is cut into 0.3 ~ 0.5cm, and insert in inducing culture and carry out Fiber differentiation, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx, cultivation cycle 25 days.The inductivity of two kinds of Chinese herbaceous peony petioles is respectively 45.0%(" lines " and plants) and 41.5%(" cattail's spike " kind).
(3) Multiplying culture of callus
To choose in above-mentioned steps the callus that not brownization, growth conditions are stable, cultivate in proliferated culture medium of transferring after it is cut into fritter in super-clean bench, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx.
Be a squamous subculture cycle with 32d, when squamous subculture is to forth generation, the proliferation times of two kinds of callus is respectively 5.3(" lines " and plants) and 4.8(" cattail's spike " kind).
The cultivation of embodiment 4 Peony Root callus
(1) sterilization of explant
The Chinese herbaceous peony of clip laboratory cultures (comprises " cattail's spike " and " lines " two kinds, original producton location Hui nationality) root, first use running water 20min, exterior earth is rinsed well, again with blade by crust scraped clean, soak rinsing 20 ~ 30min with liquid detergent dilution again, rinse well under running water.Blot with blotting paper, with 75% alcohol disinfecting 30s on superclean bench, then soak 20min with 10%NaClO, aseptic water washing 3-5 time, then blot moisture on it with sterilized filter paper.
(2) Fiber differentiation of callus
Root after sterilization being cleaned is cut into the disk that thickness is about 1mm, then hemisects, and carry out Fiber differentiation in access inducing culture, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx, cultivation cycle 25 days.The callus induction rate of two kinds of Peony Roots is respectively 83.3%(" lines " and plants) and 80%(" cattail's spike " kind).
(3) Multiplying culture of callus
To choose in above-mentioned steps the callus that not brownization, growth conditions are stable, cultivate in proliferated culture medium of transferring after it is cut into fritter in super-clean bench, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is about 1500lx.
Be a squamous subculture cycle with 32d, when squamous subculture is to forth generation, the proliferation times of two kinds of root-derived callus is respectively 3.9(" lines " and plants) and 3.3(" cattail's spike " kind).
The extraction of the two glycosides of embodiment 5 Chinese herbaceous peony
(1) take out the Chinese herbaceous peony blade of above-described embodiment squamous subculture, petiole, stem and root-derived callus, be placed in 60 DEG C of drying in oven;
(2) Chinese herbaceous peony blade, petiole, stem and root-derived callus dry product 0.3g(DW is got respectively), after being placed in mortar pulverize, with 80% methanol-water solution dipping, at 60 DEG C, ultrasonic process 30min obtains the two glycosides crude extract of Chinese herbaceous peony;
(3) two for Chinese herbaceous peony glycosides crude extract is settled to 10mL, the centrifugal 10min of 10000r/min, gets supernatant, filters, must can be used for the sample solution of sample introduction through the miillpore filter of 0.22 μm.
The assay of the two glycosides of embodiment 6 Chinese herbaceous peony
Adopt the two glycosides content of Syrups by HPLC Chinese herbaceous peony.
Filler is adopted to be Hypersil ODS2, particle diameter 5 μm, column internal diameter 4.6 μm, the C18 post of column length 250nm; Mobile phase is acetonitrile: 0.05% aqueous potassium phosphate solution (11:89), Gradient elution, flow velocity: 1.0mL/min; UV detect wavelength: 230nm; Column temperature: room temperature (about 25 DEG C).
Accurate absorption reference substance and each 20 μ L of sample solution, inject high performance liquid chromatograph and measure, chromatogram is shown in Fig. 1 ~ Fig. 4.Press callus dry product to calculate, containing Paeoniflorin (g/gDW) 0.34% in the forth generation Callus of Leaf of Cultivars of Chinese Herbaceous Peony " lines ", albiflorin 1.68%, namely containing the two glycosides 2.02% of Chinese herbaceous peony; Containing Paeoniflorin 0.1% in forth generation petiole callus, albiflorin 0.61%, namely containing the two glycosides 0.71% of Chinese herbaceous peony; Containing Paeoniflorin 0.16% in forth generation callus from stem segment, albiflorin 0.82%, namely containing the two glycosides 0.98% of Chinese herbaceous peony; Containing Paeoniflorin 2.03% in forth generation root-derived callus, albiflorin 0.29%, namely containing the two glycosides 2.32% of Chinese herbaceous peony.From above result, consider from the ability integration of multiplication capacity and the two glycosides of synthesis Chinese herbaceous peony, induce the callus produced to be suitable for producing the two glycosides of Chinese herbaceous peony by root and blade.
By the same way, the two glycosides content of Chinese herbaceous peony that " cattail's spike " plants in Chinese herbaceous peony callus is detected, result all detected the existence (the results are shown in Table 2, the two glycosides content of Chinese herbaceous peony with callus dry product quality for benchmark) of the two glycosides of Chinese herbaceous peony in corresponding four kinds of callus.
The two glycosides comparision contents of Chinese herbaceous peony in the different callus of table 2 two kinds of Chinese herbaceous peonies
| Callus of Leaf | Petiole callus | Callus from stem segment | Root-derived callus | |
| " lines " | 2.02% | 0.71% | 0.98% | 2.32% |
| " cattail's spike " | 1.98％ | 0.70% | 0.88% | 2.10％ |
Comparative example 1
Hormone combinations in proliferated culture medium is changed into 6-benzyl aminopurine 2mg/L+ methyl α-naphthyl acetate 0.2mg/L+2,4-dichlorophenoxyacetic acid 0.2mg/L, other steps are " lines " with embodiment 1(kind), when squamous subculture is to forth generation, its callus proliferation multiple is 3.9.And measuring the two glycosides content of Chinese herbaceous peony by efficient liquid phase, chromatogram is shown in Fig. 5.Press callus dry product to calculate, containing Paeoniflorin 1.95%, albiflorin 0.08%, namely containing the two glycosides 1.76% of Chinese herbaceous peony.The known plant hormone of result is larger on the impact of Chinese herbaceous peony two glycosides content thus.
Comparative example 2
Cultivate under the proliferated culture medium of callus being placed in 35 DEG C of conditions, other steps are " lines " with embodiment 1(kind), most brownization of its callus does not grow, more responsive to temperature, non-refractory.Measure the two glycosides content of Chinese herbaceous peony by efficient liquid phase, chromatogram is shown in Fig. 6.Press callus dry product to calculate, containing Paeoniflorin 1.26%, albiflorin 0.05%, namely containing the two glycosides 1.31% of Chinese herbaceous peony.The known high temperature of result makes the two glycosides content of Chinese herbaceous peony obviously reduce thus.
Claims (1)
1. produce a method for the two glycosides of Chinese herbaceous peony, it is characterized in that, comprising:
(1) sterilization of explant
The root of the Chinese herbaceous peony of clip laboratory cultures, first uses running water 20min, exterior earth is rinsed well, then with blade by crust scraped clean, then soak rinsing 20 ~ 30min with liquid detergent dilution, rinse well under running water; Blot with blotting paper, with 75% alcohol disinfecting 30s on superclean bench, then soak 20min with 10%NaClO, aseptic water washing 3-5 time, then blot moisture on it with sterilized filter paper;
(2) Fiber differentiation of callus
Root after sterilization being cleaned is cut into the disk of thickness 1mm, then hemisects, and carry out Fiber differentiation in access inducing culture, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is 1500lx, cultivation cycle 25 days;
(3) Multiplying culture of callus
To choose in above-mentioned steps the callus that not brownization, growth conditions are stable, cultivate in proliferated culture medium of transferring after it is cut into fritter in super-clean bench, cultivation temperature is 24 DEG C, and the light application time of every day is 14h, and intensity of illumination is 1500lx; It is a squamous subculture cycle with 32d;
Inducing culture: WPM medium+sucrose 30g/L+ agar 8g/L+6-benayl aminopurine 2mg/L+ methyl α-naphthyl acetate 0.2mg/L+2,4-dichlorophenoxyacetic acid 0.2mg/L, the pH value of medium is 5.2;
Proliferated culture medium: WPM medium+sucrose 30g/L+ agar 8g/L+6-benayl aminopurine 0.5mg/L+ methyl α-naphthyl acetate 1mg/L+ disleave spirit 0.1mg/L, the pH value of medium is 5.2;
(4) extraction of the two glycosides of Chinese herbaceous peony
Take out the Peony Root callus of squamous subculture, be placed in 60 DEG C of drying in oven, get dry product 0.3g; After being placed in mortar pulverize, with 80% methanol-water solution dipping, at 60 DEG C, ultrasonic process 30min obtains the two glycosides crude extract of Chinese herbaceous peony; Two for Chinese herbaceous peony glycosides crude extract is settled to 10mL, and the centrifugal 10min of 10000r/min, gets supernatant, filters through the miillpore filter of 0.22 μm;
The kind of described Chinese herbaceous peony is " cattail's spike " or " lines ".
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| CN1675992A (en) * | 2005-04-19 | 2005-10-05 | 扬州大学 | Chinese herbeceous peony invitro tissue culture method |
| CN102124946A (en) * | 2010-01-27 | 2011-07-20 | 北京林业大学 | Method for tissue culture of paeonia lactiflora |
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| CN1675992A (en) * | 2005-04-19 | 2005-10-05 | 扬州大学 | Chinese herbeceous peony invitro tissue culture method |
| CN102124946A (en) * | 2010-01-27 | 2011-07-20 | 北京林业大学 | Method for tissue culture of paeonia lactiflora |
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| 4个品种芍药愈伤组织的诱导及分化;于晓南等;《湖南农业大学学报(自然科学版)》;20110430;第37卷(第2期);第166-171页 * |
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