CN104839026A - Method for co-culturing adventitious roots of echiancea purpurea - Google Patents
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Abstract
The invention belongs to the field of biotechnologies, and particularly relates to a method for co-culturing adventitious roots of echiancea purpurea. Adventitious roots of a combination of two or more kinds of echiancea purpurea of different varieties are subjected to suspension culture in a sucrose-containing MS culture medium, so that the co-culturing of the adventitious roots of echiancea purpurea can be realized, and a lot of physiological active substances are obtained. In the aspect of producing antioxidative anti-cancer physiological active substances such as echiancea purpurea glycosides and chicoric acids and the like by using the method disclosed by the invention, the method has the characteristics of short production cycle, high efficiency, factory production, and the like; and the method also lays a theoretical and practical foundation for the development of echiancea purpurea biomedicament and series of health products by using the fermentation technology.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method of medicinal Echinacea adventive root Dual culture.
Background technology
Echinacea Echinacen spp is composite family genus echinacea herbaceos perennial, North America, original producton location.This genus has 8 kinds and several mutation, mainly contains 3 kinds, purple coneflower Echinacea purpurea, narrow leaf Echinacea Echinacea angustifolia and white Echinacea Echinacea pallida at present as drug development.As far back as Flat head period, Echinacea is just as the optimal drug for the treatment of trauma infection contamination, snake bite, toothache, be called " cure-all medicine " by locals, come in and find that Echinacea has the effects such as antitumor, antiviral, anti-oxidant, antibacterial, anti-inflammatory, its medicine is internationally recognized innate immunity conditioning agent and immunopotentiator at present.
The root of Echinacea, stem, leaf, flower, fruit all can be used as medicine, containing bioactivators such as caffeic acid derivative, polysaccharide, glycoprotein, alkylamide compound, alkaloid, phytosterol, volatile oil, polyacetylene compounds in the ethanol extract of its acrial part and root.Caffeic acid derivative is one of Echinacea main active, and 3 kinds of medicinal Echinaceas are respectively containing different caffeic acid derivative, and narrow leaf Echinacea and white Echinacea root contain the Echinacea glycosides of 0.3 ~ 1.7%; Purple coneflower root is not containing Echinacea glycosides, but Cichoric acid is its main component, and Cichoric acid is rich content in the flower and root of purple coneflower, is respectively 1.2 ~ 3.1% and 0.6 ~ 2.1%; In narrow leaf Echinacea, Cichoric acid content is very few.Recent research shows that Cichoric acid has the effect suppressing AIDS virus HIV-1 and HIV-1 integrase; Echinacea glycosides has antitumor action, has obvious inhibition to liver cancer, lung cancer, sdenocarcinoma of stomach, also has establishing-Yang, anti-ageing and effect such as treatment diabetes etc.
Along with Echinacea preparation is day by day universal in the world, the output of wild Echinacea is difficult to meet international market demand, and the ground such as America & Canada all increase its area under cultivation every year.15 provinces and cities' also successful introduction such as Beijing, Nanjing, Shenyang of China, but owing to introducing a fine variety Echinacea not through critical system ground germplasm screening, along with the growth of planting time and the expansion of geographic coverage, be easy to occur intraspecific variablity, and clinical drug effect is unstable.Therefore, a large amount of Echinacea raw material are obtained by cell engineering means imperative.
Utilize Echinacea adventive root culture technique, High-efficient Production secondary metabolite is the important leverage of Appropriate application and the wild Echinacea resource of protection, simultaneously for Echinacea modernization of Chinese medicine development provides important opportunity.
The research of initial Echinacea biotechnology lays particular emphasis on the Fast-propagation of good seed more, and recent primary study utilizes callus and adventive root to cultivate and produces its secondary metabolite.Chinese Academy Of Sciences Process Engineering Research Institute is on the basis of putting into practice for many years, purple coneflower fast seedling growing technology is established by Plant Biotechnology, and successfully set up fast growth and the high Echinacea excised root culture thing of pharmaceutical ingredient content, active constituent content and the biological effectiveness of the Echinacea root produced obviously are better than wild, demonstrate huge commercialization and are worth.But the domestic physiological activator culture studies to Echinacea, exists and only utilizes purple coneflower single variety, the limitation such as a kind of active substance of main production Cichoric acid.Inventor to the research of Echinacea adventive root, induces 3 kinds of medicinal Echinacea adventive root, and cultivates in bio-reactor from 2004.After white purple cone adventive root cultivates 30d in 5L airlift bioreactor, in adventive root, Echinacea glycosides content is 2.4 times of cultivation root; Chlorogenic acid, Cichoric acid, total caffeic acid derivative's content is 4.0 ~ 25.6 times of cultivation root.Narrow leaf Echinacea adventive root suspends and cultivates optimum medium combination is 0.5MS+IBA2mgL
-1+ 5% sucrose+5:25 (mM) ammoniacal nitrogen/nitrate nitrogen, can obtain high-biomass, total phenol and general flavone content.Purple coneflower adventive root can obtain Cichoric acid in bioreactor culture, chlorogenic acid, the caffeic acid derivative such as caftaric acid, and in the purple coneflower adventive root cultivated in 500,1000L airlift reactor, caffeic acid derivative's content is higher than cultivated plant.Above result shows, 3 kinds of indefinite root systems of medicinal Echinacea, can be used as the important materials being extracted its medicinal physiological activator by large-scale production.
But 3 kinds of medicinal Echinaceas are separately containing specific coffee acid derivative, therefore can not contain multiple caffeic acid derivative in single cultivation adventive root simultaneously, utilize 2 or 3 kind Echinacea adventive root Coculture techniques, antioxidation activity can be obtained high and altogether containing the medical science medicinal material of several physiological active substances.Up to the present, there is not yet the research report of Echinacea adventive root Dual culture.
Summary of the invention
The object of the invention is a kind of method providing medicinal Echinacea adventive root Dual culture.
For achieving the above object, the present invention adopts technical scheme to be:
A kind of method of medicinal Echinacea adventive root Dual culture, the adventive root of two or more combination in medicinal for different cultivars Echinacea is suspended to cultivate and cultivates in the MS medium containing sucrose, Echinacea adventive root Dual culture can be realized, obtain a large amount of physiological activators simultaneously.
Preferably: by purple coneflower, narrow leaf Echinacea and white Echinacea, the adventive root of two or more combination suspends to cultivate and cultivates in containing 1/4 ~ 3/4MS medium of 30 ~ 70g/L sucrose, Echinacea adventive root Dual culture can be realized, obtain a large amount of physiological activators simultaneously.
Further preferably: the adventive root of purple coneflower and white Echinacea is cultivated in containing 1/4 ~ 3/4MS medium of 30 ~ 100g/L sucrose for suspending during 3:4 to cultivate in mass ratio, Echinacea adventive root Dual culture can be realized, obtain a large amount of physiological activators simultaneously.
Most preferably: the adventive root of purple coneflower and white Echinacea is being taken up in the airlift bioreactor containing the 3/4MS medium of 50g/L sucrose for suspending during 3:4 to cultivate in mass ratio, under the air mass flow of 0.1vvm, at the temperature of 23 ± 1 DEG C, light culture 30 days, can realize Echinacea adventive root Dual culture, Dual culture adventive root dry weight is than the raising output of cultivating separately simultaneously; In Dual culture adventive root, 6 kinds of caffeic acid derivative have; Also stimulate when producing single cultivation and caffeic acid 0.12mg/g dry weight do not detected; Obtain the physiological activators such as a large amount of flavones, polyphenol, polysaccharide simultaneously.
The advantage that the present invention has:
1. the present invention adopts the best Dual culture of bioreactor culture Echinacea adventive root to be combined as purple coneflower and white Echinacea, the Dual culture adventive root dry weight of results reaches 6.78g/L, be 1.8 times of 3 kinds of Echinacea adventive root Dual culture combinations, improve 11.3% and 11.0% respectively than the dry weight of cultivating separately purple coneflower and white Echinacea.This result is one of most important achievement of the present invention, because during different cultivars Dual culture, mutual Developing restraint between kind, the cell yield gathered in the crops finally is caused to reduce, but the present invention is by screening best of breed, 2 kinds of Dual culture adventive root biomasss of results are significantly higher than single cultivation and 3 kinds of Dual culture combinations.
2. in when Dual culture purple coneflower of the present invention and white Echinacea adventive root, obtain 6 kinds of caffeic acid derivative, its chlorogenic acid content reaches 6.41mg/g dry weight, echinacoside content is 3.82mg/g dry weight, Cichoric acid content is 13.48mg/g dry weight, and total content reaches 25.05mg/g dry weight, and its total content is 1.27 times when cultivating separately purple coneflower, 1.81 times during the white Echinacea of independent cultivation, also stimulate undetected active substance caffeic acid 0.12mg/g dry weight when producing single cultivation simultaneously.
3. white Echinacea of the present invention and purple coneflower Dual culture adventive root hydroxyl radical free radical clearance rate the highest, reach 68.12%, be significantly higher than clearance rate when white Echinacea and purple coneflower are cultivated separately; The DPPH clearance rate of this Dual culture reaches 74.06%, and reducing power reaches 1.03, is all significantly higher than other combinations.
4. purple coneflower of the present invention and the best inoculative proportion of white Echinacea Dual culture adventive root are 3:4, and the control group that the adventive root dry weight now gathered in the crops is 1:1 than inoculative proportion improves 8.4%, and Cichoric acid and total caffeic acid content respectively increase by 21.1% and 23.5%.
5. adopt the method for Dual culture adventive root of the present invention, the caffeic acid derivative such as Echinacea glycosides, Cichoric acid with significant anti-oxidant antitumor action can be obtained.Adopt the inventive method to obtain caffeic acid derivative aspect, have with short production cycle, efficiency is high, can factorial praluction, also for application of fermentation technology develops Echinacea bio-pharmaceutical and series health-care products establishes theory and practice basis.
Accompanying drawing explanation
Fig. 1 carries out the water-soluble polysaccharide of Echinacea Dual culture adventive root containing spirogram for employing the inventive method that the embodiment of the present invention provides in 5L airlift bioreactor.
The hydroxyl radical free radical clearance rate figure of employing the inventive method Echinacea Dual culture adventive root in 5L airlift bioreactor that Fig. 2 provides for the embodiment of the present invention.
Employing the inventive method Echinacea Dual culture adventive root DPPH clearance rate figure in 5L airlift bioreactor that Fig. 3 provides for the embodiment of the present invention.
The reducing power figure of employing the inventive method Echinacea Dual culture adventive root in 5L airlift bioreactor that Fig. 4 provides for the embodiment of the present invention.
Employing the inventive method that Fig. 5 provides for the embodiment of the present invention is at the effect diagram of different inorganic salt concentration to the growth of Dual culture adventive root and root morphology.
Embodiment
The present invention is with purple coneflower, 3 kinds of medicinal Echinacea adventive root such as narrow leaf Echinacea and white Echinacea are research object, plan utilizes adventive root Coculture techniques in airlift bioreactor, suspended by adventive root and cultivate the regulation and control of many physical and chemical parameters, obtain Cichoric acid, Echinacea glycosides, chlorogenic acid and caffeic acid etc. the are single caffeic acid derivative that can not simultaneously have when cultivating, also has abundant flavones, the physiological activator such as polyphenol and polysaccharide, verify Dual culture adventive root anti-oxidation characteristics simultaneously, finally set up the best production system of Echinacea adventive root Dual culture, produce for application of fermentation technology exploitation Echinacea bio-pharmaceutical and industrialization thereof and a kind of new method is provided.
Adopt the Dual culture purple coneflower that obtains of the inventive method and white Echinacea adventive root dry weight to improve 11.3% and 11.0% respectively than the purple coneflower cultivated separately and white Echinacea, be purple coneflower, white Echinacea and the 3 kinds of adventive root Dual culture combinations of narrow leaf Echinacea 1.8 times; In this Dual culture adventive root, 6 kinds of caffeic acid derivative have, and total content reaches 25.05mg/g dry weight, are when cultivating separately 1.28 times of purple coneflower, are when cultivating separately 1.89 times of white Echinacea; Also stimulate undetected active substance caffeic acid 0.12mg/g dry weight when producing single cultivation; Obtain the physiological activators such as a large amount of flavones, polyphenol, polysaccharide simultaneously.The inventive method is utilized to produce the aspects such as anti-oxidant anti-tumour phological activity material Echinacea glycosides and Cichoric acid, have with short production cycle, efficiency is high, can the feature such as factorial praluction, also for application of fermentation technology exploitation Echinacea bio-pharmaceutical and series health-care products establish theory and practice basis.
Materials and methods
The purple coneflower supplying examination material to preserve for research of agricultural science institute of Daliang City biotechnology, white Echinacea and narrow leaf Echinacea 3 kinds of Echinacea adventive root.3 kinds of adventive root are all containing 3% (w/v) sucrose, and the 1/2MS solid culture medium of 1mg/L IBA carries out squamous subculture in a conventional manner, and the initial pH of medium is adjusted to 5.7 ~ 5.8 before sterilization, and sterilising conditions is 121 DEG C, 1.2kg/cm
2, 20min, the every 30d subculture of adventive root once.
Note: for ease of introducing, Pu writes a Chinese character in simplified form and replaces purple coneflower, with Pa platinite Echinacea, with Ea for narrow leaf Echinacea, hereafter identical.
Embodiment
1. Dual culture adventive root best of breed is determined
In 5L airlift bioreactor, inject 4L medium, adventive root inoculum concentration is 7g/L, carries out the screening of Dual culture adventive root best of breed.Adventive root is cultivated and is combined as, and 4 kinds of Dual culture of the independent cultivation of Ea, Pa, Pu tri-kinds and Ea+Pa, Pa+Pu, Pu+Ea, Pu+Pa+Ea, have 7 kinds and cultivate combination, and repeat for 3 times, 21 bio-reactors are cultivated simultaneously.Bioreactor culture air mass flow is 0.1vvm, culturing room's temperature 23 ± 1 DEG C, light culture 30 days.Results " Invest, Then Investigate " adventive root fresh weight, dry weight, growth rate, flavones content, polyphenol and Cichoric acid, Echinacea glycosides isoreactivity content of material and antioxidation activity, filter out the best of breed of Dual culture.
2. the best inoculative proportion of Dual culture adventive root is determined
Pa+Pu is combined as by by testing the optimum Dual culture selected above, its total inoculum concentration is 7g/L, inoculation quality combines with 7 kinds of ratios such as 6:1,5:2,4:3,1:1,3:4,2:5,1:6 respectively than Pa:Pu, in 5L airlift bioreactor, carry out inoculative proportion screening test.Condition of culture is identical with above-mentioned steps 1 with survey item.
3. in Dual culture adventive root medium, the suitableeest inorganic salt concentration is determined
Dual culture adventive root is seeded in MS inorganic salt concentration and is adjusted to respectively in the medium of 1MS, 3/4MS, 1/2MS, 1/4MS, in 5L airlift bioreactor, carry out the suitableeest inorganic salt concentration screening test.Condition of culture is identical with survey item above-mentioned steps 1.
4. in Dual culture adventive root medium, the suitableeest sucrose concentration is determined
Dual culture adventive root is seeded in the suitableeest above-mentioned 3/4MS minimal medium, its sucrose concentration added is adjusted to 0 respectively, 30,50,70,100g/L, in 5L airlift bioreactor, carry out the suitableeest sucrose concentration screening test.Condition of culture is identical with survey item above-mentioned steps 1.
Location parameter and method
Adventive root biomass estimation
Adventive root is made it to be separated with medium by stainless steel metal sieve, after rinsing well with clear water, blots surface moisture, weigh its fresh weight.Then adventive root is placed in baking oven, dries 2d at 60 DEG C to constant weight, weigh and record.Its growth rate is expressed as: results dry weight-inoculation dry weight/inoculation dry weight.
The preparation of adventive root extract
Accurately take the adventive root 0.2g being dried to constant weight, add 10mL 80% ethanol, under normal temperature, extract 2h with magnetic stirring apparatus.After centrifugation, by supernatant liquid filtering in volumetric flask, residue repeats extraction twice in the same manner, and solution is settled to 25mL the most at last.
Total polyphenols and determination of total flavonoids
Determination of Polyphenols is with reference to Folin etc.
[104]assay method, ultraviolet specrophotometer 760nm place measure its absorbance, adopt standard items to be Gallic acid.General flavone content is with reference to Dewanto etc.
[105]assay method, with ultraviolet specrophotometer 510nm place measure its absorbance, adopt standard items to be Catechin.
Caffeic acid derivative's assay
Caffeic acid derivative adopts HPLC method to measure, and concrete operation method is as follows
[106]: use XTerra RP 18 post (particle diameter is 3.0 μm, 150mm × 3mm).Mobile phase is water (A) and acetonitrile (B).Carry out gradient elution as follows: first with 10%B wash-out 40min, then 25%B wash-out 11min, 50%B wash-out 1min, then repeat 10%B wash-out 8min with 0.3mL/min flow velocity, detect in wavelength 330nm place.
Water-soluble polysaccharide assay in adventive root
Utilize phenol-dense sulphuric acid method to measure polyoses content, wavelength is that 490nm place measures its absorbance, formulates glucose standard curve, can calculate polyoses content as follows: polyoses content=XD/W × 100%
Wherein, X-sample liquid is equivalent to concentration of glucose, D-dilution factor, W-sample volume.
Dual culture adventive root Antioxidative Activity Determination
The mensuration of hydroxyl radical free radical Scavenging activity, with reference to Kong etc.
[107]method; The mensuration of reducing power, with reference to Zhu etc.
[108]method; The mensuration of DPPH free radical scavenging activity, with reference to Chen etc.
[109]method; Fe
2+the mensuration of sequestering power, with reference to Hsu etc.
[110]method.
Statistical analysis
This test each process all carry out 3 groups parallel repeat cultivate, polyphenol, flavones, water-soluble polysaccharide and caffeic acid derivative's assay are 3 repetitions.Data, by the experiment of ANOVA and Duncansmultiple range testes (DMRT) reciprocal cross, adopt spss17.0 software (IBMinstitute, USA) to carry out Treatment Analysis.
Results and analysis
1. Echinacea adventive root Dual culture best of breed is determined
In 5L airlift bioreactor, Dual culture Pa+Pu combines adventive root growing way the best, adventive root fresh weight, the dry weight of results can be respectively 50.39g/L, 6.78g/L, adventive root growth rate 9.9, polyphenol and Flavone content reach 37.08mg/g dry weight and 34.42mg/g dry weight, are significantly higher than to cultivate separately to combine with 3 kinds of Dual culture.Most important achievement is that the dry weight of Pa+Pu combination adventive root improves 11.3%, 11.0% and 60.7% respectively than Pu, Pa, Ea dry weight when cultivating separately.Between general different cultivars during Dual culture, because of mutual Developing restraint, the cell yield gathered in the crops finally is caused to reduce, but the combination of this Dual culture, the adventive root biomass of results is significantly higher than the combination (see table 1) of cultivating separately.
The growth of Dual culture adventive root and polyphenol, accumulation of flavonoids situation in table 1 5L airlift bioreactor
Note: 5% significance level, same letter represents without significance.
In Dual culture adventive root, it is the highest that Pa+pu combination produces caffeic acid derivative's content, its chlorogenic acid content reaches 6.41mg/g dry weight, Echinacea glycosides content is 3.82mg/g dry weight, Cichoric acid content is 13.48mg/g dry weight, total content reaches 25.05mg/g dry weight, and its total content is 1.28 times when cultivating separately Pu, cultivates separately 1.89 times (see table 2) during Pa.
The Echinacea adventive root caffeic acid derivative accumulation of adventive root combination in table 2 5L airlift bioreactor
Note: 5% significance level, same letter represents without significance.
Meanwhile, there is significant difference in the water-soluble polysaccharide content in 5L airlift bioreactor between different Dual culture combination.The polyoses content that Pu and Pa two kinds cultivates separately adventive root is close, all about 2.1%, is significantly higher than Ea kind; The polyoses content of Pa+Ea combination is 4.58%, is significantly higher than other combinations, but the biomass of this combination adventive root and caffeic acid derivative's content are starkly lower than Pa+Pu; Secondly for Pa+Pu combination reaches 2.34%, far above Pu+Ea and Pa+Ea+Pu combination (see Fig. 1).
Pa+Pu Dual culture combination Scavenging action to hydroxyl free radical is the highest, reaches 68.12%, is significantly higher than clearance rate when Pa and Pu cultivates separately; Its DPPH clearance rate is 74.06%, reducing power 1.03, is all significantly higher than other combinations (see Fig. 2 ~ 4).
2. the best inoculative proportion of Dual culture adventive root is determined
Table 3 different vaccination ratio on Dual culture adventive root growth and polyphenol, Flavonoids Accumulation impact
Note: 5% significance level, same letter represents without significance.
When the Pu kind proportion accessed is larger, the differentiation of Dual culture adventive root is less, and upgrowth situation is not good enough, and institute obtains the fresh weight of adventive root, dry weight is lower, illustrates that Pa kind grows Pu adventive root and has considerable influence.And along with the increase gradually of Pa kind proportion, Dual culture adventive root biomass also promotes thereupon, as Pu:Pa=3:4, the fresh weight of adventive root, dry weight, growth rate all reach maximum, be respectively 68.35g/L, 25.97g/L, 8.99, advantage is remarkable, and its dry weight improves 8.4% than contrast 1:1 ratio group, but increase Pa kind proportion again, the biomass of Dual culture adventive root presents downward trend (see table 3) on the contrary.
Inoculative proportion all has remarkable impact to all kinds of caffeic acid derivative in adventive root combination.Along with the change of Pu, Pa adventive root inoculation proportion, in root, caffeic acid derivative's total content presents the variation tendency first raising and reduce afterwards substantially, and chlorogenic acid, echinacoside, Cichoric acid three compounds that wherein content is comparatively given prominence to all present similar Changing Pattern (see table 4).
Table 4 different vaccination ratio is on the impact of caffeic acid derivative's accumulation in adventive root combination
Note: 5% significance level, same letter represents without significance.
When Pu:Pa is 3:4, caffeic acid derivative's total content reaches the highest, is 28.40mg/g dry weight, improves 23.52% than 1:1 contrast, and its Cichoric acid content is than contrast raising 21.1%; Secondly when being 1:6, its total content is 25.96mg/g dry weight, lower than 3:4 by 9.40%.
3. the suitableeest inorganic salt concentration of Dual culture adventive root is determined
Between 1/4MS and 3/4MS, along with inorganic salt concentration increases, adventive root biomass and growth rate also significantly increase.But when concentration brings up to 1MS, in medium, adventive root amount of growth declines on the contrary, adventive root growth retardation.When inorganic salt concentration is 3/4MS, adventive root growth is significantly better than other process, and now fresh weight is 52.56g/L, and dry weight reaches 6.43g/L, growth rate 8.2 (see Fig. 5).
Polyphenol and flavones content from adventive root, in 1/4MS medium, the overall growing way of adventive root is poor, but wherein total phenol content and general flavone content are relatively high, higher than the content in the adventive root that 1/2MS and 1MS cultivates, but lower than 3/4MS process.The total phenol content of 3/4MS adventive root reaches 31.74mg/g dry weight, and general flavone content reaches 31.60mg/g dry weight, is significantly higher than other medium (see table 5).
Table 5 inorganic salt concentration is on the impact of Pa/Pu assembled growth and polyphenol, Flavonoids Accumulation
Note: 5% significance level, same letter represents without significance.Note:In rows，values followed by the same letterdo not show significant difference at P\0.05.
Inorganic salt concentration also has considerable influence to caffeic acid derivative's accumulation in Dual culture adventive root.In the 3/4MS that Dual culture adventive root growing way is best, total content reaches 24.01mg/g dry weight, is 3.2 times (table 6) of the poorest 1MS medium content of growing way.
Table 6 inorganic salt concentration is on the impact of caffeic acid derivative's content in Echinacea Dual culture adventive root
Note: 5% significance level, same letter represents without significance.Note:In rows，values followed by the same letter do not showsignificant difference at P\0.05.
Inorganic salt concentration is be the highest to Scavenging action to hydroxyl free radical in 3/4/MS concentration, reach 70.12% on the impact of Dual culture adventive root hydroxyl radical free radical clearance rate, is significantly higher than other and processes; DPPH clearance rate is 70.65%, and in 1MS and 1/2MS concentration, DPPH clearance rate is lower, active poor; Adventive root reducing power variation tendency is identical with DPPH, and in 3/4/MS concentration, absorbance is the highest, reaches 0.95, is significantly higher than other process.
4. the suitableeest sucrose concentration of Dual culture adventive root is determined
Different sucrose 0,30,50,70,100g/L on the growth of Dual culture adventive root and polyphenol, Flavonoids Accumulation impact significantly.Do not containing in the medium of sucrose (0g/L), gained adventive root fresh weight and dry weight are minimum, and growth rate is negative value, show that medium lacks carbon source, adventive root can not grow, and in root, polyphenol, flavonoids also signs of degradation occur, and content processes well below other.
And along with the increase gradually of sucrose concentration, adventive root biomass, growth rate and polyphenol, flavonoid content also improve thereupon.As sucrose concentration 50g/L, adventive root fresh weight, dry weight, growth rate and total polyphenols, Flavonoids content are all the highest, be significantly higher than other process, now adventive root fresh weight is 75.68g/L, dry weight reaches 7.18g/L, growth rate is 9.3, and Determination of Polyphenols reaches 53.49mg/g dry weight (DW), and general flavone content reaches 43.95mg/g dry weight.But along with sucrose concentration be increased to 70g/L, 100g/L time, the fresh weight of adventive root, dry weight, Polyphenols and flavonoid content present downward trend (see table 7) on the contrary.
Table 7 sucrose concentration on adventive root growth and phenols, flavonoids Metabolite Accumulation impact
Note: 5% significance level, same letter represents without significance.Note:In rows，values followed by the same letterdo not show significant difference at P\0.05.
Sucrose concentration is between 0 ~ 100g/L, and caffeic acid derivative's total content presents the Changing Pattern first improving and then reduce with sucrose concentration increase, and all kinds derivatives monomer all has similar variation tendency.Sucrose concentration is 50g/L, in adventive root, total caffeic acid derivative's content (30.81mg/g dry weight) reaches the highest, its Content of Chlorogenic Acid reaches 7.21mg/g dry weight, Echinacea glycosides is 4.51mg/g dry weight, Cichoric acid is 17.66mg/g dry weight, total content is 13 times of sugar-free culture-medium, exceeds 92.92% than 30g/L.Between sucrose concentration 30g/L and 70g/L processes, total caffeic acid derivative's content is without significant difference, and each derivative content is also comparatively close to (see table 8).
Table 8 different vaccination ratio is on the impact of caffeic acid derivative's accumulation in the combination of Echinacea adventive root
Note: 5% significance level, same letter represents without significance.Note:In rows，values followed by the same letterdo not show significant difference at P\0.05.
Claims (4)
1. the method for a medicinal Echinacea adventive root Dual culture, it is characterized in that: the adventive root of two or more combination in medicinal for different cultivars Echinacea is suspended to cultivate and cultivates in the MS medium containing sucrose, Echinacea adventive root Dual culture can be realized, obtain a large amount of physiological activators simultaneously.
2. by the method for medicinal Echinacea adventive root Dual culture according to claim 1, it is characterized in that: the adventive root of two or more combination in purple coneflower (Echinacea purpurea), narrow leaf Echinacea (Echinacea angustifolia) and white Echinacea (Echinacea pallida) is suspended and cultivates in the 1/4 ~ 3/4MS medium containing 30 ~ 100g/L sucrose, Echinacea adventive root Dual culture can be realized, obtain a large amount of physiological activators simultaneously.
3. by the method for medicinal Echinacea adventive root Dual culture according to claim 2, it is characterized in that: the adventive root of purple coneflower and white Echinacea is cultivated in the 1/4 ~ 3/4MS medium containing 30 ~ 100g/L sucrose for 3:4 suspends in mass ratio, Echinacea adventive root Dual culture can be realized, obtain a large amount of physiological activators simultaneously.
4. by the method for medicinal Echinacea adventive root Dual culture according to claim 3, it is characterized in that: the adventive root of purple coneflower and white Echinacea is cultivated 30 days for 3:4 suspends to cultivate taking up the airlift bioreactor containing the 3/4MS medium of 50g/L sucrose in mass ratio, Echinacea adventive root Dual culture can be realized, obtain a large amount of physiological activators simultaneously.
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| CN110786241A (en) * | 2019-11-28 | 2020-02-14 | 大连市现代农业生产发展服务中心(大连市农业科学研究院) | Culture method for improving accumulation of chicoric acid in liquid culture echinacea adventitious roots |
| IT201900004119A1 (en) * | 2019-03-21 | 2020-09-21 | Demethra Biotech S R L | Phytocomplex and extract of meristematic cell line selected from Echinacea purpurea |
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| WO2020188535A1 (en) * | 2019-03-21 | 2020-09-24 | Demethra Biotech S.R.L. | Phytocomplex and extract of a meristematic cell line selected from echinacea purpurea |
| CN110786241A (en) * | 2019-11-28 | 2020-02-14 | 大连市现代农业生产发展服务中心(大连市农业科学研究院) | Culture method for improving accumulation of chicoric acid in liquid culture echinacea adventitious roots |
| CN110786241B (en) * | 2019-11-28 | 2022-03-22 | 大连市现代农业生产发展服务中心(大连市农业科学研究院) | Culture method for improving accumulation of chicoric acid in liquid culture echinacea adventitious roots |
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