CN110786241B - Culture method for improving accumulation of chicoric acid in liquid culture echinacea adventitious roots - Google Patents
Culture method for improving accumulation of chicoric acid in liquid culture echinacea adventitious roots Download PDFInfo
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- CN110786241B CN110786241B CN201911191087.4A CN201911191087A CN110786241B CN 110786241 B CN110786241 B CN 110786241B CN 201911191087 A CN201911191087 A CN 201911191087A CN 110786241 B CN110786241 B CN 110786241B
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- echinacea
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- chicoric acid
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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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- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention relates to a culture method for improving accumulation of chicoric acid in echinacea purpurea adventitious roots, belonging to the technical field of biology. The main technical scheme is as follows: a culture method for improving accumulation of chicoric acid in echinacea purpurea adventitious roots comprises the following steps: carrying out liquid culture on echinacea adventitious roots, and adding a sterile aspartame solution in the process of culturing for 0-600 hours, wherein the final concentration of the aspartame solution is 20-200 mg/L. The aspartame solution is prepared by dissolving the aspartame solution in deionized water, adjusting the pH value of the solution to 3.5-4.5 and sterilizing. The method has the advantages of simple operation, short production period and low production cost, and is convenient for industrial large-scale production and application of the chicoric acid, and the content of the chicoric acid in the echinacea adventitious roots can be obviously improved by the aspartame with the concentration of 20-200 mg/L.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture method for improving accumulation of chicoric acid in liquid culture echinacea adventitious roots.
Background
Echinacea purpurea spp is perennial herb of Echinacea of Compositae, and is native to North America. The main 3 varieties of Echinacea purpurea currently under development as drugs, Echinacea purpurea, Echinacea angustifolia and Echinacea pallida, are included. The echinacea purpurea is a medicinal plant with wide application, is an internationally recognized immunomodulator and has obvious effect of enhancing the immunity of organisms. The method has the advantages that the generation of secondary products by culturing the echinacea adventitious roots becomes the content of key research in several years, the adventitious roots cultured by artificial suspension culture grow rapidly, the culture period is short, and the active ingredients are stable, so that the adventitious roots cultured by artificial suspension culture technology are applied to the production and processing of natural products, natural product raw materials can be obtained in a large amount in a short period, and sufficient secondary metabolites are extracted from the natural product raw materials to meet the market demand, so that the increasingly prominent practical problems of insufficient resources, low quality and the like of wild natural products are effectively solved.
Chicoric acid is a hydroxycinnamic acid and is the most abundant active ingredient in echinacea. As the main active ingredient of echinacea, the chicoric acid mainly has the following uses: chicoric acid has antioxidant, immunity enhancing and antiinflammatory effects, and can inhibit hyaluronidase, protect collagen from free radicals which can cause its degradation, and inhibit HIV-1 integrase. Chicoric acid has various uses, but the extraction rate of the chicoric acid from echinacea or adventitious roots of the echinacea is low, so that the chicoric acid is difficult to meet the market demand.
Disclosure of Invention
The invention aims to overcome the defect of low content of chicoric acid in the tissue culture method of the echinacea purpurea adventitious roots in the prior art, and provides a culture method for improving the accumulation of the chicoric acid in the liquid culture echinacea purpurea adventitious roots.
The technical scheme of the invention is as follows: a culture method for improving accumulation of chicoric acid in echinacea purpurea adventitious roots comprises the following steps: carrying out liquid culture on echinacea adventitious roots, and adding a sterile aspartame solution in the process of culturing for 0-600 hours, wherein the final concentration of the aspartame solution is 20-200 mg/L.
Further, the aspartame solution is prepared by dissolving the aspartame solution in deionized water, adjusting the pH value of the solution to 3.5-4.5 and sterilizing.
Further, the liquid culture is cultured in a bioreactor or a triangular flask.
Further, the final concentration of the aspartame solution is 150 mg/L.
Further, the sterilization is steam sterilization or filtration sterilization.
Further, the steam sterilization is performed at the temperature of 121-.
Further, the filtration sterilization is filtration sterilization through a sterile filter membrane of 0.22 μm.
Further, the echinacea varieties are echinacea angustifolia, echinacea alba or echinacea purpurea.
Further, after culturing 480-.
The invention has the beneficial effects that: the method has the advantages of simple operation, short production period and low production cost, and is convenient for industrial large-scale production and application of the chicoric acid, and the content of the chicoric acid in the echinacea adventitious roots can be obviously improved by the aspartame with the concentration of 20-200 mg/L.
Detailed Description
The invention is further illustrated and explained with reference to specific examples, wherein the materials and equipment used are conventional in the art unless otherwise specified.
1. The preparation, culture and collection method of the culture medium comprises the following steps:
taking out the adventitious roots of the echinacea cultured by 480-one for 600 hours in an aseptic environment, shearing the adventitious roots into pieces, inoculating the pieces into an airlift bioreactor or a triangular flask filled with a liquid culture medium for culturing, adding an aseptic aspartame solution at any time of 0-600 hours in the culture process, and adjusting the final concentration of the aspartame in the liquid culture medium to be 20-200 mg/L. After culturing the adventitious roots for 720-960 hours, the echinacea adventitious roots with higher content of chicoric acid can be obtained.
The sterile aspartame solution is prepared by dissolving the aspartame solution in deionized water, adjusting the pH value of the solution to 3.5-4.5 and sterilizing.
2. The method for detecting the content of the chicoric acid comprises the following steps:
(1) chromatographic conditions
Chicoric acid is measured by an HPLC method, and the liquid chromatography conditions are as follows: column Waters Sunfire C18 column (particle diameter 4.5 μm, 250 mm. times.3 mm); mobile phase A: water (0.1% phosphoric acid) and acetonitrile B; gradient elution procedure: 0-23min, A: 90%, 23-28min A: 95 to 50 percent; 28-30min, A: 50%, 30-35min, A: 10 percent. The flow rate is 1mL/min, the sample injection volume is 10uL, and the detection wavelength is 330 nm.
(2) Preparation of Standard solutions
Precisely weighing 5mg of the cichoric acid standard substance, dissolving the cichoric acid standard substance in methanol, and then dissolving the cichoric acid standard substance in a 5mL volumetric flask to obtain a cichoric acid reference substance stock solution. The stock solution was filtered through a 0.45 μm filter and stored in a sealed manner for further use.
(3) Sample solution preparation
Accurately weighing 0.2g of dry adventitious roots of Echinacea purpurea, adding 20mL of methanol into a 50mL triangular flask with a plug, performing ultrasonic extraction (250W and 40kHz) for 60min, performing centrifugal precipitation, repeatedly extracting for 1 time, mixing supernatants, and diluting to 50mL for detection by high performance liquid chromatography.
Example 1
(1) Culturing
Taking out the adventitious roots of the echinacea purpurea cultured for 480 hours in a sterile environment, cutting into pieces, and connecting to an airlift bioreactor filled with a liquid culture medium, wherein the liquid culture medium comprises the following components in parts by weight: 3/4MS, sucrose 4 wt%, pH5.8, indolebutyric acid 1mg/L, and ventilation 1VVM, culturing at 25 + -1 deg.C, adjusting pH to 4.5 after aspartame is dissolved, sterilizing at 123 deg.C for 30min, adding into culture medium for culturing adventitious roots for 0 hr, and adjusting aspartame final concentration to 50, 100, 150, and 200mg/L respectively. After culturing for 840 hours, harvesting was performed, and each treatment was repeated 3 times, using an adventitious root to which aspartame solution was not added in the medium as a Control (CK).
(2) Content detection
Drying harvested echinacea adventitious roots to constant weight, weighing 0.2g, adding 20mL of methanol into a 50mL triangular flask with a plug, carrying out ultrasonic extraction (250W and 40kHz) for 60min, centrifuging, repeatedly extracting precipitates for 1 time, combining two supernatants, and fixing the volume to 50mL for detection by a high performance liquid chromatograph. The liquid chromatography conditions were as follows: column Waters Sunfire C18 column (particle diameter 4.5 μm, 250 mm. times.3 mm); mobile phase A: water (0.1% phosphoric acid) and acetonitrile B; gradient elution procedure: 0-23min, A: 90%, 23-28min A: 95 to 50 percent; 28-30min, A: 50%, 30-35min, A: 10 percent. The flow rate is 1mL/min, the sample injection volume is 10uL, and the detection wavelength is 330 nm. Precisely weighing 5mg of chicoric acid standard substance, dissolving with methanol, diluting to a constant volume in a 5mL volumetric flask to obtain 1000 μ g/mL of chicoric acid reference substance mother liquor, diluting to 10 μ g/mL, 25 μ g/mL, 50 μ g/mL, 75 μ g/mL and 100 μ g/mL respectively, and injecting. Get the standard curve y 41192x + 123713R2When the area is 0.9999, y is the area of the peak of the chicoric acid HPLC chart; and x is the concentration of chicoric acid and has a unit of mu g/mL. Calculation of chicoric acid content in adventitious roots ═ (As-123713)/41192 × 50 × 10-30.2mg/g DW.As: peaks of samples in HPLC chartArea, mg/g DW: mg/g dry weight.
(3) Analysis of results
The chicoric acid concentration was calculated and the significance of the differences was analyzed by the variance Dunnans Multiple Range Tests (DMRT) method, the results of which are shown in table 1.
TABLE 1 Effect of different final concentrations of Aspartame on chicoric acid content in Echinacea purpurea adventitious roots
As can be seen from Table 1, the content of chicoric acid in the adventitious roots of echinacea into which aspartame was added was significantly higher than that of the control, wherein the content of chicoric acid in the adventitious roots of echinacea with aspartame at the final concentration of 150mg/L was the highest and could reach 40.30mg/g DW, which is 1.46 times that of the control 27.46mg/g DW; the chicoric acid content in the added adventitious roots with less aspartame (50mg/L) can also reach 31.15mg/g DW, which is improved by 13.4 percent compared with the control. The experiment proves that the chicoric acid content in the adventitious roots of the echinacea purpurea can be obviously improved by adding the aspartame with certain concentration.
Example 2
(1) Culturing
Taking out the adventitious roots of the echinacea angustifolia cultured for 480 hours in a sterile environment, cutting into pieces, and then connecting into a triangular flask filled with a liquid culture medium, wherein the formula of the liquid culture medium is as follows: 1/2MS, 3 percent of sucrose, pH6.0, 2mg/L of indolebutyric acid, and shake culture at 80r/min at 25 +/-1 ℃. The pH value of the dissolved aspartame is adjusted to 3.5, the dissolved aspartame is filtered and sterilized by a 0.22 mu m sterile filter membrane and then is added into a culture medium for culturing adventitious roots for 240 hours, and the final concentration of the aspartame is adjusted to 20 mg/L. After culturing for 840 hours, harvesting was performed, and each treatment was repeated 3 times, using adventitious roots without addition of aspartame solution as a control.
(2) Detection of
The detection method was the same as in example 1.
(3) Analysis of results
The concentration of chicoric acid was calculated and analyzed by the t-test method (paired t-test). As can be seen from Table 2, the chicoric acid content (1.63mg/g DW) in the adventitious roots of Echinacea angustifolia with added aspartame was about 1.23 times higher than that of the control (1.32mg/g DW), and the difference between the two was very significant (p < 0.01). The experiment proves that the chicoric acid content in the adventitious roots of the echinacea angustifolia cultured in the liquid can be obviously improved by adding the aspartame, and the result is shown in a table 2.
TABLE 2 Effect of Aspartame on chicoric acid content in Echinacea angustifolia adventitious roots
Example 3
(1) Culturing
Taking out the adventitious roots of the white echinacea cultured for 600 hours in a sterile environment, cutting into pieces, and connecting into a triangular flask filled with a liquid culture medium, wherein the formula of the liquid culture medium is as follows: 3/4MS, 4 wt% of sucrose, pH5.9, 1mg/L of indolebutyric acid, and shake culturing at 80r/min at 25 +/-1 ℃. Dissolving aspartame, adjusting pH to 4.0, filtering and sterilizing with 0.22 μm sterile filter membrane, adding into adventitious root culture medium cultured for 360 hr, and adjusting aspartame final concentration to 100 mg/L. After 960 hours of culture, the roots were harvested and the adventitious roots were used as a control without addition of aspartame solution.
(2) Detection of
The detection method was the same as in example 1.
(3) Analysis of results
The analysis was performed as in example 2, and the results are shown in Table 3. As can be seen from Table 3, the chicoric acid content (6.71mg/g DW) in the white Echinacea adventitious roots with aspartame added is about 1.27 times higher than that of the control (5.27mg/g DW), and the difference between the two reaches a very significant level (p < 0.01). The experiment proves that the chicoric acid content in the adventitious roots of the white echinacea by liquid culture can be obviously improved by adding the aspartame.
TABLE 3 Effect of Aspartame on chicoric acid in Echinacea purpurea adventitious Roots
Example 4
(1) Culturing
Inoculating adventitious roots of Echinacea purpurea cultured for 480 hours to a sterile environment, taking out, cutting into pieces, and placing in an airlift bioreactor filled with a liquid culture medium, wherein the liquid culture medium comprises the following components in parts by weight: 3/4MS, sucrose 4 wt%, pH5.8, indolebutyric acid 1mg/L, and ventilation 1VVM, culturing at 25 + -1 deg.C, adjusting pH to 4.5 after aspartame is dissolved, sterilizing at 121 deg.C for 20min, and adding into adventitious root culture medium cultured for 600 hr until aspartame final concentration is 150 mg/L. After culturing for 840 hours, the roots were harvested and the adventitious roots were used as a control without addition of aspartame solution.
(2) Detection of
The detection method was the same as in example 1.
(3) Analysis of results
The analysis method is the same as example 2, the result is shown in Table 4, the chicoric acid content (32.95mg/g DW) in the white echinacea adventitious root added with aspartame is about 1.16 times higher than that of the control (28.37mg/g DW), and the two reach the very significant difference level (p is less than 0.01). The experiment proves that the chicoric acid content in the adventitious roots of the echinacea purpurea can be obviously improved by adding the aspartame.
TABLE 4 Effect of Aspartame on chicoric acid in Echinacea purpurea adventitious roots
The foregoing examples are provided for illustration and description of the invention only and are not intended to limit the invention to the scope of the described examples. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that many variations and modifications may be made in accordance with the teachings of the present invention, which variations and modifications are within the scope of the present invention as claimed.
Claims (8)
1. A culture method for improving accumulation of chicoric acid in echinacea purpurea adventitious roots is characterized by comprising the following steps: carrying out liquid culture on the echinacea adventitious roots, and adding a sterile aspartame solution in the process of culturing for 0-600 hours; the aspartame solution is prepared by dissolving the aspartame solution in deionized water, adjusting the pH value of the solution to 3.5-4.5 and sterilizing, wherein the final concentration of the aspartame solution is 50-200 mg/L.
2. The culture method for increasing chicoric acid accumulation in Echinacea purpurea adventitious roots according to claim 1, wherein the liquid culture is cultured in a bioreactor or in an Erlenmeyer flask.
3. The culture process for increasing chicoric acid accumulation in adventitious roots of echinacea according to claim 1, wherein the final concentration of the aspartame solution is 150 mg/L.
4. The culture method for increasing chicoric acid accumulation in echinacea purpurea adventitious roots according to claim 1, wherein the sterilization is steam sterilization or filtration sterilization.
5. The method as claimed in claim 4, wherein the steam sterilization is performed at 121-123 deg.C for 20-40 min.
6. The culture method for increasing chicoric acid accumulation in Echinacea purpurea adventitious roots according to claim 4, wherein the filter sterilization is filter sterilization through a 0.22 μm sterile filter membrane.
7. The culture process for increasing chicoric acid accumulation in adventitious roots of echinacea purpurea according to claim 1, wherein the echinacea variety is echinacea angustifolia, echinacea alba or echinacea purpurea.
8. The method as claimed in claim 1, wherein the adventitious roots of Echinacea purpurea cultured for 480-600 hours are removed under sterile environment, cut into pieces, and inoculated into a bioreactor or an Erlenmeyer flask for culturing.
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