CN106386810B - New use of chrysanthemum indicum polysaccharide in promoting synthesis of atractylenolide in rhizoma Atractylodis Macrocephalae - Google Patents
New use of chrysanthemum indicum polysaccharide in promoting synthesis of atractylenolide in rhizoma Atractylodis Macrocephalae Download PDFInfo
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Abstract
The invention discloses a new application of chrysanthemum indicum polysaccharide in promoting the growth period of bighead atractylodes rhizome to increase the accumulation of the content of atractylenolide. The chrysanthemum indicum polysaccharide can be prepared into a plant inducer, and is applied to promoting the metabolic accumulation of atractylenolide components in the atractylenolide plants, improving the active ingredients of atractylenolide, and increasing the quality of atractylenolide. The inducer of the invention is supplemented with common medicinal excipient, and can be prepared into dosage forms such as aqua, powder and the like.
Description
Technical Field
The invention relates to accumulation of wild chrysanthemum polysaccharide for promoting growth period of bighead atractylodes rhizome and increasing content of atractylenolide, belonging to the technical field of agricultural biology.
Background
Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala A, a perennial herb of Atractylodes of Compositae, and is mainly produced in Zhejiang, Anhui, Hunan, Jiangxi and other places, wherein the quality of Atractylodes macrocephala in Zhejiang is excellent, and the lactone component of Atractylodes macrocephala is the main pharmacodynamic component of Atractylodes macrocephala, and has obvious prevention and control effects on human gastric cancer and intestinal diseases. The low yield phenomenon of secondary metabolites is one of the core problems restricting the large-scale application of natural products. Although researchers at home and abroad research on the problem of low yield of secondary metabolites, including excellent strain breeding, culture technology improvement, synthesis of key enzymes for synthesizing target components and the like, the key is to search for effective inducers to promote biosynthesis of the metabolites.
The Chrysanthemum indicum polysaccharide is one of main active ingredients of Chrysanthemum indicum (Chrysanthemum indicum L), is composed of 6 monosaccharides of rhamnose, arabinose, xylose, mannose, glucose and galactose, and has the effects of improving immunity, reducing blood fat, resisting oxidation, inducing resistance (patent number:) and the like. However, the relation between the chrysanthemum indicum polysaccharide and secondary metabolites is not reported in research, and the research finds that the chrysanthemum indicum polysaccharide can promote the metabolic accumulation of atractylenolide components in atractylenolide plants, improve the active ingredients of atractylenolide, and increase the quality. The application of the chrysanthemum indicum polysaccharide in inducing the increase of the times of atractylenolide belongs to the first discovery.
Disclosure of Invention
The invention aims to provide a novel elicitor for improving the accumulation of atractylenolide, in particular to a chrysanthemum indicum polysaccharide accelerant and a preparation method thereof. Therefore, the largehead atractylodes rhizome has high content of atractylenolide, and the largehead atractylodes rhizome planted in non-Zhejiang areas also has high content of genuine components, thereby improving the economic benefit and market value of the largehead atractylodes rhizome.
The preparation method of the atractylenolide accelerator comprises the following steps:
(1) preparing wild chrysanthemum polysaccharide: a. collecting wild chrysanthemum, drying in an oven at 60-70 ℃, crushing, and sieving with a 80-100 mesh sieve;
b. adding pure water 15-20 times the weight of the wild chrysanthemum powder into the wild chrysanthemum powder, decocting for 2-3 times (2-3 hr each time), collecting decoction, removing residues, mixing filtrates, and filtering to obtain wild chrysanthemum aqueous extract solution;
c. concentrating under reduced pressure at low temperature to obtain water extract solution with specific gravity of 1.1-1.31;
d. adding 1% active carbon into the concentrated solution for decolorization for 2-3 times, and performing suction filtration to obtain decolorized concentrated solution;
e. slowly adding 90-95% ethanol 6-8 times of the volume of the concentrated and decolorized wild chrysanthemum aqueous extract solution, and standing overnight at 4 ℃;
f. after the precipitate is separated out, centrifuging the mixed solution at 3000-;
g. adding distilled water with the volume amount of 75-80 times of the mass of the crude polysaccharide into the wild chrysanthemum crude polysaccharide, and adding trichloroacetic acid with the volume same as that of the distilled water: and (2) severely shaking n-butanol (the volume ratio is 1: 8-10) for 20-25min, standing, layering, taking down clear liquid, removing the n-butanol layer at the upper layer and the middle layer of impure protein, adding 2NNaOH to be neutral, evaporating to dryness, repeating the step (g) for multiple times, and removing glycoprotein to obtain the pure chrysanthemum indicum polysaccharide.
The chrysanthemum indicum polysaccharide can also be purchased by market, the purity can be 10% -99%, and the chrysanthemum indicum polysaccharide can be prepared into an applicable concentration according to actual conditions.
The above flos Chrysanthemi Indici polysaccharide is supplemented with common medicinal excipient, and made into dosage forms such as aqua, powder, etc., which can be sprayed on crop plants for increasing the contents of atractylenolide II and atractylenolide III in Atractylodis rhizoma and improving the quality of Atractylodis rhizoma.
The application of the above flos Chrysanthemi Indici polysaccharide as elicitor for improving the content of effective components of Atractylodis rhizoma is characterized in that the above flos Chrysanthemi Indici polysaccharide can be made into dosage forms such as aqua, powder, granule, etc. with common medicinal excipient, and the application concentration of the promoter is 1-200g/L, preferably 1-20 g/L.
The application of the chrysanthemum indicum polysaccharide as an elicitor for improving the content of active ingredients of the bighead atractylodes rhizome is characterized in that the chrysanthemum indicum polysaccharide promotes the accumulation and formation of lactone ingredients of the bighead atractylodes rhizome in the seedling stage by seed soaking, root irrigation and spraying.
The above-mentioned wild Chrysanthemum polysaccharide is used as an agricultural disease-resistant inducer, and is characterized by that said wild Chrysanthemum polysaccharide can be extracted from the whole plant including root, stem and flower of Chrysanthemum indicum (Chrysanthemum indicum L), Chrysanthemum morifolium (C.boreale Mak.) or Chrysanthemum indicum (C.lavandula esculentum (Fisch.) Mak.) of Compositae.
The invention has the beneficial effects that: according to the invention, a bighead atractylodes rhizome sample plant is selected, and the promotion effect of chrysanthemum indicum polysaccharide on the accumulation of atractylenolide in bighead atractylodes rhizome is respectively examined. The results show that: the chrysanthemum indicum polysaccharide biological agent can effectively induce the accumulation of atractylenolide which is a pharmacodynamic substance of white atractylodes rhizome in the growth period of the atractylenolide, improves the quality of the atractylenolide, provides an effective means for the high-quality and high-quality production of the atractylenolide, and shows good market prospect. The invention opens up a new application of chrysanthemum indicum polysaccharide as an inducer for promoting the improvement of the quality in the planting of the bighead atractylodes rhizome, has low cost, convenient use and obvious effect, and has good development and utilization values in traditional Chinese medicine agriculture and wide application space.
Drawings
FIG. 1: HPLC chromatogram of atractylenolide I, atractylenolide III and atractylenolide II in Atractylodes macrocephala; wherein a and b are control CK; c and d are groups of applying polysaccharides of chrysanthemum indicum
The following experimental examples are intended to further illustrate the invention
Experimental example 1 Effect of Chrysanthemum indicum polysaccharide on promoting accumulation of atractylenolide in Atractylodes macrocephala Koidz
In the experiment, largehead atractylodes rhizome is taken as an object, and the influence of wild chrysanthemum polysaccharide on atractylenolide is researched.
1.1 extraction of Chrysanthemum indicum polysaccharide
(1) Collecting Chrysanthemum indicum L, oven drying in oven, pulverizing, sieving with 80-100 mesh sieve, and weighing 500 g;
(2) respectively adding petroleum ether with volume amount of 6-8 times of the mass of the wild chrysanthemum powder into the wild chrysanthemum powder, refluxing for 1-2 times, 1-2 hours each time, taking out, filtering, heating the filter residue in water bath to volatilize the residual petroleum ether, and obtaining the decolorized wild chrysanthemum powder;
(3) adding pure water with a volume amount 18-20 times of the mass of the wild chrysanthemum powder into the decolorized wild chrysanthemum powder respectively, decocting for 2-3 times, each time for 2-3 hours, taking juice, removing residues, combining filtrates, and filtering to obtain a wild chrysanthemum aqueous extract solution;
(4) concentrating under reduced pressure at low temperature to specific gravity of 1.15-1.25:
(5) slowly adding 90-95% ethanol 6-8 times of the volume of the concentrated water extract solution of wild chrysanthemum, and standing overnight at 4 ℃;
(6) after the precipitate is separated out, centrifuging the mixed solution at 3000-4500rpm for 12-15min, taking the precipitate, and drying the precipitate by using a freeze-drying method to obtain the crude chrysanthemum polysaccharide;
(7) adding distilled water with the volume amount of 75-80 times of the mass of the crude polysaccharide into the wild chrysanthemum crude polysaccharide, and adding trichloroacetic acid with the volume same as that of the distilled water: and (3) severely shaking n-butanol (the volume ratio is 1: 8-10) for 20-25min, standing, layering, taking down clear liquid, removing the n-butanol layer at the upper layer and the middle layer of impure protein, adding 2NNaOH to neutrality, evaporating to dryness, repeating the step (7) for 8-9 times, removing glycoprotein to obtain 13.56g of pure wild chrysanthemum polysaccharide, wherein the purity is 99% by a sulfuric acid-phenol method.
(8) Preparing a wild chrysanthemum polysaccharide aqueous solution: taking a proper amount of the wild chrysanthemum polysaccharide, preparing aqueous solutions of 10mg/ml, 2mg/ml and 0.4mg/ml respectively, and storing in a sealed manner for later use.
1.2 Effect of wild Chrysanthemum polysaccharide on the lactone content of Atractylodes macrocephala
(1) Design of field experiment
The experiment was set up with 7 treatments: preparing aqueous solutions of 10mg/ml polysaccharide medicament (ETH), 2mg/ml polysaccharide medicament (ETM) and 0.4mg/ml polysaccharide medicament (ETL) respectively, and adopting two methods of spraying on leaf surfaces (ETH leaves, ETM leaves and ETL leaves) and irrigating roots (ETH roots, ETM roots and ETL roots) respectively; clear water blank Control (CK). Each treatment was repeated 3 times.
(2) Experimental sample Collection
The test fields are located in a Deqing test base of Zhejiang agriculture and forestry university and a Pingshan test base of Zhejiang agriculture and forestry university, and the test time is 12 months in 2013 to 11 months in 2014. And sowing the soaked atractylodes macrocephala seeds in a test field in 12 months in 2012, and manually weeding and manually picking buds in the test period without using any other herbicide or bactericide. Spraying the mixture once every 10 days from 5 months in 2014 for 3 times, and taking sterile water as a control. Respectively collecting samples of an experimental base in 2014 10 months in the collection period of the bighead atractylodes rhizome, randomly collecting 5 strains in each treatment, drying at 50 ℃ after struggling, and storing in a refrigerator for later use.
(3) Determination of autoproteolactone components in a sample
i chromatographic conditions
A chromatographic column: sun Fire C18(250 mm. times.4.6 mm, 5 μm); mobile phase methanol (A) and water (B), gradient elution program is 0-14min, 73% -89% A; 14-15min, 89% -95% A; 15-22min, 95% -100% A; 22-26min, 100% A. The volume flow is 1mL/min, the sample injection amount is 20 mu L, the column temperature is 30 ℃, the detection wavelength of atractylenolide I, III is 220nm, and the detection wavelength of atractylenolide II is 276 nm.
The measurement of the Atractylodis rhizoma samples and standards was carried out according to this procedure.
ii preparation of test solution of Atractylodes macrocephala Koidz
Slicing the prepared rhizoma Atractylodis Macrocephalae sample, pulverizing, sieving with 200 mesh sieve to obtain rhizoma Atractylodis Macrocephalae powder, precisely weighing 0.5g, placing in a 10ml volumetric flask, adding 8ml methanol, ultrasonically extracting for 30 minutes, standing, diluting to 10ml with methanol, shaking, and filtering with organic microporous membrane (0.45 μm) to obtain sample solution.
(4) Data processing and analysis
The data were analyzed using SPSS13.0 software.
(5) Results and analysis
As shown in figure 1, the contents of atractylenolide I, II and III in the Atractylodes macrocephala koidz are obviously improved compared with the control group after the application of the chrysanthemum indicum polysaccharide water solution. Table 1 shows that the content of atractylenolide in the treated group of the polysaccharide of wild chrysanthemum in the plain and the test field of Deqing are higher than that in the control group. Wherein the ETH leaf, ETH root and ETL root groups achieved significantly different levels. The high-dosage combination adopting root irrigation treatment and foliage spraying has the best effect.
TABLE 1 Effect of Chrysanthemum indicum polysaccharide on lactone components in Atractylodes macrocephala
Note: all data are the mean of 3 replicates ± sem, with significant differences at the level of 0.05 for different lower case letters and at the level of 0.01 for different upper case letters after the same column of data
The following examples can all achieve the effects of the above experimental examples
Detailed Description
Example 1: method for extracting chrysanthemum indicum polysaccharide
(1) Collecting flos Chrysanthemi Indici (C.boreale Mak.) and oven drying, pulverizing, sieving with 100 mesh sieve, and weighing 1 kg;
(2) respectively adding petroleum ether with volume amount of 6-8 times of the mass of the wild chrysanthemum powder into the wild chrysanthemum powder, refluxing for 1-2 times, 1-2 hours each time, taking out, filtering, heating the filter residue in water bath to volatilize the residual petroleum ether, and obtaining the decolorized wild chrysanthemum powder;
(3) adding pure water with a volume amount 18-20 times of the mass of the wild chrysanthemum powder into the decolorized wild chrysanthemum powder respectively, decocting for 2-3 times, each time for 2-3 hours, taking juice, removing residues, combining filtrates, and filtering to obtain a wild chrysanthemum aqueous extract solution;
(4) concentrating under reduced pressure at low temperature to obtain water extract solution with specific gravity of 1.15-1.25;
(5) slowly adding 90-95% ethanol 6-8 times of the volume of the concentrated water extract solution of wild chrysanthemum, and standing overnight at 4 ℃;
(6) after the precipitate is separated out, centrifuging the mixed solution at 3000-4500rpm for 12-15min, taking the precipitate, and drying the precipitate by using a freeze-drying method to obtain the crude chrysanthemum polysaccharide;
(7) adding distilled water with the volume amount of 75 times of the mass of the crude polysaccharide into the wild chrysanthemum crude polysaccharide, and adding trichloroacetic acid with the volume equal to that of the distilled water: and (3) severely shaking n-butanol (the volume ratio is 1: 8-10) for 20min, standing, layering, taking down clear liquid, removing the n-butanol layer at the upper layer and the impure protein at the middle layer, adding 2NNaOH to neutrality, evaporating to dryness, repeating the step (7) for 8 times, removing glycoprotein to obtain 26.76g of pure wild chrysanthemum polysaccharide, and determining the purity of the wild chrysanthemum polysaccharide to be 98% by adopting a sulfuric acid-phenol method for later use.
Example 2: preparation of aqueous formulations
Taking the chrysanthemum indicum polysaccharide extracted in the example 1, adding a proper amount of water to prepare an aqueous solution containing 100g/L of chrysanthemum indicum polysaccharide, shaking up, sealing and storing. The atractylenolide is sprayed on leaf surfaces in the seedling stage of the growth of the atractylenolide, and compared with a control group, the atractylenolide content can be obviously improved.
Example 3: application of powder
The chrysanthemum indicum polysaccharide (with the purity of 98%) is prepared according to the method of example 1, dried, processed into fine powder and sealed for storage. When the composition is used temporarily, the composition is prepared into an aqueous solution containing 50g/L of chrysanthemum indicum polysaccharide, and the aqueous solution is sprayed on the leaves of the largehead atractylodes rhizome, so that the content of the largehead atractylodes rhizome lactone components can be obviously improved compared with that of a control group.
Example 4: application of aqueous solution
Repeating the steps (1) to (6) of the example 1 to obtain the wild chrysanthemum crude polysaccharide, adding distilled water with the volume amount of 80 times of the mass of the crude polysaccharide into the wild chrysanthemum crude polysaccharide, and adding trichloroacetic acid with the volume equal to that of the distilled water: and (2) severely shaking n-butanol (the volume ratio is 1: 8-10) for 25min, standing, layering, taking down clear liquid, removing the n-butanol layer at the upper layer and the impure protein at the middle layer, adding 2NNaOH to neutrality, evaporating to dryness, repeating the operation for 4 times, removing glycoprotein to obtain 38g of wild chrysanthemum polysaccharide, and determining the purity of the wild chrysanthemum polysaccharide to be 50% by adopting a sulfuric acid-phenol method. Adding distilled water to obtain a solution containing 1g/L of flos Chrysanthemi Indici polysaccharide, shaking, and storing. In the seedling stage of the growth of the largehead atractylodes rhizome, the roots are irrigated, and compared with a control group, the result can obviously improve the content of atractylenolide in the largehead atractylodes rhizome.
Example 5 preparation of wild Chrysanthemum polysaccharide
(1) Collecting the whole grass of coreopsis tinctoria (C.lavandula efolium (Fisch.) Mak.), drying in an oven, pulverizing, sieving with 80 mesh sieve, and weighing 2kg for use;
(2) respectively adding petroleum ether with volume amount 6-8 times of the mass of the powder into the above coreopsis tinctoria powder, refluxing for 1-2 times, each for 1-2 hr, taking out, filtering, heating the filter residue in water bath to volatilize the residual petroleum ether, and obtaining decolorized coreopsis tinctoria powder;
(3) adding pure water with a volume amount of 20 times of the mass of the powder into the decolorized coreopsis tinctoria powder, decocting for 2-3 times, each time for 2-3 hours, collecting the juice, removing residues, combining the filtrates, and filtering to obtain a water extract solution of coreopsis tinctoria;
(4) concentrating under reduced pressure at low temperature to obtain water extract solution with specific gravity of 1.10-1.30:
(5) slowly adding 90-95% ethanol 9 times of the volume of the concentrated water extract solution of the coreopsis tinctoria, and standing overnight at 4 ℃;
(6) after the precipitate is separated out, centrifuging the mixed solution at 3000-4500rpm for 12-15min, taking the precipitate, and drying the precipitate by using a freeze-drying method to obtain the crude chrysanthemum polysaccharide;
(7) adding distilled water with the volume amount 50 times of the mass of the crude polysaccharide into the wild chrysanthemum crude polysaccharide, and adding trichloroacetic acid with the volume equal to that of the distilled water: and (3) severely shaking n-butanol (the volume ratio is 1: 8-10) for 20min, standing, layering, taking down clear liquid, removing the n-butanol layer at the upper layer and the impure protein at the middle layer, adding 2NNaOH to neutrality, evaporating to dryness, repeating the step (7) for 6 times, removing glycoprotein to obtain 26.76g of pure coreopsis tinctoria polysaccharide, and determining the purity of the coreopsis tinctoria polysaccharide to be 59% by adopting a sulfuric acid-phenol method for later use.
Example 6
Taking the chrysanthemum indicum polysaccharide extracted in the example 6, adding a proper amount of water to prepare a solution containing 90g/L chrysanthemum indicum polysaccharide, shaking up, sealing and storing. The atractylenolide is sprayed on leaf surfaces in the seedling stage of the growth of the atractylenolide, and compared with a control group, the atractylenolide content can be obviously improved.
It should also be noted that: the applicant has tested. The inducer prepared by taking the chrysanthemum indicum polysaccharide as the inducer shows that the significant accumulation of the atractylenolide component in the largehead atractylodes rhizome is induced, and is related to the application dosage and the application method, so the lower limit value of the application concentration can be reduced to 0.1g/L containing the chrysanthemum indicum polysaccharide, and the upper limit value of the application concentration can be adjusted up according to the actual situation.
Claims (5)
1. The application of chrysanthemum indicum polysaccharide in promoting the synthesis of secondary metabolites is characterized in that the secondary metabolites are atractylenolide in atractylenolide.
2. Use of chrysanthemum indicum polysaccharide according to claim 1 for promoting the synthesis of secondary metabolites, wherein said chrysanthemum indicum polysaccharide is extracted from the rhizomes and flowers of chrysanthemum indicum belonging to the family of the Compositae.
3. Use of chrysanthemum indicum polysaccharide to promote the synthesis of secondary metabolites according to claim 1, wherein it is applied as top dressing or base fertilizer by seed soaking or spraying treatment or added to other fertilizers as an inducer for promoting the accumulation of atractylenolide components of plants in different seedling stages.
4. Use of chrysanthemum indicum polysaccharide in promoting the synthesis of secondary metabolites according to any one of claims 1 to 3, wherein the chrysanthemum indicum polysaccharide is supplemented with common pesticide auxiliary agents to prepare an inducer, and the inducer is prepared into one of water aqua, powder, granules or water emulsion.
5. The use of chrysanthemum indicum polysaccharide in promoting the synthesis of secondary metabolites according to claim 1, wherein the chrysanthemum indicum polysaccharide is extracted from rhizomes or flowers of chrysanthemum indicum belonging to the family Compositae, and the method comprises the following steps:
step 1: collecting buds or stems of wild chrysanthemum, drying in an oven, pulverizing, and sieving with 80-100 mesh sieve;
step 2: taking the wild chrysanthemum powder, and obtaining wild chrysanthemum crude polysaccharide by adopting a water-alcohol precipitation method;
and step 3: adding distilled water with the volume amount of 75-80 times of the mass of the crude polysaccharide into the wild chrysanthemum crude polysaccharide, adding a mixed solution of trichloroacetic acid and n-butyl alcohol with the volume equal to that of the distilled water, wherein the volume ratio of the trichloroacetic acid to the n-butyl alcohol is 1: 8-10, violently shaking for 20-25min, standing, layering, taking down clear liquid, removing an upper n-butyl alcohol layer and a middle layer hybrid protein, adding 2N NaOH to be neutral, and evaporating to dryness;
and 4, step 4: repeating the step 3, removing glycoprotein to obtain flos Chrysanthemi Indici polysaccharide with certain purity, drying, and sealing.
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| 野菊茎叶及其根际土壤浸提液对白绢病菌与白术种子萌发的化感效应;李倩 等;《广东农业科学》;20140210(第3期);第83-87页 * |
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