CN106699561B - Method for quickly obtaining chlorogenic acid crude extract - Google Patents
Method for quickly obtaining chlorogenic acid crude extract Download PDFInfo
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- CN106699561B CN106699561B CN201710025255.7A CN201710025255A CN106699561B CN 106699561 B CN106699561 B CN 106699561B CN 201710025255 A CN201710025255 A CN 201710025255A CN 106699561 B CN106699561 B CN 106699561B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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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
- A01H4/008—Methods for regeneration to complete plants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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Abstract
The invention discloses a method for quickly obtaining a crude extract of chlorogenic acid, which comprises the steps of preparing a culture medium and sterilizing the culture medium; sterilizing roots and stems of the Wusuli pyrrosia leaf; inoculating the explant; culturing rhizome callus; induction of rhizome callus; proliferation of callus of rhizome; extracting effective components from the calli of the Wusulliwei. Can quickly extract the active ingredients of the Wusuliwavawei, and solves the problem of insufficient development resources.
Description
Technical Field
The invention belongs to the technical field of anti-inflammatory drug preparation, and relates to a method for quickly obtaining a crude extract of chlorogenic acid.
Background
Wusuli warvir [ Lepisorus ussuriensis (Regel et Maack) Ching ] is a fern and dragon orthopaedics warvir plant, namely Shicha, Tucha, Huanyang grass, Dashiwei and the like. In China, it is distributed in Anhui, Henan, Shandong, Hebei, Liaoning, Jilin, Heilongjiang. The data record that the main active component of the Wusuliwavawei is chlorogenic acid, has the effects of clearing heat and removing toxicity, promoting urination, relieving cough, stopping bleeding and the like, and can be used for treating dysuresia, stranguria with urine, lung heat cough, asthma, sore throat, pyocutaneous disease and pyogenic infections, rheumatism pain and incised wound bleeding. Wusulevir grows on mountain trunks or rocks at an altitude of about 800m, and plants are short (10 cm in height). The plant is only applied in folks in the mountainous areas of the producing areas at present to treat various inflammatory pains with good curative effect. However, the distribution area is narrow, the requirement on the growth environment is strict, the plants are short and small, and the yield is not large, so that the method for quickly obtaining the effective components by adopting the tissue culture method is particularly important for ensuring the development, utilization and research of the effective components.
Disclosure of Invention
In order to achieve the purpose, the invention provides a method for quickly obtaining a crude extract of chlorogenic acid, which can quickly extract the active ingredients of the Wusuleviwei and solve the problem of insufficient development resources.
The technical scheme adopted by the invention is that the method for quickly obtaining the crude extract of the chlorogenic acid specifically comprises the following steps:
step 1, preparing a culture medium and sterilizing the culture medium;
step 3, inoculating the explants;
a. induction of rhizome callus;
1/2MS is used as a minimal medium, and 0.3 mg.L of 6-BA is added respectively-1、2,4-D 3mg·L-1、IBA 1mg·L-1As a growth regulator of the culture medium, the agar content of the culture medium is 7.0gL-1The content of sucrose is 30.0 g.L-1The pH is 5.8; inoculating the small rhizome blocks cut into 1cm on a culture medium, placing in a constant temperature incubator, and culturing at 25 + -2 deg.C in dark for 30 days;
b. proliferation of callus of rhizome;
MS is taken as a basic culture medium, and 0.3 mg.L of 6-BA is added-1、2,4-D 3mg·L-1、IBA 1mg·L-1The agar content of the culture medium as a growth regulator is 7.0 g.L-1The content of sucrose is 30.0 g.L-1The pH value is 5.8; cutting the callus into small pieces of 0.5cm × 0.5cm, inoculating on culture medium, placing in constant temperature incubator, dark culturing at 25 + -2 deg.C for 25 days;
step 5, extracting effective components from the Wusuliwavawei callus;
grinding Calvatamia ussuriensis root and stem callus, adding 70% ethanol, ultrasonic extracting for 1h with a material-liquid ratio of 1:20, concentrating, extracting with n-butanol, and concentrating to obtain extract.
Further, the step 1 is performed according to the following steps:
1/2MS is used as a minimal medium, and 0.5 mg.L of 6-BA is added-1、2,4-D 3mg·L-1、IBA 1mg·L-1The agar content of the culture medium as a growth regulator is 7.0 g.L-1The content of sucrose is 30.0 g.L-1Adding double distilled water to dissolve, adjusting the pH value of the culture medium to 5.8 +/-0.2 by using NaOH and HCl solutions, putting the culture medium into a heating sleeve to heat, and continuously stirring by using a glass rod to completely dissolve the culture medium; sealing the bottle mouth with a sealing film, placing into a vertical pressure steam sterilizer, sterilizing at 101kpa for 20min, then disconnecting the power supply, opening a deflation valve, taking out the culture medium after the air pressure is reduced to the standard atmospheric pressure, and placing the sterilized culture medium in an aseptic operation platform; the culture medium is poured into the culture dish while the culture medium is hot to prevent the culture medium from solidifying, the thickness of the culture medium poured into the culture dish is 1.5cm, and the culture medium is cooled for standby after the culture medium in the culture dish is solidified.
Furthermore, the sterile operating table needs to be irradiated by an ultraviolet lamp for 30min before use, so that the sterile operating table is in a sterile state.
Further, the step 2 is performed according to the following steps:
washing rhizome of WUSURIEWEI with tap water for 10min, placing into a beaker, introducing into a sterile inoculation chamber, adding 75% alcohol for sterilizing for 30s, washing with sterile water for 1 time, and adding 0.1% HgCl2Soaking and sterilizing the rootstock with the solution for 5min, washing with sterile water for 5-6 times, placing the completely sterilized explant in sterile filter paper, sucking off water on the surface of the explant, and cutting the rootstock into 1cm segmentsAnd (5) carrying out sterile inoculation.
Further, the step 3 is performed according to the following steps:
and (3) carrying out alcohol lamp disinfection treatment on all the tools to be used, fully contacting the rhizome explants on a culture medium, covering the rhizome explants with a cover, sealing the rhizome explants with a sealing film, culturing the explant-inoculated culture dish at 25 +/-2 ℃, and carrying out static observation.
The invention has the beneficial effect that the traditional method for using the medicinal material is to pick the overground part of the medicinal material and make tea or wine for drinking. The picking is time-consuming and labor-consuming and the yield is low because the growing environment is limited and the picking can be carried out only once every year. The method is not limited by geographical position, time and the like, and can reduce manpower and material resources and the like. Pharmacological experiments also show that the anti-inflammatory pharmacological action of the crude extract of the callus active components of the Wusuliwavawei with a certain dosage has no obvious difference with that of the wild Wusuliwavawei.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Figure 1 is a graph of the standard curve of chlorogenic acid.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for rapidly obtaining a crude chlorogenic acid extract specifically comprises the following steps:
step 1, preparation and sterilization of culture medium
Preparation of a culture medium: 1/2MS is used as a minimal medium, and 0.3 mg.L of 6-BA is added-1、2,4-D 3mg·L-1、IBA1mg·L-1The agar content of the culture medium as a growth regulator is 7.0 g.L-1The content of sucrose is 30.0 g.L-1Adding double distilled water to dissolve, adjusting pH of the culture medium to 5.8 + -0.2 with NaOH and HCl solution, heating in a heating jacket, and stirring with a glass rod to dissolve the culture medium completely. Sealing the bottle mouth with sealing film, placing into a vertical pressure steam sterilizer, and sterilizing at 101kpa for 20 min. And (3) after 20min, disconnecting the power supply, opening a deflation valve, taking out the culture medium after the air pressure is reduced to the standard atmospheric pressure, and placing the sterilized culture medium in a sterile operating platform. The sterile operation table is irradiated by ultraviolet lamp for 30min before use, so that the sterile operation table is in sterile state. The culture medium is poured into the culture dish while the culture medium is still hot, the solidification of the culture medium is prevented, the thickness of the culture medium poured into the culture dish is about 1.5cm, and the culture medium in the culture dish is cooled for standby after solidification.
Washing rhizome of WUSURIEWEI with tap water for 10min, placing into a beaker, introducing into a sterile inoculation chamber, adding 75% alcohol for sterilization for 30s, washing with sterile water for 1 time, and adding 0.1% HgCl2Soaking and disinfecting the rootstocks for 5min by using the solution, washing the rootstocks for 5-6 times by using sterile water, placing the completely disinfected explants in sterile filter paper, sucking water on the surfaces of the explants, and cutting the rootstocks into small sections of about 1cm for sterile inoculation.
Step 3, inoculation of explants
And (3) carrying out alcohol lamp disinfection treatment on all the tools to be used, fully contacting the rhizome explants on a culture medium, covering the rhizome explants with a cover, sealing the rhizome explants with a sealing film, culturing the explant-inoculated culture dish at 25 +/-2 ℃, and carrying out static observation. The above operations are carried out in a sterile manner at all times, so as to prevent bacteria from infecting and influencing the growth of the bacteria during the culture process.
a. Induction of rhizome callus
1/2MS is used as a minimal medium, and 0.3 mg.L of 6-BA is added respectively-1、2,4-D 3mg·L-1、IBA 1mg·L-1As a growth regulator of the culture medium, the agar content of the culture medium is 7.0gL-1The content of sucrose is 30.0 g.L-1The pH value is about 5.8; cutting rhizome into small pieces of about 1cm, inoculating to culture medium, placing in constant temperature incubator, dark culturing at 25 + -2 deg.C for 30 days.
b. Proliferation of rhizome callus
MS is taken as a basic culture medium, and 0.3 mg.L of 6-BA is added-1、2,4-D 3mg·L-1、IBA 1mg·L-1The agar content of the culture medium as a growth regulator is 7.0 g.L-1The content of sucrose is 30.0 g.L-1The pH value is 5.8; cutting the callus into small pieces of about 0.5cm × 0.5cm, inoculating on culture medium, placing in constant temperature incubator, dark culturing at 25 + -2 deg.C for 25 days.
Step 5, extracting effective components from the callus of the Wusuliwavawei
Grinding the callus of the rhizome of the Wusuliwavawei, adding 70% ethanol (the material-liquid ratio is 1:20), ultrasonically extracting for 1h, concentrating, extracting with n-butanol, and concentrating into an extract, namely the Wusuliwavawei active ingredient concentrate.
There are many examples of techniques for obtaining effective components of medicinal plants by tissue culture, and experimental techniques are well established. The callus culture is carried out on the Wusuliwai which is an anti-inflammatory drug with good curative effect, the weight of an explant can be increased by more than 10 times after 25 days, and the explant is cultured in vitro and is not limited by seasons, so that the problem of insufficient development resources is solved, and resource guarantee is provided for developing the Wusuliwai into an anti-inflammatory traditional Chinese medicine in future.
The chlorogenic acid content in the callus of the Wusuliwavawei is measured by an ultraviolet spectrophotometry. The measurement method and the results are as follows:
1. preparation of test solution
Precisely weighing 0.2g of callus, placing the callus in a mortar for grinding, adding distilled water for ultrasonic extraction for 1h, filtering and concentrating, and then using ethanol for volume determination in a 25mL volumetric flask for later use.
2. Preparation of control solution
Precisely weighing chlorogenic acid reference substance 4.77mg, adding ethanol, and dissolving in 10mL volumetric flask to obtain 0.477 mg/mL-1The control solution of (4).
3. Determination of detection wavelength by ultraviolet spectrophotometry
Selecting the wavelength for measuring the content of the chlorogenic acid, obtaining an absorption photometry of a chlorogenic acid standard solution by full-wavelength scanning, and obtaining that the chlorogenic acid has a relatively flat maximum absorption peak at 324nm, so 324nm is selected as the detection wavelength of the chlorogenic acid.
4. Investigation of Linear relationships
Precisely sucking 3.2.2.2 chlorogenic acid standard solutions 0.08, 0.1, 0.15, 0.2, 0.25, and 0.3mL respectively, placing in 10mL volumetric flask, and making into 3.816 μ g/mL-1、4.77μg·mL-1、7.155μg·mL-1、9.54μg·mL-1、11.92μg·mL-1、14.31μg·mL-1The standard solution was used as a spare, water was taken as a blank, absorbance values of 6 samples were measured at 324nm, and a standard curve was drawn with the absorbance (A) as ordinate and the concentration (C) as abscissa.
5. Content determination of chlorogenic acid in callus
a. Examination result of linear relationship
Drawing standard curve with absorbance (A) as ordinate and concentration (C) as abscissa, and drawing obtained chlorogenic acid standard curve chart, as shown in FIG. 1, performing regression analysis to obtain y of 0.050x +0.011, and R20.993(n is 6), and the chlorogenic acid reference substance is 3.816-14.31 mug.mL-1The linear relationship within the range is good.
b. Determination result of chlorogenic acid content in test solution
Performing callus culture according to the optimal orthogonal experimental condition of chlorogenic acid content extracted from Wusuliwavawei callus to obtain chlorogenic acid content of 3.318mg g-1。
Research shows that the content of the above-ground chlorogenic acid in the wild Usuliwavavir is 2.635mg g-1Callus of its rhizomeThe chlorogenic acid content in the tissue is higher than that of wild Wusulevivir.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (1)
1. A method for rapidly obtaining a crude chlorogenic acid extract is characterized by comprising the following steps:
step 1, preparing a culture medium and sterilizing the culture medium; 1/2MS is used as a basic culture medium, 6-BA 0.5 mg.L < -1 >, 2, 4-D3 mg.L < -1 >, IBA 1 mg.L < -1 > are added as culture medium growth regulators, the agar content of the culture medium is 7.0 g.L < -1 >, the sucrose content is 30.0 g.L < -1 >, double distilled water is added for dissolution, the pH value of the culture medium is adjusted to 5.8 +/-0.2 by NaOH and HCl solution, the culture medium is placed into a heating sleeve for heating, and a glass rod is used for continuous stirring to completely dissolve the culture medium; sealing the bottle mouth with a sealing film, placing into a vertical pressure steam sterilizer, sterilizing at 101kpa for 20min, then disconnecting the power supply, opening a deflation valve, taking out the culture medium after the air pressure is reduced to the standard atmospheric pressure, and placing the sterilized culture medium in an aseptic operation platform; pouring the culture medium into a culture dish while the culture medium is hot to prevent the culture medium from solidifying, wherein the thickness of the culture medium poured into the culture dish is 1.5cm, and cooling the culture medium in the culture dish for later use after the culture medium in the culture dish is solidified;
step 2, disinfecting roots and stems of the oriental wormwood;
step 3, inoculating the explants;
step 4, culturing rhizome callus;
a. induction of rhizome callus;
1/2MS is used as a basic culture medium, 6-BA 0.3 mg.L < -1 >, 2, 4-D3 mg.L < -1 > and IBA 1 mg.L < -1 > are respectively added as culture medium growth regulators, the agar content of the culture medium is 7.0gL < -1 >, the sucrose content is 30.0 g.L < -1 >, and the pH is 5.8; inoculating the small rhizome blocks cut into 1cm on a culture medium, placing in a constant temperature incubator, and culturing at 25 + -2 deg.C in dark for 30 days;
b. proliferation of rhizome callus;
MS is taken as a basic culture medium, 6-BA 0.3 mg.L-1, 2, 4-D3 mg.L-1 and IBA 1 mg.L-1 are added as culture medium growth regulators, the agar content of the culture medium is 7.0 g.L-1, the sucrose content is 30.0 g.L-1, and the pH value is 5.8; cutting the callus into small pieces of 0.5cm × 0.5cm, inoculating on culture medium, placing in constant temperature incubator, dark culturing at 25 + -2 deg.C for 25 days;
step 5, extracting effective components from the Wusuliwavawei callus;
grinding Callicarpa kwangsiensis rhizome callus, adding 70% ethanol, performing ultrasonic extraction for 1h, wherein the material-liquid ratio is 1:20, concentrating, extracting with n-butanol, and concentrating into extract;
irradiating the sterile operating table with ultraviolet lamp for 30min before use to keep the sterile operating table in sterile state;
the step 2 is carried out according to the following steps: washing roots and stems of Wusuli pyrrosia lingua with tap water for 10min, putting the roots and stems into a beaker and carrying into a sterile inoculation chamber for later use, pouring 75% alcohol for disinfection for 30s, washing the roots and stems with sterile water for 1 time, soaking and disinfecting the roots and stems with 0.1% HgCl2 solution for 5min, washing the roots and stems with the sterile water for 5-6 times, putting the completely disinfected explants in sterile filter paper, sucking water on the surfaces of the explants, and cutting the roots and stems into small sections of 1cm for sterile inoculation;
the step 3 is carried out according to the following steps: and (3) carrying out alcohol lamp disinfection treatment on all the tools to be used, fully contacting the rhizome explants on a culture medium, covering the rhizome explants with a cover, sealing the rhizome explants with a sealing film, culturing the explant-inoculated culture dish at 25 +/-2 ℃, and carrying out static observation.
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| CN104255522A (en) * | 2014-10-14 | 2015-01-07 | 南京帝道农业科技有限公司 | Rapid propagation method for plant regeneration of pyrrosia sheareri (Bak.) Ching |
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| CN104255522A (en) * | 2014-10-14 | 2015-01-07 | 南京帝道农业科技有限公司 | Rapid propagation method for plant regeneration of pyrrosia sheareri (Bak.) Ching |
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| 乌苏里瓦韦的抗炎活性部位筛选及抗炎活性成分研究;王丽红;《医药卫生科技辑》;20150215;第36-39，61-64页 * |
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