CN113234110B - Method for preparing lobetyolin, atractyloide III and syringin from radix codonopsis based on PRE-HPLC - Google Patents
Method for preparing lobetyolin, atractyloide III and syringin from radix codonopsis based on PRE-HPLC Download PDFInfo
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- CN113234110B CN113234110B CN202110512573.2A CN202110512573A CN113234110B CN 113234110 B CN113234110 B CN 113234110B CN 202110512573 A CN202110512573 A CN 202110512573A CN 113234110 B CN113234110 B CN 113234110B
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Abstract
The invention discloses a method for preparing lobetyolin, atractyloide III and syringin from codonopsis pilosula based on PRE-HPLC, which comprises the following steps: (1) single medicine extraction: pulverizing the codonopsis pilosula, adding 12-15 times of ethanol solution, carrying out ultrasonic extraction for 2-3 times at 500W under 400-60 min each time, carrying out rough filtration after extraction is finished, and combining filtrates to obtain a codonopsis pilosula extracting solution; (2) concentrating and drying: vacuum concentrating the filtered extractive solution under reduced pressure, and drying; (3) preparation and purification: ultrasonically dissolving the dried extract, filtering with 0.22 μm microporous membrane, and preparing liquid phase; (4) and (3) fraction collection: collecting lobetyolin, atractyloide III and syringin fractions respectively; (5) concentrating and drying: the collected fractions were concentrated under reduced pressure and dried. The method has the characteristics of convenience in operation, good reproducibility, short period, simultaneous preparation of 3 monomer components and the like, and the prepared monomer components have the characteristic of high purity.
Description
Technical Field
The invention relates to the technical field of chemical component separation and purification, and particularly relates to a method for quickly separating and preparing lobetyolin, atractyloide III and syringin from a codonopsis pilosula extract based on high-efficiency preparative liquid chromatography.
Background
Dangshen, the dried root and rhizome of Codonopsis pilosula (Franch.)) and Codonopsis pilosula (Codonopsis pilosula Nannf. var. modesta (Nannf.) l.t.shen) or Codonopsis pilosula (Codonopsis tangshen (tandshen Oliv.) belonging to the family of platycodiaceae, the name of dangshen was originally found in "materia medica", namely: "according to ancient herbal clouds: shen xu Shang Dang is good. All the herbs in all the generations are recorded and collected, and are common Chinese herbal medicines collected in Chinese pharmacopoeia. Collected in autumn, washed and dried in the sun. This herb is sweet in flavor and neutral in nature. Has effects in invigorating spleen, replenishing qi, quenching thirst, invigorating spleen, tonifying lung, nourishing blood, and promoting salivation. Can be used for treating deficiency of spleen-lung qi, anorexia, listlessness, cough, asthma, deficiency of qi and blood, sallow complexion, palpitation, short breath, thirst due to body fluid consumption, and internal heat. Laziness in speaking, weakness of limbs, poor appetite, qi deficiency, deficiency of both qi and fluid, deficiency of both qi and blood, and sallow complexion due to blood deficiency.
The codonopsis pilosula has the following effects: 1. sedation and antipyresis: can improve memory, relieve convulsion and relieve fever. 2. The function of reducing blood pressure: can dilate peripheral blood vessel. Increase blood flow, improve cardiac function and protect vascular endothelium. 3. The hypoglycemic effect: against insulin hypoglycemia. 4. Enhancing the hematopoietic function: it has effects in increasing erythrocyte and hemoglobin, reducing leukocyte, and reducing blood lipid. 5. Spasmolysis: can inhibit isolated gastrointestinal stress. Thereby promoting the healing of gastric mucosa ulcer. 6. Liver protection: has effects of protecting liver against liver injury, and promoting absorption of chylomicron. 7. The antidiuretic effect is as follows: can reduce the excretion of urine of rats and cause the contraction of isolated uterus of white rats. 8. And (3) fatigue resistance: can improve the swimming ability of animals and resist fatigue. The codonopsis pilosula can increase the animal weight by 23%. 9. The anti-tumor effect is as follows: the decoction and cyclophosphamide have the effects of inhibiting tumor and reducing metastasis.
Researches show that the codonopsis pilosula contains complex and various chemical components, including saccharides, glycosides, polyacetylenes, alkaloids, terpenes, phenylpropanoids, steroids, flavonoids, lignans, acids and the like, wherein 8 components and compounds obtained by separating the components have obvious pharmacological activity. The codonopsis pilosula active ingredient has various active ingredients such as regulating organism immunity, resisting oxidation, protecting gastrointestinal mucosa, resisting ulcer, inhibiting bacteria, resisting cancer, delaying aging and the like, and the research on the codonopsis pilosula active ingredient is mainly focused on polysaccharide, alkynedione glycosides, alkaloid and the like at present.
The lobetyolin, atractyloide III and syringin are a plurality of main functional components in the codonopsis pilosula. According to pharmacological research, the lobetyolin not only has the effects of resisting cancers, bacteria, inflammation and the like, but also has a good protection effect on gastric mucosa injury; the atractyloide III has obvious anti-inflammatory and anti-tumor functions, has a function of adjusting gastrointestinal tract functions and can promote the absorption of nutrient substances; syringin is a potent anti-hepatotoxin drug, which restores the enzymatic activity of microsomal enzyme system and inhibits lipid peroxidation to promote metabolism of hepatotoxin and improve liver function for normalization. The pharmacological action of the codonopsis pilosula total alkaloids is mainly to the nervous system, and the codonopsis pilosula total alkaloids enhance Nerve Growth Factor (NGF) to induce PC12 cells to extend a large number of protrusions by amplifying certain upstream steps of a mitogen activated protein kinase dependent signal pathway; the codonopsis pilosula total alkali can obviously improve the memory injury effect of mice caused by scopolamine.
At present, the extraction and separation modes for obtaining the functional components from the codonopsis pilosula medicinal materials are as follows: generally, countercurrent extraction, ultrasonic extraction, extraction and the like are adopted, or target components are modified and modified in a certain mode and then are further separated, and in the later stage, separation and purification are further carried out by means of a combination of a chromatographic column and medium-low pressure preparation, and monomer compounds with higher purity are obtained by means of separation and purification of supercritical fluid extraction and the like, but most of the methods are complicated in process steps, large in reagent consumption amount and relatively low in monomer yield, only 1-2 monomer components can be obtained at a time, and if multiple monomer components are required to be obtained, different conditions or methods are required for carrying out multiple operations, so that the method generally has the defects of longer period, complex process, lower efficiency, higher cost, single separation component and the like.
Disclosure of Invention
The invention aims to provide a method for separating and preparing high-purity lobetyolin, atractyloide III and syringin monomers from codonopsis pilosula, which is convenient, efficient, simple in process and easy to popularize.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing lobetyolin, atractyloide III and syringin from radix Codonopsis based on PRE-HPLC comprises the following steps:
(1) Single medicine extraction: pulverizing the codonopsis pilosula, adding 12-15 times of ethanol solution, carrying out ultrasonic extraction for 2-3 times at 500W under 400-60 min each time, carrying out rough filtration after extraction is finished, and combining filtrates to obtain a codonopsis pilosula extracting solution;
(2) concentrating and drying: vacuum concentrating the filtered extractive solution under reduced pressure, and drying;
(3) preparation and purification: adding 100-200 times of 70-80% ethanol into the dried extract, ultrasonically dissolving, filtering with a 0.22 μm microporous filter membrane, and preparing a liquid phase;
(4) and (3) fraction collection: collecting with fraction collector, collecting the fraction of radix Codonopsis alkynoside in 12-14min, collecting the fraction of syringin in 19-21min, and collecting the fraction of atractylenolide III in 37-38 min;
(5) concentrating and drying: the collected fractions were concentrated under reduced pressure and dried.
Preferably, the concentration of the ethanol solution in the step (1) is 70-80%.
Preferably, the drying temperature in the step (2) is 40-50 ℃.
Preferably, the chromatographic column in the step (3) is: the chromatographic column (Amethyl C18-H,21.2 × 250mm,10 μm) has the following specific chromatographic conditions: the mobile phase is acetonitrile: and (3) carrying out gradient elution on water according to the following table, wherein the flow rate is 8-15 mL/min, the detection wavelength is 214-225 nm, the detection wavelength is 267-275 nm, and the column temperature is 25-35 ℃.
Preferably, the gradient elution conditions are:
preferably, the drying temperature in the step (5) is 40-50 ℃.
Compared with the prior art, the invention has the following advantages:
firstly, the process is convenient and fast: most of the preparation methods of the active ingredients of the dangshen mainly carry out adsorption and impurity removal in a mode of resin chromatographic column after solvent countercurrent extraction, or gradually purify by other solvents so as to achieve the purpose of separating and purifying the active ingredients in the dangshen; the method is simple in process and clear in flow, and greatly reduces the separation and purification period.
Secondly, separating efficiently: compared with other methods for preparing the codonopsis monomer, the method for preparing the codonopsis monomer only needs to dissolve the codonopsis extract by means of a mobile phase, then pass through a membrane, detect and separate on a preparation liquid phase, can simultaneously separate and purify three monomer components, has high separation efficiency, can achieve the effect of separating three components at a time, and greatly improves the separation efficiency compared with methods for repeatedly utilizing resin, reagent and the like in other methods.
Thirdly, the product purity is high: after pretreatment, the codonopsis pilosula extract is separated and purified by a preparative liquid chromatograph, so that monomer components with the product purity of more than 95% can be obtained, and the codonopsis pilosula extract is low in impurity and high in product purity.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
Example 1
A method for preparing lobetyolin, atractyloide III and syringin from radix Codonopsis based on PRE-HPLC comprises the following steps:
(1) extracting medicinal materials: pulverizing 100g radix Codonopsis, adding 12 times volume of 65% ethanol solution, each time 1200mL, ultrasonic extracting at 400W for 60min for 3 times, coarse filtering after extraction, collecting, and mixing filtrates.
(2) Concentrating and drying: concentrating the above extractive solution at 45 deg.C under reduced pressure, and drying to obtain radix Codonopsis crude extract 9.9 g.
(3) Preparation and purification: adding a proper amount of dried codonopsis pilosula extract into 100 times of 70% ethanol solution for ultrasonic dissolution, then filtering the mixture through a 0.22-micron microporous filter membrane to obtain a sample to be prepared, and finally separating and preparing the sample through a preparation liquid phase, wherein the chromatographic conditions are as follows: the chromatographic column is a C18 reversed phase chromatographic column, and the mobile phase is methanol: 0.1% formic acid was eluted according to the following gradient at a flow rate of 8mL/min at a detection wavelength of 214nm and 267nm at a column temperature of 25 ℃.
(4) Fraction collection: collecting fractions in 12-13min as lobetyolin, collecting fractions in 19-20min as syringin, and collecting fractions in 37-38min as atractyloide III;
(5) concentrating and drying: the fractions collected above were concentrated under reduced pressure and dried under vacuum (temperature 40 ℃ C.), and the purities of the fractions were determined to be 97.6%, 96.1% and 96.3%, respectively (corresponding to the order of the fractions in (4), the same applies hereinafter).
Example 2
A method for preparing lobetyolin, atractyloide III and syringin from radix Codonopsis based on PRE-HPLC comprises the following steps:
(1) extracting medicinal materials: taking 100g of radix codonopsitis medicinal material, crushing, adding 70% ethanol solution with 14 times volume of the medicinal material, performing ultrasonic extraction for 45min at 450W for 1400mL each time, extracting for 3 times in total, performing rough filtration after extraction is finished, collecting and combining filtrates.
(2) Concentrating and drying: concentrating the above extractive solution at 60 deg.C under reduced pressure, and drying to obtain radix Codonopsis crude extract 10.4 g.
(3) Preparation and purification: taking a proper amount of dried codonopsis pilosula extract, adding a 65% ethanol solution in an amount which is 150 times that of the dried codonopsis pilosula extract, carrying out ultrasonic dissolution, then, obtaining a sample to be prepared after passing through a 0.22-micron microporous filter membrane, and finally, carrying out separation preparation through a preparation liquid phase, wherein the chromatographic conditions are as follows: the chromatographic column is a C18 reversed phase chromatographic column, and the mobile phase is acetonitrile: the water was eluted in a gradient manner at a flow rate of 15mL/min, at a detection wavelength of 220nm and 270nm, at a column temperature of 30 ℃.
(4) And (3) fraction collection: collecting fractions in 13-14min as lobetyolin, collecting fractions in 20-21min as syringin, and collecting fractions in 37-38min as atractyloide III;
(5) concentrating and drying: the fractions collected above were concentrated under reduced pressure and dried under vacuum (temperature 45 ℃ C.), and the purity of each fraction was determined to be 96.2%, 96.0% and 96.7%, respectively.
Example 3
A method for preparing lobetyolin, atractyloide III and syringin from radix Codonopsis based on PRE-HPLC comprises the following steps:
(1) extracting medicinal materials: taking 100g of radix codonopsitis medicinal material, crushing, adding 75% ethanol solution with 15 times volume of the medicinal material, performing ultrasonic extraction for 60min at 500W for 3 times each time by 1500mL, roughly filtering after the extraction is finished, collecting and combining filtrate.
(2) Concentrating and drying: concentrating the above extractive solution at 50 deg.C under reduced pressure, and drying to obtain radix Codonopsis crude extract 10.7 g.
(3) Preparation and purification: adding a proper amount of dried codonopsis pilosula extract into a 75% ethanol solution 200 times the amount of the dried codonopsis pilosula extract, performing ultrasonic dissolution, then passing through a 0.22-micron microporous filter membrane to obtain a sample to be prepared, and finally performing separation preparation through a preparation liquid phase, wherein the chromatographic conditions are as follows: the chromatographic column is a C18 reversed phase chromatographic column, and the mobile phase is acetonitrile: the water was eluted in a gradient manner at a flow rate of 10mL/min, at a detection wavelength of 225nm and 275nm, at a column temperature of 30 ℃.
| Time (min) | Acetonitrile (%) | Water (%) |
| 0 | 7 | 93 |
| 10 | 17 | 83 |
| 20 | 27 | 73 |
| 30 | 37 | 63 |
| 40 | 47 | 53 |
| 45 | 7 | 93 |
(4) And (3) fraction collection: collecting the fraction of radix Codonopsis alkyne glycoside at 12.5-13.5min, collecting the fraction of syringin at 19.5-20.5min, and collecting the fraction of atractyloide III at 37-38 min;
(5) concentrating and drying: the fractions collected above were concentrated under reduced pressure and dried under vacuum (temperature 50 ℃ C.), and the purity of each fraction was measured to be 95.7%, 96.1% and 95.3%, respectively.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A method for preparing lobetyolin, atractyloide III and syringin from radix codonopsis based on PRE-HPLC is characterized by comprising the following steps:
(1) single medicine extraction: pulverizing the codonopsis pilosula, adding 12-15 times of ethanol solution, carrying out ultrasonic extraction for 2-3 times at 500W under 400-60 min each time, carrying out rough filtration after extraction is finished, and combining filtrates to obtain a codonopsis pilosula extracting solution;
(2) concentrating and drying: vacuum concentrating the coarse filtered extractive solution under reduced pressure, and drying;
(3) Preparation and purification: adding 100-200 times of 70-80% ethanol into the dried extract, ultrasonically dissolving, filtering with a 0.22 mu m microporous filter membrane, and separating with a preparation liquid phase; the chromatographic column adopted in the separation process is an Amethyl C18-H chromatographic column, the specification of the chromatographic column is 21.2 multiplied by 250mm, and the particle size is 10 mu m; the specific chromatographic conditions are as follows: the mobile phase is acetonitrile and water, gradient elution is carried out, the flow rate is 8-15 mL/min, the detection wavelength is 214-225 nm and 267-275 nm double waves, and the column temperature is 25-35 ℃;
the conditions for gradient elution were as follows:
(4) fraction collection: collecting with fraction collector, collecting radix Codonopsis alkyne glycoside in 12-14min, collecting syringin in 19-21min, and collecting atractyloide III in 37-38 min;
(5) concentrating and drying: the collected fractions were concentrated under reduced pressure and dried.
2. The method according to claim 1, wherein the concentration of the ethanol solution in the step (1) is 70% to 80%.
3. The method according to claim 1, wherein the drying temperature in the step (2) is 40 to 50 ℃.
4. The method according to claim 1, wherein the drying temperature in the step (5) is 40-50 ℃.
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| CN101177440B (en) * | 2007-10-12 | 2011-01-12 | 成都普思生物科技有限公司 | Method for rapid purification of codonopsis pilosula lobetyolin monomer from codonopsis pilosula medicinal materials |
| CN106749444A (en) * | 2016-11-23 | 2017-05-31 | 甘肃中医药大学 | A kind of separation method of Lobetyolin in Codonopsis and atractylenoide Ⅲ |
| CN109839459A (en) * | 2019-02-22 | 2019-06-04 | 遵义医学院 | A kind of Radix Codonopsis multi-components content determination method based on the superposition of HPLC multi-wavelength |
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