CN103467425A - Method for separating quercetin from oxytropis glabra - Google Patents

Abstract

The invention discloses a method for separating quercetin from oxytropis glabra, and the method comprises the following steps: (1) crushing oxytropis glabra; (2) extracting a total oxytropis glabra extract by using an ultrasonic assistance method; (3) separating oxytropis glabra flavones by using a macroporous resin adsorption method; (4) preparing a coarse quercetin product by virtue of acid hydrolysis; (5) recrystallizing quercetin; (6) detecting the purity of the quercetin. The method disclosed by the invention has the characteristics of simple preparation process, low cost and high product purity, and is suitable for promotion and application.

Description

A method for isolating quercetin from Oxytropis miniflora

technical field

The invention belongs to the technical field of medicine and health products, and relates to a method for isolating quercetin from Oxytropis chinensis.

Background technique

Oxytropis glabra DC is a plant belonging to the Leguminosae genus Oxytropis, widely distributed in Xinjiang. According to the Mongolian Pharmacopoeia, Oxytropis florida is an important raw material of Chinese and Mongolian medicines. It has anesthesia, sedative and analgesic effects, and is mainly used for toothache, joint pain, insomnia, forgetfulness, neurasthenia and skin itching. Such as Oxytropis floret 4.5g, Acanthopanax bark 6g and Lycium barbadensis 9g, decocted in water, can cure joint pain.

Flavonoids are one of the active ingredients in Oxytropis floribunda, which contains quercetin. Quercetin (quercetin), the chemical name is 3,3',4',5,7-pentahydroxyflavone (3,3',4',5,7-pentahydroxyflavone), also known as quercetin, quercetin, Can be used as medicine. It has good expectorant and cough-relieving effects, and has a certain anti-asthma effect. In addition, it has anti-oxidation and free radical scavenging, cardiovascular protection, coronary heart disease prevention, myocardial ischemia-reperfusion injury prevention, anti-inflammation, anti-allergy, anti-virus and sedation, anti-tumor, and cancer prevention. It can be used to treat chronic bronchitis, and also has an auxiliary therapeutic effect on patients with coronary heart disease and hypertension. So far, there is no patent application for the isolation of quercetin from Oxytropis floret.

Contents of the invention

The purpose of the present invention is to overcome the defects of the above-mentioned technology, and provide a method and application for isolating quercetin from Oxytropis florida, which is convenient for operation and has high product purity.

Its specific technical plan is:

A method for isolating quercetin from Oxytropis chinensis, comprising the following steps:

(1) Grinding of Oxytropis florida: Take the above-ground part of Oxytropis florida, dry in the shade, crush, pass through a 20-mesh sieve, and set aside;

(2) Ultrasonic-assisted extraction of the total extract of Oxytropis florida: Weigh the sample of Oxytropis florida, add methanol with a volume fraction of 100% according to the ratio of material ratio 1:15g/mL, and ultrasonically extract for 90min, ultrasonic power 800W, temperature 45°C, After the extraction is completed, filter, the filtrate is concentrated by a rotary evaporator, and dried in a constant temperature drying oven to obtain the total extract of Oxytropis florida;

(3) Separation of Oxytropis flavonoids: fully dissolve the total extract of Oxytropis florida with water; add 1/3 volume of deionized water to the chromatography column, and then deionize the D-101 macroporous adsorption resin with deionized water Transfer to a chromatographic column, add 70% ethanol solution, the volume is twice the volume of the resin, and the flow rate is 1 times the volume of the resin/min, then wash with deionized water until there is no alcohol smell, and then carry out the aqueous solution of the total extract of Oxytropis florida Gradient elution; the best adsorption process conditions for macroporous resin are: pH value 4.0, temperature at room temperature, flow rate 1.0BV/h; elute with 10% to 100% ethanol, collect 50% to 85% ethanol eluate, Concentrated by a rotary evaporator and dried by a freeze dryer to obtain a brown-yellow powder;

(4) Separation of crude quercetin: Weigh about 1.0 g of echinoclavone florets, add 60 mL of hydrolyzate with a volume ratio of V _methanol : V _water : V _{hydrochloric acid} = 35:5:10, and heat in a water bath at 80°C for 90 minutes. After cooling, filter and dry at 60°C to obtain the crude quercetin product in the form of yellow needles;

(5) Recrystallization of quercetin: Take about 1.0 g of the crude quercetin prepared in step (4), add 100 mL of petroleum ether and 20 mL of deionized water, heat and stir at 80°C for 30 min, filter while hot, and deionize with 20 mL of deionized water Wash the precipitate with water for 3 times, dry at 60°C to obtain quercetin; weigh 0.5g of quercetin (1), add 100mL methanol solution, dissolve at 80°C, filter, let the filtrate cool to crystallize, filter to obtain crystals, and use 20mL The precipitate was washed three times with deionized water, and dried at 60°C to obtain quercetin with high purity.

Compared with prior art, the beneficial effect of the present invention is:

The method of the invention has the advantages of simple operation, less consumption of organic solvent, low production cost, stable process, low energy consumption, less pollution and high purity of the obtained product, which is suitable for laboratory and industrial preparation. Through the detection of the purity of quercetin: the purity of the isolated quercetin was detected by the inspection of the residue on ignition, the detection of clarity and the detection of high performance liquid chromatography, and the results showed that the purity of the isolated quercetin was >98%.

Description of drawings

Figure 1 is the HPLC chart of quercetin.

Detailed ways

The technical solution of the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments

Oxytropis floret extract test:

In order to comprehensively investigate the influencing factors of the extraction of Oxytropis flavonoids, using the characteristics of "equilibrium dispersion" of the orthogonal method, ultrasonic time, methanol concentration, and material ratio were selected as the investigation factors, and combined with the experience of multiple extractions, three levels were selected for each factor Arrange experiments, take the yield of flavonoids as an objective index to measure the extraction efficiency, and optimize the best extraction process.

Table 1 Orthogonal experimental design

Table 2 Extraction test results of Oxytropis flavonoids

According to the largest K value in the orthogonal test, the best level of each factor is selected: the concentration of methanol is 100%, the ultrasonic time is 90min, and the ratio of solid to liquid is 1:15.

At a certain temperature and pH, the extract of Oxytropis florida passes through the resin chromatography column at a certain flow rate, and the content of the total flavonoids in the effluent is measured; the flow rate, temperature, and pH of the sample solution are subjected to single-factor experiments to determine the optimum Optimum adsorption process conditions.

Table 3 Effect of pH value of sample solution on adsorption of macroporous resin

Table 4 The influence of sample solution temperature on the adsorption of macroporous resin

Table 5 The effect of sample flow rate on the adsorption of macroporous resin

According to the single factor test results, and shorten the test time as much as possible, the optimum adsorption process conditions are determined as follows: pH value 4.0, temperature room temperature, flow rate 1.0BV/h.

The preparation of quercetin requires severe hydrolysis conditions, but improper control of the conditions can easily lead to the degradation of the flavonoid ring. Single factor experiments were carried out on the four main factors of hydrolysis liquid composition, material ratio, hydrolysis time and temperature.

Table 6 The influence of hydrolyzate composition on the purity of quercetin

Table 7 The influence of material ratio on the purity of quercetin

Table 8 The influence of hydrolysis temperature on the purity of quercetin

Table 9 The influence of hydrolysis time on the purity of quercetin

According to the single factor test results and test cost, the best test conditions are: hydrolyzate composition V _methanol : V _water : V hydrochloric _acid = 35:5:10, hydrolysis time 90min, solid-liquid ratio 1:60g/mL; hydrolysis temperature 80℃ .

Take 1.5g of quercetin sample and place it in a crucible, add 1.0mL of sulfuric acid to dissolve it, heat it at low temperature until the sulfuric acid evaporates to dryness, then completely ash it at 550°C, transfer it to a desiccator, let it cool to room temperature, and weigh it accurately. have to. The burning residue of quercetin is 0.038%, which meets the drug inspection standard of the Ministry of Health (≤0.05%); the quercetin sample is made into 2% methanol solution, and it is still clear after standing for a week. The non-melting substance inspection method of the fixed method is checked, and it is clarified in ammonia solution, water and absolute ethanol, and all meet the requirements; the HPLC chromatographic conditions of quercetin are: octadecylsilane bonded gel silica gel is used as a filler, and the mobile phase is acetonitrile- Methanol (90:10), flow rate 1mL/min, detection wavelength 226nm, column temperature 30°C; detection method: Accurately weigh 1.0mg of quercetin, add 10mL of methanol to fully dissolve it, then dilute to 25mL with methanol, perform HPLC The injection volume was 10 μL, and the purity of the sample was determined by the normalization method, and the result was that the purity was >98%.

A preparation method of quercetin flavonoids in Oxytropis florifolius, comprising the following steps: precisely weighing 5 parts of 1000.00 g of Oxytropis florifolius powder, and extracting according to the best extraction process, the method of steps (3)-(5) Quercetin was separated to obtain 10.423mg, 10.532mg, 10.630mg, 10.445mg, and 10.533mg of quercetin crystals respectively; as shown in Figure 1, the purity of quercetin was found to be 98.62%, 99.01%, 98.84%, 98.40%, 98.74%, the average purity is 98.72%, and the coefficient of variation is 2.63%. The results showed that the preparation method had better precision, and the extracted quercetin had higher purity.

The above is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed in the present invention can obviously obtain the simplicity of the technical solution. Changes or equivalent replacements all fall within the protection scope of the present invention.

Claims (1)

1. the method for a separating meletin from glabrous crazyweed, is characterized in that, comprises the following steps:

(1) glabrous crazyweed is pulverized: get the glabrous crazyweed over-ground part, dry in the shade, pulverize, standby after mistake 20 mesh sieves;

(2) the Ultrasound-assisted method is extracted the glabrous crazyweed general extractive: take the glabrous crazyweed sample, the methyl alcohol ultrasonic extraction 90min that adds volume fraction 100% according to the ratio of material ratio 1:15g/mL, ultrasonic power 800W, temperature 45 C, filtration after extraction completes, filtrate adopts Rotary Evaporators concentrated, and the thermostatic drying chamber drying, obtain the glabrous crazyweed general extractive;

(3) separation of glabrous crazyweed flavones: glabrous crazyweed general extractive water is fully dissolved; To the deionized water that adds 1/3 volume in chromatography column, then D-101 type macroporous adsorbent resin is transferred in chromatography column with deionized water, add 70% ethanolic soln, volume is 2 times of resin volume, flow velocity is 1 times of resin volume/min, then with deionized water, is washed till without carrying out the gradient elution of the glabrous crazyweed general extractive aqueous solution after the alcohol flavor; Macroporous resin optimal adsorption processing condition are: pH value 4.0, temperature room temperature, flow velocity 1.0BV/h; Ethanol elution with 10%～100%, collect 50%～85% ethanol eluate, and concentrated through Rotary Evaporators, the freeze drier drying, obtain the brown color powder;

(4) separation of Quercetin crude product: take the about 1.0g of glabrous crazyweed flavones, adding volume ratio is V _{methyl alcohol}: V _water: V _{hydrochloric acid}the hydrolyzed solution 60mL of=35:5:10, in 80 ℃ of heating in water bath 90min, cooled and filtered, 60 ℃ of oven dry obtain the thick product of yellow needle-like Quercetin;

(5) recrystallization of Quercetin: get the about 1.0g of Quercetin crude product prepared by step (4), add 100mL sherwood oil and 20mL deionized water, in 80 ℃ of heated and stirred 30min, filtered while hot, precipitate 3 times with the 20mL deionized water wash, in 60 ℃ of oven dry, obtain Quercetin; Take Quercetin (1) 0.5g, add the 100mL methanol solution, in 80 ℃ of dissolvings, filter, filtrate lets cool crystallization, filters to obtain crystal, with the 20mL deionized water wash, precipitates 3 times, in 60 ℃, dries to obtain the high Quercetin of purity.

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