A method for separating and purifying dihydroquercetin from Amomum villosum

Abstract

The present invention discloses a method for separating and purifying high-purity dihydroquercetin from Amomum villosum, which includes drying, pulverizing, thermally refluxing and circulating extracting, concentrating, crystallizing, recrystallizing, eluting with a chromatographic column, collecting fractions and freeze-drying the medicinal materials of Amomum villosum to obtain pure dihydroquercetin with a purity higher than 99.50%. The raw materials used in the present invention include various components such as the pericarp, fruit and seed mass of Amomum villosum. The raw materials are widely sourced and easily obtainable. The extraction equipment used is common, the extraction solvent is low in cost, and the purification process is convenient and short in cycle, providing a new method for the preparation of high-purity dihydroquercetin.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification of effective components of Chinese medicinal materials, and particularly relates to a method for separating and purifying dihydroquercetin from Amomum villosum. Background Art

[0002] Amomum villosum is a perennial herbaceous plant that grows in valleys under forests and shady and wet places. Its fruit is a common traditional Chinese medicine, which can promote qi and regulate the middle energizer, stimulate the appetite, nourish the spleen and relieve diarrhea, regulate qi and prevent miscarriage, and is often regarded as a tonic by people. China is rich in Amomum villosum resources. As a Chinese medicinal material with both medicinal and edible uses, Amomum villosum has high medicinal value and edible value. Dihydroquercetin, also known as taxifolin, is a dihydroflavonoid compound with various natural activities. It belongs to the vitamin P family and has very significant antioxidant ability. It is a natural antioxidant and is known as the "king of scavenging free radicals". Dihydroquercetin is also an important intermediate for pharmaceutical synthesis. Modern medical research shows that dihydroquercetin has important applications in tumor treatment and the prevention and treatment of cardiovascular and cerebrovascular diseases, and is an extremely precious ingredient for drugs and health foods.

[0003] Chinese Patent Application CN202111178720.3 discloses a method for extracting and purifying dihydroquercetin with high purity and high yield. The raw materials used include but are not limited to Pinaceae plants such as larch and Douglas fir. Alcohol extraction and ester extraction are used, and then it is dissolved in boiling water, filtered, concentrated, and recrystallized. The present invention can obtain dihydroquercetin with a purity > 99%, but the larch and other Pinaceae plants in the raw materials used have a long growth cycle and relatively scarce sources.

[0004] Chinese Patent Application CN201710605163.6 discloses a method for extracting dihydroquercetin from sorghum bran by subcritical water extraction. Using sorghum bran as the raw material, subcritical water extraction, macroporous resin adsorption and elution, and recrystallization are adopted. The extraction equipment and process used in the present invention are simple and green and are suitable for industrial production, but the highest purity of the extracted dihydroquercetin is 97.68%, and there is still room for improvement in purity.

[0005] Chinese Patent Application CN201210412622.6 discloses a method for extracting dihydroquercetin from larch tree roots. Using larch tree roots (200 mm below the ground surface) as the raw material, crushing treatment (becoming wood chips), leaching with a mixed solution of water and acetonitrile, hydrochloric acid acidolysis, extraction with methyl tert-butyl ether and dioxane, and recrystallization are adopted. The extraction process of the present invention is complex, and the solvents used cause great environmental pollution.

[0006] Chinese Patent Application for Invention CN200910311351.3 discloses a method for separating and purifying dihydroquercetin monomer. Using Engelhardtia chrysolepis Hance leaves as raw materials, glycosides of dihydroquercetin are obtained by alcohol extraction, and then dihydroquercetin is obtained by strong acid hydrolysis. Macroporous resin is used for enrichment and elution, and finally recrystallization is carried out. The dihydroquercetin obtained by this invention has a relatively high purity (99.2%), but the production process is relatively complicated and the production cycle is long. Summary of the Invention

[0007] The object of the present invention is to provide a method for separating and purifying dihydroquercetin from Amomum villosum Lour., which can obtain pure dihydroquercetin from Amomum villosum Lour., with easily available raw materials for extraction, common extraction equipment, cheap and easily available extraction solvents, and simple and short-cycle purification operations. The process method described in the present invention is to dry, crush, extract by heat reflux circulation, concentrate, crystallize, recrystallize, elute with a chromatographic column, collect fractions, and freeze-dry the medicinal materials of Amomum villosum Lour. to obtain pure dihydroquercetin with a purity higher than 99.50%.

[0008] The technical solution provided by the present invention is as follows:

[0009] A method for separating and purifying dihydroquercetin from Amomum villosum Lour., comprising the following steps:

[0010] Step 1, soaking: Put Amomum villosum Lour. into an oven for drying, take the undersize material after ultrafine pulverization, and dissolve and soak it with ethanol.

[0011] Step 2, extraction: The soaking solution is extracted by heat reflux circulation and concentrated to obtain a crude extract of dihydroquercetin.

[0012] Step 3, recrystallization: The crude extract is dissolved, crystallized, and recrystallized to obtain dihydroquercetin crystals with relatively high purity.

[0013] Step 4, drying and pulverization: The obtained crystals are dried and pulverized to obtain dried dihydroquercetin (purity ≥ 95%).

[0014] Step 5, purification: The dried material is dissolved, eluted with a chromatographic column, and fractions are collected to obtain a fraction solution of dihydroquercetin.

[0015] Step 6, freeze-drying: The obtained eluate is freeze-dried to obtain pure dihydroquercetin.

[0016] Preferably, in Step 1, the drying temperature of the medicinal materials is 30 - 55 °C, and the drying time is 5 - 8 hours.

[0017] Preferably, in Step 1, the mesh number of the pharmacopoeia sieve used is 30 - 80 meshes.

[0018] Preferably, in the first step, the ethanol concentration used is 40-95%, and the volume ratio of the medicinal material to the extraction solvent is 1:15-30.

[0019] Preferably, in the first step, the soaking time is 4-24 hours.

[0020] Preferably, in the second step, the temperature for heat reflux cycle extraction is 60-85°C, the extraction times are 1-2 times, and the extraction time is 1-2 hours.

[0021] Preferably, in the second step, the extract is concentrated to 1 / 2-1 / 4 of the original volume.

[0022] Preferably, in the third step, the dissolving agent used includes one or more of pure water, an ethanol solution with a concentration of 30%, a methanol solution with a concentration of 30%, or an aqueous solution of ethyl acetate with a concentration of 30%.

[0023] Preferably, in the third step, the volume ratio of the concentrated solution to the dissolving agent is 1:2-5.

[0024] Preferably, in the third step, the crystallization time is 24-48 hours.

[0025] Preferably, in the third step, the solvent used for recrystallization includes one or more of pure water, an ethanol solution with a concentration of 30%, a methanol solution with a concentration of 30%, or an aqueous solution of ethyl acetate with a concentration of 30%.

[0026] Preferably, in the third step, the mass ratio of the recrystallized crystal to the solvent is 1:2-5.

[0027] Preferably, in the third step, the recrystallization temperature is -4-5°C, the recrystallization time is 48-72 hours, and the recrystallization times are 1-3 times.

[0028] Preferably, in the fifth step, the dried product is dissolved using one or more of ethanol, methanol, or formic acid.

[0029] Preferably, in the fifth step, the model of the chromatographic column used is C 18 、C 8 、C 30 or a CN column.

[0030] Preferably, in the fifth step, the mobile phase of the chromatographic column is one of methanol-phosphoric acid aqueous solution, acetonitrile-phosphoric acid aqueous solution, acetonitrile-aqueous solution, acetonitrile-acetic acid aqueous solution, or methanol-aqueous solution, and the ratio of the aqueous solution is 0.1%, 0.3%, 0.5%, 1.5%.

[0031] Preferably, in the fifth step, the flow rate of the mobile phase is 8-12 mL / min.

[0032] Preferably, in the fifth step, the column temperature of the chromatographic column is 20-30 °C, and the detection wavelength is 200-600 nm.

[0033] Preferably, in the fifth step, the fractionation liquid between 24.5-26.5 min is collected.

[0034] Preferably, in the sixth step, the drying method of the fractionation liquid is vacuum freeze-drying or spray freeze-drying to obtain pure dihydroquercetin.

[0035] The present invention has the following beneficial effects:

[0036] 1) Uniqueness: Dihydroquercetin is extracted, separated and purified from Amomum villosum for the first time.

[0037] 2) Wide raw material source: The resources of Amomum villosum in China are rich, avoiding the problems of resource shortage or limitation.

[0038] 3) Convenient extraction process: Common extraction equipment, low cost of extraction solvent, simple purification process operation and short cycle.

[0039] 4) High purity: The purity of dihydroquercetin obtained by this method is higher than 99.50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the high performance preparation liquid chromatogram of dihydroquercetin standard product;

[0041] Figure 2 It is the high performance preparation liquid chromatogram of dihydroquercetin in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention will be described in detail below with reference to specific implementation examples, so as to facilitate researchers in the field to implement it with reference to this specification.

[0043] Example 1

[0044] Take dried and crushed Amomum villosum medicinal material, add 60% ethanol with a solvent multiple of 15 times, fully mix and soak for 8 hours, heat and reflux for 2 times under 70°C, extract for 2 hours, and concentrate under reduced pressure to obtain a crude extract, add 4 times of 30% ethanol solution with a volume fraction to the crude extract to dissolve it, filter it, take the supernatant and place it at room temperature for crystallization for 48 hours to obtain mother liquor 1 (supernatant) and dihydroquercetin crystal 1; use 4 times of 30% ethanol solution with a volume fraction to completely dissolve the crystals, place it at low temperature for the first recrystallization for 48 hours When the reaction mixture was stirred for 2 hours, a mother liquor 2 (supernatant) and dihydroquercetin crystals 2 were obtained; 4 times the volume fraction of 20% ethanol solution was used to completely dissolve the crystals, and the crystals were placed at low temperature for a second recrystallization for 72 hours to obtain mother liquor 3 (supernatant) and dihydroquercetin crystals 3, mother liquor 3 was combined with mother liquor 2, and 2 times the volume fraction of 20% ethanol solution was added to completely dissolve the crystals, and the crystals were placed at low temperature for a third recrystallization for 48 hours to obtain high-purity dihydroquercetin crystals; the crystals were dried and crushed to obtain dihydroquercetin dry matter with a purity of 95.2%.

[0045] Take an appropriate amount of the dried product and redissolve it in methanol solution and use gradient elution to obtain the 18 The elution was performed by reverse column chromatography, the column temperature was 26°C, and the eluent consisting of methanol and 0.1% phosphoric acid aqueous solution in a volume ratio of 5-20:80-95 was used for elution from 0 to 19 minutes, and the eluent consisting of methanol and 0.1% phosphoric acid aqueous solution in a volume ratio of 20-30:70-80 was used for elution from 20 to 40 minutes, and the eluent consisting of methanol and 0.1% phosphoric acid aqueous solution in a volume ratio of 40-50:50-60 was used for elution from 40 to 60 minutes. The flow rate was 8-12 mL / min, and the fraction between 24.5 and 26.5 minutes was collected and detected, and the detection wavelength was 360 nm. The high-efficiency preparative liquid chromatograms of dihydroquercetin standard and dihydroquercetin in this embodiment are shown as follows: Figure 1-2 The collected fractions were freeze-dried to obtain a pure dihydroquercetin product with a content of 99.50%.

[0046] Example 2

[0047] Take the dried and pulverized Amomum villosum Lour. medicinal materials, add 50% ethanol with a solvent multiple of 30 times, mix well and soak for 8 hours, then heat under reflux and circulate for extraction 2 times at 70 °C for 2 hours each time. Concentrate under reduced pressure to obtain a crude extract. Add 4 times the volume of 30% ethanol solution to the crude extract to dissolve it and filter. Take the supernatant and crystallize it at room temperature for 48 hours to obtain mother liquor 1 (supernatant) and dihydroquercetin crystal 1; use 4 times the volume of 30% ethanol solution to completely dissolve the crystals, and perform the first recrystallization at low temperature for 72 hours to obtain mother liquor 2 (supernatant) and dihydroquercetin crystal 2g; use 4 times the volume of 30% ethanol solution to completely dissolve the crystals, and perform the second recrystallization at low temperature for 72 hours to obtain mother liquor 3 (supernatant) and dihydroquercetin crystal 3. Combine mother liquor 3 and mother liquor 2, then add 2 times the volume of 30% ethanol solution to completely dissolve the crystals, and perform the third recrystallization at low temperature for 48 hours to obtain dihydroquercetin crystals; dry and pulverize the crystals to obtain a dihydroquercetin dry product with a purity of 95.1%.

[0048] Take an appropriate amount of the dry product and redissolve it in an aqueous solution of methanol, then use gradient elution and elute with a C 8 reverse-phase chromatographic column. The column temperature of the chromatographic column is 24 °C. Use an eluent composed of acetonitrile and 0.3% phosphoric acid aqueous solution in a volume ratio of 5 - 20:80 - 95 to elute from 0 - 19 min, use an eluent composed of acetonitrile and 0.3% phosphoric acid aqueous solution in a volume ratio of 20 - 30:70 - 80 to elute from 20 - 40 min, and use an eluent composed of acetonitrile and 0.3% phosphoric acid aqueous solution in a volume ratio of 40 - 50:50 - 60 to elute from 40 - 60 min. The flow rate is 8 - 12 mL / min. Collect and detect the fraction between 24.5 - 26.5 min, and the detection wavelength is 360 nm. Freeze-dry the collected fraction to obtain a pure dihydroquercetin product with a content of 99.59%.

[0049] The above embodiments are only examples of the preferred embodiments of the present invention and do not cover all the scopes of the invention embodiments. Those of ordinary skill in the art, without departing from the spirit and scope of the present invention, make various changes and modifications, which are all considered to be within the scope of the claims of the present invention.

Claims

1. A method for separating and purifying dihydroquercetin from Amomum villosum Lour., which is characterized in that, it comprises the following steps: Step 1, soaking: Put Amomum villosum Lour. into an oven for drying, take the undersize material after ultrafine pulverization, and dissolve and soak it with ethanol; Step 2, extraction: The soaking solution is subjected to heat reflux cycle extraction and concentration to obtain a crude extract of dihydroquercetin; Step 3, recrystallization: The crude extract is dissolved, crystallized, and recrystallized to obtain dihydroquercetin crystals with higher purity. Among them, the solvents used include one or several of pure water, ethanol solution with a concentration of 30%, methanol with a concentration of 30%, or aqueous acetic acid ethyl ester solution with a concentration of 30%; the volume ratio of the crude extract to the solvent is 1:2 - 5; the crystallization time is 24 - 48 hours; the recrystallization temperature is -4 - 5 °C, the recrystallization time is 48 - 72 hours, and the number of recrystallization times is 1 - 3 times; Step 4, drying and pulverization: Dry and pulverize the obtained crystals to obtain the dried product of dihydroquercetin; Step 5. Purification: The dried product is dissolved, eluted through a chromatographic column, and fractions are collected to obtain a dihydroquercetin fraction solution. Among them, the dried product is dissolved using one or several reagents of ethanol, methanol, or formic acid; the model of the chromatographic column used is C 18 , C 8 , C 30 or a CN column; the mobile phase of the chromatographic column is one of methanol-phosphoric acid aqueous solution, acetonitrile-phosphoric acid aqueous solution, acetonitrile-aqueous solution, acetonitrile-acetic acid aqueous solution, or methanol-aqueous solution, and the ratio of the aqueous solution is 0.1%, 0.3%, 0.5%, 1.5%; the flow rate of the mobile phase is 8-12 mL / min; the column temperature of the chromatographic column is 20-30 °C, the detection wavelength is 200-600 nm, and the fraction solution between 24.5-26.5 min is collected; Step 6, freeze-drying: Subject the obtained eluate to freeze-drying to obtain pure dihydroquercetin.

2. The method according to claim 1, which is characterized in that, in the said Step 1, the drying temperature of the medicinal material is 30 - 55 °C, the drying time is 5 - 8 hours; the mesh number of the pharmacopoeia sieve used is 30 - 80 meshes; the volume concentration of the ethanol used is 40 - 95%, the volume ratio of the medicinal material to the extraction solvent is 1:15 - 30, and the soaking time is 4 - 24 hours.

3. The method according to claim 1, which is characterized in that, in the said Step 2, the heat reflux cycle temperature is 60 - 85 °C, the extraction times are 1 - 2 times, and the extraction time is 1 - 2 hours; the extraction solution is concentrated to 1 / 2 - 1 / 4 of the original volume.

4. The method according to claim 1, which is characterized in that, in the said Step 4, the recrystallized crystals are subjected to vacuum drying, the temperature of the vacuum drying ≤ 65 °C, and the vacuum degree ≤ 0.1 MPa.

5. The method according to claim 1, which is characterized in that, in the said Step 6, the drying method of the distillate solution is vacuum freeze-drying or spray freeze-drying to obtain pure dihydroquercetin.

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