CN113651789B - Method for separating galangin from galangal ketone by high performance liquid chromatography - Google Patents
Method for separating galangin from galangal ketone by high performance liquid chromatography Download PDFInfo
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- CN113651789B CN113651789B CN202111017611.3A CN202111017611A CN113651789B CN 113651789 B CN113651789 B CN 113651789B CN 202111017611 A CN202111017611 A CN 202111017611A CN 113651789 B CN113651789 B CN 113651789B
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
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- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
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Abstract
The invention belongs to the technical field of separation and purification of flavonoid compounds, and discloses a method for separating galangin from galangin by utilizing high performance liquid chromatography. The method comprises the following steps: s1: extracting rhizoma Alpiniae Officinarum with alcohol, and purifying the extract to obtain purified product containing galangin and kaempferide; s2: separating galangin from purified product containing galangin and kaempferide by high performance liquid chromatography; conditions for high performance liquid chromatography separation: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of chloroform to ethyl acetate to methanol is (1-5) to (0.5-4) to (1-4). The method is simple, the extraction rate of the galangal ketone compounds is high, the separation degree of galangin is high, and the retention time is short.
Description
Technical Field
The invention belongs to the technical field of separation and purification of flavonoid compounds, and particularly relates to a method for separating galangin from galangin by utilizing high performance liquid chromatography.
Background
The galangal is rhizome part of galangal belonging to genus galangal of family Zingiberaceae, and the chemical components of the medicinal material are complex. Wherein one of the active ingredients is a high-yield curcumone compound, has the effects of resisting ulcer, diarrhea and the like, and can be used in the pharmaceutical field. The galangal ketone compound mainly comprises galangin, kaempferide, etc. The galangin flavonoid has antitumor, antibacterial, and antiinflammatory effects. In order to further investigate each active ingredient in the high-yield curcumone compound, it is necessary to separate each active ingredient.
However, since the galangin and kaempferide have similar structures, when the galangin in the galangin compound is separated by a conventional method, the degree of separation of the galangin and kaempferide is insufficient, and still needs to be further improved, so that the substance is more thoroughly separated from other substances. In addition, the retention time of the existing separation method is long, the separation rate is slow, and for large-scale operation, the purification analysis effect of such a low rate is definitely not practical.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a method for separating galangin from galangin by utilizing high performance liquid chromatography. The method is simple, efficient, high in galangin separation degree and short in retention time.
The aim of the invention is achieved by the following technical scheme:
a method for separating galangin from galangal ketone by high performance liquid chromatography, comprising the following steps:
s1: extracting rhizoma Alpiniae Officinarum with alcohol, and purifying the extract to obtain purified product containing galangin and kaempferide;
s2: separating galangin from purified product containing galangin and kaempferide by high performance liquid chromatography; conditions for high performance liquid chromatography separation: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of chloroform to ethyl acetate to methanol is (1-5) to (0.5-4) to (1-4).
The volume ratio of chloroform, ethyl acetate and methanol in the mobile phase is preferably (1-5): 4:1 or 4: (0.5-2) to 1 or 4 to 1-4; more preferably (2-5) to 4:1.
The extraction in the step S1 refers to alcohol extraction of galangal; the alcohol is ethanol or more than one of methanol, preferably a mixed solution of methanol and ethanol; the volume ratio of the methanol to the ethanol is 1: (0.5-2), and is preferably 1:1.
The extraction is carried out under the condition of ultrasonic assistance; the power of the ultrasonic wave is 100-200W; the times of extraction are 2-6 times, and the time of each extraction is 30-60 min. The extraction temperature is normal temperature.
The purification refers to purification by macroporous adsorption resin; during purification, the eluent is ethanol water solution, and the volume concentration of ethanol in the ethanol water solution is 70-90%, preferably 85%; the eluting flow rate of the eluent is 0.4 BV/h-0.6 BV/h. After elution, the filtrate was collected, concentrated and dried.
During purification, the concentration of the extract is 0.8-1.2 mg/mL.
The invention adopts a high performance liquid chromatography mode, utilizes the improved mobile phases of chloroform, ethyl acetate and methanol to separate and purify the galangin in the galangin, and keeps the separation degree at 1.68 better than the separation degree of the mixed solution of the methanol and the phosphoric acid (reaching about 1.4) through the selection of the mobile phases. In addition, by selection of this mobile phase, the retention time of galangin was advanced to 41.1 minutes.
The separation degree is also called resolution, and is often used as an index of the total separation efficiency of a column, and is denoted by R, in order to determine the separation of a separation substance from a chromatographic column in the column. R is equal to the ratio of the difference between the retention time of adjacent chromatographic peaks to the peak-to-average value of the two chromatographic peaks, and represents the separation degree of the two adjacent peaks, and the larger R represents the better separation of the two adjacent components.
Compared with the prior art, the invention has the following advantages:
the method is simple, the extraction rate of the high yield curcuminoids is high, the separation degree of galangin is high, and the retention time is short.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but embodiments of the present invention are not limited thereto.
The method for separating galangin from galangal ketone by utilizing high performance liquid chromatography comprises the following steps:
s1: pulverizing rhizoma Alpiniae Officinarum, sieving, extracting with alcohol, and purifying to obtain purified product (namely galangin) containing galangin and kaempferide;
s2: separating galangin from purified product containing galangin and kaempferide by high performance liquid chromatography.
The extraction in the step S1 refers to ultrasonic extraction of galangal in alcohol; the alcohol is ethanol or more than one of methanol, preferably a mixed solution of methanol and ethanol; the volume ratio of the methanol to the ethanol is 1: (0.5-2), and is preferably 1:1. The power of the ultrasonic wave is 100-200W; the times of extraction are 2-6 times, and the time of each extraction is 30-60 min. The extraction temperature is normal temperature. During ultrasonic extraction, the mass volume ratio of the galangal to the alcohol is 1g to (2-40) mL.
Through experimental comparison, when the ultrasonic extraction is carried out by using the methanol and the ethanol alone, the content of the galangin in the extracted solution is lower than 40%, and the content of the extract obtained after the mixed solvent of the methanol and the ethanol is adopted is more than 45%.
The purification refers to purification by macroporous adsorption resin; during purification, the eluent is ethanol water solution, and the volume concentration of ethanol in the ethanol water solution is 80-90%, preferably 85%; the eluting flow rate of the eluent is 0.4 BV/h-0.6 BV/h. After elution, the filtrate was collected, concentrated and dried. The macroporous adsorption resin is used for purifying flavonoid compounds.
Conditions for high performance liquid chromatography separation in step S2: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of chloroform to ethyl acetate to methanol is (1-5) to (0.5-4) to (1-4).
The volume ratio of chloroform, ethyl acetate and methanol in the mobile phase is preferably (1-5) to 4:1 or 4: (0.5-2) to 1 or 4 to 1-4; more preferably (2-5) to 4:1. The purified product of galangin is subjected to a high performance liquid chromatography separation process in step S2 using 5 parts or more of the sample, and the yield is calculated and averaged, and the relative standard deviation is ensured to be 3% or less.
The chromatographic column selected in the high performance liquid chromatography separation in the step S2 is C18, and the flow rate is 5-8 mL/min; the detection wavelength is 360nm; the sample injection amount is 700 mu L; the column temperature is normal temperature.
The high performance liquid chromatography adopts Agilent company equipment, the chromatographic column C18 is Shimadzu ODS-3,5um,4.6mm 250mm, the ultrasonic instrument adopts the Innovative instrument company equipment; the original galangal is purchased from the Tongren Tang drug store.
Example 1
A method for separating galangin from galangin by high performance liquid chromatography, comprising the steps of:
s1: pulverizing dried rhizoma Alpiniae Officinarum, sieving with 10 mesh sieve, extracting rhizoma Alpiniae Officinarum powder with ultrasound in mixed solution of methanol and ethanol at volume ratio of 1:1, mixing extractive solutions, and obtaining coarse extract of rhizoma Alpiniae Officinarum ketone; the ultrasonic auxiliary extraction is carried out at normal temperature, the power of the ultrasonic is 150W, the time of each extraction is 45min, and the times of extraction are 3 times; the total content of galangin in the crude extract of galangin is 45.5%;
s2: purifying the crude extract of the galangal obtained in the step S1 by an adsorption resin column (macroporous adsorption resin is specifically macroporous adsorption resin prepared from styrene and divinylbenzene, such as HPD-600 macroporous adsorption resin), so as to obtain a purified galangal product, wherein the purified galangal product contains galangal He Shannai; the concentration of the galangin flavone crude extract is 1.0mg/mL, the eluent is ethanol water solution, the ethanol volume ratio of the ethanol water solution is 85%, and the elution flow rate is controlled to be 0.5BV/h; after elution, collecting, concentrating and drying the collected filtrate, wherein the drying temperature is 35-40 ℃;
s3: separating the purified galangin product by high performance liquid chromatography, wherein the detection wavelength is 360nm, and the mobile phase adopts chloroform, ethyl acetate and methanol; the chromatographic column used in the liquid chromatography is C18, and the flow rate is 7.0mL/min; the detection wavelength is 360nm; the sample injection amount is 700 mu L; the column temperature is normal temperature.
In the embodiment, in step S3, mobile phases (chloroform, ethyl acetate and methanol) with different proportions are selected for testing, and the volume ratio of each mobile phase corresponds to the volume ratio of chloroform, ethyl acetate and methanol; mobile phase 1: the volume ratio is 4:4:1, mobile phase 2: the volume ratio is 2:4:1, mobile phase 3: the volume ratio is 1:4:1, mobile phase 4: the volume ratio is 4:2:1, mobile phase 5: the volume ratio is 4:1:1, mobile phase 6: the volume ratio is 4:0.5:1, mobile phase 7: the volume ratio is 4:4:4, mobile phase 8: the volume ratio is 4:4:2.
The degree of separation obtained for mobile phases 1-8 is: 1.68, 1.62, 1.59, 1.58, 1.59, 1.60, 1.56, 1.54.
The corresponding galangin retention times of mobile phase 1-mobile phase 8 are 41.1, 41.3, 41.6, 41.5, 41.6, 42.0 and 41.8min respectively.
The purification and separation process of Gao Liangsu medicinal materials is divided into three basic steps, wherein the first step is an initial crushing and sieving step, and then the medicinal materials are further extracted and filtered by means of adsorption resin, so that the purified medicinal materials are obtained. Then, separating and purifying galangin in galangin by high performance liquid chromatography, and by improved mobile phase chloroform, ethyl acetate and methanol, wherein the volume ratio of chloroform, ethyl acetate and methanol is 4:4:1, and the separation degree is kept at 1.68 and the retention time of galangin is advanced to 41.1 minutes by selecting the mobile phase. In contrast, when the mixed solution of methanol and phosphoric acid was used, the degree of separation of the mixed solution of methanol and phosphoric acid reached about 1.4, and the retention time was 43.2 minutes.
Comparative example 1
This comparative example differs from example 1 in that: in the high performance liquid chromatography separation, the mobile phase is ethyl acetate and methanol with the volume ratio of 1:1.
Comparative example 2
This comparative example differs from example 1 in that: in the high performance liquid chromatography separation, the mobile phase is chloroform and methanol with the volume ratio of 1:1.
Comparative example 3
This comparative example differs from example 1 in that: in the high performance liquid chromatography separation, the mobile phase is chloroform and ethyl acetate with the volume ratio of 1:1.
The degree of separation of comparative examples 1 to 3 was less than 1.4, wherein the degree of separation of comparative example 1 was 1.30, the degree of separation of comparative example 2 was 1.29, and the degree of separation of comparative example 3 was 1.35.
When the high performance liquid chromatography is adopted to separate the galangin, the purity of the separated galangin is 99% or more.
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (7)
1. A method for separating galangin from galangal ketone by high performance liquid chromatography, which is characterized in that: the method comprises the following steps:
s1: extracting rhizoma Alpiniae Officinarum with alcohol, and purifying the extract to obtain purified product containing galangin and kaempferide;
s2: separating galangin from purified product containing galangin and kaempferide by high performance liquid chromatography; conditions for high performance liquid chromatography separation: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of chloroform, ethyl acetate and methanol is 4:4:1.
2. The method for separating galangin from homocurcuminones by high performance liquid chromatography according to claim 1, wherein: the extraction in the step S1 refers to alcohol extraction of galangal; the alcohol is more than one of ethanol or methanol.
3. The method for separating galangin from homocurcuminones by high performance liquid chromatography according to claim 2, wherein: the alcohol is a mixed solution of methanol and ethanol; the volume ratio of the methanol to the ethanol is 1: (0.5-2).
4. A method for separating galangin from homocurcuminones by high performance liquid chromatography according to claim 3, wherein: the volume ratio of the methanol to the ethanol is 1:1.
5. The method for separating galangin from homocurcuminones by high performance liquid chromatography according to claim 1, wherein:
the purification refers to purification by macroporous adsorption resin; during purification, the eluent is ethanol water solution, and the volume concentration of ethanol in the ethanol water solution is 70-90%; the eluting flow rate of the eluent is 0.4 BV/h-0.6 BV/h.
6. The method for separating galangin from homocurcuminones by high performance liquid chromatography according to claim 5, wherein: the volume concentration of ethanol in the ethanol water solution is 85%;
after eluting with eluent, collecting filtrate, concentrating, and drying.
7. The method for separating galangin from homocurcuminones by high performance liquid chromatography according to claim 1, wherein: the extraction is carried out under the condition of ultrasonic assistance; the power of the ultrasonic wave is 100-200W; the times of extraction are 2-6 times, and the time of each extraction is 30-60 min; the extraction temperature is normal temperature.
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| KR950000685B1 (en) * | 1991-07-10 | 1995-01-27 | 주식회사 태평양 | Stabilization method of galangin |
| CN102048993B (en) * | 2009-10-29 | 2012-05-30 | 中国中医科学院中药研究所 | Application of galangal extract in preparation of medicine for inhibiting calcium channel |
| CN101810812B (en) * | 2010-03-11 | 2011-11-30 | 闫明 | Use of rhizoma galangae effect part for preparing medicament for treating leucoderma |
| CN101824020A (en) * | 2010-05-26 | 2010-09-08 | 南京泽朗农业发展有限公司 | Method for extracting galangin from galangal |
| CN104341379A (en) * | 2013-08-02 | 2015-02-11 | 上海友思生物技术有限公司 | Galangin extraction method |
| CN104557834B (en) * | 2015-01-21 | 2016-08-17 | 聊城大学 | A kind of isolated and purified pinocembrin, Chrysin and method of Galangin from China's Water extracts of propolis |
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