阅读说明：本技术 一种利用高效液相色谱分离高良姜黄酮中高良姜素的方法 (Method for separating galangin from galangal flavone by high performance liquid chromatography ) 是由 侯红瑞 吴文 李泽玲 云娜 岑水斌 于 2021-08-31 设计创作，主要内容包括：本发明属于黄酮类化合物分离纯化的技术领域,公开了一种利用高效液相色谱分离高良姜黄酮中高良姜素的方法。方法：S1：将高良姜进行醇提取,将提取物进行纯化,获得含有高良姜素和山奈素的纯化物；S2：采用高效液相色谱分离出含有高良姜素和山奈素的纯化物中高良姜素；高效液相色谱分离的条件：流动相为氯仿、乙酸乙酯和甲醇；氯仿、乙酸乙酯和甲醇的体积比为(1～5)∶(0.5～4)∶(1～4)。本发明的方法简单、高良姜黄酮类化合物提取率高,高良姜素的分离度高,保留时间短。(The invention belongs to the technical field of separation and purification of flavonoid compounds, and discloses a method for separating galangin from galangal flavone by using high performance liquid chromatography. The method comprises the following steps: s1: extracting rhizoma Alpiniae Officinarum with alcohol, and purifying to obtain purified extract containing galangin and kaempferide; s2: separating galangin from purified product containing galangin and kaempferide by high performance liquid chromatography; conditions of high performance liquid chromatography separation: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of the chloroform to the ethyl acetate to the methanol is (1-5) to (0.5-4) to (1-4). The method is simple, the galangal flavonoid compound extraction rate is high, the galangin separation degree is high, and the retention time is short.)

1. A method for separating galangin from galangal flavone by using high performance liquid chromatography is characterized by comprising the following steps: the method comprises the following steps:

s1: extracting rhizoma Alpiniae Officinarum with alcohol, and purifying to obtain purified extract containing galangin and kaempferide;

s2: separating galangin from purified product containing galangin and kaempferide by high performance liquid chromatography; conditions of high performance liquid chromatography separation: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of the chloroform to the ethyl acetate to the methanol is (1-5) to (0.5-4) to (1-4).

2. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 1, wherein the method comprises the steps of: the volume ratio of chloroform to ethyl acetate to methanol in the mobile phase is (1-5) to 4: 1 or 4: 0.5-2: 1 or 4: 1-4.

3. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 2, wherein the step of separating galangin from galangin comprises the steps of: and the volume ratio of the chloroform to the ethyl acetate to the methanol in the mobile phase is (2-5) to 4: 1.

4. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 1, wherein the method comprises the steps of: the extraction in step S1 is to extract galangal with alcohol; the alcohol is more than one of ethanol or methanol.

5. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 4, wherein the step of separating galangin from galangin comprises the following steps: the alcohol is a mixed solution of methanol and ethanol; the volume ratio of the methanol to the ethanol is 1: (0.5-2).

6. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 5, wherein the step of separating galangin from galangin comprises the following steps: the volume ratio of the methanol to the ethanol is 1: 1.

7. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 1, wherein the method comprises the steps of:

the purification is carried out by adopting 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 elution flow rate of the eluent is 0.4 BV/h-0.6 BV/h.

8. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 7, wherein the step of separating galangin from galangin comprises the following steps: the volume concentration of ethanol in the ethanol water solution is 85 percent;

eluting with eluent, collecting filtrate, concentrating, and drying.

9. The method for separating galangin from galangin by high performance liquid chromatography as claimed in claim 1, wherein the method comprises the steps of: the extraction is carried out under the condition of ultrasonic wave assistance; the power of the ultrasonic wave is 100-200W; the extraction times are 2-6 times, and the extraction time is 30-60 min each time; the extraction temperature is normal temperature.

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 galangal flavone by using high performance liquid chromatography.

Background

Alpinia officinarum is the rhizome of galangal belonging to the genus Alpinia of the family Zingiberaceae, and the chemical composition of this herb is relatively complex. Wherein one of the effective components is galangal flavone compound, has the effects of resisting ulcer, resisting diarrhea and the like, and can be used in the field of pharmacy. The galangal flavone compounds mainly include galangin, kaempferide, etc. The galangin flavone compound has antitumor, antibacterial and antiinflammatory effects. In order to further study each active ingredient in galangal flavone compounds, it is necessary to separate each active ingredient.

However, due to the structural similarity between galangin and kaempferide, when galangin is separated from galangin compounds by conventional methods, the separation degree of galangin and kaempferide is insufficient, and further improvement is still needed to realize more thorough separation of the galangin from other substances. In addition, the existing separation methods have long retention times and slow separation rates, and the effectiveness of such low-rate purification assays is undoubtedly not practical for large-scale batch operations.

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 galangal flavone by using high performance liquid chromatography. The method is simple and efficient, and has high galangin separation degree and short retention time.

The purpose of the invention is realized by the following technical scheme:

a method for separating galangin from galangal flavone by high performance liquid chromatography comprises the following steps:

s1: extracting rhizoma Alpiniae Officinarum with alcohol, and purifying to obtain purified extract containing galangin and kaempferide;

s2: separating galangin from purified product containing galangin and kaempferide by high performance liquid chromatography; conditions of high performance liquid chromatography separation: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of the chloroform to the ethyl acetate to the methanol is (1-5) to (0.5-4) to (1-4).

The volume ratio of chloroform to ethyl acetate to methanol in the mobile phase is preferably (1-5): 4: 1 or 4: (0.5-2) to 1 or 4: 4 to (1-4); more preferably (2-5) to 4: 1.

The extraction in step S1 is to extract galangal with alcohol; the alcohol is more than one of ethanol or methanol, preferably a mixed solution of methanol and ethanol; the volume ratio of the methanol to the ethanol is 1: 0.5-2, and preferably 1: 1.

The extraction is carried out under the condition of ultrasonic wave assistance; the power of the ultrasonic wave is 100-200W; the extraction times are 2-6 times, and the extraction time is 30-60 min each time. The extraction temperature is normal temperature.

The purification is carried out by adopting 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 elution flow rate of the eluent is 0.4 BV/h-0.6 BV/h. After elution, the filtrate was collected, concentrated and dried.

The concentration of the extract is 0.8-1.2 mg/mL during purification.

The invention adopts a high performance liquid chromatography mode, and utilizes improved mobile phases of chloroform, ethyl acetate and methanol to separate and purify the galangin in the galangin, and the separation degree is kept at 1.68 and is better than that of a mixed solution of methanol and phosphoric acid (about 1.4) through the selection of the mobile phases. In addition, the retention time of galangin is advanced to 41.1 min by the selection of the mobile phase.

The separation degree is also called resolution, and in order to determine the separation condition of the separated substance in the chromatographic column, the separation degree is usually used as the total separation efficiency index of the column and is represented by R. R is equal to the ratio of the retention time difference of adjacent chromatographic peaks to the width 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 and beneficial effects:

the method is simple, the galangal flavonoid compound extraction rate is high, the galangin separation degree 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 the embodiments of the present invention are not limited thereto.

The method for separating galangin from galangal flavone by using high performance liquid chromatography comprises the following steps:

s1: pulverizing rhizoma Alpiniae Officinarum, sieving, extracting with alcohol, and purifying to obtain purified extract (i.e. galangin flavone) 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 is to perform ultrasonic extraction on the galangal in alcohol; the alcohol is more than one of ethanol or methanol, preferably a mixed solution of methanol and ethanol; the volume ratio of the methanol to the ethanol is 1: 0.5-2, and preferably 1: 1. The power of the ultrasonic wave is 100-200W; the extraction times are 2-6 times, and the extraction time is 30-60 min each time. 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 test comparison, the galangin content in the extract obtained by the ultrasonic extraction with methanol and ethanol alone is lower than 40%, and the content of the extract obtained by adopting a mixed solvent of methanol and ethanol is more than 45%.

The purification is carried out by adopting 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 elution 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 the flavonoid compound.

Conditions for the high performance liquid chromatography in step S2: the mobile phase is chloroform, ethyl acetate and methanol; the volume ratio of the chloroform to the ethyl acetate to the methanol is (1-5) to (0.5-4) to (1-4).

The volume ratio of chloroform to ethyl acetate to methanol in the mobile phase is preferably (1-5) to 4: 1 or 4: (0.5-2) to 1 or 4: 4 to (1-4); more preferably (2-5) to 4: 1. The purified galangin product is subjected to high performance liquid chromatography separation process in step S2 using 5 or more samples, and the yield is calculated and averaged, and the relative standard deviation is guaranteed to be below 3%.

In the step S2, the chromatographic column selected in the high performance liquid chromatography is C18, and the flow rate is 5-8 mL/min; the detection wavelength is 360 nm; the sample injection amount is 700 mu L; the column temperature was normal temperature.

The high performance liquid chromatography adopts Agilent company equipment, a chromatographic column C18 is Shimadzu ODS-3, 5um, 4.6mm x 250mm, and an ultrasonic instrument adopts Schauk Yunhua apparatus company equipment; the rhizoma Alpiniae Officinarum original medicinal material is purchased from Homon's pharmacy.

Example 1

A method for separating galangin from galangin by high performance liquid chromatography comprises the following steps:

s1: pulverizing dried rhizoma Alpiniae Officinarum, sieving with 10 mesh sieve, performing ultrasonic-assisted extraction on the rhizoma Alpiniae Officinarum powder in mixed solution of methanol and ethanol at volume ratio of 1: 1, and mixing extractive solutions to obtain rhizoma Alpiniae Officinarum flavone crude extract; performing ultrasonic-assisted extraction at normal temperature, wherein the ultrasonic power is 150W, the extraction time is 45min each time, and the extraction times are 3 times; the total galangin content of the coarse galangin extract is 45.5%;

s2: purifying the coarse galangin extract obtained in step S1 with adsorbent resin column (macroporous adsorbent resin is macroporous adsorbent resin prepared from styrene and divinylbenzene, such as HPD-600 macroporous adsorbent resin) to obtain galangin purified product containing galangin and kaempferide; the concentration of the galangin flavone crude extract is 1.0mg/mL, the eluent is ethanol water solution, the volume ratio of ethanol in the ethanol water solution is 85%, and the elution flow rate is controlled to be 0.5 BV/h; after elution, collecting, concentrating and drying the collected filtrate, wherein the drying temperature is 35-40 ℃;

s3: separating galangin purified 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 7.0 mL/min; the detection wavelength is 360 nm; the sample injection amount is 700 mu L; the column temperature was normal temperature.

In the present example, in step S3, different proportions of mobile phases (chloroform, ethyl acetate and methanol) were selected and tested, 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: 1, and the ratio of mobile phase 2: the volume ratio is 2: 4: 1, and the ratio of mobile phase 3: the volume ratio is 1: 4: 1, and the ratio of mobile phase 4: the volume ratio is 4: 2: 1, and the ratio of mobile phase 5: the volume ratio is 4: 1, and the mobile phase is 6: the volume ratio is 4: 0.5: 1, and the ratio of mobile phase 7: the volume ratio is 4: 4, and the ratio of mobile phase 8: the volume ratio is 4: 2.

The degrees of separation obtained for mobile phase 1 to mobile phase 8 were: 1.68, 1.62, 1.59, 1.58, 1.59, 1.60, 1.56, 1.54.

The retention time of galangin in mobile phase 1-8 is 41.1, 41.3, 41.6, 41.5, 41.6, 42.0, and 41.8min, respectively.

The purification and separation process of the homoleptic medicinal material is divided into three general basic steps, wherein the first step is an initial crushing and sieving step, and then the medicinal material is further extracted and filtered in an adsorption resin mode, so that the purified medicinal material is obtained. Then, the galangin in the galangin is separated and purified by means of high performance liquid chromatography and improved mobile phases of chloroform, ethyl acetate and methanol, wherein the volume ratio of the chloroform to the ethyl acetate to the methanol is 4: 1, through selection of the mobile phases, the separation degree is kept at 1.68, and the retention time of the galangin is advanced to 41.1 minutes. In contrast, when a mixture of methanol and phosphoric acid was used, the separation degree of the mixture 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: during 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: during 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: during the high performance liquid chromatography separation, the mobile phase is chloroform and ethyl acetate, and the volume ratio is 1: 1.

The degrees of separation of comparative examples 1-3 are all below 1.4, with the degree of separation of comparative example 1 being 1.30, the degree of separation of comparative example 2 being 1.29, and the degree of separation of comparative example 3 being 1.35.

The invention adopts high performance liquid chromatography to separate galangin, and the purity of the separated galangin is 99% or more.

It is to be understood that the described embodiments are merely a few embodiments of the 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.