阅读说明：本技术 一种从牛肝菌中提取分离甜菜碱脂的方法 (Method for extracting and separating betaine ester from bolete ) 是由 周莉 朱津瑞 杨富 于 2021-09-10 设计创作，主要内容包括：本发明公开了一种从牛肝菌中提取分离高纯度甜菜碱脂的方法,包括：步骤(1)、牛肝菌干燥后粉碎,得到牛肝菌粉末,以丙酮为提取试剂,浸泡过夜,过滤除去滤渣,得到丙酮提取液；丙酮提取液减压蒸馏除去溶剂,得到粗提物；步骤(2)、粗提物用水分散,依次加入石油醚和乙酸乙酯进行萃取,得到石油醚萃取物、乙酸乙酯萃取物；步骤(3)、将乙酸乙酯萃取物用丙酮溶解,过氨基固相萃取柱,依次以己烷、氯仿、乙酸乙酯进行洗脱,分别收集己烷洗脱液、氯仿洗脱液、乙酸乙酯洗脱液；氯仿洗脱液、乙酸乙酯洗脱液进行减压浓缩,分别得到以DGTS和DGCC为主的氯仿洗脱物,以LDGTS和DGTS为主的乙酸乙酯洗脱物,甜菜碱脂纯度大于99％。(The invention discloses a method for extracting and separating high-purity betaine ester from bolete, which comprises the following steps: drying and crushing bolete to obtain bolete powder, soaking the bolete powder in acetone serving as an extraction reagent overnight, and filtering to remove filter residues to obtain an acetone extracting solution; distilling the acetone extract under reduced pressure to remove the solvent to obtain a crude extract; dispersing the crude extract with water, and sequentially adding petroleum ether and ethyl acetate for extraction to obtain a petroleum ether extract and an ethyl acetate extract; dissolving the ethyl acetate extract with acetone, passing through an amino solid phase extraction column, sequentially eluting with hexane, chloroform and ethyl acetate, and respectively collecting hexane eluent, chloroform eluent and ethyl acetate eluent; concentrating chloroform eluate and ethyl acetate eluate under reduced pressure to obtain chloroform eluate mainly containing DGTS and DGCC, and ethyl acetate eluate mainly containing LDGTS and DGTS, wherein purity of betaine ester is greater than 99%.)

1. A method for extracting and separating high-purity betaine fat from bolete is characterized by comprising the following steps:

drying and crushing bolete to obtain bolete powder, soaking the bolete powder in acetone serving as an extraction reagent overnight, and filtering to remove filter residues to obtain an acetone extracting solution; distilling the acetone extract under reduced pressure to remove the solvent to obtain a crude extract;

dispersing the crude extract with water, and sequentially adding petroleum ether and ethyl acetate for extraction to obtain a petroleum ether extract and an ethyl acetate extract;

dissolving the ethyl acetate extract with acetone, passing through an amino solid phase extraction column, sequentially eluting with hexane, chloroform and ethyl acetate, and respectively collecting hexane eluent, chloroform eluent and ethyl acetate eluent; and concentrating the chloroform eluate and the ethyl acetate eluate under reduced pressure to obtain chloroform eluate mainly containing DGTS and DGCC and ethyl acetate eluate mainly containing LDGTS and DGTS.

2. The method for extracting and separating high-purity betaine lipid from bolete as claimed in claim 1, wherein in the step (1), the mass-to-volume ratio of bolete powder to acetone is 1:40 g/mL.

3. The method for extracting and separating high-purity betaine lipid from bolete as claimed in claim 1, wherein in the step (2), the mass ratio of the crude extract to water is 1: 5-1: 6; the volume ratio of the petroleum ether to the water is 1: 1-1: 2 during each extraction; the volume ratio of the ethyl acetate to the water is 1: 1-1: 2 in each extraction.

4. The method for extracting and separating high-purity betaine esters from bolete as claimed in claim 1, wherein the amino solid phase extraction column in step (3) is activated with acetone of 1 column capacity before use.

5. The method for extracting and separating high-purity betaine lipid from bolete as claimed in claim 1, wherein in the step (3), the mass volume of the ethyl acetate extract and the acetone is 1: 30-1: 40g/mL or kg/L.

6. The method for extracting and separating high-purity betaine lipid from bolete as claimed in claim 1, wherein in the step (3), the amount of hexane is 8-8.5 column capacity; the amount of chloroform is 13-13.5 column capacity; the dosage of the ethyl acetate is 24-25 column volumes.

7. The method for extracting and separating high-purity betaine lipid from bolete as claimed in claim 1, comprising: determining the type and purity of betaine ester by high performance liquid chromatography-mass spectrometry; wherein, the conditions of the high performance liquid chromatography are as follows: c8 column (Agilent, Eclipse Plus C8,5 μm, 4.6X 250mm), mobile phase water and acetonitrile, flow rate of 1mL/min, elution gradient as follows:

Technical Field

The invention belongs to the field of extraction and separation, and particularly relates to a method for extracting and separating high-purity betaine ester from bolete.

Background

Betaine lipids are a class of glycerolipids that are phosphorus-free and are widely found in algae, bryophytes, fungi, and some simple protozoa and photosynthetic bacteria. Three different types of betaine esters are known in seaweed, namely: 1, 2-diacylglycerol-O-4 '- (N, N, N-trimethyl) homoserine [1, 2-diacylglycerol-O-4' - (N, N, N-trimethyl) homoserine, DGTS ], 1, 2-diacylglycerol-O-2 '- (N, N, N-trimethyl) -beta-alanine [1, 2-diacylglycerol-O-2' - (N, N, N-trimethyl) -beta-alanine, DGTA ], and 1, 2-diacylglycerol-3-O- (carboxymethyl) choline [1, 2-diacylglycerol-3-O- (N-hydroxymethyl) choline, DGCC ].

Betaine esters can be used as a biomarker related to the growth and salt tolerance of microalgae. In addition, researches show that the betaine lipid has anti-inflammatory activity, can be used as a potential anti-inflammatory agent source, and provides a new idea for development of products such as medicines and health care products.

Betaine lipids can be extracted from algal cells or plant material by conventional lipid analysis techniques. The traditional method for separating betaine ester is two-dimensional thin-layer chromatography, and a solvent system of the method is developed and improved for many times, and organic solvents such as chloroform, methanol and the like are generally adopted. The method has many reports on the typical separation of betaine ester, but the operation is complicated, and the content of the obtained product is not high.

The Boletus edulis is a large-scale fungus of Boletaceae of Agaricales, is famous for its fleshy texture, and is very similar to Boletus edulis, and the germplasm resources of Boletus edulis of China are very rich, and mainly distributed in areas such as cloud, precious, Dian, Sichuan, Guangdong, etc., and the variety is as many as 390, and 199 of them are edible. The bolete is not only rare wild edible fungus, but also has good edible value and certain medicinal effects, such as enhancing immunity, improving microcirculation of organisms and the like. The main chemical components of bolete include terpenoids, steroids, phenols, polysaccharides, alkaloids and the like, and in addition, a large amount of betaine ester is contained. Since the components of Boletus edulis are complex, purification of betaine esters becomes difficult, and few reports have been made on the extraction of betaine esters from fungi. Therefore, a method which can extract and separate high-purity betaine ester from fungi and is simple to operate is needed.

Disclosure of Invention

Screening 7 kinds of edible fungus in Yunnan province, namely, dried shiitake mushroom (Lacciaia laccata), Termitomyces albuminosus (Termitomyces eurrhiza), Tricholoma matsutake (Tricholoma matsutake), Boletus deliciosus (Boletus edulis), chana-ellus cibarius Fr.), Thelephora ganbajun Zang (Thermopara ganbajun Zang) and Sarcodon aspratum, and determining the Boletus as a raw material for extracting and separating the betaine fat according to the screening result and the combination cost. The invention aims to provide a method for extracting and separating high-purity betaine ester from bolete, which is simple and convenient to operate.

TABLE 1 betaine lipid content in fungi

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

a method for extracting and separating high-purity betaine ester from bolete comprises the following steps:

drying and crushing bolete to obtain bolete powder, soaking the bolete powder in acetone serving as an extraction reagent overnight, and filtering to remove filter residues to obtain an acetone extracting solution; distilling the acetone extract under reduced pressure to remove the solvent to obtain a crude extract;

dispersing the crude extract with water, and sequentially adding petroleum ether and ethyl acetate for extraction to obtain a petroleum ether extract and an ethyl acetate extract;

dissolving the ethyl acetate extract with acetone, passing through an amino solid phase extraction column, sequentially eluting with hexane, chloroform and ethyl acetate, and respectively collecting hexane eluent, chloroform eluent and ethyl acetate eluent; and concentrating the chloroform eluate and the ethyl acetate eluate under reduced pressure to obtain chloroform eluate mainly containing DGTS and DGCC and ethyl acetate eluate mainly containing LDGTS and DGTS.

In the step (1), the mass-to-volume ratio of the boletus powder to the acetone is 1:40g/mL, namely 2000mL of acetone is used for every 500g of boletus powder.

In the step (2), the mass ratio of the crude extract to water is 1: 5-1: 6; the volume ratio of the petroleum ether to the water is 1: 1-1: 2 during each extraction; the volume ratio of the ethyl acetate to the water is 1: 1-1: 2 in each extraction.

Specifically, adding water into the crude extract, uniformly dispersing, adding petroleum ether for extraction, taking out the upper layer which is petroleum ether and the lower layer which is a water phase, continuously adding the petroleum ether, and repeating for multiple times until the extraction is complete; combining the petroleum ether extract, and recovering the solvent by reduced pressure distillation to obtain a petroleum ether extract; after the petroleum ether is completely extracted, adding ethyl acetate into the water phase for extraction, taking the upper layer of ethyl acetate and the lower layer of water phase out, continuously adding ethyl acetate, and repeating for multiple times until the ethyl acetate is completely extracted; the ethyl acetate extracts were combined and the solvent was recovered by distillation under reduced pressure to give an ethyl acetate extract.

In the step (3), the mass volume of the ethyl acetate extract and the acetone is 1: 30-1: 40g/mL or kg/L.

The amino solid phase extraction column was activated with 1 column volume of acetone before use.

The amount of the hexane is 8-8.5 column volumes; the amount of chloroform is 13-13.5 column capacity; the dosage of the ethyl acetate is 24-25 column volumes.

As a further preferable technical scheme of the method for extracting and separating the high-purity betaine lipid from the bolete, the method comprises the following steps: determining the type and purity of betaine ester by high performance liquid chromatography-mass spectrometry; wherein, the conditions of the high performance liquid chromatography are as follows: c8 column (Agilent, Eclipse Plus C8,5 μm, 4.6X 250mm), mobile phase water and acetonitrile, flow rate of 1mL/min, elution gradient as follows:

the invention has the beneficial effects that:

the method is quick and simple and convenient to operate, the betaine ester in the bolete is extracted by acetone, part of small-polarity impurities are removed by extraction, the betaine ester is obtained by separating through an amino solid-phase extraction column, and the purity of the betaine ester is verified by using high performance liquid chromatography-mass spectrometry: DGTS, DGCC and LDGTS were detected in chloroform and ethyl acetate eluates, and betaine lipid purity was greater than 99%.

Drawings

FIG. 1 is a flow chart of the operation of the present invention.

FIG. 2 shows the results of the concentration of the eluents from the amino solid phase extraction column.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

As shown in fig. 1, betaine esters were extracted and isolated from bolete:

(1) crude extraction of betaine esters in bolete

Drying and crushing bolete to obtain about 1500g of bolete powder, adding 2000mL of acetone into 6L of acetone per 500g of bolete powder, soaking overnight, and filtering to obtain acetone extract; the acetone extract was distilled under reduced pressure to remove the solvent, whereby about 55g of a crude extract was obtained.

(2) And extraction of the extract

Adding 300mL of water into the crude extract for uniform dispersion, adding 200mL of petroleum ether for extraction, taking out the upper layer which is the petroleum ether and the lower layer which is the water phase, and carrying out reduced pressure distillation to recover the solvent; this was repeated several times until the petroleum ether extraction was complete to give about 30g of petroleum ether extract. After the petroleum ether is completely extracted, adding 200mL of ethyl acetate into the water phase for extraction, taking out the upper layer which is ethyl acetate and the lower layer which is the water phase, and carrying out reduced pressure distillation to recover the solvent; this was repeated several times until the extraction with ethyl acetate was complete to give about 1g of ethyl acetate extract.

(3) Purifying the mixture

Before loading, the amino solid phase extraction column was previously activated with 6mL of acetone.

0.06g of ethyl acetate extract is taken, fully dissolved in 2mL of acetone, passed through an amino solid phase extraction column (500mg/6mL, namely 500mg of silica gel matrix aminopropyl bonded phase is filled, the column capacity is 6mL), eluted by 50mL of hexane, 80mL of chloroform and 150mL of ethyl acetate in sequence, and collected once per 10mL of eluent, and then spotted on a silica gel plate (a developing agent is dichloromethane: ethyl acetate is 2:3V/V, phosphomolybdic acid is developed), and the hardened fruit shows: eluting with the same solvent, and mixing according to the plate result to obtain hexane eluate, chloroform eluate, and ethyl acetate eluate; the hexane eluate, chloroform eluate and ethyl acetate eluate were concentrated under reduced pressure to remove the solvent, to give hexane eluate 0.005g, chloroform eluate 0.01g and ethyl acetate eluate 0.03 g.

(4) And checking the purity

Silica gel plates (specification 50mm x 100mm, thickness 0.2-0.25mm) were spotted with dichloromethane ethyl acetate 2:3V/V as developing agent and phosphomolybdic acid developed to fully develop the compound on the silica gel plates (fig. 2), showing that the hexane eluate was essentially free of betaine esters.

Detecting the purity of betaine ester in chloroform eluate and ethyl acetate eluate by high performance liquid chromatography-mass spectrometry: respectively taking chloroform eluate and ethyl acetate eluate, dissolving with methanol (chromatogram purity) (mass volume ratio of chloroform eluate to methanol is 1:20g/mL, and mass volume ratio of ethyl acetate eluate to methanol is 1:20g/mL), filtering with 0.22 μm nylon filter, and analyzing the type and purity of betaine lipid contained in the filtrate by liquid chromatography-mass spectrometry.

High performance liquid chromatography conditions: c8 column (Agilent, Eclipse Plus C8,5 μm, 4.6X 250mm), water and acetonitrile as mobile phase, flow rate of 1mL/min, room temperature column, 2 μ L sample, elution gradient as shown in Table 2.

TABLE 2 elution gradient procedure

Mass spectrometry conditions: ESI source condition settings: spray voltage (3.2Kv), sheath gas flow rate (8.4arb), sheath gas N2 (purity > 95%; 35a.u), assist gas N2 (purity > 95%; 10a.u), capillary temperature: 325 ℃, auxiliary gas heating temperature: 350 ℃, assist gas flow rate: 24.2arb, collision energy: 25/30/35eV, the primary mass spectrum scanning range m/z is between 200 and 1000, and the scanning mode is as follows: positive and negative ions are separately scanned.

The results of hplc-ms analyses are shown in tables 3 and 4, and it is understood that the chloroform eluate is mainly DGTS and DGCC, and the ethyl acetate eluate is mainly LDGTS and DGTS.

TABLE 3 high performance liquid chromatography-Mass Spectrometry results of chloroform eluate

TABLE 4 high performance liquid chromatography-Mass Spectrometry results of Ethyl acetate eluate

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. That is, all equivalent changes and modifications made according to the contents of the claims of the present invention should be the technology of the present invention.