阅读说明：本技术 一种从橄榄叶中同时提取橄榄苦甙、山楂酸和齐墩果酸的方法 (Method for simultaneously extracting oleuropein, maslinic acid and oleanolic acid from olive leaves ) 是由 宁丹 曹慧璋 易宇阳 贺玉婷 于 2021-06-04 设计创作，主要内容包括：本发明公开了一种从橄榄叶中同时提取橄榄苦甙、山楂酸和齐墩果酸的方法,包括以下步骤：将橄榄叶粉碎后用乙醇提取,将提取液固液分离,得到上清液和沉淀物；将上清液上大孔吸附树脂柱,用水和乙醇依次进行洗脱,收集乙醇洗脱液,即得橄榄苦甙提取物；将沉淀物加入乙醇提取后固液分离,收集滤渣；取滤渣加入碱水进行提取,提取液固液分离,收集滤液；将所得的滤液使用阴离子交换树脂进行纯化,依次使用水、碱性乙醇溶液洗脱,收集碱性乙醇洗脱部分,浓缩,醇沉后固液分离,所得上清液即为山楂酸,所得沉淀物即为齐墩果酸。本发明方法可以同时获得山楂酸、齐墩果酸及橄榄苦甙,简单,高效,对环境友好,易实现工业化生产。(The invention discloses a method for simultaneously extracting oleuropein, maslinic acid and oleanolic acid from olive leaves, which comprises the following steps: pulverizing folium Canarii albi, extracting with ethanol, and performing solid-liquid separation to obtain supernatant and precipitate; loading the supernatant on macroporous adsorbent resin column, sequentially eluting with water and ethanol, and collecting ethanol eluate to obtain oleuropein extract; adding ethanol into the precipitate, extracting, performing solid-liquid separation, and collecting filter residue; adding alkaline water into the filter residue for extraction, carrying out solid-liquid separation on the extracting solution, and collecting the filtrate; purifying the obtained filtrate with anion exchange resin, sequentially eluting with water and alkaline ethanol solution, collecting alkaline ethanol eluate, concentrating, precipitating with ethanol, and performing solid-liquid separation to obtain supernatant as crataegolic acid and precipitate as oleanolic acid. The method can simultaneously obtain the maslinic acid, the oleanolic acid and the oleuropein, is simple, efficient and environment-friendly, and is easy to realize industrial production.)

1. A method for simultaneously extracting oleuropein, maslinic acid and oleanolic acid from olive leaves is characterized by comprising the following steps:

s1, crushing olive leaves, extracting with ethanol, filtering the extracting solution, removing ethanol from the filtrate, and performing solid-liquid separation to obtain supernatant and precipitate;

s2, putting the supernatant obtained in the step S1 on a macroporous adsorption resin column, sequentially eluting with water and ethanol, and collecting ethanol eluent to obtain oleuropein extract;

s3, adding ethanol with the volume concentration of 10-30% into the precipitate obtained in the step S1 for extraction, carrying out solid-liquid separation, and collecting filter residue;

s4, adding alkaline water into the filter residue obtained in the step S3 for extraction, carrying out solid-liquid separation on the extracting solution, and collecting the filtrate;

s5, purifying the filtrate obtained in the step S4 by using anion exchange resin, eluting by using water and alkaline ethanol in sequence, and collecting the alkaline ethanol eluent;

s6, concentrating the alkaline ethanol eluent obtained in the step S5, adding acid to adjust the pH value, filtering, adding ethanol into filter residues, precipitating with ethanol, and performing solid-liquid separation to obtain supernate, namely maslinic acid; the obtained precipitate is oleanolic acid.

2. The method according to claim 1, wherein in step S1, the ratio of olive leaves to ethanol is 1 g: 6-15 mL.

3. The method according to claim 1, wherein in step S1, the volume concentration of ethanol is 40-90%; the extraction is heating reflux extraction, the extraction temperature is 60-85 ℃, the extraction times are 2-3 times, and the extraction time is 1-3 hours each time.

4. The method according to claim 1, wherein in step S3, the feed-to-liquid ratio of the precipitate to ethanol is 1 g: 6-12 mL, wherein the extraction is heating reflux extraction, the extraction temperature is 50-90 ℃, the extraction time is 0.5-2 hours, and the extraction times are 1-3.

5. The method according to claim 1, wherein in step S4, the feed-liquid ratio of the filter residue to the alkaline water is 1 g: 6-15 mL.

6. The method of claim 1, wherein in step S4, the alkaline water is one of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution and potassium carbonate solution.

7. The method according to claim 1, wherein in step S4, the pH of the alkaline water is 9-14; preferably, the pH value of the alkaline water is 12-13.

8. The method according to claim 1, wherein in step S5, the pH of the alkaline ethanol is 9-13, the volume concentration is 50-90%, and the elution amount is 2-4 BV.

9. The method according to claim 1, wherein in step S6, the acid is one of hydrochloric acid, sulfuric acid and nitric acid, preferably the acid is hydrochloric acid.

10. The method according to claim 1, wherein in step S6, the volume concentration of ethanol is 80-90%, preferably the volume concentration of ethanol is 90%, and the addition amount is 3-6 BV.

Technical Field

The invention belongs to the technical field of biological extraction, and particularly relates to a method for simultaneously extracting oleuropein, maslinic acid and oleanolic acid from olive leaves.

Background

The olive leaf is dried leaf of Olea europaea L (Olea-africana, Olea-africana L.) of Oleaceae, and has flowering period of 4-5 months and mature fruit of 10-12 months. It has strong resistance to microbial attack, can grow for hundreds of years, and has been used as folk medicine by people in Mediterranean region to treat fever and other diseases such as malaria. The folium Canarii albi is rich in oleuropein, hydroxytyrosol, oleanolic acid, maslinic acid, and flavone. The hawthorn acid radical is shown by research: the maslinic acid has high safety to human bodies, and modern pharmacology shows that maslinic acid has multiple pharmacological activities of resisting bacteria, diminishing inflammation, reducing blood sugar and blood fat, resisting HIV (human immunodeficiency virus), resisting tumors, resisting osteoporosis and the like. Oleuropein has effect in scavenging superoxide anion free radical (O)₂-. g) hydrogen peroxide (H)₂O₂) NO and peroxynitroso anion (ONOO-), and also has anticancer, antithrombotic, arteriosclerosis preventing and neuroprotective effects, and thus is widely used in the fields of pharmaceuticals, health foods, skin care products, and the like. Oleanolic acid has been marketed for many years as an anti-hepatitis agent, and researches show that oleanolic acid has an immunity promoting effect, so that a method capable of simultaneously extracting three kinds of oleuropein, maslinic acid and oleanolic acid is difficult to exist in the prior art, and the yield of the extracted oleuropein, maslinic acid and oleanolic acid can be more than 90%.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a method for simultaneously extracting oleuropein, maslinic acid and oleanolic acid from olive leaves, which is simple and efficient, and the obtained oleuropein, maslinic acid and oleanolic acid have high purity and high yield.

According to one aspect of the invention, the method for simultaneously extracting oleuropein, maslinic acid and oleanolic acid from olive leaves is provided, and comprises the following steps:

s1, crushing olive leaves, extracting with ethanol, filtering the extracting solution, removing ethanol from the filtrate, and performing solid-liquid separation to obtain supernatant and precipitate;

s2, putting the supernatant obtained in the step S1 on a macroporous adsorption resin column, sequentially eluting with water and ethanol, and collecting ethanol eluent to obtain oleuropein extract;

s3, adding ethanol with the volume concentration of 10-30% into the precipitate obtained in the step S1 for extraction, carrying out solid-liquid separation, and collecting filter residue;

s4, adding alkaline water into the filter residue obtained in the step S3 for extraction, carrying out solid-liquid separation on the extracting solution, and collecting the filtrate;

s5, purifying the filtrate obtained in the step S4 by using anion exchange resin, eluting by using water and alkaline ethanol in sequence, and collecting the alkaline ethanol eluent;

s6, concentrating the alkaline ethanol eluent obtained in the step S5, adding acid to adjust the pH value, filtering, adding ethanol into filter residues, precipitating with ethanol, and performing solid-liquid separation to obtain supernate, namely maslinic acid; the obtained precipitate is oleanolic acid.

In some embodiments of the present invention, in step S1, the ratio of olive leaves to ethanol is 1 g: 6-15 mL.

In some embodiments of the invention, in the step S1, the volume concentration of the ethanol is 40% to 90%, the extraction is heating reflux extraction, the extraction temperature is 60 ℃ to 85 ℃, the extraction times are 2 to 3, and the extraction time is 1 to 3 hours each time.

In some embodiments of the present invention, in step S1, the solid-liquid separation is centrifugation.

In some embodiments of the present invention, in step S1, the ethanol is removed by distillation under reduced pressure.

In some embodiments of the present invention, in the step S2, the volume concentration of ethanol is 50-70%.

In some embodiments of the present invention, in the step S2, the model of the macroporous adsorbent resin column is AB-8. The AB-8 type macroporous resin is low in polarity, the average pore diameter is 13-14 nm, and the specific surface area is 480-520 m²(ii) in terms of/g. Because oleanolic acid and maslinic acid are weak polar substances, have certain hydrophobicity and weak capability of generating hydrogen bonds, the oleanolic acid and the maslinic acid are easy to be nonpolar and weak and extremely weakAdsorbed by the resin. Secondly, when the macroporous adsorption resin adsorbs substances, the adsorbed components are diffused to the inner surface of the resin hole through the aperture of the resin and are adsorbed, when the aperture of the resin is smaller than the aperture of the adsorbed effective component molecules, the effective component molecules can not enter the resin and can only be adsorbed on the outer surface of the resin, when the aperture of the resin is larger than the aperture of the effective component molecules, the effective component can be adsorbed on the outer surface of the resin and also can be adsorbed on the inner surface of the resin, and for the maslinic acid and the oleanolic acid, the molecular weights are 472 and 456 respectively, so that the adsorption effect of the AB-8 macroporous resin is better. The ratio of the diameter to the height of the resin column is preferably 1:4 to 8, and more preferably 1: 6.

In some embodiments of the present invention, the step S2 further comprises drying the oleuropein extract to obtain oleuropein product, preferably, the drying is spray drying.

In some embodiments of the present invention, in step S3, the feed-to-liquid ratio of the precipitate to the ethanol solution is 1 g: 6-12 mL, wherein the extraction is heating reflux extraction, the extraction temperature is 50-90 ℃, the extraction time is 0.5-2 hours, and the extraction times are 1-3.

In some embodiments of the present invention, in the step S3, it is preferable that the volume concentration of the ethanol is 20% to 30%.

In some embodiments of the present invention, in the step S3, the solid-liquid separation is one of filtration and centrifugation.

In some embodiments of the present invention, in step S4, the feed-liquid ratio of the filter residue to the alkaline water is 1 g: 6-15 mL.

In some embodiments of the invention, the base water is one of a sodium hydroxide solution, a potassium hydroxide solution, a sodium carbonate solution, a potassium carbonate solution; preferably, the alkaline water is a sodium hydroxide solution, and more preferably, the alkaline water is a 0.1mol/L sodium hydroxide solution.

In some embodiments of the invention, the alkaline water has a pH of 9 to 14; preferably, the pH value of the alkaline water is 12-13.

In some embodiments of the present invention, in the step S4, the extraction is heating reflux extraction, the extraction temperature is 50 ℃ to 90 ℃, the extraction time is 0.5h to 2h, and the extraction times are 1 to 3 times.

In some embodiments of the present invention, in the step S4, the solid-liquid separation is one of filtration and centrifugation.

In some embodiments of the present invention, in the step S5, the model of the anion exchange resin is 201X 7.

In some embodiments of the present invention, the volume of the water in the step S5 is 1-5 BV.

In some embodiments of the invention, in the step S5, the pH of the alkaline ethanol is 9-13, the volume concentration is 50% -90%, and the elution amount is 2 BV-4 BV.

In some embodiments of the invention, the alkaline ethanol solution is adjusted in pH with sodium hydroxide.

In some embodiments of the present invention, in the step S6, the acid is one of hydrochloric acid, sulfuric acid and nitric acid, and preferably, the acid is hydrochloric acid.

In some embodiments of the present invention, in the step S6, the pH is in the range of 5 to 7.

In some embodiments of the present invention, in the step S6, the volume concentration of the ethanol is 80% to 90%, preferably the volume concentration of the ethanol is 90%, and the addition amount is 3BV to 6 BV.

In some embodiments of the present invention, in step S6, ethanol is added, stirred and dissolved at 60 to 80 ℃, and then subjected to alcohol precipitation.

In some embodiments of the invention, in the step S6, the obtaining of maslinic acid further includes concentrating the supernatant under reduced pressure until the volume concentration of ethanol is 50% -70%, precipitating a precipitate, and drying the precipitate to obtain maslinic acid.

In some embodiments of the invention, the condition for separating out the precipitate is 4-30 ℃ and the precipitate is placed for 2-10 h.

According to the embodiment of the invention, at least the following beneficial effects are achieved: the method adopts the scheme that after the extraction of an ethanol aqueous solution, oleuropein is separated out by utilizing the solubility difference of the oleuropein, maslinic acid and oleanolic acid; then, the impurities with larger polarity are removed by extracting with ethanol solution, the content of the sample loading solution is improved, and the adsorption capacity of the resin is increased; through the extraction of alkaline water, maslinic acid and oleanolic acid form salt in an alkaline environment, so that the solubility of maslinic acid and oleanolic acid in water is increased, and after the impurities are removed by using ethanol, the extraction of the alkaline water is performed, so that the solubility of the alkaline water to maslinic acid and oleanolic acid can be increased, and the extraction transfer rate of maslinic acid and oleanolic acid is improved; primarily purifying the maslinic acid and the oleanolic acid by using ion exchange resin, dissolving the maslinic acid and the oleanolic acid by combining high-concentration ethanol, standing the mixture to obtain high-purity oleanolic acid, and concentrating the high-purity oleanolic acid to separate out maslinic acid so as to achieve the aim of purification. After separating out the oleuropein crude product, the invention uses low-concentration ethanol to remove impurities, uses aqueous alkali solution to extract, uses anion exchange resin to purify, dissolves and separates out alcohol, and then concentrates and separates out to obtain high-purity maslinic acid and oleanolic acid, and has short production period and high yield. The scheme of the invention can simultaneously obtain the maslinic acid, the oleanolic acid and the oleuropein, does not use other toxic and harmful organic reagents except ethanol, is environment-friendly, has strong process operability and low cost, and is easy to realize industrial production.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a high performance liquid chromatogram of an oleuropein sample in a test example of the present invention;

FIG. 2 is a high performance liquid chromatogram of a maslinic acid sample in a test example of the present invention;

FIG. 3 is a high performance liquid chromatogram of an oleanolic acid sample in a test example of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

The olive leaf raw material is from Gansu Longnan, wherein the content of oleuropein in the raw material is as follows: 6.0%, maslinic acid content: 0.51%, oleanolic acid content: 1 percent.

Example 1

In the embodiment, oleuropein, oleanolic acid and maslinic acid are simultaneously prepared in olive leaves, and the specific process comprises the following steps:

(1) pulverizing 100kg of olive leaf raw materials into powder, and adding the powder which is 1g of olive leaf material liquid: 15mL of 80% ethanol, 2 times of extraction at 85 ℃ and 2 h/time of extraction. Collecting all extractive solutions, concentrating under reduced pressure until no alcohol smell is observed, and centrifuging to obtain supernatant and precipitate.

(2) Purifying the supernatant with AB-8 resin, sequentially eluting with 3BV pure water and 2BV 60% ethanol, and collecting 60% ethanol eluate.

(3) Concentrating 60% ethanol eluate, and spray drying to obtain 9.06kg powder (oleuropein content 65.11%).

(4) Adding the precipitate obtained in the step (1) into a solution with the addition and precipitation material liquid ratio of 1 g: extracting with 6mL of 30% ethanol at 70 deg.C for 0.5 hr for 2 times, and collecting the residue. Adding an additive into the filter residue, wherein the ratio of the additive to the filter residue is 1 g: 10mL of alkaline water with the pH value of 12, the extraction temperature of 80 ℃, the extraction time of 1h, the extraction times of 1 time, the filtration of the extracting solution and the collection of the filtrate. The filtrate was purified using 201X7 resin, followed by 4BV pure water and 3BV 70% ethanol (pH 10 adjusted with sodium hydroxide) and ethanol water eluate was collected.

(5) Concentrating the 70% ethanol eluate, adjusting pH to 6 with acid, filtering, adding 3BV 90% ethanol into the residue, stirring at 80 deg.C for dissolving, standing at 10 deg.C for 10 hr, centrifuging, drying the precipitate, and pulverizing to obtain 0.99kg powder with oleanolic acid content of 98.11%.

(6) Concentrating the centrifugate under reduced pressure until the concentration of ethanol is 70%, standing at 20 deg.C for 4 hr, oven drying the residue, and pulverizing to obtain 0.5kg of powder with maslinic acid content of 98.55%.

The oleuropein purity is 65.11% and yield is 98.31% according to high performance liquid chromatography; the purity of oleanolic acid is 98.55%, the yield is 97.13%, the purity of crataegolic acid is 98.55%, and the yield is 96.6%.

Example 2

In the embodiment, oleuropein, oleanolic acid and maslinic acid are simultaneously prepared in olive leaves, and the specific process comprises the following steps:

(1) pulverizing 100kg of olive leaf raw materials into powder, and adding the powder which is 1g of olive leaf material liquid: 10mL of 60% ethanol, and extracting at 85 deg.C for 3 times, wherein the extraction time is 1 h/time. Collecting all extractive solutions, concentrating under reduced pressure until no alcohol smell is detected, and centrifuging to obtain clear liquid and precipitate.

(2) Purifying the supernatant with D101 resin, sequentially eluting with 2BV pure water and 3BV 70% ethanol, and collecting 70% ethanol eluate.

(3) Concentrating 70% ethanol eluate, and spray drying to obtain 9.16kg powder (oleuropein content 64.21%).

(4) Adding the precipitate obtained in the step (1) into a solution with the addition and precipitation material liquid ratio of 1 g: extracting with 10mL of low-concentration 20% ethanol for 1 time for 1h at 50 deg.C, filtering the extractive solution, and collecting the residue. Adding an additive into the filter residue, wherein the ratio of the additive to the filter residue is 1 g: 10mL of alkaline water with pH of 13, extracting at 90 deg.C for 0.5h for 2 times, filtering the extractive solution, and collecting the filtrate. The filtrate was separated and purified with 201X7 resin, and 5BV of pure water and 3BV of 80% ethanol (pH 10 adjusted with sodium hydroxide) were used in this order to collect the ethanol eluate.

(5) Concentrating the ethanol eluate obtained in step (4), adding acid into the concentrated solution to adjust pH to 7, filtering, adding 5BV 90% ethanol into the filter residue, stirring and dissolving at 70 deg.C, standing at 15 deg.C for 8h, centrifuging, drying the precipitate, and pulverizing to obtain 1.0kg of powder with oleanolic acid content of 98.05%.

(6) Concentrating the centrifugate under reduced pressure until the concentration of ethanol is 50%, standing at 20 deg.C for 6 hr, oven drying the residue, and pulverizing to obtain 0.51kg powder containing maslinic acid 98.10%.

The oleuropein purity is 64.21% and yield is 98.02% according to high performance liquid chromatography; the purity of oleanolic acid is 98.05%, the yield is 98.05%, the purity of crataegolic acid is 98.10%, and the yield is 98.10%.

Example 3

In the embodiment, oleuropein, oleanolic acid and maslinic acid are simultaneously prepared in olive leaves, and the specific process comprises the following steps:

(1) pulverizing 100kg of olive leaf raw materials into powder, and adding the powder which is 1g of olive leaf material liquid: 15mL of 50% ethanol, and extracting at 85 ℃ for 3 times for 2 h/time. Collecting all extractive solutions, concentrating under reduced pressure until no alcohol smell is detected, and centrifuging to obtain clear liquid and precipitate.

(2) Purifying the supernatant with D101 resin, sequentially eluting with 3BV pure water and 4BV 50% ethanol solution, and collecting 50% ethanol eluate.

(3) Concentrating 50% ethanol water eluate, and spray drying to obtain 9.09kg powder (oleuropein content 65.05%).

(4) Adding the precipitate obtained in the step (1) into a mixture of an additive and a precipitate material in a liquid ratio of 1 g: extracting with 8mL of 30% ethanol water solution with low concentration for 2 times, extracting for 0.5 h/time, extracting at 90 deg.C, filtering the extractive solution, and collecting the residue. Adding an additive into the filter residue, wherein the ratio of the additive to the filter residue is 1 g: 15mL of alkaline water with the pH value of 12, the extraction temperature of 80 ℃, the extraction time of 0.5 h/time, the extraction times of 1 time, the extraction solution filtration and the filtrate collection. The filtrate was separated and purified with 201X7 resin, and the ethanol eluate was collected by sequentially using 4BV of pure water and 3BV of 80% ethanol (pH 12 adjusted with sodium hydroxide).

(5) Concentrating the ethanol eluate obtained in step (4), adding acid into the concentrated solution to adjust pH to 5, filtering, adding 6BV 90% ethanol into the filter residue, stirring and dissolving at 85 deg.C, standing at 4 deg.C for 10h, centrifuging, drying the precipitate, and pulverizing to obtain 0.98kg of powder with oleanolic acid content of 98.56%. Concentrating the centrifugate under reduced pressure until the concentration of ethanol is 60%, standing at 10 deg.C for 5 hr, oven drying the residue, and pulverizing to obtain 0.50kg powder containing maslinic acid 98.39%.

According to the determination of high performance liquid chromatography, the purity of oleuropein is 65.05%, and the yield is 98.55%; the purity of oleanolic acid is 98.56%, the yield is 96.58%, the purity of crataegolic acid is 98.39%, and the yield is 96.46%.

Comparative example 1

A method for simultaneously preparing oleuropein, maslinic acid and oleanolic acid from olive leaves is different from the method in the embodiment 3 in that 50% ethanol is used for extraction in the step (4), an extracting solution is collected, and the extracting solution is concentrated. 9.09kg of powder with oleuropein content of 65.11% was obtained, 0.3kg of powder with oleanolic acid content of 98.22% was obtained, and 0.20kg of powder with crataegolic acid content of 98.22% was obtained.

The oleuropein purity is 65.11% and yield is 98.6% according to high performance liquid chromatography; the purity of oleanolic acid is 98.22%, the yield is 29.46%, the purity of crataegolic acid is 98.22%, and the yield is 38.5%.

Comparative example 2

A method for simultaneously preparing oleuropein, maslinic acid and oleanolic acid from olive leaves is different from the method in the embodiment 3 in that the extraction of 30% ethanol is not adopted in the step (4), and the filter residue is directly extracted by alkaline water. 9.09kg of powder with oleuropein content of 65.11% was obtained, 0.51kg of powder with oleanolic acid content of 98.22% was obtained, and 0.30kg of powder with crataegolic acid content of 98.22% was obtained.

The oleuropein purity is 65.11% and yield is 98.6% according to high performance liquid chromatography; the purity of oleanolic acid is 98.22%, the yield is 50.09%, and the purity of crataegolic acid is 98.22%, the yield is 57.77%.

Test examples

1. Screening of impurity removal concentration of ethanol

The invention scheme also screens the concentration of the low-concentration ethanol in the step (4) in the example 1, and only differs from the example 1 in that the precipitates obtained in the step (1) are respectively extracted by using water and ethanol with the volume concentration of 10%, 20%, 30% and 50%.

According to the determination of the high performance liquid chromatography, the experimental result shows that the 50% ethanol is adopted for removing impurities, so that a part of crataegolic acid and oleanolic acid can be dissolved, and the yield of the crataegolic acid and the oleanolic acid is reduced; 10% ethanol is adopted for removing impurities, and the ethanol is extremely difficult to recover, so that the resource waste is caused; water is adopted for impurity removal, and as part of impurities are insoluble in water, the impurity removal effect is poor, and the purity of the obtained maslinic acid and oleanolic acid is obviously reduced; and more impurities can be removed by adopting 20-30% of ethanol, and more maslinic acid and oleanolic acid can be reserved.

2. Detection of oleuropein, maslinic acid and oleanolic acid by high performance liquid chromatography

Oleuropein, maslinic acid and oleanolic acid prepared in examples 1 to 3 and comparative examples 1 to 2 were measured and detected by high performance liquid chromatography. The HPLC adopts Agilent 1260ll, and the chromatographic test method is shown in table 1 and table 2:

TABLE 1 conditions for the liquid phase assay of maslinic acid

TABLE 2 Oleuropein liquid phase assay conditions

Chromatographic column	WONDASILTMC18 column (250 mm. times.4.6 mm, 5 μm)
		Sample volume	10μL
Flow rate of flow	1.0mL/min
		Column temperature	30℃
Wavelength of light	254nm
		Temperature of sample plate	Prepared for clinical use
Mobile phase	Acetonitrile: water 26:74(V: V)

TABLE 3 Olea acid solution phase determination conditions

Chromatographic column	WONDASILTMC18 column (250 mm. times.4.6 mm, 5 μm)
		Sample volume	10μL
Flow rate of flow	1.0mL/min
		Column temperature	30℃
Wavelength of light	205nm
		Temperature of sample plate	Prepared for clinical use
Mobile phase	Acetonitrile: 0.1% phosphoric acid aqueous solution (V: V))＝80:20(V:V)

The experimental results are shown in figures 1-3, and it can be seen from the figures that the scheme of the invention successfully prepares oleuropein, crataegolic acid and oleanolic acid, and the prepared oleuropein, crataegolic acid and oleanolic acid have high purity and low impurity content.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.