CN102532217B - Method for purifying and separating high-content oleuropein from olive leaves - Google Patents
Method for purifying and separating high-content oleuropein from olive leaves Download PDFInfo
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- CN102532217B CN102532217B CN201110439186.7A CN201110439186A CN102532217B CN 102532217 B CN102532217 B CN 102532217B CN 201110439186 A CN201110439186 A CN 201110439186A CN 102532217 B CN102532217 B CN 102532217B
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Abstract
The invention discloses a method for purifying and separating high-content oleuropein from olive leaves, which comprises cutting olive leaves, extracting at a low temperature, filtering, concentrating or directly performing chromatography, concentrating at a low temperature, and drying to obtain a product with oleuropein content higher than 80%. The invention adopts a certain concentration of ethanol or water as an extraction solvent for low-temperature extraction, and utilizes resin tandem or secondary adsorption chromatography for separation and purification to obtain the product with oleuropein content higher than 80%. The invention has the advantages of low energy consumption, short production period, environmental friendliness and a simple operation, and is especially suitable for large-scale industrial production.
Description
Technical field
The present invention relates to extract the method that separates high-content oleuropein from leaf of Fructus oleae europaeae.
Background technology
Fructus oleae europaeae belongs to Oleaceae Olea aiphyllium, is that world-renowned woody oleiferous plants is held concurrently really with seeds, and Cultivar has higher edibleness, containing enriching kind of high-quality edible plant oil---Fructus oleae europaeae oil, is famous subtropical fruit tree and Important Economic forest.Mainly be distributed in European Mediterranean country and California of American, Greece, Italy, Tunisia, Spain are for concentrating the place of production.Municipalization introducing culture is economized in China western part and Gansu, the Northwest, Shaanxi, Sichuan, Chongqing, Fujian, Guangxi, Guangdong, Hunan etc.The existing Fructus oleae europaeae in the whole nation exceedes 1,600 ten thousand strains.Wudu District, Longnan City, Gansu Province is current national maximum oil olive base.
In olive leaf, mainly contain the composition such as secoiridoid and glycosides, flavones and glycosides thereof, biflavone and glycosides thereof, low point of Chinese tannin, secoiridoid is main active ingredient.In Olive leaf P.E, main component is the pained material of iridoids, and what its activity was the highest is oleuropein.
Oleuropein is a kind of bimolecular structure phenols and Flavonoid substances, have natural anti-oxidant, antitumor, delay senility, strengthen cardiovascular function, treatment chronic prostatitis, strengthening immunity, conciliation endocrine system, protect the important nourishing function such as liver, anti-inflammatory, also have reducing blood-fat simultaneously, fall the pharmacological action of blood chaff, be widely used in the fields such as food, cosmetic and bio-pharmaceuticals.
The oleuropein of high-content is mainly used in skin care product, can protect skin cells not to be subject to ultraviolet injury, effectively maintains the tender and elasticity of skin, reaches the effect of holding up skin, tender skin.Microherb high-content oleuropein 80% is special, and for skin care product design, its effective constituent is high, of light color, is applicable to very much cosmetic formulations design.
Olive leaf extractive technique mainly includes machine solvent extraction techniques, CO_2 supercritical technology, ultrasonic extraction technology and microwave-assisted extraction technique etc.Organic solvent extraction has that foreign matter content is high, solvent consumption is large, cost is high, pollute the shortcomings such as large.SCF-CO 2 has and extracts the advantages such as content is high, pollution-free, but have high input, cost is high.Involve microwave extracting although ultrasonic and there is the advantages such as energy-conservation, efficient, on producing, be difficult to realize large-scale application.
Bibliographical information or the disclosed oleuropein extraction and separation technology of patent are more at present, but oleuropein content reaches the more than 80% rare report of separating and purifying technology.The patent " extracting method of Olive leaf P.E and technique " (CN 1827127A) of the patent " a kind of Olive leaf P.E extracting method " (CN 1583052A) of Yang Yinke application and Sun Haoyuan application, the product oleuropein that two kinds of patents separate is low, and has all added exogenous material; The patent of Wang Chengzhang, high rosy clouds application " being rich in the preparation method that the leaf of Fructus oleae europaeae of high purity oleuropein extracts " (CN 101003557A), the product that this patent system is standby, content is higher than 50%, but far below 80%; Yao from the strange patent " technique for producing extractive of olive growing leaves " that waits application (CN101049328A), the product that this patent separates, content is 2%～6%; Yao is from the strange patent " olive leaf extract and preparation method " (CN 101084945A) that waits application, although content the highest reach 95%, product yield is low, the production cycle is long, needs standing 24～36 hours; In addition, in Wang Chengzhang etc. " introduce a fine variety Oleuropein in A Si leaf of Fructus oleae europaeae and extract separation and Structural Identification ", adopt repeatedly organic solvent extraction, silica gel column chromatography and HPLC preparative chromatography, the oleuropein that to have isolated content be 95%, but this method is limited to the greatest extent a small amount of sample preparation.In the sample that US Patent No. 5714150 and US98101563 separate, oleuropein content is all lower, is no more than 40%.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art, provide a yield high, a kind of method that separates high-content oleuropein of extracting from leaf of Fructus oleae europaeae of environmental protection.
From leaf of Fructus oleae europaeae, extract the method that separates high-content oleuropein, concrete steps are as follows:
(1) by leaf of Fructus oleae europaeae chopping, for being switched to 1～2cm;
(2) leaf of Fructus oleae europaeae of chopping is added to purified water or aqueous ethanolic solution, extract at low temperature; Described middle extract at low temperature temperature is 20～70 DEG C; Described extraction solvent is purified water, and volume is 12～16 times; Described extraction solvent or be 40～80% ethanol, volume is 10～12 times;
(3) by after concentrated the solution after extracting or directly by the chromatography column by two kinds of macroporous adsorbent resin series connection; Maybe the solution after extracting concentrated rear or directly carry out primary sorption, then carrying out second adsorption after desorbed solution is concentrated;
(4) by purified water, resin is cleaned; Describedly by purified water, resin is cleaned, volume is 8～20 times of resin column volume;
(5) with 5%～80% fatty alcohol wash-out resin, in the time that effluent liquid fatty alcohol concentration reaches 5%, start to collect effluent liquid; Described fatty alcohol is C
1-C
3primary alconol and secondary alcohol, preferred alcohol, described fatty alcohol elution volume is 1-5 times of column volume;
(6) cryoconcentration elutriant; Described low temperature is 30-60 DEG C, and described concentrated solution concentration is 18～25%;
(7) concentrated solution is freezing or spraying is dry; Can obtain oleuropein.
Beneficial effect:
In the present invention, compound yield is higher than prior art, and the present invention compared with prior art, it is advantageous that:
1. adopt extract at low temperature technology, greatly reduce and extract required energy consumption, reduced the oxidation of oleuropein, improved yield, also guaranteed the quality of product simultaneously;
2. adopt the column chromatography of series connection or the separate mode of secondary chromatography, without using noxious solvent (as ethyl acetate, chloroform, sherwood oil etc.), guaranteed the quality safety of product, greatly simplify technical process, simple to operation, production cost is low simultaneously, is beneficial to suitability for industrialized production;
3. the oleuropein content of producing according to production technique provided by the invention detects and can reach 80-90% through HPLC, can meet the market requirement in the fields such as modern functions food, healthcare products, has the good market advantage.
Embodiment
In order to make technique means of the present invention, creation characteristic, workflow, using method reach object and effect is easy to understand, below in conjunction with specific embodiment, further set forth the present invention.
Embodiment 1
Leaf of Fructus oleae europaeae raw material is cut into 1～2cm; Be placed in 14 times to the purified water of its weight, 60 DEG C of extractions, filter, filtrate taking 0.8 times of column volume/hour flow velocity by model the macroporous resin as HP-20, adsorb completely, pure water is caught up with 5mins, closes 15mins.Then clean resin by 10 times of purified water to column volume; Again by resin LSA-8 or LSA-10 series connection, with 2 times of 30% ethanol to column volume with 1 times of column volume/hour flow velocity resolve, in the time that being 5%, effluent liquid alcohol concn starts to collect desorbed solution, being concentrated into concentration lower than 60 DEG C is 20%, concentrated solution adopts cryodesiccated method to be dried, and obtains olive leaf extract.Detect oleuropein content through HPLC and reach 85.26%.
Embodiment 2
Leaf of Fructus oleae europaeae raw material is cut into 1～2cm; Be placed in 12 times to 40% ethanol of its weight, 50 DEG C of extractions, filter, be concentrated into without alcohol taste lower than 60 DEG C, concentrated solution taking 0.8 times of column volume/hour flow velocity by model the macroporous resin as HP-20, adsorb completely, pure water is caught up with 5mins, closes 15mins.Then clean resin by 12 times of purified water to column volume; Again by resin LSA-8 or LSA-10 series connection, with 3 times of 40% ethanol to column volume with 1 times of column volume/hour flow velocity resolve, in the time that being 5%, effluent liquid alcohol concn starts to collect desorbed solution, being concentrated into concentration lower than 60 DEG C is 18%, concentrated solution adopts spray-dired method to be dried, and obtains olive leaf extract.Detect oleuropein content through HPLC and reach 82.19%.
Embodiment 3
Leaf of Fructus oleae europaeae raw material is cut into 1～2cm; Be placed in 10 times to 60% ethanol of its weight, 45 DEG C of extractions, filter, be concentrated into without alcohol taste lower than 60 DEG C, concentrated solution taking 0.8 times of column volume/hour flow velocity by model the macroporous resin as HP-20, adsorb completely, pure water is caught up with 5mins, closes 15mins.Then clean resin by 10 times of purified water to column volume; Again with 2.5 times of 25% ethanol to column volume with 1 times of column volume/hour flow velocity resolve, in the time that effluent liquid alcohol concn is 5%, start to collect desorbed solution, be concentrated into without alcohol taste lower than 60 DEG C; And then taking 0.8 times of column volume/hour flow velocity by model the macroporous resin as XAD-16, adsorb completely, pure water is caught up with 5mins, closes 15mins.Then clean resin by 10 times of purified water to column volume; Again with 2 times of 60% ethanol to column volume with 1 times of column volume/hour flow velocity resolve, in the time that being 5%, effluent liquid alcohol concn starts to collect desorbed solution, it is 22% that desorbed solution is concentrated into concentration lower than 60 DEG C, and concentrated solution adopts cryodesiccated method to be dried, and obtains olive leaf extract.Detect oleuropein content through HPLC and reach 88.72%.
Embodiment 4
Leaf of Fructus oleae europaeae raw material is cut into 1～2cm; Be placed in 15 times to the purified water of its weight, 55 DEG C of extractions, filter, filtrate taking 0.8 times of column volume/hour flow velocity by model the macroporous resin as XAD-16, adsorb completely, pure water is caught up with 5mins, closes 15mins.Then clean resin by 12 times of purified water to column volume; Again with 3 times of 80% ethanol to column volume with 1 times of column volume/hour flow velocity resolve, in the time that effluent liquid alcohol concn is 5%, start to collect desorbed solution, be concentrated into without alcohol taste lower than 60 DEG C; And then taking 0.8 times of column volume/hour flow velocity by model the macroporous resin as HP-20, adsorb completely, pure water is caught up with 5mins, closes 15mins.Then clean resin by 10 times of purified water to column volume; Again with 2 times of 60% ethanol to column volume with 1 times of column volume/hour flow velocity resolve, in the time that being 5%, effluent liquid alcohol concn starts to collect desorbed solution, being concentrated into concentration lower than 60 DEG C is 20%, and concentrated solution adopts spray-dired method to be dried, and obtains olive leaf extract.Detect oleuropein content through HPLC and reach 84.60%.
More than show and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (1)
1. from leaf of Fructus oleae europaeae, extract the method that separates high-content oleuropein, it is characterized in that, concrete steps are as follows:
(1) by leaf of Fructus oleae europaeae chopping, be switched to 1～2cm;
(2) leaf of Fructus oleae europaeae of chopping is added to purified water or aqueous ethanolic solution, extract at low temperature, and extract at low temperature temperature is 20～70 DEG C; The volume of purified water is 12～16 times of leaf of Fructus oleae europaeae; Ethanol is 40～80% ethanol, volume is leaf of Fructus oleae europaeae 10～12 times;
(3) by after concentrated the solution after extracting or directly by the chromatography column by two kinds of macroporous adsorbent resin series connection; Maybe the solution after extracting concentrated rear or directly carry out primary sorption, then carrying out second adsorption after desorbed solution is concentrated;
(4) by purified water, resin is cleaned, purified water volume is 8～20 times of resin column volume
(5) with 5%～80% fatty alcohol wash-out resin, in the time that effluent liquid fatty alcohol concentration reaches 5%, start to collect effluent liquid; Fatty alcohol is ethanol, and described fatty alcohol elution volume is 1～5 times of column volume;
(6) cryoconcentration elutriant, described low temperature is 30～60 DEG C, described concentrated solution concentration is 18～25%;
(7) concentrated solution is freezing or spraying is dry.
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| CN103333213B (en) * | 2013-07-24 | 2015-10-07 | 桂林茗兴生物科技有限公司 | The method of oleuropein is extracted from leaf of Fructus oleae europaeae |
| CN104311613A (en) * | 2014-09-30 | 2015-01-28 | 桂林三宝药业有限公司 | Method for extracting oleuropein from olive leaves |
| CN105011162A (en) * | 2015-07-02 | 2015-11-04 | 罗新华 | Olive leaf essence as well as making technology and application thereof |
| CN106565803A (en) * | 2016-10-10 | 2017-04-19 | 广西来宾绿翔生物科技有限公司 | Extraction process for oleuropein |
| CN109758498B (en) * | 2019-03-21 | 2021-08-17 | 湖南朗林生物资源股份有限公司 | Preparation method of olive leaf extract |
| CN117137138A (en) * | 2023-07-13 | 2023-12-01 | 南京林业大学 | Application of oleuropein in improving comfort level after drinking and alcoholic beverage |
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| WO2008136037A2 (en) * | 2007-05-04 | 2008-11-13 | Università Degli Studi Magna Graecia Di Catanzaro | Chemical-catalytic method for the peracylation of oleuropein and its products of hydrolysis |
| CN101781344A (en) * | 2009-12-13 | 2010-07-21 | 成都普思生物科技有限公司 | Method for preparing olive leaf extract |
| CN101955503A (en) * | 2010-05-25 | 2011-01-26 | 苏州派腾生物医药科技有限公司 | Preparation method of oleuropein |
| CN102060893A (en) * | 2009-11-13 | 2011-05-18 | 中国科学院兰州化学物理研究所 | Process method for synchronously preparing oleuropein and olive total flavonoids from olive leaves |
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| JP2002128678A (en) * | 2000-10-17 | 2002-05-09 | Tama Seikagaku Kk | Method for producing extract composition containing oleuropein |
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| WO2008136037A2 (en) * | 2007-05-04 | 2008-11-13 | Università Degli Studi Magna Graecia Di Catanzaro | Chemical-catalytic method for the peracylation of oleuropein and its products of hydrolysis |
| CN102060893A (en) * | 2009-11-13 | 2011-05-18 | 中国科学院兰州化学物理研究所 | Process method for synchronously preparing oleuropein and olive total flavonoids from olive leaves |
| CN101781344A (en) * | 2009-12-13 | 2010-07-21 | 成都普思生物科技有限公司 | Method for preparing olive leaf extract |
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