CN111440064A - Enrichment, purification and preparation method of oleocanthal in extra virgin olive oil - Google Patents
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Firstly, picking mature olive fruits for 24 hours as raw materials, and physically squeezing at 25 ℃ to prepare special virgin olive oil with acidity less than 0.8; then obtaining the total polyphenol extract through Centrifugal separation and Extraction (CPE); then obtaining a concentrated oleocanthal sample by Centrifugal Partition Chromatography (CPC) combined with thin layer Chromatography and preparative liquid Chromatography; and finally, carrying out structure identification and purity analysis by utilizing nuclear magnetic resonance and mass spectrometry technologies, so as to prepare high-purity oleocanthal.
Description
Technical Field
The invention relates to the technical field of daily supplies, and relates to an enrichment, purification and preparation method of oleocanthal in extra virgin olive oil.
Background
The olive oil contains a large amount of monounsaturated fatty acid such as oleic acid, and polyunsaturated fatty acid such as linoleic acid, linolenic acid, and the like, and also contains various vitamins, antioxidants and the like, wherein the content of the unsaturated fatty acid is up to 82-87%. The special virgin olive oil is olive oil with acidity not more than 0.8, is prepared by physically squeezing fresh olive fruits within 24h after picking to obtain pure natural juice, and then performing oil-water separation to obtain the olive oil with the best quality, and does not contain any preservative or additive component. Over the past decade, many epidemiological studies have correlated the intake of extra virgin olive oil with an extended life expectancy and a reduced incidence of age-related diseases. This is because the oleocanthal rich in extra virgin olive oil is a powerful non-steroidal anti-inflammatory drug, similar to ibuprofen, and has therapeutic effects on neurodegenerative diseases, joint degenerative diseases and cancer, and research shows that it can play an important role in the treatment of alzheimer's disease.
Alzheimer's disease is the most common neurodegenerative disease affecting the elderly population, and is the most common pathological type in senile dementia. The early stage mainly comprises obvious memory decline, the daily life ability is gradually lost, mental symptoms and behavior disorder are accompanied, the state of the disease is progressive, and the disease is often swallowed difficultly, bedridden, about ten years of illness and death due to complications such as infection and the like in the late stage. In European and American countries, the incidence rate of the senile dementia is 5% in the elderly over 65 years old, the incidence rate of the senile dementia is over 30% in the elderly over 85 years old, and the incidence rate of the senile dementia of the elderly 65 years old and over in China is 5.56%. At present, symptomatic treatment drugs mainly consist of acetylcholinesterase inhibitors (donepezil, rivastigmine and galantamine) and a glutamate receptor antagonist. However, these treatments have limited symptomatic improvement and do not have the effect of curing or modifying the disease. Studies have shown that the addition of oleocanthal-rich extra virgin olive oil as a medical food can broaden the therapeutic effect of donepezil, which cannot be achieved by just increasing the dosage of donepezil, as this increase may trigger undesirable cholinergic side effects. Medical food is defined herein as a special class of products intended to manage a disease or a disease with unique nutritional requirements for a particular diet through medical evaluation and based on accepted scientific principles.
However, the special virgin olive oil has obvious pungent and bitter irritant sensory characteristics when being directly eaten, so that the domestic special virgin olive oil is not too much to be directly eaten, and simultaneously, because the price is high, unsaturated fatty acid, oleocanthal and the like are easy to oxidize and deteriorate, so that the domestic related market has a large blank at present. In order to avoid the problems and keep the activity of the oleocanthal, and facilitate the later research of relevant work which can be used for the domestic Alzheimer disease, the invention is a novel preparation method for enriching and purifying the oleocanthal from the special virgin olive oil, and the method is used for enriching and purifying the oleocanthal from the picking and squeezing of fresh olive fruits and the enrichment and purification of total polyphenol.
The invention applies a Centrifugal Partition Chromatography (CPC) technology, invented by Nunogaki, the CPC belongs to one of modern countercurrent Chromatography, the principle is liquid-liquid extraction, namely, a special rotary stainless steel column is adopted, the stationary phase and the mobile phase of the column are both liquid, the tail end of the CPC column is provided with a rotary sealer, so that a solvent enters a rotor, the rotary column body is stacked by stainless steel discs with thousands of connected separation cavities, the special design ensures that the stationary phase solvent is retained under Centrifugal force, and the mobile phase solvent can flow through and complete extraction. At present, the method is widely applied to separation and purification of foreign natural active products due to high separation speed and high efficiency, but the method is only reported in China at present, so that the method is used for enriching and purifying oleocanthal on the basis of the method. In addition, Centrifugal separation Extraction (CPE) is also used in the present invention as a high productivity unsupported liquid-liquid separation technique to separate several natural metabolites, such as saponins, glucosinolates, alkaloids, phenolic acids and polyphenols, from plant extracts. The main difference between CPE and CPC depends on the length of the separation column. For equivalent column capacity, the CPE rotor contains fewer compartments of larger volume, which are connected in series by larger pipes.
Disclosure of Invention
The invention aims to provide an enrichment and purification preparation method of oleocanthal in extra virgin olive oil.
Another object of the present invention is to provide a method for preparing the above medical food additive for preventing and improving alzheimer's disease.
The invention is implemented according to the following scheme:
the invention comprises the following raw materials: freshly picked mature olive fruits.
Step 1, taking freshly picked mature olive fruits, carrying out physical squeezing to obtain pure natural juice of the olive fruits, and then carrying out oil-water separation to obtain special virgin olive oil;
step 2, mixing the extra-grade virgin olive oil with n-hexane, injecting the mixture into a CPE (chlorinated polyethylene) column, setting a flow rate of 50-100 m L/min and a rotation speed of 200-1000 rpm/min for a mobile phase and a stationary phase respectively to be methanol, distilled water, n-hexane and ethyl acetate, and repeating for 5-10 times, wherein each time lasts for about 30-60 min;
step 3, evaporating the obtained water phase to dryness under vacuum at 40 ℃ to obtain a viscous extract, extracting to remove residual fat, concentrating to obtain a total polyphenol extract, and refrigerating at 4 ℃ for later use;
step 4, dissolving the total polyphenol extract with methanol, injecting the dissolved total polyphenol extract into a CPC column, setting the flow rate of a mobile phase and the stationary phase to be 20-100 m L/min, setting the rotation speed to be 300-900 rpm/min, and collecting the mobile phase and the stationary phase once every 10m L to obtain an extraction sample solution;
step 5, according to the thin layer chromatography analysis of the extracted sample solution, concentrating the similar Rf value samples under vacuum at 40 ℃ to obtain an extract containing oleocanthal, and refrigerating at 4 ℃ for standby;
step 6, taking an oleocanthal extract, dissolving the oleocanthal extract in methanol, performing preparative liquid phase purification, performing gradient elution by using acetonitrile and water as mobile phases, collecting samples according to different peak-off times, and performing thin-layer chromatography analysis;
step 7, comparing the thin-layer chromatography analysis result with an oleocanthal standard substance, finding a sample corresponding to an oleocanthal peak, immediately concentrating at the water bath temperature of 40 ℃, and then putting the sample in a vacuum drier for overnight;
and 8, taking a dried oleocanthal concentrated sample, dissolving the sample with deuterated methanol, performing structural identification and preliminary purity analysis by using a nuclear magnetic resonance technology, comparing the obtained product with a known standard product, wherein the obtained product is feasible if the purity is more than 95%, otherwise, repeating the steps 6-8, and finally obtaining the final purity by using mass spectrometry.
The preparation method for enriching and purifying the oleocanthal in the extra virgin olive oil also comprises the following steps:
according to the preparation method for enriching and purifying the oleocanthal in the extra virgin olive oil, the fresh olive fruits are physically squeezed at the temperature of below 25 ℃ within 24 hours of squeezing in the first step, and the olive oil acidity is less than 0.8.
In the CPE column in the second step and the fourth step of the preparation method for enriching and purifying the oleocanthal in the special virgin olive oil, the proportion of the mobile phase to the stationary phase n-hexane, ethyl acetate, methanol and distilled water is 4: 1: 2: 3, 3: 2: 3, 2: 3: 2: 3, or one or more than one of 1: 4: 2: 3.
In the third step of the enrichment, purification and preparation method of the oleocanthal in the special virgin olive oil, the extracting agent is one or more of methanol, acetonitrile, n-hexane and petroleum ether.
According to the enrichment and purification preparation method of oleocanthal in extra-virgin olive oil, in the fifth step, a thin-layer chromatography developing agent is dichloromethane and methanol in a volume ratio of 90: 10-95: 5.
According to the enrichment and purification preparation method of the oleocanthal in the extra-grade virgin olive oil, the mobile phase of the prepared liquid phase in the sixth step is acetonitrile and water in a ratio of 7: 3-1: 9.
The method is simple and convenient in experimental operation but needs to be careful, a large amount of pure oleocanthal can be quickly prepared, and the method is very efficient.
Drawings
FIG. 1 is a diagram of a CPC device;
Detailed Description
The following examples are illustrative of the present invention and should not be construed as limiting thereof.
Examples
Step 1, taking fresh and picked mature olive fruits of 24 hours, namely a variety of Luxingx, physically squeezing at 25 ℃ to obtain pure natural juice, and then carrying out oil-water separation to obtain special virgin olive oil;
step 2, mixing the extra-grade virgin olive oil with n-hexane, injecting the mixture into a CPE cylinder, setting the flow rate of the mobile phase and the stationary phase to be methanol, distilled water, n-hexane and ethyl acetate respectively, setting the flow rate to be 60m L/min, setting the rotation speed to be 300rpm/min, and repeating the operation for 6 times, wherein each time lasts for about 50 min;
step 3, evaporating the obtained water phase to dryness in vacuum at 40 ℃ to obtain a viscous extract, extracting the viscous extract by using methanol and n-hexane in a volume ratio of 1: 1 to remove residual fat, concentrating the extract to obtain a TPF extract, and refrigerating the TPF extract at 4 ℃ for later use;
step 4, dissolving the TPF extract in methanol, injecting the dissolved TPF extract into a CPC cylinder, setting the flow rate of a mobile phase and a stationary phase to be methanol, distilled water, n-hexane and ethyl acetate to be 4: 1: 2: 3, 3: 2: 3 and 2: 3: 2: 3 respectively, setting the flow rate to be 50m L/min, setting the rotation speed to be 500rpm/min, collecting the TPF extract once every 10m L to obtain an extraction sample solution, wherein the CPC device diagram is shown in the following figure 1;
step 5, according to the thin layer chromatography analysis of the extraction sample solution, the developing agent is dichloromethane and methanol with the volume ratio of 95: 5, and the similar Rf value is concentrated in vacuum at 40 ℃ to obtain an extract containing oleocanthal, and the extract is refrigerated at 4 ℃ and is placed for standby;
step 6, taking an oleocanthal extract, dissolving the oleocanthal extract in methanol, performing preparative liquid phase purification, wherein a mobile phase is acetonitrile and water, a gradient elution volume ratio is shown in the following table 1, collecting samples according to different peak-out times, and simultaneously performing thin-layer chromatography analysis;
step 7, comparing the thin-layer chromatography analysis result with an oleocanthal standard substance, finding a sample corresponding to an oleocanthal peak, immediately concentrating at the water bath temperature of 40 ℃, and then putting the sample in a vacuum drier for overnight;
and 8, taking a dried oleocanthal concentrated sample, dissolving the oleocanthal concentrated sample by using deuterated methanol, performing structural identification and purity analysis by using a nuclear magnetic resonance technology, comparing the obtained product with a known standard product, wherein the purity is over 95 percent, and further performing hydrogen spectrum analysis to obtain the product with the purity of 97 percent.
TABLE 1 preparation of liquid phase gradient elution mobile phase
| Time/min | Water/%) | Acetonitrile/%) | Flow rate/(m L/min) | |
| 1 | Initial | 70 | 30 | 10 |
| 2 | 20.00 | 60 | 40 | 10 |
| 3 | 25.00 | 50 | 50 | 10 |
| 4 | 30.00 | 10 | 90 | 10 |
| 5 | 35.00 | 70 | 30 | 10 |
Claims (6)
1. An enrichment and purification preparation method of oleocanthal in extra virgin olive oil comprises the following steps:
firstly, taking freshly picked mature olive fruits, carrying out physical squeezing to obtain pure natural juice of the olive fruits, and then carrying out oil-water separation to obtain special virgin olive oil;
secondly, mixing the extra-grade virgin olive oil with n-hexane, injecting the mixture into a CPE column, setting the flow rate of a mobile phase and the stationary phase to be 50-100 m L/min, setting the rotation speed to be 200-1000 rpm/min, repeating the steps for 5-10 times, and repeating the steps for 30-60 min each time, wherein the mobile phase and the stationary phase are methanol, distilled water, n-hexane and ethyl acetate respectively;
thirdly, evaporating the obtained water phase to dryness under vacuum at 40 ℃ to obtain a viscous extract, extracting to remove residual fat, concentrating to obtain a total polyphenol extract, and refrigerating at 4 ℃ for later use;
fourthly, dissolving the total polyphenol extract with methanol, injecting the dissolved total polyphenol extract into a CPC column body, setting the flow rate of a mobile phase and the stationary phase to be 20-100 m L/min, setting the rotation speed to be 300-900 rpm/min, and collecting the mobile phase and the stationary phase once every 10m L to obtain an extraction sample solution;
fifthly, according to the thin layer chromatography analysis of the extracted sample solution, concentrating the similar Rf value samples under the vacuum of 40 ℃ to obtain an extract containing oleocanthal, and refrigerating and standing at 4 ℃ for later use;
sixthly, taking the oleocanthal extract, dissolving the oleocanthal extract in methanol, performing preparative liquid phase purification, performing gradient elution by using acetonitrile and water as mobile phases, collecting samples according to different peak-out times, and performing thin-layer chromatography analysis;
seventhly, comparing the thin-layer chromatography analysis result with an oleocanthal standard substance, finding a sample corresponding to an oleocanthal peak, immediately concentrating at the temperature of 40 ℃ water bath, and then putting the sample in a vacuum drier for overnight;
and eighthly, taking the dried oleocanthal concentrated sample, dissolving the oleocanthal concentrated sample in deuterated methanol, performing structural identification and preliminary purity analysis by using a nuclear magnetic resonance technology, comparing the obtained product with a known standard product, wherein the obtained product is feasible if the purity is more than 95%, otherwise, repeating the steps 6-8, and finally obtaining the final purity by using mass spectrometry.
2. The method for the enrichment, purification and preparation of oleocanthal in extra virgin olive oil as claimed in claim 1, wherein the fresh olive fruits are physically pressed at a temperature below 25 ℃ within 24h of the first step of the method, and the olive oil acidity is less than 0.8.
3. The method according to claim 1, wherein the ratio of n-hexane, ethyl acetate, methanol and distilled water in the CPE column in the second and fourth steps is 4: 1: 2: 3, 3: 2: 3, 2: 3: 2: 3, 1: 4: 2: 3.
4. The method for preparing oleocanthal from extra virgin olive oil according to claim 1, wherein the extractant in the third step is one or more selected from methanol, acetonitrile, n-hexane, and petroleum ether.
5. The method for the enrichment, purification and preparation of oleocanthal in extra virgin olive oil as claimed in claim 1, wherein the volume ratio of dichloromethane to methanol is 90: 10-95: 5 as the developing agent of thin layer chromatography in the fifth step.
6. The method for preparing oleocanthal from extra virgin olive oil according to claim 1, wherein the mobile phase of the liquid phase prepared in the sixth step is acetonitrile to water at ratio of 7: 3-1: 9.
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| CN202010400909.1A Pending CN111440064A (en) | 2020-05-13 | 2020-05-13 | Enrichment, purification and preparation method of oleocanthal in extra virgin olive oil |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024023550A1 (en) * | 2022-07-27 | 2024-02-01 | Gorgio International Holding Llc | Malt supplement containing oleocanthal |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103483196A (en) * | 2013-09-30 | 2014-01-01 | 桂林茗兴生物科技有限公司 | Method for extracting oleocanthal from olive oil |
| CN110554129A (en) * | 2019-09-12 | 2019-12-10 | 浙江省农业科学院 | screening method of potential anti-inflammatory active ingredients in extra-virgin olive oil |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103483196A (en) * | 2013-09-30 | 2014-01-01 | 桂林茗兴生物科技有限公司 | Method for extracting oleocanthal from olive oil |
| CN110554129A (en) * | 2019-09-12 | 2019-12-10 | 浙江省农业科学院 | screening method of potential anti-inflammatory active ingredients in extra-virgin olive oil |
Non-Patent Citations (1)
| Title |
|---|
| APOSTOLIS ANGELIS: "An integrated process for the recovery of high added-value compounds from olive oil using solid support free liquid-liquid extraction and chromatography techniques", 《JOURNAL OF CHROMATOGRAPHY A》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024023550A1 (en) * | 2022-07-27 | 2024-02-01 | Gorgio International Holding Llc | Malt supplement containing oleocanthal |
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Application publication date: 20200724 |