CN109400658B - Method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves - Google Patents
Method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves Download PDFInfo
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- CN109400658B CN109400658B CN201811420341.9A CN201811420341A CN109400658B CN 109400658 B CN109400658 B CN 109400658B CN 201811420341 A CN201811420341 A CN 201811420341A CN 109400658 B CN109400658 B CN 109400658B
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
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Abstract
The invention provides a method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves, which comprises the steps of soaking the olive leaves in water, refluxing and extracting, and concentrating an extracting solution to be used as a sample solution; uniformly mixing n-hexane, ethyl acetate, methanol and water, and standing for layering; then separating the upper phase solution and the lower phase solution to be respectively used as eluent; adsorbing the sample solution by macroporous resin, washing water-soluble impurities and removing washing liquid by using a resin column after adsorption is finished; eluting with lower phase eluent, and collecting effluent; finally eluting with upper phase eluent, and collecting effluent liquid. Oleuropein is mainly in the upper phase eluent, and hydroxytyrosol is mainly in the lower phase eluent. After the extracting solution is subjected to primary resin treatment, the oleuropein content reaches more than 45%, and the recovery rate reaches more than 90%; the content of the hydroxytyrosol reaches about 3 percent, and the recovery rate reaches more than 85 percent.
Description
Technical Field
The invention relates to a method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves, and belongs to the technical field of natural product separation.
Background
Olea europaea (L.) patOleaeuropaeaL.) as an economic crop, the economic crop is mainly planted in Greek, Italy and other countries along the coast of the Mediterranean land at first, and then introduced into China, and is mainly distributed in Yunnan, Sichuan, Gansu, Guizhou and other places. Olive leaf is a major by-product of the olive growing industry and is discarded every year when trimming and harvesting fruit. This not only pollutes the environment, but also causes a serious waste of resources. Therefore, it is necessary to comprehensively utilize and develop olive leaves.
The active substances in olive leaves mainly include polyphenols, flavonoids, secoiridoid, biflavones, etc., wherein Oleuropein (OL) and Hydroxytyrosol (HT) are two main polyphenols. According to the reports of the literature, oleuropein and hydroxytyrosol have good antioxidation, can effectively remove free radicals and oxides, and are considered as one of natural antioxidants. In addition, oleuropein also has antifungal, antiviral, anticancer and blood sugar lowering effects; the hydroxytyrosol has effects of preventing arteriosclerosis, hypertension, and cerebral hemorrhage, lowering blood pressure, and resisting and preventing cancer. Therefore, the extract containing oleuropein and hydroxytyrosol is expected to be a new raw material for health care products, medicines and cosmetics. At present, no report is found on the process method for simultaneously separating and purifying oleuropein and hydroxytyrosol from olive leaves.
Disclosure of Invention
The invention provides a method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves, thereby making up the defects of the prior art.
The method for synchronously separating and purifying oleuropein and hydroxytyrosol comprises the following steps:
(1) extraction: the olive leaves are used as raw materials, are extracted twice by water reflux, are mixed, cooled, filtered and concentrated to the concentration of 0.05-0.2 kg per liter, and the solution is used as a sample loading solution; the olive leaves and water are subjected to reflux extraction according to a solid-liquid mass ratio of 1: 5-1: 20, and the extraction time is 1.5-2 h each time.
(2) Preparation of an eluent: uniformly mixing n-hexane, ethyl acetate, methanol and water, and standing for layering; then separating the upper phase solution and the lower phase solution to be respectively used as eluent; the volume ratio of the n-hexane to the ethyl acetate to the methanol to the water is 1 (8-10) to 1-2 to 8-10.
(3) Separating and purifying by macroporous resin: adsorbing the sample solution by macroporous resin, washing water-soluble impurities and removing washing liquid by using a resin column after adsorption is finished; eluting with lower phase eluent, and collecting effluent; finally eluting with upper phase eluent, and collecting effluent liquid. Oleuropein is mainly in the upper phase eluent, and hydroxytyrosol is mainly in the lower phase eluent.
The macroporous resin is selected from styrene matrix macroporous resin, such as D101, LSA-21, HPD450, LSA-40, LSD001 and HPD600, and the diameter-height ratio of the resin column is 1: 5-1: 20.
The volume of the sample loading liquid is 3 BV-15 BV, and the flow rate is 2 BV/h-20 BV/h; the volume of the lower phase eluent is 3 BV-8 BV, and the flow rate is 2 BV/h-5 BV/h; the volume of the eluent on the upper phase is 3 BV-8 BV, and the flow rate is 3 BV/h-8 BV/h.
After the extracting solution is subjected to primary resin treatment, the oleuropein content reaches more than 45%, and the recovery rate reaches more than 90%; the content of the hydroxytyrosol reaches about 3 percent, and the recovery rate reaches more than 85 percent.
Compared with the prior art, the invention has the following advantages:
1. the oleuropein and the hydroxytyrosol are separated by one step to obtain high-purity oleuropein and high-purity hydroxytyrosol which can be used as raw materials of anti-aging, blood pressure lowering, blood sugar lowering, cancer prevention and anti-cancer medicines, health-care food and cosmetics;
2. the production process uses a green solvent, is environment-friendly, simple in process route, low in cost and high in efficiency, and is suitable for industrial production;
3. the macroporous resin has good regeneration performance and can be regenerated and reused.
Detailed Description
The method for simultaneous separation and purification of oleuropein and hydroxytyrosol according to the present invention is further illustrated by the following specific examples.
Example 1
(1) Extraction: weighing 200g of olive leaves, placing in a 5L round bottom flask, adding 3L of water, heating in an electric heating jacket, refluxing under normal pressure for 2h, and pouring out the extract. Adding 2.4L water into the residue, refluxing under normal pressure for 2 hr, pouring out the extractive solution, and discarding the residue. Mixing the two extractive solutions, cooling, filtering, concentrating the filtrate under reduced pressure, adding water to desired volume of 2L to make the concentration of the extractive solution per liter equal to 0.1g of olive leaf, and shaking to obtain a sample solution;
(2) preparation of an eluent: placing 20 mL of n-hexane, 180 mL of ethyl acetate, 20 mL of methanol and 180 mL of water in a 1000 mL separating funnel, shaking for several minutes, standing for layering, and separating an upper phase from a lower phase to obtain an eluent;
(3) separating and purifying by macroporous resin: taking LSA-2112g of macroporous resin, and filling the macroporous resin into a glass column with the inner diameter of 1.5cm and the column height of 27cm by a wet method, wherein the ratio of the inner diameter to the height is 1:14, and 1 BV =12 mL. Adsorbing the sample loading liquid 7 BV obtained in the step (1) on a column at the flow rate of 10 BV/h, and discarding the effluent liquid; and then 5 BV of water is added into the resin column after adsorption to wash and remove impurities, and the water washing liquid is discarded. Then 5 BV of prepared lower phase eluent is used for elution at the flow rate of 2 BV/h, and hydroxytyrosol is mainly eluted at the time; finally, 5 BV of prepared upper phase eluent is used for eluting at the flow rate of 6 BV/h, and oleuropein is mainly eluted at the moment;
(4) analysis by High Performance Liquid Chromatography (HPLC): the peak area ratio of hydroxytyrosol in the eluent of the lower phase and the upper phase is 17.81, the purity is improved from 0.46% to 2.96%, and the recovery rate is 88%. The peak area ratio of oleuropein in the eluents of the upper phase and the lower phase is 27.71, the purity is improved from 11.40% to 47.59%, and the recovery rate is 93%.
Example 2
(1) Extraction: the same as example 1;
(2) preparation of an eluent: placing 20 mL of n-hexane, 160 mL of ethyl acetate, 20 mL of methanol and 160 mL of water in a 1000 mL separating funnel, shaking for several minutes, standing for layering, and separating an upper phase from a lower phase to obtain an eluent;
(3) separating and purifying by macroporous resin: and (3) taking macroporous resin HPD 45012 g, and filling the macroporous resin HPD 45012 g into a glass column with the inner diameter of 1.5cm and the column height of 27cm by a wet method, wherein the ratio of the inner diameter to the height is 1:14, and 1 BV =12 mL. Adsorbing the sample loading liquid obtained in the step (1) by using an 8 BV upper column at the flow rate of 12 BV/h, and discarding the effluent liquid; and then 6 BV of water is added into the resin column after adsorption to wash and remove impurities, and the water washing liquid is discarded. Then eluting with 7 BV of prepared lower phase eluent at the flow rate of 3 BV/h, wherein hydroxytyrosol is mainly eluted; finally, 7 BV of prepared upper phase eluent is used for eluting at the flow rate of 5 BV/h, and oleuropein is mainly eluted at the moment; (4) analysis by High Performance Liquid Chromatography (HPLC): the peak area ratio of hydroxytyrosol in the eluent of the lower phase and the upper phase is 13.31, the purity is improved from 0.46% to 2.72%, and the recovery rate is 85%. The peak area ratio of oleuropein in the eluates of the upper and lower phases is 23.31, the purity is increased from 11.40% to 45.53%, and the recovery rate is 92%.
Example 3
(1) Extraction: the same as example 1;
(2) preparation of an eluent: placing 30 mL of n-hexane, 300 mL of ethyl acetate, 50 mL of methanol and 240 mL of water in a 1000 mL separating funnel, shaking for several minutes, standing for layering, and separating an upper phase from a lower phase to obtain an eluent;
(3) separating and purifying by macroporous resin: and (3) taking macroporous resin LSD 00120 g, and filling the macroporous resin LSD 00120 g into a glass column with the inner diameter of 1.5cm and the column height of 27cm by a wet method, wherein the ratio of the inner diameter to the column height is 1:14, and 1 BV =20 mL. Adsorbing the sample loading liquid 6 BV obtained in the step (1) on a column at a flow rate of 9 BV/h, and discarding the effluent liquid; and then 8 BV of water is added into the resin column after adsorption to wash and remove impurities, and the water washing liquid is discarded. Then eluting with 6 BV of mixed lower phase eluent at the flow rate of 4 BV/h, wherein hydroxytyrosol is mainly eluted; finally, 6 BV of prepared upper phase eluent is used for eluting at the flow rate of 6 BV/h, and oleuropein is mainly eluted at the moment;
(4) analysis by High Performance Liquid Chromatography (HPLC): the peak area ratio of hydroxytyrosol in the eluent of the lower phase and the upper phase is 15.46, the purity is improved from 0.46% to 2.83%, and the recovery rate is 80%. The peak area ratio of oleuropein in the eluates of the upper and lower phases is 23.67, the purity is increased from 11.40% to 48.53%, and the recovery rate is 89%.
Claims (8)
1. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves comprises the following steps:
(1) extraction: the olive leaves are used as raw materials, are extracted twice by water reflux, are mixed, cooled, filtered and concentrated to the concentration of 0.05-0.2 kg per liter, and the solution is used as a sample loading solution;
(2) preparation of an eluent: uniformly mixing n-hexane, ethyl acetate, methanol and water, and standing for layering; then separating the upper phase solution and the lower phase solution to be respectively used as eluent; the volume ratio of the n-hexane to the ethyl acetate to the methanol to the water is 1 (8-10) to 1-2 to 8-10;
(3) separating and purifying by macroporous resin: passing the sample solution through styrene matrix macroporous resin, after adsorption, washing the resin column with water to remove water-soluble impurities and discarding washing solution; eluting with lower phase eluent, and collecting effluent; finally eluting with upper phase eluent, and collecting effluent liquid; oleuropein is mainly in the upper phase eluent, and hydroxytyrosol is mainly in the lower phase eluent.
2. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves as claimed in claim 1, which is characterized in that: in the extraction in the step (1), the olive leaves and water are subjected to reflux extraction according to a solid-liquid mass ratio of 1: 5-1: 20.
3. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves as claimed in claim 1, which is characterized in that: in the extraction in the step (1), the extraction time is 1.5-2 h each time.
4. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves as claimed in claim 1, which is characterized in that: the styrene matrix macroporous resin is D101, HPD450, LSA-21, LSA-40, LSD001, HPD 600.
5. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves as claimed in claim 1, which is characterized in that: in the step (3), the ratio of the diameter to the height of the resin column is 1: 5-1: 20.
6. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves as claimed in claim 1, which is characterized in that: in the step (3), the volume of the sample loading liquid is 3 BV-15 BV, and the flow rate is 2 BV/h-20 BV/h.
7. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves as claimed in claim 1, which is characterized in that: in the step (3), the volume of the lower phase eluent is 3 BV-8 BV, and the flow rate is 2 BV/h-5 BV/h.
8. The method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves as claimed in claim 1, which is characterized in that: in the step (3), the volume of the upper phase eluent is 3 BV-8 BV, and the flow rate is 3 BV/h-8 BV/h.
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| CN111642597A (en) * | 2020-07-13 | 2020-09-11 | 中国科学院兰州化学物理研究所 | Medlar and olive tea beverage and preparation method thereof |
| CN111875650B (en) * | 2020-08-10 | 2022-05-20 | 中国科学院兰州化学物理研究所 | Preparation and application of boric acid functionalized resin |
| CN115779002B (en) * | 2022-11-10 | 2023-09-19 | 东莞职业技术学院 | Preparation method of olive leaf active site for inhibiting food-borne AGEs |
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