Method for separating and purifying oleuropein from olive leaves

Abstract

The invention discloses a method for separating and purifying oleuropein from olive leaves. Olive leaves are used as a raw material, and instantaneous high-temperature steam is used for treating the raw material by referring to the idea of a tea fixation technology to prevent enzymatic reaction; the solvent ratio of high-purity ethanol to ethyl acetate is respectively selected in the enriching and refining processes, so that the enzyme loses catalytic action, the degradation of oleuropein is reduced and eliminated, the yield of oleuropein is improved, the extraction cost is reduced, the productivity of oleuropein is effectively improved, and the industrial development is promoted.

Description

Technical Field

[0001] The invention relates to quality process optimization of olive leaf extract, and in particular to a technology for reducing the decomposition effect of endogenous enzymes in olive leaves on oleuropein, a main effective component of olive leaf extract. Background Art

[0002] Olive (Olea europaea L.) is an oil-bearing crop of the genus Oleaceae, native to the Mediterranean region. [1] , was introduced to my country in the 20th century and is mainly planted in Gansu, Sichuan, Yunnan and other places [2-3] Olives are favored because their fruits can be used to make olive oil rich in monounsaturated fatty acids, but there is relatively little research on olive leaves. Studies have shown that olive leaves are rich in a variety of antioxidant active ingredients, such as oleuropein, flavonoids, lignans, etc. [4-5] , and the content is much higher than that in olive fruit, among which the content of oleuropein and flavonoids in leaves is significantly higher than that in fruits and bark. [6] Olive leaves have broad application prospects in the fields of sterilization, anti-virus, anti-tumor and prevention and treatment of cardiovascular and cerebrovascular diseases. [7-9] .

[0003] Oleuropein is a secoiridoid polyphenol compound and the most abundant component in olive polyphenols.

[10] , which contributes the most important antioxidant effect to olive polyphenols [11-12] Oleuropein can effectively prevent spots, wrinkles and other phenomena caused by cell aging due to fat oxidation.

[13] , reducing the levels of inflammatory factors in cells and mice [14-15] , reduce the degree of skin damage in patients with psoriasis

[16] , which has a potential promoting effect on certain growth factors in mouse skin tissue

[17] It can also inhibit the activity of tyrosinase, a key enzyme in the synthesis of melanin, thereby achieving the effect of whitening. [18-19] In summary, oleuropein can be added to cosmetics as a whitening, anti-inflammatory, antioxidant, and anti-aging ingredient, and is a natural plant ingredient with great potential and value.

[0004] During the growth and development of olives, the content of oleuropein often changes. Olive leaves contain a variety of hydrolases that decompose oleuropein, resulting in a low content of oleuropein in the raw material extract. [20-21] Among them, β-glucosidase hydrolyzes oleuropein to form the aldehyde form of oleuropein and the di-dealdehyde form of decarboxymethyl oleuropein, forming a large amount of hydroxytyrosol, thereby degrading oleuropein

[22] Endogenous esterases hydrolyze the ester bond of oleuropein to produce glucose derivatives - hydroxytyrosol (3,4-DHPEA) and enolic acid

[23] ; Polyphenol oxidase catalyzes the oxidation of o-dihydroxyphenol to o-quinone, which continuously condenses to form a typical dark pigment of enzymatic browning

[24] Therefore, the decrease of oleuropein content and the large production of hydroxytyrosol are important indicators of the deterioration of olive leaf extract.

[0005] In order to overcome the technical difficulties of oleuropein extraction, the main measures currently taken in China include traditional cold and hot extraction, Soxhlet extraction, and new extraction technologies such as ultrasound-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction.

[25] In recent years, a new extraction technology, decompression extraction, has emerged. Its key technical point is to produce a tumbling effect, which protects heat-sensitive substances while being more energy-efficient; it provides a low-oxygen vacuum environment to prevent oxidizable substances from oxidizing and decomposing, and this method has a relatively high extraction efficiency.

[26] .

[0006] References

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[0008] [2] Deng Yu, Liu Ting, Liang Fang. Current situation and countermeasures of the development of China's olive industry[J]. Economic Forest Research, 2015, 33(2):3. DOI:10.14067 / j.cnki.1003-8981.2015.02.030.

[0009] [3] Shi Zongming, Sun Weibang, Qi Zhilin, et al. Study on the suitable habitat of olive in China[J]. Journal of Plant Taxonomy and Resources, 2011, 33(5):9. DOI:10.3724 / SP.J.1143.2011.11077.

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[0013] [7]Susalit E, Agus N, Effendi I, et al.Olive(Olea europaea)leaf extracteffective in patients with stage-1hypertension:Comparison with Captopril[J].Phytomedicine,2011,18(4):251-258.DOI:10.1016 / j.phymed.2010.08.016

[0014] [8]De Bock M,Derraik JG,Brennan CM,et al.Olive(Olea europaea L.)leaf polyphenols improve insulin sensitivity in middle-aged overweight men: arandomized,placebo-controlled,crossover trial[J].PloS ONE,2013,8(3):e57622.DOI:10.1371 / journal.pone.0057622.

[0015] [9]Park S, Choi Y, Um SJ, et al. Oleuropein attenuates hepatic steatosis induced by high-fat diet in mice[J]. Journal of Hepatology, 2011, 54(5):984-993.DOI:10.1016 / j.jhep.2010.08.019.

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[10] Esti M,Cinquanta L,La Notte E.Phenolic Compounds in DifferentOlive Varieties[J].Journal of Agricultural&Food Chemistry,1998,46(1):32.DOI:10.1021 / jf970391+.

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[11] Kong Weibao, Wang Junlong, Qi Huifang, et al. Research progress on the chemical composition, pharmacological effects and extraction technology of olive polyphenols[J]. 2020.DOI:10.12166 / j.zgyz.1003-7969 / 2020.11.006

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[12] Chang Q, Su MH, Chen QX, etal. Physicochemical Properties and Antioxidant Capacity of Chinese Olive (Canarium album L.) Cultivars [J]. Journal of Food Science, 2017. DOI: 10.1111 / 1750-3841.13740.

[0019]

[13] Wang Chengzhang, Gao Caixia, Jiang Chengying. Chemical composition and processing and utilization of olive[J]. Journal of Forestry Science and Technology Development, 2006, 20(2):4. DOI:10.3969 / j.issn.1000-8101.2006.02.001

[0020]

[14] Nediani C,Ruzzolini J,Romani A,etal.Oleuropein,a BioactiveCompound from Olea europaea L.as a Potential Preventive and Therapeutic Agentin Non-Communicable Diseases[J].Antioxidants,2019,8(12).DOI:10.3390 / antiox8120578.

[0021]

[15] Taskan MM, Yuce HB, Karatas O, et al. Evaluation of the effect of oleuropein on alveolar bone loss, inflammation, and apoptosis in experimental periodontitis[J]. Journal of periodontal research, 2019, 54(6): 624-632. DOI: 10.1111 / jre.12662.

[0022]

[16] El-Gogary RI, Ragai MH, Moftah N, etal. Oleuropein as a noveltopical antipsoriatic nutraceutical:formulation in microemulsion nanocarrierand exploratory clinical appraisal[J].DOI:10.1080 / 17425247.2021.1932813

[0023]

[17] Tao,Tong,Nahyun,etal.Topical Application of Oleuropein InducesAnagen Hair Growth in Telogen Mouse Skin[J].PLOS ONE,2015,10(6).DOI:10.1371 / journal.pone.0129578

[0024]

[18] Chen Jing, Wei Jianteng, Pei Dong, et al. Study on the inhibitory effect of oleuropein on tyrosinase[J]. Natural Product Research and Development, 2021, 33(12):6.

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[19] Yang Yang, Lu Jun, Sun Bingjie, et al. Kinetic study on the inhibitory activity of resveratrol on tyrosinase in vitro[J]. Journal of Food and Biotechnology, 2011, 30(4):4. DOI:CNKI:SUN:WXQG.0.2011-04-029.

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[20] Mazzuca S, Spadafora A, Innocenti AM. Cell and tissue localization of β-glucosidase during the ripening of olive fruit (Olea europaea) by in situactivity assay [J]. Plant Science, 2006, 171(6): 726-733. DOI: 10.1016 / j.plantsci.2006.07.006.

[0027]

[21] Briante R,Patumi M,Limongelli S,etal.Changes in phenolic and enzymatic activities content during fruit ripening in two Italian cultivars of Olea europaea L.[J].PLANT SCIENCE LIMERICK,2002,162(5):791-798.DOI:10.1016 / S0168-9452(02)00022-5.

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[22] Bu Wenwen, Liu Changjin, Tian Shifu. Comparison of preparation of hydroxytyrosol from olive leaf extract by hydrochloric acid method and β-glucosidase method [J]. Food Industry Science and Technology, 2011(7):5. DOI:CNKI:SUN:SPKJ.0.2011-07-055.

[0029]

[23] LIMERICK,2002,162(5):791-798.DOI:10.1016 / S0168-9452(02)00022-5.Ramírez E,Brenes M,García P,et al.Oleuropein hydrolysis in natural greenolives:Importance of the endogenous enzymes.Food Chem.2016,206:204-209.DOI:10.1016 / j.foodchem.2016.03.061

[0030]

[24] Peng Shiqing. Research progress of plant polyphenol oxidase[J]. Tropical Agricultural Sciences, 2000(3):6. DOI:10.3969 / j.issn.1009-2196.2000.03.014.

[0031]

[25] Xie Pujun, Huang Lixin, Zhang Caihong, et al. Research progress in oleuropein extraction technology[J]. Biomass Chemical Engineering, 2012, 46(3): 6. DOI: 10.3969 / j.issn.1673-5854.2012.03.007.

[0032]

[26] Xie Pujun. Research on the technology and mechanism of high-value utilization of olive leaves[D]. Beijing: Chinese Academy of Forestry, 2015. DOI: 10.7666 / d.Y2903791. Summary of the invention

[0033] On this background, the present invention optimizes parameters such as temperature and duration by drawing on the concept of tea leaf fixing technology, adopts instantaneous high-temperature steam to treat raw materials, reduce leaf water content, passivate enzyme activity, and prevent enzymatic reaction; select the solvent ratio of high-purity ethanol to change the enzyme molecular structure, lose activity, and lose catalytic effect. Therefore, the technical problem to be solved by the present invention is to provide a method for separating and purifying high-content oleuropein from olive leaf raw materials whose β-glucosidase activity is reduced and inhibited. The method eliminates all enzyme cutting, effectively prevents the hydrolysis of oleuropein and the yellowing of hydroxytyrosol, and well improves the extraction efficiency of oleuropein, saves production costs, and improves production capacity.

[0034] The technical scheme provided by the present invention is as follows: 1) taking olive leaf raw material, subjecting it to instantaneous high-temperature steam sterilization treatment, and then using anhydrous ethanol to preliminarily extract oleuropein therein to obtain oleuropein ethanol extract;

[0035] (2) subjecting the oleuropein ethanol extract obtained in step (1) to UF filtration and RO reverse osmosis concentration to recover ethanol, and then extracting with ethyl acetate to obtain an ethyl acetate extract;

[0036] (3) The ethyl acetate extraction part in the above step (2) is dried and then enriched by a macroporous resin column: the decomposition solvent is 95% ethanol, and the ethanol is recovered by high-efficiency external circulation to obtain a preliminarily enriched oleuropein dry powder;

[0037] (4) The oleuropein dry powder initially enriched in step (3) above is prepared into a column feed solution, which is separated and purified by a silica gel column: the analytical solvent is ethyl acetate, the analytical solution is collected in sections, the ethyl acetate is recovered, the sample is dissolved in 95% ethanol, and the sample is freeze-dried to obtain a high-content oleuropein product.

[0038] In a specific embodiment, in step (1), the instantaneous high temperature treatment is instantaneous 100±5°C high temperature steam killing for 8-12 minutes, which reduces the water content of the leaves, inactivates enzyme activity, and prevents enzymatic reactions, thereby inhibiting the enzymatic hydrolysis of oleuropein and retaining its content.

[0039] In a specific embodiment, it is characterized in that: in step (1), the high-purity ethanol extraction temperature is 55-65°C, the solid-liquid ratio is 1:6-10, the number of extractions is 1-4 times, and each extraction takes 1-2 hours;

[0040] The extraction temperature of high-purity ethanol was 60°C, the solid-liquid ratio was 1:8, the number of extractions was 2 times, and each extraction lasted 1.5 hours.

[0041] In a specific embodiment, in step (2), the volume of ethyl acetate extraction is 1:4-6, the number of extractions is 2-5 times, the solvent is recovered under reduced pressure, and the mixture is concentrated to 1-1.2 degrees Baume; preferably, the volume of ethyl acetate extraction is 1:5, the number of extractions is 3 times, the solvent is recovered under reduced pressure, and the mixture is concentrated to 1.10-1.15 degrees Baume.

[0042] In a specific embodiment, in step (3), the amount of macroporous adsorption resin used is 8-12 times the amount of sample loaded, the column flow rate is 0.8-1.2BV / h, 95% ethanol is analyzed for 2-5 column volumes, 2 fractions are collected, and the analysis flow rate is 1BV / h; preferably, the amount of macroporous adsorption resin used is 10 times the amount of sample loaded, the column flow rate is 1BV / h, 95% ethanol is analyzed for 3 column volumes, 2 fractions are collected, and the analysis flow rate is 1BV / h.

[0043] In a specific embodiment, in step (3), the macroporous adsorption resin is of type LX-20B.

[0044] In a specific embodiment, in step (4), the oleuropein dry powder is initially enriched to prepare a column feed solution with a concentration of 6-15%, preferably a column feed solution with a concentration of 9-11%; the sample is dissolved in 95% ethanol to a concentration of 15-25%;

[0045] The amount of silica gel used for silica gel column chromatography is 10-20 times the amount of sample loaded, the flow rate on the column is 0.5-1.5BV / h, ethyl acetate is analyzed for 3-5 column volumes, 1 fraction is collected, and the analysis flow rate is 1BV / h; preferably, the amount of silica gel used for silica gel column chromatography is 15 times the amount of sample loaded, the flow rate on the column is 1BV / h, and ethyl acetate is analyzed for 4 column volumes.

[0046] In a specific embodiment, when recovering the solvent in steps (2), (3) and (4), the temperature should be controlled below 60° C. and the vacuum degree should be controlled below -0.09 MPa.

[0047] In a specific implementation, the following steps are also included:

[0048] (5) The oleuropein product obtained in the above step (4) is subjected to HPLC detection and external standard method quantitative analysis to obtain the final content.

[0049] Specifically, during HPLC detection in step (5), the mobile phase equilibrium system time reaches 2 hours.

[0050] The present invention has the following beneficial effects:

[0051] The present invention uses high-purity ethanol as an extraction solvent through instantaneous high-temperature steam fixing treatment, effectively improves the extraction efficiency of oleuropein, and reduces the degradation of oleuropein. Through optimized extraction and enrichment processes, the production cost is reduced, the yield of oleuropein is improved, and the output of the product is increased. Oleuropein is a natural product with multiple biological activities such as whitening, anti-inflammatory, anti-oxidation, and anti-aging, and its high-purity products have a wide range of application prospects in the fields of cosmetics, health products, and medicine. The present invention is innovative in the quality process optimization of olive leaf extract, especially in the technical aspects of reducing the decomposition effect of endogenous enzymes on oleuropein, which is conducive to promoting the development of the olive leaf extract industry and provides technical support and market potential for related industries. The present invention can not only improve the quality and market competitiveness of the product itself, but also promote the technological progress and industrial upgrading of related industries, bringing positive social and economic impacts.

[0052] In summary, the process of the present invention can reduce and inhibit the activity of β-glucosidase in olive leaf raw materials, thereby reducing and eliminating the degradation of oleuropein, increasing the yield of oleuropein, reducing the cost of extraction, effectively increasing the production capacity of oleuropein, and being conducive to promoting industrial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the HPLC spectrum of 90% oleuropein. DETAILED DESCRIPTION

[0054] In order to further illustrate the present invention, the following describes the embodiments of the present invention in detail through specific examples. It should be noted that these examples are only used for illustration and are not intended to limit the scope of protection of the present invention.

[0055] Embodiment 1: A method for separating and purifying oleuropein from olive leaf raw material comprises the following steps:

[0056] 1. Pretreatment of olive leaf raw materials:

[0057] Select olive leaves, remove impurities, and air-dry at room temperature until the moisture content is less than 10%. The dried olive leaves are subjected to instant high-temperature steam sterilization treatment at a temperature of 100±5℃ for 10 minutes to reduce the moisture content of the leaves and inactivate enzyme activity.

[0058] 2. Extraction of oleuropein:

[0059] After weighing the olive leaves that had been treated with greening, anhydrous ethanol (99.7% ≥) was added at a solid-liquid ratio of 1:8 (weight ratio), and the mixture was extracted at 60° C. for 1.5 hours, and the extraction was repeated twice. After the extraction, the ethanol was recovered by UF filtration and RO reverse osmosis concentration to obtain an oleuropein extract.

[0060] 3. Ethyl acetate extraction

[0061] Ethyl acetate was added to the extract obtained in step 2, and the extract was extracted at a volume ratio of 1:5 (leaf extract: ethyl acetate), and the extraction was performed 3 times, each time for 30 minutes. After the extraction was completed, the solvent was recovered under reduced pressure, the rotary evaporation temperature was 55° C., and the extract was concentrated to a concentrate with a Baume degree of 1.10-1.15.

[0062] 4. Macroporous adsorption resin column chromatography enrichment

[0063] The concentrate in step 3 was enriched by LX-20B macroporous resin column, the amount of macroporous adsorption resin was 1.8 kg, the amount of sample was 180 g, and the column flow rate was 1 BV / h. 95% ethanol was used as the analytical solvent, 3 column volumes were analyzed, 2 fractions were collected, the analytical flow rate was 1 BV / h, and ethanol was recovered by efficient external circulation to obtain the initially enriched oleuropein dry powder.

[0064] 5. Silica gel column purification and freeze drying

[0065] The oleuropein dry powder obtained by the enrichment in step 4 was dissolved in 95% ethanol and prepared into a column feed liquid with a concentration of 0.5-1% (weight ratio). Separation and purification was carried out by silica gel column, the silica gel dosage of silica gel column chromatography was 450g, the sample load was 30g, ethyl acetate was used as the analytical solvent, 4 column volumes were analyzed, 1 fraction was collected, the analytical flow rate was 1BV / h, and the analytical solution containing oleuropein was collected in sections. The collected analytical solution was subjected to solvent recovery, and the sample was dissolved in 95% ethanol, and dried by freeze drying technology to obtain a high-content oleuropein product.

[0066] 6. HPLC detection and quantitative analysis

[0067] The freeze-dried oleuropein product was dissolved in a 50% methanol solution and tested by HPLC. The oleuropein was quantitatively analyzed using the external standard method. The final product content was 90%, the extraction yield was 96%, and the recovery rate was 85%. The spectrum is shown in Figure 1 shown.

[0068] According to the test results, the raw material β-glucosidase is 2283.0U / g. Through the process of the present invention, the enzyme in the final extract is completely inactivated and the enzyme activity of β-glucosidase cannot be detected.

Claims

1. A method for separating and purifying high-content oleuropein from olive leaves, characterized in that The steps include: (1) taking olive leaf raw material, subjecting it to instantaneous high-temperature steam sterilization treatment, and then using anhydrous ethanol to preliminarily extract oleuropein therein to obtain oleuropein ethanol extract; (2) subjecting the oleuropein ethanol extract obtained in step (1) to UF filtration and RO reverse osmosis concentration to recover ethanol, and then extracting with ethyl acetate to obtain an ethyl acetate extract; (3) The ethyl acetate extraction portion in the above step (2) is dried and then enriched by a macroporous resin column: the decomposition solvent is 95% ethanol, and the ethanol is recovered by an efficient external circulation to obtain a preliminarily enriched oleuropein dry powder; (4) The oleuropein dry powder initially enriched in step (3) above is prepared into a column feed solution, which is separated and purified by a silica gel column: the analytical solvent is ethyl acetate, the analytical solution is collected in sections, the ethyl acetate is recovered, the sample is dissolved in 95% ethanol, and the sample is freeze-dried to obtain a high-content oleuropein product.

2. The method according to claim 1, characterized in that: In step (1), the instantaneous high temperature treatment is instantaneous high temperature steam sterilization at 100±5℃ for 8-12min, which reduces the water content of the leaves, inactivates enzyme activity, and prevents enzymatic reactions, thereby inhibiting the enzymatic hydrolysis of oleuropein and retaining its content.

3. The method according to claim 1, characterized in that: In step (1), the high-purity ethanol extraction temperature is 55-65°C, the solid-liquid ratio is 1:6-10, the number of extractions is 1-4 times, and each extraction takes 1-2 hours; The extraction temperature of high-purity ethanol was 60°C, the solid-liquid ratio was 1:8, the number of extractions was 2 times, and each extraction lasted 1.5 hours.

4. The method according to claim 1, characterized in that: In step (2), the volume of ethyl acetate extraction is 1:4-6, the number of extractions is 2-5 times, the solvent is recovered under reduced pressure, and the mixture is concentrated to 1-1.2 degrees Baume; preferably, the volume of ethyl acetate extraction is 1:5, the number of extractions is 3 times, the solvent is recovered under reduced pressure, and the mixture is concentrated to 1.10-1.15 degrees Baume.

5. The method according to claim 1, characterized in that: In step (3), the amount of macroporous adsorption resin used is 8-12 times the amount of sample loaded, the column flow rate is 0.8-1.2 BV / h, 95% ethanol is used for analysis for 2-5 column volumes, 2 fractions are collected, and the analysis flow rate is 1 BV / h; preferably, the amount of macroporous adsorption resin used is 10 times the amount of sample loaded, the column flow rate is 1 BV / h, 95% ethanol is used for analysis for 3 column volumes, 2 fractions are collected, and the analysis flow rate is 1 BV / h.

6. The method according to claim 5, characterized in that: In step (3), the macroporous adsorption resin is model LX-20B.

7. The method according to claim 1, characterized in that: In step (4), the oleuropein dry powder is initially enriched to prepare a column feed solution with a concentration of 6-15%, preferably a column feed solution with a concentration of 9-11%; the sample is dissolved in 95% ethanol to a concentration of 15-25%; The amount of silica gel used for silica gel column chromatography is 10-20 times the amount of sample loaded, the flow rate on the column is 0.5-1.5 BV / h, ethyl acetate is analyzed for 3-5 column volumes, 1 fraction is collected, and the analysis flow rate is 1 BV / h; preferably, the amount of silica gel used for silica gel column chromatography is 15 times the amount of sample loaded, the flow rate on the column is 1 BV / h, and ethyl acetate is analyzed for 4 column volumes.

8. The method according to claim 1, characterized in that: When recovering the solvent in steps (2), (3) and (4), the temperature should be controlled below 60°C and the vacuum degree should be controlled below -0.09 MPa.

9. The method according to claim 1, characterized in that: The following steps are also included: (5) The oleuropein product obtained in the above step (4) is subjected to HPLC detection and external standard method quantitative analysis to obtain the final content.

10. The method according to claim 9, characterized in that During HPLC detection in step (5), the mobile phase equilibrium system time reaches 2 hours.

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