Olive fruit extract as well as preparation method and application thereof

Abstract

The invention provides an olive fruit extract as well as a preparation method and application thereof, and belongs to the technical field of anti-aging, the preparation method of the olive fruit extract comprises the following steps: performing crude extraction on fresh olive fruit powder by utilizing a supercritical fluid extraction mode, performing enzymolysis on the crude extract by utilizing cellulase and pectinase, and performing freeze drying to obtain the olive fruit extract. And precipitating the enzymatic hydrolysate with methanol and then purifying. The olive fruit extract prepared by the invention is rich in natural active ingredients, and the olive fruit extract is proved to have remarkable anti-aging and life-prolonging effects in a living body by taking caenorhabditis elegans as a model, and the action mechanism of the olive fruit extract in the living body is further analyzed. Compared with other chemical drugs, the olive fruit extract has the characteristics of low dosage and remarkable effect, so that the olive fruit extract has great application potential in the aspect of preparing anti-aging products.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-aging, and in particular to an olive fruit extract, a preparation method thereof and application thereof in the preparation of anti-aging products. Background Art

[0002] Aging is a biological process in which the intrinsic physiological functions gradually deteriorate over time, which will lead to a decrease in the body's metabolic capacity, damage repair ability and stress resistance, thus affecting physical health. Therefore, aging will lead to an increased susceptibility to age-related diseases or chronic diseases, such as cancer, diabetes, cardiovascular disease, Alzheimer's disease, Parkinson's disease, etc., which has a significant impact on human physical and mental health.

[0003] The increasing global aging has placed a serious burden on the medical and health system and the social economy. Although aging is irreversible, it can be delayed and the quality of human life can be improved. At present, studies have found that many natural active ingredients from plants with low toxicity, few side effects and a wide range of sources have anti-aging effects. Therefore, using natural active ingredients from plants to develop products, delay aging, improve human health and improve the quality of life has become a safe and effective strategy.

[0004] Olive is a sweet and nutritious plant that can be used as both medicine and food. In my country, it is mainly used as fresh food or to make fruit tea and candied fruit. Pharmacological studies have found that olives have anti-Helicobacter pylori, anti-influenza, anti-diabetes, anti-inflammatory, and anti-tumor effects. However, the natural active ingredients in olives have not been fully analyzed, and the anti-aging activity and molecular mechanism of olive extracts in vivo have not been reported. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention has developed an olive fruit extract that can significantly delay aging. Not only the active ingredients in the olive fruit extract are analyzed by high performance liquid chromatography-mass spectrometry, but also the Caenorhabditis elegans is used as a model to explore the mechanism of action of the olive fruit extract in anti-aging in vivo, laying a foundation for the application of olive fruit extract in the preparation of anti-aging and longevity products.

[0006] In a first aspect, the present invention provides a method for preparing an olive fruit extract, comprising the following steps: S1. Using CO2 as solvent, the fresh olive fruit powder is roughly extracted by supercritical fluid extraction; S2, dissolving the crude extract obtained in step S1 in water, and adding cellulase and pectinase for enzymolysis; S3. The enzymatic hydrolysate is precipitated with methanol, and the obtained product is purified and dried to obtain the olive fruit extract.

[0007] Preferably, in the above preparation method, the preparation method of fresh olive fruit powder is: after removing the pits of fresh olive fruits, vacuum drying at 40° C., cooling, and crushing to 60 meshes by low-temperature airflow.

[0008] Preferably, in the above preparation method, the conditions of supercritical fluid extraction are: temperature 35-40°C, pressure 15-30MPa, CO2 flow rate 25-30kg / h, and extraction time 3-4h.

[0009] Preferably, in the above preparation method, the conditions for enzymatic hydrolysis are: pH 4-7, temperature 40-60°C, mass ratio of cellulase to pectinase 1:150-1:250, and enzymatic hydrolysis time 2-5h. Through enzymatic hydrolysis, the olive fruit extract is further hydrolyzed, which can effectively increase the dissolution of active ingredients.

[0010] Preferably, in the above preparation method, the purification method is: fractionation treatment with n-hexane, ethyl acetate and n-butanol in sequence.

[0011] In a second aspect, the present invention provides an olive fruit extract. Through the analysis of the natural active ingredients in the olive fruit extract, it is found that the olive fruit extract is rich in flavonoids such as isoferulic acid, ferulic acid, golden holly (yellow), gallic acid, aloe-emodin, chlorogenic acid, isorhamnetin, quercetin, sinapinic acid, kaempferol, polyphenols, phenolic acids, flavonoid carbon glycosides and other substances.

[0012] In a third aspect, the present invention provides the use of the olive fruit extract in the preparation of anti-aging products, wherein the products include but are not limited to medicines, feed additives, food additives, etc.

[0013] The present invention uses Caenorhabditis elegans as an anti-aging model to study the mechanism of action of the olive fruit extract prepared by the present invention in anti-aging. Specifically, the olive fruit extract achieves the purpose of anti-aging and life extension by activating the IIS signaling pathway, DAF-16 / FOXO and SKN-1 / Nrf2 transcription factors and upregulating the expression of their downstream target genes, and has at least the following effects: a1) Improve movement ability, such as increasing the nematode's swallowing rate and bending and swinging frequency; a2) Reduce the deposition of lipofuscin in the body; a3) improving resistance to oxidative stress and heat stress; a4) Increase SOD enzyme activity and GSH content in the body.

[0014] In some embodiments of the present invention, by using an effective dose of olive fruit extract to treat Caenorhabditis elegans, not only can the average lifespan of Caenorhabditis elegans in natural state, oxidative stress state and heat stress state be significantly increased; the swallowing rate and bending swing frequency of Caenorhabditis elegans are significantly improved; in addition, the accumulation level of lipofuscin in Caenorhabditis elegans is significantly reduced, and the superoxide dismutase (SOD) enzyme activity and reduced glutathione (GSH) level in Caenorhabditis elegans are increased. Further studies have found that olive fruit extract can promote the nuclear entry of DAF-16 and SKN-1, upregulate the expression of DAF-16 and SKN-1, and thus activate genes in related signaling pathways, such as: sod-3 , hsp- 16.2 , gcs-1, cpr-5, gst-32, ugt-8, vit-3, fat-7 .

[0015] In a fourth aspect, the present invention provides an anti-aging drug containing the olive fruit extract prepared by the present invention.

[0016] It is understandable that the medicine may further include pharmaceutically acceptable excipients, and the medicine may be in the form of tablets, granules, capsules, powders, liquids or jelly.

[0017] Compared with the prior art, the beneficial effects of the present invention are: The olive fruit extract prepared by the invention is rich in natural active substances such as flavonoids such as isoferulic acid, ferulic acid, galangal (yellow), gallic acid, aloe-emodin, chlorogenic acid, isorhamnetin, quercetin, sinapinic acid, kaempferol, polyphenols, phenolic acids, flavonoid carbon glycosides, etc.; and using Caenorhabditis elegans as an anti-aging model, the function of the olive fruit extract in delaying aging in a living body is discovered for the first time, and the mechanism by which the olive fruit extract exerts an anti-aging effect in the body is further revealed, specifically including: improving exercise capacity, reducing lipofuscin deposition, improving resistance to oxidative stress and heat stress, improving SOD enzyme activity and GSH level in the body, activating IIS signaling pathway, transcription factors DAF-16, SKN-1 and their downstream gene expressions related to life extension, etc.

[0018] Compared with other chemical synthetic drugs, olive fruit extract has the characteristics of small dosage and significant effect, so it has good application prospects in the preparation of anti-aging products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 This is a graph showing the test results of the effects of olive fruit extracts of different concentrations on the average lifespan of wild-type Caenorhabditis elegans N2 in the natural state in the examples of the present invention; Figure 2 This is a graph showing the test results of the effect of the olive fruit extract on the average lifespan of wild-type Caenorhabditis elegans N2 under oxidative stress conditions in an embodiment of the present invention; Figure 3 This is a graph showing the test results of the effect of the olive fruit extract on the average lifespan of wild-type Caenorhabditis elegans N2 under heat stress conditions in an embodiment of the present invention; Figure 4 The graph is a test result of the effect of the olive fruit extract on the swallowing rate (left) and bending swing frequency (right) of wild-type Caenorhabditis elegans in the examples of the present invention; Figure 5 This is a graph showing the test results of the effect of the olive fruit extract on the accumulation level of lipofuscin in wild-type Caenorhabditis elegans in an embodiment of the present invention; Figure 6 Figure 2 shows the test results of the effects of olive fruit extract on wild-type Caenorhabditis elegans SOD enzyme activity (left) and GSH level (right) in the embodiments of the present invention; Figure 7 This is a graph showing the test results of the effects of the olive fruit extract on the nuclear entry of DAF-16 and SKN-1 in Caenorhabditis elegans in the examples of the present invention; Figure 8 This is a graph showing the detection results of the effect of the olive fruit extract on the expression levels of the downstream target genes DAF-16 and SKN-1 in Caenorhabditis elegans in an example of the present invention. DETAILED DESCRIPTION

[0021] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0023] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0024] The wild type (N2) and transgenic line TJ356 [zIs356 IV (daf-16p::daf-16a / b::GFP +rol-6 (su1006))] of Caenorhabditis elegans used in the following examples were purchased from the Caenorhabditis Genetics Center (CGC) of the University of Minnesota; uracil-deficient Escherichia coli (OP50) was from the laboratory of Professor Luo Huairong of Southwest Medical University; GSH and SOD detection kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0025] Example 1 In this example, an olive fruit extract is prepared, and the preparation method thereof comprises the following steps: (1) Crushed olives.

[0026] The fresh olive fruits are pitted, dried in vacuum at 40°C, cooled, and then crushed to 60 meshes by low-temperature air flow crushing to obtain fresh olive fruit powder.

[0027] (2) Olive fruit crude extraction.

[0028] Place fresh olive fruit powder in a supercritical fluid extractor, use CO2 as solvent, control the temperature at 36°C, the pressure at 18MPa, the carbon dioxide flow rate at 26kg / h, the extraction time at 3.5h, collect the extract, and obtain a crude olive fruit extract.

[0029] (3) Enzymatic hydrolysis.

[0030] The crude olive fruit extract was dissolved in water, the pH value was adjusted to 6, cellulase and pectinase were added at a mass ratio of 1:200, the enzymatic hydrolysis temperature was controlled at 46°C, and the enzymatic hydrolysis was performed for 2.5 hours. The enzymatic hydrolyzate was concentrated by rotary concentrator to obtain the olive fruit extract concentrate.

[0031] (4) Preparation of freeze-dried powder.

[0032] The concentrate was extracted three times with 100% methanol to obtain an olive powder sample. The olive fruit powder was then dissolved in water, and n-hexane was added at a volume ratio of 1:1, and fractionated. The water layer obtained after fractionation was further fractionated by adding ethyl acetate at a volume ratio of 3:1 to obtain a water layer. Then n-butanol was added at a volume ratio of 1:1 for fractionation to obtain a water layer. Finally, the obtained water layer was concentrated and freeze-dried to obtain olive freeze-dried powder.

[0033] Example 2 In this example, an olive fruit extract was prepared. The preparation method thereof was different from that in Example 1 in that the conditions for crude extraction in step (2) were as follows: the temperature was controlled at 38°C, the pressure was controlled at 25 MPa, the carbon dioxide flow rate was 30 kg / h, and the extraction time was 4 h.

[0034] Example 3 In this example, an olive fruit extract was prepared. The preparation method thereof was different from that in Example 1 in that the conditions for enzymatic hydrolysis in step (3) were as follows: the pH value was adjusted to 5, the mass ratio of cellulase to pectinase was 1:180, the enzymatic hydrolysis temperature was 48°C, and the enzymatic hydrolysis time was 3 h.

[0035] Example 4 This example tests the effect of olive fruit extract on the average lifespan of Caenorhabditis elegans in natural conditions. The experimental process is as follows: (1) Prepare M9 buffer and S-complete liquid medium.

[0036] M9 buffer formula: Add 0.3g KH2PO4, 0.96g Na2HPO4·12H2O, and 0.5g NaCl to every 100mL distilled water, and add 0.1mL 1M MgSO4 after sterilization.

[0037] Formula of S-complete liquid medium (10mL): Add 0.1mL 1M potassium citrate buffer, 0.1mL metal ion buffer, 30µL1M CaCl2, 30µL 1M MgSO4, 30µL cholesterol to 10mL S-based buffer, then filter through a 0.22µm organic filter, and then add 50mM FuDR to a final concentration of 150µM.

[0038] (2) To examine the effects of different concentrations of olive fruit extract on Caenorhabditis elegans.

[0039] Liquid culture medium was added to a 96-well plate at 100 µL S-complete medium per well, with DMSO added as a blank control group and different concentrations of olive fruit extract (60 µg / mL, 120 µg / mL, 180 µg / mL, prepared in Example 1) added as a treatment group. According to 10 to 15 nematodes per well, nematodes that grew synchronously to the L4 stage were picked with a needle and placed in the S-complete medium, and the time of picking nematodes was set as day 0. Then M9 buffer was added to the outermost circle of the 96-well plate, sealed and placed in a 20°C incubator for culture. The growth of nematodes was observed, and the survival of nematodes was recorded every two days until all nematodes died. The experiment was repeated three times.

[0040] GraphPad software and the log-rank (Mantel−Cox) analysis method were used to draw survival curves and analyze data. The experimental results are shown in Figure 1 As shown in Table 1. The results showed that olive fruit extract could significantly prolong the average lifespan of Caenorhabditis elegans under natural conditions, and the lifespan extension effect was concentration-dependent, among which 120µg / mL olive fruit extract had the best effect of prolonging lifespan, extending it by 29.38%.

[0041] Table 1 Effects of different concentrations of olive fruit extract on the average lifespan of wild-type Caenorhabditis elegans N2 under natural conditions

[0042] Example 5 Based on the results in Example 4, this example tested the effect of 120 μg / mL olive fruit extract on the average lifespan of Caenorhabditis elegans under oxidative stress. The experimental process is as follows: Referring to the treatment method in Example 4, DMSO (blank control group) and 120µg / mL olive fruit extract were added to culture nematodes. The nematodes were collected on the 7th day of culture. According to 10 to 15 nematodes per well, the nematodes were picked with a needle into a 96-well plate so that there were more than 100 nematodes in each treatment group. The nematodes were then exposed to 1mM hydrogen peroxide solution for oxidative stress experiments. The survival of the nematodes was recorded every 2 hours until all the nematodes died. Each group of experiments was repeated three times. GraphPad software and the log-rank (Mantel-Cox) analysis method therein were used to draw survival curves and analyze data.

[0043] Test results such as Figure 2 As shown in Table 2, it was found that 120 µg / mL olive fruit extract could significantly extend the average lifespan of C. elegans under oxidative stress.

[0044] Table 2 Effects of olive fruit extract on the lifespan of wild-type Caenorhabditis elegans N2 under oxidative stress

[0045] Example 6 In this case, the effect of 120 µg / mL olive fruit extract on the average lifespan of Caenorhabditis elegans under heat stress was tested. The experimental steps are as follows: Referring to the treatment method in Example 4, DMSO (blank control group) and 120µg / mL olive fruit extract were added to culture nematodes. The nematodes were collected on the 7th day of culture. According to 10 to 15 nematodes per well, the nematodes were picked with a needle into a 96-well plate so that there were more than 100 nematodes in each treatment group. During heat stress, the nematodes were cultured at 37°C, and the survival of the nematodes was recorded every 2 h until all the nematodes died. Each group of experiments was repeated three times. GraphPad software and the log-rank (Mantel−Cox) analysis method were used to draw survival curves and analyze data.

[0046] Test results such as Figure 3 As shown in Table 3, it was found that 120 µg / mL olive fruit extract could significantly extend the average lifespan of C. elegans under heat stress.

[0047] Table 3 Effects of olive fruit extract on the lifespan of wild-type Caenorhabditis elegans N2 under heat stress

[0048] Example 7 This example tests the effect of 120 µg / mL olive fruit extract on the motility of Caenorhabditis elegans (including swallowing rate and bending swing). The experiment includes the following steps: The nematodes were treated and cultured according to the method in Example 4. The nematodes in different treatment groups were collected on the 3rd, 6th and 9th days of culture, respectively, and transferred to new NGM plates. The bending swing frequency and swallowing rate were measured under a stereo microscope. The bending swing frequency refers to the number of times the nematode body bends within 20 seconds. The swallowing rate refers to the number of times the pharynx of the nematode pumps within 20 seconds. During the experiment, more than 30 nematode data were recorded for each group, and the experiment was repeated three times.

[0049] The experimental results are as follows Figure 4 As shown: On the 3rd, 6th and 9th days, 120µg / mL olive fruit extract can significantly improve the motility of nematodes.

[0050] Example 8 Lipofuscin is an important indicator of aging. This example shows the effect of 120µg / mL olive fruit extract on the accumulation level of lipofuscin in Caenorhabditis elegans.

[0051] The method for determining the lipofuscin accumulation level is as follows: nematodes were cultured according to the method in Example 4, nematodes in different treatment groups were collected on the 10th day, and the nematodes were anesthetized with NaN3 solution after washing with M9 three times. Then, an inverted fluorescence microscope was used to take pictures, and the data were analyzed and processed with Image J software. The number of nematodes in each group was greater than 30.

[0052] The experimental results are as follows Figure 5 As shown: 120µg / mL olive fruit extract can significantly reduce the accumulation level of lipofuscin in nematodes, indicating that olive fruit extract can extend the lifespan of nematodes Example 9 This example shows the effect of 120µg / mL olive fruit extract on SOD activity and GSH levels in Caenorhabditis elegans.

[0053] Referring to Example 4, the experimental steps of this example are specifically as follows: on the third day of culture, the nematodes were collected and washed three times with M9, the worm bodies were ground and crushed on ice, and the supernatant was collected by centrifugation at 12000 rpm / min for 5 min at 4°C, and the SOD enzyme activity, GSH level, and protein content of the supernatant were determined according to the instructions of the kit.

[0054] The experimental results are as follows Figure 6 As shown: 120µg / mL olive fruit extract significantly increased SOD enzyme activity and GSH levels in nematodes.

[0055] Example 10 The DAF-16 and SKN-1 genes are transcriptional regulatory factors related to life span, which can regulate the growth, development, metabolism, life span, health status, etc. of Caenorhabditis elegans. This example detects the effect of 120 μg / mL olive fruit extract on the transcription factors DAF-16 and SKN-1 in Caenorhabditis elegans.

[0056] TJ356 and LD1 nematodes at the L4 stage were inoculated into S-complete medium without FuDR containing 120 µg / mL olive fruit extract and 0.6% DMSO and cultured for 2 days. The nematodes in different treatment groups were collected, washed 3 times with M9 to remove impurities, anesthetized with NaN3 solution, and photographed using a fluorescence microscope. The expression locations of DAF-16::GFP and SKN-1::GFP fluorescent proteins were recorded, and their nuclear translocation patterns in "cytoplasm", "internuclear matrix" and "nucleus" were counted. There were no less than 30 nematodes in each group, and the experiment was repeated 3 times.

[0057] The experimental results are as follows Figure 7 As shown: Compared with the control group, olive fruit extract significantly promoted the ratio of DAF-16 and SKN-1 entering the cell nucleus, thereby activating DAF-16 and SKN-1.

[0058] Embodiment 11 Based on Example 10, this example studies the effect of 120 µg / mL olive fruit extract on the expression levels of DAF-16 and SKN-1 downstream target genes in Caenorhabditis elegans. Referring to Example 4, the experimental steps of this example are as follows: After culturing the nematodes for 72 hours, all the nematodes were collected, washed three times with M9 buffer, and frozen in liquid nitrogen. After grinding the nematodes on ice, 1 mL of Trizol lysis buffer was added, vortexed and incubated at room temperature for 5 minutes, and then centrifuged at 4°C and 12,000 rpm / min for 10 minutes. Total RNA was extracted and reverse transcribed into cDNA for qRT-PCR fluorescence quantitative analysis. -ΔΔCt The method was used to calculate the relative expression of genes, and the t test was used for statistical analysis of differences. Actin-1 gene was used as the internal reference housekeeping gene, and the gene primer sequences used are shown in Table 4.

[0059] Table 4 Gene primer sequences used

[0060] The experimental results are as follows Figure 8 As shown: Compared with the control group, 120 µg / mL olive fruit extract significantly increased the expression of DAF-16 and SKN-1 downstream genes sod-3 , hsp-16.2 , gcs-1 , CPR-5 , gst-32 , ugt-8 , vit-3 and fat-7 This indicates that olive fruit extract upregulated the expression levels of downstream genes related to life extension by activating DAF-16 and SKN-1 transcription factors.

[0061] Comparative Example 1 This example provides an olive fruit extract. The difference from Example 1 is that the olive fresh fruit powder prepared in step (1) is directly dissolved in water, and in step (4) of reference Example 1, the olive fruit extract is sequentially precipitated with methanol, fractionated with n-hexane, fractionated with ethyl acetate, fractionated with n-butanol, and freeze-dried to obtain olive fruit freeze-dried powder.

[0062] Comparative Example 2 This example provides an olive fruit extract, which is prepared by a deep eutectic solvent method, specifically comprising the following steps: (1) Same as step 1 of Example 1.

[0063] (2) Extraction.

[0064] Choline chloride and sorbitol were mixed at a molar ratio of 1:2, and stirred at 80°C for 7 hours until the mixture became colorless and transparent to prepare an extraction solvent. Fresh olive fruit powder was weighed, and the extraction solvent was added at a solid-liquid ratio of 1:40 (g:mL). The mixture was extracted at 50°C and an ultrasonic frequency of 400W for 4 hours, and the obtained extract was concentrated.

[0065] (3) Preparation of freeze-dried powder.

[0066] The concentrated extract was placed in a freezing tray, pre-frozen at -20°C for 12 hours, and then dried in a freeze dryer to obtain olive freeze-dried powder.

[0067] Referring to Example 4, the effects of different concentrations of olive fruit extracts in Comparative Examples 1 and 2 on the average lifespan of Caenorhabditis elegans in the natural state were detected. The results showed that the olive fruit extracts in Comparative Examples 1 and 2 can also extend the average lifespan of Caenorhabditis elegans, but the best extension effects were 16.76% and 17.91%, respectively, which were much lower than the extracts prepared by the method of the embodiment.

[0068] The extracts prepared in Example 1 and Comparative Examples 1-2 were subjected to the following tests: 1. Determination of polyphenols, flavonoids and polysaccharides content.

[0069] (1) Preparation of gallic acid standard curve: Gallic acid was used as the standard substance and dissolved in 50% methanol to prepare standard solutions of different concentrations. 500 μL of the standard solution was mixed with 250 μL of 50% folin phenol reagent and reacted for 5 min. Then 500 μL of 5% sodium carbonate solution was added and reacted for 1 h in the dark. The absorbance was measured at 765 nm. The standard curve 1 was plotted with the concentration of gallic acid as the horizontal axis and the absorbance as the vertical axis.

[0070] (2) Drawing of rutin standard curve: Take rutin as the standard, dissolve it in 60% ethanol to prepare standard solutions of different concentrations, take 500 μL of the standard solution and mix it with 20 μL of 5% Na2NO2, let it stand for 7 min, then add 20 μL of 10% Al(NO3)3 solution, shake well and let it stand for 7 min. Add 200 μL of 1 mol / L NaOH solution, shake well and let it stand for 12 min, then dilute to 4 mL with 60% ethanol, let it stand for 10 min, and measure the absorbance at a wavelength of 510 nm. Draw standard curve 2 with the concentration of rutin standard solution as the horizontal axis and the absorbance as the vertical axis.

[0071] (3) Determination of polyphenol content in olive fruit: Take 500 μL of sample and mix it with 250 μL of 50% Folin phenol reagent. React for 5 min, then add 500 μL of 5% sodium carbonate solution. React for 1 h in the dark. Measure the absorbance at 765 nm and calculate the total phenol content in the sample according to standard curve 1. The total phenol content is expressed in mg gallic acid / g sample.

[0072] (4) Determination of flavonoids in olive fruit: Take 500 μL of sample and mix with 20 μL of 5% Na2NO2, let stand for 7 min, then add 20 μL of 10% Al(NO3)3 solution, shake well and let stand for 7 min. Add 200 μL of 1 mol / L NaOH solution, shake well and let stand for 12 min, then dilute to 4 mL with 60% ethanol, let stand for 10 min, measure the absorbance at a wavelength of 510 nm, and calculate the total flavonoids content in the sample according to standard curve 2. The total flavonoids content is expressed in mg rutin / g sample.

[0073] It can be seen from Table 5 that different extraction methods have a great influence on the total phenol and total flavonoid contents in the olive fruit extract, and the olive fruit extract prepared in Example has the highest total phenol and total flavonoid contents.

[0074] Table 5 Total phenolic and flavonoid contents in extracts obtained by different extraction methods

[0075] 2. Olive fruit extract natural active ingredients.

[0076] The components of olive fruit extract were separated by high performance liquid chromatography using an HSS T3 C18 column, and the full scan mode of Q ExactiveFocus Orbitrap LC-MS / MS (Thermo Scientific, USA) was used for qualitative and quantitative analysis. In the HPLC method, 0.04% acetic acid was used as mobile phase A, 0.04% acetonitrile was used as mobile phase B, and the gradient elution program was 5% B, the elution time was 0 min; 95% B elution for 12 min; 5% B elution for 12.1-15 min, and the flow rate was 0.35 mL / min. The mass spectrum was obtained by electrospray ionization mass spectrometry (ESI-MS), and its detailed working parameters were nebulizer gas flow rate 3L min -1 , heating gas flow rate 10 L min -1 ; Interface temperature, 550°C; DL temperature, 250°C; Heat block temperature, 400°C; Drying gas flow rate: 10 L min -1 The test results of the olive fruit extract prepared in Example 1 are shown in Table 6.

[0077] Table 6 Content of natural active ingredients in olive fruit extract

[0078] In summary, the present invention uses Caenorhabditis elegans as a model organism to study and confirm for the first time the anti-aging function of olive fruit extract in vivo. In particular, the olive fruit extract prepared by supercritical fluid extraction + enzymatic hydrolysis in the present invention has a significant effect in anti-aging and longevity. At the same time, the present invention further analyzes the components of olive fruit extract and its mechanism of action in vivo, laying a foundation for the development of related anti-aging products using olive fruit extract.

[0079] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A method for preparing an olive fruit extract, characterized in that: The following steps are involved: S1. Using CO2 as solvent, the fresh olive fruit powder is roughly extracted by supercritical fluid extraction; S2, dissolving the crude extract in water, adding cellulase and pectinase for enzymolysis; S3. The enzymatic hydrolysate is precipitated with methanol, and the obtained product is purified and dried to obtain the olive fruit extract.

2. The preparation method according to claim 1, characterized in that: The conditions of supercritical fluid extraction in step S1 are: temperature 35-40°C, pressure 15-30MPa, CO2 flow rate 25-30kg / h, and extraction time 3-4h.

3. The preparation method according to claim 1, characterized in that: The conditions for the enzymolysis in step S2 are: pH 4-7, temperature 40-60° C., the mass ratio of cellulase to pectinase is 1:150-1:250, and the enzymolysis time is 2-5 h.

4. The preparation method according to claim 1, characterized in that: The purification method is: fractionation treatment is performed with n-hexane, ethyl acetate and n-butanol in sequence.

5. An olive fruit extract prepared according to any one of claims 1 to 4.

6. Use of the olive fruit extract as claimed in claim 5 in the preparation of anti-aging products.

7. The use according to claim 6, characterized in that: The olive fruit extract achieves anti-aging effects through at least the following pathways: a1) Improve athletic ability; a2) Reduce the deposition of lipofuscin; a3) Improved resistance to oxidative stress and heat stress.

8. An anti-aging drug, characterized in that: The medicine contains the olive fruit extract according to claim 5.

9. The anti-aging drug according to claim 8, characterized in that The medicine also includes pharmaceutically acceptable excipients.

10. The anti-aging drug according to claim 8, characterized in that: The medicine is in the form of tablets, granules, capsules, powders, liquids or jelly.

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