KR100740416B1 - Process of manufacturing of olive leaf extract with physiological activities - Google Patents

Abstract

The present invention relates to a method for producing an extract of the olive leaf having a physiological activity, after pulverizing the olive leaf 20 to 30 mesh without using an organic solvent, refluxed for 5 hours at 85 ℃ by adding 80% ethanol And after the step of filtration and concentrated in vacuo at 40 ℃ to prepare an extract of the olive leaf. In addition, there is provided a method of preparing an extract of the powder by lyophilizing the olive leaf extract after the above steps.

Olive leaves

Description

Production method of olive leaf extract with physiological activity {PROCESS OF MANUFACTURING OF OLIVE LEAF EXTRACT WITH PHYSIOLOGICAL ACTIVITIES}

1 is a graph of the nitrite scavenging ability of the olive leaf extract and fractions according to Example 1.

Figure 2 is a graph of the SOD-like activity of the olive leaf extract and fractions according to Example 1.

Figure 3 is a graph measuring the peroxide value of the linoleic acid substrate of olive leaf extract, BHT, and a-tocopherol according to Example 1.

Figure 4 is a graph measuring the change of linoleic acid peroxide with the addition of olive leaf fractions.

Figure 5 is a graph measuring the peroxide change of the linoleic acid emulsion substrate of olive leaf extract, BHT, and a-tocopherol according to Example 1.

Figure 6 is a graph measuring the peroxide change of the linoleic acid emulsion substrate with the addition of olive leaf fractions.

Figure 7 is a graph measuring the change in the conjugated double acid value of the extract and BHT, a-tocopherol according to Example 1 during the heat oxidation.

Figure 8 is a graph measuring the change in the conjugated double acid value of the olive leaf fraction during heating oxidation.

Figure 9 is a graph measuring the acid value change of BHT, a-tocopherol and other extracts in Example 1 during heating oxidation.

10 is a graph measuring the acid value change of the olive leaf fraction during the heat oxidation.

11 shows the results of UV spectra of OLB5-1 and oleuropein.

12 is an HPLC chromatogram of OLB5-1 isolated from olive leaf butanol extract.

The present invention relates to a method for producing olive leaf extract having physiological activity.

Olive leaf has been used as a folk medicine for the purpose of treating malaria and high fever.In addition, it is effective for hypertension, atherosclerosis, colon cancer, inflammation, and food poisoning. Olive leaf extract lowers blood pressure or coronary artery. It has been known to increase the rate of blood flow, relieve arrhythmia, and prevent spasm of the small intestine muscles.

Mediterranean plants such as olive and grapes have higher levels of cardiovascular diseases such as hypertension, arteriosclerosis and heart disease, and bioactive compounds such as vitamins, flavonoids and polyphenols that lower the incidence of breast and colon cancer. . Recently, the increasing interest in bioactive substances in foods such as natural antioxidants is known to be due to some phenolic substances in the antioxidant effect of Mediterranean plants.

Therefore, in recent years, research on hydroxytyrosol and oleuropein among the polyphenols present in olive leaves has been actively conducted. Although a small amount of polyphenols present in olive leaves, as one of the groups of bioactive substances that are recently attracting attention, various physiological activities such as anti-cancer, anti-thrombotic, anti-inflammatory, anti-allergic and antimicrobial activities have been reported.
As a manufacturing method for the olive extract known so far, according to Korean Patent Publication No. 10-0449670 (name of the invention: a method for producing an olive powder and a composition for olive powder) according to the method for producing olive powder for taking for health food and olive The composition for powders is described. The present invention is to crush the olives and put corn flour and waxy corn starch into the stirrer to prepare olive powder to be individually packaged through a stirring mixing step, a freezing step, a drying step, a grinding step to make a composition for olive powder The present invention relates to a method for producing an olive leaf extract having a bioactivity of the present invention.

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In addition, studies on the physiological activity of olive leaves have been reported to have a significant effect on the antioxidant, antimicrobial and antihypertensive. Studies on antioxidant effects include Benavente-Garcia et al. [Benavente-Garcia, O., Castillo, J., Lorente, J., Ortuno, A. and Del Rio J.A. Antioxidant activity of phenolics extracted from Olea europaea L. leaves. Food Chem., 68 (1): 457-462 (2000)] analyzed the major phenolic substances present in olive leaf extracts and investigated their radical scavenging ability. Rutin, the flavonoid form, showed the strongest antioxidant effect, and luteolin, the flavone, also showed the strong antioxidant effect.

In addition, Somova et al. [Somova, L.L., Shode, F.O., Ramnanan, P. and Nadar, A. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves. J.Ethanopharmacology, 84: 299-305 (2003)], isolated triterpenoids from olive leaves of different cultivation sites and investigated their antihypertensive, anti-arteriosclerosis and antioxidant effects.

However, except for some studies on physiological activity, reports of olive leaves include only changes in minerals of olive leaves according to seasonal changes, changes in chemical composition of olive leaves according to drying conditions, and studies on the metabolic pathways of oleuropein. Studies on the overall biological activity of the olive leaf extract and their fractions are very incomplete.

SUMMARY OF THE INVENTION An object of the present invention is to provide an olive leaf extract manufacturing method having a physiological activity and an olive leaf extract prepared using the method. When extracting the olive leaf using the present invention, it is possible to prepare an olive leaf extract containing a large amount of polyphenol component without deteriorating the activity of the polyphenol component, and provides a safe extract that can be used as food because no organic solvent is used. can do.

Hereinafter, the present invention will be described in more detail.

Since the method of the present invention uses ethanol which is an edible alcohol without using an organic solvent, it is possible to provide a safe olive leaf extract, and also to provide an olive leaf extract containing a large amount of polyphenol without degrading polyphenol activity.
The present invention is crushed olive leaf 20 to 30 mesh (mesh); Add 80% ethanol to an Erlenmeyer flask; Reflux for 5 hours in a water bath at 85 ° C; Filtration (Whatman No. 2) step. Thereafter, the olive leaf extract may be obtained through a vacuum concentration process at 40 ° C., and may include a method of freeze-drying to prepare it as a powder.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited only to these examples.
Ethanol was used to prepare extracts that can be used in foods. Polyphenols are slightly nonpolar, so the higher the ethanol concentration, the better the extraction. However, 100% ethanol is difficult to produce as a powder. The experiment was carried out using 40% and 80% ethanol. In addition, the temperature at reflux was limited to 85 ° C, which supplies heat to extract olive leaves using ethanol, and the efficiency increases as the temperature increases, but the boiling point of ethanol is below 85 ° C. Since heating is inefficient, the said temperature value was set to an upper limit.

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Example 1

After washing the olive leaves to remove the foreign matter it is dried by hot air at 40 ℃. This is used as a sample after coarsely pulverizing to 20 to 30 mesh using a grinder (IKAM20, IKA, Germany). 10 g of crushed olive leaves were added to the flask, and 100 ml of 80% ethanol was added thereto, refluxed for 5 hours in an 85 ° C water bath, and then filtered (Whatman No. 2). 100 ml of extraction solvent was added to the remaining residue after filtration, extraction and filtering were performed in the same manner, and the same procedure was repeated twice. The filtrate obtained by extracting three times was left standing at 4 ℃ for 24 hours, centrifuged at 10,000g, concentrated under reduced pressure at 40 ℃ to prepare an olive leaf extract, after the above process, lyophilized olive leaf powder Extracts can also be prepared (Table 27).
Example 2

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After washing the olive leaves to remove the foreign matter it is dried by hot air at 40 ℃. This is used as a sample after coarsely crushing into 20 to 30 mesh using a grinder. 10 g of pulverized olive leaves were added to the flask, and 100 ml of 80% ethanol was added thereto, and the mixture was cooled to reflux for 1 hour in a 25 ° C water bath, and then filtered (Whatman No. 2). 100 ml of extraction solvent was added to the remaining residue after filtration, extraction and filtering were performed in the same manner, and the same procedure was repeated twice. The filtrate obtained by extracting three times was left at 4 ℃ for 24 hours, centrifuged at 10,000g, concentrated under reduced pressure at 40 ℃ to prepare an olive leaf extract, after the above process, lyophilized to dry the powder biological activity Olive leaf extract may be prepared. (Experiment 7)

The table below shows the results of experiments to find the proper extraction conditions effective for bioactivity by comparing the component properties of olive leaf extracts with physiological activity according to extraction temperature, extraction solvent, and extraction time.

<Examples with Different Extraction Conditions of Olive Leaves>

Extraction condition Exp. no Temp. (℃) Solvent Time (hr) One 25 D.W One 2 25 D.W 3 3 25 D.W 5 4 25 40% EtOH One 5 25 40% EtOH 3 6 25 40% EtOH 5 7 25 80% EtOH One 8 25 80% EtOH 3 9 25 80% EtOH 5 10 55 D.W One 11 55 D.W 3 12 55 D.W 5 13 55 40% EtOH One 14 55 40% EtOH 3 15 55 40% EtOH 5 16 55 80% EtOH One 17 55 80% EtOH 3 18 55 80% EtOH 5 19 85 D.W One 20 85 D.W 3 21 85 D.W 5 22 85 40% EtOH One 23 85 40% EtOH 3 24 85 40% EtOH 5 25 85 80% EtOH One 26 85 80% EtOH 3 27 85 80% EtOH 5

In order to check the effect of the extract produced through the experiment as described above was carried out the following experiment.

Test Example 1 Solid Contents of Extracts

Solid content of the olive leaf extracts having a physiological activity is measured after the freeze-dried olive leaf extract weight. The solid content of the olive leaf extracts according to the extraction conditions is shown in the table below. As the extraction temperature increases, the solid content of the olive leaf extracts tends to increase as the concentration of ethanol to be extracted increases. However, in the case of extraction time, considering the solid content and the extraction time, the condition that the solid content was most markedly high was 85 ° C., 80% ethanol, and treated for 5 hours (38%).

Test Example 2 Total Phenolic Content of Olive Leaf Extracts with Physiological Activities According to Extraction Conditions

Total phenol content was analyzed by Folin-Dennis method. In other words, Folin-Dennis reagent is added 10g sodium tungstate, 2g phosphomolybdic, 5ml pfosphoric acid into a volumetric flask, and then distilled water, and then into a conical flask to reflux for 2 hours. Specifically, 7 ml of distillate was added to the cap tube, and 1 ml of the sample dissolved in DMSO (dimethylsulfoxide) was added, and 0.5 ml of the Folin-Dennis reagent was added. After 3 minutes, 1 ml of saturated sodium carbonate anhydrous solution and 0.5 ml of distilled water were added. After adding the absorbance at 725 nm, compare with the standard solution to determine the total phenol content. Tannic acid was used as the standard solution.

Comparing the total phenolic content of the olive leaf extracts according to the extraction conditions, the ethanol concentration of the extractant increased. The extraction condition that showed the highest total phenol content was treated with 80% ethanol at 25 ° C for 1 hour, and the total phenol content was 22.1%.

Test Example 3 Electron Donating Ability of Olive Leaf Extracts with Physiological Activity According to Extraction Conditions

The electron donating ability of olive leaf extracts with physiological activity was measured using DPPH radical scavenging ability. That is, by applying the Blois method, 3 ml of a 3.5Ⅹ10 ^-3 M DPPH / EtOH solution and 0.15 ml of a sample dissolved in DMSO were mixed in a cap tube and left at room temperature for 30 minutes, and the absorbance was measured at 516 nm. The radical scavenging effect was calculated.

EDA (%) = 1- SA / CA Ⅹ 100

SA: sample absorbance

CA: control absorbance

As a result of measuring the electron donating ability of the olive leaf extracts according to the extraction conditions, the electron donating ability also increased as the ethanol concentration of the extraction solvent increased. This is the same trend as the result of the total phenolic content of the olive leaf extracts according to the extraction conditions shows a strong electron donating ability of 70.5% in the extract extracted with 80% ethanol at 25 ℃ for 1 hour. This is due to the phenolic compounds present in the olive leaf extracts having physiological activity and showed a strong radical scavenging effect by the hydroxyl group (-OH) present in the phenolic compounds structure.

<Solid Content, Total Phenolic Content and Electron Donating Ability of Olive Leaf Extracts with Physiological Activity>

Exp. no Solid content (%) Total phenol content (%) EDA ^{1) (%)} One 20.1 14.4 44.6 2 21.9 13.9 42.4 3 20.2 11.4 35.6 4 25.6 16.0 53.6 5 28.0 16.1 54.5 6 28.3 16.9 54.0 7 23.8 22.1 70.5 8 24.1 18.6 64.2 9 26.1 17.3 62.0 10 17.4 12.9 42.9 11 21.6 15.6 49.4 12 22.8 15.2 48.4 13 25.4 17.2 57.0 14 29.8 16.2 57.7 15 28.1 17.6 62.9 16 26.9 19.3 62.6 17 29.3 17.0 60.5 18 35.1 19.5 65.8 19 26.8 17.5 58.4 20 22.1 15.4 52.2 21 25.2 14.0 48.7 22 30.8 20.4 64.4 23 28.1 18.6 59.9 24 33.1 20.7 65.6 25 34.6 16.7 58.4 26 34.7 17.0 57.5 27 38.0 18.2 58.7

The following experiment is an experiment measuring the physiological activity of the extract prepared by the method according to Example 1 and the fraction fractionated again with an organic solvent according to the polarity of the solvent. This is an experiment to determine whether the polyphenol component contained in the extract of Example 1 actually has a physiological activity, by the following method.

The ethanol extract according to Example 1 was fractionated in each step using solvents of different polarities. The extract according to Example 1 was dissolved in water, put into a fractional filter, hexane was added, the hexane layer and the water layer were partitioned, and concentrated under reduced pressure at 50 ° C. to obtain a hexane fraction. In the same manner, chloroform, ethyl acetate and butanol were added sequentially to obtain chloroform, ethyl acetate, butanol and water fractions, respectively. These fractions were concentrated under reduced pressure and dried in vacuo to completely remove the solvent and then measure the content of solids.

Test Example 1 Nitrite Scavenging Ability

Nitrite scavenging activity was measured by the method of Gray and Dugan. That is, 1 mL of a certain concentration of sample was added to 1 mL of 1 mM NaNO ₂ solution, the pH of the reaction solution was adjusted to 1.2 with 0.1 N HCl (pH 1.2), and the total amount was 10 mL. After reacting this solution at 37 ° C for 1 hour, take 1 mL of each reaction solution, and mix 5 mL of 2% acetic acid solution with Griess reagent (1% sulfanilic acid and 1% naphthylamine prepared with 30% acetic acid in a 1: 1 ratio). 0.4 mL of the preparation) was added immediately before use and mixed well. After the mixture was left at room temperature for 15 minutes, the absorbance was measured at 520 nm using a UV / Vis spectrophotometer to determine the amount of residual nitrite. The blank test was performed in the same manner by adding 0.4 mL of distilled water instead of the Griess reagent. Nitrite scavenging activity is expressed as the percentage of nitrites with and without sample addition.

N (%) = 1-(A-C) / B * 100

N: nitrite scavenging ability

A: Absorbance of the sample to which 1 mM NaNO ₂ was added after reacting for 1 hour.

B: 1 mM NaNO ₂ absorbance

C: control absorbance

The above experiment was measured and compared at pH 1.2, and the results are shown in FIG. 1.

(FIG. 1, H: exion fraction, C: chlorophosity fraction, E: ethyl acetate fraction,

      B: butanol fraction, W: water fraction)

Nitrite scavenging ability according to Example 1 shows a tendency to increase nitrite scavenging ability in proportion to the concentration of the extract to 8.1-72.8% as the concentration of the extract increases to 0.01-0.5%. Among the fractions, butanol and ethyl acetate fractions were significantly higher at 7% and 75.4%, respectively, at 0.5% concentration. As a result, the nitrite scavenging ability of olive leaf fractions was proportional to the total flavonoid and total phenol content in each fraction, and the butanol and ethyl acetate fractions showed high contents of total flavonoid and total phenol. The correlation between nitrite scavenging effect is high.

Test Example 2 Antibacterial Activity

Searching for the antimicrobial effect of the extracts and fractions by solvent according to Example 1 was measured using the paper disc method. Two strains, Gram-positive bacteria and Gram-negative bacteria, were used for the antibacterial effect measurement. Each test strain was precultured in the corresponding liquid medium for 24 hours, and the plate medium was prepared by coagulation by dispensing 1.5 ml of agar sterilized with each growth medium into 20 ml of petridish and adding 0.1 ml of each test bacteria. The glass rods were applied to spread evenly on the medium. After absorbing the paper disc in which the solvent-specific fractions were injected at a certain concentration on a plate medium, 30 μl of sterilized water was injected, and incubated at 37 ° C. for 24 hours to measure the diameter (mm) of the inhibition clear zone around the paper disc. The antimicrobial effect on ethanol extracts and their respective fractions was compared and analyzed.

The results of the antimicrobial effect on the extracts and the respective fractions according to Example 1 are shown in the table below. The extract of Example 1 shows strong antibacterial effect in gram-positive bacteria Bacillus cereus, Staphylococcus aureus and gram-negative bacteria Escherichia coli, Salmonella enteritidis mode. Looking at the relationship between the extract concentration and the antimicrobial effect of Example 1, as the concentration of the extract increases, the antibacterial effect of the bacteria also tends to increase.

The antimicrobial effect of each fraction showed a strong antimicrobial effect against the bacteria only in the butanol fraction and ethyl acetate fraction.

<Antibacterial activity of extracts and fractions according to Example 1>

Microorganisms tested Conc. (μg / disc) 80% EtOH H (mm) C (mm) E (mm) B (mm) W (mm) Bacillus cereus 800 19 - - 22 23 12.5 KCCM 40935 400 13.5 - - 17 18 - 200 11 - - 12.5 13 - 100 - - - 10 10.5 - Staphylococcus aureus 800 12.5 - - 21 20 - KCCM 40307 400 11 - - 14 13 - 200 - - - 11 11 - 100 - - - - 11 - Escherichia coli 800 20 - - 21 22 12 KCCM 11234 400 16 - - 17 17.5 11 200 12 - - 12.5 12 - 100 - - - 10 10 - Salmonella enteritidis 800 16 - - 17.5 20 13 KCCM 12021 400 13 - - 13 16 11 200 11 - - 11 12 10 100 10 - - 10 11 -

H: Hexane Fraction

C: chloroform fraction,

E: ethyl acetate fraction,

B: butanol fraction,

W: water fraction.

Test Example 3 SOD-like Activity

SOD-like activity was searched to determine the radical scavenging ability in the enzyme-based system for the extract extracted in accordance with Example 1 and the respective fractions. Superoxide radical scavenging activity was measured by xanthine-xanthine oxidase cytochrome C reduction method according to Lio et al. That is, 0.2 ml of sample, 1.2 ml of 50 mM PBS (phosphate buffered saline) buffer (pH7.8), 0.2 ml of 1 mM xanthine and 0.2 ml of 0.05 mM cytochrome C were mixed. To this was added 0.2 mL of xanthine oxidase diluted so that the change in absorbance per minute at 550 nm was 0.02. The scavenging ability of superoxide anion was calculated from the following equation.

Superoxide anion scavenging activity = (1-Abs / Abc) * 100

ABC: controlled absorbance at 550nm

Abs: absorbance after sample treatment at 550 nm

Similar activity for the extract according to Example 1 and its fractions is as shown in FIG. The SOD-like activity of the extract according to Example 1 showed 21.1% of the activity at the concentration of 1 mg / mL, but showed 53.1% of the activity at the concentration of 10 mg / mL. Show a tendency.

Among the fractions, butanol and ethyl acetate fractions showed significantly higher values of 85.4% and 86.3%, respectively, at a concentration of 10 mg / mL.

(FIG. 2, H: exion fraction, C: chlorophosity fraction, E: ethyl acetate fraction,

      B: butanol fraction, W: water fraction)

Experiment 4 Measurement of Antioxidant Effect on Linoleic Acid Substrate

Antioxidant effect on linoleic acid substrate was tested with control group and comparison group. The control group used 50 g of linoleic acid without any additives, and the experimental group added the extract according to Example 1 and the respective fractions at a concentration of 0.02% (w / w) with respect to 50 g of linoleic acid. To 50 g of acid, synthetic antioxidant BHT and natural antioxidant α-tocopherol were added at a concentration of 0.02% (w / w), respectively. The peroxide value was measured and compared at 12 hours intervals while storing the prepared samples in a 55 ± 1 ℃ incubator. The induction period setting according to the change of peroxide value during storage was the time taken for the peroxide value to reach 50 meq / kg oil. The measurement solution for a predetermined amount of the sample chloroform / acetic acid in gwasanhwamulga (20: 30% v / v ) was dissolved in 30 ㎖ to liberate the I ₂ was added to a saturated KI (potassium iodide) solution of Na ₂ known concentration of I ₂ After titration with S ₂ O ₃ (sodium thiosulfate) standard solution, the amount of peroxide was calculated by the following equation.

POV (meq / kgoil) = ((A-B) * N / S) * 100

A: titration of sample, mL

B: titration of blank, mL

S: weight of sample (g)

N: normality of sodium thiosulfate solution

The results of the above experiment are as shown in FIGS. 3 and 4.

As shown in FIG. 3, the peroxide value of the 80% ethanol extract of the olive leaf at 84 hours of storage was 80.1 meq / kg oil, which was lower than that of the control which was negative control. Compared with the antioxidants used as the positive control, the peroxide values of BHT and α-tocopherol at 84 hours of storage were 65.8 and 78.7 meq / kg oil, respectively, showing a greater antioxidant effect than the control or Example 1.

As shown in FIG. 4, the peroxide value of linoleic acid with the addition of olive leaf fractions was measured, and the peroxide values of hexane, chloroform, ethyl acetate, butanol and water fractions were 88.3, 83.3, 77.5, respectively. It appears as 66.3 and 83.3 meq / kg oils and appears to inhibit peroxide production in butanol fraction and ethyl acetate fraction.

Test Example 5 Measurement of Antioxidant Effect in Linoleic Acid Emulsion System

The inhibitory effect of peroxide production on the linoleic acid emulsion substrate by the extract according to Example 1 and the respective fractions was measured. A method for preparing a lenoleic acid emulsion substrate is to dissolve 2.6 mL of linoleic acid in 100 mL of 99.5% ethanol, and then take 10 mL of each into a conical tube and extract 80% ethanol extract of olive leaf, and the concentration of each fraction and antioxidant is 0.02. After each addition to the%, 0.1mL phosphate buffer (pH7.0) was added to 10mL, 4.5mL of distilled water and stored in a 40 ± 1 ℃ constant temperature and the change of peroxide was measured at day interval. Changes in peroxide content in the linoleic acid emulsion system were measured using the thiocyanate method. As a measuring method, add 0.1 mL of each sample and 0.1 mL of 30% ammonium cyanate to 4.7 mL of 75% ethanol, and after exactly 3 minutes, add 0.1 mL of 20 mM iron (II) chloride (in 3.5% HCl) at 500 nm. Changes in absorbance were measured with a UV / Vis spectrophotometer as an indicator of peroxide content in each sample.

The results according to the extract of Example 1 are shown in Figure 5, the antioxidant effect of each fraction is as shown in Figure 6.

In FIG. 5, when the extract, BHT, α-tocopherol and control peroxide contents of Example 1 were compared, the initial storage value was 0.064, but the control group was 2.277 after 4 days of storage, and α-tocopherol was 2.209 after 1 day of storage. BHT tends to increase in peroxide content as the storage period increases to 2.18 after 25 days of storage. On the other hand, the extract according to Example 1 shows a low increase of 0.362 even after 43 days of storage, showing an excellent antioxidant effect compared to the positive control BHT or α-tocopherol.

According to Figure 6, the ethyl acetate fraction and butanol fraction shows the best effect.

 Test Example 6 Antioxidant Effect of Olive Leaf Fractions During Heating Oxidation

In order to determine the rancidity inhibitory effect of frying oil during heating oxidation according to the addition of the extract according to Example 1 and the respective fractions, the concentration of the extract and the respective fractions according to Example 1 was added to soybean oil at a concentration of 0.02% and then added to the test tube. Divided into 10g each, the change of acid value, conjugated diene value and refractive index were measured at 12 hour intervals while heating and oxidizing for 48 hours in an oil bath maintained at 180 ± 2 ℃. The antioxidant effects of the extract of Example 1 and the respective fractions were measured. Acid value was measured according to A.O.C.S process method Cd-3a-63. In other words, 5 g of the sample is accurately placed in an Erlenmeyer flask, and 100 mL of ethanol-ether mixture (1: 1. V / v) is added to dissolve it. Then, 2-3 drops of 1% phenolphthalein solution is added, and a light pink color is continued for 30 seconds with 0.1 N KOH solution. Titration until The acid value is calculated by the following equation.

Acid value = ((A-B) * F * N) / S

A: mL of standard alkali used for titration

B: mL of standard alkali used in titration comparison experiment

N: standard alkali normal concentration

S: sample weight (g)

The change of conjugated double acid value was measured according to A.O.C.S process method Ti-la 64. In other words, after taking a sample of about 90 to 130 mg in a 100 mL flask, 75 mL of isooctane was added thereto, and the fats and oils were completely dissolved and used as isooctane. If necessary, the absorbance was measured at 233 nm using a UV / Vis spectrophotometer while diluting in a range not exceeding 10 times. The conjugated double acid value of the measured sample is obtained by the following equation.

Conjugated double acid value = 0.84 * (AS / bs-K ₀ )

K ₀ : absorbance by acid or ester (acid: 0.03, ester: 0.07)

AS: absorbance measured at 233 nm

b: cell length (cm)

s: final dilution concentration (g / l) of oil used for absorbance measurement

The results of measuring the change in conjugated double acid value, acid value and refractive index of the extract of Example 1 and the respective fractions for the heating oxidation are shown in FIGS. 7 to 10 and the table below.

Heat oxidation time (hr) Samples 0 12 24 36 48  Control 1.4722 1.4729 1.4735 1.4738 1.4745  BHT 1.4722 1.4730 1.4732 1.4735 1.4736  α-tocopherol 1.4722 1.4729 1.4731 1.4734 1.4739  80% EtOH 1.4722 1.4725 1.4731 1.4732 1.4739  Hexane fr. 1.4722 1.4732 1.4730 1.4738 1.4740  Chloroform fr. 1.4722 1.4731 1.4735 1.4735 1.4739  Ethylacetate fr. 1.4722 1.4731 1.4730 1.4735 1.4739  Butanol fr. 1.4722 1.4731 1.4731 1.4735 1.4739  Water fr. 1.4722 1.4731 1.4731 1.4735 1.4739

In order to investigate the bioactive substances of olive leaves, experiments to determine the separation, purification and structure of the bioactive substances were carried out as follows.

Test Example 1

In order to separate the bioactive substance of the olive leaf, the extract according to Example 1 was fractionated with butanol, and then separated and purified by HPLC to obtain five OLB 1 to 5. OLB 5-1 and OLB 5-2 were separated and purified by performing HPLC again only on OLB5, which showed the strongest DPPH radical scavenging ability among these fractions. It can be seen from the results of FIGS. 11 and 12 that the main phenolic substance in the olive leaf is oleuropein.

Test Example 2

As a result of examining the H-NMR spectrum and the C-NMR spectrum of OLB 5-1, it can be seen that the material separated from the olive leaf butanol fraction is oleuropein having the above structure.

As described above, when preparing the olive leaf extract by the method according to the present invention, there is no decrease in activity and can provide an olive leaf extract containing a large amount of phenols, which has a variety of biological effects such as antibacterial activity, antioxidant effect As having, it can be utilized in various foods.

Claims (5)

In the method for producing an extract of olive leaves having a biological activity, Adding 80% ethanol to the sample obtained by grinding the olive leaf into 20 to 30 mesh; Reflux cooling at 85 ° C. for 5 hours, Filtration step, Method for producing an olive leaf extract having a physiological activity comprising the step of vacuum concentration at 40 ℃. In the method for producing an extract of olive leaves having a biological activity, Adding 80% ethanol to the sample ground the olive leaves in 20 to 30 mesh, Reflux cooling at 25 ° C. for 1 hour, Filtration step, Method for producing an olive leaf extract having a physiological activity comprising the step of vacuum concentration at 40 ℃. delete  Adding 80% ethanol to the sample ground the olive leaves in 20 to 30 mesh,  Reflux cooling at 85 ° C. for 5 hours and filtering;  Olive leaf extract having a physiological activity prepared by the manufacturing method according to claim 1 comprising the step of vacuum concentration at 40 ℃. Adding 80% ethanol to the sample ground the olive leaf to 20 to 30 mesh, Reflux cooling at 25 ° C. for 1 hour and filtering; Olive leaf extract having a physiological activity prepared through the manufacturing method according to claim 2 comprising the step of vacuum concentration at 40 ℃.

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