KR102176441B1 - Method for preparing green tea extract comprising high concentrated theanin and gaba - Google Patents

Abstract

The present invention comprises the steps of: (a) preparing a green tea material by drying green tea leaves; (b) extracting an alcohol extract by adding the green tea material to an aqueous alcohol solution; (c) ultrasonicating the alcohol extract; And (d) enzymatic treatment of the sonicated alcohol extract; a method for producing a green tea extract containing high concentration theanine and gaba is provided. Accordingly, while extracting theanine and gava with high efficiency, the extraction process is simple and reasonable in terms of cost, and the content of caffeine is reduced while the content of theobromine is increased, thereby preventing side effects that cause anxiety or anxiety.

Description

Manufacturing method of green tea extract containing high concentration theanine and gaba {METHOD FOR PREPARING GREEN TEA EXTRACT COMPRISING HIGH CONCENTRATED THEANIN AND GABA}

The present invention relates to a method for producing a green tea extract containing high concentrations of theanine and gaba, and more particularly, to improve the brain protective function of increasing the content of theanine and gaba and lowering the caffeine content in the green tea leaf extract. It relates to a method for producing a green tea extract that can be.

Green tea is known to contain various components such as polyphenols, protein amino acids, vitamins and minerals. Among them, studies have been actively conducted on polyphenols, particularly epigallocatechin gallate (EGCG), which are known to have excellent antioxidant and anticancer effects. Recently, however, interest in this has increased as the substances of theanine and γ-Aminobutyric acid, which are amino acids that are uniquely present in green tea, among green tea ingredients have been found to be important ingredients that determine the umami and efficacy of green tea. have. Theanine is biosynthesized from the roots of green tea through the enzymatic reaction of glutamine and ethylamine, transferred to the leaves, concentrated, and converted into polyphenols by some sunlight, but most of them exist in the form of free amino acids, and theanine and gaba are tea leaves. It is known to account for more than 50% or 0.01 to 2.0% of the total amino acids with 1 to 2% of the dry amount. Research on the efficacy of theanine so far has mainly focused on nerve-related parts, such as generating alpha waves, which are brain waves in a comfortable and stable state, to stabilize the mind and body, relieve stress, and protect nerve cells. In particular, theanine, which is studied as hormones as major regulators of dopamine and serotonin, is one of the major amino acids in green tea. Previous studies showed that it has a sedative effect by increasing brain alpha waves, and has a regulating function of dopamine and serotonin. , It has been reported that when ingesting a beverage containing green tea extract, there is a change in brain waves and an effect of improving anxiety.

Until now, methods for extracting theanine and gava have been tried from various angles, mainly after physical treatment such as hot water extraction or water extraction and ultrasonic treatment, and then using expensive analysis systems such as TLC and HPLC, showing low extraction efficiency. It was disadvantageous in terms of economy as it required expensive investment, a lot of time, and a large labor force. In addition, most of the water is used due to the hydrophilic structure of the amino acid component during extraction.At this time, it is contained in a large amount of green tea, has a high arousal effect, and when overdose, caffeine accompanied by gastrointestinal disorders or anxiety symptoms is eluted together to separate them to obtain theanine and gaba Difficulties exist.

Korean Patent Application Publication No. 10-2007-0074718

It is an object of the present invention to extract theanine and gaba with high efficiency, while the extraction process is simple and reasonable in terms of cost, and a substance that has an arousal effect and does not cause anxiety symptoms while minimizing the caffeine content accompanying anxiety symptoms and gastrointestinal disorders. It is to provide a method of extracting theanine and gaba from green tea that can increase the content of theobromine.

According to an aspect of the present invention,

(a) drying green tea leaves to prepare green tea ingredients;

(b) extracting an alcohol extract by adding the green tea material to an aqueous alcohol solution;

(c) ultrasonicating the alcohol extract; And

(D) enzymatic treatment of the sonicated alcohol extract; a method for preparing a green tea extract containing high concentration theanine and gaba is provided.

In step (a), the drying may be performed according to any one method selected from hot air drying after thickening, and green tea leaves freeze drying, hot air drying, and roasting.

Preferably, the drying may be performed by hot air drying after thickening.

After the thickening, hot air drying may be performed by hot air drying at 90 to 110°C for 1 to 3 hours after the thickening.

The green tea material of step (a) may be prepared with an aqueous green tea solution of 1 to 10% (w/v).

The alcohol of the aqueous alcohol solution in step (b) may be at least one selected from methanol, ethanol, propanol, and butanol.

The aqueous alcohol solution in step (b) may have a concentration of 40 to 80% (w/w).

The extraction of step (b) may be carried out at 20 to 30°C.

After step (b), the step of removing ethanol from the alcohol extract may be further performed.

The ultrasonic treatment in step (c) may be performed at an intensity of 25 to 35% at 700 to 750W.

The ultrasonic treatment may be performed for 5 to 15 minutes.

The enzyme treatment in step (d) was performed on the black mold ( Aspergillus niger ) derived enzymes.

Enzymatic treatment in step (d) is pectinase, polygalacturonase, pectin methyl esterase, glucanase, and arabinase. It may be carried out by one or more enzymes selected from among.

Preferably, the enzymatic treatment may be performed by at least one enzyme selected from pectinase, polygalacturonase, and pectin methyl esterase.

The enzyme treatment can be used at a concentration of 0.5 to 2% (w/v).

The method for preparing the green tea extract may reduce caffeine content and increase theobromine content compared to green tea ingredients.

According to another aspect of the present invention,

There is provided a method for preparing a pharmaceutical composition for preventing or treating degenerative brain diseases comprising a green tea extract including a method for preparing a green tea extract containing the high concentration theanine and gaba as an active ingredient.

The green tea extract contained in the pharmaceutical composition contains 2.0 to 3.0 wt% of theanine, 0.1 to 0.25 wt% of Gaba, 0.05 to 1.5 wt% of caffeine, and 0.2 to 0.5 wt% of theobromine based on the total content of the green tea extract can do.

The degenerative brain disease may be any one selected from dementia, mild cognitive impairment, multiple sclerosis, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, and Pick's disease.

According to another aspect of the present invention,

There is provided a method for preparing a brain-protecting functional food composition comprising a green tea extract including a method for preparing a green tea extract containing the high concentration theanine and gaba as an active ingredient.

The green tea extract contained in the food composition contains 2.0 to 3.0 wt% of theanine, 0.1 to 0.25 wt% of Gaba, 0.05 to 1.5 wt% of caffeine, and 0.2 to 0.5 wt% of theobromine based on the total content of the green tea extract can do.

The brain protective function may be anti-stress or mental arousal function.

The method of extracting theanine and gaba from green tea of the present invention is a method of extracting theanine and gaba from green tea with high efficiency, while the extraction process is simple and reasonable in terms of cost, and has an arousal effect, but caffeine content accompanied by anxiety symptoms and gastrointestinal disorders. It provides a method of extracting theanine and gaba from green tea that can significantly increase the content of theobromine, a caffeine-like structure, which is a useful substance that has an arousal effect and does not cause anxiety symptoms while reducing by three times or more. have.

1 is a flowchart sequentially showing a method for extracting high concentration theanine and gaba according to the present invention.
2 is a result of comparing the content of theanine and gaba by variety according to Experimental Example 1 by TLC.
3 is a result of comparing the content of theanine and gaba by variety according to Experimental Example 1 by LC-MS/MS.
4 shows the TLC analysis results of theanine and gaba present in the extract according to four treatment conditions according to Experimental Example 1.
5 is a result of comparing the content of theanine and gaba by LC-MS/MS according to four treatment conditions according to Experimental Example 1.
6 is a result of TLC analysis of theanine by sample concentration according to Test Example 2.
7 is a result of TLC analysis of theanine according to ethanol concentration of the solvent according to Test Example 2.
8 is a result of analyzing theanine by ultrasonic treatment conditions according to Test Example 2 by TLC.
9 is a result of analyzing theanine (Theanine) present in green tea extract for each enzyme type according to Test Example 3 by TLC.
10 is a result of TLC analysis of theanine present in green tea extract by enzyme concentration and reaction time of CP enzyme according to Test Example 3.
11 and 12 are HPLC analysis results according to the ultrasonic and enzyme treatment of Test Example 4.
13 shows the results of analyzing the protective effect of cranial nerve cells according to Test Example 6.
14 is an analysis result of anti-stress effect according to Test Example 6.
15 is an analysis result of anti-dementia effect according to Test Example 7.

Since the present invention can apply various transformations and have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude in advance.

1 is a flowchart sequentially showing a method for preparing a green tea extract containing high concentration theanine and gaba of the present invention. With reference to this, a method for preparing a green tea extract containing high concentration theanine and gaba of the present invention will be described.

first, Green tea leaves Dry to prepare green tea ingredients (step a).

Drying may be performed by hot air drying after thickening, and green tea leaves may be performed according to any one method selected from freeze drying, hot air drying, and roasting, and preferably, the drying may be performed by hot air drying after thickening.

After the thickening, hot air drying may be performed by hot air drying at 90 to 110°C for 1 to 3 hours after the thickening.

The green tea material is preferably prepared and used in an aqueous green tea solution of 1 to 10% (w/v).

Next, the green tea material is added to an aqueous alcohol solution to extract an alcohol extract (step b).

The alcohol of the aqueous alcohol solution may be at least one selected from methanol, ethanol, propanol, and butanol.

The aqueous alcohol solution may have a concentration of 40 to 80% (w/w), preferably 45 to 55%, more preferably about 50%.

The extraction may be performed at 20 to 30° C., preferably at room temperature.

After this step, the step of removing ethanol from the alcohol extract may be additionally performed.

Thereafter, the alcohol extract is subjected to ultrasonic treatment (step c).

The ultrasonic treatment is preferably performed at an intensity of 25 to 35% at 700 to 750W, and in this case, it is preferable to perform for 5 to 15 minutes, and more preferably at 700 to 700W at 30% intensity for 10 minutes. .

Finally, the sonicated alcohol extract Enzyme treatment (Step d).

The enzyme treatment in step (d) was performed on the black mold ( Aspergillus niger ) method for producing a green tea extract containing high concentration theanine and gaba, characterized in that carried out by a derived enzyme.

Enzymatic treatment in step (d) is pectinase, polygalacturonase, pectin methyl esterase, glucanase, and arabinase. It is preferable to carry out with at least one enzyme selected from among.

More preferably, the enzymatic treatment may be performed by at least one enzyme selected from pectinase, polygalacturonase, and pectin methyl esterase.

The enzyme treatment can be used at a concentration of 0.5 to 2% (w/v).

The method of preparing the green tea extract may be a method of decreasing the caffeine content and increasing the deobromine content compared to the green tea material.

The present invention provides a method for preparing a pharmaceutical composition for preventing or treating degenerative brain diseases comprising a green tea extract including a green tea extract containing the high concentration theanine and gaba as an active ingredient.

The green tea extract contained in the pharmaceutical composition contains 2.0 to 3.0 wt% of theanine, 0.1 to 0.25 wt% of Gaba, 0.05 to 1.5 wt% of caffeine, and 0.2 to 0.5 wt% of theobromine based on the total content of the green tea extract It is desirable to do.

More preferably, the green tea extract contained in the pharmaceutical composition is based on the total content of the green tea extract, theanine 2.3 to 2.8 wt%, gaba 0.15 to 0.22 wt%, caffeine 0.1 to 1.2 wt%, and theobromine 0.3 to It may contain 0.5 wt%.

The degenerative brain disease may be any one selected from dementia, mild cognitive impairment, multiple sclerosis, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, and Pick's disease, but the scope of the present invention is not limited thereto, and degeneration due to destruction of brain cells Brain diseases can be applied.

The pharmaceutical composition prepared according to the preparation method of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, Expanding agents, lubricants, lubricants or flavoring agents may be used.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form of the pharmaceutical composition may be a liquid, syrup, juice, suspension, emulsion, drop, or the like. For example, also, if necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. It is preferable that the pharmaceutical composition of the present invention is administered by transdermal administration.

The present invention provides a method for preparing a brain-protecting functional food composition comprising a green tea extract including a method for preparing a green tea extract containing the high concentration theanine and gaba as an active ingredient.

The green tea extract contained in the food composition contains 2.0 to 3.0 wt% of theanine, 0.1 to 0.25 wt% of Gaba, 0.05 to 1.5 wt% of caffeine, and 0.2 to 0.5 wt% of theobromine based on the total content of the green tea extract It is desirable to do.

More preferably, the green tea extract contained in the food composition is based on the total content of the green tea extract, theanine 2.3 to 2.8 wt%, gaba 0.15 to 0.22 wt%, caffeine 0.1 to 1.2 wt%, and theobromine 0.3 to It may contain 0.5 wt%.

The brain protective function may be anti-stress or mental arousal function.

The food composition prepared according to the manufacturing method of the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the food composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice creams, vitamin complexes, health supplements. And foods.

The food composition may include not only the active ingredient, but also ingredients commonly added during food production, and, for example, include proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, in addition to the active ingredients of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included.

[Example]

Example

(1) material preparation

Green tea used in the examples ( Camellia sinensis ) used domestic varieties, bohyang, chamnok, and foreign varieties, Yabukida. In April-May, when it is known that it contains large amounts of gaba and theanine, green tea leaves were collected directly from Jeollanam-do Green Tea Research Institute (Boseong) and 1) freeze-dried 2) hot air drying (1 hour at 100℃), 3) thickening After hot air drying (2 hours at 100℃ after 20kg/L steam), 4) roasting for green tea (2.3 minutes at 40℃ → 5 minutes to keep in mind → 4.3 minutes at 120℃ → 4.5 minutes at 100℃ → 80℃) It was dried in four methods of drying for 20 minutes). Thereafter, freeze-dried and hot-water extracted samples were prepared.

(2) Theanine and Gaba extract

The dried green tea leaf material was dissolved in water containing 0.1% TFA (trifluoroacetic acid) to prepare a green tea leaf sample, placed in an aqueous ethanol solution and subjected to ultrasonic treatment, centrifugation was performed at 10,000 rpm for 30 minutes at 4°C, and then 0.22 After ethanol was removed by filtering with a µm filter, theanine and gaba were extracted by enzyme treatment.

[Experimental Example]

Experimental example 1: Analysis of effects of green tea varieties and material treatment

Samples containing high concentrations of gava and theanine, which are known to have good brain protection effects in green tea, were selected. At the end of April, 3 kinds of green tea 1) Bohyang, 2) Chamnok, 3) Yabugida were collected and analyzed. Among these varieties, chamnok was used at the time of collecting the first water tea with a high theanine content. May 15), 2) Three works (April 25), 3) Middle works (May 5), 4) Master works (May 15). In addition, as important amino acids contained in green tea leaves are destroyed in a short time by degrading enzymes such as polyphenoloxydate, dry them in the following four ways to maintain their content: 1) Freeze drying 2) Hot air drying (1 at 100℃) Time), 3) Hot air drying after thickening (2 hours at 100℃ after 20kg/L steam), 4) Roasting (2 hours at 100℃ after steaming 20kg/L), 4) Roasting at 40℃ for 2.3 minutes → 5 minutes to keep in mind → 4.3 minutes at 120℃ → 4.5 minutes at 100℃ → It was cooked at 80℃ for 20 minutes). The dried green tea leaf material is dissolved in water containing 0.1% TFA (trifluoroacetic acid) at a concentration of 2% (w/v) for 30 min at 60°C for 30 min, ultrasonication 2 min, centrifugation at 10,000 rpm for 30 min 4 After performing at °C, a sample was prepared by filtering with a 0.22 μm filter. The content of the final extracted theanine and gaba was investigated using thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC).

Table 1 below shows the results of the investigation of total amino acid, theanine, and gaba content for each green tea variety.

division Theanine GABA Total amino acid (mg/g DW) Bohyang 9.22b ±0.06 0.24b　±0.01 18.95b ±0.07 Charm green 10.29a ±0.01 0.38a ±0.07 20.50a ±0.04 Swear 8.13c ±0.02 0.23b ±0.02 17.59c ±0.10

In addition, the results of comparing the content of Theanine and Gaba (γ-Aminobutyric acid, GABA) present in the extract by green tea variety (right) and first water tea time (left) by TLC are shown in FIG. 2.

In addition, the content of theanine (red dotted line) and gaba (green dotted line) present in the extract by green tea variety (right) and first water tea time (left) is LC-MS/MS with positive electro-spray ionization (+ESI). After ionization, analysis was performed in multiple reaction monitoring (MRM) mode, and the results are shown in FIG. 3.

In addition, the results of the total amino acid, theanine, and gaba content survey results for each period of the true green cultivar whose theanine and gaba content were measured relatively high among green tea varieties are shown in Table 2 below.

Charm green (collection date) Theanine GABA Total amino acid (mg/g DW) Woojeon/Gogu (4.15) 12.94a ±0.07 0.42a ±0.01 22.50a ±0.04 Sejak (4.25) 12.65b±0.12 0.39b ±0.04 21.04b ±0.06 Medium (5.5) 11.01c±0.09 0.35b ±0.07 20.87c ±0.19 Masterpiece (5.15) 8.98d±0.10 0.32b ±0.03 20.41c ±0.43

According to the TLC and LC-MS/MS analysis results of Table 1, Table 2, and FIGS. 2 and 3, among the three cultivars, the content of theanine of charm rust was 127% higher than that of Yabugida, the foreign variety, and the content of Gaba was 165% higher. Appeared. In addition, as a result of investigating the theanine content of the first water tea of the true green cultivar, the theanine content was less than 15% in the samples of medium crops or large crops that are cheaper than expensive green teas such as Ujeon or Sejak, and have large leaves. In particular, it can be seen that there is no significant difference in the Gaba content.

Fig. 4 shows the TLC analysis results of theanine and Gaba present in the extract for each of the four treatment conditions using the first water difference of the cultivar of the true green cultivar, medium and fine crops, and LC-MS/ in multiple reaction monitoring (MRM) mode after ionization. The MS analysis results are shown in FIG. 5.

In addition, the results of the investigation of theanine and gaba content for each treatment are shown in Table 3 below.

Treatment method Theanine GABA Total amino acid (mg/g DW) freezing 10.29a ±0.11 0.35a ±0.07 20.50a ±0.04 sirocco 4.80d ±0.10 0.11c ±0.10 12.23d ±0.43 Thickening + hot air 8.96b ±0.07 0.34b±0.07 16.40b ±0.30 Thinness 5.28c ±0.02 0.18c ±0.02 13.54c ±0.26

As a result of the TLC and LC-MS/MS analysis of Table 3 and FIGS. 4 and 5, in order to prevent the loss after collection of theanine, theanine during the four treatments was 10.2mg for the raw sample, compared to 10.2mg for the steam, 8.9mg for the finished green tea, and 5.2 Tea leaves steamed in mg compared to green tea contain 70% higher theanine content. The same pattern was found in the gaba content, and it was found that gaba was more than three times higher in heat-evaporated leaves compared to green tea or hot air drying.

Therefore, it is effective to extract theanine and Gaba at high concentrations to extract green tea using hot air treatment (100°C, 2 hours after 20kg/L steam treatment) after collecting medium or large crops of true green varieties.

Experimental example 2: high concentration Theanine And Gavar Analysis of extraction conditions contained

First, after extracting the true green using the heat-evaporated powder in four ways as follows, an efficient method for extracting theanine and gaba was selected. The sample was treated as follows after adding the tea leaf powder at a concentration of 2% (w/v) except for the investigation of the sample concentration condition.

-Sample concentration: 0~10%, 80℃ for 10 minutes

-Solvent conditions (ethanol added amount): 5% sample use, 0-100% ethanol concentration, extraction at 25℃ for 10 minutes

-Ultrasonic treatment: 30% (700W, 20kHz) 5 sec/1 sec 0~30 min

The results of analyzing the theanine in 5% extract by sample concentration, solvent ethanol concentration, and sonication conditions of the true green variety by TLC are shown in FIGS. 6 to 8, respectively.

In addition, the results of the analysis of theanine and gaba content by HPLC according to treatment conditions after collection of true green varieties are summarized in Table 4 below.

Treatment conditions Theanine GABA Total amino acid (mg/g DW) Sample concentration
(%) 0.5 0.80d ±0.04 0.06d ±0.08 6.03d ±0.06 One 2.10c ±0.12 0.09c ±0.16 10.12c ±0.10 5 10.16b ±0.07 0.32b ±0.08 24.42b ±0.02 10 15.26a ±0.01 0.69a ±0.11 42.05a ±0.13 Solvent condition
(%) 0 9.66e ±0.07 0.30d ±0.08 19.92d ±0.02 10 11.39cd±0.21 0.36d ±0.01 19.81d ±0.27 20 12.56c ±0.01 0.41c ±0.22 20.51bc ±0.15 50 15.02a ±0.25 0.69a ±0.08 25.44a ±0.04 80 13.13b ±0.03 0.52b ±0.02 22.58b ±0.23 100 7.96e ±0.12 0.13e ±0.31 13.46e ±0.19 ultrasonic wave
time
(min) 0 09.66d ±0.09 0.28d ±0.09 19.92e ±0.02 One 10.71d ±0.11 0.32d ±0.10 20.86c ±0.10 3 12.55b ±0.18 0.40bc ±0.22 22.99b ±0.33 5 14.37b ±0.25 0.69b ±0.06 23.31b ±0.06 10 17.66a ±0.06 0.97a ±0.01 23.99a ±0.12 30 12.72e ±0.22 0.39c ±0.22 16.23d±0.01

Looking at each sample concentration, 5 to 10% optimally contained 10.16 mg/g theanine, which is 41% of the total amino acid content in the case of 5%, and 35% theanine extraction efficiency was shown in 10%.

As a result of extraction and irradiation for 10 minutes at 25℃ by adding 5% green tea with 0~100% of the extraction solvent for each ethanol content, extraction with 50% ethanol solvent contains 15 mg/g theanine (60% of total amino acids). The amount of theanine extracted was shown. In addition, there was little change in the theanine content up to the ethanol concentration of 50 to 80%, but at 90% and 100%, the theanine content of the extract was significantly reduced to a level of 0.5 times compared to the extract with 50% ethanol.

The ultrasonic extraction conditions were treated for 0 to 30 minutes with a 5 second on/1 second off cycle at 30% (700W, 20 kHz) intensity, and the highest content of theanine and gaba were extracted during 10 minutes treatment, and ultrasonic treatment for a longer time. Showed a result of significantly reducing the content by decomposing the components of theanine and gaba.

Experimental example 3: according to enzyme treatment Theanine Gaba extraction

In order to examine the degree of extraction of theanine and gaba from green tea according to the types of enzymes, eight types of commercial complex enzymes listed in Table 5 below were used.

Fig. 9 shows the results of TLC analysis of theanine present in the 2% extract using 2% (w/v) concentration for each enzyme type, and the results of the analysis by TLC are shown in FIG. Fig. 10 shows the results of analyzing theanine in the 2% extract by TLC.

According to this, the results of 8 commercial enzyme treatments showed that CP and UF were most effective in the production of theanine and gaba. In addition, as a result of secondary treatment of only the two enzymes, the CP enzyme showed the best effect on extracting theanine and Gaba.

Therefore, the effect of extracting theanine and gaba according to the enzyme concentration and reaction time was analyzed using CP enzymes (containing Pectinase, polygalacturonase, and pectin methyl esterase). As a result, when 0.5~2% CP enzyme was added, the decomposition efficiency was the highest, and the reaction time was 3~6 hours, and the efficiency was high. If it was out of the range, the extraction amount of the target component tended to decrease.

The contents of theanine and Gaba extract according to the CP enzyme treatment conditions were investigated in the hot air-dried green tea after the thickening of the examples, and the results are shown in Table 6 below.

Treatment conditions Theanine GABA Total amino acid (mg/g DW) Enzyme concentration
(%) 0 8.46f ±0.10 0.26f ±0.03 20.11f ±0.30 0.05 9.23e ±0.0 0.27e ±0.02 20.83f ±0.12 0.1 11.10cd ±0.61 0.29de ±0.01 22.19e ±0.10 0.25 11.93cd ±0.80 0.30d ±0.08 23.38d ±0.02 0.5 15.20b ±0.45 0.43b ±0.31 24.22b ±0.05 One 18.81a ±0.01 0.85a ±0.21 26.09a ±0.14 2 13.17c ±0.33 0.36c ±0.04 22.61c ±0.06 Reaction time
(1% concentration)
(time) 0 08.71e ±0.66 0.29d ±0.04 19.13d ±0.03 1h 12.19d ±0.45 0.33d ±0.02 20.28d ±0.07 3h 19.22a ±0.08 0.88a ±0.06 25.73a ±0.02 6h 18.12b ±0.63 0.83b ±0.07 24.44b ±0.11 18h 16.05c ±0.02 0.42c ±0.10 22.74c ±0.43

In the case of CP enzyme treatment, the concentration of the enzyme treated on green tea leaves showed little difference at 0~0.1%, the theanine and Gaba contents increased at 0.5% or more, and especially the highest at 0.5~1% enzyme treatment.

In addition, in the case of 1% enzyme treatment, the content difference is small for 1 hour or less, and the highest theanine and gaba contents are shown when reacted for 3 to 6 hours, and the theanine and gaba contents tend to decrease after 6 hours. Showed. In summary, when the enzyme CP was added to green tea leaves, the extracts of theanine and Gaba were highest when reacted at 37°C for 3-6 hours at a concentration of 0.5-1%.

Test example 4: according to ultrasonic and enzyme treatment Theanine And Gaba Analysis of extraction conditions

After thickening the green tea leaves collected in early May, a 5% sample of hot air-dried powder was extracted with 50% ethanol at 25℃ for 10 minutes, and then the ethanol was removed using a rotary evaporator, and water was added to fill the lost amount. After examining the optimal extraction conditions through ultrasonic and enzyme treatment, the results are shown in Table 7 below, and the HPLC analysis results are shown in FIGS. 11 and 12.

Extraction conditions Theanine GABA Caffeine theobromine (mg/g DW) Untreated group 9.66d±0.25 0.30d±0.12 30.36a±2.16 0.12d±0.12 Treatment group 1: 50% ethanol extraction 15.02c±0.44 0.69bc±0.22 21.77b ±4.20 0.92c±0.15 Treatment group 2: 50% ethanol + ultrasound 18.97b±0.71 0.97b±0.25 19.86c±1.55 1.12b±0.10 Treatment group 3: 50% ethanol + ultrasound + enzyme treatment (3h) 25.48a±0.71 1.76a±0.29 11.72d ±0.55 3.82a±0.22

Here, the untreated group was extracted with water at 80℃ for 10 minutes, the 50% ethanol extraction of treatment group 1 was extracted with 50% ethanol at 25℃ for 10 minutes, and the ultrasonic treatment of treatment groups 2 and 3 was 700W and 30% intensity ' 5 seconds treatment + 1 second stop cycle' was carried out for 10 minutes, and the enzyme treatment of treatment group 3 was reacted at 37°C for 3 hours after adding CP (0.1~2.2 U/ml) enzyme at a concentration of 1%. .

According to this, when extracting by adding 50% ethanol of treatment group 1 rather than untreated group, theanine increased by 64% from 9.6mg to 15mg, and the Gaba content increased by 2.3 times from 0.3 to 0.69mg. In addition, theanine and gaba components were increased in treatment group 2 to which ultrasonic treatment was added and treatment group 3 to which ultrasonic treatment + enzyme treatment was added. Compared to untreated, theanine increased by 3 times and gaba by 6 times. On the other hand, the amount of caffeine present in green tea decreased by 3 times in the treatment group 3 compared to the untreated group, but the content of theobromine having a chemical structure similar to that of caffeine was rather increased. Both caffeine and deobromine are ingredients that exhibit an arousal effect, but caffeine exhibits side effects such as anxiety, anxiety, or gastrointestinal disorders, whereas deobromine exhibits an arousal effect and does not exhibit side effects such as anxiety, anxiety, or gastrointestinal disorders. It is not a beneficial ingredient.

Test example 5: Theanine And Gaba's Analysis of optimal yield

The experimental plan is 20 combinations of green tea sample content (%), ultrasonic treatment time (minutes), and enzyme treatment concentration (%), three factors that are considered as important independent variables (Xi) for the production conditions by implementing the central synthesis planning method. It was investigated using. The optimal theanine and gaba generation conditions were predicted as the range of overlapping parts when superimaging contour maps such as green tea sample content (%), ultrasonic treatment time (minute), and enzyme treatment concentration (%) obtained by reaction surface analysis. . The statistical program Design-Expert 6.0.11, was used to obtain this regression analysis and graph. Table 8 below summarizes the experimental plans for the three independent variables.

Extraction requirements -1.682 -One 0 One 1.682 Green tea sample concentration (%) 0.8 2.5 5 7.5 9.2 Ultrasonic treatment time (min) 0.27 3 7 11 13.73 Enzyme treatment concentration (%) 0.06 0.3 0.65 One 1.24

The three extraction requirements were investigated using 20 combinations as shown in Table 9 below. 20 reaction conditions are green tea sample concentration 0.8% ~ 9.2% (w/v), ultrasonic treatment is 700W 30% intensity, 5 seconds on / 1 second off cycle conditions for 0.3 ~ 13.7 minutes, CP enzyme concentration is 0.06% ~ 1.24 The content of theanine and gaba produced by treatment with% was investigated by HPLC.

Run No. Codded value Actual value Sample conc Steaming Enzyme conc Sample conc
(%) Ultrasonication
(min) Enzyme conc
(%) One -One -One -One 2.50 3.00 0.30 2 One -One -One 7.50 3.00 0.30 3 -One One -One 2.50 11.00 0.30 4 One One -One 7.50 11.00 0.30 5 -One -One One 2.50 3.00 1.00 6 One -One One 7.50 3.00 1.00 7 -One One One 2.50 11.00 1.00 8 One One One 7.50 11.00 1.00 9 -1.682 0 0 0.80 7.00 0.65 10 1.682 0 0 9.20 7.00 0.65 11 0 -1.682 0 5.00 0.27 0.65 12 0 1.682 0 5.00 13.73 0.65 13 0 0 -1.682 5.00 7.00 0.06 14 0 0 1.682 5.00 7.00 1.24 15 0 0 0 5.00 7.00 0.65 16 0 0 0 5.00 7.00 0.65 17 0 0 0 5.00 7.00 0.65 18 0 0 0 5.00 7.00 0.65 19 0 0 0 5.00 7.00 0.65 20 0 0 0 5.00 7.00 0.65

As a result of this experiment, in the present invention, the optimum conditions for gaba and theanine were powdered green tea leaves with a sample concentration of 5%, ultrasonic treatment (30% (700W, 20kHz) for 7 minutes, and 0.7% of CP enzyme was added. By reacting for 3 hours, it was found that 25.48 mg/g theanine and 1.76 mg/g gaba were produced.

Test example 6: Brain nerve cells Protection and Anti-stress Effect analysis

SH-SY5Y cells (KCLB 22266, Korean Cell Line Bank, Seoul, Korea) were used in the experiment to confirm the neuronal protective effect. Cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. Brain nerve cells (SH-SY5Y) were cultured in RPMI-1640 medium containing 1% antimicotics/antibiotics and 10% FBS, and seeded to 10 ⁴ -10 ⁶ cells/ml in a 96-well plate. After the green tea extract containing theanine and Gaba were treated at 10, 25, 50 mg/ml concentration. Cells in which stress was induced by treatment with 100 mM glutamate were measured for cytotoxicity using MTT. As a positive control, 1-10 mg Theanine and 10 mg GABA, which are known to have high cerebral sedation, were used, and absorbance was measured at a wavelength of 570 nm to investigate the protective effect of brain cells through cell viability. To measure the content of the stress hormone Cortisol, the cell culture supernatant was centrifuged at 10,000xg for 10 minutes before use. The experiment was carried out at 4°C, and the protein content was irradiated with the BCA Kit and 2.5µg/µl was used, and the anti-stress effect was performed by Cortisol Elisa Kit (Calbiotech) according to the manufacturer's instructions.

Figure 13 shows the results of the analysis of the protective effect of cranial nerve cells. According to this, the neuroprotective effect of green tea extract containing high theanine and Gaba using cranial nerve cells (SH-SY5Y) showed 28% and 86% brain cell survival rates at concentrations of 10 mg/ml and 50 mg/ml. In particular, these results showed a higher survival rate than 72% observed when the theanine was treated with 10 mg of pure theanine, and showed better brain protection than theanine 10 mg at a concentration of 50 mg/ml of green tea extract.

In addition, the anti-stress effect analysis results are shown in FIG. 14. According to this, the anti-stress effect of green tea extract containing high theanine and Gaba using cranial nerve cells (SH-SY5Y) is similar to that of theanine or 10 mg of pure Gaba substance at a concentration of 25mg-50mg/ml of green tea extract. It was found that the anti-stress effect was high because the content of was less than 15 ng and showed a low cortisol production rate.

Test example 7: Anti-dementia Effect analysis

The enzyme Acetylcholinesterase (AChE) breaks down acetylcholin, a neurotransmitter in the brain, and is known to cause the production of amyloid plaques and neurofibrillary tangles, which are the triggers of Alzheimer's disease. Brain nerve cells (SH-SY5Y) were treated with 100 mM glutamate to treat the stress-induced cells by concentrations of theanine and gaba, and then 10,000 xg of the cell culture supernatant was centrifuged for 10 minutes and used. In addition, the anti-dementia effect was measured to the extent of inhibiting the AChE enzyme that degrades acetylcholine, a cranial nerve transport material. The green tea extract containing theanine and gaba of the present invention was used at concentrations of 10, 50, and 100 mg/ml, and at this time, based on the anti-dementia drug Tacrine 10uM and theanine standard 1, 10 mg as standard substances. The antidementia effect was investigated. Acetylcholinesterase (AChE) Inhibition assay kit (Sigma) was performed according to the manufacturer's instructions, and the results are shown in FIG. 15. According to this, it was found that the green tea extract, compared to tacrine, which is known as a treatment for degenerative brain diseases such as dementia through the inhibition of AChE enzyme, showed an anti-dementia effect of about 50% at a concentration of 100 mg/ml, indicating that it is effective in treating dementia.

In the above, embodiments of the present invention have been described, but those of ordinary skill in the art will add, change, delete or add components within the scope not departing from the spirit of the present invention described in the claims. Various modifications and changes can be made to the present invention by means of the like, and it will be said that this is also included within the scope of the present invention.

Claims (22)

(a) drying green tea leaves to prepare green tea ingredients;
(b) extracting an alcohol extract by adding the green tea material to an aqueous alcohol solution;
(c) ultrasonicating the alcohol extract; And
(d) enzymatic treatment of the sonicated alcohol extract; and a method for producing a green tea extract comprising,
The enzyme treatment of step (d) is performed by an enzyme derived from black mold ( Aspergillus niger ),
The enzymes include pectinase, polygalacturonase, and pectin methyl esterase,
The manufacturing method of the green tea extract is a method of manufacturing a green tea extract containing a high concentration of theanine and gaba, characterized in that the caffeine content is reduced and the deobromine content is increased compared to the green tea material. The method of claim 1,
In step (a), the drying is performed according to any one method selected from hot air drying, freeze drying, hot air drying, and roasting after thickening.The method for producing a green tea extract containing high concentration theanine and gava. The method of claim 2,
The drying is a method for producing a green tea extract containing high concentration theanine and gaba, characterized in that the drying is performed by hot air drying after thickening. The method of claim 3,
After the thickening, hot air drying is performed by hot air drying at 90 to 110°C for 1 to 3 hours after the thickening. A method for producing a green tea extract containing high concentration theanine and Gaba. The method of claim 1,
The green tea material of step (a) is a method for producing a green tea extract containing a high concentration of theanine and gaba, characterized in that it is prepared with an aqueous green tea solution of 1 to 10% (w/v). The method of claim 1,
The method for producing a green tea extract containing high concentration theanine and gaba, characterized in that the alcohol of the aqueous alcohol solution of step (b) is at least one selected from methanol, ethanol, propanol and butanol. The method of claim 1,
The method for producing a green tea extract containing high concentration theanine and gaba, characterized in that the alcohol aqueous solution of step (b) has a concentration of 40 to 80% (w/w). The method of claim 1,
The extraction of step (b) is a method for producing a green tea extract containing high concentration theanine and gaba, characterized in that carried out at 20 to 30 ℃. The method of claim 1,
After the ultrasonic treatment in step (c) and before the enzyme treatment in step (d), the step of removing ethanol from the alcohol extract is additionally performed.The method for producing a green tea extract containing high concentration theanine and gaba. The method of claim 1,
The ultrasonic treatment of step (c) is a method for producing a green tea extract containing high concentration theanine and gaba, characterized in that the ultrasonic treatment is carried out at an intensity of 25 to 35% at 700 to 750W. The method of claim 10,
The method for producing a green tea extract containing high concentration theanine and gaba, characterized in that the ultrasonic treatment is performed for 5 to 15 minutes. delete delete delete The method of claim 1,
The enzyme treatment is a method for producing a green tea extract containing a high concentration of theanine and gaba, characterized in that used in a concentration of 0.5 to 2% (w / v). delete A pharmaceutical composition for preventing or treating degenerative brain diseases comprising a green tea extract comprising a method for preparing a green tea extract containing high concentration theanine and gaba of any one of claims 1 to 11 and 15 as an active ingredient Method of manufacturing. The method of claim 17,
The green tea extract contained in the pharmaceutical composition contains 2.0 to 3.0 wt% of theanine, 0.1 to 0.25 wt% of Gaba, 0.05 to 1.5 wt% of caffeine, and 0.2 to 0.5 wt% of theobromine based on the total content of the green tea extract Method for producing a pharmaceutical composition for preventing or treating degenerative brain diseases, characterized in that. The method of claim 17,
The degenerative brain disease is any one selected from dementia, mild cognitive impairment, multiple sclerosis, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, and Pick's disease. Method for producing a pharmaceutical composition for preventing or treating degenerative brain diseases. A method for producing a brain-protecting functional food composition comprising as an active ingredient a green tea extract comprising a method for preparing a green tea extract containing the high concentration theanine and gaba of any one of claims 1 to 11 and 15. The method of claim 20,
The green tea extract contained in the food composition contains 2.0 to 3.0 wt% of theanine, 0.1 to 0.25 wt% of Gaba, 0.05 to 1.5 wt% of caffeine, and 0.2 to 0.5 wt% of theobromine based on the total content of the green tea extract A method for producing a functional food composition for brain protection, characterized in that. The method of claim 20,
The brain protection function is a method of manufacturing a brain protection functional food composition, characterized in that the anti-stress or mental arousal functionality.

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