JP5786169B2 - Fermented tea, extract from fermented tea and medicinal composition containing the same - Google Patents

Description

The present invention relates to a fermented tea produced by fermentation of tea leaves, an extract from the fermented tea, and a medicinal composition containing the extract.
In particular, the present invention is a fermented tea that is excellent in flavor and contributes to health, and further, an extract that may be used as a medicament for prevention or treatment of various diseases extracted and separated from the fermented tea. The present invention relates to a medicinal compound and a medicinal composition containing the extract or medicinal compound, and belongs to tea production technology, health food preparation technology, and pharmaceutical preparation technology.

Tea as a luxury item has been used habitually for a long time and all over the world.
Most of them are pouring hot water into tea leaves and drinking the leachate.
Since the exudate contains various components extracted from tea leaves, it has a unique aroma and taste and has a specific function.
Therefore, research on the components of tea leaves has been conducted extensively, and the following is also known regarding the components.

That is, the characteristic components of tea are caffeine and tannin-based catechins.
This caffeine gives a person an excitatory action, has a bitter taste, and also has a diuretic action.
The catechin is the most abundant tea ingredient and is a tea astringent ingredient.
In addition to these, amino acids typified by theanine, vitamins typified by vitamin C, chlorophylls, inorganic components such as potassium and calcium, and fragrance components such as dimethyl sulfide, green leaf alcohol, and terpene alcohol are widely known. ing.

It is also known that these components change at the stage of tea production from tea leaves.
For example, kelp fried tea and roasted green tea contain a large amount of nitrogen-containing compounds such as pyrazine and pyrrole. (See encyclopedia).

As described above, since tea leaves contain a lot of various useful components, many attempts have been made since long ago to extract the components efficiently and to process the tea leaves to obtain more active ingredients. .
In particular, regarding fermented tea, for example, in JP-A-2002-370994 (Patent Document 1), blood sugar is extracted by hot water extraction from black tea, that is, black tea aged by a post-fermentation method using black koji mold or the like. It has been reported that value-inhibiting substances have been obtained.

  JP-A-2005-341876 (Patent Document 2) reports that a tea having a maltase inhibitory effect can be obtained by adding koji mold or koji to tea leaves and fermenting them.

On the other hand, the inventors of the present application found that a medicinal composition capable of preventing deterioration of liver function and the like can be obtained from fermented tea leaves by extraction with acetic acid, and filed a patent application.
The contents are disclosed in Japanese Patent Application Laid-Open No. 2007-238584 (Patent Document 3).

JP 2002-370994 A (Claims) Japanese Patent Laying-Open No. 2005-341876 (Claims) JP 2007-238584 A (Claims)

The invention described in Patent Document 3 extracts an active ingredient in tea leaves, uses it as a raw material for health foods and pharmaceuticals, and can use tea leaves more efficiently and tea leaves (cutting leaves) that cannot be used for tea making. It aims at providing a composition with high property.
The active ingredient in Patent Document 3 can be used in a powder state, as acetic acid or water or an ethanol solution, and is prepared from cut leaves that have not been used. Therefore, it is environmentally friendly and ecological. But it is excellent.

  In particular, this medicinal composition prevents deterioration of liver function (AST, ALT), increase of HDL cholesterol (good cholesterol), prevention of increase of blood sugar level, prevention of decrease of platelets, reduction of red blood cell deformability due to oxidative stress. It has excellent medical effects such as prevention.

  In such a situation, the inventors further pursued the characteristics of various components of fermented tea (post-fermented tea and black tea), and examined the preparation conditions of fermented tea, the changes in the components contained by the raw tea leaves, etc. In order to make more effective use of the widely used tea leaves, we conducted an extensive study on the active ingredients in tea leaves and the changes caused by the fermentation conditions.

As a result, the inventors of the tea leaves fermented with black koji, depending on the fermentation conditions, the structure has not yet been elucidated, but the compound that does not exist in conventional tea leaves is produced and exists, The tea leaves in which this compound existed were tasty, and this compound was separated by extraction with water, particularly hot water, cold water or dilute acetic acid, and found that they had medicinal properties, completing the present invention. It has been made.

That is, the invention according to claim 1 of the present invention is
A fermented tea prepared by subjecting raw tea leaves to aerobic fermentation at a temperature of 30 to 45 ° C. for a period of 3 to 6 days using black koji, followed by steaming.

The invention according to claim 2 of the present invention is
The raw tea leaves were separated from the fermented tea prepared by steaming after the aerobic fermentation using black koji at a temperature of 30 to 45 ° C for a period of 3 to 6 days. It is a featured hot water extract.

The invention according to claim 3 of the present invention is
A medicinal composition comprising the extract according to claim 2 .

The fermented tea according to the present invention has a great effect on erythrocyte deformability due to the presence of a medicinal compound.
Therefore, it is not only effective as a tea for maintaining health, but also has a flavor that surpasses that of fermented tea prepared from gyokuro and can further utilize tea leaves as a healthy beverage with high flavor.
Moreover, according to the present invention, fermented tea containing such a medicinal compound in a high content can be obtained in 3 to 6 days.

In particular, the fermented tea according to the present invention contains medicinal compounds having various functions.
The extract from the fermented tea, and further purified medicinal compounds, have the following excellent medicinal properties and can be effectively used as raw materials for health foods and pharmaceuticals. Is high.
1. 1. Prevention of reduced red blood cell deformability 2. Antioxidant activity, radical scavenging activity α-Glucosidase / Lipase inhibitory activity

  Since the medicinal compound according to the present invention can be used in a powder state or as acetic acid or water or an ethanol solution, it is efficient when used as a health food or a medicine in order to exhibit the above effects. It can be used effectively and effectively.

Since the medicinal composition according to the present invention contains the extract or medicinal compound, it can be used as a medicament for prevention or treatment of various diseases.

It is a HPLC chart of the extract of a fermented tea sample 1 day after a fermentation process. It is a HPLC chart of the extract of a fermented tea sample 3 days after a fermentation process. It is a figure which shows the change of the component of the extract by a fermentation process period. It is a figure which shows the detection result of the extract by the fluorescence detection method of catechin. It is a figure which shows the detection result of the extract by the detection method of the polymerization catechin which uses 70% acetone. It is a figure which shows the detection result of X component in thiol decomposition. It is a figure which shows the result of the HPLC analysis of the 4.5% acetic acid extract of fermented tea. It is an ultraviolet absorption spectrum of each peak of FIG. It is a fractional drawing by Bio-Gel column of 4.5% acetic acid extract of fermented tea. It is a fraction drawing by Bio-Gel column of ethyl acetate extraction from fermented tea acetic acid extract. It is a HPLC chart of 3 fractions of FIG. FIG. 11 is a diagram of adsorption chromatography using a Sepdex LH column (2 × 27 cm) of fraction 5 in FIG. 10. It is a HPLC chart of the fraction which has absorption in 300 nm of FIG. 6 is an HPLC chart of X component purified by HPLC of fraction 5. It is an ultraviolet absorption spectrum of the X component purified by HPLC of fraction 5. It is a crystal photograph of X component. It is a figure which shows the erythrocyte deformability fall inhibitory effect of X component. It is a figure which shows the antioxidant activity of X component. It is a figure which shows DHHP radical scavenging activity of X component. It is a figure which shows the enzyme inhibitory activity of X component.

The fermented tea of the present invention is prepared by a conventionally known fermentation method using black potato.
Although the method itself does not have any exceptionally new conditions, it is necessary that the medicinal compound discovered by the inventors this time be produced and the fermented tea maintain a state containing the medicinal compound.

As the fermentation condition for maintaining the state in which the fermented tea contains a medicinal compound, the most important condition is a fermentation period.
In the case of a long period of time, it takes several months to produce excellent black tea, and in Patent Document 2, it is preferably about 3 weeks (paragraph 0030). The preferred fermentation period is 3 to 10 days, more preferably 3 to 6 days, whereby a fermented tea containing a high content of medicinal compounds is obtained.
The fermentation period is 3 to 6 days.
Aerobic fermentation is employed for the fermentation.

As the raw tea leaves used in the present invention, it is preferable to use the second tea from the point of the flavor of the obtained fermented tea, that is, the tea leaves from which the buds appearing after the first tea harvested around May are harvested around June. .
The fermented tea using the second tea has an excellent flavor as compared with the fermented tea using the gyokuro, the first tea, the third tea, and the autumn / winter bancha.

Other fermentation conditions, that is, raw material black koji, temperature during fermentation (normal temperature 30 to 45 ° C.), moisture during fermentation (usually 25 to 35%), etc. Generally, conditions for preparing fermented tea using black koji By performing according to the above, the desired fermented tea and medicinal compounds can be obtained.

  Hereinafter, fermented tea according to the present invention, extracts from the fermented tea, medicinal compounds contained therein, and medicinal compositions containing the extract or medicinal compound will be described in detail based on Examples. explain.

<Preparation of fermented tea>
Gyokuro, Ichibancha, Nibancha, Sanbancha, and Akiyubancha were used as raw tea leaves, and fermented tea was prepared by the following steps.

240 kg of raw tea leaves (raw tea) was put into a drum rotary automatic koji making apparatus, and 210 kg of water was added while stirring by rotating the drum to swell the raw tea leaves.
Separately, a mixture of 30 kg of raw tea leaves (powdered tea) and 210 g of seed koji prepared with black koji was added to the swollen raw tea leaves and fermented at a set temperature of 35 ° C. for 6 days with stirring.
The temperature rise during fermentation was suppressed using an air cooling fan, and efforts were made to maintain the set temperature.
The fermented tea leaves were made into fermented tea as a product through steam sterilization at a temperature of 100 ° C. and drying at a temperature of 55 ° C.

<Sensory test of fermented tea>
About the fermented tea obtained from each raw material, when the flavor test by five men and women was conducted by the 5-point method (5: good, 3: normal, 1: bad), the results shown in Table 1 below were obtained. It was.
As is clear from Table 1, in the raw tea leaves (raw tea), gyokuro is rated the most, followed by the most tea.
In the fermented tea adjusted under the above conditions, the evaluation of gyokuro was greatly reduced, and instead, fermented tea made from Nibancha has the best flavor.

<Inhibition of reduced red blood cell deformability of fermented tea>
Among the fermented teas described above, the raw materials are gyokuro, nibancha, and fall / winter bancha, and the raw material, fermented tea and the fermented tea are further fermented for 15 days under normal fermentation conditions for secondary fermented tea. Table 2 shows the erythrocyte deformability reduction-inhibiting effect of the hot water extract measured by the method described below.
The numbers in the table represent the relative change (%) between the addition of the oxidizing agent AAPH (B), which reduces the red blood cell deformability, and the addition of AAPH and the sample solution (C).

<Erythrocyte deformability measuring method>
5. Centrifuge the blood collected 10.0 ml in a blood collection tube containing 1.0 ml of 3.8% sodium citrate solution at 2500 rpm × 10 minutes to precipitate red blood cells, wash, add HEPES, and 6. A 0% erythrocyte suspension was prepared.
After adding HEPES ((A) 3.0 ml, (B) 2.40 ml, (C): 2.34 ml) to 3.0 ml of 6.0% erythrocyte suspension and preincubating at a temperature of 37.0 ° C. In (B), 0.6 ml of 500 mM AAPH solution is added, and in (C), 0.6 ml of 500 mM AAPH solution and 0.06 ml (10 mg / 2 ml) of extract solution of fermented tea are added. Incubated for 45 minutes at a temperature of 37.0 ° C.
Then, it cooled on ice until it measured, and the measurement was performed after incubating again for 7 minutes at the temperature of 25.0 degreeC.
The above (A) is a control.

In the measurement, a nickel mesh holder was connected to a vertically standing glass tube via a Tygon tube, and physiological saline adjusted with a HEPES buffer was used from a height of 15 cm (height: h). The erythrocyte suspension prepared as described above is filtered.
Around the glass tube, constant temperature water is refluxed to keep the sample at a constant temperature.
A pressure (P) transducer installed at the zero level of the glass tube continuously detects the pressure drop during the filtration of the sample, and this is taken into a personal computer through an amplifier and an AD converter, and the flow rate (flow: Q) is calculated.
The flow rate is converted from pressure to height (P = pgh), the differential value (dh / dt) of the height-time (ht) curve is taken, and this is multiplied by the cross-sectional area (a) of the glass tube. (Q = dh / dt · a).

Using the pressure-flow curve of control x (HEPES buffer-adjusted saline: Newtonian fluid) that does not contain blood cells as a control, the pressure-flow curve of the erythrocyte suspension was examined, and at a certain pressure (usually 100 mm · H ₂ O). The erythrocyte deformability was evaluated based on the flow rate (%) of the erythrocyte suspension relative to the flow rate of the control solution.

<Changes in components of fermented tea extract>
During the preparation of fermented tea based on Nibancha, the tea leaves sampled every day were subjected to hot water extraction, and the resulting extract was analyzed by HPLC.

FIG. 1 is an extract of the sample after 1 day, and FIG. 2 is an HPLC chart of the extract of the sample after 3 days.
Changes in the components of the extract obtained based on these results are shown in FIG.
In addition, the symbol in a figure shows the following compounds.
GA; gallic acid EC; epicatechin ECG; epicatechin gallate EGC; epigallocatechin EGCG; epigallocatechin gallate

X, Y, and Z are thought to be polyphenols, but are currently unknown components, and the amount of the X component is particularly large.
The red blood cell deformability of these extracts was as shown in Table 3 below.
Although not shown in the table, the steaming treatment performed after fermentation also improved the flavor and the effect of suppressing the decrease in red blood cell deformability. For example, what was fermented for 4 days showed 37% It increased to 79% after steaming.

<Identification of X component>
As is apparent from FIG. 3, in the figure, except for the X component, most of the other components have maximum values after 1 to 2 days from the start of fermentation, and thereafter tend to decrease.
In contrast, component X was not found on day 1 but increased significantly from day 2 and had a maximum on day 5 and was shown in Table 3. It was presumed that the compound was strongly related to erythrocyte deformability.

At present, the X component has not been identified, but the following data confirmed that it is not catechin, polymerized catechin, or hydrolyzed tannin.
1) In the catechin fluorescence detection method (280 nmEx, 310 nmEm), as shown in FIG. 4, the presence of catechin is not observed in the extract.
2) In the method for detecting polymerized catechin using 70% acetone, polymerized catechin is not detected as seen in FIG.
3) As shown in FIG. 6, catechin is not detected by thiol decomposition of the X component.
In addition, it is considered that the gallic acid-derived compound is hydrolyzed tannin, but when 4M hydrochloric acid is used and the X component is hydrolyzed at 85 ° C for 5 hours, some phenols are produced by decomposition. It was confirmed that the acid was not hydrolyzable tannin.

<Extraction and purification of X component>
The extraction and purification of the X component was performed according to the following procedure.
First, 200 g of fermented tea was used, 4.5% acetic acid was used as an extraction medium, and 72.6 g of extract (yield: 36.3%) was obtained by concentration under reduced pressure and lyophilization.
This extract was subjected to HPLC analysis as described above, and the results are shown in FIG. 7, FIG.
The ultraviolet absorption spectrum of each peak is shown.
In FIG. 7, peaks a to c are EGC, Cafein, and EC from the elution positions and absorption spectra, respectively, and peak d is the X component under the conditions of FIG. I understood.

The results of fractionation of the extract using a Bio-Gel P-10 column (2 × 28 cm) and a 1% aqueous acetic acid solution as a developing solution are shown in FIG. 9 and Table 4.
As a result of analyzing each fraction by HPLC in the same manner as described above, it was found that the X component was contained in fraction 5 together with the brown catechin polymer.

Fraction 5 containing the X component obtained by the above fractionation was extracted with ethyl acetate.
As a result, it was confirmed that the X component was extracted into the ethyl acetate layer and the catechin polymer remained in the aqueous layer. After concentrating the X component-containing ethyl acetate, deionized water was added to dilute the solution, and the Bio-Gel P FIG. 10 shows an elution pattern when applied to a −10 column (Laboratories, Inc .: 3 × 28 cm) and developed with a 1% aqueous acetic acid solution.
As a result of performing HPLC analysis on the fractions 4 to 6 in the same manner as described above, it was found that all contained X component as shown in FIG.

The supernatant obtained by adding methanol to lyophilized fraction 5 and stirring and centrifuging is applied to a Sephadex LH column (GE Healthcare Biosciences: 2 × 27 cm) equilibrated in advance with methanol. Deployed.
The elution pattern is shown in FIG. 12, and the HPLC pattern of the fraction having absorption at 300 nm is shown in FIG.

When this fraction was purified by TFA-MeCN HPLC in the same manner as described above, a purified X component was obtained as shown in FIG.
The ultraviolet absorption spectrum was as shown in FIG.
This fraction was dried under reduced pressure, deionized water was added, and the mixture was warmed and dissolved, and then cooled. As a result, crystals as shown in FIG. 16 were obtained.

The function of the obtained X component was measured, and the results are shown in FIGS.
The contents of FIGS. 17 to 20 are as follows.
From these results, it is clear that the X component is a compound having excellent medicinal properties, and in the present invention, this X component is referred to as a medicinal compound.
-Fig. 17: Erythrocyte deformability-Fig. 18: Antioxidant activity-Fig. 19: DHHP radical scavenging activity-Fig. 20: Enzyme inhibitory activity Measurement methods other than erythrocyte deformability are as follows.

<Measurement method of antioxidant activity>
The antioxidant activity of each sample obtained above was measured by the following method according to the method of Miller et al. Using the action of the linoleic acid oxide to discolor β-carotene.
The fading degree of β-carotene when the linoleic acid-β-carotene solution is added to the spectrophotometer test tube cell into which the sample has been dispensed and stirred and reacted in a thermostat at a temperature of 50 ° C. for 20 minutes. Was determined by the absorbance at 470 nm, the decrease in absorbance by the synthetic antioxidant butylhydroxyanisole (BHA) was measured, and the antioxidant activity of the sample was expressed by the concentration of BHA giving the same decrease as the sample.

<Radical scavenging activity measurement method>
An equal amount of DPPH (1,1-diphenyl-2-picrylhydrazyl) solution, MES buffer, and 20% ethanol were added to the sample solution, and the mixture was reacted at room temperature for 20 minutes, and then the absorbance at 520 nm was measured. .
A calibration curve for DPPH radical scavenging activity was prepared using ascorbic acid (AsA), which was similarly obtained, and the radical scavenging activity per sample g was expressed in terms of AsA equivalent (μmol / g or mmol / g). .

<Method for measuring enzyme inhibitory activity>
After adding 25 μl of small intestine α-glucosidase solution or pancreatic lipase solution to 50 μl or 25 μl of the sample solution and incubating at a temperature of 37 ° C. for 10 minutes, α-D-glucose or oleate (substrate) solution of 4-methylumbelliferone 25 μl or 50 μl was added and incubated.
After stopping the reaction by adding 0.2 molar Na ₂ CO ₃ solution or 0.1 molar citrate buffer (pH 4.2), the fluorescence of the produced 4-methylumbelliferone was measured at 450 nm. The inhibition rate was calculated by measurement.

  The fermented tea in this invention, the extract from this fermented tea, the medicinal compound contained therein, and the medicinal composition containing the extract or medicinal compound have the above-mentioned excellent characteristics, And since the said extract and medicinal compound can be supplied as a non-toxic solvent solution of powder or water, acetic acid or ethanol, this invention is widely used in the health food industry and the pharmaceutical industry including the tea manufacturing industry. It is highly likely that

Claims (3)

A fermented tea prepared by subjecting raw tea leaves to aerobic fermentation at a temperature of 30 to 45 ° C. for a period of 3 to 6 days using black koji and then steaming. The raw tea leaves were separated from the fermented tea prepared by steaming after the aerobic fermentation using black koji at a temperature of 30 to 45 ° C for a period of 3 to 6 days. Characteristic hot water extract. A medicinal composition comprising the extract according to claim 2 .