JPWO2015002209A1 - Antihypertensive agent, saccharification inhibitor, and method for producing the same, containing coffee bean extract as an active ingredient - Google Patents

Abstract

The present invention is a coffee bean extract obtained through a subcritical treatment step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature, When the inhibitory activity rate of angiotensin converting enzyme is 50%, the increase in blood pressure containing a coffee bean extract whose active ingredient is a solid content concentration (IC50 value) of 0.16 mg / mL or less as the coffee bean extract It relates to the agent. The present invention further relates to saccharification inhibitors and methods for producing them. ADVANTAGE OF THE INVENTION By this invention, the more active antihypertensive agent or saccharification inhibitor derived from coffee beans, the manufacturing method thereof, and the manufacturing method of the coffee bean extract which has an antihypertensive action and an antisaccharification action are provided. The

Description

  The present invention relates to an antihypertensive agent containing, as an active ingredient, an extract obtained by subcritical processing of coffee beans. Furthermore, the present invention relates to a saccharification inhibitor containing, as an active ingredient, an extract obtained by subcritical processing of coffee beans. Furthermore, this invention relates to the manufacturing method of the extract of coffee beans which has such a blood pressure increase inhibitory effect or saccharification inhibitory effect from coffee beans.

  Coffee has long been enjoyed as a favorite beverage around the world. Generally, coffee is provided as a beverage that enjoys flavors typified by aroma and taste extracted from coffee beans with water, hot water, and the like. In recent years, attention has also been paid to the physiologically active action of coffee, and the blood pressure lowering action caused by chlorogenic acid contained in coffee has been reported (Patent Documents 1 and 2).

JP 2008-48728 A JP 2007-31392 A

  However, extraction methods such as hot water for obtaining the physiological activity of lowering blood pressure or suppressing blood pressure increase or saccharification from coffee have a limit in the amount of extractable dissolved component concentration typified by polyphenol. Therefore, when evaluation of suppression of blood pressure increase or suppression of glycation was performed, the suppression rate did not become a certain value or less. For this reason, it has been estimated that the functions such as the blood pressure increase inhibiting action and the glycation inhibiting action cannot be sufficiently exhibited.

  Therefore, it was necessary to examine methods other than the hot water extraction method for obtaining the physiological activity of the blood pressure increase inhibitory action or the saccharification inhibitory action from the coffee beans.

  As a result of intensive studies on the coffee by the present inventors, an extraction composition liquid and an extraction liquid obtained by hydrolyzing green coffee beans and / or roasted coffee beans in a subcritical state (above the saturated vapor pressure at each temperature) The inventors have found characteristics of the production method, and found that a certain amount or more of a dissolved component coexisting composition liquid typified by polyphenol is obtained, and functions such as an antihypertensive action and a saccharification inhibiting action are sufficiently exhibited. As a result, it was found that the extract obtained by the subcritical processing of the coffee beans of the present invention has a blood pressure elevation inhibitory action and a saccharification inhibitory action superior to the extract obtained by the conventional hot water extraction method.

That is, the gist of the present invention is as follows.
[1] An antihypertensive agent comprising a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
Blood pressure containing, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.16 mg / mL or less when the angiotensin converting enzyme (ACE) inhibitory activity rate is 50% Elevation inhibitor;
[2] A saccharification inhibitor containing a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
A saccharification inhibitor containing, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.06 mg / mL or less when the production inhibition rate of the saccharification product is 50%;

[3] crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a blood pressure increase-inhibiting action, comprising a step of recovering the coffee bean extract by treating it under a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature. And
[4] crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A process for producing a coffee bean extract having a saccharification-inhibiting action, comprising a step of recovering the coffee bean extract by a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature; About.

  The extract obtained by subcritical processing of the coffee beans of the present invention has a blood pressure increase inhibitory action and a saccharification inhibitory action superior to those of an extract obtained by a conventional hot water extraction method. Further, the extraction method does not require extra steps such as adsorbent treatment and heat sterilization treatment, and addition of acid, alkali, and enzyme, and an extract can be obtained by a simple technique at a relatively low temperature.

FIG. 1 is a graph showing the relationship between the amount of sample solution and the inhibition rate for ACE inhibitory activity measurement. FIG. 2 is a graph showing the relationship between the amount of sample liquid and the inhibition rate related to the measurement of the inhibition rate of glycation product production. FIGS. 3A and 3B are graphs showing the dissolved component concentration and polyphenol concentration of each sample. FIGS. 4-1 and 4-2 are graphs showing the amount of extract (μL) of each sample that inhibits the activity of angiotensin converting enzyme by 50%. FIG. 5 is a graph showing the amount of extract (μL) of each sample that inhibits the production of a saccharification product by 50%.

  The coffee bean extract of the present invention is an extract obtained by hydrolyzing green coffee beans and / or roasted coffee beans in a subcritical state (above the saturated vapor pressure at each temperature). As a specific subcritical state, the temperature is 140 to 200 ° C., the pressure is 0.15 to 1.8 MPa, and the subcritical treatment step is performed at a pressure equal to or higher than the saturated vapor pressure at each temperature. The coffee bean extract obtained through this subcritical treatment step has a blood pressure increase inhibitory effect and a saccharification inhibitory effect. In the present specification, the saturated vapor pressure at each temperature preferably means the saturated water vapor pressure at each temperature.

  The coffee bean extract preferably has a dissolved component concentration of 30 mg / mL or more when the extract is prepared by a specific method described below. Furthermore, specifically, it is desirable that the concentration of the dissolved component is 30 mg / mL or more when the ratio of coffee beans to water is 1: 8 (mass ratio). The dissolved component concentration is measured by a heat drying method. The heat drying method means that the sample is dried under certain conditions, the component that volatilizes is moisture, the remaining component is the dried product (dissolved component), and the sample contains (mass of dried product) / (water before drying) Volume). Actually, volatile components other than moisture (alcohol, etc.) are also removed, but they are measured on the assumption that they are extremely small and hardly affect the measurement results.

  The dissolved component is a mixture of polyphenols, saccharides, proteins, minerals, etc., and in the dissolved components, by-products other than polyphenols, saccharides, proteins, minerals, etc. are obtained, and these byproducts When a product can cope with combined functionality with different actions (for example, immediate action and delayed action, functional action and functional auxiliary action) for suppressing blood pressure rise and glycation inhibition, The inventors estimated. Based on this estimation, it was also estimated that a highly functional extract can be obtained by hydrolyzing green coffee beans and / or roasted coffee beans in a subcritical state (above the saturated vapor pressure at each temperature).

  Further, the polyphenol concentration in the aforementioned extract is desirably 6.8 mg / mL or more when the extract is prepared by a specific method described later. Furthermore, specifically, the polyphenol concentration in the extract when the ratio of coffee beans to water is 1: 8 (mass ratio) is desirably 6.8 mg / mL or more. When it is 6.8 mg / mL or more, it becomes easy to obtain the effect on suppression of blood pressure increase and suppression of glycation. The polyphenol concentration can be measured by the Folin-Ciocalteu method. The Folin-Ciocalteu method is a mixture of a Folin-Ciocalteu solution and a sodium carbonate solution, which will be described later, mixed with a coffee extract, and the absorbance at 765 nm of this mixture is measured. This is a method of calculating the amount of polyphenol by the amount corresponding to chlorogenic acid (mg / mL) from the calibration curve according to the above.

  When the coffee extract is prepared by a specific method to be described later, the dissolved component concentration is preferably 30 mg / mL or more, and the polyphenol concentration is more preferably 6.8 mg / mL or more. More specifically, the coffee extract in the case of coffee beans and water in a ratio (mass ratio) of 1: 8 has a dissolved component concentration of 30 mg / mL or more and a polyphenol concentration of 6.8 mg / mL. The above is more desirable. By satisfying these two concentrations, it becomes easy to obtain effects related to suppression of blood pressure increase and suppression of glycation.

  One of the inventions of the present application is an effective use of a coffee bean extract whose solid content concentration (IC50 value) is 0.16 mg / mL or less when the inhibitory activity rate of angiotensin converting enzyme (ACE) is 50%. It is a blood pressure increase inhibitor contained as a component.

  The antihypertensive effect in the present invention is determined by measuring the inhibitory activity of angiotensin converting enzyme (ACE) that causes an antihypertensive effect by the measurement method described later, and the activity when the inhibitory activity rate of angiotensin converting enzyme (ACE) is 50%. It is defined by the solid content concentration (IC50 value) of the extract showing inhibition.

  Here, angiotensin converting enzyme (ACE) will be described. Angiotensin converting enzyme is an enzyme that exists on the surface of vascular endothelial cells and converts angiotensin I into angiotensin II. An angiotensin converting enzyme inhibitor (ACE inhibitor) that prevents this action is used as a pressor suppressant or a therapeutic agent for heart failure. ACE is an abbreviation for angiotensin-converting enzyme. Angiotensin is one of the peptides present in blood, and there are four types of angiotensin I to IV. Among these, angiotensin II-IV has the effect | action which contracts the smooth muscle of an arteriole and raises blood pressure, and the effect | action which accelerates | stimulates the secretion of aldosterone in adrenal cortex. Angiotensin II-IV has a particularly strong blood pressure raising action. Angiotensin I has no such physiological activity.

  Angiotensin converting enzyme (ACE) has a function of converting angiotensin I into angiotensin II having a pressor action in the renin-angiotensin system which is one of the blood pressure regulating mechanisms in the body. The blood pressure in the body is adjusted by increasing the blood pressure with angiotensin II. The ratio of inhibiting the conversion of angiotensin I to angiotensin II is called an angiotensin converting enzyme (ACE) inhibitory activity rate.

  In the present invention, when the ratio of inhibiting the conversion of angiotensin I to angiotensin II is 50%, that is, the angiotensin converting enzyme (ACE) inhibitory activity rate is 50%, the solid content concentration in the coffee extract (IC50 value) Perform an evaluation at.

  In general, the antihypertensive action in the present invention is evaluated by measuring the activity inhibition rate of angiotensin converting enzyme (ACE) that causes the antihypertensive action by the measurement method described later. It is usually defined by what percentage of ACE inhibitory activity is exhibited at a constant solid content concentration, and the lower the concentration is desirable. Usually, in order to facilitate comparison between samples, it is defined by the solid content concentration (IC50 value) of the extract showing inhibitory activity at an inhibitory activity rate of 50% of angiotensin converting enzyme (ACE). (Reference: “Science of Food Functionality”, edited by Science Editor of Food Functionality, published by Industrial Technology Service Center Co., Ltd.)

The measurement of the activity inhibition rate of angiotensin converting enzyme (ACE) will be described based on the measurement example performed this time. ACE inhibitory activity is measured using a kit of ACE-Kit WST manufactured by Dojindo Laboratories. First, four or five diluted samples having different dilution rates or different concentrations of the extract are prepared using the coffee bean extract. Specifically, 0.04 μL (sample A1), 0.12 μL (sample B1), 0.20 μL of a coffee extract (here, 45.3 mg / mL) whose dissolved component concentration was measured in advance. (Sample C1) and four samples having different sample liquid amounts from 0.28 μL (sample D1) are prepared. Water (for example, pure water) is added to each sample, and a diluted sample is prepared so as to be 20 μL. Further, 20 μL of water (sample E1) to which no coffee extract is added is also prepared as a standard sample for the diluted sample. To each diluted sample A1 to E1 (liquid volume: 20 μL), 20 μL of the substrate buffer attached to the kit is added, and then 20 μL of Enzyme Working solution included in the kit is added, and the total volume of the diluted sample is reduced to 60 μL ( = 0.06 mL), a measurement sample of the ACE activity inhibition rate is prepared. Thereafter, a sample for measuring the ACE activity inhibition rate is cultured at 37 ° C. for 40 minutes to produce angiotensin converting enzyme. After the culture, 200 μL of the Indicator Working solution included in the kit is added to each well and allowed to stand at room temperature for 10 minutes. Thereafter, the activity of angiotensin converting enzyme is calculated from the absorbance at 450 nm using an absorbance plate reader Multiscan JX (Thermo Scientific). The inhibition rate of angiotensin converting enzyme activity is determined from the measured absorbance using the following formula.
Inhibition rate (%) = {(absorbance of sample E1) − (absorbance of diluted sample) / (absorbance of sample E1)} * 100
The inhibition rate of each sample is shown in Table 1 below.

Based on the results obtained in Table 1, the amount of sample solution is plotted on the horizontal axis and the inhibition rate (%) is plotted on the vertical axis (FIG. 1). At that time, a straight line is drawn in the horizontal direction from the point of the inhibition rate 50% on the inhibition rate axis, and an intersection point X is obtained as an intersection with the plot. When a straight line is drawn from this intersection X in the vertical direction, an intersection T1 (0.182 μL (0.182 × 10 ⁻³ mL)) with the sample liquid volume axis is obtained.

The solid content concentration (IC50 value) of the sample with an inhibitory activity rate of 50% of the angiotensin converting enzyme is obtained from the amount of the sample solution obtained at the intersection T1 by the following equation.
Solid content concentration (IC50 value) [mg / mL]
= (Extracted solution amount [mL] at 50% inhibitory activity of angiotensin converting enzyme) * Dissolved component concentration [mg / mL] / Measurement sample total amount (0.06 [mL])
= (0.182 × 10 ⁻³ mL) * (45.3 mg / mL) / (0.06 mL)
= 0.137 mg / mL

  The measurement result of this sample is an example, and it is necessary to change the liquid volume of the diluted sample according to the sample extraction amount, dissolved component concentration, and the like. The amount of the sample solution at an angiotensin converting enzyme inhibitory activity rate of 50% is obtained according to the above description.

  As a feature of the active ingredient of the coffee bean extract, the solid concentration (IC50 value) of the extract showing an activity inhibition rate of 50% of angiotensin converting enzyme (ACE) is 0.16 mg / mL or less. Is mentioned. When the solid content concentration (IC50 value) is 0.16 mg / mL or less, the effect of suppressing blood pressure increase is exhibited to the maximum. On the other hand, if the solid content concentration (IC50 value) exceeds 0.16 mg / mL, it is difficult to exert the effect of suppressing blood pressure rise, which is not preferable.

  Further, when the activity inhibition rate of angiotensin converting enzyme (ACE) is 50%, the solid content concentration (IC50 value) of the extract showing activity inhibition is 0.001 mg / mL, which is the lower limit due to the measurement limit. Up to this lower limit value, the effect of suppressing blood pressure increase can be confirmed.

  Therefore, in the blood pressure increase inhibitor of the present invention, the IC50 value relating to ACE inhibition is more preferably an extract of coffee beans of 0.157 mg / mL or less, and further preferably an extract of coffee beans of 0.150 mg / mL or less. .

  The coffee bean extract exhibiting such excellent action can be obtained through a subcritical processing step described later.

  The saccharification-inhibiting action in the present invention is determined by measuring the production inhibition rate that suppresses the production of a reduced saccharified substance that causes an aging phenomenon by the measurement method described later. When the production inhibition rate of the saccharification product is 50%, It is defined by the partial concentration (IC50 value).

  Saccharification is one of the aging mechanisms in the body, and is a reaction in which sugar and protein are combined to produce a saccharified product. The rate at which the production of a saccharified product due to this saccharification reaction is suppressed is referred to as the saccharified product production inhibition rate. In the present invention, when the production inhibition rate of the saccharification product in which sugar and protein bind is 50%, that is, when the production inhibition rate of the saccharification product is 50%, the evaluation is performed with the solid content concentration (IC50 value) of the coffee bean I do.

  The saccharification-inhibiting action in the present invention is generally evaluated by measuring the production inhibition rate that suppresses the production of a reduced saccharified substance that causes an aging phenomenon by a measurement method described later. Usually, it is defined by what percentage of production inhibition is shown at a constant solid content concentration, and the lower the concentration is desirable. It is generally and widely used in pharmaceutical research that it is defined by the solid content concentration (IC50 value) of the extract at a glycation product production inhibition rate of 50%, which facilitates comparison between samples. (Reference: M. Hori et al., Anti-Aging Medicine, 9 (2012) 135.)

  The production inhibition rate measurement of a saccharification product for evaluating the saccharification inhibiting action will be described based on the measurement example performed this time. First, four or five diluted samples having different dilution rates or different concentrations of the extract are prepared using the coffee bean extract. Specifically, the coffee bean extract (here 27.2 mg / mL) whose dissolved component concentration has been measured in advance is 1.33 μL (sample A2), 2.00 μL (sample B2), 2 Five samples having different sample liquid amounts are prepared: .67 μL (sample C2), 4.00 μL (sample D2), and 5.33 μL (sample E2). Water (for example, pure water) is added to each sample, and a diluted sample is prepared so as to be 100 μL. In addition, 100 μL of water (sample F2) to which no coffee extract is added is also prepared as a standard sample for the diluted sample. Each diluted sample (100 μL) is mixed with 200 μL of 10 mg / mL bovine serum albumin (BSA), 500 μL of 0.1M phosphate buffer, 100 μL of distilled water and 100 μL of 2M fructose, and a measurement sample adjusted to a total volume of 1 mL is prepared. . Thereafter, using an Infinite F200-microplate reader (TECMAN), the fluorescence derived from the reduced saccharified substance is measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 440 nm, and the fluorescence intensity of the reduced sugar substance in the diluted sample is measured. Then, the production | generation inhibition rate of the reducing sugar substance in a dilution rate sample is calculated | required with the following calculation formulas.

Inhibition rate (%) = {(fluorescence intensity of sample F2) − (fluorescence intensity of diluted sample) / (fluorescence intensity of sample F2)} * 100

  The inhibition rate of each diluted sample is shown in Table 2 below.

Based on the results obtained in Table 2, the amount of sample solution is plotted on the horizontal axis, and the inhibition rate (%) is plotted on the vertical axis (FIG. 2). At that time, a straight line is drawn in the horizontal direction from the inhibition rate 50% from the inhibition rate axis, and an intersection point Y is obtained as an intersection with the calibration curve. When a straight line is drawn from the intersection Y in the vertical direction, an intersection T2 (2.64 μL (2.64 × 10 ⁻³ mL)) with the sample liquid volume axis is obtained.

From the amount of the extract obtained at the intersection T2, the solid content concentration (IC50 value) of the sample at a glycation product production inhibition rate of 50% is obtained by the following equation.
Solid content concentration (IC50 value) [mg / mL]
= (Extracted solution volume [mL] at 50% inhibition rate of glycation product production) * Solution component concentration [mg / mL] / Measurement sample total volume (1 [mL])
= (2.64 × 10 ⁻³ mL) * (27.2 mg / mL) / (1 mL)
= 0.0718 mg / mL

  The measurement result of this sample is an example, and it is necessary to change the liquid volume of the diluted sample according to the sample extraction amount, dissolved component concentration, and the like. The amount of sample solution at a glycation product production inhibition rate of 50% is obtained according to the above description.

  A feature of the active ingredient of the coffee bean extract is that the solid concentration (IC50 value) of the coffee extract at a glycation product production inhibition rate of 50% is 0.06 mg / mL or less. By making this solid content concentration (IC50 value) 0.06 mg / mL or less, the decrease in saccharification inhibition can be exhibited to the maximum.

  The solid content concentration (IC50 value) of the coffee extract at a glycation product production inhibition rate of 50% is 0.001 mg / mL, which is the lower limit for reasons such as the measurement limit. Up to this lower limit, the efficacy of saccharification inhibition can be confirmed.

  On the other hand, if the solid content concentration (IC50 value) exceeds 0.06 mg / mL, the effect of inhibiting saccharification becomes difficult to be exhibited, which is not preferable.

  Therefore, in the saccharification inhibitor of the present invention, the IC50 value relating to the inhibition of saccharification product production is more preferably an extract of coffee beans of 0.0597 mg / mL or less, and an extract of coffee beans of 0.0590 mg / mL or less. Further preferred.

  The coffee bean extract exhibiting such excellent action can be obtained through a subcritical processing step described later. In addition, the blood pressure elevation inhibitor and glycation inhibitor of the present invention are effective for various animals, and for mammals (eg, humans, monkeys, horses, cows, goats, sheep, dogs, cats, mice, etc.). It works more effectively.

Next, the manufacturing method of this invention is demonstrated.
As one aspect of the production method of the present invention,
Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a blood pressure increase-inhibiting action, comprising a step of recovering the coffee bean extract by treating it under a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature. It is.

As another aspect of the production method of the present invention,
Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a saccharification-inhibiting action, comprising a step of recovering the coffee bean extract by treating it at 1.8 MPa and a subcritical condition at a pressure equal to or higher than a saturated vapor pressure at each temperature. is there.

  In the present invention, the water used for the subcritical treatment with water, that is, the hydrolysis reaction, can be used in a liquid state or a gas state as long as it is a high-temperature water treatment. The temperature is desirably 100 ° C. or higher, and the desired reaction field is that the reaction proceeds more easily in the liquid state than in the gas. Therefore, water in a so-called subcritical state in which the liquid state is forcibly forced in a container close to a sealed state. Is preferred.

  The subcritical process is to extract a predetermined component from an extraction raw material by bringing a subcritical fluid brought into a subcritical state under a predetermined temperature and pressure into contact with the extraction raw material. For example, when water is raised to a pressure of 22.12 MPa and a temperature of 374.15 ° C., it shows a state where it is neither liquid nor gas. This point is called the critical point of water, and hot water at a temperature and pressure lower than the critical point is called subcritical water. Subcritical water has an excellent component extraction action and hydrolysis action due to a decrease in dielectric constant and an improvement in ionic product.

  More specifically, coffee beans and water as an extractant are placed in a pressure vessel such as metal or ceramics to bring them into a closed state, and a subcritical state (a mixture of ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to 1.8 MPa, and a pressure equal to or higher than the saturated vapor pressure at each temperature) for a certain period of time to obtain a coffee bean extract, which is used as a subcritical processing extract Yes.

  Examples of the extraction agent used for the subcritical treatment include, in addition to water, ethylene, ethane, propane, carbon dioxide, methanol, ethanol, and mixtures thereof. Among these, it is most preferable to use water from the viewpoint of safety.

  The temperature for carrying out the subcritical treatment of coffee beans is preferably between 140 and 200 ° C, more preferably between 140 and 180 ° C. It is because dissolved components represented by polyphenol are easily generated by adjusting to this temperature range. If the temperature of the subcritical treatment is less than 140 ° C., it may be difficult to generate dissolved components. If the temperature of the subcritical treatment exceeds 200 ° C., the dissolved components in the coffee beans are decomposed, and the amount of dissolved components generated is reduced.

  The processing pressure in the subcritical processing of coffee beans is that pressure: 0.15 to 1.8 MPa and a pressure equal to or higher than the saturated vapor pressure at each temperature. For example, “at or above the saturated vapor pressure at each temperature” means that at 160 ° C., 0.62 MPa or more, and at 200 ° C. or more, 1.55 MPa or more. This is because by using this pressure, dissolved components typified by polyphenol are easily generated. If the subcritical processing pressure is less than the saturated vapor pressure at each temperature, it is difficult to generate dissolved components, and processing time may be required. The upper limit of the subcritical processing pressure is not particularly limited to the above, but it is desirable to suppress the pressure to around 20 to 30 MPa due to the specifications of the high pressure apparatus.

  The subcritical processing time of the coffee is preferably between 3 and 30 minutes. If the subcritical processing time is less than 3 minutes, it is difficult to produce dissolved components typified by polyphenols. If the subcritical processing time exceeds 30 minutes, the polyphenol in the coffee beans will be decomposed, and the amount of polyphenol produced will be reduced.

  The subcritical processing time of roasted coffee beans is more preferably 3 to 15 minutes, and more preferably 3 to 9 minutes. It is because it becomes easy to produce | generate the dissolved component represented by polyphenol by setting it as the range of this processing time. If the subcritical processing time exceeds 15 minutes, the polyphenols in the coffee beans tend to be decomposed and the production amount of polyphenols tends to be reduced.

  The subcritical processing time of green coffee beans is more preferably between 10 and 30 minutes. It is because it becomes easy to produce | generate the dissolved component represented by polyphenol by setting it as the range of this processing time.

  At this time, as an example of hydrolysis conditions by subcritical processing of the coffee bean extract, temperature: 140-200 ° C., pressure: 0.15-1.8 MPa, and a pressure equal to or higher than the saturated vapor pressure at each temperature, The treatment time is desirably 3 to 30 minutes, more preferably 3 to 15 minutes. It is because it becomes easy to produce | generate the dissolved component represented by polyphenol by performing on these conditions.

  After performing the subcritical process, the pressure in the pressure vessel is returned to atmospheric pressure, and the subcritical process product and the container washing water are recovered. Distilled water is added to the two recovered materials to adjust the total amount, and a prepared liquid is obtained so that the ratio of coffee beans: distilled water becomes a constant ratio. This adjustment liquid can be filtered and the obtained filtrate can be used as a subcritical processing liquid.

  At this time, filtration of the adjustment liquid can be performed by a general method. Specifically, filtration using a filter paper or a coffee filter, decantation, screw press, roller press, rotary drum screen, belt screen, vibration, It can be performed by any one of a screen, a multi-plate vibration filter, vacuum dehydration, pressure dehydration, belt press, centrifugal concentration dehydration, and multi-disc dehydration.

The coffee beans used in the present invention may be raw beans or roasted beans.
Roasted coffee beans are roasted coffee beans to an appropriate roasting degree. The coffee beans can be roasted by a method such as a direct fire method, a hot air method, or a semi-hot air method. The roasting method, temperature, and time may be appropriately selected in order to achieve a desired roasting degree.

  The type of green coffee beans is not particularly limited, and may be any of Arabica and Robusta. More specifically, the coffee beans may be selected from, but not limited to, Brazil, Colombia, Vietnam, Tanzania and Mocha. One kind of coffee beans may be used alone, or a plurality of kinds may be blended and used.

  The pulverization of roasted coffee beans can be carried out using any type of pulverizer such as a roll grinder type, a flat cutter type, or a conical cutter type. Here, for suppressing blood pressure increase, either raw beans or roasted beans may be used, but it is more preferable to use raw beans. For suppressing saccharification, either raw beans or roasted beans may be used, but it is more preferable to use roasted beans.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited at all by these Examples.

Example 1
100 g of roasted and crushed coffee beans and 500 g of distilled water were placed in a pressure-resistant container having a volume of 2 L, and subcritical processing was performed at a processing temperature of 140 ° C., a processing pressure of 0.36 MPa, and a processing time of 3 minutes.

  After performing the subcritical treatment, the pressure in the pressure vessel was returned to atmospheric pressure, and the subcritical treatment product and the vessel washing water were collected. Distilled water was added to this recovered material to adjust the total amount to 900 g, and an adjusted liquid was obtained so that coffee beans: distilled water = 1: 8 (mass ratio). This adjustment liquid was filtered with a coffee filter (commercially available), and the obtained filtrate was used as a subcritical treatment liquid.

Example 2
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 140 ° C., a processing pressure of 0.36 MPa, and a processing time of 9 minutes.

Example 3
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 160 ° C., a processing pressure of 0.62 MPa, and a processing time of 6 minutes.

Example 4
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 180 ° C., a processing pressure of 1.0 MPa, and a processing time of 3 minutes.

Example 5
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 180 ° C., a processing pressure of 1.0 MPa, and a processing time of 9 minutes.

Example 6
In a pressure-resistant container having a volume of 2 L, 100 g of crushed green coffee beans and 500 g of distilled water were placed, and subcritical processing was performed at a processing temperature of 165 ° C., a processing pressure of 0.70 MPa, and a processing time of 30 minutes.

  After performing the subcritical treatment, the pressure in the pressure vessel was returned to atmospheric pressure, and the subcritical treatment product and the vessel washing water were collected. Distilled water was added to this recovered material to adjust the total amount to 900 g, and an adjusted liquid was obtained so that coffee beans: distilled water = 1: 8 (mass ratio). This adjustment liquid was filtered with a coffee filter (commercially available), and the obtained filtrate was used as a subcritical treatment liquid.

Example 7
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 6 except that the subcritical processing was performed at a processing temperature of 180 ° C., a processing pressure of 1.0 MPa, and a processing time of 20 minutes.

Example 8
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 6 except that the subcritical processing was performed at a processing temperature of 195 ° C., a processing pressure of 1.4 MPa, and a processing time of 10 minutes.

Example 9
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 6 except that the subcritical processing was performed at a processing temperature of 195 ° C., a processing pressure of 1.4 MPa, and a processing time of 30 minutes.

Comparative Example 1 Hot Water Extraction 40 g of roasted and ground coffee beans and 320 g of distilled water (coffee beans: distilled water = 1: 8 (mass ratio)) were prepared, and a commercial coffee maker (TOPVALUE, product number: TCO) was prepared. -Hot water extraction was performed using -CA6). Coffee beans and distilled water were set in a coffee maker, and distilled water heated to 95 ° C. was dropped onto the coffee beans on the paper filter for coffee. The hot water extract was obtained by recovering the coffee bean extract that passed through this filter.

Comparative Example 2 Extraction with hot water 20 g of ground coffee beans and 160 g of distilled water (raw coffee beans: distilled water = 1: 8 (mass ratio)) were prepared, and a commercial coffee maker (manufactured by TOPVALUE, product number: TCO- Hot water extraction was performed using CA6). Coffee beans and distilled water were set in a coffee maker, and distilled water heated to 95 ° C. was dropped onto the coffee beans on the paper filter for coffee. The hot water extract was obtained by recovering the coffee bean extract that passed through this filter.

Physical property value identification results The physical property values of two types of extraction liquids obtained in Examples 1 to 9 and Comparative Examples 1 and 2 were measured. The measured physical property values were polyphenol concentration and dissolved component concentration.

The polyphenol concentration was measured by the Folin-Ciocalteu method.
1 mL of Folin-Ciocalteu solution (MP Biomedicals, Inc.) diluted twice with distilled water, 1 mL of each extract, and 5 mL of 0.4 M aqueous sodium carbonate solution were mixed. The mixture was held at 50 ° C. for 5 minutes and then cooled with room temperature water for 30 minutes. Then, the light absorbency in 765 nm of each liquid mixture was measured. At this time, a calibration curve was prepared using a chlorogenic acid aqueous solution, and the amount of polyphenol was calculated as the amount corresponding to chlorogenic acid (mg / mL). The results are shown in FIGS. 3-1 and 3-2.

The dissolved component concentration was measured by a heat drying method.
Dispense 10 mL of each extract into a ceramic container, dry in a dryer at 105 ° C. overnight, evaporate the water in the extract, and determine the concentration of dissolved components (mg / mg) from the weight difference before and after drying the container. mL) was calculated. The results are shown in FIGS. 3-1 and 3-2.

  FIG. 3-1 and FIG. 3-2 show that the subcritical process can extract more dissolved components and polyphenols from coffee beans than the hot water extraction process.

ACE inhibitory activity test The ACE inhibitory activity test of two types of extracts, a subcritical processing solution and a hot water extract, was performed.

1. Sample preparation Two types of extracts were prepared: a subcritical processing solution and a hot water extract. The prepared extract is shown in Table 3.

2. Measurement of ACE inhibitory activity ACE inhibitory activity was measured using ACE-Kit WST manufactured by Dojindo Laboratories.
Predetermined amounts (hereinafter referred to as extract amounts) of test samples of the subcritical treatment liquid and hot water extract prepared in 1 above were collected and appropriately diluted with distilled water. The dilution rate was set so that the measured inhibition rate was 50%, and 4 or 5 dilution solutions were prepared for each sample. 20 μL of each diluted sample solution and 20 μL of substrate buffer included in the kit are added to each well of the microplate, and then 20 μL of Enzyme Working solution included in the kit is added to make a total volume of 60 μL (= 0.06 mL). An angiotensin converting enzyme was produced by incubation at 40 ° C. for 40 minutes. After culturing, 200 μL of the Indicator Working solution included in the kit was added to each well and allowed to stand at room temperature for 10 minutes. Thereafter, the inhibitory activity of angiotensin converting enzyme was measured at an absorbance of 450 nm using an absorbance plate reader Multiscan JX (Thermo Scientific), and the inhibition rate at each dilution rate was determined. Further, from the behavior of the obtained inhibition rate, the amount of the extract at 50% inhibition rate of the angiotensin converting enzyme was determined. Moreover, the solid content concentration (IC50 value) of each sample having an inhibitory activity rate of 50% of angiotensin converting enzyme was determined from the amount of the obtained extract using the following formula.

IC50 [mg / mL] = (Amount of extract (mL) at 50% inhibitory activity of angiotensin converting enzyme) * Dissolved component concentration [mg / mL] /0.06 [mL]

3. Evaluation Results The above test results are summarized in FIG. 4-1, FIG. 4-2, and Table 4.

  For reference, the results of a similar ACE inhibition test with peptides having angiotensin converting enzyme inhibitory activity disclosed in paragraphs 0018 to 0019 of Japanese Patent No. 3149199 are shown in Table 4. In paragraphs 0020 to 0021 of the publication, it is described that as a result of oral administration of this peptide to spontaneously hypertensive rats, 100 mg of oral administration showed a sufficient antihypertensive effect. Since the IC50 values of Examples 1 to 5 are sufficiently lower than this reference value, it is possible to expect a more sufficient blood pressure increase suppressing effect than this peptide.

  From the above results, it can be seen from Table 4 that Examples 1 to 5 have an excellent blood pressure increase suppressing effect as compared with Comparative Example 1. From Table 4, it can be seen that Examples 6 to 9 have superior blood pressure elevation inhibiting action as compared with Comparative Example 2.

Saccharification inhibition test A saccharification inhibition test was conducted on two types of extracts, a subcritical processing solution and a hot water extract.
The saccharification inhibition test measures the production of reducing saccharified substances such as amino groups and lipids and glucose of biological proteins. Formation and accumulation of a reduced saccharified substance in the skin easily causes a decrease in elasticity and flexibility of the skin and yellowing, which is a cause of aging as a result. Therefore, the saccharification inhibitory action is positioned as an anti-aging agent, and if the production of a reduced saccharified substance (saccharification product) can be suppressed, it becomes an index that the saccharification inhibitory action is excellent.

1. Sample preparation Two types of extracts were prepared: a subcritical processing solution and a hot water extract. The prepared extract is shown in Table 5.

2. Saccharification suppression measurement Each test sample of the subcritical processing solution and hot water extract prepared in 1 above was serially diluted with distilled water. The dilution rate was set to about 50 to 200 times so that the measured inhibition rate value exceeded or below 50%, and a diluted solution of 4 or 5 points was prepared for each sample. 100 μL of each of these diluted sample solutions was mixed with 200 μL of 10 mg / mL bovine serum albumin (BSA), 500 μL of 0.1 M phosphate buffer, 100 μL of distilled water, and 100 μL of 2M fructose, and adjusted to a total volume of 1 mL. Thereafter, the resultant was incubated at a temperature of 60 ° C. for 40 hours to produce a reduced saccharified substance. Thereafter, using an Infinite F200-microplate reader (TECMAN), the fluorescence derived from the reduced saccharified substance was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 440 nm, and the production inhibition rate of the reduced sugar substance at each dilution rate was determined. Further, from the behavior of the inhibition rate obtained, the amount of the extract was obtained when the production inhibition rate of the saccharification product was 50%. Moreover, the solid content concentration (IC50 value) of each sample having a production inhibition rate of saccharification product of 50% was determined from the amount of the obtained extract using the following formula. As a comparative control, aminoguanidine having a saccharification inhibiting action was used. For aminoguandi, the solid content concentration (IC50 value) with an inhibition rate of glycation product production of 50% was determined by the same procedure as the test sample.

IC50 [mg / mL] = (Extracted solution amount [mL] at 50% inhibition rate of glycation product production) * Dissolved component concentration [mg / mL] / 1 [mL]

3. Evaluation Results The results of the saccharification inhibition test are shown in FIG.

  From the above results, it can be seen that Examples 1 to 5 have an excellent saccharification inhibiting action as compared with Comparative Example 1. Further, compared with the value of aminoguanidine used as a control, Comparative Example 1 shows an activity equivalent to that of aminoguanidine, whereas Examples 1 to 5 show an activity exceeding that of aminoguanidine. I understand.

  Since the coffee bean extract of the present invention has a high blood pressure rise inhibitory effect and a high saccharification inhibitory effect, it can be used as a raw material for pharmaceuticals or health foods such as cosmetic ingredients and supplements.

Claims (14)

An antihypertensive agent containing a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
Blood pressure containing, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.16 mg / mL or less when the angiotensin converting enzyme (ACE) inhibitory activity rate is 50% An increase inhibitor.   The blood pressure elevation inhibitor according to claim 1, wherein the coffee beans are green coffee beans and / or roasted coffee beans.   The blood pressure increase inhibitor according to claim 1 or 2, wherein a treatment time of the subcritical treatment is 3 to 30 minutes.   The blood pressure increase inhibitor according to any one of claims 1 to 3, wherein a treatment time of the subcritical treatment is 3 to 15 minutes. A saccharification inhibitor containing a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
A saccharification inhibitor comprising, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.06 mg / mL or less when the production inhibition rate of a saccharification product is 50%.   The saccharification inhibitor according to claim 5, wherein the coffee beans are green coffee beans and / or roasted coffee beans.   The saccharification inhibitor according to claim 5 or 6, wherein a treatment time of the subcritical treatment is 3 to 30 minutes.   The saccharification inhibitor according to any one of claims 5 to 7, wherein a treatment time of the subcritical treatment is 3 to 15 minutes. Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a blood pressure increase-inhibiting action, comprising a step of recovering the coffee bean extract by treating it under a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature. .   The method for producing a coffee bean extract having a blood pressure increase-suppressing effect according to claim 9, wherein the treatment time of the subcritical treatment is 3 to 30 minutes.   The manufacturing method of the coffee bean extract which has the blood pressure rise inhibitory effect of Claim 9 or 10 whose processing time of the said subcritical process is 3 to 15 minutes. Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a saccharification-inhibiting action, comprising a step of recovering the coffee bean extract by a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature.   The method for producing an extract of coffee beans having a saccharification-inhibiting action according to claim 12, wherein the subcritical processing time is 3 to 30 minutes.   The method for producing a coffee bean extract having a saccharification-inhibiting action according to claim 12 or 13, wherein the treatment time of the subcritical treatment is 3 to 15 minutes.

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