JP2017031069A - Hydrangea macrophilla var. thunbergii extract and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain Hydrangea macrophilla var. thunbergii extract which is extracted in a larger amount per unit as compared with Hydrangea macrophilla var. thunbergii extract obtained by conventional hot water extraction with hot water at 100°C or less, and which is excellent in sugar absorption inhibiting function, anti-saccharification function, and the like.SOLUTION: The present invention relates to Hydrangea macrophilla var. thunbergii extract having a sugar absorption suppressing function and saccharification suppressing function, the extract obtained by the subcritical treatment of Hydrangea macrophilla var. thunbergii as a raw material, with water as an extracting solvent, at a temperature of 120-160°C, at a pressure equal to or higher than the saturated vapor pressure of each temperature; and to a production method thereof.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an amateur extract and a method for producing the same.

  Conventionally, it has been known that armatures and armature extracts have excellent functionality such as suppression of sugar absorption and suppression of protein glycation. In particular, attention is focused on diabetes prevention / treatment functions and aging prevention functions such as a sugar absorption inhibitory function and a saccharification inhibitory function.

  Patent Document 1 discloses an armature extract obtained by hot water extraction at a temperature of 100 ° C. or lower in a range of the boiling point of a solvent at room temperature or normal pressure using a solvent such as water. Is used as an α-glucosidase inhibitor. Patent Document 2 describes that an armature extract extracted from an armature at 60 ° C. with a solvent such as water or ethanol is used as an inhibitor of the Maillard reaction.

JP-A-2005-104871 JP 2007-254344 A

  Patent documents 1 and 2 which are prior arts describe an armature extract obtained by hot water extraction with hot water at 100 ° C. or lower. The prior art has a problem that the extraction efficiency of the amateur extract is not good. That is, it is desired to improve the extraction efficiency of the amateur extract. Moreover, it is desired that the amacha extract comprehensively suppresses saccharification. Specifically, it is also desired to obtain an armature extract having a further excellent sugar absorption suppressing function (α-glucosidase inhibitory activity) and blood protein glycation suppressing function (CML production inhibitory activity).

  The present invention has a larger amount of extract per unit amount than the conventional hamcha extract obtained by hot water extraction with hot water at 100 ° C. or less, and is excellent in saccharide absorption inhibition function, anti-glycation function, etc. The purpose is to obtain.

  The present invention is obtained by a subcritical process using armature as a raw material and using water as an extraction solvent at a temperature of 120 to 160 ° C. and a pressure equal to or higher than a saturated vapor pressure at each temperature, and suppresses sugar absorption and saccharification of protein in blood The present invention relates to an amateur extract having a suppressing function and a manufacturing method.

  It is preferable to perform a solid-liquid separation step after the subcritical treatment step.

  It is desirable that the processing time of the subcritical processing is 5 to 60 minutes.

  The armature extract of the present invention is superior to the armature extract obtained by hot water extraction with hot water at 100 ° C. or less, and has an excellent ability to suppress sugar absorption per unit amount of extract and to suppress glycation of blood proteins. It is an extract. In addition, according to the method for producing an amacha extract of the present invention, compared to an amacha extract obtained by hot water extraction, the sugar absorption inhibition function per extract unit amount and the glycation inhibition function of blood proteins were excellent. An amateur extract can be produced.

It is a graph which shows the CML production | generation inhibitory activity of an Example and a comparative example. It is a graph which shows the alpha-glucosidase inhibitory activity of an Example and a comparative example.

  The armature extract of the present invention is an armature extract produced by a production method including a subcritical processing step of subcritically processing a raw material amateur under a predetermined condition.

Raw material The raw material for the raw material of the extract of the present invention is the genus Hydrangea hydrangea, Hydrangea var. In the present invention, flowers, spikes, pericarps, fruits, stems, leaves, branches, branches and leaves, stems, bark, rhizomes, root barks, roots, seeds or whole plants can be used as raw materials, but leaves and / or branches It is preferable to use the tip.

  The state of the armature used for the extraction step described later is not particularly limited, and a raw state, a dried state, a powdered state, or the like is preferably used. From the reason that the extraction efficiency is excellent, it is preferable to use a dried pulverized product pulverized by a mixer or the like until the particle size becomes 1 to 3 mm after drying. In addition, when using a raw material, it is preferable to wash | clean dirt, such as soil, before an extraction process.

Manufacturing method The armature extract of the present invention is an armature extract obtained through a subcritical treatment process in which a raw material armature is subcritically treated under predetermined conditions.

Extraction Step The extraction step is a step of obtaining a subcritical processed product by subjecting the amateur material to subcritical processing. The subcritical treatment is a process in which a subcritical fluid as an extraction solvent brought into a subcritical state under a predetermined temperature and pressure is brought into contact with a raw material to be extracted (in the present invention, an armature) to thereby obtain predetermined components from the extracted raw material. Is extracted. 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. This subcritical water has an excellent component extraction action and hydrolysis action due to a decrease in dielectric constant and an improvement in ionic product.

  In the subcritical treatment of the present invention, water is used as an extraction solvent from the viewpoint of safety. When water is used as the extraction solvent used for the subcritical treatment, it can be used in a liquid state or a gas state as long as the water treatment is performed at a high temperature. That is, water vapor may be supplied to the subcritical processing tank, water may be supplied, or both of them may be supplied. The temperature of water or water vapor is desirably 100 ° C. or higher, and the desired reaction field is more likely to proceed in a liquid state than in a gas. The use of water in a critical state is preferred. More specifically, it can be obtained by putting the armature and the extraction solvent water in a pressure vessel such as metal or ceramics, making it close to a sealed state, and making contact with each other for a certain time or more in a subcritical state of water. The resulting extract can be a subcritical process.

  The subcritical processing temperature is preferably between 120 and 160 ° C, more preferably between 125 and 155 ° C, because an armature extract containing a large amount of functional components can be obtained. When the temperature of the subcritical treatment is less than 120 ° C., the extraction becomes insufficient and the functionality of the extract tends to be difficult to improve. Moreover, when the temperature of subcritical processing exceeds 160 degreeC, it will decompose excessively and there exists a tendency for the effect of a functional component to fall.

  The subcritical processing pressure is preferably carried out at a saturated vapor pressure or higher at each temperature. The saturated vapor pressure at each temperature can be determined by referring to the Japan Society of Mechanical Engineers steam table (1968). Examples of saturated vapor pressure at intervals of 10 ° C. between 120 to 160 ° C. are 120 ° C .: 0.20 MPa (2.02 at), 130 ° C .: 0.27 MPa (2.75 at), 140 ° C .: 0.36 MPa (3. 69 at), 150 ° C .: 0.48 MPa (4.85 at), and 160 ° C .: 0.62 MPa (6.30 at). By setting the pressure to be equal to or higher than the saturated vapor pressure, it becomes easy to efficiently obtain the functional component of the armature. In addition, although the upper limit of the pressure of a subcritical process is not specifically defined, it is preferable to suppress to around 20-30 MPa on the specification of a high voltage | pressure apparatus.

  The subcritical processing time is preferably 5 to 60 minutes, more preferably 10 to 30 minutes. By setting this processing time range, it becomes easy to efficiently obtain the functional component of the armature. If the subcritical processing time is less than 5 minutes, the resulting extract tends to be insufficient. Moreover, when subcritical processing time exceeds 60 minutes, an extract will decompose | disassemble excessively and there exists a tendency for the effect of a functional component to fall.

  That is, when the extraction solvent is water, the extraction conditions by the subcritical processing of the armature are a processing temperature of 120 to 160 ° C., a processing pressure equal to or higher than the saturated vapor pressure of each temperature, and a processing time of 5 to 60 minutes. By performing under this condition, the functional component of the armature can be extracted efficiently.

Solid-liquid separation step and drying step The solid-liquid separation step is a step of separating the subcritical processed product into an extract and a raw material residue (solid matter). Specific solid-liquid separation processes include filtration using filter paper, centrifugation, decantation, screw press, roller press, rotary drum screen, belt screen, vibrating screen, multi-plate vibrating filter, vacuum dehydration, pressure dehydration, Belt press, centrifugal concentration dehydration, multiple disk dehydration and the like can be mentioned. Among these, filtration is preferable because the operation is simple and the separation efficiency is excellent.

  The extract obtained by the solid-liquid separation process can be used as it is as an armature extract containing the functional components of armature, or it can be used for the intended purpose in a concentrated state. It is desirable to dry it. A solid armature extract is obtained by drying the extract. As a drying method, a general drying method can be used. Naturally, it is allowed to stand naturally, heat transfer drying such as box drying or spray drying which is a heating system, internal heat drying such as microwave drying, non-heating system Freeze drying, vacuum drying, suction drying, pressure drying, ultrasonic drying, and the like are possible. Of course, it is acceptable to dry using a general and simple oven and thermostat.

  Moreover, you may perform the impurity removal process using adsorption agents, such as activated carbon and an ion exchange resin, before a drying process suitably. The armature extract thus obtained can be used for the intended purpose as it is, or other ingredients may be formulated and blended to be processed into a powder, capsule or tablet form, such as water, alcohol, etc. Of course, it may be dissolved in a soluble solvent and used in liquid form.

  Moreover, in order to selectively obtain a component having high functionality, the extract obtained through the solid-liquid separation step and the drying step may be extracted with an organic solvent (alcohols, ketones, acetones, etc.). Of these, ethanol is preferred because it has little effect on the human body and is easy to use in foods and cosmetics.

  As an evaluation method of sugar absorption suppression in an amateur extract, α-glucosidase inhibition evaluation is exemplified, and as an evaluation method of saccharification suppression, CML (carboxymethyllysine) production inhibition evaluation is exemplified.

  The α-glucosidase inhibition evaluation evaluates the suppression of sugar absorption in the extract, and α-glucosidase is an enzyme that catalyzes a reaction that hydrolyzes the α-1,4-glucoside bond of sugar, In the process, it has the role of breaking down disaccharides into monosaccharides. The small intestine absorbs monosaccharides decomposed by enzymes, and this absorption increases the blood sugar level in the body. By inhibiting the role of this enzyme, disaccharides are less likely to be broken down into monosaccharides, so the degree of absorption of monosaccharides in the small intestine is reduced, and the blood sugar level in the body is less likely to rise. Expected to suppress rapid increase in blood glucose level after meals. In α-glucosidase inhibition evaluation, α-glucosidase inhibition rate (%) is calculated and expressed as mg / ml as IC50 value (final concentration). At this time, the smaller the IC50 value, the better the function of suppressing sugar absorption.

  In the evaluation of inhibition of CML generation, non-fluorescent CML (carboxymethyl lysine) is one of AGEs (final glycation end products), and is generated by oxidative cleavage of an Amadori compound. Specifically, it is produced by the reaction of glyoxal generated from lipid peroxidation or the like, or glycolaldehyde generated from hypochlorous acid and serine and a lysine residue. When an amateur extract inhibits the production of CML, which is one type of AGEs, the saccharification reaction can be suppressed, and it is expected to prevent saccharification risks such as skin deterioration and arteriosclerosis. In the evaluation of CML production inhibition, the CML production inhibition rate (%) is calculated and expressed as mg / ml as IC50 value (final concentration). At this time, the smaller the IC50 value, the better the function of inhibiting saccharification reaction.

Amateur extract The amateur extract of the present invention has excellent functionality for a sugar absorption inhibitory function (α-glucosidase inhibitory activity) and a blood protein glycation inhibitory function (CML production inhibitory activity). Therefore, as a first step, the extract of the present invention makes it difficult for the sugars in the food to be decomposed by the sugar absorption suppression function, and reduces the sugars that enter the blood in the body, thereby reducing the reaction between the sugars and the proteins in the body. As a second step, it is presumed that the production itself of one type of CML of AGEs (final glycation end product), which is a reaction between sugar and protein, is also suppressed by the glycation-inhibiting function in the body. Thus, it can act on saccharification suppression in two stages.

  The armature extract of the present invention is an armature extract that is superior in the function of suppressing sugar absorption per unit amount and the function of inhibiting glycation of blood proteins, compared to the armature extract obtained by hot water extraction. It can be suitably used as an agent, a therapeutic agent for diabetes, a slimming agent, an anti-aging agent and the like.

  The present invention will be described based on examples, but the present invention is not limited to the examples.

<Preparation of amateur extract>
The preparation process of the amateur extract of an Example and a comparative example is demonstrated.

Preparation of dried crushed hamaches After the leaves of Indonesian hamache (Hydrangea serrata var. Thunbergii) were washed with water, they were completely dried in an oven to a mixer (BM-RS08 made by Elephant Mark Mahobin Co., Ltd.) Then, the mixture was pulverized until the particle diameter became 1 to 3 mm to obtain a dry crushed product of armature.

Example 1 (subcritical treatment, 125 ° C.)
In a pressure-resistant container having a volume of 2 L, 40 g of dry crushed material of the armature was put, and subcritical processing was performed at a processing temperature of 125 ° C., a processing pressure of 0.2 MPa, and a processing time of 30 minutes (including a temperature rising time of 3 minutes). After the subcritical processing was completed, the exhaust valve was operated to return to atmospheric pressure in 5 minutes, and the processed product (slurry) in the processing can was collected. The recovered slurry was subjected to suction filtration with a cellulose filter paper (pore size: 1 μm, ADVANTEC 5C) to obtain a filtrate (subcritical extract). Then, the subcritical extract was dried with a freeze dryer (FDU-2200 manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain 16.2 g of a subcritical extract in a powder state.

Example 2 (subcritical treatment, 155 ° C.)
Subcritical processing, solid-liquid separation and freeze-drying were performed in the same manner as in Example 1 except that the subcritical processing conditions were a processing temperature of 155 ° C., a processing pressure of 0.5 MPa, and a processing time of 30 minutes. 20.3 g of critical extract was obtained.

Comparative Example 1 (subcritical treatment, 165 ° C.)
Subcritical processing, solid-liquid separation and freeze-drying were performed in the same manner as in Example 1 except that the subcritical processing conditions were a processing temperature of 165 ° C., a processing pressure of 0.7 MPa, and a processing time of 30 minutes. 20.1 g of critical extract was obtained.

Comparative Example 2 (subcritical treatment, 175 ° C.)
Subcritical processing, solid-liquid separation and freeze-drying were performed in the same manner as in Example 1 except that the subcritical processing conditions were a processing temperature of 175 ° C., a processing pressure of 0.9 MPa, and a processing time of 30 minutes. 21.4 g of critical extract was obtained.

Comparative Example 3 (subcritical treatment, 195 ° C.)
Subcritical processing, solid-liquid separation and freeze-drying were performed in the same manner as in Example 1 except that the subcritical processing conditions were a processing temperature of 195 ° C., a processing pressure of 1.4 MPa, and a processing time of 30 minutes. 24.8 g of critical extract was obtained.

Comparative example 4 (hot water treatment)
In a 2 L beaker, 40 g of dried hamaches and 1000 ml of water were placed and subjected to hydrothermal treatment at a treatment temperature of 90 ° C. and atmospheric pressure for a treatment time of 120 minutes to obtain a hydrothermally treated product. After the hot water treatment time, suction filtration was performed with a cellulose filter paper (pore size: 1 μm, ADVANTEC 5C) to obtain a filtrate (hot water extract). Then, the hot water extract was dried by a freeze dryer (FDU-2200 manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain 17.2 g of powdered hot water extract.

<Evaluation>
The following evaluation was performed about the extract prepared by each Example and the comparative example.

Amateur extract amount As an extract amount, it was calculated by the amount of extract (mg) per 1 g of dry pulverized product.

CML Production Inhibitory Activity Test 100 μL of a solution obtained by dissolving each extract in distilled water so that the concentration of each extract is 100 mg / ml and diluting with a 0.20 μm membrane filter is appropriately diluted with distilled water, 8 mg / mL human serum albumin (Sigma-Aldrich Corporation) 200 μL, 0.1 M phosphate buffer (pH 7.4) 500 μL, distilled water 100 μL, and 2 M glutose solution 100 μL were mixed to adjust the total volume to 1 mL. After that, those mixed solutions incubated at a temperature of 60 ° C. for 40 hours [Sample (S)] were used as a sample solution of the saccharification reaction product for the CML production inhibitory activity test.
As a control, the same amount of distilled water added instead of the 2M glucose solution [sample blank (SB)], the same amount of distilled water added instead of the sample [control (C)], the sample and 2M What added the same amount distilled water instead of the glucose solution [control blank (CB)] was also made to react on the same conditions.

Using the measurement kit: CircuLex CML / Nε- (carboxymethyl) lysine ELISA KIT (Cyclex Co., Ltd.) described in JP 2013-253072 A and the like, the CML production inhibitory activity of each example and comparative example (protein The glycation inhibiting function) was measured.
The following reagents were prepared and used.
Washing solution: 10 × Wash Buffer attached to the measurement kit diluted 10-fold with purified water Primary antibody solution: 1.25 ml of purified water added to the Primary Antibody attached to the measurement kit and mixed well, 400 μl of the mixture Was diluted 30-fold with Sample Dilution Buffer attached to the kit, and standard solutions 1 to 7 and blank were used for preparing a calibration curve, and CML-HSA Standard attached to the kit was diluted (standard solution 1: 7. 0 μg / ml, standard solution 2: 3.5 μg / ml, standard solution 3: 1.75 μg / ml, standard solution 4: 0.875 μg / ml, standard solution 5: 0.438 μg / ml, standard solution 6: 0. (219 μg / ml, standard solution 7: 0.109 μg / ml, blank: 0 μg / ml)

In each well of the microplate, 60 μl of the sample solution diluted 4-fold with the Sample Dilution Buffer and 60 μl of the primary antibody solution were added and mixed. 100 μl of the obtained mixed solution was dispensed into each well of the antigen solid phase plate, sealed with a plate seal, and reacted at 25 ° C. for 1 hour while shaking at 60 rpm. After completion of the reaction, the reaction solution in each well was removed, and washing with a washing solution was performed four times, and then water was removed to such an extent that it was not completely dried. Thereafter, 100 μl of the secondary antibody solution (HPR Conjugated Detection Antibody) attached to the kit was dispensed into each well, sealed with a plate seal, and reacted at 25 ° C. for 1 hour while shaking at 60 rpm. After the reaction, the reaction solution in each well was removed, and washing with a washing solution was performed four times, and then water was removed to such an extent that it was not completely dried. Then, 100 μl of color developing agent (Substrate Reagent) attached to the kit was dispensed, mixed, shielded from light with aluminum foil, and allowed to stand at 25 ° C. for 20 minutes. After standing, the reaction stop solution was added in the order in which the color formers were added to stop the reaction. Within 30 minutes after stopping the reaction, the absorbance at a measurement wavelength of 450 nm / reference wavelength of 540 nm is measured with a microplate reader (Multiscan JX manufactured by Thermo Fisher Scientific Co., Ltd.), and the CML amount is calculated from the calibration curve of the standard solution. did.
[Calculation of CML production inhibition rate and IC50 value]
Using each CML amount measurement result, the CML production inhibition rate (%) of each armature extract was calculated by the following formula, and the IC50 value (final concentration) was obtained. Table 1 and FIG. 1 show the average value of the values measured at n = 3. In addition, the error bar in a figure shows the standard deviation of 3 times of measurement.
Formula: ((C-CB)-(S-SB)) / (C-CB) × 100

α-Glucosidase Inhibitory Activity Test The sugar absorption inhibition function (α-glucosidase inhibitory activity) of each Example and Comparative Example was measured by the following method.
The following reagents were prepared and used. In addition, all the phosphate buffers used the same thing of pH = 7.0 and 0.1M.
Albumin solution: 10 mM phosphate buffer solution containing 0.2% by weight of bovine serum-derived albumin Enzyme solution: Albumin solution substrate solution containing 0.25 unit / ml α-glucosidase (manufactured by Wako Pure Chemical Industries): 1 mM p-nitro Phosphate buffer solution stopping solution containing phenyl-α-D-glucoside (manufactured by Wako Pure Chemical Industries): 0.2 M sodium carbonate aqueous solution sample solution: dissolved in distilled water so that the concentration of each extract is 100 mg / ml A solution obtained by diluting a solution filtered through a 0.20 μm membrane filter with distilled water as appropriate [Sample (S)]
In each well of the microplate, 25 μl of each sample solution, 25 μl of substrate solution and 25 μl of phosphate buffer were put in order. Further, 25 μl of enzyme solution was added and allowed to stand at 37 ° C. for 15 minutes, and then 200 μl of a reaction stop solution was added. Thereafter, the absorbance (S) at 405 nm was measured using an absorbance plate reader Multiscan JX (Thermo Fisher Scientific Co., Ltd.).
[Control (C)]
Absorbance (C) was measured by reacting under the same conditions as (S) except that 25 μl of distilled water was used instead of 25 μl of the sample solution.
[Sample blank (SB)]
Absorbance (SB) was measured by reacting under the same conditions as (S) except that 25 μl of phosphate buffer was used instead of 25 μl of enzyme solution.
[Control blank (CB)]
The reaction was performed under the same conditions as in (S) except that 25 μl of distilled water was used instead of 25 μl of the sample solution, and 25 μl of phosphate buffer was used instead of 25 μl of the enzyme solution, and the absorbance (CB) Was measured.
[Calculation of α-glucosidase inhibition rate and IC50 value]
Using each absorbance measurement result, the α-glucosidase inhibition rate (%) of each armature extract was calculated by the following formula, and the IC50 value (final concentration) was determined. The average value of the values measured at n = 3 is the result and is shown in Table 1 and FIG. In addition, the error bar in a figure shows the standard deviation of 3 times of measurement.
Formula: ((C-CB)-(S-SB)) / (C-CB) × 100

  From the results of Table 1 and FIG. 1 and FIG. 2, the armature extract of the present invention has a function of suppressing sugar absorption per unit amount and a function of inhibiting glycation of blood proteins, compared to the armature extract obtained by hot water extraction. It turns out that it is an excellent amateur extract. In addition, there was no correlation with extraction amount and functional evaluation, and it was confirmed that there is no influence in the contribution to the sugar absorption suppression function and the glycation suppression function of blood protein even if there is much extraction amount.

Claims (5)

An amateur extract having a sugar absorption inhibiting function and a saccharification inhibiting function, obtained by subcritical processing using an amateur as a raw material and water as an extraction solvent at a temperature of 120 to 160 ° C. and a pressure equal to or higher than the saturated vapor pressure at each temperature. . The armature extract according to claim 1, wherein a processing time of the subcritical processing is 5 to 60 minutes. Amateur extraction with sugar absorption suppression and saccharification suppression function including subcritical treatment process using amateur as raw material and water as extraction solvent at a temperature of 120-160 ° C and a pressure equal to or higher than the saturated vapor pressure at each temperature. Manufacturing method. The manufacturing method of the armature extract of Claim 3 which performs a solid-liquid separation process after the said subcritical process process. The method for producing an armature extract according to claim 3, wherein a processing time of the subcritical processing is 5 to 60 minutes.

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