KR102590469B1 - Immature fruit of diospyros kaki liquid extractor and method for extracting liquid of immature Fruit of diospyros kaki with anti-allergic and anti-inflammatory effects using the liquid extractor, and extract extracted therefrom - Google Patents

Abstract

The present invention provides a liquid extractor for immature persimmons that can be used in various ways by extracting the extract containing tannin contained in immature persimmons in its natural state without chemicals, and a liquid extractor for immature persimmons with anti-allergy and anti-inflammatory effects using the liquid extractor. It relates to a liquid extraction method and the extract extracted thereby, comprising a washing step (S100) of removing the stem of an immature persimmon and washing it in running water, and a cutting step (S200) of cutting the washed immature persimmon into pieces of 1 to 3 cm in size. ) and 100 kg of the cut immature persimmons are put into the upper container (10) equipped with an electric heating oven (60) outside the container, and connected to the upper container (10) using the filter assy (40) to prevent leakage. After fixing, a heating step (S300) of applying power to the electric heat furnace 60 and heating it to incinerate for 5 to 7 days at 600 to 700°C, and temporarily storing the extract generated as the heating step progresses. At the same time, a natural cooling step (S400) of natural cooling, a supernatant separation and discharge step (S500) of separating and discharging the supernatant when the extract is filled to the position of the discharge port 21 while natural cooling is performed, and the separated supernatant is discharged to the outside. A low-temperature ripening step (S600) of aging by storing at low temperature for 5 to 7 days in a low-temperature storage tank (80) operated by application of power, and obtaining the low-temperature aged extract (70) by weight (g). It consists of an extraction extraction/packaging step (S700).

Description

Liquid extractor of immature persimmon, method of extracting liquid of immature persimmon with anti-allergy and anti-inflammatory effect using the liquid extractor, and extract liquid extracted thereby {Immature fruit of diospyros kaki liquid extractor and method for extracting liquid of immature Fruit of diospyros kaki with anti -allergic and anti-inflammatory effects using the liquid extractor, and extract extracted therefrom}

The present invention relates to a liquid extractor of immature persimmons, a liquid extraction method of immature persimmons with anti-allergic and anti-inflammatory effects using the liquid extractor, and an extract extracted thereby. More specifically, it relates to an extract containing tannin contained in immature persimmons. It relates to a liquid extractor for immature persimmons that can be extracted in its natural state without chemicals and used for various purposes, a liquid extraction method for immature persimmons with anti-allergy and anti-inflammatory effects using the liquid extractor, and the extract extracted thereby.

Common persimmon ( Diospyros Kaki Thunb.) is a material that originates from Korea, China, and Japan and is mass-produced domestically. It is less damaged by pests and diseases and is a material used since ancient times for its various effects. It has antibacterial properties, durability, breathability, stiffness, UV protection, and preservative effect without any special processing. It is known to have various functional properties and a variety of bioactive substances such as dietary fiber, carotenoids, and catechins.

Persimmon fruit is an alkaline food rich in sugars such as glucose and fructose, and is used as dried persimmons and persimmons. In Korea, it is widely cultivated in Gyeongsang-do, Jeolla-do, and Chungcheongbuk-do, where the temperature is relatively warm, and contains a large amount of a unique ingredient called 'Diosprin'. I'm doing it.

Persimmon leaves ( Diospyros) Kaki Leaves) is effective for adult diseases such as high blood pressure, arteriosclerosis and heart disease, and chronic diseases such as diabetes, gastric ulcer and duodenal ulcer.

In addition, persimmon leaf tea is rich in chlorophyll and vitamins A, C, and D, which are known to have cancer prevention effects, and also contains high amounts of vitamin B1, folic acid, and pantothenic acid, so persimmon leaf tea is recommended as a good food for preventing adult diseases.

Persimmons are classified into sweet persimmons, which are completely removed from the water surface during growth, and immature persimmons, in which the persimmons remain.

Regardless of the variety, an astringent taste (sapmi) appears in immature fruit due to tannin, an antioxidant, and as the fruit ripens, the tannin content gradually decreases. Tannin has various biological activities, antibacterial and antiviral effects, wastewater treatment effect of tannin isolated from astringent persimmon skin, immune function revitalization effect, and has anticancer and tumor cell proliferation inhibitory effects due to active oxygen scavenging effect, etc. indicates.

Tannin isolated from persimmon leaves has an anti-mutagenic effect, and kakishibu, a persimmon juice containing tannin, has been reported to have a heavy metal removal effect and a tyrosinase inhibitory effect. Unripe fruit has an anti-diabetic function, an effect to improve neutral lipid metabolism, and an effect to improve cholesterol metabolism. has

Accordingly, there is a demand for functional products that can improve allergy relievers and skin allergies by extracting the tannin contained in immature persimmons in a natural state without adding chemicals, and Patent Application No. 10-2014-0021196 (invention of (Name: Anti-inflammatory composition using extract of unripe persimmon fruit and composition for improving atopic dermatitis) is differentiated from the present invention in that ethanol is added when extracting unripe persimmon fruit.

1. Patent Application No. 10-2014-0021196 (Title of Invention: Anti-inflammatory composition using unripe persimmon fruit extract and composition for improving atopic dermatitis)

The present invention was developed in response to the above-mentioned need, and the tannin component contained in immature persimmons, which has anti-allergy and anti-inflammatory effects, is extracted in its natural state and can be used in various ways as an allergic reliever and a functional product that can improve skin allergies. The purpose of the present invention is to provide a liquid extractor for immature persimmons that can extract liquid from immature persimmons, a liquid extraction method for immature persimmons with anti-allergy and anti-inflammatory effects using the liquid extractor, and an extract extracted thereby.

In order to achieve the above object, the liquid extractor for immature persimmons of the present invention is coupled and fixed to an upper container (10) provided with a space inside to fill the cut immature persimmons, and at the inlet of the upper container (10). , a filter assembly (40) in which filtration filters (41, 42), which can filter foreign substances of particles and particulates in two stages, are integrally fixed, and are fixed in a symmetrical direction of the upper container (10), and the extract liquid is temporarily A lower container 20 having an outlet 21 through which the supernatant is discharged while being stored, a coupling link 30 that couples and secures the upper container 10 and the lower container 20 to seal them, and A heat exchanger 60 is fixed in a form that surrounds the outer surface of the upper container 10 and radiates heat in the direction of the upper container 10 by application of an external power source 50, and through the outlet 21. It is characterized by including a low-temperature storage tank (80) for storing the discharged extract (70) at a low temperature.

In addition, a band-shaped floating material discharge prevention fence 90 is formed on one side of the outlet 21 of the lower container 20 to prevent floating material from being discharged through the outlet 21.

In addition, the liquid extraction method of immature persimmons with anti-allergy and anti-inflammatory effects using a liquid extractor of immature persimmons includes a washing step (S100) of removing the stems of immature persimmons and washing them in running water, and In the cutting step (S200) of cutting into sizes of 3 cm to 3 cm, 100 kg of the cut immature persimmons are put into the upper container (10) equipped with an electric heating oven (60) outside the container, and the filter assembly (40) is used to A heating step (S300) of attaching and fixing to the upper container 10 to prevent leakage, and then applying power to the electric heat furnace 60 to incinerate at 600 to 700°C for 5 to 7 days, and the heating A natural cooling step (S400) in which the extract liquid generated as the step progresses is temporarily stored and naturally cooled at the same time, and a supernatant separation and discharge step in which the supernatant is separated and discharged when the extract liquid fills up to the discharge port 21 while the natural cooling is performed. (S500) and a low-temperature ripening step (S600) in which the separated supernatant is stored at low temperature in a low-temperature storage tank (80) operated by application of an external power source for 5 to 7 days to ripen, and the low-temperature aged extract ( 70) is characterized in that it consists of an extract obtaining/packaging step (S700) in which the extract is obtained and packaged by weight (g).

The liquid extractor of immature persimmon according to the present invention having the above-described characteristics, the liquid extraction method of immature persimmon with anti-allergy and anti-inflammatory effects using the liquid extractor, and the extract extracted thereby, as long as the tannin component contained in the immature persimmon is not destroyed. It is easy to extract in its natural state, and the tannin component extracted in its natural state can be used as a variety of functional foods.

1 is a block diagram showing the manufacturing process of a preferred embodiment of the present invention.
Figure 2 is a device configuration diagram showing a preferred embodiment of the present invention.
Figure 3 is HPLC chromatogram of gallic acid standard material (A) and immature persimmon ethanol extract (B)
Figure 4 is a calibration curve of gallic acid standard product
Figure 5 shows HPLC chromatograms of water extract (A), 70% EtOH extract (B) and EtOH extract (C).
Figure 6 is an ESI-MS spectrum of [MH]-ions for the extract.
Figure 7 is a result table of ABTS radical scavenging ability of immature persimmon ethanol extract.
Figure 8 is a result table of electron donating ability of immature persimmon extract
Figure 9 is a table showing the results of superoxide dismutase (SOD)-like activity of immature persimmon extract.
Figure 10 is a table of cell viability results of IPEE in RBL-2H3 cells.
Figure 11 Table of results of the ability of IPEE to inhibit β-hexosaminidase release from RBL-2H3 cells stimulated with IgE antigen complexes
Figure 12 is a result table of the effect of IPEE treatment on IL-4 levels of inflammation in RBL-2H3 cells.
Figure 13 is a cell viability result table of RAW 264.7 cells.
Figure 14 is a cell viability result table of LPS and IPEE in RAW 264.7 cells.
Figure 15 is a table of the inhibition rate of nitric oxide secretion by IPEE in RAW 264.7 cells.
Figure 16 is a result table of the effect of IPEE treatment on TNF-α levels of inflammation in RAW 264.7 cells.
Figure 17 is a result table of the effect of IPEE treatment on IL-6 levels of inflammation in RAW 264.7 cells.
Figure 18 is a table of calculated opacity values of IPEE for BCOP analysis.
Figure 19 is a table of calculated permeability values of IPEE for BCOP analysis.
Figure 20 is a table of calculated IVIS values of IPEE for BCOP analysis.

Hereinafter, according to the attached drawings and preferred embodiments, the liquid extractor of immature persimmon provided by the present invention, the liquid extraction method of immature persimmon with anti-allergy and anti-inflammatory effects using the liquid extractor, and the extract extracted thereby will be described.
The term 'immature persimmon' refers to an unripe persimmon with a diameter of 2 to 5 cm or less that is inedible for about 4 to 6 weeks after the persimmon flower blooms and is subject to fruit thinning because the size of the fruit becomes small when left unattended, These immature persimmons have high tannin and polyphenol contents.
In particular, the 'tannin' component is detected in very small amounts in persimmons at the harvest time, but a large amount can be detected in immature persimmons, and this institute seeks to extract these 'tannin' components from 'immature persimmons.'

First, as shown in Figure 2, the liquid extractor for immature persimmons provided by the present invention and the liquid extractor include an upper container (10) provided with a space inside which can be filled with cut immature persimmons, and the upper container (10). ), and is configured to have a lower container (20) that is fixed in the symmetrical direction and has an outlet (21) that allows the supernatant to be discharged while temporarily storing the extract.

At the inlet of the upper container 10, a filter assembly 40 in which filtration filters 41 and 42, which can filter foreign substances of particles and particulates in two stages, is integrally fixed is fixed to the inlet, so that the filter assembly removes foreign substances. It performs a double filtration function and a stopper function at the same time.

The upper container 10 and the lower container 20 must be sealed to ensure airtightness, and the coupling link 30 performs this sealing function, and a solid fixation can be provided through this coupling link. .

The outer circumferential surface of the upper container 10 is provided with an electric heat furnace 60 fixed in a form that surrounds the outer circumferential surface. The electric heat furnace 60 generates heat by applying an external power source 50, and the upper container 10 generates heat by applying an external power source 50. It is configured to transmit heat of 600 to 700°C to the inside of the container, which is at the level where the immature persimmons filled inside the upper container are incinerated.

As described above, the extract extracted by heat generation is temporarily stored in the lower container 20. At this time, some natural cooling occurs until the extract liquid fills up to the vicinity of the outlet 21 formed on one side of the lower container 20, and the outlet (20) A low-temperature storage tank 80 is configured to store the extract 70 discharged through 21) at a low temperature.

In addition, a band-shaped suspended matter discharge prevention fence 90 is provided on one side of the outlet 21 of the lower container 20 to prevent suspended matter from being discharged through the outlet 21, so that it is formed on the surface of the extract liquid. It is easy to obtain/package after filtering out any suspended matter.

In addition, the liquid extraction method of immature persimmons with anti-allergenic and anti-inflammatory effects using a liquid extractor of immature persimmons involves removing the stems of immature persimmons and washing them under running water (S100).

The washed immature persimmon was cut into pieces of 1 to 3 cm (S200), and 100 kg of the cut immature persimmon was put into the upper container (10) equipped with an electric heating oven (60) outside the container, and then filtered into the filter assembly (40). ) is used to fix it to the upper container 10 to prevent leakage, and then apply power to the electric heating oven 60 and heat it to incinerate for 5 to 7 days at 600 to 700°C (S300).

Of course, the temperature conditions and heating period may vary depending on the amount of immature fruit put into the upper container, but they can be sufficiently resolved through repetition.

As the heating step progresses, the extracted liquid generated is temporarily stored and naturally cooled (S400), and when the extract liquid fills up to the discharge port 21 while the natural cooling is performed, the supernatant is separated and discharged (S500).

The separated supernatant is stored and aged at low temperature for 5 to 7 days in a low-temperature storage tank (80) operated by application of an external power source (S600), thereby preventing deterioration and at the same time ensuring the complete integrity of the immature fruit filled in the upper container. It can be maintained without any additional work until it is incinerated.

The extract 70 aged at low temperature as described above is obtained and packaged by weight (g) (S700) to complete the packaging of the extract, and the packaged extract can be used in various ways as functional foods.

It was confirmed that the extract extracted as above can be expected to be practical as an allergic dermatitis alleviator with antioxidant, anti-allergy, and anti-inflammatory effects, as confirmed in the examples below.

[Example 1]

As a result of HPLC quantitative analysis, as shown in Figure 3, gallic acid standard was detected at 6.7 minutes, and gallic acid in IPEE was also detected at 6.7 minutes.

In addition, as shown in Figure 4, the gallic acid standard solution showed excellent linearity in the range of 5 to 500 μg/mL, and the gallic acid content per 10 mg/mL of persimmon EtOH extract was 0.522±0.004% on average. Confirmed.

As a result of HPLC qualitative analysis, as shown in FIG. 5, gallic acid was detected in the hot water, EtOH, and 70% EtOH extracts of immature persimmons, and it was confirmed that gallic acid was present as shown in FIG. 6.

[Example 2]

As a result of the antioxidant activity evaluation (total polyphenol and total flavonoid content), the total polyphenol content and total flavonoid content of the immature persimmon extract were analyzed, and the total polyphenol content was 428.3 mg/g, and the flavonoid content was 31.1 mg/g. It was confirmed.

In addition, as shown in Figure 7, the evaluation of ABTS ⁺ radical scavenging activity shows that in the antioxidant measurement experiment using ABTS radical, the ABTS free radical generated in the potassium persulfate reaction is removed by the antioxidant contained in the sample, discoloring the radical's characteristic blue-green color. The ABTS radical scavenging activity of the immature persimmon extract increased in a concentration-dependent manner as the concentration of the substance increased, and at a concentration of 250 μg/mL, it showed antioxidant activity similar to that of ascorbic acid, a positive control. gave

In addition, as shown in Figure 8, DPPH electron donating ability evaluation can estimate antioxidant activity through electron donating ability because it receives hydrogen or electrons from antioxidant substances to form an irreversibly stable molecule. When it comes into contact with a substance with antioxidant activity, it becomes radical. This is erased. At this time, DPPH's unique purple color has the property of being discolored, and the electron donating ability can be measured by colorimetric difference in color. The electron donating ability of IPEE showed that its activity increases in a concentration-dependent manner as the concentration of the substance increases. It showed 73.7% electron donating ability at a concentration of 1,000 μg/mL.

Additionally, SOD-like activity, as shown in Figure 9 Superoxide radical scavenging activity was confirmed to scavenge superoxide anion radical generated by autoxidation of pyrogallol. As the concentration of IPEE extract increased, the activity showed a concentration-dependent increase, and IPEE showed 54.5% active oxygen scavenging ability at a concentration of 1,000 ppm, which is not considered to be low compared to ascorbic acid.

[Example 3]

Anti-allergy evaluation (cytotoxicity evaluation) using RBL-2H3 was performed as a result of MTT assay measurement to confirm the effect of the test substance on the survival and proliferation of the test substance in RBL-2H3 cells, as shown in Figure 10, IPEE was 125, 250, When treated at a concentration of 500 μ, the results were 93.4%, 85.1%, and 81.4%, respectively. Therefore, subsequent experiments were performed at a concentration of 500 μ or less, which is the concentration range that does not affect cell survival and proliferation.

In addition, as shown in Figure 11 as a result of β-hexosaminidase measurement, it is common to measure the amount of histamine released from mast cells sensitized to IgE to confirm the effect of the test substance on degranulation of RBL-2H3 mast cells. Since the concentration of histamine released from cells was low and the measurement process was complicated, β-hexosaminidase concentration was measured. β-hexosaminidase exists in granules where histamine is stored in mast cells or basophils and is released out of the cells through allergic reactions, so it is used to measure allergy inhibitors as an indicator of degranulation. As a result of confirming the effect of IPEE on the amount of β-hexosaminidase production in RBL-2H3 cells, as shown in Figure 11, considering that the inhibitory ability of β-hexosaminidase secreted in the control group sensitized by DNP-IgE and DNP-HSA was 0%, IPEE Inhibitory effects of 83.05 ±6.6, 60.69 ±3.08, and 52.58 ±3.08% were confirmed at concentrations of 500, 250, and 125 μ. As the concentration of the test substance increased, the amount of β-hexosaminidase secretion decreased.

In addition, the results of the protein expression inhibitory activity of the allergy factor IL-4 are shown in Figure 12. In all RBL-2H3 cells treated with IPEE, IL-4 decreased compared to the IgE + HSA group, and in particular, a significant decrease was confirmed in the group treated at a concentration of 200 μg/mL.

[Example 4]

Anti-inflammatory evaluation (cytotoxicity evaluation) results using RAW 264.7 cells, as shown in Figure 13 IPEE showed a high cell survival rate of over 80% at concentrations ranging from 31.25 to 500 μ, and 82.6% at the highest concentration of 500 μ. Therefore, subsequent experiments were performed at a concentration of 500 μ or less, which is a concentration range that does not affect cell survival and proliferation.

In addition, the cell survival rate measurement results according to LPS treatment are as shown in Figure 14. Compared to the cell survival rate of the LPS group of 77.8%, IPEE showed a cell survival rate of 80.6% at a concentration of 500 μg/mL.

In addition, the results of measuring the NO inhibition rate in RAW 264.7 cells showed that as the concentration of IPEE increased, the NO inhibition rate increased in a concentration-dependent manner, as shown in Figure 15, showing a high NO production inhibition rate. The test substance showed a NO inhibition rate of 94.5% at the highest concentration of 1,000 μ.

In addition, the results of the inhibitory effect of IPEE on the expression levels of TNF-α and IL-6 by LPS showed that, as shown in Figures 16 and 17, all RAW 264.7 cells treated with IPEE had TNF-α, IL-6 decreased, and in particular, the group treated at a concentration of 500 μg/mL showed a significant decrease.

[Example 5]

In the ocular mucosa irritation test evaluation using the BCOP assay, the opacity was as shown in Figure 18. Ethanol used as a positive control showed very high corneal opacity, and IPEE showed a corneal opacity close to 0, similar to distilled water, a negative control.

Additionally, the transmittance is as shown in Figure 19. The cornea of the positive control group showed a transmittance close to 1.8, but IPEE showed a very low transmittance, close to 0, similar to the negative control group, at all concentrations.

In addition, the result of calculating the eye irritation index (IVIS) using the opacity and permeability of the cornea is as shown in Figure 20. The eye irritation index was determined based on the standards of OECD test guideline 437, and in OECD test guideline 437, IVIS is If it is 55 or higher, it is classified as an irritating substance, and if it is less than 3, it is classified as a non-irritating substance. In this study, all concentrations of IPEE showed an IVIS value of 3 or less in the cornea. Like distilled water, which is a negative control, IPEE is considered to be a non-irritating substance according to the results of the eye mucosa irritation test.

As confirmed in the above examples, the immature persimmon extract was analyzed for content In the experiment, high tannin content was confirmed, antioxidant, anti-allergy and anti-inflammatory effects and their mechanistic aspects were confirmed, and safety against irritation was also confirmed. Based on this, immature persimmons are useful in the development of anti-inflammatory and anti-allergy functional products. It can be said that it provided basic data on the practical feasibility of a natural material.

Although the embodiments of the present invention have been described, those skilled in the art will be able to understand the present invention by adding, changing, deleting or adding components without departing from the spirit of the invention as set forth in the patent claims. Various modifications and changes may be made, and this will also be included within the scope of the rights of the present invention.

10: upper container 20: lower container
21: outlet 30: coupling link
40: Filter Assay 41, 42: Filtration Filter
50: External power source 60: Electric heating oven
70: Extract 80: Low-temperature storage tank
90: Fence to prevent floating material discharge
S100: Washing step S200: Cutting step
S300: Leakage prevention step S400: Combination fixation step
S500: Heating step S600: Natural cooling step
S700: Low-temperature ripening step S800: Obtaining/packaging step

Claims (4)

delete delete A washing step (S100) of washing under running water while removing the stem of the immature persimmon; and,
A cutting step (S200) of cutting the washed immature persimmon into 1 to 3 cm pieces; and,
100kg of the cut immature persimmons are put into the upper container (10) equipped with an electric heating furnace (60) outside the container, and filtration filters (41, 42) that can filter particles and foreign substances in two stages are installed. A leak prevention step (S300) of preventing leakage of contents by fixing the integrally fixed filter assembly (40) to the upper container (10) to prevent leakage; and,
The lower container (20), which has an outlet (21) through which the extract is temporarily stored and the supernatant is discharged, is fixed to the bottom of the upper container (10), and the upper/lower parts are coupled and fixed so as to be airtight using the coupling link (30). Container combination fixing step (S400); and,
A heating step (S500) of applying power to the electric heat furnace 60 provided outside the upper container after fixing the upper/lower container to heat at 600 to 700° C. for 5 to 7 days; and,
A natural cooling step (S600) in which the extract produced as the heating step proceeds is temporarily stored in the lower container 20 and simultaneously cooled naturally; and,
When the extract liquid is filled to the position of the outlet 21 during the natural cooling, the supernatant is separated and discharged, and the suspended matter is discharged while preventing simultaneous discharge through the band-shaped suspended matter discharge prevention fence 90. A low-temperature ripening step (S700) in which the extract 70 is introduced into a low-temperature storage tank 80 operated by the application of an external power source and stored at low temperature for 5 to 7 days and simultaneously ripened; and,
An extract obtaining/packaging step (S800) in which the low-temperature aged extract (70) is obtained and packaged by weight (g);
A liquid extraction method of immature persimmon, characterized in that it consists of: delete