KR101265876B1 - Urushiol reduction disposed lacquer, lacquer extract by using the same and Method of manufacturing the same - Google Patents

Abstract

The present invention relates to a urushiol-reduced lacquer leaf, a lacquer leaf extract using the same, and a method for preparing the same, and specifically, to a lacquer leaf and an extract of the lacquer leaf reduced by urushiol by treating the lacquer with electron ionization energy. Since the lacquer extract of the present invention reduces urushiol causing allergy reactions, it can be used as a composition of pharmaceutical components, and in particular, it can be used as an anticancer component as it has excellent scavenging ability against nitrite in the body, known as an anticancer substance. Moreover, since it is excellent in nitric oxide scavenging ability, it is applicable to pharmaceutical compositions, such as an anti-inflammatory component. And, the manufacturing method of the lacquer extract of the present invention has the advantage that can be mass-produced.

Description

 Urushiol-reduced lacquer, extracts thereof, and preparation method thereof {Urushiol reduction disposed lacquer, lacquer extract by using the same and Method of manufacturing the same}

The present invention relates to a lacquer that effectively reduces urushiol by using electron ionization energy, an extract of the lacquer, and a preparation method thereof.

In general, lacquer refers to the sap coming from the lacquer tree when the tree of the lacquer family is wounded. The lacquer family includes 60 genera and 400 species distributed in subtropical or tropical regions, and in Korea, there are 5 species of lacquer trees, lacquer trees, and rhododendrons.

Such lacquer has been used in Korea, China, Japan, etc. for a long time as a coating material for metal and woodworking, and it is also used as a medicinal herb because it has the effect of promoting blood circulation in the human body, tonic effect, pain resistance, and Jinhae. come. In addition, recently, there have been reports of extracting an anticancer substance (MU2) from lacquer.

In case of directly ingesting lacquer having various pharmacological effects in the sap state, there is a problem in that side effects such as rash and itching are caused by a toxic substance called urushiol (urshiol).

Reducing the side effects of lacquer can be ingested with chicken, jujube, chestnut, astragalus, etc. with lacquer and detoxification of lacquer using mushrooms, but these methods are not suitable for mass production. There is.

Therefore, the present inventors have studied the method of effectively reducing urushiol from lacquer, and using the uricol-reduced lacquer and its extracts in mass production and commercially using it, and treating the lacquer with electron ionization energy can effectively reduce urushiol. It was found that the present invention was completed. Accordingly, an object of the present invention is to provide a novel urushiol reduced lacquer, extracts thereof and a method for producing the same.

The present invention for solving the above problems relates to a method for producing urushiol reduced lacquer, the step of water treatment of the lacquer; And treating the water-treated lacquer with electron ionization energy.

The present invention also relates to urushiol reduced lacquer prepared by the above production method.

In addition, the present invention relates to a method for producing a lacquer extract, the step of water treatment of the lacquer; Treating the water-treated lacquer with electron ionization energy; And boiling the mixture of lacquer and solvent treated with the electron ionization energy, and filtering the insoluble material to generate an extract.

In addition, the present invention is characterized by another urushiol reduced lacquer extract prepared by the above production method.

In addition, the present invention relates to a pharmaceutical composition, characterized in that it comprises the lacquer extract.

The manufacturing method of the lacquer of the present invention can effectively reduce the urushiol, by using this can be mass-produced lacquer, lacquer extract with reduced urushiol. In addition, the lacquer extract of the present invention does not cause an allergic reaction, has little toxicity, and has anti-cancer and anti-inflammatory effects, and can be applied to various pharmaceutical compositions, food compositions, and the like.

1 is a schematic diagram of a manufacturing process of urushiol reduced lacquer according to the present invention.
2 is a HLPC measurement result of the gamma ray-treated lacquer was carried out in Experimental Example 1.
3 is a measurement result of the urushiol content of the gamma ray-treated lacquer conducted in Experimental Example 1.
4 is a HLPC measurement result of the electron beam-treated lacquer carried out in Experimental Example 1.
5 is a measurement result of the urushiol content of the lacquer treated with electron beams carried out in Experimental Example 1.
6 is a cytotoxicity measurement results of the gamma-irradiated lacquer leaf extract performed in Experimental Example 7.
7 is a cytotoxicity measurement results of the lacquer extract treated with electron beam irradiation performed in Experimental Example 7.
8 is a result of measuring anticancer activity of the gamma-ray irradiation-treated lacquer extract performed in Experimental Example 8.
9 shows the results of anticancer activity of the lacquer extract treated with electron beams in Experiment 8
10A and 10B are cytotoxicity measurement results for the anti-inflammatory activity test conducted in Experimental Example 9, A is a cytotoxicity measurement result of gamma-irradiated lacquer leaf extract, and B is a cytotoxicity measurement of the lacquer leaf extract treated with electron beam irradiation. The result is.
11 is an anti-inflammatory activity test result of the gamma-irradiated lacquer leaf extract performed in Experimental Example 9, A is a graph measuring the nitric oxide production, B is a graph measuring the nitric oxide removal rate.
12 is an anti-inflammatory activity test result of the electron beam irradiation treated lacquer extract performed in Experiment 9, A is a graph measuring the nitric oxide production, B is a graph measuring the nitric oxide removal rate.

The term used in the present invention, 'lacpi' means dried lacquer bark, 'lacquer' means dried lacquer leaves. In addition, the term "lacquer" used in the present invention is meant to include at least one selected from lacquer and lacquer, and the "lacquer extract" is meant to include at least one selected from the lacquer extract and lacquer extract.

Hereinafter, the present invention will be described in detail.

Radiation such as electron beams and gamma rays can cause a wide variety of chemical changes in various biological cells. Water molecules forming the environment around the living body are hydrogen molecules (H ·), hydroxyl radicals (OH ·), and water-soluble electrons (e _s ⁻ ). By the action of converting into a primary radical, such as The present invention applying such a conversion action by the radicalization of the urushiol contained in the lacquer in low temperature, short time, the polymerization of unstable radicals so that the lacquer no longer causes an allergy reaction to the living body. And, in order to maximize the radical conversion reaction, the present reaction is subjected to electron ionization energy (radiation) treatment after lacquer moisture treatment.

Such a method of manufacturing lacquer of the present invention comprises the steps of treating the lacquer with moisture; And treating the water-treated lacquer with electron ionization energy; reducing urushiol present in the lacquer.

In the step of water treatment of the lacquer, the water treatment method is not particularly limited, for example, by spraying water on the lacquer, or impregnated the lacquer in water, it is sufficient that the lacquer can sufficiently absorb the water.

The electron ionization energy treatment may be performed by irradiating the lacquer with at least one electron ionization energy selected from gamma ray irradiation and electron beam irradiation, thereby radicalizing the water contained in the lacquer, and a radical product thereof radicalizes the urushiol of the lacquer. It is possible to reduce the urushiol contained in.

The gamma-irradiation may irradiate gamma-rays at a dose rate of 1 kGy, 2 kGy, 5 kGy or 10 kGy per hour, preferably so that the total absorbed dose is 1 kGy, 2 kGy, 5 kGy or 10 kGy in the water-treated lacquer. Gamma rays can be irradiated at a dose rate of 10 kGy per hour, and more preferably gamma rays are irradiated at a dose rate of 10 kGy per hour such that the total absorbed dose is 5 kGy or 10 kGy. In addition, the gamma irradiation method may be any method capable of irradiating gamma rays at the dose rate and the total absorbed dose, and is not particularly limited in the present invention, for example, source 300,000 Ci, Co-60 gamma irradiation We can check gamma ray with facility.

The electron beam irradiation may be irradiated with an electron beam at a dose rate of 1 kGy, 2 kGy, 5 kGy or 10 kGy per hour, preferably so that the total absorbed dose to 1 kGy, 2 kGy, 5 kGy or 10 kGy The electron beam can be irradiated at a dose rate of 10 kGy per hour, and more preferably, the electron beam is irradiated at a dose rate of 10 kGy per hour such that the total absorbed dose is 5 kGy or 10 kGy. The electron beam irradiation method may use any method capable of irradiating an electron beam with the dose rate and the total absorbed dose, and is not particularly limited in the present invention. For example, 14.5 using a 2.5MeV transformer electron accelerator may be used. The electron beam can be irradiated with a beam current of mA.

And, in the present invention, the lacquer may include at least one selected from lacquer and lacquer.

In addition, the present invention can be used to prepare a lacquer extract with reduced urushiol using the lacquer prepared by the manufacturing method of the lacquer, it will be described below.

Method for producing urushiol reduced lacquer extract of the present invention

Water treatment of lacquer; Treating the water-treated lacquer with electron ionization energy; And boiling the mixture of lacquer and solvent treated with the ionization energy, and filtering the insoluble material to produce an extract. The method may further include concentrating the extract. .

The step of treating the lacquer with water and the step of treating with the electron ionization energy are the same as described above.

In the step of producing the extract, the solvent may be used at least one selected from water and ethanol, preferably ethanol. The water may be any distilled water, ionized water (DI water), etc., as long as it is edible water without impurities.

In addition, the mixture is a mixture of the lacquer treated with the electron ionization energy and the solvent in a weight ratio of 1: 5 to 1:15, preferably in a 1: 5 to 1:12 weight ratio, and 1: 5 weight ratio When used, the amount of solvent is so small that the extraction may not be possible due to the evaporation of the solvent when the extract is prepared by applying heat such as hydrothermal reaction, and when the solvent is used in excess of 1:15 weight ratio, the extraction time is too long. There is.

In the step of producing the extract, the method of boiling the mixture may be a method of heating above the boiling point temperature of the mixture including the solvent, and is not particularly limited. However, hot water extract is preferably applied for 3 to 5 hours at 95 ~ 110 ℃, and ethanol (95%) extract is attached to the reflux cooling device at atmospheric pressure for 2 to 3 hours at 75 ~ 90 ℃ to heat.

The lacquer extract of the present invention prepared by the above manufacturing method is reduced in urushiol causing an allergic reaction and does not cause an allergic reaction.There is no toxicity, and the lacquer extract is variously applied to pharmaceutical compositions such as herbal medicines, food compositions, etc. Can be used.

Inflammation is a biological response in response to damage caused by physical and chemical stimuli, and is an important factor in the causes of various diseases such as arthritis, asthma, multiple sclerosis and colitis. Macrophages play an important role in innate and acquired immune responses and regulate various inflammatory mediators, including nitric oxide (NO) and inflammatory cytokines. In addition, lipopolysaccharide (LPS), known as endotoxin, is present in the extracellular membrane of Gram-negative bacteria and is known to increase inflammatory cytokines in macrophages or monocytes such as RAW 264.7. In addition, NO formation plays an important role in killing bacteria or removing tumors, but NO produced excessively by inducible nitric oxide synthase (iNOS) causes inflammation and damages tissues, genetic mutations, and nerve damage. Cause. The lacquer extract of the present invention can be applied to pharmaceutical compositions such as anti-inflammatory agents by inhibiting the production of nitric oxide and having an anti-inflammatory effect.

In addition, amines and nitrites react in vivo to produce nitrosamines, which are carcinogenic substances. The lacquer extract of the present invention has an excellent effect of eliminating nitrites, which can be applied to pharmaceutical compositions such as anticancer drugs. .

Hereinafter, the present invention will be described in detail with reference to a preferred embodiment so that those skilled in the art can easily practice the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

Example  1: gamma irradiation treatment Lacquer  Produce

After 1 kg of lacquer leaves were impregnated with distilled water at 20 ° C. for 1 hour, the lacquer leaves were treated with sufficient moisture, and the water-treated lacquers were 300,000 Ci, Co-60 gamma-irradiating facilities (point source AECL, IR-79 Gamma rays were irradiated so that the total absorbed dose of gamma rays was 1 kGy as shown in Table 1 at a dose rate of 10 kGy per hour. Next, the gamma-ray treated lacquer was lyophilized and ground, and then cryogenically stored at -80 ° C.

Example  2 to 4

     The same procedure as in Example 1, except that Examples 2 to 4 were performed so that 2kGy, 5kGy and 10kGy per hour.

division
(Unit: kGy) Example 1 Example 2 Example 3 Example 4 Gamma rays
Total absorbed dose One 2 5 10

Example  5: electron beam irradiation treatment Lacquer  Produce

After 1 kg of lacquer leaves were impregnated with distilled water at 20 ° C. for 1 hour, the lacquer leaves were treated with sufficient moisture, and the water-treated lacquer leaves were then subjected to a 2.5 MeV transformer type electron accelerator (ELV-8 model, EB Tech Co., Ltd.). The electron beam was irradiated with a beam current of 14.5 mA, and gamma rays were irradiated such that the total absorbed dose was 1 kGy as shown in Table 2 at a dose rate of 10 kGy per hour. Next, the electron beam treated lacquer was lyophilized and ground, and then cryogenically stored at -80 ° C.

Example  6 to 8

The same procedure as in Example 1, except that Examples 2 to 4 were performed so that 2kGy, 5kGy and 10kGy per hour.

division
(Unit: kGy) Example 5 Example 6 Example 7 Example 8 Electron beam
Total absorbed dose One 2 5 10

Experimental Example  1: Electron ionized energy treated Lacquer Urushiol  Content measurement experiment

(1) gamma-rayed Lacquer Urushiol  Content measurement experiment

HLPC analysis was performed to determine the amount of urushiol remaining after treatment from each of the gamma-rayed lacquers prepared in Examples 1 to 4 above. In addition, lacquer leaves without gamma ray treatment were also subjected to HLPC analysis.

To this end, 10 g of the lacquered leaves treated with each of the electron ionization energy prepared in the above example was mixed with 50 ml of n -hexane ( n -hexane), and then shaken with the mixed solution, and the lacquer in the bark at 25 ° C. for 3 hours. crude urushiol) was extracted from the lacquer.

Next, the extract was concentrated with a rotary vaccum evaporator, and then the concentrate, in which n-hexane was completely evaporated, was dissolved in 10 ml of a 85% methanol solution, followed by a membrane filter (0.45 µm, manufactured by Chrom Tech). ), And then passed through a Sep-Pak (Sep-pak, C ₁₈ type, Waters) was used as a sample for HLPC analysis.

The HLPC analysis conditions were used by Sycam (Germany) pump (pump, S2100), autosampler (autosampler, S5200), column oven (Column oven, S4011), photodiode array UV detector (S3210). Then, the column (Column) was done using GROM's Grom-SIL 120 ODS-4 HE (4 × 250mm, id 5㎛), was used to attach the C ₁₈ guard column (C ₁₈ guard column). The analysis temperature was performed at 25 ° C., 85% methanol was used as the eluent, the flow rate of the eluent was 1 ml / min, and the injection volume was detected at 272 nm with 20 μl. Table 3, Figure 2 and shown in FIG. In FIG. 2, 1 is not gamma-ray-processed, and 2-5 each means Example 1-4. As can be seen from Figure 2, 3 and Table 3 it can be seen that the content of urushiol decreases as the total absorbed dose of gamma rays increases.

division Gamma rays
Total absorbed dose Of extract
Urushiol Content (mg%) From lacquer
Urushiol removal rate (%) - 0kGy 176.12 - Example 1 1kGy 19.11 89.2 Example 2 2kGy 17.58 90.0 Example 3 5kGy 15.96 90.9 Example 4 10kGy 7.83 95.6

(2) electron beam treatment Lacquer Urushiol  Content measurement experiment

The HLPC analysis was performed in the same manner as in (1) to determine the remaining urushiol content from each of the electron beam treated lacquer leaves prepared in Examples 5 to 8, and the results are shown in Tables 4, 4 and 5 below. Shown in In FIG. 4, 1 has not carried out the electron beam processing, and 2-5 each means Example 5-8. As can be seen from Figure 4, Figure 5 and Table 4 it can be seen that the content of urushiol decreases as the total absorbed dose of the electron beam increases.

division Electron beam
Total absorbed dose Of extract
Urushiol Content (mg%) From lacquer
Urushiol removal rate (%) - 0kGy 176.12 - Example 5 1kGy 48.72 70.3 Example 6 2kGy 45.49 74.2 Example 7 5kGy 27.18 84.6 Example 8 10kGy 19.95 88.7

Comparing Table 3 and Table 4, which are the results of the HLPC analysis experiment, when the same dose was irradiated, when comparing the removal rate of the urushiol of the gamma ray and the electron beam, the electron beam showed 89% of the removal rate at 10kGy, but the gamma ray was irradiated. When the gamma ray was treated with the removal rate of 96%, the removal rate was higher. This is considered to be because the transmittance of the electron beam is weaker than that of the gamma ray. The removal rate of urushiol was more than 15% when the low dose of 1kGy and 2kGy electrons and gamma rays were irradiated, respectively. When irradiated with high doses below, there was little difference between the electron beam and gamma ray treatment.

However, both gamma ray and electron beam treatment showed a very high urushiol removal efficiency of 70% or more, and in particular, as the total absorbed dose increased, the urushiol removal rate also increased significantly, and lacquer treatment using the electron ionization energy of the present invention. We can see that is very efficient.

Manufacturing example  1: gamma-irradiated Lacquer  Preparation of extract

(1) Preparation of Extract Using Water as Solvent

5 g of the gamma-rayed lacquer leaves prepared in Examples 1 to 4 were mixed in a flask equipped with a reflux condenser, and then extracted by hot water treatment at 100 ° C. for 5 hours, followed by filtering the extract to extract lacquer leaves. Each was prepared.

(2) Preparation of Extract Using Ethanol as Solvent

10 mg of gamma-rayed lacquer prepared in Examples 1 to 4 and 100 ml of ethanol (95%) were mixed in a flask equipped with a reflux condenser, and then extracted by applying heat at 80 ° C. for 3 hours. The extract was filtered to prepare lacquer extract.

Manufacturing example  2: electron beam treated Lacquer  Preparation of extract

(1) Preparation of Extract Using Water as Solvent

10 mg of the electron beam-treated lacquer leaves prepared in Examples 5 to 8 and 100 ml of water were mixed in a flask equipped with a reflux condenser, and then extracted by hot water treatment at 100 ° C. for 5 hours, followed by filtering the extracts. Each extract was prepared.

(2) Preparation of Extract Using Ethanol as Solvent

10 mg of the electron beam treated lacquer leaves prepared in Examples 5 to 8 and 100 ml of ethanol (95%) were mixed in a flask equipped with a reflux condenser, and then extracted by applying heat at 80 ° C. for 3 hours. The extract was filtered to prepare lacquer extract.

Experimental Example  2 : Lacquer  Determination of Total Polyphenol Content in Extracts

Polyphenol content of each gamma ray-treated lacquer extract prepared in Preparation Example 1 using water or ethanol was measured by adding 5 ml of sodium carbonate (Na ₂ CO ₃ , 2%) to 0.5 ml of each extract, followed by 2 minutes of mixing. After standing, 0.5 ml of 50% Folin-Ciocalteu's reagent was added thereto and left for 30 minutes, and then absorbance was measured at 750 nm. At this time, catechol was used to prepare the standard curve, and the polyphenol content was converted into the amount of catechol (mg / 100ml,%). The results are shown in Table 5 below. In addition, the polyphenol content of each of the electron beam treated lacquer extract prepared using Preparation Example 2 using water or ethanol was measured, and the results are shown in Table 6 below.

Gamma-ray total absorbed dose
(kGy) Total Polyphenol Content (mg%) Solvent (water) Solvent (ethanol) 0 63.56 78.5 One 21.47 44.43 2 18.41 44.20 5 22.10 39.67 10 20.42 26.17

Electron beam total absorbed dose
(kGy) Total Polyphenol Content (mg%) Solvent (water) Solvent (ethanol) 0 63.56 78.5 One 17.56 17.65 2 17.73 18.47 5 19.80 17.11 10 23.60 21.33

Looking at Table 5 and Table 6, the total polyphenol content of the lacquer extract without treatment with electron ionization energy showed a higher polyphenol content in the ethanol extract than the hydrothermal extract.

      In the case of the gamma-ray irradiated lacquer extract of Table 5, the total polyphenol content was reduced by more than 50%, and in the case of the lacquered extract treated with the electron beam of Table 6, the total polyphenol content was increased as the total electron beam absorbed dose was increased. Although this showed a slight increase, it can be seen that the total polyphenol content is reduced by more than 70%.

There was no difference in the total polyphenol contents in the hot water extracts when irradiated with gamma rays or electron beams. However, in the ethanol extracts, the lacquer leaves irradiated with gamma rays showed a higher polyphenol content than the lacquer leaves irradiated with electron beams. However, there was no proportional relationship between the electron ionization energy dose and the polyphenol content.

Experimental Example  3: Lacquer  Total flavonoids in the extract ( flavonoid ) Content measurement

In Preparation Example 1, the total flavonoid content of each gamma ray-treated lacquer extract prepared using water or ethanol was measured. That is, 10 ml of diethyleneglycol was added to 1 ml of the culture extract and mixed well, and 0.1 ml of 1N sodium hydroxide (NaOH, w / v) was mixed well and reacted in a water bath at 37 ° C. for 1 hour, followed by absorbance at 420 nm. Was measured. For the background experiment, instead of the sample solution, each extraction solvent was used. At this time, the routine was used to prepare the standard curve, and the total flavonoid content was converted into the amount of rutin (Rutin, mg / 100mL,%), and the results are shown in Table 7 below. It was.

In addition, the total flavonoid content of each of the electron beam treated lacquer extract prepared using Preparation Example 2 using water or ethanol was measured, and the results are shown in Table 8 below.

Gamma-ray total absorbed dose
(kGy) Total flavonoid content (mg%) Solvent (water) Solvent (ethanol) 0 330.1 807.5 One 205.5 513.3 2 228.7 584.6 5 216.6 577.2 10 204.5 538.2

Electron beam total absorbed dose
(kGy) Total flavonoid content (mg%) Solvent (water) Solvent (ethanol) 0 330.1 807.5 One 180.3 306.9 2 186.4 432.6 5 206.9 370.1 10 229.4 378.8

Looking at Table 7 and Table 8, the total flavonoid content of the lacquer leaf extract and the treated lacquer extract untreated with ionization energy showed a higher flavonoid content in the ethanol extract than the hot water extract.

It is known that lacquer leaves contain a lot of flavonoids, and the total content of flavonoids was reduced by 46% in hot water extracts and 62% in ethanol extracts when compared with lacquer extracts irradiated with gamma and electron beams. . In addition, the lacquer extracts irradiated with gamma rays and electron beams showed a similar tendency to decrease of polyphenols.

Experimental Example  4 : Lacquer  Antioxidant Activity of Extracts

In Preparation Example 1, the antioxidant activity of each gamma ray-treated lacquer extract prepared using water or ethanol was measured. That is, 800 μl of 100 mM Tris-HCl (pH 7.4) buffer solution was well mixed into 200 μl of each extract, and then 1 ml of 0.5 mM DPPH (2, 2-Diphenyl-1-picryhydrazyl) methanol solution was added thereto and mixed. Next, the reaction was left at 25 ° C. for 20 minutes, filtered (0.45 μm), and the absorbance was measured at 517 nm. At this time, the pure experiment was used for each solvent instead of the sample solution, and the antioxidant activity was expressed as the concentration of the sample (50% inhibition concentration, IC ₅₀ ) showing a 50% absorbance reduction for the blank experiment, the results are shown in Table 9 Shown in In addition, the antioxidant activity of each of gamma-ray-treated lacquer extracts prepared using water or ethanol in Preparation Example 2 was measured, and the results are shown in Table 10 below.

Gamma-ray total absorbed dose
(kGy) IC ₅₀ (μg / ml) Solvent (water) Solvent (ethanol) 0 26.89 13.34 One 45.07 23.75 2 50.99 24.29 5 43.90 26.93 10 64.27 32.01 BHA 14.09 BHT 83.62

Electron beam total absorbed dose
(kGy) IC ₅₀ (μg / ml) Solvent (water) Solvent (ethanol) 0 26.89 13.34 One 80.89 27.72 2 70.26 33.51 5 84.24 32.78 10 95.59 32.49 BHA 14.09 BHT 83.62

Looking at the antioxidant activity measurement results of Table 9 and Table 10, it can be seen that the antioxidant activity is much lower than the lacquer extract treated with electron ionization energy compared to the untreated lacquer extract. In addition, the antioxidant activity of the ethanol extract was better than that of the hot water extract, and when the gamma ray was irradiated rather than the electron beam irradiation, the radical scavenging ability was high.

Experimental Example  5: Lacquer  Measurement of Nitrite Scavenging Activity of Extracts

Nitrite scavenging activity of each gamma ray-treated lacquer extract prepared in Preparation Example 1 using water or ethanol was measured, and the results are shown in Table 11 below. In addition, the nitrite scavenging activity of each gamma ray-treated lacquer extract prepared in Preparation Example 2 using water or ethanol was measured, and the results are shown in Table 12 below.

The measuring method was to add 1 ml of extract to 2 ml of 1 mM sodium nitrate (NaNO ₂ ) solution, and then add pH of the reaction solution to 1.2, 3.0 and 5.0 using 0.1 N HCl and 0.2 M citric acid buffer, respectively. The reaction solution was adjusted to 10 ml. Next, the solution was reacted at 37 ° C. for 1 hour, and then 1 ml of each reaction solution was prepared, which was prepared with 5 ml of 2% acetic acid solution and 30% acetic acid, respectively, 1% sulfanilic acid and 1% naphthylamine ( Griess reagent prepared by mixing Naphthylamine in a 1: 1 volume ratio was prepared immediately before use, 0.4 ml was added and mixed well, and the mixture was left at room temperature for 15 minutes and absorbance was measured at 520 nm to calculate the amount of residual nitrite. In this case, 0.4 ml of distilled water was added instead of the Griess reagent, and the nitrite scavenging activity was expressed as the percentage of nitrite (%) when the compound was added or not.

menstruum Gamma-ray total absorbed dose (kGy) Nitrite scavenging activity (%) pH1.2 pH3.0 pH5.0 water 0 99.7 99.6 17.5 One 99.4 71.9 11.3 2 97.3 70.4 10.0 5 97.4 68.1 6.5 10 85.2 54.5 11.4 ethanol 0 100.3 N.D. 30.8 One 98.5 99.8 43.7 2 99.1 71.4 38.3 5 98.5 171.8 57.3 10 81.3 N.D. 55.2

menstruum Total absorbed dose of electron beam (kGy) Nitrite scavenging activity (%) pH1.2 pH3.0 pH5.0 water 0 99.7 99.6 17.5 One 97.3 60.5 11.8 2 99.8 73.6 10.2 5 99.7 74.0 16.9 10 99.1 76.8 13.5 ethanol 0 100.3 N.D. 30.8 One 88.3 N.D. 30.4 2 48.5 N.D. N.D. 5 43.9 N.D. N.D. 10 73.4 N.D. 17.3

The nitrite scavenging ability of the lacquer extract treated with electron ionization energy was slightly higher than that of ethanol extract in the hot water extract at pH 1.2, and all the samples showed high scavenging ability over 90% except the sample irradiated with gamma rays at 10 kGy. At pH 3.0, 54% to 76% of hot water extracts and almost no ethanol extracts were detected. At pH 5.0, nitrite scavenging activity was about 15% in hot water extracts, and ethanol extracts exhibited about 30% scavenging activity. . Irradiation of electron beams and gamma rays on lacquer leaves did not show any difference from irradiated extracts.

     Although there is a slight difference in each extract, it can be confirmed that most of the pH increases as the nitrite scavenging ability decreases as the pH is increased.

Nitrite, which is widely used for processing and storage of foods, especially as aquatic products or meat products, is used as a toxin production inhibitor, color developing agent, and anti-scattering agent itself.It is toxic and consumes more than a certain concentration, so that hemoglobin in blood is oxidized. Methemoglobin is formed to induce methemoglobinosis, and nitrites react with amines such as secondary and tertiary amines present in protein foods, pharmaceuticals and pesticides to produce nitrosamines (nitrosamines). It is reported. In addition, nitrosamine formation process is known to occur easily at low pH. The water extract of lacquer treated with electron ionization energy shows more than 90% removal at pH 1.2, similar to the pH condition in the stomach of the human body, thus providing effective nitrite scavenging activity in vivo. It is thought to inhibit the production of nitrosamine.

Experimental Example  6: Lacquer  Tyrosinase of extracts ( tyrosinase ) Inhibitory activity measurement

Tyrosinase inhibitory activity of each gamma ray-treated lacquer extract prepared using Preparation Example 1 using water or ethanol was measured, and the results are shown in Table 13 below. In addition, the tyrosinase inhibitory activity of each gamma ray-treated lacquer extract prepared using Preparation Example 2 using water or ethanol was measured, and the results are shown in Table 14 below.

The measurement method was a mushroom tyrosinase (110 units / ml) in a mixture of 0.2 ml of 0.175 M phosphate buffer (pH 6.8), 0.2 ml of 5 mM L-DOPA solution and 0.5 ml of extract sample solution, which had been pre-adjusted in a 35 ° C. water bath. , Sigma) 0.1 ml of the solution was added and reacted at 35 ° C. for 2 minutes, and the absorbance at 475 nm was measured (S _abs ), and the absorbance was measured by adding 0.1 ml of distilled water instead of the enzyme solution (B _abs ). Instead of the sample solution, 0.5 ml of distilled water was added, and the absorbance was measured using the value (C _abs ).

Gamma-ray total absorbed dose
(kGy) Tyrosinase inhibitory activity efficiency (%) Solvent (water) Solvent (ethanol) 0 91.4 46.1 One 53.6 90.6 2 56.9 67.2 5 58.2 64.8 10 66.6 62.5

Electron beam total absorbed dose
(kGy) Tyrosinase inhibitory activity efficiency (%) Solvent (water) Solvent (ethanol) 0 91.4 46.1 One 64.7 34.4 2 58.1 60.2 5 83.9 83.6 10 85.0 71.9

Looking at Table 13 and Table 14, the hydrothermal extract of lacquer leaves treated with electron beam or gamma-ray irradiation showed lower efficiency of tyrosinase inhibitory activity than the irradiated extract. However, it showed more than 50% tyrosinase inhibitory activity. Through this process, some of the tyrosinase inhibitory active substances are removed in the manufacturing process of the lacquer extract of the present invention, it can be confirmed that the effective range of tyrosinase inhibitory substances remain. In addition, it was confirmed that the tyrosinase inhibitory activity increased as the absorbed dose of the electron beam or gamma ray increased. Through this, it can be confirmed that the lacquer extract of the present invention has excellent tyrosinase activity inhibiting ability, and can be used as a product or a whitening product for preventing browning of food.

Experimental Example  7: Lacquer  Cytotoxicity of Extracts

      In order to verify the cytotoxicity of the hot water extract of lacquer treated with electron ionization energy, the cells were treated with a concentration of 50 µg / well in a normal cell line NIH3T3 for 48 hr, and then gamma or electron beams prepared in Preparation Examples 1 and 2, respectively. The cytotoxicity was measured by inoculating the lacquer extract treated with irradiation, and the results are shown in FIGS. 6 and 7, respectively.

Although no cytotoxicity was observed at 50 μg / well in the no-irradiation region of the electron beam (FIG. 7) and gamma ray (FIG. 6), the lactose extract treated with electron ionization energy confirmed about 10% cytotoxicity. Cytotoxicity was not observed in the lacquer leaf extracts irradiated with electron ionization energy, which may have the effect of cytotoxicity by gamma and electron beam irradiation.

Experimental Example  8 : Lacquer  Anticancer Activity of Extracts

A549 (lung cancer), AGS (stomach cancer), HT-29 (colon Cancer), HepG2 (liver cancer) cells were used for anticancer activity measurement experiments. In addition, as a control (labeled C in FIGS. 8 and 9), NIH3T3 cells, which are normal cells, were used, and anticancer activity was measured using a WST analyzer (Enhanced Cell viability Assay Kit, Daeil, Korea). 8 and 9 are shown.

Looking at the anti-cancer activity of the lacquer leaf extract prepared by using an ethanol solvent in the lacquer treated with electron beam irradiation as shown in FIG. Had no effect on growth. It can be seen that the untreated lacquer extract does not affect the growth rate of cancer cells. 8 and 9, gamma rays showed better growth inhibition rate in lung cancer cells (A549), but gastric cancer cells (AGS), liver cancer cells (HepG2), and colon cancer cells (HT-29) showed that cancer cell growth was observed. It was more effective at suppressing it.

The lacquer leaf extracts treated with gamma rays (FIG. 8) and electron beams (FIG. 9) showed superior anticancer activity in the three types of cancer cells except lung cancer cells, compared with the lacquer leaf extracts without electron ion energy treatment. Through this experiment, it can be seen that it can be used as a pharmaceutical composition such as anticancer agent of the lacquer extract of the present invention.

Experimental Example  9: Lacquer  Determination of Anti-inflammatory Activity of Extracts

Oxidation known to play an important role in inducing inflammation in order to measure the anti-inflammatory activity of urushiol-reduced lacquer extracts prepared using water (H ₂ O) solvents of Preparation Example 1 (1) and Preparation Example 2 (1) In order to measure the inhibitory effect of the production of nitrogen (nitric oxide, NO), WST assay was carried out to determine the cytotoxicity of RAW 264.7 mouse macrophage cells, the results are shown in Figs. It was.

       The cell viability of the lacquer leaf extract at 50 ㎍ / well was similar to that of the non-treated group, which did not show cytotoxicity. Was found to be independent of cytotoxic effects.

     Next, the effect of the lacquer extract of the present invention on the nitric oxide secreted by LPS-activated RAW 264.7 was tested using a Greries reagent, and the results are shown in FIGS. 12 and 13.

There was no difference in the NO inhibition rate of the lacquer extract treated with gamma rays (FIG. 12) and electron beams (FIG. 13). As the absorbed dose of gamma rays or electron beams increased, the NO inhibitory activity tended to increase.

The extracts of lacquer leaves irradiated with gamma rays and electron beams showed more than 50% NO inhibitory activity of 54-66% at a concentration of 50㎍ / well, which was slightly lower than that of 68% NO inhibitory activity. The results showed that the anti-inflammatory active substances were slightly reduced during the electron ionization energy treatment to reduce urushiol. In other words, the lacquer leaf extract of the present invention reduced the anti-inflammatory effect slightly than the non-electron ionized energy extract, but because the untreated extract contains a large amount of urushiol can not be used as an anti-inflammatory ingredient due to this side effect, the lacquer leaf of the present invention The extract has a very low urushiol content, so there are no side effects caused by this, and can be used as an anti-inflammatory ingredient.

Through the above examples and experimental examples, it was confirmed that by treating the lacquer with electron ionization energy proposed by the present invention, urushiol causing side effects such as allergic reactions can be effectively removed or reduced from the lacquer leaves. In addition, it was confirmed that the lacquer extract treated with electron ionization is excellent in antioxidant activity, anticancer effect, anti-inflammatory effect, etc. Through this, it was confirmed that the lacquer extract of the present invention can be used in various pharmaceutical compositions.

Claims (16)

delete delete delete delete delete delete delete delete Moisture treatment of the lacquer or lacquer; And
Treating the moisture-treated lacquer or lacquer with at least one electron ionization energy selected from gamma rays and electron beams; And
Boiling the mixture of the lacquer or lacquer and the solvent treated with the ionization energy, and filtering the insoluble material to produce an extract; Urushiol reduced and anti-inflammatory activity of the method Pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising the prepared lacquer extract. delete delete delete delete delete delete delete

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