KR20190065084A - pharmaceutical composition for antioxidant or antiinflammatory comprising extract of Lonicera caerulea L. var. edulis fruits - Google Patents

Abstract

The present invention relates to an antioxidant or anti-inflammatory pharmaceutical composition which comprises a lonicera caerulea L. var. edulis fruit enzyme extract having a liver protection effect from oxidative damage. According to the present invention, the enzyme is pectinase. The antioxidant or anti-inflammatory pharmaceutical composition further comprises at least one selected from the group consisting of chitosan and a guar gum.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for antioxidant or antiinflammatory activity, which comprises an extract of a buttock, and an antioxidant or an antiinflammatory comprising extract of Lonicera caerulea L. var. edulis fruits}

The present invention relates to a pharmaceutical composition for antioxidant and / or anti-inflammation comprising an enzyme extract of Staphylococcus aureus, a food composition, and a process for producing the enzyme extract of Staphylinobacterium.

The liver is a major metabolic organ responsible for various physiological functions including metabolism and utilization of nutrients, glucose homeostasis, detoxification, and plasma protein synthesis. Generally, liver damage is caused by exposure to a large number of toxic components from the environment, and can involve a variety of pathological changes ranging from the blood glass of mild transient liver enzymes to life-threatening liver fibrosis, cirrhosis, and liver cancer.

To date, various causes have been noted as the main cause of liver damage, and oxidative stress and inflammation are regarded as one of the most important causes. Accordingly, development of drugs capable of protecting the liver from damage has been continuously carried out, but it is still necessary to find drugs showing strong hepatic protective effects while having few side effects.

Among the drugs known to be effective against chronic hepatitis B, interferon was the most studied. The goal of antiviral therapy, including interferon, for chronic hepatitis B is to inhibit viral proliferation and to keep hepatitis activity low before the liver is at an unrecoverable level.

As a chronic hepatitis B treatment approved by the US Food and Drug Administration (FDA), lamivutin is unlike interferon, which has few side effects and is easy to use because it is taken orally. However, there are cases where the occurrence of medicinal-resistant virus reaches 50% within 3 years (14-32% of resistance incidence within 1 year, 60-70% in 5 years) I do not know much about how long it will help me.

In 2005 EnterCabier was developed and resistance rate dropped to 1.2%. However, if a patient with resistance to other drugs is switched to Enterobacter, the antiviral effect will be reduced and tolerance to Enterovir may occur.

In 2012, Tenofovir was developed and tolerance problems did not occur until now. However, adverse reactions include acid lactic acid, severe liver disease (death due to liver disease, liver hypertrophy, fatty liver), aggravation of hepatitis B infection The occurrence is known.

 Chronic hepatitis C has no symptoms and clinical course is mild, but some patients may develop liver cirrhosis or liver cancer with complications. However, there is no treatment available to eradicate the hepatitis C virus and it is difficult to expect it in the near future.

Currently, the search for liver protection drugs using natural products is actively under way.

Blue honeysuckle is a species of Lonicera caerulea var. It has been used as a traditional medicinal plant in northern Russia, China and Japan. It is a relatively uncommon medicinal fruit in North America and Europe. The nutrients are rich in ascorbic acid and phenolic components, and are rich in anthocyanins, flavonoids and low-molecular phenolic acids, which are known to have a relatively good antioxidant effect .

Recently, it has been known that the protective effect against the ionizing radiation by the oral administration of the soya nut is known through mouse experiment, and the therapeutic effect on lipid and glucose metabolism improving effect, liver protecting effect, anti-inflammatory effect and hypothyroidism is also known. It is known to exhibit the strongest antioxidative effect among colored berry species.

In addition, in vitro and in vivo experiments, the extracts of P. japonicus, which is rich in phenol components, have been shown to exhibit relatively strong antiinflammatory and wound healing promoting effects. Skin protection effects against UV rays are also well known, More detailed study is needed.

Accordingly, the present inventors have completed the present invention by confirming that Lonicera caerulea L. var. Edulis extract has antioxidative and anti-inflammatory effects.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for antioxidant and / or anti-inflammation which comprises an enzyme extract of Staphylococcus aureus.

Another object of the present invention is to provide an antioxidant and / or anti-inflammatory food composition containing an enzyme extract of Staphylococcus aureus.

Another object of the present invention is to provide a method for preparing an antioxidant and / or anti-inflammatory pharmaceutical composition comprising an enzyme extract of Staphylococcus aureus.

The present invention relates to a pharmaceutical composition for antioxidant or antiinflammatory action comprising an enzyme extract of royal jelly, and a method for producing the same.

Hereinafter, the present invention will be described in more detail.

As used herein, the term " shallow tree " refers to a shrub with an iris, 1 to 2 meters high, and the leaves are opposite, oval or oblong. In May and June, white flowers bloom on the axil and the fruit is harvested with black berries. It is distributed in Korea, Sakhalin, Manchuria, Tibet, etc. It picks up fruit in the scarf and eats it, it is made into the juice, and sometimes it drinks. Pharmacological actions are known to be effective for astringent, antimicrobial, diuretic, hypertension, stomach diseases or liver diseases. The main ingredients are bioflavonoid, vitamin B3, betaine ), Catechine, and anthocyanin.

One example of the present invention relates to a pharmaceutical composition for antioxidant or anti-inflammation comprising Lonicera caerulea L. var. Edulis fruit extract.

The enzyme may be, but is not limited to, pectinase.

The farinaceous enzyme extract may be in the form of a solution, a concentrate or a powder.

The pharmaceutical composition may further comprise one or more selected from the group consisting of chitosan and guar gum.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition comprising a pharmaceutically effective amount of an enzyme extract of Nutmeg variety and / or a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve efficacy or activity of the enzyme extract of the above-described royal jellyfish.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical compositions according to the present invention can be administered to mammals, including humans, in a variety of routes. The mode of administration may be any conventional manner and may be administered, for example, by oral, skin, intravenous, intramuscular, subcutaneous, and the like routes, preferably orally.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 100 to 400 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets, capsules or gels (e.g., hydrogels), and may additionally contain dispersing or stabilizing agents .

Another example of the present invention relates to an antioxidant and / or anti-inflammatory food composition comprising a Lonicera caerulea L. var. Edulis fruit enzyme extract.

The farinaceous enzyme extract may be in the form of a solution, a concentrate or a powder.

The food composition may additionally comprise chitosan and / or guar gum.

The content of the enzyme extract of P. japonicus as an active ingredient contained in the food composition is not particularly limited depending on the form of the food, the intended use, etc., and may be, for example, 0.01 to 15% , And the health beverage composition may be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

When the food is a beverage, there is no particular restriction on the liquid ingredient besides the isotonic acid extract as an essential ingredient at the indicated ratio, and various flavors or natural carbohydrates, such as ordinary beverages, .

The natural carbohydrate may be selected from the group consisting of monosaccharides such as disaccharides such as glucose and fructose such as maltose and sucrose and polysaccharides such as dextrin and cyclodextrin, , Sorbitol, and erythritol.

Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, A salt thereof, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like.

In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Another example of the present invention relates to a method for preparing a royal jellyfish enzyme extract comprising the step of contacting an enzyme to a Lonicera caerulea L. var. Edulis fruit.

The enzyme may be, but is not limited to, pectinase.

The contacting of the enzyme may be performed for 2 to 2.5 hours, but is not limited thereto.

The method for preparing the pharmaceutical composition may further include adding at least one selected from the group consisting of chitosan and guar gum.

As a specific example, the manufacturing method may include the following steps.

A preparation step of heating and crushing the Lonicera caerulea L. var. Edulis fruit at 40 to 60 캜;

An enzyme treatment step in which an enzyme is contacted with a nut of a houseplant so that it is treated for 1 to 5 hours;

A precipitate removing step for removing the precipitate from the enzyme-treated soya nut;

A second heating step of heating the soymilk removed from the sediment to 60 to 80 캜;

Adding an additive to each of the at least one selected from the group consisting of chitosan and guar gum in an amount of 0.001 to 0.1 wt.

The heating in the preparation step may be performed at 45 to 55 占 폚, but is not limited thereto.

The enzyme may be, but is not limited to, pectinase.

The pectinase may be 0.01 to 1.0 wt%, but is not limited thereto.

The enzyme treatment step may be performed for 2 to 2.5 hours, but is not limited thereto.

The precipitate removal step may be performed by centrifugation, but is not limited thereto.

But are not limited to, 0.001 to 0.1% by weight, 0.001 to 0.01% by weight, 0.001 to 0.005% by weight or 0.005 to 0.1% by weight, for example, 0.005 to 0.01% by weight of the chitosan or guar gum.

The present invention relates to a pharmaceutical composition for antioxidant and / or anti-inflammation comprising an enzyme extract of Staphylococcus aureus, a food composition, and a method for producing an enzyme extract of the above-described Stable nuts. have.

FIG. 1 is a standard curve of an indicator substance of an enzyme extract of Staphylococcus aureus according to an embodiment of the present invention.
2 is a graph showing the DPPH scavenging ability of the hot-water extract and the enzyme extract of P. thunbergii according to an embodiment of the present invention.
FIG. 3 is a graph showing the HepG2 cell safety (MTT assay) of the hot-water extract and the enzyme extract of Staphylococcus aureus according to an embodiment of the present invention.
4 is a graph showing the effect of Nrf2-dependent antioxidant transcription factor (hHO-1, hGCLC, hNQO-1) on the expression of Nutrient Fruit Extract according to an embodiment of the present invention.
FIG. 5 is a graph showing an effect of increasing the antioxidant enzymes of the royal jelly extract according to an embodiment of the present invention.
FIG. 6 is a graph showing the anti-inflammatory effect (inhibitory effect of NO production on LPS-induced inflammation) of Thymus nuts extract according to an embodiment of the present invention.
FIG. 7 is a graph showing the cytoprotective effect of LPS-induced cell damage of the extract of Staphylococcus aureus according to an embodiment of the present invention.
FIG. 8 is a graph showing the AST and ALT measurement results of Sweet Potato fruit extract according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Production example. Cell culture

Human hepatocyte hepatocyte replacement cells, HepG2 cells, and mouse-derived macrophage replacement cells, Raw264.7 cells, were obtained from ATCC (Rockville, Md., USA). Recombinant HepG2 cells stably expressing ARE-luciferase were prepared according to established methods (Moon, SY, Lee, JH, Cho, IJ, Kim, YW, stress via activation of the extracellular signal-regulated kinase / NF-E2-related factor 2 pathway. Biol Pharm Bull. 2014; 37: 1633-40).

Specifically, the ARE reporter gene pGL4.37 [luc2P / ARE / Hygro] (Promega, Madison, Wis., USA) was introduced into HepG2 cells using Fugene HD transfection reagent (Promega) Cells with antibiotic resistance were selected using hygromycin and used for reporter gene analysis. Cells were cultured in a 6-well plate at a density of 1 × 10 5 cells / cell to 70-80% of the cultured cell surface. All cells were cultured in a 10% FBS (fetal bovine serum), 50 units / mL penicillin, / mL Streptomycin-containing DMEM (Dulbecco's modified Eagle's medium) at 37 ° C and 5% CO2.

Comparative Example. Production of hot-water extract of nutshell (BHw)

100 g of the dried powder of royal jellyfish was added to 1 liter of distilled water, stirred well and then refluxed for 3 hours at an extraction temperature of 90 to 95, and the filtrate was separated. Then, the extract was concentrated under reduced pressure at 55 to 65, 10 to 50 mmHg, and 5 to 6 hours. Then, it was lyophilized at -40 캜, 5 to 10 mmHg for 48 hours to obtain 20.8 g of dried powder.

Examples. Production of Enzyme Extract of Butterfly Nuts (BHe)

The soymilk was heated at 45 to 55 ° C for 3 minutes, pulverized by a grinder, and treated with 0.05% by weight of pectinase (Weissbiotch, Germany) in the nutshell. Then, the skin and seeds were removed by continuous centrifugation at 4,000 rpm, and chitosan and guar gum were added to the centrifugation liquid at a ratio of 0.005%, respectively. Then, the resultant was filtered through a filter press at 1500 rpm / min and concentrated under reduced pressure at 65 ° C for 1 minute under conditions of 50 ° C and 0.092 MPa. Then, it was sterilized at 90 to 95 캜 for 15 to 30 seconds, and lyophilized at -40 캜, 5 to 10 mmHg, for 48 hours to obtain a powder of a crude thyme nutrient enzyme extract (yield: 16.5%).

Experimental Example 1. Analysis of Cyanidine-3-Glucoside Content

The sample (250 mg) was precisely weighed into a 10-mL volumetric flask and homogenously shaken to dissolve in a mobile phase, which was then filtered through a 0.45-μm syringe filter. The analysis conditions are shown in Tables 1 and 2 below, and the results are shown in FIG. 1 and Table 3.

[Equation 1]

C3G content (ug / mg) = (concentration value of test solution X total amount of test solution) / sample weight

column Shiseido, C18, 4.6 mm x 250 mm, 5 m Detector UV 516 nm Column temperature 30 ℃ Dose 10 μl Mobile phase condition - Mobile phase A: 5% Formic acid in H2O
- Mobile phase B: Acetonitrile
- Flow rate: 1.0 mL / min

time
(minute) Mobile phase A
(vol%) Mobile phase B
(vol%) 0 90 10 15 90 10 16 0 100 22 0 100 23 90 10 30 90 10

Sample
(Amount taken) No. Peak area Con.
(ug / mL) Average
(ug / mL) RSD
(%) content
(ug / mg) content
(%) Average content
(%) Test_1
(260.8 mg) One 1081.92261 34.98754 35.09986 0.29 1.3459 0.13 0.13 2 1088.09692 35.18515 3 1086.27673 35.12690 Test_2
(246.5 mg) One 1012.71759 32.77255 32.73258 0.13 1.3279 0.13 2 1010.05933 32.68747 3 1011.62946 32.73772 Test_3
(253.0 mg) One 1034.12317 33.45766 33.65662 0.52 1.3303 0.13 2 1042.39880 33.72253 3 1044.49597 33.78965

As shown in FIG. 1 and Table 3, it was confirmed that the content of cyanidine-3-glucoside was 1.32 to 1.34 mg / g.

Experimental Example 2. Measurement of DPPH radical scavenging ability

DPPH radical scavenging activity was measured according to the method of Kim et al. (2003) (Kim DO, Chun OK, Kim YJ, Moon HY, Lee CY. Quantification of polyphenols and their antioxidant capacity in fresh plums. : 6509-15.)

Specifically, the DPPH solution diluted to 150 M in ethanol was reacted with 100, 300 ug / mL BHw, BHe or 100 uM trolox solution for 30 minutes at room temperature and absorbance was measured at 570 nm using an automated microplate reader The results are shown in FIG. 2 and Table 4.

[Equation 2]

Radical Scavenging Activity (%) = [1- (SS ₀ ) / (CC ₀ )] X 100

S and S0 represent the absorbance of a DPPH solution with or without a sample. C and CO represent the absorbance of a solvent with or without a sample.

DPPH scavenging activity (%) Control BHw (ug / ml) BHe (ug / ml) 100 300 100 300 0 ± 2.846 33.907 ± 5.881 ** 86.053 ± 2.197 ** 42.706 ± 6.100 ** 92.961 ± 2.193 **

BHw: Hot Water Extract of Swallowtail

BHe: Enzymatic Extract of Butterfly Nut

* P <0.05 compared with control

** P <0.01 compared with control

As shown in FIG. 2 and Table 4, the radical scavenging activity of DPHH was increased in a concentration dependent manner at 100 and 300 mg / mL of BHw and BHe, respectively. Compared with the conventional hot water extract (BHw) , And 20.6% and 7.4%, respectively. The radical scavenging activity by BHw at 100 and 300 ug / mL was 33.91 ± 5.88 and 86.91 ± 2.20%, respectively. The radical scavenging activities by BHe at 100 and 300 ug / mL were 42.71 ± 6.10 and 92.96 ± 2.19%, respectively.

These results indicate that the extracts of DPPH showed higher antioxidant activity than DPPH extracts at 100 and 300 ug / ml concentration.

Experimental Example 3. Measurement of cell viability

HepG2 or Raw264.7 cells were cultured in a 24-well plate at a concentration of ⁵ × 10 ⁴ , depleted of serum for 12 hours, and then treated with 10-300 ug / mL of BHw and BHe for 24 hours. HepG2 cells were pretreated with 10-300 ug / mL of BHw and BHe for 1 h and then treated with 150 uM t -BHP for 12 h to evaluate the cytoprotective effect against t- BHP-induced oxidative stress. Raw 264.7 cells were pretreated with 10-300 ug / mL of BHw and BHe for 1 hour and then treated with 1 ug / mL LPS for 18 hours to evaluate the cytoprotective effect against LPS induced stress. The resulting formazan crystals were dissolved in 200 dimethylsulfoxide, and the absorbance was measured using an automated microplate reader at 570 nm to calculate the cell survival rate. The results are shown in FIGS. 3 and 5 Respectively.

[Equation 3]

Relative Cell Viability (% of control) = (absorbance of treated sample) / (absorbance of control) X 100

HepG2 cell viability Control 100 ± 6.647 BH dose (ug / ml) 3 10 30 100 300 BHw 110.811
± 3.478 110.849
± 1.769 110.250
± 3.886 105.385
± 4.656 97.206
± 13.973 BHe 99.260
± 3.862 97.866
± 6.691 101.517
± 2.292 100.100
± 4.217 100.177
± 3.827

BHw: Hot Water Extract of Swallowtail

BHe: Enzymatic Extract of Butterfly Nut

As can be seen from FIG. 3 and Table 5, no change in cell viability by BH treatment was observed compared to the control cells. Cell viability by BHw at 3, 10, 30, 100, and 300 ug / mL was 110.81 ± 3.48, 110.85 ± 1.77, 110.25 ± 3.89, 105.39 ± 4.66, and 97.21 ± 13.97% 100, and 300 ug / mL of BHe were 99.26 ± 3.86, 97.87 ± 6.69, 101.52 ± 2.29, 100.10 ± 4.22, and 97.21 ± 13.97%, respectively.

This indicates that the nutrient extract is safe without toxic effects on the cells. It is generally considered that changes of about 10% are safe. BHw showed a change of about 10%, but BHe could be judged to be safer with a change of less than 3%.

Experimental Example 5. Separation of Total RNA and Real-time PCR Analysis

Of total RNA from the treated cells Trizol reagent (Invitrogen, Carlsbad, CA, USA) utilized was isolated according to the manufacturer's method, a cDNA by reverse transcription of RNA of 2 ug using an oligo-d (T) ₁₆ primer a . Real-time PCR was performed on Bio-Rad's CFX96 thermal cycler using HO-1, GCLC, and NQO-1 specific primers and SyBr Green Ex-Taq (Takara, Shiga, Japan) and glyceraldehyde-3-phosphate and dehydrogenase gene. The specificity of the reaction was verified by melting curve analysis after the PCR reaction, and the results are shown in FIG. 4 and Table 6.

Nr2 dependent antioxidant gene expression hNQO1 hHO-1 hGCLC Control 1 ± 0.000 1 ± 0.000 1 ± 0.000 BHw 1.467 ± .410 1.197 + - 0.237 1.340 0.209 BHe 2.514 + - 0.349 * 1.779 ± 0.065 ** 1.452 + - 0.223 *

BHw: Hot Water Extract of Swallowtail

BHe: Enzymatic Extract of Butterfly Nut

* P <0.05 compared with control

** P <0.01 compared with control

As shown in FIG. 4 and Table 6, the antioxidative effect of Nrf2-dependent antioxidant transcription factors hHQO1, hHO-1, and hGCLC increased by dose-dependent increase of antioxidant effect. BHe extract by enzyme extraction could have a higher antioxidant effect by increasing the expression of antioxidant transcription factor than the conventional extraction method (BHw).

Experimental Example 6. Analysis of SOD and CAT activity

The activity of SOD and CAT in HepG2 cells treated with 300 ug / mL of BHw and BHe for 24 hours was measured by using a kit of Cayman Chemical Co. (Ann Arbor, MI, USA) 5 and Table 7.

CAT 1 U was defined as the enzyme activity capable of degrading 1 mmol of H 2 O 2 and SOD 1 U was defined as the amount of enzyme used to remove 50% of superoxide radicals.

SOD activity (U / mg protein) CAT activity (uM / mg protein) control 16.547 ± 2.689 347.727 ± 125.208 BHw 13.736 ± 6.834 506.491 + - 145.696 BHe 33.772 ± 6.868 ** 802.154 ± 139.135 **

** P <0.01 compared with control

As shown in FIGS. 5 and 7, SOD activity was increased by BHw and BHe, and CAT activity was increased by both BHw and BHe. Only the increase by BHe was statistically significant (Figure 6). The SOD activity of the control cells and the BHw and BHe treatment at 300 ug / mL were 16.55 ± 2.69, 13.74 ± 6.83, and 33.77 ± 6.87 U / mg of protein, respectively. The BHw and BHe treatments of control cells and 300 ug / SOD activity was 347.73 ± 125.21, 506.49 ± 145.70, and 802.15 ± 139.14 uM / mg of protein, respectively.

The extracts of the soya nutrients increased the antioxidant activity of SOD and catalase. BHw extract showed a decrease in SOD activity compared to control, but BHe showed a significant increase in SOD activity compared to control, indicating that BHe had a better SOD activity than BHw.

BHw extract increased catalase activity, but there was no significant difference. However, the BHe extract showed a significant increase in catalase activity compared to the control, indicating that BHe had better catalase activity than BHw.

Experimental Example 7. Measurement of NO production amount

Raw264.7 cells were pretreated with 100, 300 ug / mL of BHw and BHe for 1 hour, treated with 1 ug / mL of LPS for 18 hours, and the same amount of Griess reagent was added to the recovered culture. An automated microplate reader , And the NO production amount was measured. The results are shown in Figs. 6 to 7 and Tables 8 to 9.

NO production (fold) control LPS BHw (ug / ml) BHe (ug / ml) 100 300 100 300 1 ± 0.003 2.189 + - 0.193 ** 2.285 + 0.142 2.021 ± 0.158 2.021 ± 0.158 ## 1.650 0.073 ##

^** P <0.01 compared with control

^# P <0.01 compared with LPS control, ^# P <0.05 compared with LPS control

NO production (relative) control LPS BHw (ug / ml) BHe (ug / ml) 100 300 100 300 100
± 2.231 54.476
± 1.920 ** 94.388
± 6.653 90.142
± 3.172 ## 106.310
± 1.298 ## 106.943
± 4.624 ##

^** P <0.01 compared with control

^# P <0.01 compared with LPS control, ^# P <0.05 compared with LPS control

As shown in FIGS. 6 to 7 and Tables 8 to 9, LPS significantly increased NO production by about 2.19 times compared with control cells, and BHe pretreatment inhibited NO production in a concentration-dependent manner. NO production by BHw pretreatment at 100 and 300 ug / mL was 2.29 ± 0.14 and 2.02 ± 0.16 fold, respectively, compared to the control cells. NO production by BHe pre-treatment at 100 and 300 ug / 1.75 + 0.13, and 1.65 + 0.07, respectively.

LPS-induced NO production tended to decrease in BHw, but BHe showed a dose-dependent decrease, suggesting that BHe inhibits NO production (anti-inflammatory effect).

LPS significantly inhibited cell viability by about 54.48% compared with control cells, and BHw and BHe pretreatment increased cell viability. The cell viability by BHw pretreatment at 100 and 300 ug / mL was 94.39 ± 6.65 and 90.14 ± 3.17%, respectively, compared to the control cells. The cell survival rates by BHe pretreatment of 100 and 300 ug / mL were 106.31 ± 1.30, 106.94 + 4.62%.

In LPS-induced cytotoxicity (approximately 55% of deaths), BHw exhibited a dose-independent cytoprotective effect, but BHe showed a significant dose-dependent cytoprotective effect and protected the cells closer to the normal state than BHw And showed good cell protection effect.

Experimental Example 8. Measurement of liver protection effect

The liver protection effect was measured from the oxidative damage of the extracts of P. thunbergii after the oral administration of extract powder (BHw, enzyme extract: BHe) for 7 days and the last day of CCl4 administration.

Experimental group

Normal control group: Olive oil (solvent) group after sterilized water.

CCl4 Control group: 0.5 ml / kg of CCl4 after sterile distilled water administration.

silymarin treated group: 0.5 mg / kg of CCl4 after administration of silymarin 100 mg / kg.

BHw 200mg / kg administration group: BHw 200mg / kg administration and CCl4 0.5ml / kg administration group.

BHe 200mg / kg administration group: BHe 200mg / kg administration and CCl4 0.5ml / kg administration group.

Body weight

groups Last 7th test substance administration 24 hrs after CCl4 treatment [B] * Intact   36.14 + 1.14   33.72 + 1.46 CCl4   36.05 ± 1.34   31.04 1.57a Silymarin   36.18 + - 1.27   32.79 ± 1.62d BHw   36.19 + 1.30   32.60 ± 1.28 d BHe   36.18 ± 1.67   33.12 ± 1.88 c

a p <0.01 and b p <0.05 compared with intact control by LSD test

c p <0.01 and d p <0.05 as compared with CCl4 control by LSD test

There was a significant weight loss in the CCl4 control group compared to the intact control group. In the silymarin control group and BHw BHe group (BHw BHe), significant weight loss inhibition effect was shown as compared with CCl4, . Especially, weight loss inhibition effect was the best in BHe.

Items (Unit)

Groups Lipid Peroxidation (nM of MDA / mg protein) GSH Contents (nM / mg protein) Enzyme Activity SOD (U / mg protein) CAT (U / mg protein) Intact   1.49 ± 0.86  38.48 + - 10.33 418.83 + - 135.08 242.68 ± 102.48 CCl4   8.31 + 1.44a   3.78 ± 1.21 c  58.76 ± 27.74 c  45.60 ± 18.65 c Silymarin    5.00 ± 1.13ab  17.59 ± 3.71 cd 184.74 ± 72.99 cd 121.85 ± 22.91 cd BHw   6.05 ± 1.10ab  10.74 ± 2.89 cd 126.68 ± 35.63 cd  88.15 + - 21.04 cd BHe   4.45 ± 1.45ab  21.41 ± 5.08 cd 223.66 +/- 55.75 cd 133.21 ± 20.74 cd

The lipid peroxidation by CCl4 was significantly inhibited by silymarin and the test substance. In the group of BHw and BHe treated with the extract of Nodong fruit, the inhibition of lipid peroxidation and the increase of antioxidant enzyme appeared. BHe extract inhibited lipid peroxidation by -26.44% more than BHw extract. In addition, BHe extract increased the antioxidative enzymes GSH, SOD and catalase activities by 99.35, 76.56, and 51.12%, respectively, compared with BHw extract. Therefore, the BHe extract showed better lipid peroxidation inhibition and antioxidant effect than the conventional BHw extract.

Items (Unit)


Groups General histomorphometry Histological Activity Index
(Scores; Max = 10) Percentages of degenerative regions (% / mm2) Numbers of degenerative hepatocytes (cells / 1000 hepatocytes) Numbers of inflammatory cells infiltrated (cells / mm2) Intact 0.40 + - 0.52 2.53 + - 1.95  29.60 ± 19.41  43.40 ± 16.60 CCl4 8.40 ± 1.07a 79.82 ± 10.06 c 809.50 ± 100.54c 269.10 + - 74.04 c Silymarin 4.40 + 0.84ab 44.22 ± 11.44 cd 446.90 ± 108.91 cd 73.50 ± 15.86 cd BHw 5.00 ± 1.33ab 54.92 ± 12.47 cd 563.30 ± 136.44 cd 102.20 ± 25.42 cd BHe 3.50 ± 1.27ab 38.32 + - 11.02 cd 407.40 ± 121.06 cd 62.40 ± 23.80 d

Histological examination of the liver revealed that the degenerative area ratio, degenerative hepatocyte count, and inflammatory infiltration cell number were increased in the CCl4 group compared to the normal control group, but these degenerative and degenerative indexes were significantly decreased in the silymarin and test substance treated groups. BHe has higher histological activity idesx, percentage of degenerative regions, Numbers of degenerative hepatocytes, and numbers of inflammatory cells infiltrated by BHw compared to BHw -30. -30.23, -27.68, and -38.94%, respectively. Therefore, BHe extracts can protect liver vesicle damage by CCL4 more effectively than BHw extracts.

Items (Unit)

Groups Positive cells by immunohistochemistry (cells / 1000 hepatocytes) Cleaved caspase-3 Cleaved Poly (ADP-ribose) polymerase Nitrotyrosine 4-Hydroxynonenal Intact 2.00 ± 1.41 4.10 ± 2.88 17.20 ± 10.12 15.80 ± 8.38 CCl4 718.20 ± 108.68a 714.40 ± 111.79 c 707.10 + - 101.38 c 757.10 ± 120.07 c Silymarin 383.00 + - 104.97ab 288.80 ± 64.00 cd 331.50 ± 115.94 cd 440.20 ± 135.73 cd BHw 488.90 ± 119.27ab 470.40 ± 126.21 cd 486.90 ± 108.44 cd 535.70 ± 107.30 cd BHe 310.10 ± 111.16ab 196.90 ± 62.99 cd 291.60 ± 69.14 cd 264.10 ± 70.92 cd

The cleaved caspase-3 (cleaved poly (ADP-ribose) polymerase) was increased by CCl4, but cell death was decreased by silymarin and test substance. In addition, nitrotyrosine and 4-hydroxynonenal, which are indicators of lipid oxidation by CCl4, increased, but lipid oxidation indexes decreased by silymarin and test substance. BHe extract reduced the levels of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase, nitrotyrosine, and 4-hydroxynonenal by -36.57, -58.14, -40.11 and -50.70%, respectively. Therefore, BHe extracts showed better hepatocyte protective effect by effectively reducing the cell death index and lipid oxidation indexes as compared with the BHw extract.

The AST and ALT levels of the liver were higher in the CCl4 group, but the AST and ALT levels were significantly lower in the silymarin and test substance groups. In the BHe extract administration group, AST was -23.2% and ALT was -65.1% lower than BHw extract administration group. Therefore, it can be seen that the BHe extract shows a more effective liver protection effect than the BHw extract.

Statistical analysis

All data were expressed as mean and standard deviation from three experiments. Multiple comparison tests were performed for groups with different doses. The equilibrium test was performed using the Levene test and the least-significant differences (LSD) multiple comparisons were used for group comparisons. If the equilibrium is not satisfied, the Kruska-Wallis H test, a nonparametric test, was performed with the Levene test.

Dunnett's tests were performed to determine specific pairs between groups when there were significant differences in the Kruskal-Wallis H test. Differences were considered significant at p <0.05 level. Statistical analysis was performed using SPSS for Windows (14.0K, SPSS Inc., Chicago, IL, USA).

Claims (10)

An antioxidant or anti-inflammatory pharmaceutical composition comprising Lonicera caerulea L. var. Edulis fruit extract. 2. The pharmaceutical composition according to claim 1, wherein the enzyme is pectinase. The pharmaceutical composition according to claim 1, wherein the composition further comprises at least one selected from the group consisting of chitosan and guar gum. An antioxidant or anti-inflammatory food composition comprising Lonicera caerulea L. var. Edulis fruit extract. 5. The food composition of claim 4, wherein the enzyme is pectinase. The food composition according to claim 4, wherein the composition further comprises at least one selected from the group consisting of chitosan and guar gum. A method for preparing a royal jellyfish enzyme extract comprising the step of contacting an enzyme to a fruit of Lonicera caerulea L. var. Edulis. 8. The method according to claim 7, wherein the enzyme is pectinase. The method according to claim 7, wherein the method further comprises adding at least one selected from the group consisting of chitosan and guar gum. 8. The process according to claim 7, wherein the enzyme is 0.01 to 1.0% by weight, based on palm kernel.

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