KR101773322B1 - PHARMACEUTICAL COMPOSITION CONTAINING ELAEAGNUS MULTRIFLORA Thunb. EXTRACT FOR IMMUNE-ENHANCING - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing an Elaeagnus multiflora leaf extract as an active ingredient for immune enhancement, and provides a pharmaceutical composition for immune enhancement or a functional food composition, capable of enhancing immunity of a living body by inducing cytokine production of IL-2 (Interlukin-2), IL-4 (Interlukine-4), or IL-10 (Interlukine-10) and by activating immune cells which directly counter tumor cells.

Description

[0001] PHARMACEUTICAL COMPOSITION CONTAINING ELAEAGNUS MULTRIFLORA [0002] Thunb. EXTRACT FOR IMMUNE-ENHANCING}

The present invention relates to a composition for enhancing an immune response comprising an extract of Valium Borealis as an active ingredient and more specifically to a composition for preventing and treating diseases caused by toxicity and side effects using a natural extract of Elaeagnus multriflora Thunb. To a pharmaceutical composition for enhancing immunity.

While recent economic developments have led many people to enjoy abundant life, westernization of diet and decrease in activity have put them at risk for weight gain, atherosclerosis, hypertension and diabetes. These chronic degenerative diseases are recognized as a serious problem not only because of the rapid increase in the number of patients but also because the age of disease is gradually lowered.

In addition, many chemicals that have been created to enhance the quality of life of humans have reduced immunity and have caused unexpected diseases. As a result, there is an increasing tendency to discover new materials or discover their pharmacological effects by approaching new foods or ingredients that are traditionally found beneficial to our body. Until now, there have been an increasing number of cases of finding anticancer substances from natural products, which are simply known as ornamental plants, or developing therapeutic agents.

Elaeagnus multriflora Thunb., On the other hand, is known as a garden that is planted in the garden and eats its fruit. The garden borealum is an Elaeagnus plant which is native to various parts of Korea. It is known as a yamanda (桜 桃) or an ape (四月 子). It is also often called house borealis and common boreal trees, and young branches are covered with reddish brown scaly fur.

It looks like a bamboo tree, but it is called a garden bamboo tree because it grows because its fruit is large. When it is used as a medicine, it is mainly used as a bath. Leaves are alternate long oval with length of 3 ~ 10cm. Both ends are narrow and sharp, with no sawtooth on edge. On the front, there are scaly hairs at the time of childhood, but gradually disappear, and the back side is characterized by a mixture of white scaly hairs and brown scaly hairs. Flowers bloom in April ~ May with light yellow color, and 1 ~ 2 are on axilla. Flowers have white and brown scales hairs. The bottom of the calyx tube is rapidly narrowed and surrounds the ovary. The end is divided into 4 pieces. There are 4 stamens and 1 pistil. In July, a long elliptical nucleus with a length of 1.5cm is hanging down and ripen red. The water pipe is a deciduous form and grows up to 2 ~ 3m in height.

It is similar in appearance to Elaeagnus umbellata Thunberg, but the tree is distinguishable from the garden tree by its small twigs with white scaly hairs and its fruit with a length of less than 1 cm.

Recently, we have been studying the efficacy of natural herbs such as yew tree, which is traditionally known, and have tried to develop substances that have little side effects and are useful for prevention and recovery of diseases. However, the nutritional value, There is a lack of relevant information on active functionality.

Korean Patent Laid-Open Publication No. 10-2016-0012540 discloses a method for producing a fermented acetic acid composition using a vitamin tree and a fermented acetic acid composition obtained therefrom (1) including cereals, vitamin leaf extract, koji, modified koji and water Comprising the step of fermenting a second mixture comprising an alcoholic fermentation product obtained in the step of fermenting the first mixture with alcohol, a herbicide and a vitamin Nut fruit juice, wherein the content of the vitamin tree leaf extract contained in the first mixture is in the range And 2% by weight based on 100% by weight of the second mixture, and the content of vitamin Nut fruit juice contained in the second mixture is 6% by volume based on 100% by volume of the total amount of the second mixture. And a fermented acetic acid composition obtained therefrom. Korean Patent Laid-Open Publication No. 10-2014-0140902 discloses that the extract of Sadukazu Fruit has an effect of inhibiting liver damage induced by alcohol and promoting decomposition of acetaldehyde, which is a hangover inducing substance caused by alcohol metabolism, Compositions are disclosed for pharmaceutical compositions for inhibiting liver damage or hangover containing diatomaceae fruit extract which can be used as a composition for preventing alcoholic liver damage or hangover relief. Korean Patent No. 10-1220519 discloses a method for inhibiting the signal transduction system specifically induced by IL-6 by including as an active ingredient quercetin 3-glucoside-7- Thereby preventing and treating diseases mediated by IL-6, as well as a composition capable of preventing and treating infectious diseases of rhinovirus. In Korean Patent No. 10-1276141, diatomaceous earth extract is added to diatomaceous earth aloe extract of algae, and after storing diatomaceous earth at low temperature, it is filtered to effectively remove substances having low lipid and solubility and effectively remove , Extracts with low lipid content and low solubility do not remain, so they do not form sediments over time, and have excellent antioxidative and antimicrobial activities. They are useful as antioxidants or medicines for preventing skin aging, cosmetics and health foods. The present invention relates to a method for preparing a seaweed extract, and a process for producing the same. However, these prior art documents have shown that induction of cytokine production by IL-2 (Interlukin-2), IL-4 (Interlukine-4) or IL-10 (Interlukine-5) of the present invention, The present invention is different from the composition and effect of the pharmaceutical composition for immunity enhancement, which comprises an extract of Gardenia borealum having an immunopotentiating activity by activating a cell (NK cell) as an active ingredient.

It is intended to provide a composition for immune enhancement which can be safely used without toxicity and side effects by using a garden borealis leaf extract, which is a natural resource of Korea.

In order to achieve the above object, the present inventors have found that induction of cytokine production of IL-2 (Interlukin-2), IL-4 (Interlukine-4) or IL-10 (Interlukine-10) The present invention provides a pharmaceutical composition or functional food composition having an immunostimulatory function, which comprises as an active ingredient an extract of Gardenia borealum having an immunoenhancing activity by activating NK cells.

The present invention enhances the activity of a natural killer cell (NK cell) directly against tumor cells by extracting a composition for enhancing immunity using an extract of Gardenia borealum as an active ingredient, and in vitro and in vivo Can be usefully used as a pharmaceutical composition and a functional food composition useful for immunity enhancement by increasing the secretion amount of immunocytokines such as mouse splenocytes IL-2, IL-4, and IL-10.

FIG. 1 is a graph showing changes in the splenocyte proliferative activity against the extracts of Y. bovis leaf.
FIG. 2 is a graph showing changes in IL-2 cytokine levels of spleen cells against the extracts of Y. bovis leaf.
FIG. 3 is a graph showing changes in the level of IL-4 cytokine of spleen cells against the extract of Y. bovis leaf.
FIG. 4 is a graph showing changes in the level of IL-10 cytokine in spleen cells against the extract of Y. bovis leaf.
FIG. 5 is a graph showing changes in the IL-4 cytokine level of Balb / c mouse serum against the extract of Y. bovis leaf.
FIG. 6 is a graph showing changes in the level of IL-10 cytokine of Balb / c mouse serum against the extract of Y. barkii leaf.
FIG. 7 is a graph showing changes in the level of NK cell activation effect on the extract of Y. barkii leaf.

The present invention relates to a pharmaceutical composition for enhancing immunity comprising an extract of Gardenia borealum Leaf as an active ingredient and is useful as an anti-inflammatory agent for the treatment of inflammation caused by cytokines of IL-2 (Interlukin-2), IL-4 (Interlukine- The present invention relates to a pharmaceutical composition for enhancing immunity, which can induce cytokine production and activate a natural killer cell (NK cell) directly against tumor cells. Hereinafter, the structure and effects of the present invention will be described in more detail with reference to specific examples and comparative examples in order to facilitate understanding of the present invention.

1. Production of garden boreal leaf extract

Gardenia borealis leaf extract is prepared by solubilizing it from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. In one embodiment, 100 g of dried gardenia leaves were washed with distilled water, 500 mL of distilled water was added, and the mixture was heated and extracted at 100 ° C for 3 hours. After filtration using a filter paper (Watts 41), the filtrate was concentrated under reduced pressure and freeze-dried at -50 ° C for 48 hours using a freeze dryer. According to the above method, 16.8 g (16.8%) of the hot-water extract of Yoshimura bamboo leaf was obtained according to the extraction solvent and used as a sample of the following experimental example.

2. Measurement of splenocyte proliferation by Y. barki leaf extract

Male Balb / c mice at 6 weeks of age were sacrificed by cervical dislocation, and the spleen was sterilized aseptically and crushed on a 100 mesh mesh to form single cells.

The mononuclear cell layer was taken and spleen cells were prepared by centrifugation three times for 5 minutes at 12,000 rpm. The spleen cells were diluted with RPMI 1640 medium containing 10% heat-inactivated FBS to a concentration of 2 × 10 ⁵ cells / mL and dispensed in a volume of 100 μL into a 96-well plate. After 30 minutes, 5 ug / mL of lipopolysaccharide (LPS) and 0.1 ug / mL of ConA (Concanavalin A) were added to the extracts of 0, 50, 100 and 200 ug / And cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours. The WST-1 (Water Soluble Tetrazolium Salts) solution was added at a concentration of 5 mg / mL in the dark condition. After 2 hours of reaction, the OD value was measured at 440 nm.

Fig. 1 shows the effect of splenocyte proliferative activity on the hot-water extract of the garden boreal tree leaf. T cells and B cells were treated with Con A (0.1 ug / mL) and LPS (5 ug / mL) as control cells. The control group was found to induce normal growth, and it was confirmed that splenocyte proliferative activity was increased at 50, 100 and 200 ug / mL of Gardenia borealis leaf extracts compared with no treatment group.

3. Quantification of splenocyte cytokine secretion by bark extract

The concentration of cytokines (IL-2, IL-4, IL-10) secreted by splenocytes was measured by ELISA. The amount of cytokine produced during the 24 hour reaction was measured using a DuoSet sandwich ELISA Mouse kit (R & D system, USA). The primary antibody, which was characterized for each cytokine, was diluted in PBS and dispensed into 100-μl aliquots on a 96-well plate for ELISA for one day. The next day, the primary antibody was washed with washing buffer, In order to fill the other space, the buffer was put in and treated for 2 hours and washed with the washing buffer. Standard and sample are added to each well in 100 μl of each well for 2 hours. After completion of reaction, wash with washing buffer, dispense 100 μl of diluted buffer in each well and treat for 2 hours. At the end of this procedure, the plate was washed with the buffer solution for washing, and 100 μl of the substrate for color development was reacted. Then, the absorbance was measured at 570 nm in an ELISA reader and the amount of cytokine produced in the cells was calculated using a standard curve.

Figures 2, 3, and 4 show the amounts of splenocytes IL-2, IL-4, and IL-10 cytokines secreted to the extracts of Y. bovis leaf. T cells and B cells were treated with Con A (0.1 ug / mL) and LPS (5 ug / mL) as control cells. As shown in FIG. 2, it was confirmed that the control group was induced to normal. In the case of IL-2 secretion, it was confirmed that the extract of Y. bovis leaf increased the secretion amount in a dose-dependent manner.

As shown in FIG. 3, IL-4 production was remarkably increased more than twice as much as that of the control group at a concentration of 50 ug / mL or more of Y. bovis leaf extract. As shown in FIG. 4, IL-2 and IL-4, respectively, compared with control group.

4. Quantification of cytokine secretion from Balb / c mouse serum by Y. bark leaf extract

Six to eight week old male Balb / c mice were adapted for one week at 23 ° C, 40-60% humidity, 12 hours of light-dark cycle, and then 100 mg / kg of Y. bark extract was added for 4 days 4, 6, and 8 hours after injection of 2 mg / kg of Concanavalin A (ConA) as a positive control in the tail vein of the mice on the 4th day. Immunocytochemicals such as IL-10 were measured for absorbance at a wavelength of 450 nm using an ELISA reader.

Figures 5 and 6 show the amount of cytokine secreted from the Balb / c mouse serum against the Y. bark leaf extract. As shown in FIG. 5, the amount of IL-4 secretion did not increase with time in the untreated group, whereas the amount of IL-4 secretion increased significantly at 4 hours in the group treated with 100 mg / kg of bovine leaf extract Respectively.

FIG. 6 is a graph showing changes in the level of IL-10 cytokine of Balb / c mouse serum against the extract of Y. barkii leaf. The amount of IL-10 produced in the untreated group did not increase with time, whereas the amount of IL-10 secreted at the 6th hour in the 100 mg / mL treated group of bovine leaf extract was significantly increased compared to the untreated group I could.

5. Measurement of the activity of Natural Killer cells (hereinafter abbreviated as NK-cells)

The YAC-1 cells and mouse spleen cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin at 37 ° C, After incubation in 5% CO ₂ incubator,

Balb / c mouse splenocytes were inoculated into a 96-well plate (50 μl, 1 × ^{10 6} , ⁵ × 10 ⁵ cells / ml) and 50 μl of YAC-1 cancer cells (2 × 10 ⁵ cells / ml). 50, 100, and 200 ug / ml were added to the extracts of Bombyx mori L. and cultured in a 5% CO ₂ incubator at 37 ° C for 4 hours. 45 minutes before the end of the reaction, 10X lysis buffer was added and incubated again, followed by centrifugation at 2,500 rpm for 4 minutes.

50 μl of the supernatant was transferred to a 96-well plate, and 50 μl of a substrate mix was added, followed by shading for 30 minutes at room temperature. After stopping the reaction by adding 50 ul of stop solution to each well, the absorbance was measured at 490 nm using a microplate reader, and the activity of NK-cells was calculated by the following equation.

FIG. 7 is a graph showing changes in the level of NK cell activation effect on the extract of Y. barkii leaf. Compared with the control group, the extracts of Y. bokus leaf showed the NK cell activity in a concentration dependent manner. Especially, the group treated with 200 ug / Of NK cell activity.

6. Yam tree leaf  Toxicity to extracts

The composition containing the gardenia borealis leaf extract can be formulated into powders, granules, tablets, capsules, suspensions, emulsions, syrups, transdermal preparations, suppositories or sterile injectable solutions, etc., and can be applied to the subject by oral administration or injection, Yam tree leaf extract may be applied in an amount of 10 to 2000 mg / kg body weight per day. In order to confirm this, the SD rats (male, 6 weeks old, Orient Bio Co., Ltd.) were administered 300 mg / kg / day of 2000 mg / kg per day through oral administration to the garden boreal tree leaf extract The mortality and body weight were measured for 14 days.

Mortality and body weight changes of the rats administered with the gardenia borealis leaf extract according to this comparative example are shown in the following Tables 1 to 2, respectively. Table 1 shows the mortality of rats treated with Y. bark extract and Table 2 shows the body weight change of rats treated with Y. bark leaf extract.

Mortality of rats treated with Gardenia borealis leaf extract Group Dose (mg / kg B.W) Mortality Yam tree leaf
extract 300 0%
(0/3) ^a 2000 0%
(0/3)

a: Number of dead animals / Number of animals tested

Changes in body weight of rats treated with Gardenia borealis leaf extract Group Dose
(mg / kg BW) Day of administration 0 days 1 day 3 days 7 days 14 days Yam tree leaf
extract 300 209.4
± 7.0 231.3
± 6.1 233.3
± 3.7 242.6
± 1.4 255.7
± 1.5 2000 213.9
± 10.3 242.3
± 7.9 239.5
± 13.6 248.7
± 14.4 264.8
± 12.2

As shown in Table 1, yam bora leaf extracts did not affect the survival rate of rats regardless of the dose. In addition, as shown in Table 2 above, the experimental group treated with Y. borealis leaf extract showed normal weight gain. The results showed that the extract of Y. bovis leaf showed no acute toxicity until oral administration of 2000 mg / kg, and that Y. bovis leaf extract could also be used as a pharmaceutical composition for enhancing immunity and functional food.

The pharmaceutical composition for enhancing immunity comprising the extract of the gardenia bittern leaf of the present invention as an active ingredient is useful for inducing cytokine production of IL-2 (Interlukin-2), IL-4 (Interlukine-4) or IL- (NK cells) directly against tumor cells, and thus can be used industrially as it is useful for treating human health and diseases.

Claims (8)

A pharmaceutical composition for immune enhancement characterized by comprising Elaeagnus multriflora Thunb. Leaf extract as an active ingredient The pharmaceutical composition for immunotherapy according to claim 1, wherein the gardenia borealis leaf extract is an extract soluble in water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. [Claim 3] The pharmaceutical composition for immune enhancement according to claim 2, wherein the pharmaceutical composition for immunomodulation is prepared by containing a gardenia bittern leaf extract in an amount of 0.1 to 2000 mg / kg of body weight per day. The pharmaceutical composition according to claim 3, wherein the composition is formulated as a powder, granule, tablet, capsule, suspension, emulsion, syrup, transdermal preparation, suppository or sterile injectable solution. 3. The pharmaceutical composition for immunotherapy according to claim 1 or 2, wherein the gardenia leaf extract has an immunopotentiating activity by activating natural killer cells directly against tumor cells. delete delete delete

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