KR102250488B1 - Composition for protecting hepatic damage on heavy metal comprising extracts from Elaeagnus umbellata leaves - Google Patents

Abstract

The present invention relates to a composition for protecting liver damage by heavy metals containing a bodhi tree (Elaeagnus umbellata ) leaf extract as an active ingredient, and in detail, to prepare an ethanol extract of bodhi tree leaf, and its toxicity from heavy metals (cadmium or lead) Confirmed relief. According to the present invention, when Cadmium is treated to induce toxicity to HepG2 hepatocytes pre-treated with Bodhi tree leaf extract, cytotoxicity and the amount of cadmium uptake in cells are reduced compared to HepG2 hepatocytes not treated with Bodhi tree leaf extract. It can be used in pharmaceutical compositions and health functional foods useful for preventing and treating liver damage caused by heavy metals by confirming the possibility of mitigating liver damage caused by cadmium of the bodhi tree leaf extract.

Description

Composition for protecting hepatic damage on heavy metal comprising extracts from Elaeagnus umbellata leaves}

The present invention is a bodhi tree ( Elaeagnus umbellata ) It is for a composition for protecting liver damage by heavy metals containing leaf extract as an active ingredient.

Bodhi tree ( Elaeagnus umbellata Thunb. Autumn olive) is a deciduous broad-leaved shrub belonging to the Elaeagnus genus of the Elaeagnus family, and is widely distributed in Gaya-myeon, Hapcheon-gun in Korea, and is known as a barley dung tree, black dung, and pink pop bodhi tree in the private sector. It is grown in areas such as scavengers, castles buckthorn with garden lime (Elaeagnus multiflora Thunb.) And 7-8 are cultivated in areas such as Hwacheon Chuncheon on April orange circular shape with rounded fruit that is harvested from June to July linden trees Unlike (aka vitamin tree, Hippophae rhamnoides L.), the fruit opens like a cluster of grapes from October to November and ripens in red. Fruits are 1cm long and have a bit astringent and sweet taste, and the leaves are alternate, oval, and the upper part is dark green. The fruit, leaves, and roots of the bodhi tree are called unnaeja in China, and are used as dysentery, hemostasis, and branching agents. Meanwhile, in Korea, the fruit of the bodhi tree is called Mokbanha (木半夏) in oriental medicine, and is known to be effective in diarrhea, bleeding, and indigestion in terms of efficacy. It is used a lot for the treatment of the back.

There are not many studies on the efficacy of bodhi tree, so antibacterial activity of fruit, leaf, and flower extracts, analysis of general components and lycopene content of fruit, antioxidant and cancer cell proliferation inhibitory effect of fruit extract, and comparison of antioxidant activity of fruit and leaf solvent fractions It has been reported, and as a study related to Tulbori, there have been reports on the analysis of useful components of leaves, antioxidant and anti-cancer, whitening effect of fruit extract, anti-platelet and anti-inflammatory.

Bodhi tree has no systematic research, and although it is permitted for food, it is not used as a food material at all. Accordingly, in the present invention, the functionality of bodhi tree leaves (liver protection effect against heavy metals) was attempted to improve utilization as a high value-added biomaterial.

Korean Patent Registration No. 10-1873297 (registered on June 26, 2018)

Accordingly, an object of the present invention is to provide a composition for protecting liver damage by heavy metals containing a leaf extract of Elaeagnus umbellata as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating liver damage caused by heavy metals containing a bodhi tree leaf extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving liver damage caused by heavy metals containing a bodhi tree leaf extract as an active ingredient.

The present invention is a bodhi tree ( Elaeagnus umbellata ) For a composition for protecting liver damage by heavy metals containing leaf extract as an active ingredient, in detail, ethanol extract of bodhi tree leaves was prepared, and toxicity relief from heavy metals (cadmium or lead) thereof was confirmed. According to the present invention, when Cadmium is treated to induce toxicity to HepG2 hepatocytes pre-treated with Bodhi tree leaf extract, cytotoxicity and the amount of cadmium uptake in cells are reduced compared to HepG2 hepatocytes not treated with Bodhi tree leaf extract. It can be used in pharmaceutical compositions and health functional foods useful for preventing and treating liver damage caused by heavy metals by confirming the possibility of mitigating liver damage caused by cadmium of the bodhi tree leaf extract.

1 shows the cytoprotective effect of bodhi tree leaf extract in cadmium-treated HepG2 cells.
2 shows the inhibitory effect of bodhi tree leaf extract on cadmium uptake in HepG2 cells.
3 shows the protective effect of bodhi tree leaf extract against lead toxicity in HepG2 cells.

The present invention provides a pharmaceutical composition for preventing or treating liver damage caused by heavy metals containing a leaf extract of Elaeagnus umbellata as an active ingredient.

Preferably, the heavy metal may be cadmium or lead, but is not limited thereto.

The bodhi tree leaf extract of the present invention may be prepared according to a conventional method known in the art, that is, under the conditions of a conventional temperature and pressure, using a conventional solvent, preferably water, C1 to C4 It is extracted using alcohol or a mixed solvent thereof, and more preferably, it can be extracted using 70% ethanol.

When the composition of the present invention is a pharmaceutical composition, the pharmaceutical formulation form of the pharmaceutical composition is a granule, powder, coated tablet, tablet, capsule, suppository, syrup, juice, suspension, emulsion, drop or injectable liquid and active compound. It can be formulated as a sustained-release formulation. The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants. Alternatively, solubilizing agents such as flavoring agents may be used. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition, including at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterilized and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention is in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered as. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be adjusted according to various factors, including the rate of secretion, duration of treatment, and drugs used concurrently.

In addition, the present invention provides a health functional food composition for preventing or improving liver damage caused by heavy metals containing a bodhi tree leaf extract as an active ingredient.

Preferably, the heavy metal may be cadmium or lead, but is not limited thereto.

The bodhi tree leaf extract of the present invention may be prepared according to a conventional method known in the art, that is, under the conditions of a conventional temperature and pressure, using a conventional solvent, preferably water, C1 to C4 It is extracted using alcohol or a mixed solvent thereof, and more preferably, it can be extracted using 70% ethanol.

When the composition of the present invention is a health functional food composition, the health functional food composition may be provided in the form of a powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is a food or food additive other than the active ingredient. It is used together with, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health functional food composition may be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it should be less than the above range. It is clear that the active ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, and vitamin complexes.

In the present invention, liver diseases that may occur due to liver damage caused by heavy metals may be acute hepatitis, chronic hepatitis, fatty liver disease, cirrhosis, liver fibrosis, liver cancer, drug-induced hepatitis, jaundice, etc., but are limited thereto. no.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

< Example 1> Preparation of bodhi tree leaf extract

The dried bodhi tree leaves are crushed, and ethanol solvent conditions (0%, 20%, 50% 70%, 100%), temperature conditions (RT{25℃}, 60℃, 80℃), time conditions (3, 6) , 12, 24, 48 hours). The extracted sample was filtered through Advantec (No. 5A, Tokyo, Japan) filter paper, concentrated using a vacuum concentrator, freeze-dried, and used as a sample for analysis.

< Example 2> Antioxidant against Bodhi tree leaf extract and liver protection against heavy metals

Antioxidant evaluation of Bodhi tree leaf extract was confirmed by measuring DPPH radical scavenging activity, ABTS radical scavenging activity, polyphenol content, and flavonoid content. HepG2 cells were used to confirm the liver-protective effect on bodhi tree leaf extract.

1. Bodhi tree leaves ethanol and Hydrothermal Antioxidant effect of extract

(1) Experiment method

1-1) ABTS radical scavenging activity

14 mM ABTS reagent and 4.9 mM potassium persulfate solution were mixed in a 1:1 volume ratio, and then reacted at room temperature for 24 hours. Into a 1.5 ml tube, 180 μl of ABTS reagent and 20 μl of a solution obtained by diluting Bodhi tree leaf extract were added to react. After reacting in the dark for 10 minutes at room temperature, absorbance was measured using an ELISA reader device (734 nm).

1-2) DPPH radical scavenging activity

A 0.2 mM DPPH reagent was prepared, and a solution obtained by diluting the Bodhi tree leaf extract was added to a 96-well plate. And 100 μl of DPPH reagent was reacted in each well. After reacting in the dark for 30 minutes at room temperature, absorbance was measured using an ELISA reader device (517 nm).

1-3) Polyphenol content

After adding 60 μl of a 2-fold diluted Folin reagent to a 96-well plate, 60 μL of a diluted solution of bodhi tree leaf extract was added and allowed to stand for 3 minutes. Then, 60 μl of a 10% Na ₂ CO ₃ solution was added and reacted. After the reaction at 30° C. for 1 hour, absorbance was measured using an ELISA reader device (700 nm).

1-4) Flavonoid content

In a 1.5 ml tube, add 860 μl of 100% EtOH, 20 μl of 10% aluminum nitrate, and 20 μl of 1 M potassium acetate, and add 100 μl of a solution obtained by diluting various concentrations of bodhi tree leaf extract. Made it. After reaction at room temperature for 40 minutes, absorbance was measured using an ELISA reader device (415 nm).

(2) Experiment result

As a result of antioxidant evaluation of Bodhi tree leaf extract, all bodhi tree leaf extracts showed excellent ABTS radical scavenging activity, DPPH radical scavenging activity, polyphenol and flavonoid content (Table 1).

2. HepG2 Protective Effect of Bodhi Tree Leaf Extract against Heavy Metal (Cadmium) in Hepatocytes

(1) Experiment method

2-1) Protective Effect of Bodhi Tree Leaf Extract against Cadmium Toxicity in HepG2 Cells

MTT assay was performed to measure the cell protective ability of Bodhi tree leaf extract against cadmium-treated HepG2 cells. HepG2 cells were dispensed into a 24-well plate at 2×10 ⁵ cells/well and incubated for 20 to 24 hours in a 5% CO ₂ incubator, and then treated according to the concentration of bodhi tree leaf extract, and then 20 in a _{5% CO 2 incubator.} It was pre-incubated for ~24 hours. After treatment so that the final concentration of CdCl ₂ was 10 μM, it was reacted for 24 hours, 50 μl of MTT (5 mg/ml) solution dissolved in PBS was added to each well and reacted in an incubator for 4 hours. Thereafter, the supernatant was removed, and 200 μl of DMSO was added to each well to dissolve the generated formazan crystal, and the absorbance was measured with an ELISA reader at a wavelength of 550 nm. The measured formazan production was expressed as a percentage (%) compared to the normal cell value.

2-2) Inhibitory Effect of Bodhi Leaf Extract on Cadmium Uptake in HepG2 Cells

ICP-MS (Agilent 7500cx) was used to confirm the inhibitory effect of cadmium uptake on HepG2 cells of bodhi tree leaf extract. HepG2 cells were dispensed to a 6-well plate at 5×10 ⁵ cells/well and pre-incubated in a 5% CO ₂ incubator for 20 to 24 hours. After treatment by concentration of bodhi tree leaf extract, it was incubated for 20 to 24 hours in a _{5% CO 2 incubator.} After treatment so that the final concentration of CdCl ₂ was 10 μM, the reaction was performed for 24 hours, washed twice with PBS, and the cells were collected with PBS. Count the number of cells as 1×10 ⁶ cells/ml, dilute with 1.8 ml of aqua regia (hydrochloric acid 3: nitric acid 1) and 18 ml of distilled water, and react at 80°C for 3 hours. After filtering, cadmium in the solution was measured using ICP-MS.

(2) Experiment result

2-1) Cytoprotective Effect of Bodhi Tree Leaf Extract in HepG2 Cells Treated with Cadmium

As a result of measuring the cytoprotective effect of bodhi tree leaf extract in cadmium-treated HepG2 cells, it was found that the cell viability was increased in a concentration-dependent manner in both bodhi tree leaf extracts (FIG. 1).

In the extraction conditions according to the extraction solvent ratio, the cell protection ability of the bodhi tree leaf extract under the conditions of 70% ethanol, RT (room temperature), and 6 hours was the highest at 500 μg/ml at 92.06%, and the 70% ethanol extraction condition was selected.

In the extraction conditions according to the extraction temperature, the cell protection ability of the bodhi tree leaf extract under the conditions of 80℃, 70% ethanol, and 6 hours was the highest at 500μg/ml at 94.08%, and the extraction temperature condition was selected as 80℃.

In the extraction conditions according to the extraction time, the cell protection ability of the bodhi tree leaf extract under the conditions of 6 hours, 70% ethanol, and 80℃ was the highest, and the extraction time condition was selected as 6 hours.

Accordingly, it was confirmed that the extraction condition of the bodhi tree leaf extract was the optimum condition in the extraction condition of 70% ethanol, 80°C, and 6 hours.

2-2) Inhibitory Effect of Bodhi Leaf Extract on Cadmium Uptake in HepG2 Cells

As a result of measuring the uptake inhibitory effect of bodhi tree leaf extract in cadmium-treated HepG2 cells, cadmium ^{content was measured as 4.76 ppb in 1×10 6} cells that were not treated with cadmium, and 33.76 ppb when cadmium was treated. It was confirmed that cadmium was measured and uptaken.When the bodhi tree leaf extract was treated, it was measured as 23.69 ppb at a concentration of 250 μg/ml, confirming that the uptake amount was reduced by 29.89%, and 500 μg. At a concentration of /ml, it was measured as 13.72 ppb, confirming that the uptake amount was reduced by 59.40%, and it was confirmed that the uptake amount decreased in a concentration-dependent manner. It can be seen that the bodhi tree leaf extract reduces the uptake of Cd in HepG2 hepatocytes (FIG. 2).

3. HepG2 Protective Effect of Bodhi Tree Leaf Extract against Heavy Metals (Lead) in Hepatocytes

(1) Experiment method

3-1) Protective Effect of Bodhi Tree Leaf Extract against Lead Toxicity in HepG2 Cells

In order to measure the cytoprotective ability of Bodhi tree leaf extract against lead-treated HepG2 cells, inhibition of MDA (malondialdehyde) production was measured. HepG2 cells were dispensed into a 24-well plate at 2×10 ⁵ cells/well and incubated for 20 to 24 hours in a 5% CO ₂ incubator, and then treated according to the concentration of bodhi tree leaf extract, and then 20 in a _{5% CO 2 incubator.} It was pre-incubated for ~24 hours. After treatment so that the final concentration of PdCl ₂ was 100 μM, the cells were cultured for 24 hours, and the cells were collected and suspended in 0.2 N HCl to hydrate the cells at 60° C. for 80 minutes. Protein was quantified using BCA assay, and the color development produced by reacting MDA present in 2 mg of the same amount of protein with 0.4 mM methyl phenylindole was measured with an ELISA reader.

(2) Experiment result

As a result of measuring the inhibitory effect of MDA production of bodhi tree leaf extract in lead-treated HepG2 cells, MDA production increased by 70% compared to cells not treated in lead-treated HepG2 cells, and bodhi tree leaf extract was treated. In this case, it could be confirmed that 46.87% production was inhibited at a concentration of 500 μg/mL, and it was confirmed that the amount of MDA production decreased depending on the concentration of the bodhi tree leaf extract (FIG. 3).

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

Bodhi tree ( Elaeagnus umbellata ) A pharmaceutical composition for preventing or treating liver damage caused by cadmium or lead containing ethanol extract from leaves as an active ingredient. delete delete delete Health functional food composition for preventing or improving liver damage caused by cadmium or lead containing ethanol extract of bodhi tree leaves as an active ingredient. delete delete delete

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