KR20140145570A - Pharmaceutical Compositions for Prevention or Treatment of Disease Causing Aging of Vascular Endothelial Cell Comprising Extract of Physalis Angulata - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a Physalis angulata L. extract for preventing or treating diseases caused by vascular endothelial cell aging. Since the pharmaceutical composition comprising a Physalis angulata L. extract for preventing or treating diseases caused by vascular endothelial cell aging of the present invention can effectively enhance fibroblast or vascular endothelial cell proliferation and inhibit SA-β-galactosidase activity to thereby suppress vascular endothelial cell aging, the composition can be useful to prevent diseases caused by vascular endothelial cell aging such as cardiovascular disease, chronic renal disease, chronic inflammatory dermatitis, atheroma inflammation, and Parkinson′s disease.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating diseases induced by vascular endothelial cell senescence,

The present invention relates to a physiological saline solution The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by vascular endothelial cell senescence comprising an extract of angulata L., and more particularly to a pharmaceutical composition for enhancing fibroblast or vascular endothelial cell proliferation and inhibiting SA-β-galactosidase The present invention relates to a pharmaceutical composition for preventing or treating a disease caused by vascular endothelial cell senescence, which comprises an extract of Pseudomonas aeruginosa inhibiting vascular endothelial cell senescence through activity inhibition.

The delay in the aging process, without degenerative disease, healthy and long-lasting without disease is an ongoing effort to find a substance that inhibits aging by the wind of all people. Oxidation by active oxygen species such as superoxide, hydrogen peroxide, hydroxyl group and singlet oxygen is one of the factors promoting aging. Free radicals generated by the metabolites of reactive oxygen species act on proteins, biomembranes, and DNA in vivo and cause oxidative damage. Many studies have been conducted on antioxidant components to suppress these effects, Researches are being actively conducted at home and abroad to search for new materials having action. However, since the aging phenomenon is not only caused by oxidation but is known to be influenced by numerous factors such as telomerase, cell replication ability, gene damage and recovery ability, it is necessary to evaluate the antioxidative effect of natural products and drugs, A method of using aging is needed.

Cell senescence is a phenomenon in which normal somatic cells can no longer divide after a certain number of divisions, contributing to the aging of individuals and tissues, and is an important mechanism for inhibiting abnormal cell proliferation and cancer formation. Cell senescence is caused by the shortening of the telomere, which is the end of the chromosome, due to repeated somatic cell division, or by the increased activity of cancer gene or cancer suppressor gene, excessive oxidative stress, cytotoxic substances such as ultraviolet radiation or radiation, And the like.

The senescence-associated β-galactosidase (SA-β-gal) activity increases with the morphological features that aged cells become larger, flattened, increase in heterochromatin in the nucleus, (IGFBPs), interleukin-6 (interleukin-6), and transgene-like growth factor-binding proteins (IGFBPs) (TGF-beta), interferon, and the like.

Cell senescence not only contributes to the aging of individuals and tissues, but also plays an important role in the pathogenesis of various diseases. Aging cells are frequently observed in inflammatory lesions such as rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin injured tissue, and arteriosclerotic vascular tissue. In addition, cell senescence is observed in proliferative hyperplasia, hepatitis, and liver cancer.

As the aging cells accumulate, the aging cells do not divide well. Therefore, not only the damaged tissues are properly restored but also the enzymes that decompose the surrounding tissues or the inflammatory cytokines are secreted, accelerating the damage of the tissues. Contributes to the pathogenesis.

Since cellular aging is considered to be an essential cause of aging, efforts have been made to develop methods for delaying cellular aging. Among them, some researches have been conducted to search for substances capable of regulating cell senescence and to utilize them for the prevention and treatment of diseases.

Some of the substances known to regulate cell senescence include resveratrol, which is known to increase SIRT1 activity, and retinoic acid, which is used to delay cellular aging in keratinocytes. In addition, studies have been reported on the identification of substances capable of regulating cell senescence from plant extracts and their use in cosmetics and health functional foods.

 On the other hand, chrysanthemum is a perennial herbaceous plant belonging to the branch, and it is called a physalis herba, a cleavage. It has a uterine contraction effect on the root of the eel root, and has a diuretic effect on the fruit. As for the herbaceous plant, the plant and roots have been used as antipyretic drugs, analgesic drugs for disintegration, diuretics and transdermal drugs, It is used as an analgesic, and the fruit has been used for nose cold, rubella, gonorrhea and hypertension. As such, it has been known that there are various functions in acorns, but scientifically proven results are limited.

Previous studies on acorns have reported on the amino acid sequences of physalin A, B, physalin T and ferredoxin in relation to structural analysis. From the roots of acorns The results of the inhibition activity of glycosidase and the effect of whitening activity on the extract of Ulmus kirilowii were investigated. However, the results of inhibiting the cell senescence of kelp extract were reported In particular, the research on the efficacy of the extract of Quercus acutissima has been insufficient.

DISCLOSURE OF THE INVENTION The present invention was conceived in order to solve the above-mentioned problems, and it was found that, in researching herbal medicine extract effective for inhibiting cell senescence, There is provided a pharmaceutical composition for preventing or treating diseases induced by vascular endothelial cell senescence comprising as an active ingredient.

In order to solve the above-described problems,

Enhances the proliferation of fibroblasts or vascular endothelial cells, inhibits the aging of vascular endothelial cells through inhibition of SA-β-galactosidase activity,

It contains fruit extract of Physalis angulata L. as an active ingredient,

There is provided a pharmaceutical composition for preventing or treating diseases caused by at least one type of vascular endothelial cell senescence selected from the group consisting of cardiovascular diseases, chronic renal diseases, chronic inflammatory skin diseases, atherosclerotic inflammation and Parkinson's disease.

According to a preferred embodiment of the present invention, the extract of Pseudomonas aeruginosa can be extracted with at least one solvent selected from the group consisting of water, alcohol, ethyl acetate, chloroform and hexane.

According to another preferred embodiment of the present invention, the composition may contain a gruel extract at a concentration of 5 to 130 μg / ml.

The pharmaceutical composition for prevention or treatment of disease caused by vascular endothelial cell senescence comprising the extract of P. acutissima according to the present invention can effectively enhance fibroblast or vascular endothelial cell proliferation and inhibit SA-? -Galactosidase activity Can be effectively used for the prevention of diseases caused by vascular endothelial cell senescence such as cardiovascular diseases, chronic kidney diseases, chronic inflammatory skin diseases, atherosclerotic inflammation and Parkinson's disease.

1 is a graph showing the results of MTT analysis in fibroblasts (HDF) and vascular endothelial cells (HUVEC).
2 is an optical microscope photograph and a graph showing evaluation of SA-beta-gal activity inhibitory activity in vascular endothelial cells (HUVEC).

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

As described above, the acolyte has conventionally been studied in terms of the amino acid sequence of physalin and ferredoxin, the inhibitory activity of glycosidase from the root of acorn, the whitening activity of the acorn extract, However, no effect on the inhibition of cell senescence by the extract of Quercus mongolica was reported. Especially, the effect of extract of Quercus mongolica extract was insufficient.

 Accordingly, the present invention improves the proliferation of fibroblasts or vascular endothelial cells, inhibits the aging of vascular endothelial cells through inhibition of SA-β-galactosidase activity,

It contains fruit extract of Physalis angulata L. as an active ingredient,

There is provided a pharmaceutical composition for preventing or treating diseases caused by at least one type of vascular endothelial cell senescence selected from the group consisting of cardiovascular diseases, chronic renal diseases, chronic inflammatory skin diseases, atherosclerotic inflammation and Parkinson's disease.

According to the present invention, the composition containing the extract of Pseudomonas aeruginosa as an active ingredient was subjected to MTT analysis to show no cytotoxicity and increased cell viability, and SA-β-galactosidase (SA-β-galactosidase) Activity was inhibited. It was confirmed that the extract of Quercus acutissima extract directly inhibited cell senescence.

The earth ground cherry is specifically earth ground cherry (Physalis angulata L.), P. minima L., P. wrightii Gray , and the like, and may be a single or mixed form thereof, Physalis angulata L. extract may be the most effective inhibitor of cell senescence.

 Preferably, the extract is extracted from the ground almond fruit. In addition to the fruit, other parts may have insufficient effect of inhibiting cell senescence, and it is most preferable to use the ground fruit.

The extract may be extracted with a solvent such as water, alcohol, ethyl acetate, chloroform, hexane, or the like, more preferably a C _1-4 lower alcohol. The extract may be applied by a general solvent extraction method using the above-mentioned solvent, or it may be a fraction purified by using column chromatography. The method for producing the extract according to the present invention can be applied to any plant extraction method known to those of ordinary skill in the art. For example, extraction with water, alcohol or mixed solvent, extraction with hot water or room temperature But is not limited thereto.

The composition containing the extracted extract of Pseudomonas aeruginosa as an active ingredient did not show cytotoxicity in cells such as fibroblasts (HDF) and vascular endothelial cells (HUVEC), and the cell proliferation was significantly increased, and SA-β - inhibit senescence-associated β-galactosidase activity and inhibit cell senescence. In particular, it can be confirmed that the extract of Pseudomonas externa has a remarkably superior cytotoxic effect on vascular endothelial cells (HUVEC). (See Table 2 and Fig. 2)

The composition for inhibiting cell senescence of the present invention may contain the above extract of Pseudomonas aeruginosa at a concentration of 5 to 130 μg / ml. The solvent of the composition may be water, ethanol, propyl alcohol, DMSO, and the like, although it is not particularly limited as long as it does not interfere with the cell aging inhibitory effect of the extract. When the concentration of the extract is lower than 5 μg / ml, the effect of inhibiting SA-β-galactosidase activity is insufficient and the effect of inhibiting cell senescence is deteriorated. When the concentration exceeds 130 μg / ml, There is a problem that the efficiency is lowered. The concentration of the extract of Phellodendus rubella extract having no cytotoxic effect and exhibiting excellent cell senescence inhibiting effect may be more preferably 70 to 120 占 퐂 / ml.

The pharmaceutical composition can be administered to mammals such as rats, mice, livestock, humans, and the like through various routes including oral, transdermal, subcutaneous, intravenous or muscular. In addition, the pharmaceutical composition comprising the composition for suppressing cell senescence comprising the extract of Pseudomonas exocuta according to the present invention as an active ingredient can be formulated into various formulations. In the case of formulation, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient (e. G., Starch, sucrose, lactose and gelatin) Can be mixed and prepared. In addition to simple excipients, lubricants can also be used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, glycerol, gelatin and the like may be used.

The dosage of the pharmaceutical composition may be varied depending on the age, sex, condition of the subject, the degree of absorption of the active ingredient in the body, the rate of inactivation and the rate of excretion, and the drugs used in combination. A daily dose that can be used without side effects can be administered in single or divided doses in an amount of 0.1 to 100 mg / kg.

When a pharmaceutical composition comprising a composition for suppressing cell senescence comprising the extract of the present invention as an active ingredient is administered to a human body, it can be safely used because it is a natural extract, so that there is less concern about side effects than other synthetic drugs .

The pharmaceutical composition can be used to treat or prevent diseases related to cell senescence and may be useful for preventing or treating diseases such as rheumatoid arthritis, osteoarthritis, hepatitis, chronic arteriosclerosis, prostatic hyperplasia, liver cancer, Can be used to prevent or treat diseases such as arteriosclerosis, cardiovascular disease, chronic kidney disease, chronic inflammatory skin disease, atherosclerotic inflammation and Parkinson's disease known to be induced.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, and should be construed to facilitate understanding of the present invention.

< Example 1 >

1. Preparation of the extract of Quercus mongolica extract

Physicis angulata L. fruit was collected from Seodong-dong, Suwon-si, Gyeonggi-do, Korea, and dried and pulverized. The extract was extracted with ethanol by an accelerator solvent extraction apparatus at 85 ℃ and then filtered. The water was removed from the freeze dryer and used for the experiment. The extract obtained from 108 g of the dried sample was 10 g, and the yield was 9.3%.

The obtained extract was dissolved in a solvent DMSO (dimethyl sulfoxide) at a concentration of 100 μg / ml to prepare a ground acorn extract composition.

2. Cell culture

Human fibroblasts (HDF) were cultured in a DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic at a density of 1 × 10 ⁵ cells in a 100 mm diameter culture dish, cultured in a 5% carbon dioxide incubator at 37 ° C. Respectively. When 80-90% of the cells were grown on the bottom of the culture dish, the cells were treated with 2.5 × Trypsin-EDTA solution and subcultured. Cells (umbraculifera) were cultured in the same manner using EGM-2 as a culture medium.

The population doubling (PD) was examined by the following equation.

PD = log ₂ F / log ₂ I (F = number of cells last, I = number of cells first).

The cells used in the experiment were 40 times in the case of human fibroblasts and 30 times or less in the cord blood.

&Lt; Example 2 >

The extract was prepared in the same manner as in Example 1, except that the extract of Pseudomonas externa was dissolved in a solvent DMSO at a concentration of 10 μg / ml.

<Experimental Example 1> Cytotoxicity measurement

1. Cell culture and treatment of extract

In a 50 ml tube, 5,000 cells / ml of fibroblasts were added to a DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic, and the number of cells in the umbilical cord blood was 10,000 cells / ml in the EGM-2 culture medium. 100 [mu] L of each well was added to each well of a culture well. Finally, 500 fibroblasts and 1,000 cells were placed in each well and cultured in a 5% CO 2 incubator at 37 ° C for one day. 100 쨉 l of a DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic and an EGM-2 culture medium were further added to each well.

As a negative control, DMSO (dimethyl sulfoxide) was treated and 5 mM N-acetylcysteine and 500 mM rapamycin were treated as a positive control.

2. MTT analysis

After the cells were cultured in a 5% CO 2 incubator at 37 ° C. for 3 days, the degree of cell growth was confirmed by MTT method. 50 μl of 0.1% MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide) solution was added to each well of a 96-well culture vessel and reacted for 3 hours at 37 ° C in a 5% CO 2 incubator After removing the culture medium and MTT solution, 100 μl of DMSO was added and the resulting crystals were dissolved. The absorbance was measured at 550 nm using a microplate reader. The results expressed in percentage (%) are shown in Table 1 and FIG.

&Lt; Experimental Example 2 > Evaluation of cytotoxicity of SA-β-galactosidase activity through cell staining

1. Induction of cell aging by adriamycin

Adriamycin is a type of anticancer drug that kills cells at high concentrations but is known to induce cell senescence at low concentrations. Human umbilical cord blood cells were plated at a density of 1.5 × 10 ⁵ cells in a 100 mm diameter culture dish and cultured in a 5% carbon dioxide incubator at 37 ° C. for 3 days. The cell culture medium was removed and the cells were washed twice with DMEM containing 1% After washing, 500 nM adriamycin was treated for 4 hours.

2. Cell culture and treatment of extract

The cells treated with adriamycin for 4 hours were separated from the culture dish with trypsin-EDTA and the cells were placed in a 24-well culture container. The umbilical cord blood was added to the EGM-2 culture medium containing 10% fetal bovine serum and 1% antibiotic in a 50 ml tube and dispensed into each well of the culture container. Finally, 5000 cells were placed in 24-well culture vessels and cultured in a 5% CO 2 incubator at 37 ° C for one day. DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic and EGM-2 medium were changed into each well, and then the extract of the extract of Pseudomonas aeruginosa was treated.

As a negative control, DMSO (dimethyl sulfoxide) was treated and 5 mM N-acetylcysteine and 500 mM rapamycin were treated as a positive control.

3. Staining of SA-β-galactosidase activity

The cells were cultured in a 5% CO 2 incubator at 37 ° C for 3 days after the treatment, and then the effect of the extract on the cell aging was examined by SA-β-gal staining. Cells aged by SA-β-gal staining are stained cytoplasmically blue.

After 3 days of treatment, the cells were washed twice with phosphate buffer, fixed with 3.7% paraformaldehyde for 1 minute, and then fixed. 5 mM potassium ferricyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ , X-gal 1 mg / ml) in a 24 well culture vessel Was added, and the mixture was wrapped in a silver foil and reacted at 37 캜 for 18 hours. After washing twice with phosphate buffered saline (PBS), the cells were stained with 1% ezine solution for 1 minute. When crystals were formed, the crystals were dissolved by DMSO and removed. After washing twice with phosphate buffer, blue-stained cells were observed under an optical microscope. The degree of SA-β-gal activity was determined by counting the number of cells stained blue in the cytoplasm among 50 to 100 cells in total, and the results are shown in Table 2 and FIG.

Cell proliferation (%) Fibroblast (HDF) Vascular endothelial cells (HUVEC) Example 1 115.6 ± 11.2 92.7 ± 4.9 Cont 100 ± 0 100 ± 0 DMSO 91.0 ± 8.9 86.2 ± 18.0 NAC 91.5 ± 10.4 102.2 ± 11.7 Rapamycin 90.6 ± 4.6 77.4 ± 3.2

The fibroblasts and vascular endothelial cells did not show cytotoxic effect on the extract of P. acutissima according to the above example, and the cell proliferation was significantly increased.

SA-β-gal staining (%) Vascular endothelial cells (HUVEC) Example 1 52.9 ± 6.2 Example 2 65.2 ± 7.9 Cont 184.9 ± 8.6 DMSO 84.4 ± 4.2 NAC 68.8 ± 7.5 Rapamycin 36.1 ± 17.9

The ground acorn seed weight composition according to the Example showed excellent SA-β-gal activity reduction, and thus showed excellent cell aging inhibitory effect.

Hereinafter, formulation examples of the pharmaceutical composition containing the extract of the present invention will be described. However, the present invention is not intended to be limited thereto but is specifically explained.

&Lt; Formulation Example 1 > Preparation of powders

300 mg of extract of ground corn, 100 mg of lactose and 10 mg of talc were mixed and packed in airtight bags to prepare powders.

&Lt; Formulation Example 2 > Preparation of tablet

The tablets were prepared by mixing 50 mg of ground corn root extract, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, followed by tableting according to a conventional preparation method.

&Lt; Formulation Example 3 > Preparation of capsules

According to a conventional capsule preparation method, a mixture of 50 mg of ground corn oil extract, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate was mixed and filled in gelatin capsules to prepare capsules.

&Lt; Formulation Example 4 > Preparation of injection

(2 ml) per acre (50 mg), sterile distilled water suitable for injection, and an appropriate amount of pH adjuster were prepared according to the usual injection preparation method.

&Lt; Formulation Example 5 > Preparation of liquid agent

In accordance with the usual preparation method of the liquid preparation, 100 mg of the extract of Phellodendrularis sieboldii, 10 g of isomerized sugar and 5 g of mannitol were dissolved in an appropriate amount of purified water and mixed with a proper amount of lemon flavor, followed by adding purified water, The solution was packed in a bottle and sterilized to prepare a liquid preparation.

Hereinafter, a preparation example of a health functional food containing the extract of the present invention will be described. However, the present invention is not intended to be limited thereto but is specifically described.

&Lt; Formulation Example 6 > Preparation of health functional foods

(Vitamin A acetate: 70 ㎍, vitamin E: 0.1 mg, vitamin B: 0.13 mg, vitamin B 2: 0.15 mg, vitamin B 6: 0.5 mg, vitamin B 12: 0.2 쨉 g, vitamin C: 10 mg, 10 mg of biotin, 1.7 mg of nicotinic acid amide, 50 엽 of folic acid and 0.5 mg of calcium pantothenate) and an appropriate amount of inorganic mixture (1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate, 55 mg of calcium, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed to prepare a granule, and a health food was prepared according to a conventional method.

Hereinafter, formulation examples of the cosmetic composition containing the extract of P. galanii according to the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

&Lt; Formulation Example 7 > Preparation of essence

Ethyl alcohol 4.00 wt%

Methyl paraben 0.10 wt%

Fragrance 0.10 wt%

Hydrotreated castor oil 0.60 wt%

DL-a-tocopheryl acetate 0.02 wt%

Glycerin 5.00 wt%

1,3-butylene glycol 3.00 wt%

0.20% by weight of hydroxyethylcellulose

Xanthan gum 0.40 wt%

0.02 wt% of E. D. T. A-2Na,

Fish-collagen 4.00 wt%

Ground cornelia extract composition 0.5 to 2.0 wt%

Distilled water balance

&Lt; Formulation Example 8 > Preparation of Cleansing Foam

6.5% by weight stearic acid

Myristic acid 28.0 wt%

3.0% by weight of self-emulsifiable glycerin monostearate

5.0% by weight of propylene glycol

Concentrated glycerin 10.0 wt%

Sodium hydroxide 7.0 wt%

0.1% by weight of sodium ethylenediaminetetraacetate

&Lt; tb &gt; &lt; SEP &gt; 15.0 wt%

Fragrance

Purified water balance

Claims (3)

Enhances the proliferation of fibroblasts or vascular endothelial cells, inhibits the aging of vascular endothelial cells through inhibition of SA-β-galactosidase activity,
It contains fruit extract of Physalis angulata L. as an active ingredient,
A pharmaceutical composition for preventing or treating diseases caused by at least one type of vascular endothelial cell senescence selected from the group consisting of cardiovascular diseases, chronic renal diseases, chronic inflammatory skin diseases, atherosclerotic inflammation and Parkinson's disease.
2. The method according to claim 1, wherein the fruit extract of the ground almond is extracted with at least one solvent selected from the group consisting of water, alcohol, ethyl acetate, chloroform and hexane. &Lt; / RTI &gt;
[Claim 5] The pharmaceutical composition according to claim 1, wherein the composition comprises a fruit extract of ground corn oil at a concentration of 5 to 130 [mu] g / ml.

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