KR100571886B1 - Composition for skin cancer prevention and therapy comprising asiatic acid - Google Patents

Abstract

The present invention relates to a composition for preventing or treating skin cancer, and more particularly, a cosmetic composition or pharmaceutical composition containing asiatic acid not only induces apoptosis but also significantly inhibits tumor formation, thereby preventing skin cancer. Or it can be very useful for treatment.

Asiatic acid, skin cancer

Description

Composition for skin cancer prevention and therapy comprising asiatic acid}

1 shows a decrease in cell viability following treatment with a composition of the present invention in SK-MEL-2 human melanoma,

Figure 2 shows the concentration-dependent apoptosis induction according to the treatment of the composition of the present invention in SK-MEL-2 human melanoma,

Figure 3 shows the activity of kerose phase-3 in SK-MEL-2 human melanoma treated with the composition of the present invention,

Figure 4 shows the degree of ROS occurring in SK-MEL-2 human melanoma treated with the composition of the present invention,

5 shows that apoptosis induced by the composition of the present invention is blocked by an antioxidant and a casease-3 inhibitor,

Figure 6 shows the expression level of Bax, Bcl-2 and p53 according to the treatment of the composition of the present invention in SK-MEL-2 human melanoma,

Figure 7 shows the anticancer effect of treatment with the composition of the present invention in DMBA-initiated and TPA-promoted mouse skin tumorigenesis.

The present invention relates to a composition for preventing or treating skin cancer, including asiatic acid, which not only induces apoptosis but also significantly inhibits carcinogen-induced tumor formation, which can be very usefully used for preventing or treating skin cancer. .

Skin cancer refers to all cancers that occur in the skin and is a relatively common cancer, but because of the low risk of life, such as stomach cancer, liver cancer, lung cancer is not considered important. It does not require complicated examinations or expensive medical equipment compared to other cancers, and can be easily detected with the naked eye and diagnosed through simple biopsy. Many skin cancer patients are simply overlooked with dermatitis or spots or boils when the first skin cancer occurs, so it is difficult to cure the disease and sometimes life is lost because of early detection of the disease.

There are many different types of skin cancers, depending on what kind of skin cells they are. Many of these skin cancers are rarely metastatic and easy to cure, while others have a poor prognosis even at an early stage. Therefore, diagnosing what kind of skin cancer is very important for the prognosis of skin cancer. It is common and occupies a large proportion of these skin cancers, including basal cell carcinoma, squamous cell carcinoma, and melanoma.

Basal cell carcinoma is the most common malignant tumor occurring in humans, including skin. It is known that the incidence rate is higher in men than in women, but recently the gap is slowing down, and often occurs after the 50s, but also in young people. This is due to the suntan for the purpose of skin beauty, but it is thought to be the destruction of the ozone layer that can filter out the range of skin cancer in the ultraviolet rays of the sun's rays. The most important trigger is ultraviolet radiation, which can occur where the trauma, scar, or burn was present. The skin symptom is one or several small nodules gradually enlarged, the central part is depressed, the area is ulcer or crust or crust, and the periphery is slightly embankment shaped. Except for easy bleeding by stimulation, there is no subjective symptoms and is often overlooked by the patient. It is primarily related to UV rays, so it is most common on the face, especially on the nose, forehead, ears and cheeks.

Squamous cell carcinoma is the second most common malignancy after basal cell carcinoma. It is mostly in the elderly population, and the incidence in men is about twice as high as in women. The causes of squamous cell carcinoma, like basal cell carcinoma, are an important cause of ultraviolet light, as well as genetic factors, various chemicals known as carcinogens, radiation, papilloma virus, and burn scars. Symptoms often occur in places exposed to ultraviolet light, such as the face or back of the hand, with a hard boil that covers the surface with ulcers.

Melanoma is a skin cancer that commonly occurs in whites, but rarely occurs in Asians and blacks, but the skin is white and frequently occurs in people with sunburn. The prognosis is worse than that of other skin cancers, so early relapses are common and can shorten life. Skin lesions appear as yellowish brown or blackish spots or nodules, usually caused by sun exposure after middle age. Occasionally it occurs at the ends of hands, toes, especially the thumb and toes, and black vertical lines can be seen on the nails. At this time, the disease should be thoroughly investigated.

As in the treatment of skin cancer, early diagnosis and resection are fundamental to the treatment of cancer in other areas. There are various methods for the treatment of basal cell carcinoma, such as surgery, cryo-surgery, electrosurgery, ionizing irradiation, curettage and surgery, but the patient's age, location, lesion size, primary or recurrence, histological characteristics, one-shot Multiple factors are factors to be considered in the selection of treatment. One method may not be ideal, but is generally chosen with reference to considerations. In the case of squamous cell carcinoma and melanoma, the various treatments are used, and chemotherapy may be required in some cases. However, advanced squamous cell carcinoma and melanoma often have a poor prognosis despite treatment, so early diagnosis and treatment are very important.

However, the therapeutic drugs for skin cancers developed so far are only to alleviate the symptoms of skin cancer, not only the basic treatment, but many side effects are known. That is, until now, there is almost no fundamental treatment for treating skin cancer.

Accordingly, the present inventors have tried to develop a new prophylactic or therapeutic agent for skin cancer, which is effective and can cause treatment even though there are few side effects, and as a result, the composition containing asiatic acid not only induces apoptosis but also causes skin tumor formation. The present invention has been completed by revealing suppression.

Therefore, the present invention not only induces apoptosis, but also significantly inhibits carcinogen-induced tumor formation, and thus a composition for preventing or treating skin cancer comprising asiatic acid, which can be very usefully used for preventing or treating skin cancer. The purpose is to provide.

The present invention provides a pharmaceutical composition for preventing or treating skin cancer containing asiatic acid as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or treating skin cancer containing asiatic acid as an active ingredient.

The asiatic acid is an ingredient contained in a quantitative extract of a plant called centella asiatica. Centella asiatica is widely used in subtropical regions of the Indian Ocean and has been used by folk medicine to treat leprosy, particularly the ulcers of leprosy.

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

The present invention relates to a composition for preventing or treating skin cancer, including asiatic acid, which not only induces apoptosis but also significantly inhibits carcinogen-induced tumor formation. .

The composition comprises 0.01 to 50% by weight of asiatic acid, preferably 0.05 to 10% by weight, more preferably 0.10 to 5% by weight.

In the present invention, as shown in Figure 1, it was confirmed that the composition of the present invention containing asiatic acid inhibits the cell survival rate of human melanoma in a concentration-dependent manner.

In FIG. 2, it was confirmed that the composition of the present invention containing asiatic acid induces apoptosis in a concentration-dependent manner by flow cytometry.

In FIGS. 3 and 5, the composition of the present invention containing asiatic acid induces the activity of kephas-3 in a concentration-dependent manner, and the activity of kephase-3 derived with the composition of the present invention containing asiatic acid is shown in FIG. It was confirmed that it is inhibited by an inhibitor of -3.

In Figure 7, it was confirmed that the composition treatment group of the present invention containing asiatic acid is reduced tumorigenicity compared to the group not treated with asiatic acid.

The compositions of the present invention can be administered in parenteral transdermal formulations during actual clinical administration. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and the like. Pharmaceutically acceptable solvents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. .

If necessary, an appropriate absorption accelerator may be further added for the purpose of promoting the transdermal absorption of the composition of the present invention. Examples of absorption accelerators include isopropyl myristate, diethyl sebacate, sorbitan monolaurate, glycerol monooleate, sodium oleate phosphate, sodium lauryl sulfate, octylphenyl ether, adducts of polyethylene glycol and octyl phenyl ether, la Uryl ether, adduct of polyethylene glycol and lauryl ether, sorbitan monooleate, adduct of polyethylene glycol and sorbitan and monooleate, lauroyl diethanolamide, lauyl sarcosine ole oil sarcosine sugar ester, lecithin , Glycyrrhetitin, urea, salicylic acid, calcium thioglycolate, lactic acid, lactate esters, olive oil, squalene, lanolin, liquid paraffin and glycerin.

Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

In the pharmaceutical composition for preventing or treating skin cancer, the effective dose of the asiatic acid of the present invention is 0.10 μg / cm ² to 5.00 μg / cm ² , preferably 0.50 μg / cm ² to 2.50 μg / cm ² , and a day It can be applied to the skin 1-6 times. However, the dosage level for a particular patient may vary depending on the weight, age, sex, health condition, diet, time of administration, severity of the disease, and the like of the patient.

In particular, the pharmaceutical composition of the present invention was administered parenterally to the parenterally, and as a result of the toxicity test, 50% lethal dose (LD ₅₀ ) by the toxicity test was found to be a stable substance of more than 0.1 mg / cm ² asiatic acid. Stability is secured.

The present invention also provides a cosmetic composition for preventing or treating skin cancer containing asiatic acid.

When Asiantic acid is used as a cosmetic, the substance may be added as it is or used with other cosmetic ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be appropriately determined according to its purpose of use (prevention, health or therapeutic treatment), and can be used according to the effective dose of the pharmaceutical composition, and the active ingredient has no problem in terms of safety. It may also be used in an amount above the above range. There is no particular limitation on the kind of the cosmetic.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Measurement of Cell Viability

1. Cell Culture

SK-MEL-2 human melanoma was used from the American Type Culture Collection (Rockville, Mass.). SK-MEL-2 cells are powdered Eagle's minimum essential medium, Sigma Chemical CO containing 10% FBS (fetal bovine serum, GIBCO), 200 IU / ml penicillin, 200 mg / ml streptomycin, 1 mM sodium pyruvate Incubated at 37 ° C. in a humidified incubator under 5% CO ₂ /95% air. At this time, the culture medium was replaced once every other day. After growing confluent, the cells were trypsinized and then subcultured.

2. Measuring Cell Viability (MTT Essay)

Cell viability was performed by the MTT staining method (J Immunol Methods, 141, 15-22, 1991). That is, cells cultured for 4 to 5 days were dispensed in a 24-well plate at a density of 5 × 10 ⁴ cells / well. The media dose of each well was set at 1 ml. As a control group, cells grown in the same medium without drug treatment were used. After incubation with 10, 20, 30, 40 and 50 μM of asiatic acid for 48 hours, 100 μl of MTT (5 g MTT / 1 in H ₂ O) was added and the cells were further incubated for 4 hours. 200 μl of dimethylsulfoxide (DMSO) was added to each cell and mixed by pipette to dissolve the reduced MTT crystals. Relative cell viability was measured by scanning with a microplate reader (Molecular Devices, Menlo Park, CA) with a 540 nm filter.

As a result, as shown in FIG. 1, it was confirmed that cell viability was suppressed in a concentration-dependent manner of asiatic acid.

Example 2 Flow Cytometry Analysis of Apoptosis

For flow cytometry analysis, cultured cells were collected and washed twice with PBS buffer (pH 7.4). After 30 min fixation with 80% ethanol, cells were washed twice and 0.1% tryptone X-100, 5 mg / ml propium iodide and 50 mg / ml ribonuclease for DNA staining Resuspend in PBS buffer containing A (pH 7.4). Cells were then analyzed by FACS Calibur (Becton Dickson, USA). More than 20,000 cases were evaluated. All histograms are measured using the cell Quest (Becton Dickson, USA) and the percentage of nucleic acid is determined by the apoptotic hypodiploid content indicator.

The normal lipid composition of the plasma membrane changes immediately after apoptosis begins. Annexin-V, a Ca ²⁺ -dependent phospholipid-binding protein with high affinity with phosphatidylserine (PS), was used for the measurement of early apoptosis (J Immunol Methods, 184, 39-51, 1995). This was analyzed using a commercial kit (Boehringer Mannheim Biochemicals, Mannheim, Germany). Cells were washed in cold PBS and resuspended in bound buffalo. Some cell suspension (500 ml) was exposed to Annexin-V-FITC. Cells were gently vortexed and incubated in the dark for 20 minutes at room temperature and then measured using FACS Calibur within 1 hour of staining.

As a result, in FIG. 2A, asiatic acid caused a decrease in phospholipid symmetry, resulting in the appearance of phosphatidylserine in the outer layer of the plasma membrane by Annexin-V binding.

2B and 2C, it was confirmed that asiatic acid induces apoptosis in a concentration-dependent manner.

<Example 3> ROS generation measurement (nitroblue tetrazolium reduction assay)

Intracellular superoxide production was measured using the conversion of nitroblue tetrazolium (NBT) to formazan. NBT was added to the cell medium to give a final concentration of 1 mg / ml. After Asiantic acid treatment, the cells were lysed and the formazan formed was lysed with 2M KOH and 1.4 ml of DMSO. The absorbance was measured on a 654 nm spectrophotometer.

In Figure 4A, the amount of superoxide generated per hour of Asiantic acid treatment was measured. Treatment was performed 30 minutes and 2 hours prior to (40 μM) treatment, and cell viability was measured. It was confirmed that the caspase inhibitor inhibited asiatic acid-induced cell death.

Example 4 Western Blotting Analysis

Cells were washed with PBS solution and centrifuged at 1,000 g for 5 minutes. Cell pellets were 50 mM Hepes (pH 7.4), 0.5% Nonidet P-40, 10% glycerol, 137 mM NaCl, 1 mM ethylene glycol-bis- (aminoethyl ether) N, N, N, N-tetraacetic acid [ethylene glycol -bis- (aminoethyl ether) N, N, N, N-tetraacetic acid, EGTA], 10 mM NaF, 1 mM benedate, 1 mM phenylmethylsulfonyl fluoride, 2 μg / ml lupepine, 2 μg / ml Lysis was performed at 4 ° C. for 15 minutes in whole cell extract buffer containing aprotinin, 1 μg / ml pepstatin A, 40 mM α-glycerophosphate, 0.1 mM dithiothreitol (DTT). . Cell lysates were centrifuged at 20,000 g for 10 min at 4 ° C., and supernatant proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and Hybond ECL at 30 V overnight. Transferred to nitrocellulose membrane (Amersham Life Science, Buckinghamshire, England).

The membrane was blocked with 5% skim milk in Tween-20 containing Tris buffered cell line (TTBS, 20 mM Tris-HCl, pH 7.6), and the primary antihuman Bax, Bcl- in TTBS containing 5% skim milk. 2, p53 antibody (Santa Cruz Biotechnology, California, USA). After incubation with a horseradish peroxidase-conjugated anti-IgG antibody, the immunodetected proteins were subjected to an enhanced chemiluminescent assay kit (Amersharm Life Science, Buckinghamshire, England). Visualization.

In FIG. 6A, maximal expression of Bax was induced at 12 hours by asiatic acid, but did not affect the expression of Bcl-2. 6B, Trolox inhibited Bax expression by Asiantic acid. 6C confirmed that asiatic acid did not contribute to p53 expression at all.

Example 5 Casephase Activity Essay

Caspase activity was measured using the ApoAlert Kephas Chlorimetric Essay Kit (BD Biosciences, CA, USA). The cell lysates were mixed with a 50 μM Ac-Asp-Glu-Val-Asp-CHO (DEVD) -pNA, DTT (10 mM) -rich reaction buffer containing the Kephase-3 substrate and incubated for 1 hour at 37 ° C. . The glass of enzyme catalyzed pNA was monitored using a microplate reader at 405 nm.

In FIG. 3, it was shown that the activity of kephase-3 was induced depending on the concentration of asiatic acid, and the activity of kephase-3 induced by asiatic acid was inhibited by an inhibitor of the cephase-3. In Figure 5 it was confirmed that the apoptoses induced by asiatic acid by the caspase inhibitors.

Thus, it was judged that asiatic acid induced apoptosis was mediated by activation of kease phase-3.

Example 6 Tumorigenesis Essay

Female 6-week-old ICR mice (Hyochang Science, Korea) were housed in a well-ventilated polypropylene cage, 10 per cage. Each mouse was fed feed and water optionally. Mice were shaved with an electric varican after applying hair removal cream on their back skin at least 2 days prior to drug treatment. Only mice that showed no signs of hair regeneration were used for tumor induction experiments.

First, mice were divided into three groups of 20 animals in each group. Shaved backs of all groups of mice were treated with one-time topical administration of DMBA (7,12-dimethylbenz [a] anthracene, 40 μg) dissolved in 0.2 ml acetone. The first group was a positive control group, which was treated with 200 µl of acetone per mouse and then treated with 5 µg of TPA (12-o-tetradecanoyl-13-phorbol acetate) dissolved in 0.2 ml of acetone. In the second group, 200 μl of acetone in 30 μM Asiantic acid was applied locally to each mouse, and then treated with 5 μg of TPA dissolved in 0.2 ml acetone every three weeks for 15 weeks. The third group was treated in the same manner as the second group except that 200 µl of acetone in which 30 µM Asiantic acid was dissolved was applied locally. Tumor development was assessed by palpation and observation every 3 days.

As a result, as shown in FIG. 7, it was confirmed that the group treated with asiatic acid had significantly less tumor occurrence than the group not treated with asiatic acid.

Experimental Example 1 Toxicity Test During Transdermal Administration

A 6-week-old female ICR mouse (Hyochang Science, Korea) was applied to the dorsal skin (4 cm ² ) once to examine the number of mice killed. Single application did not show toxicity at doses of 0.1 μg / cm ² to 10 μg / cm ² . In addition, multiple application toxicity experiments were conducted twice a week for 22 weeks by applying asiatic acid, and mice were not killed even at a dose of 0.1 mg / cm ² .

Preparation Example 1 Preparation of Transdermal Preparation

A transdermal formulation consisting of 0.25 wt% asiatic acid, 10 wt% stearic acid, 20 wt% stearic alcohol, 5 wt% butyl stearate, 7 wt% glycerin mono stearic acid ester, 10 wt% propylene glycol, and the residual purified water was prepared.

Production Example 2 Preparation of Varnishing Cream

0.25 wt% asiatic acid, 20 wt% liquid paraffin, 5 wt% lanolin, 1 wt% talc, 10 wt% solid paraffin, 0.01 wt% titanium dioxide, 2 wt% kaolin, 5 wt% sorbitan seski, oleic acid ester 10 A foundation cream was prepared, consisting of weight percent, sodium benzoate 0.1 weight percent, dibutyl hydroxytoluene 0.1 weight percent, and the remainder of the purified water.

As described above, the composition according to the present invention not only induces apoptosis, but also significantly inhibits carcinogen-induced tumor formation, and thus may be very useful for preventing or treating skin cancer.

Claims (3)

A pharmaceutical composition for transdermal administration comprising asiatic acid in an effective dose of 0.10 μg / cm 2 to 5.00 μg / cm 2 and applied to the prevention or treatment of human melanoma, which is skin cancer. The pharmaceutical composition for transdermal administration according to claim 1, wherein the effective dose of asiatic acid is 0.50 µg / cm 2 to 2.50 µg / cm 2. delete

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