WO2017034260A1 - Medicine containing natural raw material extract - Google Patents

Abstract

The present invention relates to an antioxidant or a pharmaceutical composition, containing, as an active ingredient, a composite distilled extract of bee venom, musk, and deer antlers. The present invention, unlike each individually distilled extracts of bee venom, musk, and deer antlers, exhibits an excellent antioxidant effect and can be useful for whitening, shrinking pores, and alleviating wrinkles. In addition, the present invention uses natural medicinal herbs, which have been proven to be safe, thereby being usable as a cosmetic material or a food.

Description

Medicines containing natural raw extracts

The present invention relates to an antioxidant and a pharmaceutical composition comprising a complex distillation extract of bee venom, musk and antler as active ingredients.

Bee venom is a toxin that comes from the bee's spawning tube. Raw bee venom has a specific gravity of 1.3, pH of 5.2, bitter taste, and weak fragrance. Evaporation at room temperature leaves about 30% dry matter, the composition of which is complex, and 75% of live bee venom is protein. Ingredients include potassium, sodium, magnesium, phosphorus, alanine, valine, arginine, tryptophan, methionine and histidine. Bee venom is widely used as a folk remedy and is known to have a particularly good effect on neuralgia, rheumatism and back pain (Doosan Encyclopedia).

Musk (麝香) is one of the animal flavorings, the musk deer, musk cat, musk rats are famous as a raw material for harvesting. The musk gland is located in the sachet of the subcutaneous subcutaneous region of the belly and navel of the musk deer male and is attached to the genital organs. A sachet is an egg-sized egg and weighs about 30 g. It is cut and dried to form a slightly damp purple-brown powder, sometimes mixed with granules. On the other hand, the musks of musk cats are located in the sachets of the groin in both females and males, and the secretion is classified as civet. Musk has been an oral medicine since ancient times as a strong heart, excitement, soothing agent, and when it faints, it is an inner medicine (chemical term dictionary, iljinsa).

Deer antler (鹿茸) refers to a medicinal herb that is collected by processing the horned young horns of various deer such as deer and manok. It is a weak child and has slow development or chondrosis.病) If you have hunger, taking antler not only promotes development and strengthens the skeleton, but also develops intelligence and increases the digestive absorption of the stomach. Deer antler contains a large amount of hormones and estrous hormones, which is known to have an excitement effect on sexual decline (Korea National University of Culture, Korea Research Institute of Korean Studies).

However, although bee venom, musk and antler have been proved usefully over a long time, the medical properties of the complex distillation extract have not been studied or utilized.

The matters described as the background art are only for the purpose of enhancing the understanding of the background of the present invention and should not be taken as acknowledging that they correspond to the related arts already known to those skilled in the art.

Natural herbal medicines such as bee venom, musk and antler are useful in our real life, but they have been used for limited purposes due to traditional extraction methods (for example, hot water extraction). In this regard, the present inventors made an effort such as attempting various extraction methods to develop a natural medicine using the herbal medicines. As a result, surprisingly, bee venom, musk or antler alone distillate extracts had no antioxidant effect, but in the case of their complex distillation extracts, they had anti-oxidant effects opposite to those of the distillate extracts alone, and showed an excellent skin improvement effect. The present invention has been completed.

Therefore, it is an object of the present invention to provide an antioxidant comprising a complex distillation extract of bee venom, musk and antler as an active ingredient.

Another object of the present invention to provide a cosmetic comprising the complex distillation extract as an active ingredient.

Another object of the present invention to provide a food comprising the complex distillation extract as an active ingredient.

Still another object of the present invention is to provide a method for preparing an antioxidant comprising bee venom and the complex distillation extract as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition comprising the complex distillation extract as an active ingredient.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides an antioxidant comprising a complex distillation extract of bee venom, musk and antler as an active ingredient.

According to another aspect of the present invention, the present invention provides a method for preparing an antioxidant comprising a complex distillation extract of bee venom, musk and antler as an active ingredient.

In order to develop natural antioxidants, the present inventors extracted natural herbal medicines such as bee venom, musk and antler by various extraction methods (hot water extraction, alcohol extraction, distillation extraction, etc.) and compared their effects for a long time. The natural herbal medicines were found to have a significant difference in clinical efficacy according to the extraction method, surprisingly, in the case of distillation, bee venom, musk or antler alone extracts had no antioxidant effect, but they were mixed and then distilled complex In the case of the extract, the present invention was completed by confirming that there was an anti-oxidant effect opposite to the above distilled extracts.

As used herein, the term "antioxidant" refers to a substance that inhibits the reaction of oxidation so as to eliminate the action of so-called oxidants, including hydroxyl radicals, superoxide anions, hydrogen peroxide, etc., called active oxygen or free radicals. Or, as a generic term for a substance to be attenuated, it is used in the same sense as the terms such as antioxidant composition, antioxidant, antioxidant composition.

As used herein, the term "distillation extraction" means the extraction method of mixing the herbal medicine with water (preferably, distilled water), and vaporizing it by heating or the like to obtain the active ingredient.

The manufacturing method of the present invention will be described in detail as follows:

(a) Bee venom Preparing a mixture of water, musk and antler in water

First, a mixture of bee venom, musk and antler is prepared in water. For example, 10 to 1,000 mg of bee venom (preferably lyophilized bee venom powder), 0.1 to 10 g of musk (preferably roe deer or epic) and 1.5 to 150 g of deer antler (preferably, Distilled water) to 1500 ml to prepare a mixture. Bee venom may be toxic when administered at high doses, and musk is an expensive herbal medicine that should not be mixed more than necessary.

(b) distilling the mixture to obtain a distillation extract

Next, the mixture is distilled off to obtain a distillation extract. Distillation extraction can be carried out using a variety of commercially available distillation extractors (for example, DM-100 or DM-200), for example, when using a DM-100 at a temperature of 100 to 110 ℃ 1 To extract for 5 hours.

As can be seen in FIGS. 6 and 7 of the present specification, the complex distillation extract of the present invention exhibited high antioxidant efficacy in contrast to the single distillation extract of bee venom, musk and antler, showing no antioxidant effect at all. As it increased, it showed more antioxidant efficacy, but the distillate alone did not.

According to another aspect of the present invention, the present invention provides a cosmetic or food comprising the antioxidant.

The antioxidant may be used for the purpose of whitening, pore reduction, wrinkle improvement, anti-aging, dermatitis (including atopic dermatitis), arthritis, immune strengthening, etc. may be used as a main ingredient or additives.

In the cosmetic of the present invention, the compounding ratio of the antioxidant of the present invention can be appropriately selected depending on the kind and the type, amount, form, and the like of the other components to be blended. It may comprise 0.001 to 20% by weight, preferably 0.01 to 10% by weight.

The cosmetic of this invention can be mix | blended with other various other components in the range which does not impair the desired effect of this invention. As other components, for example, purified water, emulsions, surfactants, lubricants, alcohols, gelling agents, moisturizers, buffers, preservatives, anti-inflammatory agents, thickeners, fragrances, vitamins, and the like are appropriately selected and blended. can do.

According to another aspect of the invention, the present invention provides a pharmaceutical composition comprising a complex distillation extract of bee venom, musk and antler as an active ingredient.

According to another aspect of the invention, the present invention provides a tyrosinase activity inhibitor comprising a complex distillate of bee venom, musk and antler as an active ingredient.

Tyrosinase activity inhibition experiment is a commonly performed experiment to show the melanin inhibitory effect, the complex distillation extract of the present invention showed a significant inhibitory effect on tyrosinase activity compared to the single distillation extract, Increased showed greater inhibitory efficacy (FIGS. 1-5).

According to another aspect of the invention, the present invention provides a pharmaceutical composition for the prevention or treatment of skin diseases comprising a complex distillation extract of bee venom, musk and antler as an active ingredient.

According to a preferred embodiment of the present invention, the skin disease for which the present invention is intended to prevent or treat is a skin whitening related disease.

Various medicines are currently available in Korea for the treatment of skin whitening-related diseases, and their official use as medicines is as follows:

① Domina Cream (Taeguk Pharmaceutical): Excessive pigmentation of the skin, such as: Gradual bleaching of the skin: liver spots, melanoma (freckle), freckles, senile black spots, excessive melanin pigmentation in other unnecessary areas,

② Trimina Cream (Taegeuk Pharm.): Short-term treatment of moderate to severe facial melanoma,

③ Neomina Cream (Taegeuk Pharm.): Treatment of symptoms of the following diseases: excessive melanin of the skin (brown spots), melanoma (freckle, freckles), hepatic spots, brown spots of skin after inflammation,

④ Biskin's liquid (Daehan New Farm): Gradual bleaching of excessive pigmentation skin such as: brown spots, melanoma (freckle), freckles, senile black spots, excessive melanin pigmentation in unnecessary areas.

Therefore, the pharmaceutical composition of the present invention is used for the prevention, improvement and treatment of skin whitening-related diseases such as melanosis, chloasma, blemishes, freckles, hepatic spots, brown spots of skin after inflammation and senile dark spots. It may be duly licensed and used.

According to a preferred embodiment of the present invention, the skin disease for which the present invention aims to prevent or treat is a skin wrinkle-related disease.

For the treatment of skin wrinkle-related diseases, various pharmaceutical products are also currently available in Korea, and their official use as medicines is as follows:

① Botox strain (Allergan Korea): Temporary improvement of the following upper facial wrinkles: moderate to severe severe glabellar wrinkles associated with corrugator muscle and / or procedure muscle activity, orbicularis oculi ) Moderate to severe external angle wrinkles (eye wrinkles) related to the activity, simultaneous treatment of moderate to severe eye wrinkles and brow wrinkles,

② Stea A Cream (Glaxos Mist Kline): Light aging (fine wrinkle) alleviation,

③ Emoticon Cream (Korean McNaughty): Alleviates photoaging (fine wrinkles, hyperpigmentation).

Accordingly, the pharmaceutical composition of the present invention may be applied to skin such as brow wrinkles associated with corrugator muscle or procerus muscle activity, external eye wrinkles associated with orbicularis oculi activity, and skin wrinkles due to photoaging. It may be formally licensed and used for the prevention, amelioration and treatment of wrinkle-related diseases.

According to another aspect of the present invention, the present invention provides an injection comprising the pharmaceutical composition.

In the injection of the present invention, the compounding ratio of the pharmaceutical composition of the present invention can also be appropriately selected depending on the kind and the type, amount, form, etc. of the other components to be blended. Usually, the pharmaceutical composition of the present invention is used for the total amount of the injection. And 0.1 to 99% by weight, preferably 10 to 90% by weight.

Injectables of the present invention may be blended with other various other ingredients without departing from the desired effect of the present invention. As another component, 1 type, or 2 or more types can be suitably selected and mix | blended with NaCl, NaOH, glucose, acupuncture base, other active ingredients other than the pharmaceutical composition of this invention, etc., for example.

Clinical results by a clinician composed of oriental medical doctors showed that skin whitening was improved by 14.0% to 32.1% in the patients who received the complex distillation extract of the present invention as a dermal injection, and 446 to 675 in the case of a decrease in the number of pores. It was confirmed that the pores of the dog was reduced, and in the case of wrinkle improvement, wrinkle improvement effects of 1.9% to 3.8% were confirmed (FIGS. 14 to 16).

The features and advantages of the present invention are summarized as follows:

(1) The present invention provides an antioxidant comprising a complex distillation extract of bee venom, musk and antler as an active ingredient.

(2) The present invention also provides a pharmaceutical composition comprising a complex distillation extract of bee venom, musk and antler as an active ingredient.

(3) The pharmaceutical composition of the present invention, unlike bee venom, musk and antler, alone distilled extract, shows excellent antioxidant efficacy and can be usefully used for the purpose of whitening and wrinkle improvement.

1 is a result of measuring the tyrosinase inhibitory activity of 5% distilled extract (Sample 1: distilled extract of bee venom; Sample 2: distilled extract of musk; Sample 3: distilled extract of antler; Sample 4: bee venom, musk And antler complex distillation extract).

Figure 2 is the result of measuring the tyrosinase inhibitory activity of 10% distilled extract (Sample 1: distilled extract of bee venom; Sample 2: musk distillation extract; Sample 3: distilled extract of antler; Sample 4: bee venom, musk And antler complex distillation extract).

Figure 3 is the result of measuring the tyrosinase inhibitory activity of the 20% distilled extract (Sample 1: distilled extract of bee venom; Sample 2: musk distillation extract; Sample 3: distilled extract of antler; Sample 4: bee venom, musk And antler complex distillation extract).

Figure 4 is the result of measuring the tyrosinase inhibitory activity of 40% distilled extract (Sample 1: distilled extract of bee venom; Sample 2: musk distillation extract; Sample 3: distilled extract of antler; Sample 4: bee venom, musk And antler complex distillation extract).

5 is a result of measuring the tyrosinase inhibitory activity of 50% distilled extract (Sample 1: distilled extract of bee venom; Sample 2: musk distillation extract; Sample 3: distilled extract of antler; Sample 4: beet venom, musk And antler complex distillation extract).

Figure 6 is the result of measuring the antioxidant capacity (DPPH radical scavenging activity capacity) of 50% distillation extract (Sample 1: distilled extract of bee venom; Sample 2: musk distillation extract; Sample 3: distilled extract of antler; Sample 4: Complex distillation extract of bee venom, musk and antler).

7 is a result of measuring the antioxidant activity (DPPH radical scavenging activity ability) of 60% distillation extract (Sample 1: distillation extract of bee venom; Sample 2: musk distillation extract; Sample 3: distilled extract of antler; Sample 4: Complex distillation extract of bee venom, musk and antler).

Figure 8 shows a schematic diagram and photograph showing the dorsal part of Brown guinea pig for whitening related in vivo experiments.

Figure 9 shows the treatment schedule of UV irradiation and sample for whitening related in vivo experiments.

10 shows photographs of dorsal tissue of Brown guinea pigs stained with Fontana-Masson staining. The red arrow indicates the concentration of melanin in the skin epidermis (normal group: no UVB irradiation group, UVBcont group: UVB irradiation group, Arbutin group: 1% arbutin group, JDH 150ul group: Group which processed 150 uL of the sample of manufacture example 2, JDH 300ul group: group which processed 300 uL of the sample of manufacture example 2).

11 is a photograph showing the results of the measurement of the skin surface with a contact melanin measuring device (Dermacatch) (normal group: UVB irradiated group, UVBcont group: UVB irradiated group, arb group: arbutin 1% treated group , JDH-L group: 150 uL of the sample of Preparation Example 2, JDH-H group: 300 uL of the sample of Preparation Example 2, UVBcont 'group: MTS (microneedle therapy system) Treated and irradiated with UVB, arb 'group: treated with MTS and treated with arbutin, JDH-L' group: treated with MTS and treated with 150 uL of the sample of Preparation Example 2, JDH-H 'group: MTS Treated with 300 uL sample of Preparation Example 2)

12 is a photograph of the change of the epidermal layer of the skin using H & E (Hematoxylin & eosin) staining method (normal group: UVB irradiated group, UVBcont group: UVB irradiated group, PC 1% group: a positive control Reti Neil palmitate (retinyl palmitate 1%) treated group, JDH 150 uL group: 150 uL treated sample of Preparation Example 2, JDH 300 uL group: 300 uL treated sample of Preparation Example 2).

Figure 13 is a photograph of the change of the dermal layer of the skin using Masson trichrome staining method (normal group: UVB irradiated group, UVBcont group: UVB irradiated group, PC 1% group: positive control group retinyl palmitate ( retinyl palmitate 1%), JDH 150 uL group: 150 uL of the sample of Preparation Example 2, JDH 300 uL group: 300 uL of the sample of Preparation Example 2).

14 is a clinical result confirming the whitening function using the complex extract of the present invention (Fig. 14a: 20s patients; Fig. 14b: 40s patients; Fig. 14c: 50s patients).

15 is a clinical result confirming the reduction in the number of pores using the complex extract of the present invention (Fig. 15a: patient 1 in 50's; Fig. 15b: patient 2 in 50's; Fig. 15c: patient in 60's).

Figure 16 is a clinical result confirming the improvement of wrinkles using the complex extract of the present invention (Fig. 16a: patient 1 in 40's; Fig. 16b: patient 2 in 40's; Fig. 16c: patient in 70's).

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Preparation of Experimental Materials

1. Preparation Example 1 Preparation of Sample (Distillate Extract)

Distilled extract of bee venom was poured into 100 mg of lyophilized bee venom powder (oil sealed bee venom) and poured 1,500 ml of tertiary distilled water into a distillation extractor (DM-100), extracted for 3 hours at 107 ° C, and filtered three times. ML was obtained. The distilled extract of musk was poured 1,500 ml of tertiary distilled water into 1 g of musk (our herbal medicine) and put into the distillation extractor and extracted for 3 hours at 107 ° C. and filtered three times to obtain 1,000 ml of distilled extract. The distilled extract of antler was poured 1,500 ml of tertiary distilled water into 15 g of antler (non-sealed pharmaceutical), put in a distillation extractor, extracted for 3 hours at 107 ° C., and filtered three times to obtain 1,000 ml of distilled extract. The mixed distillate extract of bee venom, musk and antler is mixed with 100 mg of bee venom powder, 1 g of musk and 15 g of antler, and then poured 1,500 ml of distilled water into the distillation extractor, extracted at 107 ° C. for 3 hours, and filtered three times. 1,000 ml of distilled extract were obtained. The distillation extract of bee venom sample 1 (Sample 1), the distillation extract of musk sample 2 (Sample 2), the distillation extract of deer antler sample 3 (Sample 3), and the mixed distillation extract of bee venom, musk and deer antler sample 4 It was designated as (Sample 4) and used for the experiment.

2. Preparation Example 2: Preparation of Pharmaceuticals

1,000 mL of Sample 4 was prepared using NaCl, NaOH, medicinal needle base (Medical Co., Ltd.), pH 7.0, and 0.9% isotonic solution, and then filtered three times with a PES 0.1 filter. Necessary vials, rubber jars and aluminum caps were sterilized, subdivided into 20 ml portions of vials, capped with rubber jars and aluminum caps, and put into autoclave and autoclaved. Foreign body test was carried out through a foreign body tester, microbiological test and endotoxin test was performed, and then labeled JDH and used for the experiment.

In vitro experiment

1. Measurement of extracellular tyrosinase inhibitory activity

Tyrosinase activity was measured by colorimetric measurement of dopachrome resulting from the action of tyrosinase. 1250 unit / mluL of enzyme (mushroom tyrosinase) purchased from Sigma (St. Louis, MO, USA), L-DOPA (dihydroxyphenylalanin) was 5 mM, 0.1 M potassium phosphate buffer (pH6.8) mixed solution as a substrate After addition with the reaction for 15 minutes at 37 ℃ was measured for absorbance using a microplate reader at 475 nm. As a control, kojic acid (KA), which is known as a melanogenesis inhibitor, was used. Tyrosinase inhibitory activity was calculated by the following formula:

% Inhibition = 1- (A-B) / (C-D) x 100

A: sample treatment tool to which enzyme solution was added

B: Sample Treatment Port Without Enzyme Liquid

C: Absorbance after adding enzyme solution and distilled water instead of sample

D: absorbance added with distilled water instead of enzyme solution and sample

In vitro tyrosinase inhibitory activity assay with 5% concentration of samples showed that sample 1 and sample 2 did not noticeably decrease tyrosinase activity, while sample 3 slightly decreased, compared to sample 4 It was found that tyrosinase activity was markedly reduced (FIG. 1).

Activity was measured by increasing the volume of the sample to 10%, 20%, 40% and 50%. In case of Sample 4, it was confirmed that the degree of inhibition of tyrosinase activity increased as the concentration was increased (FIGS. 2 to 5).

2. DPPH  Free radical scavenging activity measurement

DPPH assay was performed to measure the antioxidant activity. DPPH (2,2-diphenyl-1-pycryl-hydrazyl, sigma) was dissolved in ethanol to make concentrations (300 uM, 375 uM, 500 uM, 750 uM). As a positive control, Vitamin C was dissolved in water and used at 200 ppm. DPPH solution and samples were treated by concentration in 96 well plates, Incubation was performed at 37 ° C. for 30 minutes, and the absorbance was measured at 517 nm.

Erasure Activity (%) = (1-OD _sample / OD _control ) x 100

In DPPH Assay, the other samples showed little antioxidant activity, while sample 4 showed antioxidant activity. The scavenging activity (46.98%) was higher at the 60% concentration (Fig. 7) than the scavenging activity at the 50% concentration (Fig. 6) (30.32%).

In vivo vivo ) Experiment

1. Measure whitening efficacy

The most widely used animal model to measure the whitening effect is brown guinea pig. Brown guinea pig, which is changed by UV, shows that the change in skin is almost similar to that of human body by UV. similar. Brown guinea pigs were purchased from Saronbio, four male 6-week-old males, and then purified in a two-week test room. Examination of the health and pathogen test results of the test system showed no factors that could adversely affect the test. Only normal animals were provided for the test by observing the general symptoms during the acclimation period. This test consists of 23 ± 3 ℃, 55 ± 15% relative humidity, 10 to 20 ventilation / hr, 12 hours lighting time (8 am to 8 pm), and light intensity of 150 to 300 Lux. The experiment was conducted at the Laboratory Animal Lab, 5th Floor, College of Life Sciences. Animals were bred in a polycarbonate breeding box, one per breeding period, quarantine period, and dosing period, respectively. The breeding boxes were identified by attaching individual identification cards containing test numbers and animal numbers. Feed and water were free.

The test group consisted of the normal group not irradiated with UVB, the UVBcont group irradiated with UVB, the arb group treated with arbutin (arbutin, Sigma), JDH-L group treated with 150 uL of the sample of Preparation Example 2, Preparation Example 2 JDH-H group treated with 300 uL of sample, UVBcont 'group treated with MTS (microneedle therapy system), UVB irradiated, arb' group treated with MTS, arbutin treated, MTS JDH-L 'group treated with 150 uL of sample 2, and JDH-H' group treated with 300 μL of sample of Preparation Example 2 in total, were 9 groups, and the back portion of Brown Guinea Pig was divided into 9 parts. Experiment was performed (FIG. 8).

Shave the hairs of the brown guinea pig and shave clean with a razor. Then, an anesthetic is made by mixing zoletil: ketamine in a ratio of 3: 1, and then anesthetized by intraperitoneal injection. ) Was bonded to a rectangular plate and irradiated 3 days a week for 3 weeks at a strength of 350 mJ / cm ² / Day to irradiate a total of 3150 mJ / cm ² UVB. Treatment of Test Substances Arbutin (1%) and the sample of Preparation Example 2 were applied to the back portions of the guinea pigs divided into each test group for 3 days and 3 weeks per week after irradiation with UVB (FIG. 9).

After completion of the experiment, the melanin distribution of the entire epidermal layer was observed by Fontana-Masson staining. Fontana-Masson staining is an immunological tissue staining method that can identify melanin pigments distributed throughout the epidermal layer. As a result, it was confirmed that melanin was excessively produced in the UVBcont group compared to the normal group, and less melanin was produced in the positive control group arbutin and the sample treatment group of Preparation Example 2. In particular, it was confirmed that the melanin production of the JDH-H (Preparation Example 2: 300 uL) group was excellent enough to be similar to the distribution of the Normal group, UVB untreated group. No significant difference was found between the MTS untreated and MTS treated groups (FIG. 10).

In addition, as a result of measuring the surface of the skin with a contact melanin measuring device (Dermacatch), it was confirmed that the melanin significantly increased in the skin tissue of the group irradiated with UVB. Compared to the normal group, the UVBcont group without MTS treatment increased by 197% and the UVBcont 'group with MTS treatment increased by 220%. In the MTS-treated group, 18 days after the test substance treatment (6nd), the arb group decreased by 26%, the JDH-L group by 22%, and the JDH-H group by 31% compared to the UVBcont group. In the MTS-treated group, 18 days after the test material treatment (6nd), compared with UVBcont 'group, 32% in the arb' group, 31% in the JDH-L 'group, and 50% in the JDH-H' group ( 11).

2. Wrinkle improvement efficacy measurement

Wrinkle improvement efficacy was measured by hairless mice (Hairless mice) to cause photoaging by UVB irradiation to analyze wrinkle formation and improvement. Hairless mice were purchased from 25 animals of 6 weeks old from the central laboratory animals and divided into 5 groups. The breeding environment of the laboratory was the same as that of the whitening efficacy measurement.

The test group consisted of a normal group not irradiated with UVB, a UVBcont group irradiated with UVB, a PC 1% group treated with retinyl palmitate (1%, 300 uL) as a positive control, and a sample of Preparation Example 150. The uL-treated JDH 150 uL group, the sample of Preparation Example 2 was divided into 300 uL-treated JDH 300 uL group, the other groups except the normal group was irradiated with UV of 200 mJ / cm ² daily at week 1, Photoaging was induced by UV irradiation of 400 mJ / cm ² twice a week from week 2 to week 4. Treatment of the sample was applied for 3 days (3 weeks-4 weeks), 3 days a week after irradiation with UVB.

After the end of the experiment, the tissues of the normal group, UVBcont group, PC 1% group, JDH 150 uL group and JDH 300 uL group were stained by H & E (Hematoxylin & eosin) staining method to observe the change of the skin epidermal layer. As a result, it was observed that the outer epidermal tissue was enlarged by the effect of UV in the UVBcont group compared to the normal group. However, in the PC 1% group, JDH 150 uL group and JDH 300 uL group showed a thickness of the epidermal layer similar to the normal group, it was confirmed that the samples of Preparation Example 2 has an effect of protecting the epidermal tissue from photoaging. (FIG. 12).

In addition, in order to observe the change in the dermal layer of the skin was confirmed by staining the tissue of the normal group, UVBcont group, PC 1% group, JDH 150 uL group and JDH 300 uL group by Masson trichrome staining method. As shown in FIG. 12, collagen of the dermal layer of the skin was dyed blue. In the UVBcont group, the collagen of the dermal layer was not dense and separated from each other in a large part, which can be considered to cause skin wrinkles due to photoaging. . However, in the PC 1% group, JDH 150 uL group and JDH 300 uL group showed a dense layer of the dermal layer similar to the normal group, it was confirmed that the samples of Preparation Example 2 have an excellent effect of protecting the dermal tissue from photoaging. (FIG. 13).

Clinical trial

1. Clinical whitening confirmation

Using the sample of Preparation Example 2, three patients were subjected to clinical trials to confirm the whitening function. After washing the patient before the procedure, the skin condition of the patient was measured by DermaVision (Optobiomed Co., Ltd.), and 10 ml of the sample was evenly administered to the face of the patients by injection. The same dose was administered to the patient's face after skin measurements in the same manner three times every two weeks. During the procedure, in order to increase the reliability of measuring skin condition changes of patients, excessive stimulation on the face such as excessive exercise / health / sauna / drinking / massage was prohibited, and sunscreen was used when going out.

DERMA VISION is a device that analyzes the skin tone according to melanin and erythma pigment of the skin.When there are many melanin and erythma pigments, the skin tone appears dark (abnormal skin condition: Yellow / Red / Dark Red). Skin tone appears bright (normal skin condition: Dark Blue / Blue). After skin care or treatment, the percentage of abnormal skin color value decreases (-) and the normal skin color percentage increases (+), indicating a good effect.

A total of three administration results for patients in their 20s, 40s and 50s showed skin tone improvement effects of 14.0% to 32.1%, as shown in Table 1 below (FIGS. 14A to 14C).

Clinical results of whitening after 3 doses

Change rate after administration	Normal skin condition		Abnormal skin condition
	Dark blue	Blue	Yellow	Red	Dark red
20's patient	0.0%	+ 18.3%	-6.1%	-11.4%	-0.8%
40's patient	0.0%	+ 14.0%	-0.7%	-12.9%	-0.4%
50's patient	0.0%	+ 32.1%	-25.5%	-6.5%	-0.1%

2. Clinical confirmation of reduced pore count

In the same manner as the whitening functional confirmation clinic, three patients (two in 50's and one in 60's) were performed to confirm the reduced number of pores. Dermavision counts the reduced number of pores after treatment or care of pores, and as a result of the experiment, it was confirmed that 446 to 675 pores were reduced (FIGS. 15A to 15C).

Confirmation of pore reduction after 3 doses

	Before dosing	After administration	Reduced pore count
Patients in their 50s	12,450	12,004	446
Patients in their 50s	12,898	12,278	620
60's patient	14,319	13,644	675

3. Wrinkle improvement confirmed clinical

In the same manner as the whitening functional confirmation clinic, three patients (two 40s, one 70s) were performed to confirm the improvement of wrinkles. DERMAVISION shows the curved surface of the skin as a percentage (%) to measure the degree of wrinkles, and as a result of the experiment was able to confirm the wrinkle improvement effect of 1.9% to 3.8% (Fig. 16A to 16C). .

 Wrinkle improvement confirmed after 3 doses

	Before dosing	After administration	Wrinkle reduction rate
40s Patient1	23.4%	21.5%	1.9%
Patients in their 40s	24.2%	20.4%	3.8%
70's patient	32.8%	29.6%	3.2%

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (10)

1. Antioxidant comprising a complex distillate extract of bee venom, musk and antler.
2. Cosmetics containing the antioxidant of claim 1.
3. Food comprising the antioxidant of claim 1.
4. A method for preparing an antioxidant comprising a complex distillation extract of bee venom, musk and antler, comprising the following steps:

   (a) preparing a mixture of bee venom, musk and antler in water; And

   (b) distilling the mixture to obtain a distillation extract.
5. The method according to claim 4, wherein the step (a) comprises mixing 10 to 1,000 mg of bee venom, 0.1 to 10 g of musk and 1.5 to 150 g of antler in 1,500 ml of water.
6. The method of claim 4, wherein the step (b) is characterized in that the extraction for 1 to 5 hours at a temperature of 100 to 110 ℃.
7. A pharmaceutical composition for the prevention or treatment of skin diseases, comprising a complex distillation extract of bee venom, musk and antler.
8. The pharmaceutical composition according to claim 7, wherein the skin disease is a skin whitening-related disease or a skin wrinkle-related disease.
9. The method of claim 8, wherein the skin disease is a skin whitening-related disease selected from the group consisting of melanosis, chloasma, blemishes, freckles, hepatic spots, brown spots of skin after inflammation and senile black spots. A pharmaceutical composition characterized by.
10. 9. The skin disease of claim 8, wherein the skin disease is a brow wrinkle associated with corrugator muscle or proerus muscle activity, an external angular wrinkle associated with orbicularis oculi activity, and skin wrinkles due to photoaging. A pharmaceutical composition, characterized in that the skin wrinkle-related diseases selected from the group consisting of.

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