JP2015509527A - Method of extracting giant hornet venom and functional cosmetic composition using the same - Google Patents

Abstract

The present invention relates to a method for extracting a giant hornet venom for producing a functional cosmetic composition, a preparatory process of putting an ethanol solution with an alcohol concentration of 15% to 50% into a storage container, and a working bee among living giant hornets, 1 liter of the ethanol solution. In the storage container at a rate of 15 to 100, the lid was sealed, and then the giant hornet was introduced into the ethanol solution so that the movement was slowed down. It is characterized in that a series of processes including an aging process for aging for at least one month, preferably for one year or more, and an aging liquid separation process for separating the ripened liquid are continuously performed.

Description

  The present invention relates to a method for producing a functional cosmetic composition using giant hornet, and more specifically, to develop the effect of giant hornet on various inflammations in order to effectively obtain anti-inflammatory effects such as acne treatment as well as skin moisturization. In addition, the present invention relates to a method for extracting a giant hornet venom using a giant hornet and a functional cosmetic composition using the same.

  The giant hornet is approximately 25-37mm in size, and belongs to the largest type of hornet that inhabits in Korea. If you eat plant sap or honey, attack various insects mainly in summer and other beehives in autumn when food is insufficient, and beekeepers recognize it as a pest There were many.

  In addition to a large body, strong chin, and strong skin, even a mantis can be a prey to a giant hornet, with a powerful venom up to 600 times that of a typical bee. One is a terrible existence that kills hundreds of bees, but humans are no exception, and can be killed if stabbed by a poisonous needle several times.

  On the other hand, the field that has recently been in the spotlight is research on bee venom. From the stage of sewing needles, which have been used only in some areas, recently we have extracted venoms from hornets including not only honeybees but also highly toxic bees. It is neutralized and used to treat various disease names.

  This bee venom, which is mainly composed of protein, has an anti-inflammatory effect that is 1000 times that of antibiotics (mycines), and is not only known for existing treatments such as arthritis, but also acne, It is also used to improve skin diseases such as fine lines.

  Furthermore, in the case of wasps whose poison is 550 times stronger than the bees, they contain a substance called Peptide, but it is clear that such ingredients are effective for cardiac arrhythmias along with the poison. In Japan, wasp poison has been booming for several years.

  However, as the interest in bee venom increases in the medical and cosmetics markets, the government or company supplies equipment for extracting bee venom free of charge, which is applied to bees passing through the hive. It collects bee venom by applying electrical shock. However, such an apparatus is limited to bees, and its efficiency is extremely low. Even if it is extracted over several months, the extraction amount is only about 5 g.

  In this way, productivity is extremely low, and bees often die due to stress caused by electric shock, so beekeepers are in fact encouraging the installation of such bee poison collectors. .

  On the other hand, in the case of a giant hornet whose toxicity is 550 times higher than that of a bee, the toxicity can be changed to one that can obtain various effects obtained from a honey bee. From 550 bees, one obtained from one giant hornet can be obtained. It is said that it is the same as that obtained.

  However, giant hornets have long needles that are not useful in normal-thickness clothes and are powerful, and they are also dangerous for those who are professionally collecting them, extracting poison one by one While there is a shortcoming that the risk of encountering a danger in the process is high, the amount of honey produced in the case of capturing and using giant hornet that invades the beehive's nest box when it runs out of food from around August will cause enormous damage. It is expected that additional income will be obtained with the increase of.

Korean Patent Publication No. 10-1999-0039050 (June 05, 1999) Korean Patent Registration No. 10-1018352-0000 (February 22, 2011)

  The present invention has been developed to solve the above-mentioned problems, and is applied to the skin in a state in which components that are excellent in absorption into the skin and give direct irritation are sufficiently neutralized. It is an object of the present invention to develop a method for extracting giant hornet venom that is effective for skin moisturization, anti-inflammatory treatment, and skin whitening, and a functional cosmetic composition using the same.

In order to achieve the object as described above, the present invention provides a preparatory process in which an ethanol solution having an alcohol concentration of 15% to 50% is put in a storage container, and only a worker bee among living giant hornets is added to 15 liters of the ethanol solution. After putting the lid in the storage container at a ratio of ~ 100 and sealing the lid, the giant hornet was introduced into the ethanol solution to slow down the movement, and this was performed for 6 months or more at room temperature in a dark place. Preferably, a method of extracting giant hornet venom is provided, wherein a series of processes including an aging process for aging for 1 year or more and an aging liquid separation process for separating the ripened liquid are continuously performed.
Moreover, this invention provides the functional cosmetic composition using the giant hornet venom extract extracted by the said method.

As described above, the present invention produces a liquid using a giant hornet that is excellent in skin moisturizing effect, anti-inflammatory effect, and skin whitening effect, and can be used alone or mixed with other substances as a cosmetic. It has the effect of providing functional cosmetics that are mainly effective in skin treatment and condition improvement.
Moreover, since it acts not only on skin ecology but also on skin pigments, a whitening effect can be obtained.

  Accordingly, specific embodiments of the present invention will be described in detail so that those skilled in the art can easily understand and reproduce them.

  First, a preliminary preparation process in which an ethanol solution with an alcohol concentration of 15% to 50% is put into a storage container, and only 15 to 100 animals, preferably about 20 to 1 liter, of the living giant hornet in 1 liter of the ethanol solution. After putting the lid in the storage container at a ratio of 80 animals and sealing the lid, the process of charging the giant hornet into the ethanol solution to slow down the movement, and this is performed at room temperature in a dark place for 6 months or more, preferably Includes an aging process for aging for 1 year or more and an aging liquid separation process for separating an aged liquid.

  At this time, the alcohol content is allowed to ooze out during the time when various components contained in the giant hornet are restricted by the osmotic pressure phenomenon.On the other hand, the alcohol component soaks into the giant hornet to prevent spoilage. This is a necessary condition for aging late and is generally an ethanol solution having an alcohol concentration of 15% to 50%. In the examples of the present invention, homemade liquor (alcohol content of 30 degrees) that can be easily obtained in the market was used.

  The introduction of giant hornets usually limits and sustains the number of giant hornets that have invaded beekeeping farmers if they fail to find the nest of the giant hornet, using insect nets, etc. Therefore, it is necessary to capture and enter each time.

  On the other hand, even when a giant hornet's nest is found, after putting the living giant hornet into the storage container one by one and closing the lid of the storage container, if the movement is slowed by immersing the giant hornet in liquor, another giant hornet It is preferable to prevent the needle from being pierced by inserting a large number at a time by repeating the same process.

  At this time, the reason why the living individual is introduced is to prevent a decrease in effective medicinal effect due to, for example, a bee venom that rapidly hardens in the case of a dead wasp.

  Furthermore, the present application is limited to giant hornet wasp, and other hornets such as killer hornet, hornet hornet, hornet hornet, hornet hornet, etc., may have side effects when extracting bee venom by the method of the present application. In the present application used as an application, it must be noted that such hornets are mixed.

  Thereafter, the present invention may include an alcohol separation process in which the separated ripening liquid is heated to reduce the alcohol concentration. At this time, in the aging process, a dark place where light is minimized is preferable because it can prevent the contents from being deteriorated. In the alcohol separation process, the amount of generated odor and alcohol is reduced, and the cosmetic material is used. As a more effective use.

  This presentation of the present application shows the efficiency of the present application, the effect of collecting and selling large hornets on beekeeping farmers, and the damage of honey harvesting by hornets on the existing beekeeping farmers. It is expected to increase the income of beekeeping farmers.

  As mentioned above, bee venom is generally known to have an anti-inflammatory effect that is nearly 1000 times that of a general anti-inflammatory agent, that is, myching, which is also a type of dermatitis. For this reason, when the liquid extracted in the present application is applied to the skin, pores are opened by evaporation of alcohol, and bee venom penetrates into the skin, which has an excellent effect on various acne and stains.

  In general, external preparations such as cosmetics are products that are used continuously by healthy normal people over a long period of time on a daily basis, and are different from drugs that are used only for a certain period of time to treat specific diseases. In the case of pharmaceuticals, the value is determined by taking into account the effectiveness of treatment and the side effects caused by the treatment.On the other hand, the effectiveness of cosmetics must be examined after absolute safety is ensured. As a means for ensuring the safety of the raw material and the finished product, it is preferable that a test using human skin is essential.

  As a result, the primary irritation of the human skin to the extract according to the present invention, the eye mucous membrane irritation test is performed to verify the effectiveness of the composition, and the present invention is performed while testing the whitening effect of the human skin. It was found that this composition is effective for whitening human skin.

  In the following experimental examples, essence and lotion of a giant hornet venom extract obtained by introducing 40 hornet bees (working bees) into 1.8 liters of homemade liquor (30% alcohol) by the method of the present invention and aged at room temperature for one year. Cosmetics were manufactured and tested respectively.

1. [Primary irritation test of human skin of three kinds of giant hornet cosmetics]
1) Test Background External preparations such as cosmetics are products that are used regularly by healthy normal people over a long period of time on a daily basis, and are different from pharmaceuticals that are used for a specific period of time to treat specific diseases. In the case of pharmaceuticals, the value is determined by taking into account the effectiveness of treatment and the side effects of the treatment at the same time. On the other hand, the effectiveness of cosmetics must be examined after absolute safety is ensured.

  Skin reactions induced by external preparations including cosmetics may be acute contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, depending on the appearance of symptoms. ), Phototoxic dermatitis, photoallergic contact dermatitis, contact urticaria, and skin discomfort without macroscopic findings of inflammatory reactions Subjective irritation (Sensory Iritation) and acne, skin changes (Hyper / Hypopig) entation), local side effects, may be divided, such as in systemic side effects.

  Until now, as a means for ensuring the safety of cosmetic raw materials and finished products, tests using human skin and tests using animals have been carried out universally. Tests using human skin have the advantage of being able to foresee skin irritation under actual conditions of use, but require a long time, individual differences among test subjects, the intervention of subject's subjective factors, and strong irritation In the case of substances, testing using animals is a human being because of the limitations that cause pain to the test subject and that the difference is not clear depending on the primary exposure rather than repeated exposure when compared to substances with low irritation levels. Has been carried out as a pre-stage of the test using.

  However, animal testing at the stage of developing cosmetics has faced social criticism in the dimensions of environment and animal protection, and the EUC (European Community Cosmetics Directive) 6th amendment proposed for raw materials and products since 1998. It is decided that the animal test will be stopped and applicable alternative test methods will be applied step by step. Furthermore, in the “EU Cosmetic Direct No. 7” clause, Skin induction, Phototoxicity, Dermal absorptivity, Skin corrosivity, Mutagenicity, Eyre In 2009, the Animal Prohibition Law was enforced from 2009, and the movement of the systemicity part from 2013 A law prohibiting physical experiments is being implemented.

  As a result, up to now, in vitro test methods have been developed all over the world, and confirmation studies between countries in order to utilize such methods for actual product development are being continued.

  Until now, human body tests have been carried out by the reality that in vitro alternative tests and animal test methods cannot accurately reflect the mechanism of human skin.

2) Test purpose This test was conducted to confirm the presence or absence of primary irritation of human skin of raw materials and cosmetics.

3) Testing organization-Organization name: The Mapro Co., Ltd. / Dermatology Research Institute-Address: Address 919-1, Seocho-dong, Seocho-gu, Seoul

4) Test period From July 25, 2011 to July 28, 2011

  5) Test substance

6) Test method 6-1. Test subjects The subjects were 30 or more men or women aged 18 to 60 who met the subject selection criteria and exclusion criteria.
Each applicant signed a written agreement to respond to the details and gave a unique number with numbers.

6-1-1. Selection criteria Volunteers that meet the conditions described below are selected from the test group.
1) Men and women between the ages of 8 and 60 who are healthy without skin disease
2) Applicants who have sufficiently listened to the explanation of the purpose and content of the exam prior to the exam and voluntarily signed a written consent form
3) Those who intend to cooperate with the requirements of the experiment during the test period and to contact immediately when abnormal symptoms occur
4) Applicants who can follow-up during the test period

6-1-2. Exclusion Criteria Volunteers that meet the following criteria will be excluded from the study group.
1) Women who are pregnant, breastfeeding or planning to become pregnant within 6 months
2) When there are tattoos, scars, burns, etc. in the test area and disturbs scoring
3) If there is an infectious skin disease or other skin disease that interferes with the purpose of the study
4) If the drug currently being treated affects the skin reaction
5) Those who are severely irritated or allergic to cosmetics, pharmaceuticals, or daily light exposure
6) Person with atopic skin
7) Those taking contraceptives, antihistamines and anti-inflammatory agents
8) If you have pore keratosis or dermatitis
9) Those with severe irritation or allergies to patch tape
10) If you are participating in another study at the same time
11) If more than 4 weeks have not passed since joining the same previous study
12) In addition to the matters described above, if the clinical trial is judged to be difficult by the judgment of the clinical trial manager

6-1-3. Limitations
1) The subject kept the test site from getting wet with water while applying the patch.
2) The tester was notified when taking or using medications for physical treatment.
6-1-4. Number of test subjects and calculation basis The number of subjects was selected and enforced on the basis of the human body patch test method, which is the toxicology test method paragraph 7 (1) related to the examination of functional cosmetics and the like.

6-1-5. Test suspension and dropout criteria For subjects who do not follow the test schedule during the course of this test, after deciding whether or not to discontinue after hearing their willingness to participate, other side effects, inability to follow-up, and not following the protocol The test was interrupted when this occurred.
1) When the examiner withdraws consent to participate in the clinical trial
2) When side effects due to the test substance are serious during the test
3) When it is impossible to proceed with the test due to sudden accidents or disease
4) In addition, when it is considered that there is a problem in the execution of the test by the examiner

6-2. Test Material IQ chamber: Chemotechnique Diagnostics AB (Sweden) Micropore type: 3M / Medical-Surgical Division Microman (M250): Gilson, France
Marking pen: Skin marker Slim (Sweden)

6-3. Test method
1) The test site was washed with 70% ethanol and then dried.
2) The test substance was supplied as provided by the client.
3) After 20 μl of the test substance was dropped into the IQ chamber, it was placed on the back part, which is the test part, and fixed with a micropore tape.
4) The patch was applied for 48 hours, and after removing the patch, the test site was displayed with a skin marker, and each test site was observed after 30 minutes and 24 hours.

6-4. Judgment Criteria Observation was conducted when 30 minutes and 24 hours passed after the patch was removed, and the skin reaction was evaluated according to the criteria in Table 2 reflecting the Frosch & Kligman method and The Cosmetic, Toiletley, and Fragrance Association (CTFA) guidance. did.

6-5. Result calculation method The average reactivity of 48 hours and 72 hours was compared, and the result was judged based on the average reactivity for each substance.

7) Test results 7-1. A total of 31 participants participated in the entire study.
The average age of the subjects was 40.9 ± 9.5 years, the oldest was 53 years old, and the lowest was 20 years old.
The skin characteristics of the test subjects were investigated by questionnaire, and the results are shown in Table 3.

7-2. Results Test substances # 21, # 22, and # 23 showed a skin reaction of 1+ grade in 4 to 7 subjects during the test period (Table 4).

8) Conclusion Test substance # 22 is determined to be moderately irritating on the primary irritation side of human skin, and the other test substances (# 21, # 23) are determined to be substances of the mild irritation category.

  In addition, the inventor requested a skin irritation test (GLP) from the Korea Institute of Chemical Fusion Test and conducted a test based on the method of “Food and Drug Safety Agency Examination No. 2009-116”. Does not induce erythema, crust, swelling, etc. I. I. The index was “0.0”, and the result that it was judged as a non-irritant (Non-irritant) was obtained (Transcript Number: TBH-000407).

2. [Ocular mucosal irritation test (GLP)]
The inventor requested an eye mucous membrane irritancy test (GLP) from the Korea Institute of Chemical Fusion Test and conducted a test based on the method of “Food and Drug Safety Agency Examination No. 2009-116”. And the IAO I index was found to be “no irritation” because it showed an index of “0.7” in both the unwashed and washed groups ( Transcript number: TBH-000408).

  The eye mucous membrane irritation test shows that cosmetics have a high probability of entering the eye for whatever reason they are used, and are harmful to the eyes even if they are harmless to the skin. If present, it was tested because it is preferable to judge that it is quite dangerous as a cosmetic.

3. [Human test for whitening effect evaluation]
1) Introduction Skin color changes depending on gender, individual differences, age, region, season, and body part, or emotions such as health and stress. It is generally known that there are more pigments in men than women and in older people than young people.

  Human skin color is determined by the amount of melanin, carotene, and hemoglobin, of which melanin synthesized by melanocytes is the most critical factor.

  Melanin is synthesized in melanocytes, which are pigment cells present in the basal layer within the epidermis, and metastasizes to peripheral keratinocytes.

Melanin production is known to be generated by a complex chemical change in which tyrosine is changed to (3,4-dihydroxyphenyl) -alanine (DOPA), DOPAchrome, etc. by tyrosinase.
Hypopigmented lesions occur when melanin is produced in abnormally low amounts.
On the other hand, overproduction has been reported to form blemishes and freckles, one of women's main agony, and is also closely related to skin cancer (melanoma).

Melanin is a substance of phenols that is a complex of pigment and protein, and is divided into neuronal melanin synthesized in nerve cells and cutaneous melanin present in the skin, hair follicle, eyes, etc. Is done.
Typical whitening ingredients used in whitening cosmetics include Arbutin, Kojic acid, Ascorbic acid and the like.

Most whitening components are known to inhibit the activity of tyrosinase to suppress melanin pigment formation or reduce the produced melanin pigment to produce a whitening effect.
Two methods are used for the whitening test using the human body.

  The first is a method of measuring the effect of whitening while using a sample on a naturally generated hyperpigmentation site (eg, a stain), and the second is artificial irradiation by irradiating the skin with ultraviolet rays. And a method of measuring a whitening effect using a sample after artificial pigmentation (artificial pigmentation method).

  When using naturally occurring hyperpigment or stain sites, it takes a long time and it is difficult to find subjects with hyperpigment sites. There is an advantage that it can be measured.

  However, the whitening effect measurement method using artificial pigmentation has many advantages in recent years because it can confirm the whitening effect of a sample in a short period of about 2 to 3 months and can easily find a subject. Whitening test by artificial pigmentation method.

  In this study, a test was conducted by an artificial pigmentation method, and the whitening effect of a sample was evaluated in parallel with skin color measurement using Spectrophotometer and Mexometer and macroscopic evaluation by a dermatologist.

  In order to compare and evaluate the whitening effect of the sample “[J-007]” provided by the client with the control sample, the subject's inner skin of the lower eye was irradiated with ultraviolet rays to artificially generate a pigmentation site.

  The subject himself / herself applies the sample to the designated sample use position twice a day for 8 weeks each, and arranges the sample application position randomly so that the sample application position differs depending on the subject, The study was performed in a double blind condition.

2) Research method 2-1) Purpose of the study After inducing artificial blackening in the lower armpit of a Korean adult female subject, the sample (the giant hornet venom extract extracted by the method of the present invention) was used for 8 weeks. The skin whitening effect of the sample was evaluated using the control sample.

5-2) Research Overview In order to evaluate the whitening effect of the sample in comparison with the control sample, more than 20 adult female subjects were recruited.
After inducing the artificial blackening by irradiating the skin of the lower arm of the subject with ultraviolet rays, the instrument measurement and the naked eye evaluation were performed in parallel while the sample and the control sample were applied continuously for 8 weeks.

2-3) Schedule and Procedure The detailed schedule of this test is as shown in Table 5.

2-4) Subjects 2-4-1) Recruitment and selection of subjects After informing the subjects of the purpose and method of the experiment clearly, the subject decides to participate freely and signs the participation agreement. Participated. The specific procedure is as follows.
-IEC Korea, Inc. secures and manages appropriate subjects through a pre-executed subject recruitment survey (Medical examination ^* ).
-When the clinical experiment plan is confirmed, subjects suitable for the subject condition of this clinical trial are automatically selected from the subject database of IEC Korea.
-Randomly call selected subjects to explain the details of the clinical trials, and make sure that subjects who agree with the experimental content visit us on the first day of the experiment.

  -On the first day of the experiment, using a questionnaire (subject selection and exclusion conditions) configured to suit the research purpose and method, the research director (dermatologist) will finally select the subjects to participate in this study.

The questionnaire for final selection of subjects is configured so that subject selection conditions, exclusion conditions, restrictions, etc. are suitable for the research content, and this questionnaire survey is executed immediately before the start of research.
-Explain the study schedule, participation fee, expected risk (pruritus, depigmentation, etc.) and drop-off criteria to subjects.
The subject decides whether or not to participate in the experiment with a free will after considering the explanation, and then signs the participation agreement.
Explain to the subject that the client is eligible for appropriate compensation in accordance with the subject compensation rules specified in the contract when the side effect occurs.

-The confidentiality of subjects who participated in this experiment is guaranteed.
However, depending on the medical purpose, inform the applicant that the test data may be used to the extent that the subject's identity is not revealed.
-Subjects who have signed the clinical trial consent form will begin the experiment.

^* Medical examination: Interviewing and recruiting subjects and registering them in the database by applying general subject selection exclusion conditions of IEC Korea.

2-4-2) Number of subjects participating in the experiment 25 subjects were recruited according to the request of the client.

2-4-3) Selection conditions 2-4-3-1) General selection conditions to be applied from the subject database-Korean women-Weight: Not considered in recruitment survey for database registration, final subjects of each detailed study Exclude subjects who may interfere with research based on the judgment of the principal investigator at the time of selection.
-Those who can understand Korean and who can read the provided documents and listen to explanations by researchers.
-Persons with national health insurance-Excludes persons with extreme distortion or warping in the spine.

^* This general item was not recorded separately in the Case Report Form.
2-4-3-2 Subject selection conditions to be applied in the whitening efficacy evaluation test after artificial pigmentation The following selection conditions were recorded by the dermatologist on the day of the experiment to the subjects and recorded in the case record.
-A person who understands the contents of the study and voluntarily prepares and signs a clinical trial participation agreement-A healthy person who has no sudden / chronic physical disease including skin disease-A light skin type falls under II, III and IV (Table 7)
-Adult women aged between 18 and 60-Those who can follow-up during the study period

2-4-4) Exclusion conditions 2-4-4-1) General exclusion conditions applied in the subject database -Persons who are legally restricted by life -Persons with severe illness -Minor or domestic Those who are unable to participate in clinical research legally-Employees of IEC corea-Those who are unable to contact emergency or who cannot meet during the experiment-Those who do not meet the following conditions-During the study period Those who participate in a variety of clinical studies (those who are concerned about their participation)
-Those who have not passed the experiment re-participation restriction period: Those who have not participated in the same type of study for six months.-Health condition: This condition is strictly for ensuring the safety of the subject. Must be applied to and confirmed by questionnaire survey.
・ Women who are pregnant or breastfeeding, women who are using medically inappropriate contraceptive methods ・ Organization (main organs such as liver, lung, heart) transplantation within the last 5 years or long-term amputation Those who have undergone brain surgery, those who have surgical sequelae ・ Cardiovascular disease, endocrine system disease, digestive system disease, nervous system disease, urological disease ・ Antihistamine, steroid, allergy suppressant・ Persons who have been taking or applying drugs with similar functions for a long time ・ Persons with severe asthma ・ Persons who have experienced drug-sensitive reactions ・ For substances used in laboratories (gloves, adhesives, etc.) Those who are concerned about allergies ・ Those with the following skin diseases: urticaria, oedema, eczema, recurrent herpes, herpes zoster having erupted in he last 3months, pityriasis versicolour, common acne with a sudden rise of inflammation or nodular or cystic acne, psoriasis, ichthyosis, lichen planus, chronic lupus erythematosis, keloid scars, severe pigmentation disorders (vitiligo, chloasma, multiple lentigines, numerous or congenital nevi , Special if the of of large size), hyperhydrosis, dorsal hyperpilosi ty
・ People who have immune disorders or are receiving immunosuppressant treatment ・ People who smoke more than 10 cigarettes a day or drink more than 3 drinks daily ・ People who have sensitive or atopic skin
^* This general exclusion condition was not recorded separately in the case report form (Case Report Form) created in clinical studies. However, the experiment re-participation restriction period and current health status were recorded in the case record.

2--4-4-2) Subject exclusion conditions applied in the whitening efficacy evaluation test after artificial pigmentation The following selection conditions were recorded by the dermatologist on the subject on the day of the experiment, and recorded in the case record.
-Those who are pregnant or breastfeeding or plan to become pregnant within 3 months -Those with psychiatric or skin diseases -Those with chronic debilitating diseases such as asthma, diabetes and hypertension -To treat skin diseases Persons who have been using a topical skin preparation containing steroids for more than 1 month-Persons with sensitive, hypersensitive or atopic skin-Patients with a history of photoallergy or photosensitization-Participated in the same experiment After 6 months-Those who have used the same or similar cosmetic or medicinal product within 3 months before the start of the study-Participate in other experiments that use the same test site within 3 weeks of the start of the study -Those who have skin abnormalities such as moles, acne, erythema, dilation of capillaries, etc.-People who have undergone organ transplantation or excision within the last 5 years-Chinese herbal medicine (adjunct) within the last 3 weeks Those who took long-term Persons with gastrointestinal, immune, cardiovascular, endocrine, or urinary illnesses-Experiences that are sensitive to drugs or hypersensitivity to substances commonly used in experiments-Before the start of the study 6 Person who has undergone treatment of skin disease at the test site within a month-Person who has experienced hyperthermia for more than 24 hours within the last 8 days. Those who have plans to give vaccinations-Others who are considered unsuitable for testing at the discretion of the principal investigator

2-4-5) Restriction conditions Items restricted to subjects during the experiment.
-The use or use of aspirin, anti-inflammatory agents, antihistamines, and steroids (including traditional Chinese medicines) is prohibited during the experiment.
-Prohibition of vaccinations and treatment of immunosuppressants during the experiment are also prohibited.
-No other whitening cosmetics other than the provided sample can be used at the test site during the experimental period.
-Avoid exposing the test site to sunlight.
-Apply the sample without fail.
-Do not perform activities that deviate significantly from daily life during the experiment.
2-4-6) Subject's obligations Items to be observed by subjects for the protection of subjects and accurate research progress-Protect sample usage, restrictions, and other examination and visit dates.
-Detailed reports on all diseases and symptoms that occur during the experiment.
-Keep confidential information about this experiment until the end of the experiment.
-Make all materials involved in the experiment honest.

2-4-7) Subject dropout criteria during the course of research Even if a subject is selected according to subject selection and exclusion criteria, if the following dropout criteria occur during the course of the experiment, the judgment of the subjective person in charge and the researcher Excluded by below and excluded from the experimental results calculation.

-If a sample has not been applied for more than 3 days during the study period, it may be excluded from the study due to non-compliance with subject requirements.
-When an adverse case such as pruritus or erythema occurs at the UV irradiation site-When an unexpected adverse case or side effect occurs during the experiment-During the course of the experiment, excessive UV exposure or drinking If the assessment fails due to smoking, smoking, etc.-If other treatments that may affect the study content are to be received.-If the subject withdraws consent to participate.-If the subject cannot be followed up.-Individual subjects. If the reason for dropout occurs due to the circumstances of the study −If the researcher determines that the subject cannot continue the experiment −If the subject does not comply with the experimental restrictions or obligations

2-4-8) Regulations on the progression of human experiments on subjects-While conducting the experiments, the clinical trial manager and research personnel must do their best to ensure the safety of the subjects, and take prompt and appropriate measures when all adverse cases occur. And minimize its harmful consequences.

-If the subject reports skin irritation or adverse cases with the sample during the course of the experiment, wipe the applied sample immediately, and if symptoms do not improve, receive a dermatological assessment and appropriate treatment by a dermatologist.
-If erythema with blisters occurs on the skin surface due to excessive UV irradiation, ensure appropriate dermatological evaluation and treatment.
-In the event of other abnormal skin reactions, clinical trial directors and investigators will take appropriate measures along with dermatological assessments and document details of cases and circumstances.

2-5) Sample 2-5-1) Sample Information-Name (Sample Code) Sample: [J-007] (Sample A)
Sample containing the main component (hereinafter referred to as “sample” or “sample A”)
^* Lotion is produced from the giant hornet venom extract extracted by the method described above and provided as a sample.
-Control sample: [J-008] (Sample B)
Sample that does not contain the main component (hereinafter referred to as “control sample” or “sample B”)
-Receive the sample from the client as described above. Confirmation of the sample information is received from the client immediately before the report is prepared after the experiment.
Property sample: lotion control sample: lotion sample control number sample: A2122 (sample A)
Control sample: A2123 (Sample B)
-Immediately after receiving the sample, the sample is registered with IEC Korea and given a control number, and the sample is labeled.
-Analysis: The client must confirm that the sample is accurate and provide information about the sample (such as properties). IEC Korea did not carry out chemical analysis to confirm the stability and physical characteristics of the sample.
-Container (or packaging): Pump type plastic container-Amount of sample received: 25 samples, 25 control samples-Receiving date: September 29, 2011-Storage method: Avoid high temperature and direct sunlight 5 Store at 25 ° C. IEC corea stores samples for 180 days from the date the report is issued, and discards them unless otherwise requested by the client.

2-5-2) Method of using sample The method of use is as follows.
-Sample use position: Artificial blackening area inside the lower arm (Randomly arrange the use area for each sample)
-Sample usage: appropriate amount-Sample usage cycle: Twice a day (morning and evening)
-Sample use period: 8 weeks-Sample use method: The subject applies a specified sample at a specified position at home. Care should be taken not to leave the artificial blackening area or apply other samples with the sample applied fingers.

2-5-3) Active ingredient of sample -Sample name: [J-007]
-Component name: Hornet Extract
-Content (%): 33.3%

2-6) Test Principle and Equipment 2-6-1) Test Principle When the skin is irradiated with ultraviolet rays, the skin melanocytes activity and melanin production increase and the skin color changes to black. With the passage of time, the activity of melanocites becomes normal, and melanin gradually decreases and returns to the original color. When a substance having a whitening effect is applied, the reduction rate of melanin and the decrease in activity of melanocites proceed more rapidly due to the activity of the whitening substance, so that the color reduction rate becomes faster. It is possible to compare and confirm the whitening effect in parallel with the naked eye judgment of a trained expert by measuring the color with a color difference meter while applying a substance having a whitening effect and a substance not having the same condition for a certain period of time.
2-6-2) Ultraviolet irradiation device (Solar simulator)

  During the experiment, an artificial ultraviolet irradiation device (Solar simulator) is used to utilize uniform quality ultraviolet rays, and a xenon arc lamp that has a radiation spectrum similar to that of sunlight and does not show a peak at a specific wavelength is mounted. A solar simulator was used.

  The type of Solar simulator used in the experiment is Multi-port solar simulator 601 (Solarlight co., US) and has the following characteristics.

It is equipped with a 300 watt xenon arc lamp and emits ultraviolet rays in a circle of 8 mm in diameter through 6 liquid light guides (LLG). The intensity of ultraviolet rays radiated through the six LLGs can be individually adjusted using a diaphragm attached to the upper end of each LLG. Using a Schott WG 320 filter and a UG11 filter with a thickness of 2 mm, the wavelengths in the ultraviolet C and visible light regions were removed and used.
2-6-3) Light quantity measuring device (PMA 2100 UV meter)

  The light intensity measuring device used in this study was a PMA 2100 UV meter (Solarlight Co., US), and the light intensity was measured after binding the UVB sensor (PMA 2103) to the end of the Solar simulator LLG. The measured value is displayed in units of MED / min. 1.0 MED / min is the same light energy as 21 mJ / cm 2. The light intensity was measured immediately before each subject was irradiated with ultraviolet rays and recorded in a case record.

2-6-4) Color measurement using Spectrophotometer Spectrophotometer (registered trademark) CM2600d (Minolta, Japan) was used for instrument measurement of skin color. This device is a device that emits white light and measures the light reflected from the surface and expresses the hue according to the CIE hue standard. The measured value is displayed as an L ^* a ^* b ^* value. L ^* is a value indicating brightness, and the higher the skin color is brighter and transparent, the higher the value. a ^* indicates skin red-green, and b ^* indicates skin yellow-blue. The probe of the Spectrophotometer was 3 mm, and the average value shown after three automatic measurements was used as effective data.

2-6-5) Melanin index measurement using Mexameter (registered trademark) MX18 Mexameter (registered trademark) MX18 (Courage + Khazaka GmbH, Germany) is a skin color measuring device that uses only a narrow wavelength range (Narrow-band reflectivity spectroscopic spectrophotometer). And is suitable for measuring the amount of melanin and hemoglobin in the skin. The measured value is expressed by Melanin index (MI) and Erythema index (EI). EI is calculated based on the ratio of red and green wavelengths of reflected light, and MI is calculated based on the ratio of red light in the total reflected light. Mexameter (registered trademark) MX18 uses a diode that emits green light and red light to calculate the total amount of light emitted from the device and the amount of reflected light by a computer, and shows the result.

  In this experiment, MI was measured three times using Mexameter (registered trademark) MX18, and the average value was used as effective data. Each measured result was recorded in a case record.

2-7) Experimental process 2-7-1) Determination of minimum erythema amount (MED) After irradiating the skin with ultraviolet rays using an ultraviolet irradiation device Solar simulator Multiport 601 (Solarlight Co., USA), the existing erythema amount was confirmed. Based on the study results (Table 8), the minimum amount of erythema by skin color (ITA °) was determined. The skin color inside the lower arm of the selected subject was measured with a skin color meter (Spectrophotometer (registered trademark) CM2600d).

2-7-2) Artificial Pigment Induction Ultraviolet rays were irradiated on the inner side of the subject's lower eyelid based on the minimum amount of erythema determined by each subject. The intensity of the light intensity was 2.0 MED / min, and the test site was selected to be a clean and flat site, avoiding portions with excessive hair and skin color differences. After the minimum amount of erythema was determined, ultraviolet rays of 0.5 to 3.0 MED were cumulatively irradiated three times. The amount of UV irradiation varied depending on the degree of blackening of each subject, and the degree of blackening was determined by the naked eye judgment of the researcher and the L ^* difference value (after blackening-before blackening) of Spectrophotometer CM2600d (Table 9) .

Six days after the third ultraviolet irradiation day, the color on which the artificial pigment was deposited was measured with a Spectrophotometer, and the L ^* (brightness) value was 1 or more different from that before artificial blackening. Subjects who observed crystallization began to apply samples.

2-7-3) Measurement and naked eye evaluation Illumination was kept constant in a space where there was no air movement and direct sunlight and constant temperature and humidity conditions (22 ± 2 ° C., 50 ± 5%) were maintained. The subjects were allowed to stabilize the skin for a minimum of 15 minutes, and then measured and evaluated.

2-7-3-1) Equipment evaluation using Spectrophotometer Before artificial pigmentation, before pigmentation after artificial pigmentation, 4 weeks and 8 weeks after sample coating, L ^* a ^* b ^* values on the test section The measurement was repeated three times for each site (auto condition).

2-7-3-2) Macroscopic evaluation Two dermatologists evaluated the skin color of the test site with the naked eye based on the color evaluation standard table (Intensity score table). The color evaluation standard table indicates the brightness of the skin color from 1 point (bright and transparent) to 10 points (dark and dark) according to the skin color. An intermediate score of 0.5 was given between each score. Macroscopic evaluation was performed before artificial pigmentation, before sample application, 4 weeks and 8 weeks after sample application.

2-7-4) Photography Photographing was performed using a digital camera (Nikon D90 digital camera, Japan) in a photo room (Orion 400 strobo, Aurora light bank, Korea) prepared for clinical photography. The same researchers photographed with fixed shooting conditions such as the subject's posture, distance between the subject and the camera, angle, illuminance, exposure, aperture value, etc., so that a certain quality photo was obtained.

2-7-5) Subject final evaluation questionnaire 8 weeks after using the sample, the research supervisor asked the subject about the skin compatibility of the sample and its effect as a cosmetic, and evaluated it on a 5-point scale.

2-7-6) Evaluation of skin safety of dermatologist A dermatologist evaluated the safety of the sample before using the sample and after 4 and 8 weeks of using the sample. Visual signs related to sample use (physical signs: erythema, oedema, dryness, desquamation,...) And visual symptoms (functional signs: pickling, heightness, local・ ・) Interviewed and recorded in the case record, and then determined the association with the sample.

2-7-7) Data Analysis and Result Interpretation 2-7-7-1) Instrumental Measurement Results-The skin color lightness value L ^* and ITA ° were determined using Spectrophotometer. The difference values (ΔL ^* , ΔITA °) before and after using the sample were determined, and the statistical significance between the sample and the control sample was verified.

-Melanin index was measured using Mexameter. The difference value (ΔMI) before and after using the sample was determined, and the statistical significance between the sample and the control sample was verified.
-If the skin color change value between the sample and the control sample showed a statistically significant difference, it was interpreted as having a whitening effect on the sample.

2-7-7-2) Macroscopic evaluation results-The results obtained by the macroscopic evaluation verified the statistical significance of the sample and the control sample by obtaining the macroscopic evaluation difference values before and after using the sample (△ value).
-When two researchers showed a statistically significant difference, it was interpreted as having a whitening effect on the sample.

2-7-7-3) Statistical Analysis Method SPSS (registered trademark) 14.0 was used as the statistical analysis program. The normality of the instrument measurement result and the macroscopic evaluation result data was verified by the Shapiro-wick test 1, respectively.
Sample vs. Control sample-When normality is satisfied (p> 0.05): Paired t-test (p <0.05)
-When normality is not satisfied (p <0.05): Wilcoxon test (p <0.05)

3) Test results 3-1) Subject composition Twenty-five adult female subjects who meet the basic selection criteria for this study were recruited. On the first day of the experiment, 25 subjects were selected as subjects suitable for the experiment according to subject selection and exclusion conditions by the dermatologist, and the experiment was started. During the experiment period, 2 subjects dropped out, and the final 23 (average 37) Valid data was obtained (Table 11).

3-3) Brightness of skin color (L ^* ) measurement result The skin color brightness (L ^* ) at the site where the sample was used was measured 4 and 8 weeks after using the sample. A statistically significant difference was observed between the samples after 4 weeks and 8 weeks after the use of the sample, and it appeared that the improvement of the skin color brightness at the site where the sample A was used was superior to the site where the sample B was used (p <0. 05). (Table 13, Table 14)

  p-value: Significant probability, Paired t-test (p <0.05: there is a significant difference between the sample use site and the control sample use site)

3-4) Results of ITA ° Measurement As a result of analyzing the ITA ° of the sample use site color after 4 weeks and 8 weeks of sample use, all became brighter than before the sample use, and significant between samples after 8 weeks of sample use A difference was shown, and the amount of increase in ITA ° at the site where the sample A was used was statistically superior to the site where the sample B was used (p <0.05). (Table 15, Table 16)

3-5) Melanin index measurement result As a result of analyzing the melanin index of the skin color at the site of sample use after 4 weeks and 8 weeks of sample use, all became brighter than before the sample use. Did not appear. (Table 17, Table 18)

3-6) Results of visual inspection by experts As a result of visual evaluation of the skin color after 4 weeks and 8 weeks of sample use by two experts, evaluator 1 found that the skin color of the sample use site was significantly different between samples. However, the evaluator 2 evaluated that the skin color of the site where the sample A was used was significantly brighter than the site where the sample B was used after 4 weeks of using the sample (p <0.05). (Table 19, Table 20)

3-7) Questionnaire for final evaluation of subjects The research supervisor conducted a questionnaire evaluation on the skin compatibility and whitening efficacy of the samples on the day when the experiment was completed. As a result of analyzing the ratio of subjects who answered “very good” and “good”, the skin compatibility was the same for both sample A and B as 61%, and the whitening effect was 61% for sample A and 57% for sample B. Sample A was evaluated to be slightly superior to Sample B in whitening effect. (Table 21)

3-8) Results of skin safety assessment Dermatologists visually examine the test site 4 and 8 weeks after application of the sample, and inquire about the time of occurrence, frequency, intensity, and duration of irritation reported by the subject. The safety of the sample was finally evaluated.

  Three subjects reported a tingling sensation (No. 14: B site, No. 19: A, B site, No. 21: B site) immediately after using the sample during the sample use period. These reactions were extremely weak, with a weak response and a short duration of a few seconds to a few minutes. Even after repeated use of the sample, the symptoms did not recur after 2 weeks of use of the sample, and the sample association was judged to be low. .

In addition, no adverse cases requiring research interruption or sample usage changes were reported or observed, and the reported reaction was determined not to be a specific reaction deserving of sample safety.
As a result of interviews and observations by dermatologists, no sample-related skin hazard cases were observed, and the skin safety was judged to be “good”.

3-9) Results synthesis • Of the 25 subjects who started the experiment, 2 dropped out, and valid data for the final 23 (average 37.5 years) were obtained. Six days after the third ultraviolet irradiation day, the artificially pigmented skin color is measured with a Spectrophotometer, and the L ^* (brightness) value is one or more different from that before artificial blackening, or black that is obvious by the naked eye of a dermatologist Subjects who observed crystallization began to apply samples.
・ As a result of measuring the skin color of the artificial blackened site 4 weeks and 8 weeks after the sample application start date, the color (L ^* ) of the site where the sample was applied 4 weeks and 8 weeks after using the sample was applied to the control sample It was shown that it was significantly (p <0.05) brighter than the site, and after 8 weeks of sample use, the ITA ° value of the sample use site was shown to be significantly brighter than the control sample use site.
・ As a result of two experts visually judging the skin color of the artificial blackened site 4 weeks and 8 weeks after the sample application start date, the evaluator 1 found that the skin color of the sample use site was significantly different between samples The evaluator 2 evaluated that the skin color of the site where the sample A was used was significantly brighter than the site where the sample B was used after 4 weeks of using the sample.
-As a result of dermatologists observing the skin safety of the samples at intervals of 4 weeks during the sample use period, no adverse cases related to the samples were observed.

4) Discussion and Conclusion This test was conducted to evaluate the whitening effect of the sample “[J-007]” requested by John Wukchul (individual) in comparison with the control sample. In this study, valid data were obtained from 23 Korean female subjects aged 21 to 51 years (average age 37.5 years), and the results of an 8-week experiment were analyzed.

  In order to evaluate the whitening effect of the sample, after subjecting the subject's lower eyelid to ultraviolet irradiation by artificially inducing pigmentation, the subject himself applied twice a day for 8 weeks, The skin color of the sample application site was measured with a Spectrophotometer and a Meameter after 4 weeks and 8 weeks after the sample application, and after the sample application. In addition, two specialists (dermatologists) visually evaluated changes in skin color before and after use of the samples, 4 weeks and 8 weeks after the samples were used. A dermatologist performed a macroscopic examination to assess the skin safety of the sample.

Measurement results of Spectrophotometer CM2600d (L ^* ), skin applied with sample “[J-007]” after 4 weeks and 8 weeks of sample use, skin at a significant level (p <0.05) compared to the control sample The color has become brighter.
Therefore, the sample “[J-007]” was judged to support skin whitening when used on an artificial blackening site induced by ultraviolet irradiation for 8 weeks.

Claims (5)

  A method of extracting giant hornet venom, wherein only worker bees are selected from living giant hornets and the bees are immersed in an ethanol solution for a certain period of time.   The method of claim 1, wherein the ethanol solution has an alcohol concentration of 15% to 50%.   The method of extracting giant hornet venom according to claim 1, wherein the hornet is immersed in one liter of the ethanol solution at a rate of 15 to 100 animals.   2. The method of extracting giant hornet venom according to claim 1, wherein the wasps immersed in the ethanol solution are aged in a dark place at room temperature for 6 months or more, preferably 1 year or more.   The functional cosmetic composition using the giant hornet venom extract extracted by the method as described in any one of Claims 1-4.