KR20130102712A - Anti-heat aging effect of fermented limonia acidissima extract and its cosmetic usage to improve skin problems - Google Patents

Abstract

PURPOSE: A composition containing a fermented Limonia acidissima extract, which prevents aging caused by heat and improves skin conditions is provided to remarkably reduce skin irritation and effects to a cosmetic formulation. CONSTITUTION: A cosmetic composition contains 0.001-95 wt% of a fermented Limonia acidissima extract. The cosmetic composition is prepared by fermenting a Limonia acidissima extract with Bacillus subtilis, lactic acid bacteria, and/or yeast. The cosmetic composition is formulated in a unit dosage formulation by adding a pharmaceutically or cosmetically acceptable carrier or additive.

Description

Anti-aging effect and fermented Limonia acidissima extract and its cosmetic usage to improve skin problems}

The present invention relates to a tanaka fermentation extract and a composition for preventing heat aging containing an active ingredient thereof, and more particularly, skin improvement through pore contraction and sebum secretion inhibition, redness relief by skin cooling effect, whitening, heat It relates to a cosmetic composition having a skin aging prevention and improvement effect.

Skin aging can be largely divided into endogenous aging and photoaging. Endogenous aging is the aging phenomenon that cannot be avoided with the passage of time. Photoaging is the aging observed on the skin of the face, back of the hand, back of the neck, etc. that have been exposed for a long time by environmental factors such as sunlight, cold, wind, and environmental pollutants. Say the phenomenon. Photoaging is more closely related to rough wrinkles, pigmentation, loss of skin elasticity, and skin cancer than endogenous aging, but it can be prevented by avoiding exposure to ultraviolet rays. Recently, it has been found that infrared rays, which constitute 60% of solar energy, are one of environmental factors that promote skin aging by increasing skin temperature, and thermal aging due to infrared rays as well as ultraviolet rays has become a significant threat to skin aging.

When the temperature of the skin rises due to temperature change due to carbon dioxide greenhouse gas emission and ozone increase, so-called skin warming state, collagen decreases, the dermis layer is damaged, and the skin becomes dull and skin elasticity decreases and wrinkles Is generated. When the skin temperature rises, as a natural defense material, the heat receptor in the skin cells increases, and the generated heat dries out the skin essence except blood components. This makes nutrient delivery to the skin difficult and accelerates the drying of the skin to promote aging. In addition, ultraviolet rays transmit only to the upper part of the dermis, but 65% of near infrared rays (Infrared A, wavelength range 760 to 1440 nm) of infrared rays can be transmitted deeply to the subcutaneous tissue layer of the skin. Therefore, when repeatedly exposed to thermal energy, the skin temperature is increased and aging is generated by generating deep wrinkles on the skin.

Thanakha grows in areas such as Myanmar, Thailand, the Himalayas, and India, but the amount is very small, and Myanmar's Shuebo, Burkeok and Shan areas are known as major habitats. The Tanaka tree grows well in areas with abundant rainfall, and has excellent propagation in dry, rocky soil, sand and red soil. The wood is hard like a stone, but the bark is thin and soft. It takes about 3 to 10 years for a Tanaka tree to grow about 3 inches in diameter depending on soil and climatic conditions. A tree that has been grown for at least 10 years can be recognized for its value as a cosmetic or pharmaceutical product.

Ttanaka (Thanakha) is noticeable feature of the local hot climate and beauty by Burmese commodities have traditionally been used, is very closely linked with the lives of the residents it is reportedly the first in Southeast Asia and the original Burma. Myanmarians have been using tanaka trees for makeup and folk remedies for about 2000 years. Thick white dough was used for cosmetics at events and was usually applied to protect the skin from the sun. It has been believed that the traditional make-up using Tanaka wood has excellent effects in reducing heat from the body, preventing fever in hot climates, preventing insect bites, and protecting the skin from strong sun heat. Tanaka makeup has been a great help in maintaining their beauty, and has been used as a medicine in addition to cosmetics. Myanmar people grind the Tanaka bark well with water and apply Tanaka pure powder not only on the face but also on the whole body exposed to the sun. Tanaka has excellent cooling effect to cool and cool the skin and pore contraction to shrink pores in accordance with the characteristics of hot countries. Traditionally, folk remedies have been effective in clearing blood flow in vivo as well as skin care effects, and have excellent effects on smallpox, measles, epilepsy, malaria and fever. The fruit of Tanaka tree is widely used as a tonic, to treat smallpox and as an antidote, the leaves are effective for heart disease and cold, the roots are effective for abdominal pain.

In the West, Clarins researchers first became interested in Tanaka powder, using Tanaka powder for skin care for the first time with the discovery that it made the skin soft and clean, and is known as Limonia Acidissima and Woodapple.

Considering that the research on materials that can block skin temperature increase due to strong ultraviolet rays and solar heat due to extreme weather fluctuations has been passive for some time, the tanaka tree of the present invention can be an excellent composition having anti-aging and improvement effects. . In order to easily use Tanaka tree, which has many excellent effects, anywhere, it is necessary to combine the active ingredient of Tanaka with modern science to expand its scope as various medicine materials and cosmetics, and to use mask packs and sunscreens (scattering agents and Absorbent), face wash, powder, skin, foundation, BB cream, lotion, perfume, etc.

The patents on Tanaka are still in the introduction stages such that domestic patents on pore-contracting cosmetic compositions containing extracts of Tanaka and foreign patents on skin diseases such as UV protection, acne, and sweat bands are still in the introduction stage. There is no research on the fermentation extract of Tanaka that can maximize the active ingredient through the fermentation process.

Fermentation refers to a process in which microorganisms decompose or synthesize organic matter using their own enzymes. Representative fermentation microorganisms include Bacillus subtilis, lactobacillus, koji, yeast, mold, and acetic acid. The fermentation broth extract does not use any other solvents and, through fermentation and maturation, converts sugar components contained in natural materials into alcohols, and is effective in flavonoids, vitamin C, vitamin P, amino acids, catechins, etc. By increasing the extraction efficiency of the ingredients, it has an important meaning in attempting to improve the efficacy of natural materials and solve safety problems. The fermentation process such as Bacillus subtilis, lactic acid bacteria and yeast can be generated after extraction as well as maximizing the efficacy of natural materials in the process of converting into low molecular weight materials that are easily absorbed and converting from unstable or inactive forms into active forms. By stabilizing or reducing the toxicity of natural materials that may be irritating or toxic, they can be effectively converted into stable derivatives.

Accordingly, the present invention relates to a cosmetic composition for exploring physiologically active substances possessed by the Tanaka fermentation extract and enhancing the content of the active ingredient of the Tanaka fermentation extract through the fermentation process. It has been developed a cosmetic composition for skin improvement effect, skin cooling effect, whitening, heat aging prevention and improvement through sebum secretion inhibition.

An object of the present invention to provide a skin improvement composition for preventing and improving heat aging containing Thanakha fermented extract and the same as an active ingredient, fermentation by one or two or more of Bacillus subtilis, lactic acid bacteria, yeast The present invention provides a composition for improving and alleviating heat aging of extracts and skin containing the same as an active ingredient. More specifically, by using a fermentation process utilizing a variety of mixed strains, not only maximizes the efficacy of the fermentation extract of Tanaka, but also stabilizes or reduces toxicity, thereby improving skin through skin pore contraction and sebum secretion, and skin cooling. An object of the present invention is to provide a cosmetic composition for effect, whitening, thermal aging prevention and improvement.

The method of extracting Tanaka according to the present invention used the Tanaka fermentation extraction method which went through the fermentation process of the Tanaka general extraction method without the fermentation process and Bacillus subtilis, lactic acid bacteria, and yeast. The extraction method comprises the steps of: (a) mixing water warmed to 80 ° C. to Tanaka and then stirring; (b) naturally cooling to room temperature. Tanaka general extract obtained by the above method can obtain a Tanaka fermentation extract through a fermentation process. More specifically, the fermentation method is characterized in that it comprises a step of mixing and fermenting Tanaka using Bacillus subtilis, lactic acid bacteria, yeast. The fermentation product obtained by the above method may be subjected to the step of aging, and the fermentation product obtained therefrom or the concentrate obtained by partially or totally reducing the concentration and freeze-drying the fermentation product, or the main effect contained in the fermentation product and the concentrate. It contains the chemicals that exert themselves.

The fermentation process is characterized in that the fermentation of Bacillus subtilis, lactic acid bacteria, yeast. Adjusting the medium to pH 4.0-5.0 to about 5.0 to 7.5 and, respectively, ⁷ to 10 Bacillus subtilis, lactic acid bacteria, yeast pre-incubation - 10 ⁸ CFU / mL was added to a concentration, 25 ℃ to 50 ℃ in a temperature range of 18 hours to 120 Time incubation.

The cosmetic composition according to the present invention may contain, in addition to the above extracts and fermented products, other ingredients which may give a synergistic effect to the main effect without impairing the main effect of the present invention. The Tanaka extract and the fermented extract may be present in the composition in an amount of 0.001 to 95% by weight based on the total weight of the composition. Hereinafter, the present invention will be described in detail with reference to Examples, Comparative Examples, and Experimental Examples. However, the present invention is not limited thereto. In the present invention, the skin improvement effect, skin cooling effect, whitening, heat aging prevention and improvement effect through pore contraction action and sebum secretion of the composition containing Tanaka extract and fermented extract was confirmed, sebum secretion inhibition and pore shrinkage evaluation, Objectives were achieved through inhibition of tyrosinase activity and inhibition of melanin biosynthesis, experiments with skin temperature lowering effect (cooling effect), and evaluation of human skin irritation test.

As described above, the Tanaka fermentation extract according to the present invention is characterized in that it uses a fermentation method of mixing Bacillus subtilis, lactic acid bacteria, and yeast in Tanaka, which does not destroy the active ingredient, contaminated by various bacteria and poor fermentation. It can effectively reduce odor. The cosmetic composition containing the Tanaka fermented extract prepared by the above method has been found to have excellent skin improvement effect, skin cooling effect, whitening, thermal aging prevention and improvement effect through pore contraction and sebum secretion, and prevent skin aging due to heat and It can be used in various fields, such as cosmetics, for the purpose of improving sebum secretion and skin pore contraction.

Example 1 Preparation of Tanaka Mixed Fermented Extract

The tanaka tree was washed with purified water, dried and powdered, and then mixed with 10 times the amount of 80 ° C. distilled water and dissolved in the tanaka powder, and then the obtained extract was naturally cooled to room temperature. Adjusting the broth to pH 4.0 to 5.0 to 5.0 to 7.5 and, respectively, 10 ^7, a pre-culture of Bacillus subtilis, lactic acid bacteria, yeast - 10 ⁸ CFU / mL was added to a concentration, 25 ℃ to 50 ℃ in a temperature range of 18 hours to 120 Time incubation. To terminate fermentation, heat was applied at 80 ° C for 10 minutes to induce loss of Bacillus subtilis, lactic acid bacteria, and yeast. The precipitate was removed using a centrifuge and the supernatant was collected, or the filtered supernatant was used.

Comparative Example 1 Preparation of Tanaka General Extract

Tanaka general extract used as a control was obtained through the same extraction process without the fermentation process. That is, after washing the purified water with purified water and dried and powdered, and then dissolved by mixing and stirring 80 times distilled water 10 times the amount of Tanaka powder, the obtained extract is naturally cooled to room temperature. The precipitate was removed using a centrifuge and the supernatant was collected or the filtered supernatant was used as a Tanaka general extract.

<Experimental Example 1> Antioxidant activity

In order to measure the antioxidant activity of Tanaka fermented extract, DPPH (2.2-diphenyl-1-picryl hydrezyl) radical scavenging ability was used.

500 uM DPPH (2.2-diphenyl-1-picryl hydrezyl) solution was dissolved in ethanol and filtered. The prepared DPPH solution and the sample solution were mixed at a ratio of 1: 1 and mixed vigorously. The mixture was allowed to stand at room temperature for 20 minutes. After measuring the absorbance at 540 nm using a Microplate reader (Chromate, USA), DPPH radical removal rate (%) was calculated through Equation 1 below. At this time, 1 mM ascorbic acid was used as a positive control to remove radicals.

As shown in Table 1, in Example 1, the antioxidant activity was higher than that of the general extract of Comparative Example 1, which was not subjected to the fermentation process.

Equation 1

DPPH radical scavenging rate (%) = (A - B) / A x 100

Where A is the absorbance of the blank test solution and B is the absorbance of the test solution

 Antioxidant Activity of Tanaka General Extracts and Fermented Extracts Name of sample Treatment concentration (%) Antioxidative effect(%) Example 1 0.5 80 Comparative Example 1 0.5 65 Ascorbic acid 0.5 82

Experimental Example 2 Evaluation of Tyrosinase Activity Inhibition

In this experiment, 30 ul of 1.5 mM tyrosine (Sigma, USA) dissolved in sodium phosphate buffer (pH 6.8) was used as a substrate. Samples of the present invention were diluted with 0.05 M sodium phosphate buffer (pH 6.8) and prepared according to concentration (0.5%, 1.0%). 200ul of the sample was added to 30ul of tyrosine, and 20ul of tyrosinase was added and reacted at 37 ° C for 20 minutes, and then the absorbance was measured at 492nm.

Equation 2

% Activity inhibition = [1- comparison absorbance / control absorbance] x100

Table 2 shows the result of inhibiting activity of tyrosinase by Equation 2.

 Determination of Tyrosinase Activity Inhibition of Tanaka General and Fermented Extracts Test substance concentration (%) Melanin production inhibition rate (%) Tanaka fermented extract 0.5% 69 Tanaka fermented extract 1.0% 87 Tanaka extract 0.5% 35 Tanaka extract 1.0% 56

Experimental Example 3 Melanin Biosynthesis Inhibition Experiment

B-16 cells (rat melanoma cells, Korea Cell Line Bank) were inoculated to 6 × well plates to 1 × 10 ⁵ cells / well and cultured for 24 hours. Each well was treated with Example 1 and Comparative Example 1 0.5%, 1% and after 4 days of incubation, the culture medium of each well was centrifuged. The isolated cells were lysed with 500ul of 1N NaOH solution dissolved in DMSO (dioxyl sulfoxide) and heated in a 85 ° C water bath for 10 minutes. The collected supernatants were measured for absorbance at 492 nm. The results are shown in Table 3.

  Melanin biosynthesis inhibition assessment Test substance concentration (%) Melanin production inhibition rate (%) Tanaka fermented extract 0.5% 50.2 Tanaka fermented extract 1.0% 88.5 Tanaka extract 0.5% 34.5 Tanaka extract 1.0% 67.9

As shown in Table 3 above, it was confirmed that the Tanaka fermented extract (Example 1) was superior in the melanin production inhibitory effect compared to the Tanaka general extract.

Experimental Example 4 Pore Contraction Effect Evaluation

4-1. Pore Contraction Effect I

For the Tanaka fermented extract and the Tanaka general extract prepared in Example 1 and Comparative Example 1 were subjected to pore contraction effect using a bovine hemoglobin protein. Prepare a solution (1 mg / ml) in which PBS (phosphate buffered saline, pH 7.4) is mixed 1: 1 with hemoglobin (1 mg / ml), and shake the solution and the sample diluted in buffer solution for 1: 1 concentration for 30 seconds. After mixing, the mixture was left for 30 minutes, and the supernatant was taken to measure absorbance at 407 nm. At this time, the final concentration of the sample contained in the reaction solution was adjusted to 0.1%, 1%, 2%, 5%, a sample solvent was used instead of the sample as a control. The shrinkage effect was evaluated by measuring the amount of hemoglobin precipitate, and the results are shown in Table 4. The higher the turbidity and the higher the percent precipitation of hemoglobin, the better the pore contraction effect.

 Pore Contraction Effect Using Hemoglobin Protein Final concentration of the sample in the reaction solution (%) Precipitation of hemoglobin (%) Example 1 Comparative Example 1 0 0.4 0.1 23.5 18.5 One 56.3 41.8 2 75.4 64.5 5 88.1 75.6

As can be seen from the results of Table 4, it can be seen that the precipitation of hemoglobin increases as the concentration of the Tanaka fermentation extract increases. Therefore, it can be seen that the pore contraction effect of the Tanaka fermented extract is superior to the general extract of Tanaka through the protein coagulation effect.

4-2. Pore contraction effect II

Pore contraction effect was performed using albumin as a protein to replace the skin. After dissolving 0.3% albumin in 50 mM citric acid buffer (pH 4.5), 1 ml of each sample was added to 2 ml of 0.3% albumin, and the control group was added 1 ml of sample solvent instead of the sample. After stirring for 5 minutes, the absorbance was measured at 650 nm and the results are shown in Table 5. The higher the absorbance value, the better the pores shrinkage function.

 Pore Contraction Effect Using Albumin Protein Final concentration of the sample in the reaction solution (%) Absorbance Example 1 Comparative Example 1 0 0.007 0.1 0.7 0.5 One 1.2 1.0 2 1.8 1.3 5 2.8 1.9

Experimental Example 5 Emulsion Base Preparation

As in Example 1 and Comparative Example 1, an emulsion base including a cosmetic containing Tanaka fermented extract and Tanaka general extract was prepared. The cosmetic material used in this experiment is in the form of an emulsion type cosmetic liquid, and its composition is shown in the table. First, add the Tanaka fermented extract, the Tanaka general extract, and other contents recorded in the table by weight, and then (a) heat the phase to maintain the temperature at 90 ° C, and purified water (30%) to 90, then (a) in the water phase Add slowly and emulsify uniformly with homomixer. Thereafter, the mixture was cooled to room temperature, and the remaining (b) phase purified water was added and stirred uniformly to prepare a cosmetic liquid.

 Emulsion base manufacturing division ingredient weight% end Tanaka fermented extract and Tanaka general extract 20 glycerin 3.00 Disodium iodide 0.02 Polyglyceryl-3-methylglucoside distearate 1.5 Cetearyl alcohol 0.5 Caprylic / capric triglyceride 7.0 Polyacrylamide & C13-14 Isoparaffin & Laureth-7 0.60 antiseptic Suitable amount I Purified water to 100

Experimental Example 6 Sebum Secretion Inhibitory Effect

In order to measure the test for sebum secretion inhibitory effect according to the present invention, 30 healthy subjects in their 30s and 30s with oily skin were divided into three groups of 10 people each, using the cosmetic liquid prepared in Experimental Example 5 in the morning. After application to the face twice a day and in the evening, sebum secretion of the skin was measured using a skin oil meter (Sebum meter C + K electronic Gmbh, Germany). At this time, the control means the cosmetic liquid of Example 6 containing no Tanaka fermented extract and Tanaka general extract. The experiment was carried out at a constant temperature and humidity conditions of 25 ℃, relative humidity 45%. For oily skin, the measured value is 220 µg / cm 2 or more, and normal skin is 100 to 200 µg / cm 2. As can be seen in Table 7, the sebum inhibitory effect was excellent in Example 1 and Comparative Example 1, it was confirmed that the effect is more excellent in the Example.

 Sebum branch inhibitory effect Name of sample Days to elapsed (Oily skin mean: 220ug / cm ² ) Before application 10 days after application 20 days after application 30 days after application Control group 242 240 239 241 Example 1 242 215 198 186 Comparative Example 1 241 227 223 214

Experimental Example 7 Skin Temperature Lowering Effect Experiment (Cooling Effect)

7-1. Cooling Effect Measurement I

In order to directly measure the cooling effect of the Tanaka fermented extract and the Tanaka general extract component of the present invention, the time was increased by using a thermometer in an aqueous solution in which Tanaka fermented extract, Tanaka general extract and water were mixed at 8: 2 (v / v). The experiment was performed to measure the temperature change according to. Water was used as a control. At this time, after spraying 30ml (temperature: 22 ℃) of distilled water on each mask sheet, the temperature change was measured according to time, and the part measured by thermometer to minimize the influence of the surrounding environment was made by using aluminum foil and insulation styrofoam. Blocked me. As a result, it can be seen that the Tanaka fermented extract has an excellent temperature cooling effect compared to the Tanaka extract.

In the case of the control group, the temperature was restored to the level of 90% of the room temperature after about 10 minutes, but Example 1 and Comparative Example 1 according to the present invention were found to take about 30 minutes or more to recover to room temperature. The minimum temperature was maintained for a longer time, and then slowly rose to room temperature. It was confirmed that the Tanaka fermented extract can alleviate the heat of the skin and provide a cool and calming effect.

 Cooling effect measurement time Temperature Example 1 Comparative Example 2 Control group 0 minutes (before experiment) 22 22 22 0.5 minutes (30 seconds) 18 18 21.5 1 minute 17 17 21.5 2 minutes 16.5 17 21 3 minutes 15.5 16.5 20.5 5 minutes 15 16 20 7 minutes 15 16 20 10 minutes 15.4 16.7 20.5 15 minutes 16 17.5 21 20 minutes 17 18.2 21.7 30 minutes 19 20 22

7-2. Skin cooling effect measurement II

In order to measure the test for the skin cooling effect according to the present invention, 30 healthy subjects in their 30s and 30s, 15 minutes after applying evenly to the face using the cosmetic liquid prepared in Experiment 5, After the skin temperature cooling effect was measured using a thermometer (Thermometer, Courage + khazaka electronic GmbH, Germany).

 Skin temperature drop effect measurement Name of sample Before and after comparison values Before use after use Example 1 32.7 31.0 Comparative Example 1 32.8 31.8

Experimental Example 8 Evaluation of Human Skin Irritation Test

In order to evaluate the safety of cosmetics according to the present invention, human skin irritation test evaluation was conducted. The test evaluation was judged as a skin irritation test (patch test) as a skin irritation test of the human body. The subjects were 30 healthy subjects in their 30's and 30's according to the CTFA guidelines (The Cosmetic Toiletry and Frangrance association, Safety testing guidelines). 1 g of each of the cosmetic liquids prepared in Experimental Example 6 was added dropwise, and then a patch test was performed on the inside of the left upper arm as a test site. The patch is applied for 24 hours. After the patch is removed, the test site is marked with a marking pen. After 24 hours, 48 hours, and 72 hours at 24 hour intervals, the inflammation and skin reaction of each test site are observed. The decision was made according to the regulations of the International Contact Dermatitis Research Group (ICDRG) (Tables 10 and 11). The final readings were all negative, indicating good skin safety (Table 12).

  Criteria for skin safety readings reaction weight Criteria for judgment - 0 No reaction ± 0.5 Faint erythema + One The border is clear but weak erythema, edema and palsy formation ++ 2 Apparent erythema, papule and blister formation +++ 3 Algebraic formation

Evaluation criteria according to skin response (calculated value) Test substance
(Sample) Criteria 0.0 to 0.9 1.0 to 2.9 3.0 ~ 4.9 5.0 or higher No stimulation Light stimulus Moderate irritation River stimulation

 Primary Stimulation Experiment Results Example 1 Comparative Example 1 Application concentration (%) as is as is Positive reaction (±) - - Weak positive reaction (+) - - Probability of causing contact dermatitis (%) 0.00 0.00

 (-: No irritation)

&Quot; (3) &quot;

                        ∑ (grade x No. of responces)

Skin Reactivity = ----------------------------------------------- -x 100

3 (Maximum grade xn (No. of total subjects)

Claims (7)

Cosmetic composition comprising a Tanaka fermented extract. The cosmetic composition according to claim 1, which contains 0.001 to 95% by weight based on the dry weight of the Tanaka fermented extract. The cosmetic composition according to any one of claims 1 to 2, which is fermented to Tanaka extract alone or through a fermentation process through two or more kinds of Bacillus subtilis, lactic acid bacteria, and yeast. According to claim 1 to 2, wherein the fermented extract characterized by Tanaka fermented extract or reduced pressure concentrated and lyophilized after extraction, skin improvement, skin cooling, skin whitening, anti Thermal aging prevention and improvement cosmetic composition. The composition of any one of claims 1 to 2, further formulated into a unit dosage form further containing a pharmaceutically or cosmetically acceptable carrier or additive. The softening lotion, nourishing lotion, gel, water soluble liquid, milk lotion, nourishing cream, massage cream, essence, eye cream, oil in water (O / W) or water in water (W / O) type according to claim 1 or 2 Emulsions, flake or solid anhydrous products, oil dispersions in aqueous phase using globules, forms of lipid vesicles in ionic or nonionic form, ointments, cleansing creams, cleansing foams, cleansing water, packs or body oils Choose from color cosmetics such as basic cosmetics, oil-in-water or water-in-oil makeup bases, foundations, skin covers, lipsticks, lip gloss, face powders, two-way cakes, eye shadows, mascara, cheek colors and eyebrow pencils. Cosmetic composition for skin improvement, skin cooling, whitening, anti-aging and improvement through pore contraction action and sebum secretion suppressed, characterized in that it is. According to claim 1 to 2, cosmetics, food and pharmaceutical products for the skin improvement effect, skin cooling effect, whitening, anti-aging and anti-aging through pore contraction action and sebum secretion produced by Tanaka fermentation extract.