KR20060014203A - Magnolol and honokiol as a effective compound for acne, one of skin diseases - Google Patents

Abstract

The present invention relates to a composition for acne comprising magnolol represented by the following formula (1) and honokiol as an active ingredient. The magnolol and honokiol as a substance that inhibits the growth of propionibacterium acne and inhibits the production of inflammatory cytokines induced by propionibacterium, and thus can be usefully used for acne improvement products. have.

       <Formula 1>

Magnolol, honokiol, propionibacterium acnes, antibacterial effect, anti-inflammatory effect

Description

Magnolol and honokiol as active ingredients for acne {Magnolol and honokiol as a effective compound for acne, one of skin diseases}

The present invention relates to a substance that can be used for the treatment and management of acne, and more particularly, to inhibit the growth of propionibacterium, which is the main cause of acne, and to inhibit the production of basophilic cytokines induced by propionibacterium. It relates to a material for the treatment and management of acne by controlling acne.

Acne is known to be caused by a combination of various causes. In general, acne has increased secretion of sebaceous glands due to increased male hormone function in puberty, epithelial cells of hair follicles cause abnormal keratosis and clogged hair follicles. It is recognized that propionibacterium acnes, a fungus, actively proliferates to produce free fatty acid, which stimulates hair follicles and causes inflammation of acne. The progression of acne is exacerbated by sebum oversecretion associated with 5-alpha-reductase.

Conventionally, as a method for treating or alleviating acne, a method of using female hormones such as estrogen, which is a male hormone inhibitor, or an antimicrobial agent, has been used to suppress sebum hypersecretion. In recent years, a method of inhibiting sebum hypersecretion using a 5-alpha-reductase inhibitor has been proposed.

However, the method of using the female hormone agent is highly susceptible to skin inflammation as a side effect during the treatment of acne, and the use of female hormone has been reported as a result of the long-term administration of female hormone has been refrained from use.

In addition, typical antimicrobial agents for acne bacteria include resorcinol, sulfur, benzoyl peroxide, tetracycline, erythromycin or methacycline, but the use of such antimicrobial agents may improve acne to some extent, There are many problems in terms of side effects. For example, retinoic acid and benzoyl peroxide, which are antimicrobial agents, are highly susceptible to skin irritation and contact dermatitis, and tetracycline-containing antibiotics have been reported to have side effects due to the appearance of resistant bacteria and photosensitive effects.

Recently, in order to overcome the problems described above, cosmetic compositions for improving acne skin using natural fragrance substances such as tea tree oil or rosemary oil have been proposed. Has a pungent odor that does not meet consumer preferences.

Therefore, the inventors of the present invention have attempted to develop a single substance having excellent acne-improving effect, high stability, and no skin side effects. Magnolol and honokiol obtained during this series of processes are substances present in plants such as magnolia and have been known mainly as antibacterial and anti-inflammatory substances. In addition to these substances, it is well known that many substances obtained from plants have a strong antibacterial effect. However, no antimicrobial and acne-improving effects on the acne bacteria by magnolol and honokiol have been reported. In the present invention, magnolol and honokiol have excellent antibacterial effect on propionibacterium acnes, which is the main acne bacterium, and excellent effect on inhibiting the production of proinflammatory cytokine by propionibacterium, and also excellent in human skin safety. The invention was completed by confirming that it can be used as a material for improving acne.

An object of the present invention is to provide acne-producing cosmetics, foods, pharmaceuticals, etc. by providing a natural material that simultaneously exhibits the effect of inhibiting the phenomena caused by the antibacterial and propionibacterium propionibacterium acnes It is intended for use in the composition of the present invention.

In order to achieve the above object, the present invention provides a material for improving acne having a magnolol and honokiol of the formula (1) as an active ingredient.

  <Formula 1>

                          Magnolol Honokiol

Hereinafter, the present invention will be described in detail.

Magnolol and Honokiol used as active ingredients in the present invention as a compound present in magnolia, has been reported to have anxiety relief, anti-inflammatory, skin cancer inhibitory and antibacterial effect. However, most of the results of the antimicrobial effect are obtained from aerobic bacteria and fungi, and the antimicrobial effect is not known for anaerobes. In particular, the antimicrobial effect against propionibacterium acnes, the main cause of acne, and the inhibitory effect on the production of proinflammatory cytokines caused by propionibacterium are not known. The present invention has been completed by demonstrating that the magnolol and honokiol can be used as an active ingredient of the acne improvement composition.

The structure of magnolol and honokiol is a structure in which two benzene rings are connected by C-C bonds, which correspond to isomers (Formula 1). Magnolol and Honokiol showed strong antimicrobial activity against Propionibacterium acnes and Propionibacterium granulosum. The antimicrobial effect on these anaerobic bacteria is the first result found in the present invention. In addition, it has been shown to significantly reduce the production of interleukin-8 and TNF-alpha, which are pro-inflammatory cytokines induced by propionibacterium acnes. No application of irritation was observed when applied to human skin and it was found that it could be used in cosmetics and medicines aimed at improving acne.

Magnolol and Honokiol discovered according to the present invention can be used as a main ingredient or as an additive in the form of cosmetics, soaps, skin ointments, foods, pharmaceuticals and the like for the purpose of improving acne.

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

Example 1

Antimicrobial Activities of Magnolol and Honokiol against Propionibacterium Acnes and Propionibacterium Granulum

Agar diffusion (standardized filter-paper disk-agar diffusion method) was used to determine the antimicrobial activity of magnolol and honokiol against propionibacterium acnes and propionibacterium granulosum. This method is well known as the Kirby-Bauer method and is widely used for the determination of antimicrobial activity (see European Journal of Pharmacology, Junho Park et al, 2004, 496 (1-3), 189-195). Propionibacterium Acnes and Propionibacterium granulosum cells were cultured using brain heart infusion broth until they reached the stationary phase at 37 degrees under anaerobic conditions. Approximately 10 ⁶ bacterial cells were mixed in a 7 ml GAM agar medium containing 0.8% phytoagar and poured onto a GAM agar plate containing 1.5% phytoagar. Each test concentration of magnolol and honokiol was treated on a filter-paper disc with a diameter of 10 mm, and then placed on a GAM agar plate inoculated with bacteria and incubated at 37 degrees for 24 hours under anaerobic conditions. Antimicrobial activity was measured by measuring the size of halo). Erythromycin was used as a positive control. The size of the zona pellucida was a function of the logarithmic value of the concentrations of magnolol and honokiol, from which a standard curve was obtained (FIG. 1).

Fig. 1

The unknown concentration of the sample can be determined through the standard calibration curve shown in FIG. As shown in Table 1, Magnolol and Honokiol showed significant antimicrobial activity against Propionibacterium Acnes and Propionibacterium granulosum. However, antimicrobial activity was not higher than that of erythromycin. Honokiol showed distinct antimicrobial activity from 6.25ug / disk (0.39 mM) and 12.5ug / disk (0.78 mM) for magnolol (Table 1).

Table 1

Example 2

Minimum Inhibitory Concentration (MIC) of Magnolol and Honokiol

Experiments were performed to determine the minimum inhibitory concentration (MIC) of magnolol and honokiol. Propionibacterium acnes and propionibacterium granulose were incubated for 24 hours in 3 ml brain heart infusion broth, and at the same time, magnolol and honkyol were added, followed by incubation for 24 hours at 37 degrees under anaerobic conditions. do. Two-fold serial dilution method was used to measure the MIC of honokiol and magnolol. Here MIC is defined as the minimum concentration that inhibits the growth of microorganisms. As shown in Table 2, the MIC of magnolol for Propionibacterium acnes was 9 ug / ml (33.8 uM), and the MIC of honokiol was 4 ug / ml (15 uM). For propionibacterium granulosum, 9 ug / ml (33.8 uM) for magnolol and 3 ug / ml (11.3 uM) for honokiol was determined as MIC. It can be seen that the antibacterial activity of honokiol is higher than that of magnolol.

[Table 2]

              Example 3

Minimum Bacteriocidal Concentration (MBC) Determination of Magnol and Honokiol for Propionibacterium Acnes

Inhibition of growth of bacteria by certain substances is the result of inhibition of the proliferation of individual cells or the death of cells. In the case of growth inhibition (bacteriostatic), the growth of bacteria can be resumed by the removal of the substance, but in the case of death. In order to determine the minimum bactericidal concentration (MBC) of magnolol and honokiol, the following experiment was performed. Once in the liquid culture, go through the same process as the MIC measurement, and after the incubation a portion of the culture medium in which no growth has occurred, diluted to remove the magnolol and honokiol and incubated on a solid medium. 24 hours after the culture, the colony formation was observed. At this time, MBC means the minimum value of the magnolol or honokiol concentration of the original culture of the plate medium without colonies. Propionibacterium acnes was used to determine the MBC of magnolol and honokiol, and 1, 5, and 10 times the MIC concentrations of magnolol or honokiol for the determination of kinetics of bacterial killing. Concentration was applied. Microbial viability was measured within 2 hours. As shown in Figure 2, the rate of apoptosis is dependent on the treatment concentrations of magnolol and honokiol, 20 ug / ml (75.2 uM) for honokiol, and 45 ug / ml (169.2) for magnolol. uM) was observed to extinction propionibacterium acnes within 10 minutes (Fig. 2)

[Figure 2]

(Example 4)

Evaluation of the Production Inhibitory Effect of Anti-inflammatory Cytokines Induced by Propionibacterium Acnes by Magnolol and Honokiol

In general, inflammatory reactions caused by bacteria or bacterial components are mediated mainly by proinflammatory cytokine. ELISAs for interleukin-8 and TNF-alpha were performed to determine whether magnolol or honokiol inhibit the inflammatory response caused by propionibacterium acnes. Using THP-1 Cell Line, a Human Monocyte Cell Line

1X10 ⁶ THP-1 cells were incubated for 14 hours in the absence of serum and then treated with 100 ug / ml of propionibacterium acnes inactivated by heat treatment alone or simultaneously with magnolol and honokiol, respectively. After 48 hours incubation. The levels of interleukin-8 and TNF-alpha secreted into cell media were measured by ELISA kit (Genzyme, Mineapolis, MN). As shown in Figure 3, it was observed that the production of interleukin-8 and TNF-alpha induced by Propionibacterium acnes was reduced by both magnolol and honokiol. Overall, Honokiol showed somewhat stronger cytokine inhibition at test concentrations than Magnolol, but 10 uM of Magnolol showed stronger interleukin-8 inhibition than Honokiol at the same concentration. In the case of 20 uM magnolol, the inhibitory effect of interleukin-8 production was not observed compared to the same concentration of honokiol (FIG. 3). These results suggest that 10 uM is the optimal concentration of magnolol for Interleukin-8.

3

(Example 5)

Efficacy in the treatment of acne by external skin preparations containing magnolol and honokiol

Acne cosmetic compositions containing magnolol, honokiol and triclosan, respectively, were prepared according to the conventional methods with the component contents shown in Table 3 below. In the following Table 3, the control group is an external preparation for skin that does not contain an antibacterial and anti-inflammatory substance, and test group 1, test group 2, test group 3, test group 4, and the like, respectively, are magnolol, honokiol, magnolol + honokiol And external skin acne cosmetic compositions including triclosan, respectively.

For the preparation of external skin preparations, purified water, glycerin and butylene glycol were mixed and dissolved at 70 ° C. (water part), and the other components except the above three components and trimethanolamine were dissolved at 70 ° C. (oil part). The oil part was added to the water part and stirred with a homomixer (Tokushu Kika, Japan) to emulsify first, to which trimethanolamine was finally added. After removing the bubbles generated in the mixed solution, and cooled to room temperature to prepare a skin external preparation.

Table 3

ingredient content (%) Control Test group 1 Test group 2 Test group 3 Test group 4 Purified water 72 72 72 72 72 glycerin 8.0 8.0 8.0 8.0 8.0 Butylene glycol 4.0 4.0 4.0 4.0 4.0 Magnolol 0 1.0 0 0.5 0 Honokiol 0 0 1.0 0.5 0 Triclosan 0 0 0 0 1.0 Caprylic Capri Triglyceride 8.0 8.0 8.0 8.0 8.0 Squalane 5.0 5.0 5.0 5.0 5.0 Cetearyl Glucoside 1.5 1.5 1.5 1.5 1.5 Sorbitan stearate 0.4 0.4 0.4 0.4 0.4 Cetearyl Alcohol 1.0 1.0 1.0 1.0 1.0 Trimethanolamine 0.1 0.1 0.1 0.1 0.1 Gross weight 100 100 100 100 100

In order to measure the clinical effect on the acne treatment or alleviation of the external dermal cosmetic composition containing magnolol and honokiol, a clinical evaluation of the skin lotion of the control group, test groups 1 to 4 was performed. Thirty women in their 20s with acne are developing skin acne with the composition of the control group and test groups 1 ~ 4 twice a day for 30 days, and their acne treatment status is evaluated by visual and photographic clinical evaluation. It was determined, and the results are shown in Table 4 below.

Table 4

Good effect Effect slightly no effect Worse system Control - 2 (6.6%) 26 (86.6%) 2 (6.6%) 30 persons Test group 1 20 (66.6%) 8 (26.6%) 2 (6.6%) - 30 persons Test group 2 22 (73.3%) 7 (23.3%) 1 (3.3%) - 30 persons Test group 3 21 (70.0%) 8 (26.7%) 1 (3.3%) - 30 persons Test group 4 15 (50.0%) 14 (46.7%) 1 (3.3%) - 30 persons

From Table 4, it was found that the external preparation for skin according to the present invention containing magnolol and honokiol has an excellent acne treatment to alleviate the acne. In particular, it can be seen that the effect is excellent compared to the conventionally used triclosan.

(Example 6)

Cytotoxicity Assessment of Magnolol and Honokiol

MTT experiments were carried out using HaCaT, a human keratinocyte line and human normal fibroblasts, for the cytotoxic effects of magnolol and honokiol. A comparative experiment was performed with triclosan, which is used as an existing acne improver. In human normal fibroblasts, magnolol and triclosan did not show cytotoxicity at concentrations below 5 ug / ml. Honokiol, on the other hand, showed cytotoxicity of 58% at 5 ug / ml. HaCaT cell line, a human keratinocyte line, also shows cytotoxicity similar to that of human normal fibroblasts. In conclusion, Honokiol shows slightly higher cytotoxicity than magnolol or triclosan, but there is no significant difference in cytotoxicity compared to conventional acne improvers (FIG. 4). While magnolol and honokiol are not decomposed by light or by-products, triclosan is converted to dioxin, a highly toxic environmentally harmful substance by light. This is reported in the Journal of Photochemistry and Photobiology A: Chemistry ( Vol. 158, Issue 1 , 30 May 2003). In fact, triclosan is widely used in acne products, and the main cause of acne is the face, which is exposed to sunlight. Therefore, the triclosan used on the face is naturally changed to dioxin, which is harmful to the human body. Thus, in vitro tests showed no toxicity to honokiol, but it can be seen that it is harmful to the human body in actual use.

[Fig 4]

(Example 7)

Magnolol and Honokiol to confirm safety of human skin

In order to confirm that the magnolol and honokiol, which were found to have excellent acne-improving effect as described above, are safe for human skin, skin safety verification experiments for the test groups 1 to 4 listed in Example 5 were performed. As a test method, a skin cumulative stimulation test was performed.

Magnolol and Honokiol irritate the skin by performing a total of nine 24-hour cumulative blisters on the upper forearm every other day for 30 healthy adults using each skin preparation prepared in Example 5). Was measured.

The patching method used a fin chamber (Finn chamber, Epitest Ltd, Finland). 15ul of each said skin external preparation was dripped at the chamber, and the patch was performed. The degree of response to the skin each time was scored using the following formula, and the results are shown in Table 6 below.

Average responsiveness = [[response index x responsiveness / total number of subjects x highest score (4 points)] x 100] ÷ number of tests (9 times)

At this time, ± 1 point, + 2 points, + + 4 points in the reactivity, it is determined as a safe composition when the average reactivity is less than 3.

Test substance Number of subjects with response                                          Average reactivity 1 week                                          2 weeks                                          3 weeks                                          Primary Secondary 3rd 4th 5th 6th 7th 8th 9th ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + Control 2 - - --- --- --- --- --- --- --- --- 0.18 Test group 1 2 - - 0 - - --- --- --- --- --- --- --- 0.18 Test group 2 3-- One - - --- --- --- --- --- --- --- 0.37 Test group 3 2 - - 0 - - --- --- --- --- --- --- --- 0.18 Test group 4 3-- One - - --- --- --- --- --- --- --- 0.37 Inspector                                          30 30 30 30 30 30 30 30 30

In the above Table 1, the number of people corresponding to ±, +, ++ was 2, 0, 0, respectively, and the rest did not appear. When calculated according to the formula described above, the average reactivity of test group 1 to test group 4 was 0.18, 0.37, and 0.18, respectively. It became 0.37, and it was three or less, and judged to be a safe composition.

As shown in Table 6, the external skin preparations including magnolol (test group 1), honokiol (test group 2), magnolol + honokiol (test group 3), or the skin containing triclosan (test group 4) It did not show a distinct cumulative stimulus pattern like external preparations and was determined to be safe for human skin.

According to the present invention, at the in vitro level, magnolol and honochiol have an excellent antimicrobial activity against the main causative agents of acne, as well as the effect of inhibiting the production of proinflammatory cytokines induced by propionibacterium. In clinical trials, it also showed a pimple relief effect. Magnolol and Honokiol also proved to be safe for human use because they do not generate any toxicity or irritation in human safety tests. Therefore, the magnolol and honokiol or its complexes and derivatives can be usefully used in cosmetics, medicine and food for the purpose of improving acne.

Claims (10)

Regarding the use of at least one or more of magnolol, honokiol, a complex and derivatives thereof of Formula 1 as an active ingredient of a product for improving acne skin              <Formula 1>

                            Magnolol

       (Honokiol) When the acne skin improvement product of claim 1 is a soap, cosmetics, food, and medicinal products According to claim 1, wherein the composite is based on the composition ratio of magnolol or honokiol, and when the total weight is 100%, the composition ratio is between (1 to 99% by weight: 99 to 1%). According to claim 1 when applied to humans and animals for the purpose of improving acne skin According to claim 1, wherein magnolol and honokiol are extracted from natural products The method of claim 1 wherein the magnolol and honokiol is partially or completely synthesized The chemical substance according to claim 1, wherein other chemicals are derivatized by synthesizing magnolol and honokiol by synthesis. When the derivative of claim 6 is used to improve acne skin Soaps, cosmetics, foods, and pharmaceutical products containing the derivatives of claim 6 According to claim 2, wherein the use concentration of magnolol and honokiol is from 0.0001% by weight to 20% by weight