Anti‐acne activity of carnitine salicylate and magnolol through the regulation of exfoliation, lipogenesis, bacterial growth and inflammation

Abstract

Background

Salicylic acid has been used as an anti‐acne agent with its comedolytic property and antimicrobial activity. However, there is a limit to use for leave‐on cosmetics because of the transient skin irritation and low efficacy at neutral pH condition. We prepared a salicylic acid‐based ionic pair with _L‐carnitine (we named, IP‐BHA) overcoming the limitation of salicylic acid. We examined the effect of IP‐BHA as well as the combination effect with magnolol, a bioactive organic lignan, in order to clarify their efficacy as anti‐acne agents.

Methods

After verifying the structure of IP‐BHA, we confirmed anti‐acne activities including the regulation of exfoliation, lipogenesis, bacterial growth, and inflammation with IP‐BHA and/or magnolol.

Results

The antibacterial activity of IP‐BHA and magnolol was evaluated by determining the minimum antibacterial inhibitory concentration. Magnolol showed strong activity against Cutibacterium acnes, which was better than a medical antibiotic acne drug, clindamycin. The combined application with IP‐BHA was more effective in antibacterial activity by 2.5 times. It was confirmed that testosterone‐induced lipogenesis was significantly inhibited by treatment with IP‐BHA and magnolol, while single treatment had no significant inhibitory effect. Interestingly, MMP‐1 and VEGF were induced by C. acnes lysate in human keratinocytes. We found that these inflammatory molecules were completely inhibited by combined application of IP‐BHA and magnolol. Through ex vivo test, the dose‐dependent exfoliation effect of IP‐BHA was confirmed at pH 5.5, and the synergic exfoliation effect was shown in the combined application of IP‐BHA and magnolol. When topically applied, the emulsion containing IP‐BHA and magnolol relieved the sodium dodecyl sulfate‐induced erythema and improved inflamed acne with papule and pustule.

Conclusion

Our data demonstrate that the ionic paired salicylic acid with _L‐carnitine can overcome the limitations of salicylic acid at low concentration and natural skin pH. Based on the dual administration effects, we suggest that IP‐BHA and magnolol may be the potential agent for acne by improving inflammatory skin condition.

1. INTRODUCTION

Acne vulgaris is a common skin condition in adolescents and young adults, which affects about 85% of adolescents but can persist for more than 30 years into adulthood in some patients. ¹ , ² Acne can be classified as either comedones, which is non‐inflammatory acne, or papules, pustules, and nodules, which are inflammatory acne. ³ , ⁴ In particular, as inflammatory acne leaves erythema, hyperpigmentation, and acne scars, it can cause physical and psychological deformities, which makes continued research on causes and treatment methods important. ⁵ The main factors in the development of acne include (1) excessive sebum production, which is dependent on androgen hormones; (2) hyperkeratosis and occlusion of hair follicles; (3) colonization by Cutibacterium acnes (C. acnes) and subsequent (4) perifollicular inflammation. ⁶ , ⁷ Acne treatment often includes topical or systemic antibiotics and retinoids. A combination treatment optionally includes chemical peels, hormone therapy and laser therapy to help remove lesions. However, these treatments are often accompanied by side effects, such as microbial resistance to antibiotics, stomatitis, skin irritation and teratogenicity caused by retinoids. ⁸ Therefore, a need for potential alternative therapies acne still exists and daily applying cosmetics with anti‐acne efficacy are increasing in recent years. ⁹ , ¹⁰

Salicylic acid, known as BHA (β‐hydroxy acid), is a metabolite of acetylsalicylic acid which exhibits analgesic and anti‐rheumatic activity. For a long time it has been used as a good agent for patients with acne because of the comedolytic and bactericidal properties. ¹¹ , ¹² As an exfoliator, hydroxyl and carboxyl groups of salicylic acid can bind with calcium ions (Ca²⁺) of corneodesmosome, which induces disrupting cellular junctions and eliminating hyperkeratinized cornified cells. ¹³ In addition, as an antibacterial agent, salicylic acid is known to permeate bacterial cell wall and bind with fatty acids in cell membranes, thereby destroying the lipid bilayer. Despite the various effects of salicylic acid involved in anti‐acne, it has been shown that repeated application of salicylic acid may cause skin irritation and dryness. For this reason, the cosmetic ingredient review expert panel recommends using it with pH 3.5 or higher and 3.0% or lower concentration. Conversely, there is a limit in that the effect is significantly reduced in a weakly acidic cosmetic environment due to a decrease in skin permeability by the ionization of salicylic acid. ¹⁴ , ¹⁵ , ¹⁶ In addition, Asian countries such as Korea, China, and Japan as well as the United States and Europe restrict salicylic acid content to less than 0.5% in leave‐on type cosmetics, ¹⁷ so research for better use of salicylic acid is needed. Previous studies on salicylic acid‐based ionic pair (IP) have been tried to overcome these limitations of salicylic acid. The enhancement of skin delivery of salicylic acid by IP has been reported. ¹⁸ , ¹⁹

Magnolol is one of main bioactive neolignans of Magnolia bark extracts which have been widely used for traditional medicines in Asia. ²⁰ Numerous previous studies showed that magnolol has extensive biological activities, such as antimicrobial activity, antioxidation, antiangiogenesis, neuroprotection and anti‐inflammation. ²¹ In addition, magnolol has been known to show antibacterial effects against C. acnes that cause acne. ²² In this study, we examined antibacterial and anti‐inflammatory effects of magnolol as well as the combination effect with IP‐BHA to confirm their efficacy as anti‐acne agents.

As mentioned above, the ionic paired salicylic acid with _L‐carnitine (IP‐BHA) was prepared and its structure was analyzed in this study. We subsequently investigated the antibacterial activity, inhibition of lipogenesis and inflammatory cytokines, and exfoliation efficacy by single or combined treatment of IP‐BHA and magnolol. Finally, comparative clinical evaluation and home use trial (HUT) were conducted with product containing IP‐BHA and magnolol in moderate acne patients.

2. MATERIALS AND METHODS

2.1. Preparation and characterization of IP‐BHA

All used chemicals were purchased from Sigma‐Aldrich (St. Louis, MO). Salicylic acid and L‐carnitine were mixed at a molar ratio of 1: 2, and a small amount of purified water was added, and stirred until the solution turned homogeneous at 50°C. No further purification was performed; however, dipropyleneglycol was added to improve the low‐temperature stability. The final salicylate content of IP‐BHA was 10 wt%. Titration was performed using potassium hydroxide (KOH) when needed. Ionic pair formation and functional group analyses were performed by Fourier transform infrared (FTIR) spectra using a Vertex70, Hyperion 2000 (FTIR spectrophotometer, Bruker Instruments) over a spectral range of 4000 to 500 cm⁻¹ at a data acquisition rate of 2 cm⁻¹ per point. Opus 5.5 software (Bruker Instruments) was used for development and deconvolution of the spectra. UV/Vis absorption spectra were measured using the Synergy 2 Multi‐Mode Microplate Reader (BioTek Instrument, Winooski, VT).

2.2. Determination of minimal inhibitory concentration against C. acnes

C. acnes Ribotype 5 (Strain HL043PA1) were cultured in Reinforced Clostridial broth media (BD Diagnostics, Franklin Lakes, NJ) under anaerobic conditions (37°C, shaking at 175 rpm). Prepared samples were dissolved in water or DMSO and diluted by two‐fold serial titrations, after which 7.5 μL was added to 142.5 μL of a 7.5 × 10⁵ CFU/mL bacteria suspension and incubated for 72 h. Turbidity (OD600) was measured using the Synergy 2 Multi‐Mode Microplate Reader (BioTek Instrument, Winooski, VT). The Minimum Inhibitory concentration (MIC) was defined as the lowest concentration of an agent that achieved ≥90% growth inhibition.

2.3. Cell culture

Human epidermal keratinocyte cell line (HaCaT cell) was grown in Dulbecco's Modified Eagels's media containing 10% fetal bovine serum (FBS, Gibco; Thermo Fisher, Waltham MA), penicillin (100 U/mL, Gibco) and streptomycin (100 μg/mL, Gibco). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. Cells were sub‐cultured at approximately 80%–90% confluency. Primary sebocyte cell (SZ95 cell) was cultured and maintained in DMEM/Ham's F12 medium (Gibco) supplemented with 10% FBS and 5 ng/mL recombinant human epidermal growth factor (Invitrogen; Thermo Fisher, Waltham MA) at 37°C in a humidified atmosphere containing 5% CO₂. After the cells became sub‐confluent, they were harvested with 0.05% Trypsin‐EDTA (Gibco) and sub‐cultured. Cells obtained after the second passage were used in this study.

2.4. Lipogenesis of intracellular lipids

For assessment of sebaceous lipids, SZ95 was seeded at 1 × 10⁵ cells/well in 24 well plates (Greiner, Frickenhausen, Germany), then incubated for 24 h at 37°C and 5% CO₂. To stimulate lipogenesis, SZ95 cells were treated with a 2 × 10⁻⁸ M testosterone, for 24 h, and then cells were treated with IP‐BHA (5 μg/mL) and/or magnolol 0.5 μg/mL or vehicle for 48 h. To evaluate the lipid level, subsequently, cells were washed with phosphate buffer saline (PBS), and 4% formaldehyde was added at RT for 5 min to fix cells. After washing with PBS, 100 μl of 1 μg/mL Nile red (Sigma‐Aldrich) solution diluted in PBS was added into each well at RT for 15 min. To measure the fluorescence, the well plate was covered with aluminum foil to block the light and fluorescence was read at Ex/Em = 550/580 nm with a microplate reader (BioTek Instrument). The results are presented as percentages of the absolute fluorescence units compared to controls.

2.5. Measurement of MMP‐1 and VEGF

HaCaT cells were used to evaluate the production of matrix metalloproteinase‐1 (MMP‐1) and vascular endothelial growth factor (VEGF) by IP‐BHA and/or magnolol. To induce the expression of MMP‐1 and VEGF, lysates were prepared by culturing C. acnes (1.36 × 10⁹ CFU/mL, OD = 1.7) of ribotype 5, an acne pathogen, and treated in HaCaT cells for 30 min. To evaluate the inhibition effects, IP‐BHA and/or magnolol were pretreated in HaCaT cells for 4 h before exposed to C. acnes. Supernatants were harvested for measurements, the concentration of MMP‐1 and VEGF was determined by ELISA following the manufacturer's protocols (R&D Systems; Minneapolis, MN).

2.6. Ex vivo exfoliation assessment

To evaluate the effects on exfoliation, after pre‐marking the application site on the skin, 50 μl of IP‐BHA and/or magnolol was applied. After 3 h the skin surface was gently wiped to remove the remaining substances. D‐squame adhesive tape disks (CuDerm, Dallas, TX) were applied on pre‐marked test sites, applying a 150 g/cm² pressure using a cylindrical weight for 3 s, after which the pressure was removed and the tape peeled from the skin. Three tape strips were taken from each site. After tape stripping, the keratin surface came up to collect the tape on the polystyrene petri dish (Thermo Fisher), and then performed a keratolytic quantification. For this quantification, 1 mL of 1 M NaOH was added to each Petri dish, which was then shaken for 2 h. Seventy‐five microlitre of the solution was subsequently transferred into three wells (96‐well, Corning), and each was neutralized with an equivalent volume of 1 M HCl. Fifty microlitre of Bradford assay reagent (Bio‐Rad, Hercules, CA) was added to each well. After 15 min, the optical density at 595 nm (OD 595 nm) was measured with an empty well as background. The stratum corneum (SC) contents were determined using a γ‐glubullin standard (Bio‐Rad).

2.7. Evaluation of skin erythema recovery

For assessment of skin erythema recovery, the erythema index was evaluated in nine subjects before erythema induction, 5 days and 12 days after erythema induction. Subjects were aged 25–35 years and volunteered for evaluation of erythema inhibitory effect. Studies were conducted under the supervision of a superintendent in accordance with the Standard Operating Instructions. All studies were conducted after obtaining approval from the Institutional Review Board (IRB approval number: HEET‐20211130). Informed consent was obtained from each volunteer prior to participation (research period: 30 November – 14 December 2021). To damage the skin barrier and induce erythema, 1% sodium dodecyl sulfate (SDS) solution was applied to the forearm for 24 h. After induction of erythema, 5 wt% of IP‐BHA and 0.02 wt% of magnolol were repeatedly applied for 12 days in the morning and evening. The erythema index was measured using Mexameter (MX 18; Courage+Khazaka electronic GmbH, Germany), and the erythema recovery index was calculated as follows:

$$\begin{array}{ccc}& & {\mathrm{Erythema}\mathrm{recovery}\mathrm{index}}_t\left(\%\right)\hfill \\ & & =\frac{{\mathrm{Erythema}\mathrm{index}}_0-{\mathrm{Erythema}\mathrm{index}}_t}{{\mathrm{Erythema}\mathrm{index}}_0-{\mathrm{Erythema}\mathrm{index}}_{-1}}\times 100\hfill \end{array}$$

(t: time of measurement point, −1: time of measurement before erythema induction, 0: measurement time after erythema induction)

2.8. Clinical assessment of anti‐acne activity

In order to select appropriate subjects for anti‐acne evaluation, inclusion and exclusion criteria were set following expert clinical grading evaluations to determine eligibility. Eight male and female subjects aged 19–38 years (average 25.09 ± 5.93 years old) with acne classified as investigator's global assessment grades 2–3 and ISGA grade 1 or higher were randomly divided into two groups for evaluation. Studies were conducted under the supervision of a superintendent in accordance with the Standard Operating Instructions. Informed consent was obtained from each volunteer before participation (research period: 2 May – 3 June 2022, IRB approval number: DEF‐ACSIT015(3)−22094). The test products composed with or without (control) 5 wt% IP‐BHA and 0.02 wt% magnolol were applied morning and evening, and subjects were instructed to use no other skincare products, ointments, or drugs on their faces during the study. The study duration was 4 weeks with two evaluations: at enrolment (Baseline) and at Week 4. At each visit, all subjects were permitted to participate in the test after washing the test site with lukewarm water only on each visit, and after resting for 20 min under conditions of constant temperature and humidity, (temperature 22 ± 2°C, humidity 50% ± 5%) with constant lighting, no air movement, and no direct sunlight.

2.9. Questionnaire survey

The survey was performed on 20 volunteers with acne skin aged 20 and 35 years using the HUT method under the supervision of an experienced survey expert. Informed consent was obtained from all volunteers after application (test period: 1 to 29 March 2022). The subjects applied products containing 5 wt% IP‐BHA and 0.02 wt% magnolol for 4 weeks, and subsequently answered question regarding their skin changes over 4 weeks. No subjects exhibited any allergic reactions, such as reddening or itching of the facial skin by the treatment.

2.10. Statistics

All data were analyzed for statistical significance using the SPSS software program (IBM, NY). Normality was verified through the Shapiro‐Wilk test, and normal distribution was considered to be satisfied when the p value was 0.05 or higher. For before‐and‐after comparison, the significance of differences was analyzed using ANOVA for parametric data and the Friedman test/Wilcoxon signed‐ranks test (Bonferroni correction) for nonparametric data. All data are expressed as the mean ± standard deviation, with statistical significance set at p < 0.05. The differences between time points or groups were considered significant at p < 0.05.

3. RESULTS

3.1. Preparation and characteristics of IP‐BHA

The ionic pair system is known to increase skin affinity by reducing the overall polarity of the complex surface and increasing the lipid affinity through bonding between functional groups representing opposite charges on the molecular surface. To optimal prepare carnitine salicylate ionic pair (IP‐BHA), _L‐carnitine and salicylate were mixed in various molar ratios from 3:1 to 1:3. Through confirmation of macroscopic ionic pair stability, we verified the optimal molar ratio of _L‐carnitine: salicylate is 2: 1. The macroscopic stability of the ionic pair between carnitine and salicylic acid was confirmed by maintaining the content of salicylic acid without precipitation at a level much higher than the literature solubility in water (2.48 g/L at 25°C). Figure 1A shows the FTIR spectral change confirming the bonding between the functional groups, and the bonding between the two materials was analyzed based on their previously reported spectra data. ²³ Through the changes of IR spectrum, the forming ionic pair between carnitine and salicylate was confirmed (red dotted square). Finally, by measuring the absorbance of two individual molecules and IP‐BHA, the change in the absorption wavelength through the binding between the two molecules was confirmed (Figure 1B). Through these changes (red dotted square in black dotted line), the formation of IP‐BHA by binding between the two molecules was verified.

FIGURE 1.

Spectroscopic analyses of IP‐BHA. (A) The IR spectrum of vehicle, carnitine/salicylic acid simple mixture, and IP‐BHA. In order to observe the change due to ionic pair formation, the 900–1650 cm⁻¹ IR peak is enlarged (black dotted square). Major changes by ionic pairs appeared at 1386, 1396, 1444–1503, and 1586 cm⁻¹ (red dotted squares). (B) The absorbance spectra of salicylic acid, carnitine/salicylic acid simple mixture and IP‐BHA. In order to observe the change in the absorption pattern due to the formation of the ionic pair, the 280–350‐nm peak was magnified (black dotted square). The change in absorbance spectrum form by ionic pair is clearly shown in the red dotted square.

3.2. Antibacterial activity of IP‐BHA and magnolol

Table 1 shows the MIC test results for C. acnes of IP‐BHA and magnolol compared to antibiotics (clindamycin) and non‐antibiotics (benzoyl peroxide and salicylic acid). Carnitine, one of the components of IP‐BHA, was confirmed to have no antibacterial activity against C. acnes. The MIC against C. acnes was found to be 5000 μg/mL for IP‐BHA and 40 μg/mL for magnolol, respectively. It is known that the antibacterial activity of salicylic acid is decreased in skin pH environment (pH 5.0–6.0). However, the MIC of IP‐BHA at pH 5.5 was found to be equivalent to that of original salicylic acid. To confirm the effect of combinational application of IP‐BHA and magnolol, the MIC by simultaneous application were tested. When applied together, the MIC was decreased to IP‐BHA concentration of 80 μg/mL and magnolol concentration of 16 μg/mL. This result suggests that the combined application of IP‐BHA and magnolol can be much more effective in antibacterial activity.

TABLE 1.

Minimum inhibitory concentrations against Cutibacterium acnes bacteria measured under aerobic conditions, μg/mL.

				Antibiotics	Non‐antibiotics
Substances	IP‐BHA	Magnolol	IP‐BHA / Magnolol Combination	Clindamycin	Benzoyl peroxide	Salicylic acid
MIC	5000	40	80 / 16 (1/62.5 of IP‐BHA MIC) (1/2.5 of Magnolol MIC)	156.25	2500	4000−8000

3.3. Inhibition of lipogenesis

To confirm that IP‐BHA and/or magnolol could regulate sebum secretion through inhibition of lipogenesis, both substances were evaluated in testosterone‐induced lipogenesis assays using SZ95 cells. After confirming that lipogenesis was effectively induced through testosterone treatment, the inhibitory effect on lipogenesis was confirmed through IP‐BHA and magnolol or combination application (Figure 2). When SZ95 cells were treated with IP‐BHA or magnolol, the amount of lipid increased by testosterone was reduced by 5.9% and 3.8%, respectively, and no significant difference was found. However, when IP‐BHA and magnolol were simultaneously treated, the intracellular lipid significantly decreased to 23.4% compared with testosterone treatment.

FIGURE 2.

Effects of IP‐BHA and magnolol on SZ95 cell lipogenesis. (A) Lipogenesis of SZ95 induced by 2 × 10⁻⁸ M testosterone treatment. Induced SZ95 cell were incubated in the presence or absence of IP‐BHA (5 μg/mL) and/or magnolol (0.5 μg/mL). Lipid droplets were stained with Nile Red for the detection of neutral lipid (red). (B) Quantitative assessment of lipid synthesis (Nile Red staining). The results are presented as percentages of the absolute fluorescence units compared to controls. Data represent the results of three independent experiments. *Significant difference between the negative control (non‐treated) or positive control (testosterone treated only) at p < 0.01 (**) (IP‐BHA: IP‐BHA treated, M: magnolol treated).

3.4. Inhibition of inflammatory‐related factors, MMP‐1 and VEGF

MMP‐1 and VEGF are representative inflammatory‐related factors involved in tissue protein degradation and edema formation, respectively. In order to evaluate the efficacy of IP‐BHA and magnolol in relieving inflammation caused by C. acnes lysate, the degree of inhibition of expression concentrations was evaluated. ²⁴ A lysate of ribotype 5 (Strain HL043PA1) of C. acnes, which is known to be one of the dominant species in the skin of acne patients, was prepared and treated into the HaCaT cells. ²⁵ As a result, the expression levels of both MMP‐1 and VEGF were significantly increased by C. acnes lysate. IP‐BHA (0.1 μg/mL) and magnolol (4 μg/mL) were treated and observed to be effective in inhibiting the production of MMP‐1 and VEGF (Figure 3A and 3B). MMP‐1 decreased expression by 49.7% and 33.6% treated with IP‐BHA and magnolol, respectively. A significant decrease in expression by 81.0% was observed when both agents were simultaneously treated. VEFG was also observed to decrease the expression of both IP‐BHA (27.8%) and magnolol (21.2%), and a significant 47.3% decrease in expression was confirmed during simultaneous treatment. These results suggest that combinational application of IP‐BHA and magnolol can effectively inhibit the expression of inflammation‐related factors under C. acnes growing condition.

FIGURE 3.

IP‐BHA and magnolol inhibited Cutibacterium acnes lysate‐induced expression of MMP‐1 (A), and VEGF (B) by HaCaT cells. Cells were treated with C. acnes ribotype‐5 lysate 0.5 h after which cells were treated with IP‐BHA (0.1 μg/mL) and/or magnolol (4.0 μg/mL) for 4 h for the determination of concentration of MMP‐1 and VEGF. A control experiment without lysate treatment was conducted in parallel. Each column shows the mean ± SD. *Significant difference between the negative control (non‐treated) or C. acnes lysate treated only at p < 0.05 (*).

3.5. Keratolytic properties using stripped SC

Afterwards, we conducted evaluation through SC tape‐stripping to evaluate the keratolytic properties of IP‐BHA and magnolol as factors affecting acne vulgaris. The SC exfoliation effect increased according to the concentration of IP‐BHA, and when applied at 5% or more, it showed a significant increase compared to vehicle and no application. Eventhough it is difficult to expect the keratolytic effect of magnolol 0.02 wt%, when simultaneously applied, the exfoliating effect was increased by 35.8% compared to IP‐BHA 5 wt% application alone. Figure 4 shows the dose‐dependent SC exfoliation effect of IP‐BHA and the synergistic effect of SC exfoliation by simultaneous application of IP‐BHA and magnolol. These results verify that the combinational application of IP‐BHA and magnolol could be more effective than IP‐BHA alone on promoting SC exfoliation.

FIGURE 4.

Cumulative amount of SC removed through tape strip after 3 h of treatment with IP‐BHA (0, 2.5, 5, 10 wt%), (A) and treatment with IP‐BHA (0.5 wt%) and magnolol (0.02 wt%) (B). Mean ± SD. Average shown; n = 4. *, † denotes significant difference between the non‐treated or vehicle‐treated, respectively, at p < 0.05 (*,†), p < 0.01 (**,††) (U: untreated, V: vehicle applied, IP‐BHA: IP‐BHA applied, M: magnolol applied).

3.6. Inflammation relief via inhibition of induced erythema

To evaluate the relief of erythema caused by inflammation, erythema was induced for 24 h in six spots with 1% SDS aqueous solution on the forearms of 9 participants. After induction of erythema, each spot was randomly selected as no application, vehicle, 5% IP‐BHA emulsion, 0.02 wt% magnolol emulsion, and IP‐BHA/magnolol emulsion application. In each spot, the emulsion was applied twice daily for 12 days. The erythema index was measured before and after erythema induction. Erythema recovery rates of 46.8 and 59.9% were observed in the non‐applied group and in the vehicle‐applied group after 12 days, respectively. Conversely, the IP‐BHA applied group showed an erythema recovery rate of 74.03%, the magnolol applied group showed an erythema recovery rate of 69.64% after 12 days, and significant differences were confirmed with the no application and vehicle application groups, respectively. Although IP‐BHA and magnolol achieved excellent erythema alleviation effects when applied individually, the simultaneously applied group showed a better improvement effect. The erythema recovery rates were 81.9% on day 12 with statistical significance (Figure 5).

FIGURE 5.

Evaluation of alleviation of inflammation‐induced erythema by chemical skin barrier destruction by SDS. The results of comparison of the erythema relief effect of the emulsion containing IP‐BHA (0.5 wt%) and/or magnolol (0.02 wt%) with the group without application or vehicle application. (*comparison between groups compared to not applied, † comparison between groups compared to vehicle‐applied *, † p < 0.05, **, †† p < 0.01) (U: untreated, V: vehicle applied, IP‐BHA: IP‐BHA applied, M: magnolol applied).

3.7. Clinical and sensory assessment of anti‐acne activity by IP‐BHA and magnolol

Finally, we performed a randomized, double‐blind, placebo‐controlled clinical evaluation, as outlined in the materials and methods. During the course of treatment, compared to baseline, the group that applied the emulsion containing 5 wt% IP‐BHA and 0.02 wt% magnolol showed an improvement in the number of acne and inflammatory lesions compared to the group that applied the vehicle (Figure 6). At the 4th week, the number of papule, pustular acne was especially lower in the treated group compared to the vehicle group. No side effects were reported, and none of the participants complained of pain at the lesion site, or showed hyperpigmentation, ulceration, purpura, infection, scarring, or other serious side effects. For test subjects with acne, a survey was conducted on the sensory effect of using the product for 4 weeks with 20 subjects to confirm the degree of improvement in acne. Results were scored on a 5‐point scale (1: very worse, 2: worse, 3: no change, 4: improved, 5: much improved) and are presented in Table 2. The results of this questionnaire showed that both purulent and comedonal acne were improved, and confirmed that the alleviation and calming of inflamed areas caused by acne, and the reduction of sebum secretion were all significantly felt.

FIGURE 6.

The result of anti‐acne efficacy of 4‐week application of an emulsion containing IP‐BHA (0.5 wt%) and magnolol (0.02 wt%) to acne subjects. Anti‐acne efficacy was evaluated by dividing into non‐inflammatory acne (comedones) and inflammatory acne (divided into papule and postule depending on the severity). (*comparison of before evaluation (week 0) and after evaluation (after 4 weeks), *p < 0.05) (V: vehicle applied, IP‐BHA: IP‐BHA applied, M: magnolol applied).

TABLE 2.

The results of questionnaire survey after applying facial cream. Twenty volunteers between the ages of 20 and 35 with acne‐prone skin were surveyed about the sensory efficacy of skin changes after using an emulsion containing 5% IP‐BHA and 0.02% magnolol for 4 weeks. (1: very dissatisfied, 2: dissatisfied, 3: neutral, 4: satisfied, 5: very satisfied, *p < 0.05).

Questionnaire	Average score
Reduce the size of purulent/papule acne	3.7*
Whitehead/Blackhead reduction	3.8*
Reduction of inflamed areas caused by acne	3.8*
Improves the soothing rate of inflammation caused by acne	4.0*
Reduced sebum secretion	3.7*
Skin feels calmed due to acne improvement	3.8*

4. DISCUSSION

In this study, we assessed synergistic anti‐acne activity of IP‐BHA and magnolol through antibacterial, inhibition of lipogenesis and inflammatory factors, and exfoliating efficacy as cosmetic anti‐acne ingredients. For enhancing efficacy of salicylic acid and reduction irritation by salicylic acid, IP‐BHA was prepared and its structure was analyzed. The intermolecular bonding between salicylic acid and carnitine was analyzed through FT‐IR and absorbance spectra. In previous research, we have been conducting studies on the formation of IPs through intermolecular bonds (hydrogen bonds or dipole‐dipole interactions) and their effects to (1) increase the solubility of poorly soluble substances, or (2) increase skin permeation efficacy. ²⁶ , ²⁷ As a synergistic effect verified by applying IP‐BHA and magnolol together, MIC against C. acnes increased by 62.5 times and 2.5 times, respectively, compared to IP‐BHA and magnolol alone. Along with a direct antibacterial effect, a necessary factor for bacterial inhibition is to reduce sebum secretion. Therefore, the lipogenesis inhibitory effect of IP‐BHA and magnolol for the reduction of sebum secretion was evaluated, as a result, significant lipogenesis inhibition was confirmed by the synergistic effect of the two agents. Another requirement for an effective anti‐acne agent along with antibacteria is anti‐inflammatory efficacy. Among the inflammatory factors, MMP‐1 is known to be involved in the degradation of tissue protein and mediate inflammation in this process. VEGF is known as a substance that mediates vasodilation, swelling and pain. Inhibition of inflammatory factors exhibits an anti‐acne effect by relieving inflammation caused by acne‐bacteria. It was confirmed that IP‐BHA and magnolol showed a synergistic effect on the anti‐inflammatory effect when applied simultaneously. Based on these results, it was verified that IP‐BHA and magnolol had a significant synergistic effect in the evaluation of erythema recovery by skin barrier destruction. In a double‐blind, randomized, clinical trial to investigate the anti‐acne efficacy, the group applied with 5 wt% IP‐BHA and 0.02 wt% magnolol showed significant improvement in the treatment of inflammatory (papule and postule) acne compared to the placebo. This result suggest that IP‐BHA and magnolol could be effective at inhibiting the growth of the anaerobic C. acnes, by increasing the oxygen exposure of hyperkeratinized comedones through the exfoliation effect as well as reducing lipogenesis and inflammatory.

Salicylic acid, which is widely used as non‐antibiotic anti‐acne agent in cosmetics and over the counter (OTC) drugs, is known to show antibacterial, anti‐inflammatory, and exfoliating effects. Nevertheless this anti‐acne related efficacy, many countries regulate the content limit of leave‐on type cosmetics to 0.5%. In addition, Asians have a thin skin barrier on average and are sensitive to irritation, so they often complain of irritation in an acidic environment (under pH < 3.5) including salicylic acid, so the development of an agent that exhibits effective anti‐acne activity in the skin environment (weak acidic pH) is required. IP‐BHA can overcome many of the limitations of salicylic acid in the skin environment. This study, which investigated the simultaneous application of IP‐BHA and magnolol, showed it was possible to develop anti‐acne cosmetics within the compounding limit of salicylic acid. In addition, through the sensory evaluation with the HUT method, we verified that the reduction of sebum, the reduction of pain in the inflamed area, the recovery of erythema due to the inhibition of inflammation, and the reduction of inflammatory acne were noticeably improved.

Through this study showed that IP‐BHA and magnolol both target the main mechanisms of acne, and it is expected that they could be widely used in the cosmetic industry and to treat other dermatological problems as an acne improving material. In addition, future research on appropriate combination therapy may be able to further enhance the treatment effect for more severe acne by applying leave‐on type cosmetics. For example, using dicarboxylic acids as an exfoliating material with higher effectiveness, or using niacinamide as a material that can more effectively suppress sebum may be potential methods. In addition, it may be effective to apply laser‐based home‐used devices or to use microneedles as a delivery form of materials. Through follow‐up research, it is expected that it will be possible to develop a hypoallergenic and effective anti‐acne cosmetic containing IP‐BHA and magnolol which could be widely used academically and industrially.

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest to declare.

FUNDING INFORMATION

The authors received no specific funding for this work

ETHICS STATEMENT

The study design was approved by the appropriate ethics review board.

Kwon KC, Won JG, Kim M‐S, Shin YW, Park S‐W, Song Y‐S. Anti‐acne activity of carnitine salicylate and magnolol through the regulation of exfoliation, lipogenesis, bacterial growth and inflammation. Skin Res Technol. 2023;29:e13406. 10.1111/srt.13406

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.