.scirp.org/file/3-1050448x10.png" />(b)

Figure 3. Suppressive effect of PAC on proliferation and lipogenesis in IGF-1-stimulated SEB-1 sebocytes. (a) Cell cycle progression in SEB-1 sebocytes was analyzed by flow cytometry. Marker 1 (M1) represents apoptotic cells; (b) SEB-1 sebocytes were treated with 20 nM of IGF-1 with 1 ug/ml PAC or 1 uM EGCG treatments for 24 h. Nile red staining was performed to detect lipid (Sebum) production of SEB-1 sebocytes.

3.6. Clinical Effect of PAC-Containg Test Product on Acne Prone Skin

To investigate the efficacy of PAC-containing test product versus a control product, the test and control product were applied twice a day for a 4-weeks on 20 volunteers. The skin oil content (μg/ml), number of skin sebum, numbers of non-inflammatory and inflammatory acne lesion was measured before treatment, at 2 weeks and 4 weeks following 4 consecutive weeks treatment of test product. The results showed a significant decrease in skin oil content of 48.9%

(a) (b) (c) (d) (e)

Figure 4. Effect of Figure 4. Effects of application of PAC-containing test product on acne lesions in a 4-weeks human clinical study. Skin oil content (a); Number of skin sebum (b); Number of whiteheads (c); Number of blackheads (d); and photographs of the faces of control product group (control group) and PAC-containing test product group (PAC group) (e). *P < 0.05 compared with before treatment.

after 4 weeks and sebum secretion rate of 17.6% after 2 weeks (Figure 4(a), Figure 4(b)). Compared to before treatment, the number of whiteheads and blackheads were significantly reduced at PAC-containg test product group at 2 weeks and 4 weeks (Figure 4(c), Figure 4(d)). In addition, compared to the control product group at 4 weeks, PAC-containg test product group significant reduction of 27.2% and 11.3% in the mean number of whiteheads and blackheads. Papules and pustules were showed a tendency to decrease at 4 weeks in PAC-containing test product group compared to the control product group (Table 3). These results indicate that the PAC complex exerts an acne-mitigating effect.

3.7. Human Skin Primary Irritation Test of PAC

A skin primary irritation test in humans was conducted to evaluate the irritation potential of PAC on human skin. A human patch test is a general method that is used to determine the human skin safety of a substance for clinical applications. The patch test was usually removed after 48 hours, and readings were recorded 30 minutes after removing the strips (day 2) and after 24 (day 3) or 48 (day 4) hours, as modified by Frosch & Kligman [44] . 31 healthy volunteers aged 20 to 50 years (average 41.6 ± 6.5 yrs) were recruited for this study. During this study, none of the 31 subjects experienced a reaction based on the 48- and 72-h readings (Table 4) and PAC did not induce any skin reaction. Therefore, these data demonstrate that the PAC is within the scope of the slight-irritation and is safe to apply to human skin.

4. Discussions

Acne is characterized by multifactorial conditions, including hormonal imbalance, abnormal ductal keratinization, bacterial overgrowth, inflammation, and excessive sebum production. Despite the existence of many studies on the pathophysiological mechanisms of acne, the development of effective ingredients for treatment has been limited because such substances have shown a single effect among multiple physiological factors of acne, not on multiple aspects [47] [48] . Recently, there has been an increasing focus on developing therapeutic agents that target multiple pathogenesis of acne, with no side effects. Pretsch et al. reported that Talaromyces wortmannii extracts have an anti-microbial activity against acne-inducing bacteria including P. acnes and anti-inflammatory properties by inhibiting P. acnes-mediated activation of NF-kB and activator protein-1 (AP-1) activation, as well as IL-8 release [49] . Results from in vitro, ex vivo, and clinical studies on BGM complex composed of bakuchiol, Ginkgo biloba extract, mannitol for acne therapy showed antibacterial activity against

Table 3. Statistical analysis of whiteheads, blackheads, papule and pustule by lesion counting.

*Significantly different at P < 0.05 compared with control group.

Table 4. Human skin primary irritation test.

P. acne, antioxidation activity of squalene, anti-inflammatory activity and regulation of sebum composition [50] .

In the current study, we first report on the antibacterial activity against P. acnes of Pinus thunbergii needle extract, Artemisia annua L. extract, and Citrus junos Tanaka fruit juice and anti-inflammatory activity of Pinus thunbergii needles extract and Artemisia annua L. extract.

We also assessed antibacterial properties against P. acnes and S. epidermidis, anti-inflammatory potential of a new combined plant extract (PAC), consisting of Pinus thunbergii needle extract, Artemisia annua L. extract, and Citrus junos Tanaka fruit juice. PAC showed the lowest MIC value of 2.25 mg/ml and 0.75 mg/ml against P. acnes and S. epidermidis and was more effective than LP and AA on inhibition of P. acnes- and LPS-stimulated inflammation. In addition, we evaluated sebosupressive and clinical effect on acne prone skin of PAC whether PAC can regulate multiple pathogenic factor of acne. The results indicated that PAC had suppressive effect on differentiation and lipogenesis in IGF-1-stimulated SEB-1 cells. In clinical study, compared to the control product group at 4 weeks, skin oil content, skin sebum number, and the numbers of whiteheads and blackheads in PAC-containg test product group were significantly decreased. Furthermore, a patch test was performed to confirm the safety of PAC, and it did not show any adverse reactions on human skin.

Based on these results, this study suggests that PAC may be introduced as a potential therapeutic agent for acne vulgaris.

5. Conclusion

In summary, our results reveal that PAC is effective in suppressing multifactorial pathological conditions of acne, including antimicrobial activities against and S. epidermidis, anti-inflammatory activities, sebo-suppressive effects, and reduction of skin oil content, sebum number, and the numbers of acne lesions. Therefore, this study demonstrated that the PAC can improve acne treatment and can be provided as an effective new agent for acne therapy.

Acknowledgements

This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region.

Conflicts of Interest

The authors declare no conflicts of interest.

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