Multiple Activities of Punica granatum Linne against Acne Vulgaris

Acne is a common skin condition with sebum overproduction, hyperkeratosis, Propionibacterium acnes (P. acnes) and Staphylococcus aureus, and inflammation. Punica granatum (pomegranate) is well-known for its anti-inflammatory effects; however, few studies have discussed the anti-acne effects of pomegranate. In this study, we found that pomegranate extract (PG-E) significantly reduced P. acnes-induced edema in Wistar rat ears. Therefore, an evaluation platform using multiple pathogenic mechanisms of acne was established to explore the anti-acne effects of pomegranate. Results showed that PG-E inhibited bacterial growth and lipase activity. Through a bioguided-fractionation-isolation system, four hydrolysable tannins, punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4), were isolated. Compounds 1 and 2 had greater anti-bacterial activities and anti-testosterone-induced HaCaT proliferative effects than the others. Compounds 1, 3, and 4 displayed lipase inhibitory effects. Compound 4 decreased cyclooxygenase-2 expression and downregulated prostaglandin E2 production in heat-killed P. acnes-treated RAW 246.7 cells. In conclusion, PG-E is abundant in hydrolysable tannins that display multiple anti-acne capacities, including anti-bacterial, anti-lipase, anti-keratinocyte proliferation, and anti-inflammatory actions. Hence, PG-E has great potential in the application of anti-acne and skin-care products, and punicalagin (1), the most effective component in PG-E, can be employed as a quality control marker.


Introduction
Acne vulgaris (acne), a common skin disease, usually appears in young adolescents with a hormone imbalance [1,2]. More than 85% of teenagers are affected by acne, and most of them continue to be affected into adulthood. In the United States, acne therapies cost approximately $1 billion per year, while more than $100 million is spent on over-the-counter acne products [3,4]. Major causes of acne formation are fourfold: (1) increasing sebum production by overactive oil glands; (2) retention hyperkeratosis, which blocks skin pores; (3) activities of normal skin bacteria (Propionibacterium acnes and Staphylococcus aureus); and (4) skin inflammation [5].
In sebum production, human sebum is composed of fatty acids, triglycerides, wax esters, and so on. Skin areas rich in sebaceous glands are positively correlated with acne lesions [2]. Nowadays, one therapeutic strategy for the increased sebum production is topical use of azelaic acid, a dicarboxylic acid found in rye, wheat, and barley, which can block the synthesis of fatty acids to avoid the overproduction Punica granatum Linne (pomegranate) belongs to the Punicaceae family. In the Mediterranean area, pomegranate is commonly used as a fruit for therapeutic agents, foods, and cosmetics. However, in traditional Chinese medicine, pomegranate was recorded as an astringent agent [13]. Evidence has shown that the pharmacological effects of pomegranate are its anti-inflammatory, anti-oxidative, anti-lipoperoxidative, anti-bacterial, and anti-tumor activities [13][14][15][16][17]. In our previous study, four hydrolysable tannins, punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4), were isolated from pomegranate using column chromatography combined with in vitro anti-inflammatory-guided fractionation [13]. These four compounds and the 70% acetone pomegranate extract displayed both in vitro and in vivo anti-inflammatory effects. However, few researchers have discussed the anti-acne effects of pomegranate. Therefore, an evaluation platform was used to explore the anti-acne effects of pomegranate. In addition, the principal anti-acne components of pomegranate are also discussed in this study.

Pomegranate Attenuate P. acnes-Induced Wistar Ear Edema
In vivo anti-acne effects are difficult to assess because of a lack of animal models. We set up an animal model to simulate acne formation by directly injecting P. acnes into a rat's ear and evaluated the in vivo anti-acne effects of PG-E. Wistar rat ears exhibit edema when live P. acnes is injected. As shown in Figure 1, rat ear edema in the PG-E ointment group was significant lower than that in the vehicle control group. In this model, live P. acnes was injected into Wistar rat ears. We first suggest that PG-E can inhibit the growth of P. acnes, resulting in a lower inflammatory status and less ear edema. of pomegranate. In addition, the principal anti-acne components of pomegranate are also discussed in this study.

Pomegranate Attenuate P. acnes-Induced Wistar Ear Edema
In vivo anti-acne effects are difficult to assess because of a lack of animal models. We set up an animal model to simulate acne formation by directly injecting P. acnes into a rat's ear and evaluated the in vivo anti-acne effects of PG-E. Wistar rat ears exhibit edema when live P. acnes is injected. As shown in Figure 1, rat ear edema in the PG-E ointment group was significant lower than that in the vehicle control group. In this model, live P. acnes was injected into Wistar rat ears. We first suggest that PG-E can inhibit the growth of P. acnes, resulting in a lower inflammatory status and less ear edema. Anti-ear edema effects of pomegranate extract (PG-E) ointment against a P. acnes injection; * p < 0.05, compared to the vehicle group on the same day; Data are presented as the mean ± SD; Significance was calculated using Student's t-test by SPSS software v.15; Each group contained eight rats.

Pomegranate Displayed No Skin Irritation
As shown in previous studies, a modern therapeutic strategy for acne is to use retinoic acid or salicylic acid. However, skin irritation and desquamation are obvious side-effects of these chemical agents. In this study, a single-dose skin irritation test was performed by modifying the Draize test [18]. PG-E displayed no skin irritation in Wistar skin at 0.1-10 mg/site for 24-48 h, indicating the excellent safety of PG-E (data not shown).

Pomegranate Significantly Inhibited P. acnes and S. aureus Growth
Because PG-E significantly attenuated P. acnes-induced Wistar ear edema, we continued to explore the in vitro activities and try to discover the mechanisms. PG-E displayed stronger antibacterial activity against P. acnes than against S. aureus. The diameter of the inhibition zone of PG-E against P. acnes ranged 11.3-17.1 mm. Moreover, the inhibition zone of PG-E against S. aureus ranged 12.6-15.9 mm. We calculated the ratio of the inhibition zone compared to penicillin. Ratios of the inhibition zone of PG-E against S. aureus and P. acnes were 0.451 and 0.544, respectively (Table 2). Anti-ear edema effects of pomegranate extract (PG-E) ointment against a P. acnes injection; * p < 0.05, compared to the vehicle group on the same day; Data are presented as the mean ± SD; Significance was calculated using Student's t-test by SPSS software v.15; Each group contained eight rats.

Pomegranate Displayed No Skin Irritation
As shown in previous studies, a modern therapeutic strategy for acne is to use retinoic acid or salicylic acid. However, skin irritation and desquamation are obvious side-effects of these chemical agents. In this study, a single-dose skin irritation test was performed by modifying the Draize test [18]. PG-E displayed no skin irritation in Wistar skin at 0.1-10 mg/site for 24-48 h, indicating the excellent safety of PG-E (data not shown).

Pomegranate Significantly Inhibited P. acnes and S. aureus Growth
Because PG-E significantly attenuated P. acnes-induced Wistar ear edema, we continued to explore the in vitro activities and try to discover the mechanisms. PG-E displayed stronger anti-bacterial activity against P. acnes than against S. aureus. The diameter of the inhibition zone of PG-E against P. acnes ranged 11.3-17.1 mm. Moreover, the inhibition zone of PG-E against S. aureus ranged 12.6-15.9 mm. We calculated the ratio of the inhibition zone compared to penicillin. Ratios of the inhibition zone of PG-E against S. aureus and P. acnes were 0.451 and 0.544, respectively ( Table 2). Table 2. Anti-P. acnes and S. aureus effects of pomegranate extract (PG-E).

Pomegranate Polyphenols Caused Shrinkage and Damage in P. acnes and S. aureus
Morphological changes are a clear indicator for monitoring the process of bacterial death. SEM, a powerful electron microscope, can be used to investigate diversification of a bacterium's shape. Differences between treatments with four hydrolysable tannins in P. acnes and S. aureus are illustrated in Figure 2. As shown in Figure 2A, outer membranes of the control group were smooth and rod-shaped. However, after treatment with 1 or 2 (100 µg/mL) for 12 h, S. aureus produced around grains and exhibited bulging or deformation of its spherical shape. Intra-inclusions were found to have effluxed outside (representatives are indicated by arrows), and the bacteria were obviously swollen. In Rabie et al.'s study, the bacterial structure is an important point for its normal physiology, such as the outer membrane integrity [23]. We suggested that hydrolysable tannins in pomegranate damaged the structural integrity and bacterial proteins, leading to changes in the integrity and function of bacteria. Compounds 1 and 2 also significantly inhibited the growth of P. acnes ( Figure 2B). However, P. acnes originally had a spherical shape and displayed a smooth, complete surface. The bacterial surface exhibited obvious shrinkage, damage to the surroundings, and increased granules after treatment with 1 and 2 for 12 h. Biofilms of bacteria play an important role in their virulence and pathogenicity. Another anti-bacterial strategy is to inhibit biofilm formation. It was reported that pomegranate significantly decreases biofilm formation by S. aureus [24]. Anti-biofilm formation capacities of pomegranate will be an important target in future studies.
Int. J. Mol. Sci. 2017, 18, 141 5 of 12 P. acnes originally had a spherical shape and displayed a smooth, complete surface. The bacterial surface exhibited obvious shrinkage, damage to the surroundings, and increased granules after treatment with 1 and 2 for 12 h. Biofilms of bacteria play an important role in their virulence and pathogenicity. Another anti-bacterial strategy is to inhibit biofilm formation. It was reported that pomegranate significantly decreases biofilm formation by S. aureus [24]. Anti-biofilm formation capacities of pomegranate will be an important target in future studies.  (2), strictinin A (3), and granatin B (4); All photos were taken with scanning electron microscopy; C: control group; The concentration of 1 to 4 was 100 µg/mL; Data are from three separate experiments, the picture of one of which is shown; The arrow indicates a disruption site of bacteria; Scale bar of control group is 5 µm; Scale bar of four hydrolysable tannins against S. aureus and P. acnes is 5 µm and 2 µm, respectively.

Pomegranate Polyphenols Inhibited Lipase Activity
Lipase is an enzyme that hydrolyzes lipids. Some bacteria, such as P. acnes, can secrete lipase to resolve lipids and facilitate lipid-related nutrient absorption from an external medium. In this study, a fluorometric assay of lipase activity was administrated with and without PG-E and the four hydrolysable tannins. Compared to the control, PG-E (200 µg/mL) inhibited lipase activity by 20% as shown by a decrease in lipase substrate degradation. The anti-lipase effects of the essential oil compositions of pomegranate peel extracts have been previously reported, but the principle active principle components were not discussed [25]. In this study, three hydrolysable tannins, 1, 3, and 4, at 200 µg/mL significantly inhibited the lipase activity by 39.8%, 34.7%, and 35.2%, respectively ( Figure 3).

Pomegranate Polyphenols Inhibited HaCaT Cell Proliferation
Because keratinocyte over-proliferation is a crucial event in acne formation, testosterone was used as an inducer to stimulate HaCaT cell proliferation. Results showed that testosterone time-and dose-dependently induced HaCaT cell proliferation. Further, testosterone significantly induced proliferation of keratinocytes at 10 and 100 µg/mL for 72 h (Figure 4).  (2), strictinin A (3), and granatin B (4); All photos were taken with scanning electron microscopy; C: control group; The concentration of 1 to 4 was 100 µg/mL; Data are from three separate experiments, the picture of one of which is shown; The arrow indicates a disruption site of bacteria; Scale bar of control group is 5 µm; Scale bar of four hydrolysable tannins against S. aureus and P. acnes is 5 µm and 2 µm, respectively.

Pomegranate Polyphenols Inhibited Lipase Activity
Lipase is an enzyme that hydrolyzes lipids. Some bacteria, such as P. acnes, can secrete lipase to resolve lipids and facilitate lipid-related nutrient absorption from an external medium. In this study, a fluorometric assay of lipase activity was administrated with and without PG-E and the four hydrolysable tannins. Compared to the control, PG-E (200 µg/mL) inhibited lipase activity by 20% as shown by a decrease in lipase substrate degradation. The anti-lipase effects of the essential oil compositions of pomegranate peel extracts have been previously reported, but the principle active principle components were not discussed [25]. In this study, three hydrolysable tannins, 1, 3, and 4, at 200 µg/mL significantly inhibited the lipase activity by 39.8%, 34.7%, and 35.2%, respectively ( Figure 3).

Pomegranate Polyphenols Inhibited HaCaT Cell Proliferation
Because keratinocyte over-proliferation is a crucial event in acne formation, testosterone was used as an inducer to stimulate HaCaT cell proliferation. Results showed that testosterone timeand dose-dependently induced HaCaT cell proliferation. Further, testosterone significantly induced proliferation of keratinocytes at 10 and 100 µg/mL for 72 h (Figure 4).   Therefore, treatment with testosterone at 10 µg/mL for 72 h was used to assess which hydrolysable tannins could inhibit HaCaT cell proliferation. Result showed that four hydrolysable tannins could significantly decrease the testosterone-induced HaCaT cell proliferation at 50 µg/mL ( Figure 5). Two research teams discussed the protective effects of pomegranate against UV-induced HaCaT oxidative stress and markers of photoaging [26,27]. Taken together with results of this study, we suggest that pomegranate can act as a UV protector and growth regulator of skin keratinocytes.   Therefore, treatment with testosterone at 10 µg/mL for 72 h was used to assess which hydrolysable tannins could inhibit HaCaT cell proliferation. Result showed that four hydrolysable tannins could significantly decrease the testosterone-induced HaCaT cell proliferation at 50 µg/mL ( Figure 5). Two research teams discussed the protective effects of pomegranate against UV-induced HaCaT oxidative stress and markers of photoaging [26,27]. Taken together with results of this study, we suggest that pomegranate can act as a UV protector and growth regulator of skin keratinocytes.  Therefore, treatment with testosterone at 10 µg/mL for 72 h was used to assess which hydrolysable tannins could inhibit HaCaT cell proliferation. Result showed that four hydrolysable tannins could significantly decrease the testosterone-induced HaCaT cell proliferation at 50 µg/mL ( Figure 5). Two research teams discussed the protective effects of pomegranate against UV-induced HaCaT oxidative stress and markers of photoaging [26,27]. Taken together with results of this study, we suggest that pomegranate can act as a UV protector and growth regulator of skin keratinocytes.  Therefore, treatment with testosterone at 10 µg/mL for 72 h was used to assess which hydrolysable tannins could inhibit HaCaT cell proliferation. Result showed that four hydrolysable tannins could significantly decrease the testosterone-induced HaCaT cell proliferation at 50 µg/mL ( Figure 5). Two research teams discussed the protective effects of pomegranate against UV-induced HaCaT oxidative stress and markers of photoaging [26,27]. Taken together with results of this study, we suggest that pomegranate can act as a UV protector and growth regulator of skin keratinocytes.

Pomegranate Polyphenols Attenuated Heat-Killed P. acnes-Induced NO and PGE 2 Production by RAW 264.7 Cells
In our previous study, we showed the anti-inflammatory effects of PG-E and four hydrolysable tannins against LPS (lipopolysaccharide)-treated RAW 264.7 cells [12]. Moreover, in this study, we used HKP (heat-killed P. acnes), instead of LPS, to simulate the irritation and inflammation caused by a P. acnes infection. As shown in Figure 6A,B, HKP at 25-200 µg/mL dose-dependently and significantly induced NO (nitric oxide) and PGE 2 (Prostaglandin E 2 ) production by RAW 264.7 cells. However, a good relationship between the inflammatory response and the dose of HKP occurred at 50-200 µg/mL. Ultimately, we chose 100 µg/mL of HKP as our inflammation induction dosage. Compounds 1, 2, 3, and 4 at 50 µM displayed over 50% NO inhibitory effects against HKP-induced RAW 264.7 cells ( Figure 6C). In addition, 1 and 4 at 50 µM also significantly inhibited about 50% of PGE 2 production in HKP-treated RAW 264.7 cells ( Figure 6D).

Pomegranate Polyphenols Attenuated Heat-Killed P. acnes-Induced NO and PGE2 Production by RAW 264.7 Cells
In our previous study, we showed the anti-inflammatory effects of PG-E and four hydrolysable tannins against LPS (lipopolysaccharide)-treated RAW 264.7 cells [12]. Moreover, in this study, we used HKP (heat-killed P. acnes), instead of LPS, to simulate the irritation and inflammation caused by a P. acnes infection. As shown in Figures 6A,B, HKP at 25-200 µg/mL dose-dependently and significantly induced NO (nitric oxide) and PGE2 (Prostaglandin E2) production by RAW 264.7 cells. However, a good relationship between the inflammatory response and the dose of HKP occurred at 50-200 µg/mL. Ultimately, we chose 100 µg/mL of HKP as our inflammation induction dosage .  Compounds 1, 2, 3, and 4 at 50 µM displayed over 50% NO inhibitory effects against HKP-induced RAW 264.7 cells ( Figure 6C). In addition, 1 and 4 at 50 µM also significantly inhibited about 50% of PGE2 production in HKP-treated RAW 264.7 cells ( Figure 6D).

Pomegranate Polyphenols Attenuated Heat-Killed P. acnes-Induced IL-8 and TNF-α Production by THP-1 Cells
Cytokine, such as IL-8 (interleukin 8) and TNF-α (tumor necrosis factor-α), releases are also key points in the inflammatory status of human acne lesions [28]. As shown in Figure 7, HKP (100 µg/mL) obviously induced IL-8 and TNF-α production in a human monocytic cell line. In Figure 7A, the four hydrolysable tannins inhibited IL-8 and TNF-α production in dose-dependent manners at 50-200 µg/mL, and compound 4 had the strongest inhibitory activity. Anti-TNF-α activities of pomegranate polyphenols are shown in Figure 7B. Compounds 2, 3, and 4 exhibited significant inhibitory activities against TNF-α production, with 4 also displaying the strongest effect. This is the first time that Compound 4 has been reported to have anti-inflammatory cytokine effects.

Pomegranate Polyphenols Attenuated Heat-Killed P. acnes-Induced IL-8 and TNF-α Production by THP-1 Cells
Cytokine, such as IL-8 (interleukin 8) and TNF-α (tumor necrosis factor-α), releases are also key points in the inflammatory status of human acne lesions [28]. As shown in Figure 7, HKP (100 µg/mL) obviously induced IL-8 and TNF-α production in a human monocytic cell line. In Figure 7A, the four hydrolysable tannins inhibited IL-8 and TNF-α production in dose-dependent manners at 50-200 µg/mL, and compound 4 had the strongest inhibitory activity. Anti-TNF-α activities of pomegranate polyphenols are shown in Figure 7B. Compounds 2, 3, and 4 exhibited significant inhibitory activities against TNF-α production, with 4 also displaying the strongest effect. This is the first time that Compound 4 has been reported to have anti-inflammatory cytokine effects.

Preparation of Pomegranate Extract (PG-E)
Dried peels of pomegranate were purchased from a traditional Chinese medicine store in Taipei and identified by Hsien-Chang Chang. A voucher specimen (PG-01) was deposited in the School of Pharmacy, College of Pharmacy, Taipei Medical University (Taipei, Taiwan). Dried peels were extracted by homogenization with 70% acetone. The extracted solution was filtered, evaporated on a rotary evaporator, lyophilized, and called PG-E. PG-E and its active components were dissolved in 10% DMSO to a concentration of 10 mg/mL for subsequent experiments, stored at 4 °C, and used within 1 month. Serial dilutions of test solutions with culture medium were prepared before the in vitro assays.

P. acnes-Induced Ear Edema in Wistar Rats
The animal use protocol was reviewed and approved by the institutional Animal Care and Use Committee or Panel (IACUC/IACUP), Taipei Medical University (IACUC Approval no. LAC-97-0122). All methods involved in animal experiments were performed in accordance with the previous relevant guidelines and regulations. Female Wistar rats weighing 250 ± 20 g were bought from BioLASCO Taiwan (Yilan, Taiwan) and kept on a 12 h light/12 h dark cycle at 21 ± 2 °C with food and water ad libitum. P. acnes (8 × 10 7 colony-forming units (CFU)/20 µL) was intradermally injected into the left ear of rats to induce edema ( Figure 8). PBS (phosphate-buffered saline) was injected into the

Preparation of Pomegranate Extract (PG-E)
Dried peels of pomegranate were purchased from a traditional Chinese medicine store in Taipei and identified by Hsien-Chang Chang. A voucher specimen (PG-01) was deposited in the School of Pharmacy, College of Pharmacy, Taipei Medical University (Taipei, Taiwan). Dried peels were extracted by homogenization with 70% acetone. The extracted solution was filtered, evaporated on a rotary evaporator, lyophilized, and called PG-E. PG-E and its active components were dissolved in 10% DMSO to a concentration of 10 mg/mL for subsequent experiments, stored at 4 • C, and used within 1 month. Serial dilutions of test solutions with culture medium were prepared before the in vitro assays.

P. acnes-Induced Ear Edema in Wistar Rats
The animal use protocol was reviewed and approved by the institutional Animal Care and Use Committee or Panel (IACUC/IACUP), Taipei Medical University (IACUC Approval no. LAC-97-0122). All methods involved in animal experiments were performed in accordance with the previous relevant guidelines and regulations. Female Wistar rats weighing 250 ± 20 g were bought from BioLASCO Taiwan (Yilan, Taiwan) and kept on a 12 h light/12 h dark cycle at 21 ± 2 • C with food and water ad libitum. P. acnes (8 × 10 7 colony-forming units (CFU)/20 µL) was intradermally injected into the left ear of rats to induce edema ( Figure 8). PBS (phosphate-buffered saline) was injected into the right ear as a vehicle control [29]. One hour before the injection, PG-E ointment (10% PG-E in hydrophilic ointment) was administered (eight rats per group) on the skin surfaces of the left ear. The perimeter of the ear edema included diameter and height after P. acnes or PBS injection after 1-4 days were measured by calipers. Volume of ear edema could be calculated using multiplication by diameter, height, and circular constant. In the vehicle control group, the rat ear edema reduce volume was calculated as the original volume of ear edema after the PBS injection minus the volume of ear edema after injection on days 1, 2, 3, and 4. In the PG-E ointment group, the rat ear edema reduce volume was calculated as the original volume of ear edema after the P. acnes injection minus the volume of ear edema administrated with PG-E ointment after injection on days 1, 2, 3, and 4. right ear as a vehicle control [29]. One hour before the injection, PG-E ointment (10% PG-E in hydrophilic ointment) was administered (eight rats per group) on the skin surfaces of the left ear. The perimeter of the ear edema included diameter and height after P. acnes or PBS injection after 1-4 days were measured by calipers. Volume of ear edema could be calculated using multiplication by diameter, height, and circular constant. In the vehicle control group, the rat ear edema reduce volume was calculated as the original volume of ear edema after the PBS injection minus the volume of ear edema after injection on days 1, 2, 3, and 4. In the PG-E ointment group, the rat ear edema reduce volume was calculated as the original volume of ear edema after the P. acnes injection minus the volume of ear edema administrated with PG-E ointment after injection on days 1, 2, 3, and 4.

Skin Irritation
The skin toxicity of PG-E was evaluated from a single topical application in Wistar rats. Twentyfour hours before the test, the backs of rats were shaved free of hair (2.5 × 2.5 cm 2 at each site) and checked for any abnormalities (integrity and allergies). An appropriate concentration of PG-E (50 µL per sample) was smeared onto the shaved back of a Wistar rat. The patches were secured for a 4 h exposure period and removed with distilled water. At 24 h and 48 h after the application of PG-E, skin irritation was observed, and the extent of the evident skin allergic reaction was evaluated for each test animal. The acute primary irritation test was applied following the classic Draize test [17].
3.5. Anti-Bacterial Activities against P. acnes and S. aureus P. acnes (BCRC10723) and S. aureus (BCRC 10781) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). P. acnes was cultured in BAP with an anaerobic atmosphere using MGC AnaeroPack-Anaero and MGC AnaeroPack-Jar (Mitsubishi Gas Chemical Company). S. aureus was cultured in Bacto™ TSB (Difco). PG-E, punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4) were tested against P. acnes by the paper disc-diffusion method. All experimental procedures were performed according to our previous study with slight modification [17]. Freshly grown P. acnes was diluted with Bacto™ TSB, and 10 mL of prepared bacteria (4 × 10 8 CFU/mL) was aseptically added to 90 mL of sterilized media (TSA) at 45 °C in a water bath. The seeded agar media were immediately mixed and poured into a Petri dish. Prepared sample discs (8 mm in diameter) were added. In addition, 10 U of penicillin was used as the positive control. Plates were incubated under anaerobic conditions at 37 °C for 24 h, and inhibition zones in millimeters were measured. Each experiment was repeated in triplicate. The experimental protocol of anti-S. aureus (0.5 × 10 8 CFU/mL) activities was the same as that described above with some modifications. The anti-bacterial activity is expressed as the diameter of the inhibition zone against the test microorganisms as follows: ratio (%) = (diameter zone of sample/diameter zone of penicillin) × 100. An inoculation loop was used to collect bacteria from the clear zone on an agar plate. The inoculation loop was enriched in TSB for 24 h. At the end of incubation, TSB was put onto the surface of nutrient agar plates and incubated at 37 °C for 24 h. The minimum bactericidal concentration (MBC) was estimated as the lowest concentration of the samples where no visible growth was observed. The minimum inhibitory

Skin Irritation
The skin toxicity of PG-E was evaluated from a single topical application in Wistar rats. Twenty-four hours before the test, the backs of rats were shaved free of hair (2.5 × 2.5 cm 2 at each site) and checked for any abnormalities (integrity and allergies). An appropriate concentration of PG-E (50 µL per sample) was smeared onto the shaved back of a Wistar rat. The patches were secured for a 4 h exposure period and removed with distilled water. At 24 h and 48 h after the application of PG-E, skin irritation was observed, and the extent of the evident skin allergic reaction was evaluated for each test animal. The acute primary irritation test was applied following the classic Draize test [17].
3.5. Anti-Bacterial Activities against P. acnes and S. aureus P. acnes (BCRC10723) and S. aureus (BCRC 10781) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). P. acnes was cultured in BAP with an anaerobic atmosphere using MGC AnaeroPack-Anaero and MGC AnaeroPack-Jar (Mitsubishi Gas Chemical Company). S. aureus was cultured in Bacto™ TSB (Difco). PG-E, punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4) were tested against P. acnes by the paper disc-diffusion method. All experimental procedures were performed according to our previous study with slight modification [17]. Freshly grown P. acnes was diluted with Bacto™ TSB, and 10 mL of prepared bacteria (4 × 10 8 CFU/mL) was aseptically added to 90 mL of sterilized media (TSA) at 45 • C in a water bath. The seeded agar media were immediately mixed and poured into a Petri dish. Prepared sample discs (8 mm in diameter) were added. In addition, 10 U of penicillin was used as the positive control. Plates were incubated under anaerobic conditions at 37 • C for 24 h, and inhibition zones in millimeters were measured. Each experiment was repeated in triplicate. The experimental protocol of anti-S. aureus (0.5 × 10 8 CFU/mL) activities was the same as that described above with some modifications. The anti-bacterial activity is expressed as the diameter of the inhibition zone against the test microorganisms as follows: ratio (%) = (diameter zone of sample/diameter zone of penicillin) × 100. An inoculation loop was used to collect bacteria from the clear zone on an agar plate. The inoculation loop was enriched in TSB for 24 h. At the end of incubation, TSB was put onto the surface of nutrient agar plates and incubated at 37 • C for 24 h. The minimum bactericidal concentration (MBC) was estimated as the lowest concentration of the samples where no visible growth was observed. The minimum inhibitory concentration (MIC) was estimated as the lowest concentration of the samples where visible growth was observed.
in vivo and inhibited the growth of acne-related bacteria, i.e., P. acnes and S. aureus, down-regulated sebum production by reducing lipase activity, attenuated the inflammatory status, and avoided keratinocyte over-proliferation in vitro. Taken together, we suggest that the 70% acetone PG-E showed multiple anti-acne capacities, including anti-bacterial, anti-lipase, anti-keratinocyte proliferation, and anti-inflammatory actions. Four hydrolysable tannins, punicalagin, punicalin, strictinin A, and granatin B, were isolated from 70% acetone PG-E and exhibited different anti-acne effects. However, punicalagin, which had the highest effect on various functions, can be employed as a quality control marker. Pomegranate has great potential to be developed as a dietary supplement and for use in skin-care products.