Antiacne and Anti-Inflammatory Effects of Phenolic Compounds from Quercus acutissima Carruth. Leaves

Quercus plants are widely distributed in Korea and have been used for their antiallergic and anti-inflammatory properties to treat dermatitis. The phenolic compounds of Quercus acutissima Carruth (QA) are estimated to have antioxidant and anti-inflammatory activities, based on the results of previous studies with Quercus mongilica, Quercus stenophylla, Quercus gilva Blame., and Quercus acuta Thunb. We yield QA extract and the isolated phenolic compounds (hyperoside (1), astragalin (2), kaempferol 3–O-(6″- galloyl)–β–D–glucopyranoside (KGG) (3), quercetin 3–O-(6″-O-galloyl)-β–D–glucopyranoside (QGG) (4), pedunculagin (5), and casuarinin (6)) and were identified using NMR. Among them, KGG (3) and QGG (4) were isolated for the first time from QA. QA extract and the isolated phenolic compounds demonstrated antioxidative, anti-inflammatory, and antiacne activities in RAW 264.7 mouse macrophage cells in vitro. 3–6 demonstrated strong inhibitory activities in the DPPH scavenging and NO production assay and anti-inflammatory and antiacne activities through western blotting (NLRP3, IL-1β, and 5α-reductase). The most outstanding activity in all experiments was casuarinin (6). The study findings suggest potential therapeutic candidates for acne.


Introduction
Natural products have contributed to mankind as an important source of treatment for various diseases and are used depending on traditional medicine or alternative medicine as a result of modern medicine's failure to accurately treat certain diseases. Today, with increased interest in naturalbased drugs and functional foods, scientifc knowledge such as exploring pharmacologically active components in natural products has increased. It has been found that several natural products have analgesic and anti-infammatory effects [1][2][3][4].
Quercus is a genus of the Fagaceae family [5]with more than 600 species, many of which have medicinal properties [6,7] that efectively treat eczema, furuncles, diarrhea, and tonsillitis [7]. Te medicinal parts of the plant include the acorn [8,9], roots [10], leaves [11,12], stems [13], and bark [14]. Quercus acutissima Carruth. (QA) is a tall deciduous tree that grows up to 20 m in sunny, low mountainous regions and is widely dispersed in Korea. Its leaves are long and oval, with thorn-shaped serrations at the tip. Te antiallergic, anti-infammatory, and antiedema properties of QA have been used to treat dermatitis [15].
Acne vulgaris (AV) is a chronic infammatory disease caused by excessive androgen-induced sebum production, which is thought to play a major role in the development of AV [16,17]. Moreover, 5α-reductase activity in the skin infuences the manifestation of excess circulating levels of endogenous androgen [17,18], and sebum production is associated with the conversion of testosterone to dihydrotestosterone (DHT) through the action of 5α-reductase type 1 [19]. Inhibition of 5α-reductase type 1 (5α-R1) activity has been shown to decrease DHT levels and sebum production to help manage acne [20,21]. Te occurrence of acne, a chronic infammatory disease, is likely to be reduced if the infammatory response can be suppressed 5α-R1 activates DHT, which binds to the androgen receptor and causes acne. Inhibiting 5α-R1 expression may reverse or prevent acne.
Te NLRP3 infammasome pathway is reportedly activated by P. acnes, which has been recently associated with the pathogenesis of acne [22,23]. P. acnes reportedly induced the secretion of a major infammatory mediator, namely, interleukin-1β (IL-1β), from monocytes in a NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) -dependent manner [24,25]. Inhibiting NLRP3 and IL-1β expression may also reverse or prevent acne. However, the role of P. acnes in the infammatory response associated with AV remains unclear.
Previously we reported that phenolic compounds judged to be major components of the Quercus genus have antioxidant and anti-infammatory activity [26,27], and based on that, the goal of this study was to elucidate the structures of phenolic compounds isolated from QA leaves and further evaluate their antioxidant and anti-infammatory activities including inhibitory activity against acne-associated proteins (NLRP3, IL-1β, and 5α-R1) in an efort to identify potential treatments for acne. Tin layer chromatography (TLC) of the compounds was performed on precoated silica gel 60 F 254 plates (Merck, Darmstadt, Germany) in chloroform, methanol, and distilled water (6 : 4 : 1 volume ratio). Spots were detected under UV radiation (254 nm) and sprayed with FeCl 3 , 10% H 2 SO 4 , or anisaldehyde-H 2 SO 4 followed by heating.

Materials and Methods
Te chemical structures of the isolated compounds were determined using 1D-NMR, including 1 H-(600 MHz) and 13 C-(150 MHz) measurements, conducted using a JEOL NMR spectrometer (JEOL, Peabody, MA, USA) at Chung-Ang University.

Measurement of DPPH Radical Scavenging Activity.
Te antioxidant activity of the isolated compounds was determined by evaluating each compound's scavenging activity of the stable 1,1-diphenyl-2-picrylhydazyl (DPPH) free radical (Sigma-Aldrich, St. Louis, MO, USA). Briefy, 20 μL sample in anhydrous-ethanol was added to 180 μL DPPH solution (0.2 mM in anhydrous-ethanol). After thoroughly mixing and reacting for 30 min, 37°C. Te absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan Co. Ltd., Salzburg, Austria). L-Ascorbic acid was used as a positive control. Te free radical scavenging activity was calculated as IC 50 values were defned as the concentration that could scavenge 50% DPPH free radical.  (3). IC50 values were defned as the concentration that could inhibit 50% of NO production. 2.9. Statistical Analyses. All data were analyzed using oneway analysis of variance (ANOVA) followed by the Student-Newman-Keuls test with the SPSS software pack (IBM, Armonk, NY, USA). Diferences between groups were considered to be signifcantly diferent when the p value was less than 0.05. Values bearing diferent superscripts in the same column are signifcantly distinct from other data.  4)) and two ellagitannins (pedunculagin (5) and casuarinin (6)). Two of which (3 and 4) were isolated from QA for the frst time ( Figure 1).

Evidence-Based Complementary and Alternative Medicine 3
3.1.1. Compound 1. 1 was obtained as a yellow powder. TLC analysis revealed a spot under UV radiation, a dark blue spot was detected after FeCl 3 spraying, and a yellow spot was detected after 10% H 2 SO 4 spraying followed by heating. Te 1H-NMR spectrum of 1 exhibited an ABX-spin system with a meta-coupled aromatic signal at δ 7.89 (1H, d, J � 1. Tese results confrmed the identity of 1 as quercetin [28]. Furthermore, the 1 H and 13 C-NMR spectra of 1 suggested the presence of a galactosyl moiety with signals at  [30]. Comparing the data with values reported in the literature, 2 was identifed as kaempferol 3-O-β-D-glucopyranoside (astragalin) [31]. 3. 3 was obtained as a yellow amorphous powder. During TLC analysis, a green spot was detected after FeCl 3 spraying, whereas a yellow spot was detected after 10% H 2 SO 4 spraying followed by heating. In the 1 H-NMR spectrum, 3 was exhibited a similar to that of 2 aromatic A 2 B 2 -type system with aromatic signals at  C-6″)). Finally, a galloyl group with a large singlet signal at δH 7.04 (2H, s, H-2‴, and H-6‴) was determined to be adjacent to the 6-position of the sugar due to the downfeld-shifted 6″ protons at δH 4.33 and 4.31 [26].
3.1.5. Compound 5. 5 was obtained as a brown amorphous powder. TLC analysis revealed a brown spot after 10% H 2 SO 4 spraying followed by heating.
3.1.6. Compound 6. 6 was obtained as a brown amorphous powder. TLC analysis revealed a brown spot after 10% H 2 SO 4 spraying followed by heating.
Te 1 H-NMR spectrum of 6 showed similarity with 5 except for the presence of one galloyl group moiety at δ 7.13 (2H, S, galloyl-H), and the 13  From these results, 6 was identifed as casuarinin by comparing the spectral data with values reported in the literature [35,36].

Cell Viability.
MTT assays, based on the mitochondriadependent reduction of lightly colored tetrazolium salt to formazan of the intense violet color [39,40], were performed to assess the cytotoxic and cell viability efects of QA extract and its compounds. Te QA extract was performed at experimental doses (50, 25, 12.5, 6.25, 3.125, and 1.5625 μg/mL) and compounds were performed at experimental doses (25, 12.5, 6.25, 3.125, and 1.5625 μM) in RAW 264.7 cells. Te cell viability did not afect (>80%) at experimental doses of QA extract and compounds. Tese results suggest that the inhibitory activities against NO production and the expression of acne-associated cytokines and proteins were not associated with their cytotoxic efects (Figures 4 and 5).

Inhibitory Activity against NO Production.
Te role of nitro oxide (NO), a free radical produced in mammalian cells, has been reported to have been extensively studied. Overproduction of NO is associated with acute or chronic infammation [41,42], so inhibition of NO production can be a useful treatment strategy [43]. Inhibitory activity against NO production was evaluated in RAW 246.7 macrophage cells. QA extract (IC 50 � 16.84 ± 1.42 μg mL) exerted an inhibitory efect against NO production (Table 3, Figure 6). Among the six compounds isolated from QA, 6 (IC 50 � 34.56 ± 1.36 μM) exhibited the strongest inhibitory activity against NO production, which was signifcantly stronger than that of the positive control, namely, L-NMMA (IC 50 � 38.32 ± 0.23 μM) (Table 4, Figure 7). (NLRP3, IL-1β, and 5α-R1). Trough DPPH radical scavenging and NO production inhibitory activities guided isolation from QA, we isolated six compounds (1-6). Ten, we selected compounds (3-6) that showed potent activity compared with other compounds for the evaluation of antiacne.

Discussion
We aimed to evaluate the inhibition of the antioxidant, antiinfammatory, and acne-associated factors of the extracts of QA and isolated compounds from QA. Acne is one of the chronic diseases and is caused by various causes. Excessive sebum production causes acne, and sebum production is related to 5α-reductase. 5α-reductase changes DHT from testosterone and it has isoenzymes which are 5α-reductase type 1 and type 2 [45]. Te presence of 5α-reductase type 1 (5α-R1) in the skin may imply that the androgen regulation of sebaceous glands and sebum production requires the local conversion of testosterone to dihydrotestosterone (DHT) [46]. Skin 5α-reductase activity is a primary factor infuencing the manifestation of endogenous androgen excess [18]. Sebum production is associated with DHT which changes from testosterone by 5α-reductase type 1 [19]. Inhibition of 5α-R1 was found to help manage acne by reducing DHT levels and sebum production. Also, Activation of the NLRP3 infammasome is closely associated with various chronic diseases [47]. Te role of infammasome activation by Propionibacterium acnes in the pathogenesis of acne was only recently proposed [48]. Since NLRP3 is the frst mediator in the activation of the infammasome pathway, inhibition of NLRP3 expression may inhibit the next step of the pathway, in which caspase-1 fnally activates IL-1β [24], resulting in the development of acne. Inhibiting NLRP3 and IL-1β expression may reverse or prevent acne.
Te Quercus genus reported several types of bioactivities such as antioxidant, anti-infammatory, antimicrobial, and antibacterial. Terefore, we have confrmed from previous studies that use as a treatment for infammatory diseases by evaluating inhibition of NO production and proinfammatory cytokines from Quercus gilva Blume [49] and inhibition of 5a-reductase and anti-infammatory activity from Quercus mongolica [50].
Te results of our previous studies suggested that QA has the potential to be used as a treatment for infammatory diseases such as acne.
Our results in this current study demonstrated that the QA as well as compounds 1-6 exhibited good antioxidant ability. Especially, ellagitannins (5, 6) were the most potent. In addition, the QA and compounds exerted antiinfammatory efects. Based on these results, we selected compounds and evaluated whether compounds were able to inhibit the expression of NLRP3, IL-1β, 5α -R1 associated acne. Several studies have shown that phenolic compounds inhibit the activation of NLRP3 infammasome and inhibit the expression of various infammatory factors such as IL-1β [51,52]. In addition, our previous studies confrmed that phenolic compounds efectively inhibit 5α-reductase [50,53]. Pedunculagin (5) has the efect of the most 5α-R1 expression inhibition and casuarinin (6), and QGG (4) was demonstrated the most inhibitory efects in NLRP3 expression through western blot analysis, IL-1β was suppressed to a normal level in all compounds. Te compound that showed the most excellent activity in all biology activities was casuarinin (6). Casuarinin (6) has a structure in which one more galloyl group is attached to the pedunculagin (5), compounds (5 and 6) have previously been found in Alnus sieboldiana and Casuarina stricta [35,54], so it is demonstrated to have better activity than pedunculagin (5) due to its structural stability.

Evidence-Based Complementary and Alternative Medicine
In this study, we demonstrated the efects of the biological activity of Quercus acutissima Carruth. Leaves extracts and isolated compounds by evaluating the inhibition of expression of infammatory mediated proteins associated with acne, DPPH radical scavenging activity, and inhibition of NO production. Our results suggested that Quercus acutissima Carruth. Leave extracts and isolated compounds from QA have therapeutic functions and values as new  Cell viability (%) Figure 4: Efects of QA extract on RAW 264.7 cell viability.   Evidence-Based Complementary and Alternative Medicine 9 treatment materials for anti-infammatory healthy functional foods, acne treatments, and cosmetics for acne skin.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this paper.