Three New Isoprenylated Flavones from Artocarpus chama Stem and Their Bioactivities

Phytochemical investigation of Artocarpus chama stem was performed by chromatographic techniques, resulting from the isolation and structure elucidation of three new compounds, namely 3′-farnesyl-apigenin (1), 3-(hydroxyprenyl) isoetin (2), and 3-prenyl-5,7,2′,5′-tetrahydroxy-4′-methoxyflavone (3), and five known compounds, namely homoeriodictyol (4), isocycloartobilo-xanthone (5), artocarpanone (6), naringenin (7), and artocarpin (8). From the screening result, A. chama extract showed a potent tyrosinase inhibitory effect. Ihe isolated compounds 1, 4 and 6 also exhibited tyrosinase inhibition with IC50 of 135.70, 52.18, and 38.78 µg/mL, respectively. Moreover, compounds 3, 4, 5, 6, and 8 showed strong activity against Staphylococcus epidermidis, S. aureus, methicillin-resistant S. aureus, and Cutibacterium acnes. This study is the first report on phytochemical investigation with new compounds and biological activities of A. chama. Skin infection can cause dark spots or hyperpigmentation. The isolated compounds that showed both anityrosinase and antimicrobial activities will be further studied in in vivo and clinical trials in order to develop treatment for hyperpigmentation, which is caused by infectious diseases by microorganisms.


Introduction
Nowadays, skin whitening, or skin lightening, is popular; thus, skin-whitening agents have been searching. Melanin is an important factor that determines skin color [1]. Melanin is a polyphenolic pigment and causes dark-colored skin. It is produced in the process called melanogenesis [2]. In melanogenesis, tyrosinase enzymes, an important enzyme, catalyzes L-tyrosine to L-Dopa and to o-Dopaquinone-H + before passing the intermediate to the final melanin [3].
Tyrosinase is one of the main enzymes in the melanogenesis process. Therefore, inhibition of tyrosinase activity can decrease melanogenesis. The most commonly used treatment for all types of hyperpigmentary disorders is topical hydroquinone. It can lead side-effect reactions, such as skin irritation, dermatitis, melanocyte demolition, ochronosis, post-inflammatory pigmentation, cytotoxicity, and skin cancer [4].
Tyrosinase inhibitors from natural product might be an alternative way to provide the compounds for antityrosinase activity because plants are a rich source of bioactive chemicals that are mostly free from harmful side effects [5]. Nowadays, interest in tyrosinase inhibitors from natural sources is increasing [6][7][8][9][10]. Moraceae is the most interesting plant family to study concerning antityrosinase activity because many compounds from this family showed inhibitory activity against tyrosinase enzymes [11]. Artocarous chama, which is a plant in the Moraceae family, has been reported to show antioxidant activity [12,13] and was screened for antityrosinase and antibacterial activities [11], as well as chemical constituents, mostly flavonoids and phenolic compounds [14][15][16]. Flavonoids play a key role in newly discovered natural tyrosinase inhibitors [17,18]. The number and location of phenolic hydroxyl groups on flavonoids significantly influences the inhibition of tyrosinase activity [19][20][21].
Infection by pathogenic bacteria, for example, Cutibacterium acnes, Staphylococcus epidermidis, and S. aureus, can cause acne vulgaris or acne inflammation resulting in postinflammatory hyperpigmentation [22,23]. Therefore, effective treatment of hyperpigmentation should include antimicrobial agents.
Tyrosinase inhibition is important to reduction of melanin, may be developed into new drug to treatments for hyperpigmentation [24], and could be useful in cosmetology [25]. Tyrosinase inhibitors from natural products are getting a lot of attention. This is because plant-based tyrosinase inhibitors are cost-effective and cause fewer side effects.

Structure Elucidation of Isolated Compounds
Eight pure compounds from EtOAc extract of A. chama stem were isolated and identified by using physical properties and spectroscopic data. Then, their chemical structures were confirmed, by comparison with previous reports, as 3 [12,13] and was screened for antityrosinase and antibacterial activities [11], as well as chemical constituents, mostly flavonoids and phenolic compounds [14][15][16]. Flavonoids play a key role in newly discovered natural tyrosinase inhibitors [17,18]. The number and location of phenolic hydroxyl groups on flavonoids significantly influences the inhibition of tyrosinase activity [19][20][21]. Infection by pathogenic bacteria, for example, Cutibacterium acnes, Staphylococcus epidermidis, and S. aureus, can cause acne vulgaris or acne inflammation resulting in postinflammatory hyperpigmentation [22,23]. Therefore, effective treatment of hyperpigmentation should include antimicrobial agents.
Tyrosinase inhibition is important to reduction of melanin, may be developed into new drug to treatments for hyperpigmentation [24], and could be useful in cosmetology [25]. Tyrosinase inhibitors from natural products are getting a lot of attention. This is because plant-based tyrosinase inhibitors are cost-effective and cause fewer side effects.
Please delete this structure  were in agreement with those of compound 2, except that the carbon signal at position C-9 (24.85) was shifted up field by 45.36 ppm, compared with the carbon chemical shift of compound 2 (70.21), due to oxygenation effects, indicating a methylene group at C-9. Moreover, the signals of the B ring suggested that the methoxy group in compound 3 was possibly presented in the B ring. These findings indicated that compound 3 is was a tetrahydroxyflavone compound with a prenyl group. The flavone structure and the location of the prenyl moiety were confirmed based on the HMBC experiment ( Figure 4). Thus, the structure of compound 3 was determined to be 3-prenyl-5,7,2 ,5 -tetrahydroxy-4 -methoxyflavone, a new prenylated flavone. 407.1101, correlated with a molecular formula of C21H20O7. The 1 H-NMR and 13 C-NMR data of compound 3 were quite similar to those of compound 2, except for the replacement of the methylene signal at δH 3.01 (2H, d, J = 7.0 Hz, H-9) and the methoxyl signal at δH 3.71 (3H, s, 4′-OCH3) of ring B. The carbon signals of compound 3 were in agreement with those of compound 2, except that the carbon signal at position C-9 (24.85) was shifted up field by 45.36 ppm, compared with the carbon chemical shift of compound 2 (70.21), due to oxygenation effects, indicating a methylene group at C-9. Moreover, the signals of the B ring suggested that the methoxy group in compound 3 was possibly presented in the B ring. These findings indicated that compound 3 is was a tetrahydroxyflavone compound with a prenyl group. The flavone structure and the location of the prenyl moiety were confirmed based on the HMBC experiment ( Figure 4). Thus, the structure of compound 3 was determined to be 3-prenyl-5,7,2′,5′-tetrahydroxy-4′-methoxyflavone, a new prenylated flavone.
The results are presented in Table 1.

Cell Viability
The initial purpose was to test whether the isolated compounds could inhibit melanogenesis in cultured melanocytes without affecting cell growth. The results showed that all the isolated compounds were not toxic to B16-F1 melanoma cells. Cell viability was still more than 80% at the highest concentration of 50 µg/mL. The results are presented in Figure 5.

Std.
Water extract of A. lakoocha wood P 8.73 ± 0.69 P for positive standards.

Cell Viability
The initial purpose was to test whether the isolated compounds could inhibit melanogenesis in cultured melanocytes without affecting cell growth. The results showed that all the isolated compounds were not toxic to B16-F1 melanoma cells. Cell viability was still more than 80% at the highest concentration of 50 μg/mL. The results are presented in Figure 5.

Intracellular Antityrosinase Activity and Melanin Content
The effects of the isolated compounds at concentrations of 50 μg/mL on intracellular antityrosinase activity and melanin content of B16F1 melanoma cells was determined. The antityrosinase activity of supernatants was measured, and the results showed that Compound 6 (artocarpanone) exhibited antityrosinase activity ( Figure 6A). Moreover, melanin content showed an inverse result with antityrosinase activity; usually the increase of antityrosinase activity led to the decrease of melanin content ( Figure 6B).

Intracellular Antityrosinase Activity and Melanin Content
The effects of the isolated compounds at concentrations of 50 µg/mL on intracellular antityrosinase activity and melanin content of B16F1 melanoma cells was determined. The antityrosinase activity of supernatants was measured, and the results showed that Compound 6 (artocarpanone) exhibited antityrosinase activity ( Figure 6A). Moreover, melanin content showed an inverse result with antityrosinase activity; usually the increase of antityrosinase activity led to the decrease of melanin content ( Figure 6B).

Antimicrobial Activity Assay
From the screening of antimicrobial activity [11], ethyl acetate extracts of A. chama and S. taxoides showed potential effects against S. epidermidis, S. aureus, MRSA, and P. acnes. Thus, the ethyl acetate extracts of these plants were selected for further isolation of active compounds. Then, the MIC and MBC of isolated compounds were investigated for S. epidermidis, S. aureus, MRSA, and C. acnes. The MIC and MBC values of the isolated compounds are presented in Table 2. . Data were expressed as mean ± SD from three independent experiments. * p < 0.05, and ** p < 0.01 indicated a significant difference from the positive control group. Positive controls were arbutin and kojic acid.

Antimicrobial Activity Assay
From the screening of antimicrobial activity [11], ethyl acetate extracts of A. chama and S. taxoides showed potential effects against S. epidermidis, S. aureus, MRSA, and P. acnes. Thus, the ethyl acetate extracts of these plants were selected for further isolation of active compounds. Then, the MIC and MBC of isolated compounds were investigated for S. epidermidis, S. aureus, MRSA, and C. acnes. The MIC and MBC values of the isolated compounds are presented in Table 2.
However, only artocarpanone could reduce the amount of melanin content and exhibited intracellular antityrosinase activity on B16F1 melanoma cells. This could be due to melanogenesis processes, such as microphthalmia-associated transcription factor (MITF), because melanogenesis-related enzyme expression can activate or depress by up or down regulation of MITF activity; therefore it could exhibit a stimulatory or inhibitory effect on melanogenesis [30][31][32].

Plant Materials
Stems of Artocarpus chama Buch. Was collected from Southern Literature Botanical Garden, Songkhla Province. The plant was identified by a botanist of Southern Literature Botanical Garden, and the voucher specimen number was SKP 117 01 03 01. The sample specimen was kept at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand.

Extraction and Isolation
6.8 kg of A. chama stem powder was macerated repeatedly with petroleum ether for 3 days (3 times). Then, the extraction was filtrated, and the solvent was evaporated using a rotary evaporator under reduced pressure at 40 • C to yield a petroleum ether extract. Then, the marc was macerated three times with ethyl acetate and methanol for 3 days (3 times each) and boiled with H 2 O. Next, the solvent was removed to provide ethyl acetate, methanol, and H 2 O extracts.
Based on screening results, the ethyl acetate extract showed potent effects on antityrosinase and antimicrobial activities. Therefore, it was selected for further phytochemical investigation. First, the ethyl acetate extract (40 g) was fractionated by quick-column chromatography, and the eluates were examined by TLC using a gradient solvent system between hexane, ethyl acetate, and methanol. The fractions that showed the similar TLC fingerprints were combined. The interesting fractions, D, F, G and H, were selected to isolate and purify. The isolation step is illustrated in Scheme 1. Afterward, the chemical structures of the isolated compounds were interpreted using nuclear magnetic resonance (NMR) and other spectroscopic techniques. chromatography, and the eluates were examined by TLC using a gradient solvent system between hexane, ethyl acetate, and methanol. The fractions that showed the similar TLC fingerprints were combined. The interesting fractions, D, F, G and H, were selected to isolate and purify. The isolation step is illustrated in Scheme 1. Afterward, the chemical structures of the isolated compounds were interpreted using nuclear magnetic resonance (NMR) and other spectroscopic techniques.  The known natural products, homoeriodictyol (compound 4) [36], isocycloartobiloxanthone (compound 5) [37], artocarpanone (compound 6) [38], naringenin (compound 7) [39], and artocarpin (compound 8) [38], were identified by comparing their NMR data with previously published data.

Enzymetic Antityrosinase Activity Assay
Antityrosinase activity was determined by the dopachrome method. L-dopa was used as the substrate. Oxidation of L-Dopa to dopachrome which, represented by red color, could be detected by visible light at 492 nm [40,41]. Briefly, 140 µL of phosphate buffer pH 6.8, 20 µL of sample solution (200 µg/mL), and 2 µL of tyrosinase enzyme solution (203.3 unit/mL) were mixed in a 96-well plate at 25 • C for 10 min. Then, 20 µL of L-Dopa (0.85 mM) was added, and optical density (OD) was detected. After incubation at 25 • C for 20 min, OD was detected again. Dimethyl sulfoxide (DMSO) was used as a negative control. Kojic acid and water extract of A. lakoocha wood were used as positive controls. The percent inhibition of tyrosinase reaction was calculated by the following equation: Tyrosinase inhibition (%) = [1 − (OD 492 of sample/OD 492 of control)] × 100.

Cell Viability Assay
Cell viability was determined by Sulforhodamine B (SRB) assay. First, 5 × 10 3 cells/well were seeded in a 96-well plate, incubated for 24 h, and treated with test samples; control cells were treated with 0.5% DMSO. After 48 h incubation, cells were fixed with 10% trichloroacetic acid (TCA) and kept at 4 • C for 1 h. After being strained with 0.45% SRB, 10 mM Tris base was added and shaken to dissolve the SRB color. Optical densities were determined at 492 nm. The percent cell viability was calculated.

Intracellular Antityrosinase Activity and Melanin Content Assays
First, 3 × 10 5 cells/well were seeded in 12-well plates and allowed to adhere at 37 • C for 12 h. Cells were treated with test samples; control cells were treated with 0.5% DMSO. After 48 h incubation, cells were lysed with RIPA and centrifuged at 14,000 rpm for 20 min (4 • C) in order to separate the supernatant for measurement of tyrosinase activity, and the cell pellet was measured for melanin content.

Intracellular Antityrosinase Activity
The supernatant was collected for determination of protein content by the Bradford method, with bovine serum albumin (BSA) used as a standard. The supernatant of lysate cells and 2 mg/mL L-Dopa were added to a 96-well plate and incubated at 25 • C for 1 h. After that, optical densities were determined at 492 nm. Tyrosinase inhibition was then calculated.

Melanin Content
Cell pellets were dissolved with 1M NaOH and incubated at 55 • C for 1 h. Melanin content was calculated by comparison with the standard curve of synthetic melanin at 475 nm.

Antimicrobial Activity Assay
Microorganisms that cause skin infection, such as Staphylococcus aureus (ATTC 25923), Staphylococcus epidermidis (TISTR 517), Cutibacterium acnes (DMST 14916), and methicillinresistant Staphylococcus aureus (DMST20654),t were selected for this study. The isolated compounds were determined for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by a modified broth microdilution method [45,46]. Briefly, 20 µL of the test samples (dilution series ranging from 2560 to 1.25 µg/mL) was added in wells 1 to 12. Then, 80 µL of media was added into each well. Next, 100 µL of the inoculum (concentration 10 6 CFU/mL) was added in each well. The final concentration of inoculum in each well was 5 × 10 5 CFU/mL. Then, the cultures were incubated follow the conditions. Incubation mixtures showing positive results of inhibitory effect (MIC) were streaked on media agar, and then incubated by following the conditions. The lowest concentration that did not show any growth was taken as the MBC.

Conclusions
Three new compounds (1-3) and five known compounds (4)(5)(6)(7)(8) were isolated from the stem of A. chama. The chemical structures of the isolated compounds were identified on the basis of their physical properties and spectroscopic data. Compounds 1, 4, and 6 were affected to tyrosinase inhibitory activity, while compounds 3, 4, 5, 6, and 8 showed potential effects on antimicrobial activity.
This study is the first report on antityrosinase and antimicrobial activities of A. chama. Additionally, three new compounds were investigated. Some isolated compounds showed effects on biological activities, which can be further studied on in vivo and in clinical trials for potential use in the treatment of hyperpigmentation and/or infectious diseases caused by microorganisms.