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Natural Product Sciences 27(1) : 28-35 (2021) https://doi.org/10.20307/nps.2021.27.1.28

Anti-inflammatory, Anti-glycation, Anti- and CDK4 Inhibitory Activities of Alaternin (=7-Hydroxyemodin)

Grishma Bhatarrai1,#, Jeong-Wook Choi1,#, Su Hui Seong1,#, Taek-Jeong Nam1, Hyun Ah Jung2,*, and Jae Sue Choi1,* 1Department of Food and Life Sciences, Pukyoung National University, Busan 48513, Republic of Korea 2Department of Food Science and Human Nutrition, Jeonbuk National University, Jeonju 54896, Republic of Korea

Abstract  The aim of this study was to anatomize the therapeutic potential of alaternin (=7-hydroxyemodin) against , advanced glycation end products (AGEs) formation, tyrosinase, and two cyclin-dependent kinases (CDKs), CDK2 and CDK4, and compare its potency with emodin. Alaternin showed lower cytotoxicity and higher dose-dependent inhibition against lipopolysaccharide (LPS) induced (NO) production with half maximal inhibitory concentration (IC50) of 18.68 µM. Similarly, alaternin efficaciously inhibited biotransformation of fluorescent AGEs and amyloid cross-β structure on the bovine serum albumin (BSA)- glucose-fructose system, five times more than emodin. Interestingly, alaternin also showed selective activity against CDK4 at 170 µM, whereas emodin inhibited both CDK2 and CDK4 at a concentration of 17 and 380 µM respectively. In addition, alaternin showed dose-dependent inhibitory activity against mushroom tyrosinase with inhibition percentage of 35.84 % at 400 µM. Altogether, alaternin with pronounced inhibition against inflammatory mediator (NO), glycated products formation, and targeted inhibition towards CDK4 receptor can be taken as an important candidate to target multiple diseases. Keywords  Alaternin, inflammation, tyrosinase, AGEs, CDK4, RAW 264.7

Introduction oxidative,11 neuroprotective,12 hepatoprotective,13 and selective (MAO) inhibitory activities14 Anthraquinone comprises a large group of aromatic whereas emodin is testified to have anti-bacterial,15 anti- compounds nucleated with 9, 10-dioxoanthracene. A wide inflammatory,16,17 anti-purgative, vasorelaxant, anti-fibrotic, range of anthraquinones has been shown to exhibit and immunosuppressive effects.18 numerous biological properties like hepatoprotective,1,2 Inflammation occurs as a host defense mechanism in anti-Alzheimer’s,3 anti-inflammatory,4 lung anti-inflam- response to harmful stimuli (pathogen) and is accom- matory,5 mucin production inhibiting,6 anti-microbial,7,8 panied by inflammatory mediators. Dysregulation of and anti-cancer effects.9 Among the derivatives of inflammatory response is related to neurodegenerative anthraquinone family, alaternin and emodin are two disorders, cancer, atherosclerosis, and type 1 diabetes.19 important compounds that differ by the number and Lipopolysaccharide (LPS), a bacterial endotoxin, when position of functional group, chemically named as 1,2,3,8- presented to macrophage produces a proinflammatory tetrahydroxy-6-methyl-9,10-anthraquinone and 1,3,8-trihydroxy- signals and inflammatory mediators like 6-methyl-9,10-anthraquinone, respectively. Alaternin has been (COX)-2, NO, and ROS. Three isoforms of NO: neuronal reported with anti-diabetic,10 anti-Alzheimer’s,3 anti- NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) are the responsible for catalyzing 20 #These authors contributed equally to this work. the production of NO from L- substrate . The stimulation of iNOS in response to immunological stimuli *Author for correspondence produces NO in nanomolar concentration. Excessive Hyun Ah Jung, Department of Food Science and Human Nutrition, Jeonbuk National University, Jeonju 54896, Republic of Korea. productions of NO leads to oxidation of various cellular Tel: +82-63-270-4882; E-mail: [email protected] contents like proteins, lipids, and DNA.21,11 ROS is Jae Sue Choi, Department of Food and Life Sciences, Pukyoung produced by cells like polymorphonuclear neutrophils National University, Busan 48513, Republic of Korea. (PMNs) that are involved in host-defense mechanism are Tel: +51-629-7547; E-mail: [email protected]

28 Vol. 27, No. 1, 2021 29 well-known effectors of inflammation. The action of diseases involving oxidative stress; anti-inflammatory, ROS, , for instance, reacts with NO to form anti-AGE, anti-tyrosinase and, anti-CDK activities. The reactive nitrogen species such as peroxynitrite (ONOO) influence of structure on activities of alaternin has not that induces nitrosative stress and pro-inflammatory been fully established. Therefore, we applied a versatile burden to the cell.22 approach to explore and compare and relate the biological The formation of advanced glycation end products properties of alaternin and emodin. (AGEs) occurs via nucleophilic addition of amino group of protein or nucleic acid to the carbonyl group of a Experimental reducing sugar called Maillard reaction. In this reaction, nonenzymatic glycation of proteins or amino acids with Chemicals and reagents – Alaternin and emodin were reducing sugars results in production of a large number of isolated from Cassia obtusifolia Linn. through a column free radicals, carbonyl species, and reactive dicarbonyl chromatographic technique as mentioned by Jung et al. species.23 Several studies have highlighted anti-glycation (2016)3. CellTiter 96 Aqueous Non-Radioactive Cell activity of Cassia species.24-26 Some studies have linked Proliferation Assay Kit was purchased from Promega AGEs with chronic diseases, oxidative stress, and, Corporation (Madison, WI, USA). The bovine serum inflammatory reaction.27-29 Therefore, the discovery of albumin (BSA), LPS, D-(−)-fructose, D-(+)-glucose, glycation inhibitor can provide a therapeutic aspect to aminoguanidine hydrochloride (AG), thioflavin T, L- other related diseases like diabetes.24 DOPA, kojic acid, and mushroom tyrosinase were bought Cyclin-dependent kinases (CDKs) are one of the most from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s studied anti-cancer targets that play a vital role in cell modified essential medium (DMEM), penicillin–strep- cycle events. CDK binds to a family of regulatory tomycin, fetal bovine serum (FBS), were procured from proteins called cyclins that phosphorylates specific / Gibco-BRL Life Technologies (Grand Island, NY, USA). residue and forms activated heterodimer Primary and secondary antibodies were obtained from complex, thereby regulating cell cycle progression.30 Technology Inc. (Beverly, MA, USA) and Inhibition of such kinase can be used as an effective Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). strategy to halt uncontrolled cellular proliferation in Sodium azide and K2HPO4, were acquired from Junsei cancer.31 Some of the CDK inhibitors like palbociclib, Chemical Co. Ltd. (Tokyo, Japan) and, L-, and ribociclib, and abemaciclib (CDK4/6 inhibitors) have KH2PO4 were purchased from Jannssen Chimica (Geel already been already identified.32 The toxicity profile of Belgium), and Yakuri Pure Chemicals Co. Ltd. (Osaka, individual inhibitory drug needs to be acceptable before Japan), respectively. Cell cycle kinases, Cdk4 (sc-23,896), proceeding into combination and monotherapies. The cyclin D1, Cdk2 (sc-6248), and cyclin A were supplied inhibition specificity at selective CDK like CDK4/6 and by Takara Bio, Inc. (Takara Bio, CA, USA). 96-Well CDK2 have shown to be associated with minimal side polyvinylidene fluoride (PVDF) filter and membrane effect when compared to the non-selective ones.33,30 The (Immobilon-P) was purchased from EMD Millipore presence of ROS has also shown to control cell cycle (Millipore, CA, USA). Ultra-pure grade water was used progression by influencing the activity of cyclins and through and all graded chemicals/solvents used all over CDKs.34-36 the experiment were purchased from the commercial Tyrosinase is a copper-containing metalloenzyme sources. Unless otherwise stated, all the solvents and capable of oxidizing tyrosine to L-DOPA or o-diphenol chemicals were acquired from Sigma-Aldrich (St. Louis, (3, 4-dihydroxyphenylalanine) and subsequently converting MO, USA). L-DOPA to dopaquinone (o-quinones).37 This transformed Cell culture – RAW 264.7 cell line was purchased dopaquinone leads to the formation of melanin which is from American Type Culture Collection (Rockville, MD, responsible for skin disorders like hyperpigmentation and USA) and cultured in Dulbecco’s modified essential enzymatic browning of foods. Along with the production medium (DMEM) that comprised of penicillin (100 units/ of melanin, highly reactive DOPA-quinones are also mL), streptomycin (100 μg/mL), and 10% heat- described to damage neurons and cause cell death.38 inactivated fetal bovine serum (FBS). Cells were grown to Our previous study demonstrated the anti-oxidant,39 70-80% confluency and then trypsinized and seeded for human monoamine oxidase inhibitory14 and hepato- experiments. Cells were maintained at 37 ºC in a 13 proprotective nature of alaternin. This study was humidified atmosphere of 5% CO2 at all times. undertaken to evaluate the potency of alaternin against Cell viability assay – The survival of LPS stimulated 30 Natural Product Sciences

RAW 264.7 cell was determined using MTS assay CS analyzer. method as reported by Lee et al.40 Briefly, the formation Evaluation of glycated end products formation  To of formazan product from tetrazolium compound was quantify the anti-glycation activity of alaternin, previously measured using CellTiter 96 Aqueous Non-Radioactive reported method41 was followed. Firstly, 10 mg/mL BSA Cell Proliferation Assay. RAW 264.7 cells (1.5 × 104 was dissolved in 50 mM sodium phosphate buffer (pH cells/well) were seeded in 96-well plate supplemented 7.4) and added with 0.2 M fructose, 0.2 M glucose, and with 10% FBS and incubated for 24 h at 37 ºC. After 0.02% bacterial growth inhibitor, sodium azide. This attachment of cell to the plate, various concentration of AGE reaction solution (3.8 mL) was mixed with 200 µL alaternin diluted in serum-free DMEM was added and of different concentration of test compounds and a incubated for 1h. It was followed by addition of LPS standard AG. The reaction mixtures were incubated at (1 µg/mL) and incubation for another 24 h. Then, MTS 37 ºC for 3 weeks. This mixture was used to determine solution (10 µL) was added on each well and was AGE and amyloid cross β-structure inhibitory activities. incubated again. After 30 minutes at 37 ºC, absorbance Determination of fluorescent AGE inhibition  The was taken using a Gen5 ELISA (Bio-Tek, Houston, TX, amount of inhibited AGE product was determined every 7 USA) at 490 nm. Experiments were carried out in triplicate. days for three weeks using a microplate reader (Dual Survival of murine macrophage cell was calculated as cell scanning SPECTRAmax Molecular Devices Co., Sunny- viability (%) = OD570 (treated cell)/OD570 (vehicle control) vale, CA, USA) with respective excitation and an emission × 100. wavelength of 350 and 450 nm. NO production assay  RAW 264.7 macrophages Determination of amyloid cross β-structure inhi- (1.5 × 104 cells/well) were seeded in a 96-well plate as bition  The inhibition of quaternary β-structure was mentioned previously for 24 h at 37 ºC, followed by determined by a slight modification of a previously addition of predetermined concentration of alaternin. reported method42. Briefly, 64 µM thioflavin T (100 µL) After 1 h, LPS (1 µg/mL) was added and incubated for was mixed with 0.1 M phosphate buffer solution (pH 7.4) another 24 h. After incubation, 100 μL of Griess reagent and 10 µL of glycated samples, then kept under incubation and the stable NO was measured by using MTS at the for 1h at 25 ºC. The fluorescence intensity was calculated optical density of 490 nm. using 435 nm as excitation and 485 nm as emission Western blot analysis  RAW 264.7 macrophages (5 wavelength. ×105 cells/mL) were seeded in 100-mm culture dishes in Evaluation of cyclin-dependent kinase inhibition  the presence or absence of LPS (1 µg/mL) with or without The ability of alaternin to downregulate cell cycle kinase samples. After 18 h, cells were washed twice with ice- was determined by the formerly stated procedures.43-45 cold PBS and treated with lysis buffer (50 mM Tris-HCl, Determination of CDK2 inhibition  CDK2 was 150 mM NaCl, 1% Nonidet P-40, 1% Tween 20, 0.1% obtained as a combination of (His) labeled

SDS, 1 mM Na3VO4, 10 µg/mL leupeptin, 50 mM NaF, human CDK2 protein and cyclin A protein. and 1 mM PMSF, pH 7.5) on ice for 30 min. Lysed expressing His-CDK2 was expressed in an insect infected solution was centrifuged at 14,000 × g at 4 ºC for 20 min with baculovirus. Along with CDK2 complex, 32P labeled and protein content was determined using Bradford assay. ATP, diluted sample and retinoblastoma protein (Rb) were Proteins were applied to SDS-PAGE, transferred to PVDF used to initiate the reaction. Precisely, assay was membrane, and immediately blocked using 1% BSA in carried out in 20 µL reaction mixture containing the Tris-buffered saline supplemented with 0.1% Tween-20 active kinase enzyme with unit activity of 57 mole/ min

(pH 7.4) (TBST) at room temperature for 60 min. After and Km of 940 µM. The substrate was purified by C- washing the membrane for 3 times with TBST buffer, terminal region protein with specific substrate specificity primary antibody diluted as 1: 1,000 in non-fat dry milk for (Rb). After 10 min, the reaction was ceased by (50 g/L) was used to incubate membrane overnight at addition of 80 µL of 12% phosphoric acid and passed to 4 ºC. Again, the membrane was washed thrice with TBST 96-well PVDF filter prewetted with 50% EtOH, followed buffer and treated with a horseradish peroxidase (HRP)- by removal of 32P ATP by addition of 10 mM tris-HCl conjugated secondary antibody diluted with non-fat dry containing 0.1 M NaCl. Obtained filtrate was thoroughly milk (50 g/L) at 1:2,000 for 2 h at room temperature. dried and radioactivity was measured using phosphoi- Signals were detected in enhanced chemiluminescence magner reader. western blot analysis kit (Thermo Fisher Scientific) and Determination of CDK4 inhibition  To evaluate photo documentation of the bands were done by ATTO kinase inhibitory activity, CDK4 expressed as a complex Vol. 27, No. 1, 2021 31

Fig. 1. Chemical structure of alaternin and emodin. of CDK4 and cyclin D1 protein linked to glutathione-S- (GST) on the N-terminal of CDK4 obtained from a sf21 insect cell was taken. Experimental condition for CDK4 was identical to CDK2 except for the difference in CDK4/ cyclin D1 instead of CDK2/ cyclin A and the use of purified retinoblastoma substrate specific for CDK4. Evaluation of mushroom tyrosinase inhibition  Anti-tyrosinase activity of alaternin and emodin was determined using L-tyrosine as substrate following previously reported methods46,47 and with slight modifi- cations. Absorbance was measured with a fluorescence Fig. 2. Effect of alaternin on cell viability in LPS (1 μg/mL) microplate reader (Molecular Devices, Sunnyvale, CA, treated RAW 264.7 cells. Cell viability was measured by MTS assay. Values are expressed as the mean ± SD of triplicate USA). Results were calculated as IC50. experiments. Statistics  All results are expressed as mean ± standard deviation (SD) of triplicate samples. Statistical significance was analyzed using one-way analysis of seases.48,49 As a response to inflammatory stimulant like variance (ANOVA) (Systat Inc., Evaston, IL) and was LPS; NO and ROS are generated. Interaction of NO with noted at p < 0.05. superoxide generates an extremely potent oxidant, ONOO which protonates to peroxinitrous acid in the acidic Result and Discussion environment and again splits homolythically to form two 50 strong radicals, OH· and NO2·. As shown in Fig. 2, Anthraquinones are an important class of organic alaternin in LPS (1 µg/mL) treated RAW 264.7 cells at 5- compound, present in both higher and lower plants, and 30 µM showed cell viability of >80% which is comparable can also be obtained synthetically. In order to understand with the untreated control. and expand knowledge about anthraquinones, we took LPS (1 µg/mL) considerably induced NO production one of the well-known derivatives, alaternin as repre- and this stimulation was one up to 24 h. Pretreatment with sentative candidates to evaluate the activity. Additionally, alaternin inhibited this effect at the rate of 249.28, 224.36, we tried to understand the structure-based mechanism of 212.61, 147.56, and 131.23% at 10, 15, 20, 25, and 30 this compound by comparing its activity to structurally µM concentration, respectively giving an IC50 of 18.68 similar anthraquinone derivative, emodin. Chemical µM. LPS, on the other hand, induced NO production by structures of alaternin and emodin are shown in Fig. 1. 266.48% at 1 µg/mL (Fig. 3). Further, western blot (Fig. Inflammation is a natural reaction of a mammalian cell 4), shows the shows that alaternin at 15 and 30 µM against harmful pathogen and stimuli wherein immune supressed the expression of both iNOS and COX-2 in cell produces inflammatory mediators. If this process RAW 264.7 cells, thereby illustrating the molecular occurs for a greater interval, it can cause chronic inflam- mechanism of NO inhibition by alaternin. Several in- mation, which commences numerous diseases like arthritis, vestigations have highlighted the role of ROS in cancer, diabetes, cardiovascular, and neurological di- inflammatory conditions.51-57 Moreover, the significant 32 Natural Product Sciences

anti-oxidative potential of alaternin to scavenge ONOO has already been reported.11 Studies also revealed that alaternin downregulates the action of 2,2-diphenyl-1- picrylhydrazyl (DPPH) ,58 free radicals,39 and exhibits (SOD) like activity.59 Anti- inflammatory activity of emodin has also been reported in rat model17 and rat kidney (NRK-52E) cells.60 Emodin has also been shown to reduce the induction of iNOS and COX-2 in LPS stimulated RAW 264.7 cells and exert anti-inflammatory activity via NF-κB (nuclear factor-κB) suppression61,16 among other emodin-type anthraquinones, Fig. 3. Effect of alaternin on LPS-induced (NO) in RAW owing to the presence of C-3 hydroxyl group. Alaternin 264.7 cells. Cells pretreated with indicated concentrations of and emodin are also reported to inhibit peroxidation of tested compounds for 1 h were stimulated with LPS (1 μg/mL) linoleic acid and only alaternin has been found to inhibit for 24 h. The cultured media were used to measure the amount of 39 NO production. ap < 0.001 indicates a significant difference from oxidation and nitrogen mediated reactions. On the basis the unstimulated control group, bp < 0.001 indicate significant of above outcome, the presence an hydroxyl group (C-3 differences from the LPS-stimulated control. and C-7) in alaternin could be responsible in showing iNOS and COX-2 inhibition. AGE/ RAGE signaling cascade results in production of cytokines, and other proinflammatory mole- cules that induce inflammation. Activation of the receptor of AGE (RAGE) also produces ROS in a dependent manner.23,62,29 The higher degree of glycation predisposes proteins to oxidative damage as the addition of glucose or glycated collagen can catalyze the polyunsaturated lipid vesicles resulting in lipid peroxyl radicals and hydro- peroxides formation.63,64,28 Increased AGE/ RAGE ratio Fig. 4. Effect of alaternin on the production of COX-2 and iNOS can also be used as a biomarker for assessing risk of protein in RAW 264.7 cells. Cytosolic lysates from Raw 264.7 developing certain age-related disease.65 As shown in Fig. cells stimulated with LPS (1μg/mL) for 24 h were separated by SDS-PAGE and the protein levels of iNOS, COX-2, and β-actin 5A, on the third week, with the inhibition of 29.51, 33.05, were detected by western blot. 54.24, and 84.04% for 2, 10, 20, and 100 µM, alaternin dose-dependently inhibited the production of AGE in

Fig. 5. The inhibitory effect of alaternin, emodin and aminoguanidine (AG) on the formation of fluorescent AGE (A) and amyloid cross-β structures (B) in the BSA-glucose-fructose system. Results are expressed as mean ± SEM of duplicate experiments. ap < 0.001 indicates a significant difference from BSA group, bp < 0.05, cp < 0.01, and dp < 0.001 indicate significant differences from the control group. Vol. 27, No. 1, 2021 33

Fig. 7. The inhibitory effect of alaternin and emodin on mushroom tyrosinase. Results are expressed as mean ± SD of triplicate experiments.

have demonstrated the association of AGE/ RAGE signaling with immune inflammatory processes like diabetes66,67 and renal complication,68 neurological disorders like Alzheimer’s,69 induction of retinal vascular per- meability,70 inactivation of NO, and aging.71 In this experi- ment, emodin inhibited fluorescent AGE and amyloid cross β-structures, supporting previous findings,26,72 however, the extent of glycated product inhibition was markedly higher for alaternin. Even though the mechanism by which these anthraquinones inhibit AGE is Fig. 6. The inhibitory effect of alaternin and emodin on CDK2 yet to be established, it is clearly seen that the presence of (A) and CDK4 (B). Results are expressed as mean ± SD of an extra hydroxy function at the positions C-7, as duplicate experiments. evidenced by alaternin, plays a key role in inhibiting glycation. comparison to emodin (2.13, 7.90, 21.64, and 58.51% for ROS like (H2O2) can also be found 10, 20, 100, and 200 µM, respectively) and AMG (49.01% in the melanin biosynthesis pathway.73 Therefore, we for 900 µM). Similarly, in Fig. 5B, on the third week, tested the anti-tyrosinase activity of alaternin and emodin. alaternin with an inhibition percentage of 17.00% (2 µM), However, our anthraquinones were less potent tyrosinase 43.38% (10 µM), 83.53% (20 µM), and 93.73% (100 µM) substrate (Table 1 and Fig. 7). Nonetheless, the result of dose-dependently inhibited the formation of cross-β this study supports earlier reports,59,74 where the addition structure, whereas emodin exhibited inhibition of 51.42% of hydroxyl group, as in alaternin and emodin at C-3 (10 µM), 76.29% (20 µM), 81.28% (100 µM), and 82.77% showed faint tyrosinase inhibitory activity, and hence (200 µM). At concentration 900 µM, AMG also inhibited explaining the reason behind the activity of these anthra- amyloid cross-β glycation by 119.88%. Various findings quinones.

Table 1. Mushroom tyrosinase inhibitory activities of alaternin Tyrosinase inhibition (%) ± SEM Sample 100 µM 200 µM 400 µM Alaternin 16.88 ± 2.21 27.58 ± 3.12 35.84 ± 4.61 Emodin 97.62 ± 4.25 32.26 ± 2.36 51.02 ± 4.83 Kojic acida,b 96.40 ± 2.34 73.89 ± 3.06 97.22 ± 0.67 a Inhibitory activity of kojic acid was evaluated at the concentration of 4, 20, and 100 µM. b Positive control. 34 Natural Product Sciences

Prolonged oxidative attack by ROS and chronic further evaluation. It can also be concluded that the inflammation is related to increased risk of several presence of catechol moiety is critical for increasing the cancers.35,36,75-77 As shown in Fig. 6, the inhibition of potency of alaternin. However, the precise underlying CDK2 for emodin was obtained as 17 µM whereas mechanism of these anthraquinones remains to be clarified alaternin did not show notable inhibition while both and a detailed toxicological and preclinical studies are alaternin and emodin with the respective concentration of necessary to confirm and promote the rational use of 170 and 380 µM inhibited the by CDK4. alaternin on radical related injuries. Recent studies have shown that the presence of ROS influences regulation of CDKs by governing transcription, Acknowledgments translation, ubiquitin-mediated degradation, reverse phosphorylation, all of which are necessary for expression This research was supported by the Basic Science and functioning of a cell.34,78,35,79 The absence of selective Research Program through the National Research CDK inhibition has been shown to be associated with Foundation of Korea (NRF) funded by the Ministry of serious adverse effects.30,80 Among tested compounds, Science (2012R1A6A1028677). alaternin selectively inhibited CDK4, while emodin suppressed both kinases, however, the inhibition against References CDK2 was more than 20-fold higher than CDK4. The inability of non-selective CDK inhibitor to discriminate (1) Ali, M. Y.; Jannat, S.; Jung, H. A.; Min, B. S.; Paudel, P.; Choi, J. S. between healthy and cancerous tissue has created a need J. Food Biochem. 2018, 42, e12439. (2) Paudel, P.; Jung, H. 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