Devendra Sir Cover Page copy.jpg BIBECHANA 17 (2020) 20-27 20 BIBECHANA ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher: Department of Physics, Mahendra Morang A.M. Campus, TU, Biratnagar, Nepal Structure-activity relationship and MM2 energy minimized conformational analysis of quercetin and its derivatives in the DPPH• radical scavenging capacity Gan B. Bajracharya 1* , Mohan Paudel 2 , Rajendra K. C. 2 , Sajan L. Shyaula 1 1 Faculty of Science, Nepal Academy of Science and Technology (NAST), Khumaltar, Lalitpur, Nepal 2 Department of Chemistry, Tri-Chandra Multiple Campus, Tribhuvan University, Kathmandu, Nepal *Email: ganbajracharya@yahoo.com; gan.bajracharya@nast.gov.np Article Information: Received: August 13, 2019 Accepted: September 7, 2019 Keywords: Antioxidant 1,1-Diphenyl-2-picrylhydrazyl Flavonoid Free radical IC50 SAR ABSTRACT Antioxidant activity of quercetin (1) and its derivatives (2-15) was evaluated by using DPPH assay and IC50 values were calculated. Dihedral angles α of C3-C2- C1’-C6’ chain and β of O1-C2-C-1’-C2’ chain between AC and B rings of these flavones were determined by using MM2 energy minimized structures. Structure- activity relationship study revealed that quercetin (1), quercetin-5-methyl ether (2), quercetin-3’-methyl ether (3) and quercetin-3’,5-dimethyl ether (4) displaying a high antioxidant activity (IC50 = 47.20-119.27 μM) possess similar dihedral angles (α 11.1-11.5º and β 6.3-6.6º). Mono- and/or di-methoxy substituent(s) at 3’ and 5 positions of the flavone are most suitable for the preservation of the antioxidant capacity while retaining conformational geometry. 1. Introduction Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen ( 1 O2), superoxide (O2•–), hydroxyl radicals (OH•), etc. are generated during oxygen metabolism in biological systems. Imbalance between generation and elimination of ROS leads cellular aging, mutagenesis, carcinogenesis, immunodeficiency syndrome, diabetes, coronary heart diseases, neurodegenerative diseases, etc. [1-3]. Dietary flavonoids are considered as powerful antioxidants since they act as radical scavengers, metal chelators and enzyme inhibitors, and produce the beneficial health effects [4-7]. In several publications, the antioxidant activity of flavonoids have been evaluated using 1,1-diphenyl- 2-picrylhydrazyl radical (DPPH•) [8], hydroxyl radical (HO•) [9], superoxide (O2• ˗ ) [10], peroxyl radical (ROO•) [11], and hypochlorite (HOCl/OCl ⁻ ) [12], and structure-activity relationships (SAR) of flavonoids in antioxidant activity have been documented [1, 11-27]. Hydrogen or electron- donating properties of flavonoids are considered to be the basis of their antioxidant activity [1, 25]. This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons.org/licenses/by-nc/4.0/ DOI: https://doi.org/10.3126/bibechana.v17i0.25208 http://nepjol.info/index.php/BIBECHANA mailto:ganbajracharya@yahoo.com mailto:gan.bajracharya@nast.gov.np https://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.3126/bibechana.v17i0.25208 Gan B. Bajracharya, Mohan Paudel et al / BIBECHANA 17 (2020) 20-27 21 Structural requirement in flavonoids for the hydrogen-donation by single-electron transfer includes ortho-dihydroxyl substituent in the B ring and C2-C3 double bond in conjugation with C-4 carbonyl group in the C ring, which expands electron delocalization for radical stabilization and determines the co-planarity of the hetero ring. Quercetin, a pentahydroxy flavone (1), seems to be a paradox which satisfies these structural variations and so efficiently captures free radicals thereby exhibiting high antioxidant activity. In case of absence of a catecholic structure in B ring, the antioxidant activity is compensated by 3- and/or 5- hydroxyl substituent(s). Blocking or removing the 3-OH group decreases antioxidant property. The dihedral angle of the B ring with respect to remaining structure in flavones is considered for strong influence on radical scavenging activity [18, 27]. However, the roles of chemical structure and underlying molecular phenomena have stayed elusive [23, 28]. The nature of rapid conjugation with glucuronosyltransferases and sulfotransferases present in the small intestine halts quercetin (1) to reach the malignant organs through gastrointestinal tract in oral therapy [29]. When the hydroxyl groups in polyphenols are methylated, the resulting compounds are much less prone to glucuronidation and sulphation, and hence are more metabolically stable increasing their bioavailablity. These underlying facts promoted us to commence a research program to study the SAR of quercetin derivatives for possible enhancement of antioxidant capacity. 2. Experimental Chemicals and equipments Quercetin (1), DPPH and gallic acid were purchased from Sigma-Aldrich and were used as received. Quercetin derivatives (2-15) used in this work were previously synthesized in our laboratory [30]. Spectrophotometric analysis was performed with a Cary 60 UV-Visible spectrophotometer (Agilent Technologies). DPPH radical scavenging assay The DPPH assay was performed according to the procedure described by Brand-Williams et al. with a slight modification [31]. To generate free DPPH• radical, 11.7 mg of DPPH in methanol (300 mL) was stirred overnight at 0 °C and used immediately. Sample solutions of the compounds 1-15 of different concentrations (5, 25, 50, 100, 250 and 500 μM in acetone) were prepared. Each sample solution (0.5 mL) was mixed with freshly prepared DPPH• solution (2.5 mL). A control solution was prepared by mixing acetone (0.5 mL) and DPPH• solution (2.5 mL). The content was shaken well, kept in dark at room temperature for 30 min and then absorbance was measured at 517 nm against the blank solution consisting acetone (0.5 mL) and MeOH (2.5 mL). The percentage of inhibition was calculated by the equation: I (%) = (1 – Asample / Acontrol) × 100 where, Asample and Acontrol are the absorbance values of the reaction mixture with and without sample, respectively. Thus obtained data of % inhibitions at different concentrations were computed to calculate IC50 values by employing the equation: IC50 = (50 μM - c) / m where, IC50 = concentration causing 50% inhibition of absorbance, c = intercept, m = slope of a linear curve describing dependence of % inhibition with concentration. Computational analysis Energy minimized structure of quercetin (1) and synthesized quercetin derivatives (2-15) were produced by using Molecular Mechanics Part 2 (MM2) calculation, Cambridgesoft’s Chem3D Pro 12.0.2.1076. Thus optimized geometry of the structures were studied for determining the dihedral angles between the planes of the AC-ring and the B-ring employing the angels of C3-C2-C1’-C6’ (α) and O1-C2-C1’-C2’ (β). Gan B. Bajracharya, Mohan Paudel et al / BIBECHANA 17 (2020) 20-27 22 3. Results and Discussion DPPH assay measures the antioxidant capacity of a compound in terms of its ability to donate hydrogen atom to the free DPPH• radical. The DPPH• radical, which shows absorption at 517 nm, is reduced to the corresponding hydrazine when it reacts with hydrogen donors. DPPH assay is considered a valid and easy assay to evaluate the SAR of antioxidants [32]. Some of the compounds used in this study were not completely soluble in MeOH; therefore, standard procedure was slightly modified by using acetone as a dissolving solvent to prepare the sample solutions. In the DPPH assay, six different concentrations of each compound were used in order to obtain IC50 value. Linear regression curve of concentration versus percentage of inhibition for each compound was plotted where R 2 values were found nearly equal to 1. The slope and intercept values of linear regression curve were used to obtain the IC50 value and the result is presented in Table 1. The IC50 value of quercetin (1) in DPPH assay was reported by several groups and it ranged from 1.82 to 119.60 μM [26, 33-39]. In this study, compared to the parent quercetin 1 (IC50 = 47.20 µM), its monomethylated derivatives 2 (IC50 = 52.24 µM) and 3 (IC50 = 52.45 µM) have retained the antioxidant capacity in the DPPH assay (Table 1). The dimethylated derivative 4 also displayed antioxidant property with IC50 value of 119.27 µM. The antioxidant capacity was gradually reduced as the number of substituents other than hydroxyl group is increased. Although 3,5-diOH and/or 3’,4’-diOH groups are important for scavenging of DPPH• radical; however, this study showed that one (or two) of these hydroxyl group(s) can be replaced with methoxy group(s). This conclusion is also supported by Moalin et al., who have reported that substitutions of methoxy group at 5 and/or 7 position(s) in ring A of quercetin (1) retain the antioxidant capacity in ABTS assay [27]. Acetylation of free hydroxyl groups reduced the antioxidant capacity; however, quercetin 3,3’,4’,5-tetraacetate (5) and quercetin 3,3’,4’,5,7- pentaacetate (6) derivatives have exhibited a mild antioxidant activity with IC50 values of 516.26 and 790.57 µM, respectively. These results also indicated that a free 7-OH group in a flavone has a crucial role. Remaining quercetin derivatives (7-15) were found to be lost their DPPH• radical scavenging capacity displaying IC50 value >2000 µM. Olejniczak and Potrzebowski have reported that a small perturbation of geometry has a great influence on bond orders in ring B in density functional theory (DFT) calculation of quercetin (1) [40]. And, the change in conformers may significantly change the susceptibility for the formation of radicals and consequently alters the biological property. Although molecular structure of quercetin (1) (and its derivatives as well) can be represented by exact conformation; however, the presence of many flexible hydroxyl groups and hydrogen bonding networks in the system leads a wrong conclusion in the crystal lattice provided by X-ray diffractometry (XRD). Later, Filip et al. have performed the conformational calculation of quercetin (1) by employing the molecular mechanics (MM) level of theory that combined with previous XRD and solid state NMR data [41, 42]. It was concluded that small changes of conformation and hydrogen bonding pattern greatly influence the bond order parameters of quercetin (1). The authors have further mentioned that no matter how precisely the conformational analysis is performed, the most important is finding of the most probable molecular conformation. The MM2 energy minimization calculation can identify the more stable conformation [43]. Therefore in the present study, the MM2 calculations of the compounds 1-15 were performed. In the case of quercetin (1), the orientation of the hydroxyl groups at 3 and 3’ carbon atoms in a planar arrangement of A, B and C rings produces anti and syn conformers. Herein, energy minimized structures of compounds 1-15 were computed by keeping 3 and 3’ substituents at anti fashion. The optimal conformations of the compounds obtained are depicted in Figure 1 and their dihedral angles are given in Table 1. Gan B. Bajracharya, Mohan Paudel et al / BIBECHANA 17 (2020) 20-27 23 Table 1: IC50 values of quercetin scaffolds (1-15) in DPPH• free radical scavenging activity. The dihedral angles α = 11.5º and β = 6.6º were found in MM2 energy minimization calculation of quercetin (1), which exhibited a high antioxidant property (IC50 = 47.20 µM) (Table 1, Figure 1). Interestingly, in comparison with quercetin (1), quercetin-5-methyl ether (2) (α 11.2º and β 6.4º), quercetin-3’-methyl ether (3) (α 11.1º and β 6.3º) and quercetin-3’,5-dimethyl ether (4) (α 11.2º and β 6.3º) showed similar dihedral angels and they were also highly efficient in scavenging of DPPH• radical with the IC50 values of 52.24, 52.45 and 119.27 µM, respectively. The decrease in dihedral angles α and β decreased the antioxidant property as shown by quercetin-3,3’,4’,5-tertaacetate (5) (α = 9.2º, β = 4.6º, IC50 = 516.26 µM) and quercetin- 3,3’,4’,5,7- pentaacetate (6) (α = 7.3º, β = 3.9º, IC50 = 790.57 µM). The MM2 calculation of remaining compounds (7-15) showed that they exist in more or less planar conformation. Therefore, a slightly twisted conformation between rings B and C with dihedral angles α 11.5-7.3º and β 6.6-3.9º plays a pivotal role in preserving the antioxidant capacity of parent compound quercetin (1) and perfect planarity is merely not necessary [27]. While modifying the structure of a flavone in search of the molecule with enhanced antioxidant activity, preservation of its planarity is therefore a must. 4. Conclusion In conclusion, we have evaluated the DPPH• free radical scavenging capacity of quercetin (1) and its derivatives (2-15). Quercetin (1) and its methoxyl derivatives 2, 3 and 4 were found as highly efficient antioxidants. The acetyl derivatives 5 and 6 exhibited a mild antioxidant capacity while other remaining derivatives (7-15) were found to be inefficient. Dihedral angles α of C3-C2-C1’-C6’ chain (11.5-7.3º) and β of O1-C2-C-1’-C2’ chain Compound Substituents Dihedral angles IC50 (μM) R 1 R 2 R 3 R 4 R 5 α° β° Quercetin (1) OH OH OH OH OH 11.5 6.6 47.20 Quercetin 5-methyl ether (2) OH OMe OH OH OH 11.2 6.4 52.24 Quercetin 3’-methyl ether (3) OH OH OH OMe OH 11.1 6.3 52.45 Quercetin 3’,5-dimethyl ether (4) OH OMe OH OMe OH 11.2 6.3 119.27 Quercetin 3,3’,4’,5-tetraacetate (5) OAc OAc OH OAc OAc 9.2 4.6 516.26 Quercetin 3,3’,4’,5,7-pentaacetate (6) OAc OAc OAc OAc OAc 7.3 3.9 790.57 Quercetin 3,3’,4’,7-tetraacetate (7) OAc OH OAc OAc OAc 7.3 1.7 >2000 Quercetin 3,3’,4’,7-tetrabenzyl-5-methyl ether (8) OBn OMe OBn OBn OBn 3.4 1.3 >2000 Quercetin 3,4’,7-tribenzyl ether (9) OBn OH OBn OH OBn 2.8 0.9 >2000 Quercetin 3,3’,4’,7-tetrabenzyl ether (10) OBn OH OBn OBn OBn 2.7 1.1 >2000 Quercetin 3,4’,7-tribenzyl-3’,5-dimethyl ether (11) OBn OMe OBn OMe OBn 2.6 0.9 >2000 Quercetin 3,4’,7-tribenzyl-3’-methyl ether (12) OBn OH OBn OMe OBn 2.3 0.8 >2000 Quercetin 3,3’,4’,5,7-pentamethyl ether (13) OMe OMe OMe OMe OMe 1.7 0.5 >2000 Quercetin 3,3’,4’,7-tetramethyl ether (14) OMe OH OMe OMe OMe 1.5 0.4 >2000 Quercetin 3,4’,7-trimethyl ether (15) OMe OH OMe OH OMe 1.4 0.3 >2000 Gan B. Bajracharya, Mohan Paudel et al / BIBECHANA 17 (2020) 20-27 24 (6.6-3.9º) between AC and B rings as can be found in quercetin (1) are suitable for the preservation of antioxidant capacity. Mono- and/or di-methoxy substituent(s) at 3’ and 5 positions of the flavone are most suitable for the preservation of the antioxidant capacity. Free 7-OH group of a flavone also plays a crucial role in preserving of the antioxidant capacity. Fig. 1: Three dimensional energy minimized structure of quercetin (1) and its derivatives (2-15) with their dihedral angles α and β. Gan B. Bajracharya, Mohan Paudel et al / BIBECHANA 17 (2020) 20-27 25 Acknowledgements The World Academy of Sciences (TWAS) is gratefully acknowledged for supporting our research program by purchasing precious instruments (Process Station Personal Synthesizer PPS-CTRL 1 and Microwave Synthesis Reactor Monowave 300) through the research grant no. 13- 198 RG/CHE/AS_G. References [1] K. E. Heim, A. R. Tagliaferro, D. J. Bobilya, Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships, J. Nutr. Biochem. 13 (2002) 572–584. https://doi.org/10.1016/S0955-2863(02)00208-5. [2] İ. Gülçin, Antioxidant activity of food constituents: an overview, Arch. Toxicol. 86 (2012) 345–391. https://doi.org/10.1007/s00204-011-0774-2. [3] J. Emerit, M. Edeas, F. Bricaire, Neurodegenerative diseases and oxidative stress, Biomed. Phrmacother. 58 (2004) 39–46. https://doi.org/10.1016/j.biopha.2003.11.004. [4] C. Kandaswami, E. Middleton, Free radical scavenging and antioxidant activity of plant flavonoids, Free Radicals in Diagnostic Medicine (D. Amstron, Ed.), Plenum Press, New York, 1994. [5] R. J. Nijveldt, E. van Nood, D. E. C. van Hoorn, P. G. Boelens, K. van Norren, P. A. M. van Leeuwen, Flavonoids: a review of probable mechanisms of action and potential applications. Am. J. Clin. Nutr. 74 (2001 )418–425. https://doi.org/10.1093/ajcn/74.4.418. [6] A. L. Miller, Antioxidant flavonoids: structure, function and clinical usage. Altern. Med. Rev. 1 (1996) 103–111. https://pdfs.semanticscholar.org/5e51/cec57096d03 df56dd7538c30bf32c91f6dfa.pdf?_ga=2.8434229.1 61730912.1565674832-1063744325.1529383604. [7] P. G. Pietta, Flavonoids as antioxidants, J. Nat. Prod. 63 (2000) 1035–1042. https://doi.org/10.1021/np9904509. [8] F. Nanjo, K. Goto, R. Seto, M. Suzuki, M. Sakai, Y. Hara, Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical, Free Radic. Biol. Med. 21 (1996) 895–902. https://doi.org/10.1016/0891-5849(96)00237-7. [9] Y. Hanasaki, S. Ogawa, S. Fukui, The correlation b e t w e e n a c t i v e o x y g e n s s c a v e n g i n g a n d antioxidative effects of flavonoids, Free Radic. Biol. Med. 16 (1994) 845–850. https://doi.org/10.1016/0891-5849(94)90202-X. [10] J. Robak, R. J. Gryglewski, Flavonoids are scavengers of superoxide anions, Biochem. Pharmacol. 37 (1988) 837–841. https://doi.org/10.1016/0006-2952(88)90169-4. [11] A. J. Dugas Jr., J. Castañeda-Acosta, G. C. Bonin, K. L. Price, N. H. Fischer, G. W. Winston, Evaluation of the total peroxyl radical-scavenging capacity of flavonoids:structure-activity relationships, J. Nat. Prod. 63 (2000) 327–331. doi: https://doi.org/10.1021/np990352n. [12] O. Firuzi, P. Mladěnka, R. Petrucci, G. Marrosu, L. Saso, Hypochlorite scavenging activity of flavonoids, J. Pharm. Pharmacol. 56 (2004) 801– 807. https://doi.org/10.1211/0022357023556. [13] A. Arora, M. G. Nair, G. M. Strasburg, Structure- activity relationships for antioxidant activities of a series of flavonoids in a liposomal system, Free Radic. Biol. Med. 24(1998)1355–1363. https://doi.org/10.1016/S0891-5849(97)00458-9. [14] G . R . M . M . H a e n e n , J . B . G . P a q u a y , R . E . M . K o r t h o u w e r , A . B a s t , P e r o x y n i t r i t e scavenging by flavonoids, Biochem. Biophys. Res. Commun. 236 (1997) 591–593. https://doi.org/10.1006/bbrc.1997.7016. [15] M. Furusawa, T. Tanaka, T. Ito, A. Nishikawa, N. Yamazaki, K.-I. Nakaya, N. Matsuura, H. Tsuchiya, M. Nagayama, M. Iinuma, Antioxidant activity of hydroxylflavonoids. J. Health Sci. 51 (2005) 376– 378. https://doi.org/10.1248/jhs.51.376. [16] J. P. Hu, M. Calomme, A. Lasure, T. de Bruyne, L. Pieters, A . V l i e t i n c k , D. A. V a n d e n B e r g h e , Structure-activity relationship of flavonoids with superoxide scavenging activity. Biol. Trace Elem. Res.47(1995)327–331. https://doi.org/10.1007/BF02790134. [17] E. Mikamo, Y. Okada, M. Semma, Y. Ito, T. Morimoto, M. Nakamura, Studies on structural correlation with antioxidant activity of flavonoids. Nippon Shokuhin Kagaku Gakkaishi 7 (2000) 97– 101. [18] S. A. B. E. van Acker, M. J. de Groot, D.-J. van den Berg, M. N. J. L. Tromp, G. D. den Kelder, W. J. F. van der Vijgh, A. Bast, A quantum chemical e x p l a n a t i o n o f t h e antioxidant activity of flavonoids. C h e m . R e s . T o x i c o l. 9 (1996) 1305– 1312. https://doi.org/10.1021/tx9600964. [19] T. Yokozawa, C. P. Chen, E. Dong, T. Tanaka, G.-I. Nonaka, I. Nishioka, Study on the inhibitory effect of tannins and flavonoids against the 1,1-diphenyl-2 picrylhydrazyl radical. Biochem. Pharmacol. 56 (1998) 213–222. https://doi.org/10.1016/S0006-2952(98)00128-2. https://doi.org/10.1016/S0955-2863(02)00208-5 https://doi.org/10.1016/S0955-2863(02)00208-5 https://doi.org/10.1016/j.biopha.2003.11.004 https://doi.org/10.1093/ajcn/74.4.418 https://pdfs.semanticscholar.org/5e51/cec57096d03df56dd7538c30bf32c91f6dfa.pdf?_ga=2.8434229.161730912.1565674832-1063744325.1529383604 https://pdfs.semanticscholar.org/5e51/cec57096d03df56dd7538c30bf32c91f6dfa.pdf?_ga=2.8434229.161730912.1565674832-1063744325.1529383604 https://pdfs.semanticscholar.org/5e51/cec57096d03df56dd7538c30bf32c91f6dfa.pdf?_ga=2.8434229.161730912.1565674832-1063744325.1529383604 https://doi.org/10.1021/np9904509 https://doi.org/10.1016/0891-5849(96)00237-7 https://doi.org/10.1016/0891-5849(94)90202-X https://doi.org/10.1016/0006-2952(88)90169-4 https://doi.org/10.1021/np990352n https://doi.org/10.1211/0022357023556 https://doi.org/10.1016/S0891-5849(97)00458-9 https://doi.org/10.1006/bbrc.1997.7016 https://doi.org/10.1248/jhs.51.376 https://doi.org/10.1007/BF02790134. https://doi.org/10.1021/tx9600964 https://doi.org/10.1016/S0006-2952(98)00128-2 Gan B. Bajracharya, Mohan Paudel et al / BIBECHANA 17 (2020) 20-27 26 [20] E. Middleton Jr., C. Kandaswami, T. C. Theoharides, The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer, Pharmacol. Rev. 52 (2000) 673–751. https://www.semanticscholar.org/paper/The-effects- of-plant-flavonoids-on-mammalian-cells%3A- Middleton- Kandaswami/07d75a864bc661a0079b418c69cd0b4 c38c9385d. [21] A. S. Pannala, T. S. Chan, P. J. ƠBrien, C. A. Rice- Evans, Flavonoid B-ring chemistry and antioxidant activity: fast reaction kinetics. Biochem. Biophys. Res.Commun.282(2001)1161–1168. https://doi.org/10.1006/bbrc.2001.4705. [22] J. W. Chen, Z. Q. Zhu, T. X. Hu, D. Y. Zhu, Structure-activity relationship of natural flavonoids in hydroxyl radical-scavenging effects. Acta Pharmacologica Sinica 23 (2002) 667–672. [23] D. Amić, D. Davidović-Amić, D. Bešlo, N. Trinajstić, Structure-radical scavenging activity relationships of flavonoids. Croatica Chemica Acta 76(2003)55–61. fulir.irb.hr/753/1/CCA_76_2003_055_061_amic.pdf [24] A. Lugasi, J. Hóvári, K. V. Sági, L. Bíró, The role of antioxidant phytonutrients in the prevention of diseases. Acta Biologica Szegediensis 47 (2003) 119–125. [25] D. Amić, D. Davidović-Amić, D. Bešlo, V. Rastija, B. Lučić, N. Trinajstić, SAR and QSAR of the antioxidant activity of flavonoids, Curr. Med. Chem. 14 (2007) 827–845. https:// www.academia.edu/16318419/SAR_and_QSAR_of _the_Antioxidant_Activity_of_Flavonoids . [26] D. Kruzlicova, M. Danihelova, M. Veverka. Quantitative structure-antioxidant activity relationship of quercetin and its new synthesized derivatives. Nova Biotechnologica et Chimica 11 (2012) 37–44. https://doi.org/10.2478/v10296-012-0004-1. [27] M. Moalin, G. P. F. van Strijdonck, M. Beckers, G. J. Hagemen, P. J. Borm, A. Bast, G. R. M. M. Haenen, A planar conformation and the hydroxyl groups in the B and C rings play a pivotal role in the antioxidant capacity of quercetin and quercetin derivatives, Molecules 16 (2011) 9636–9650. https://doi.org/10.3390/molecules16119636. [28] N. Cotelle, Role of flavonoids in oxidative stress. Curr. Top. Med. Chem. 1(2001)569–590. https://doi.org/10.2174/1568026013394750. [29] R. R. J. Arroo, V. Androutsopoulos, K. Beresford, K. Ruparelia, S. Surichan, N. Wilsher, G. A. Potter, Phytoestrogens as natural prodrugs in cancer prevention: dietary flavonoids, Phytochem. Rev. 8 (2009) 375–386. https://doi.org/10.1007/s11101-009-9128-6. [30] G. B. Bajracharya, M. Paudel, R. K. C., Insight into the structure elucidation of flavonoids through UV- visible spectral analysis of quercetin derivatives using shift reagents, J. Nepal Chem. Soc. 37 (2017) 55–64. [31] W. Brand-Williams, M. E. Cuvelier, C. Berset, Use of a free radical method to evaluate antioxidant activity, LBT – Food Sci. Technol. 28 (1995) 25– 30. https://doi.org/10.1016/S0023-6438(95)80008-5. [32] C. Sánchez-Moreno, Review: methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci. Technol. Int. 8 (2002)121–137.: https://doi.org/10.1106/108201302026770. [33] Y. Lu, T. J. Khoo, C. Wiart, Antioxidant activity determination of citronellal and crude extracts of Cymbopogon citratus by 3 different methods, Pharmacol.Pharm.5(2014)395–400. https://doi.org/10.4236/pp.2014.54047. [34] D. Rusmana, R. Wahyudianingsih, M. Elisabeth, Balqis, Maesaroh, W. Widowati, Antioxidant activity of Phyllanthus niruri extracts, rutin and quercetin, Indonesian Biomed. J. 9 (2017) 84–90. https://doi.org/10.18585/inabj.v9i2.281. [35] R. B. Pereira, C. Sousa, A. Costa, P. B. Andrade, P. Valentão, Glutathione and the antioxidant potential of binary mixtures with flavonoids: synergisms and antagonisms, Molecules 18 (2013) 8858–8872. https://doi.org/10.3390/molecules18088858. [36] P. K. Srimathi, K. Vijayalakshmi, Investigation of antioxidant potential of quercetin and hesperidin: an in vitro approach. Asian J Pharm Clin Res 10 (2017) 83–86.: https://doi.org/10.22159/ajpcr.2017.v10i11.20260. [37] M. Majewska, M. Skrzycki, M. Podsiad, H. Czeczot. Evaluation of antioxidant potential of flavonoids: an in vitro study. Acta Pol. Pharm. – DrugRes.68(2011)611–615. www.ptfarm.pl/pub/File/Acta_Poloniae/2011/4/611. pdf. [38] J. M. A. Farboodniay, M. R. Moein, S. Ahmadi, In vitro free radical scavenging activity and total phenolic and flavonoid content of Spathe extracts from 10 cultivar varieties of Phoenix dactylifera L. Int. J. Pharmacog. Phytochem. Res. 8 (2016) 1481– 1486. https://ijppr.com/volume8issue9/ [39] A. Seyoum, K. Asres, F. K. El-Fiky. Structure- radical scavenging activity relationships of flavonoids. Phytochemistry 67 (2006) 2058–2070. https://doi.org/10.1016/j.phytochem.2006.07.002. [40] S. Olejniczak, M. J. Potrzebowski, Solid state NMR studies and density functional theory (DFT) calculations of conformers of quercetin, Org Biomol Chem 2 (2004) 2315–2322. https://www.semanticscholar.org/paper/The-effects-of-plant-flavonoids-on-mammalian-cells%3A-Middleton-Kandaswami/07d75a864bc661a0079b418c69cd0b4c38c9385d https://www.semanticscholar.org/paper/The-effects-of-plant-flavonoids-on-mammalian-cells%3A-Middleton-Kandaswami/07d75a864bc661a0079b418c69cd0b4c38c9385d https://www.semanticscholar.org/paper/The-effects-of-plant-flavonoids-on-mammalian-cells%3A-Middleton-Kandaswami/07d75a864bc661a0079b418c69cd0b4c38c9385d https://www.semanticscholar.org/paper/The-effects-of-plant-flavonoids-on-mammalian-cells%3A-Middleton-Kandaswami/07d75a864bc661a0079b418c69cd0b4c38c9385d https://www.semanticscholar.org/paper/The-effects-of-plant-flavonoids-on-mammalian-cells%3A-Middleton-Kandaswami/07d75a864bc661a0079b418c69cd0b4c38c9385d https://doi.org/10.1006/bbrc.2001.4705 https://doi.org/10.1006/bbrc.2001.4705 https://doi.org/10.1006/bbrc.2001.4705 https://doi.org/10.2478/v10296-012-0004-1 https://doi.org/10.3390/molecules16119636 https://doi.org/10.2174/1568026013394750 http://dx.doi.org/10.1007/s11101-009-9128-6 https://doi.org/10.1016/S0023-6438(95)80008-5 https://doi.org/10.1106%2F108201302026770 http://dx.doi.org/10.4236/pp.2014.54047 http://dx.doi.org/10.4236/pp.2014.54047 https://doi.org/10.3390/molecules18088858 https://doi.org/10.22159/ajpcr.2017.v10i11.20260. http://www.ptfarm.pl/pub/File/Acta_Poloniae/2011/4/611.pdf. http://www.ptfarm.pl/pub/File/Acta_Poloniae/2011/4/611.pdf. https://ijppr.com/volume8issue9/ https://doi.org/10.1016/j.phytochem.2006.07.002 Gan B. Bajracharya, Mohan Paudel et al / BIBECHANA 17 (2020) 20-27 27 https://doi.org/10.1039/b406861k. [41] X. Filip, I.-G. Grosu, M. Micalus, C. Filip, NMR crystallography methods to probe complex hydrogen bonding networks: application to structure elucidation of anhydrous quercetin, Cryst Eng Comm,15(2013)4131–4142. https://doi.org 10.1039/C3CE40299A. [42] X. Filip, C. Filip, Can the conformation of flexible h y d r o x y l groups be constrained by simple NMR crystallography approaches? The case of the quercetin solid forms, Solid State Nucl. Magn. Reson.65(2015)21–28. https://doi.org/10.1016/j.ssnmr.2014.10.006. [43] M. J. Dudek, J. W. Ponder, Accurate modeling of the intramolecular electrostatic energy of proteins. J. Comput.Chem.16(1995)791–816. https://doi.org/10.1002/jcc.540160702. https://doi.org/10.1039/b406861k https://doi.org/10.1039/b406861k https://doi.org/10.1016/j.ssnmr.2014.10.006 https://doi.org/10.1002/jcc.540160702