Rutin

Title: Rutin as Deoxyribonuclease I Inhibitor

Authors: Ana Kolarevic, Aleksandra Pavlovic, Aleksandra Djordjevic, Jelena Lazarevic, Sasa Savic, Gordana Kocic, Marko Anderluh, and Andrija Smelcerovic

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Ana Kolarevic,a Aleksandra Pavlovic,b Aleksandra Djordjevic,b Jelena Lazarevic,c Sasa Savic,d Gordana Kocic,e Marko Anderluh,f and Andrija Smelcerovic*,c
a Department of Pharmacy, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000 Nis, Serbia
b Department of Chemistry, Faculty of Science and Mathematics, University of Nis, Visegradska 33, 18000 Nis, Serbia
c Department of Chemistry, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000 Nis, Serbia, e-mail: [email protected] (or [email protected])
d Faculty of Technology, University of Nis, Bulevar Oslobodjenja 124, 16000 Leskovac, Serbia
e Institute of Biochemistry, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000 Nis, Serbia
f Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Askerceva cesta 7, 1000 Ljubljana, Slovenia

DNase I inhibitory potential of water extract of nine Hypericum species (H. umbellatum, H. barbatum, H. rumeliacum, H. rochelii, H. perforatum, H. tetrapterum, H. olympicum, H. hirsutum, H. linarioides) and the most important Hypericum secondary metabolites (hypericin, hyperforin, quercetin and rutin) was investigated. All examined Hypericum extracts inhibited DNase I with IC50 below 800 µg/ml, whereby H. perforatum was the most potent (IC50 = 391.26 ± 68.40 µg/ml). Among the investigated Hypericum secondary metabolites, rutin inhibited bovine pancreatic DNase I in a non-competitive manner with IC50 value of 108.90 ± 9.73 μM. DNase I inhibitory ability of rutin was further confirmed on DNase I in rat liver homogenate (IC50 = 137.17 ± 16.65 μM). Due to the involvement of DNase I in apoptotic processes the results of this study indicate the importance of frequent rutin and H. perforatum consumption in daily human nutrition. Rutin is a dietary component that can contribute to male infertility prevention by showing dual mechanism of sperm DNA protection, DNase I inhibition and antioxidant activity.

Keywords: Hypericum, rutin, DNase I inhibition, molecular docking, apoptosis, male infertility

Introduction

Deoxyribonucleic acid (DNA) is one of the most important macromolecules in living organisms, containing genetic instructions necessary for their growth, development, functioning and reproduction. Various effects can cause DNA degradation leading to different disorders and disease conditions. Higami and Shimokawa[1] demonstrated that aging enhances DNA fragmentation and activates apoptotic machinery in several types of intact cells. It was shown on both rat and human heart that aging accelerates DNA fragmentation in myocytes, causing a significant myocyte loss, and consequentially myocardial dysfunction and failure in the elderly,[2–4] probably as a result of higher activity of Ca2+-dependent DNase I.[5] Deoxyribonuclease I (DNase I) inhibitors could have protective role towards the DNA. However, the number of organic DNase I inhibitors, whether natural or synthetic, is relatively small.[6] Our recent papers have been focused on finding the novel ones.[7–10]

Plants of the genus Hypericum L. (Hypericaceae) have been used as traditional medicinal plants worldwide.[11] Species of this genus were primarily used for cervical injuries, internal purification, gastrointestinal ailments, flatulence, pulmonary ailments, throat inflammations, bedwetting, renal stones and as roborantium for strengthening the corpus.[12] The main constituents of the Hypericum species are naphthodianthrones, phloroglucinol derivatives, and flavonoids.[13]

The aim of this study was to investigate the inhibition of DNase I by water extracts of the aerial parts of nine Hypericum species (H. umbellatum, H. barbatum, H. rumeliacum, H. rochelii, H. perforatum, H. tetrapterum, H. olympicum, H. hirsutum, H. linarioides) collected in Southeast Serbia, as well as the inhibition of DNase I by the well-known bioactive compounds present in Hypericum extracts: naphthodianthrones represented by hypericin, phloroglucinol derivatives represented by hyperforin, and flavonoids represented by quercetin and rutin (Figure 1).[13] To the best of our knowledge this is the first paper reporting the activity of Hypericum, or any plant extract in general, on DNase I inhibition.

Figure 1. Studied secondary metabolites from Hypericum species.

Results and Discussion

DNase I Inhibition and Molecular Docking

Water extracts of nine studied Hypericum species inhibited bovine pancreatic DNase I with IC50 values below 800 µg/ml (Table 1). The most potent DNase I inhibition was obtained by water extract of H. perforatum (IC50 = 391.26 ± 68.40 µg/ml), while H. hirsutum showed the weakest DNase I inhibition (IC50 = 736.30 ± 90.60 µg/ml). Water solution of crystal violet, used as positive control, inhibited bovine pancreatic DNase I with IC50 value of 143.56 ± 13.90 µg/ml.

Since the different activities within the investigated Hypericum extracts are due to different chemical composition, DNase I inhibitory effect of the most important Hypericum secondary metabolites (hypericin, hyperforin, quercetin and rutin) was also investigated. Among them, rutin inhibited bovine pancreatic DNase I with an IC50 value of 108.90 ± 9.73 μM, while quercetin, hypericin and hyperforin showed no inhibitory activity within the investigated concentrations (IC50 > 200 µM). Crystal violet, dissolved in DMSO, exhibited weaker DNase I inhibition (IC50 = 362.95 ± 44.37 μM) compared to rutin. Based on the obtained Lineweaver-Burk plots, rutin inhibited DNase I activity in a non-competitive manner (Figure 2).

Figure 2. Lineweaver-Burk plots for the inhibition of DNase I by rutin with DNA as a substrate.

In order to obtain a more complete picture of rutin as the most potent DNase I inhibitor among the investigated compounds, we further evaluated its ability to inhibit DNase I in rat liver homogenate. As a result, rutin inhibited rat liver DNase I with an IC50 value of 137.17 ± 16.65 μM. Moreover, rutin inhibited DNase I with IC50 values similar to those obtained for synthetic DNase I inhibitors that we recently published.[7–10]

Docking studies (Figure 3) were used to get insight into possible binding mechanism of rutin into bovine DNase I (PDB code: 2DNJ). The suggested rutin binding mode reveals just slight overlap between rutin and DNA, which suggests a partial DNA-competitive binding mode that probably does not prevent DNA from binding to the DNase I. Namely a large portion of the DNA could still bind to DNase I, as seen in the Figure 3a, but could prevent the catalytic action of the enzyme. This is in line with the recent publication of Singh et al.[14] who predicted that the DNase active site is probably formed by two cationic residues, which could correspond to the two arginines (Arg 9 and Arg 73) that are in close contact with the docked pose of rutin, as seen in the Figure 3b. Alternatively, since docking was done without DNA molecule in the binding site and since overlap is quite negligible, rutin could in fact stabilise DNA-DNase I complex thus rendering the enzyme inactive.[14]

Figure 3. Result of rutin docking into DNase I (PDB code: 2DNJ): a) the highest scored docked pose of rutin (green sticks) in the binding site of the DNase I (solid surface, coloured by atom charge) with the DNA molecule and GlcNAc (both in sticks, coloured by atom colour); b) zoomed look of rutin in the DNase I binding site with explicit intermolecular interactions.

Total Flavonoid Content and Rutin Determination in Hypericum Extracts

Due to DNase I inhibitory potential of rutin, the total flavonoid content of the studied Hypericum extracts was also determined (Table 1). The highest total flavonoid content was found in H. perforatum (41.18 ± 0.40 mg/g plant material) which simultaneously showed the most potent DNase I inhibition, while H. hirsutum contained the lowest level of total flavonoids (2.30 ± 0.08 mg/g plant material) and also showed the weakest DNase I inhibition. However, there is no direct correlation between obtained IC50 values of the studied Hypericum extracts and their total flavonoid contents (Table 1).

Using UHPLC-DAD-HESI-MS/MS rutin was detected in three of nine studied Hypericum extracts, H. perforatum, H. tetrapterum, and H. olympicum
(Table 1). The highest content of rutin was determined in H. perforatum extract (15.80 mg/g plant material) which was the most potent DNase I inhibitor.

Potential Therapeutic Applications

Rutin, the glycoside consisting of flavonolic aglycone quercetin and disaccharide rutinose, occurs naturally in variety of daily dietary sources. The highest content was found in food such as capers,[15] black olive,[16] buckwheat,[17] asparagus,[18] black tea,[19] black and red raspberry, blackberry,[20] green currant[21] and plums[22]. Rutin has demonstrated a number of pharmacological activities, including neuroprotective,[23] cardioprotective,[24] anticancer,[25] antiasthmatic,[26] antiarthritic,[27] nephroprotective,[28] hepatoprotective,[29] antihyperglycaemic and antioxidant[30] activities. Rutin also increases thyroid iodide uptake and thus might be useful as an adjuvant in radioiodine therapy.[31] Our results indicate that consummation of rutin and H. perforatum, as DNase I inhibitors, may prevent or slow down age-induced DNA fragmentation and thus protect elderly from development of many diseases caused by age-induced apoptosis.
Yamada et al.[32] suggested that high mobility group box1 (HMGB1) is released during apoptosis as a result of nucleosomal DNA fragmentation catalyzed by DNase γ (DNase I family member), and consequently causes inflammatory responses. Additionally, Yoo et al.[33] found that rutin potently inhibits HMGB1 release. Regarding our results and the results obtained by Yamada et al.[32] and Yoo et al.[33] it could be concluded that rutin, as DNase I inhibitor, inhibits HMGB1 release probably via suppression of DNA fragmentation, and might become novel lead compound for development of new pharmaceuticals against HMGB1 release-related disorders, such as sepsis, septic shock, cerebral and myocardial ischemia.

As oxidative stress is considered one of the main factors contributing to the development of male infertility, antioxidants play an important role in prevention and therapy of such condition.[34] Investigating the total antioxidant activity, reducing power, superoxide anion scavenging activity, DPPH radical scavenging activity, hydrogen peroxide scavenging activity and lipid peroxidation assay, Yang et al.[30] showed that rutin has powerful antioxidant capacity. Additionally, Moretti et al.[35] indicated to the role of rutin in decreasing of lipid peroxidation in human sperm. Since apoptotic DNA fragmentation in sperm cells may also contribute to the development of male infertility, inhibition of DNase I, one of the main endonucleases involved in DNA fragmentation during apoptosis, represents another possible mechanism for prevention of male infertility.[7] Based on our results, rutin is, in addition to ascorbic acid,[7] another dietary antioxidant able to inhibit DNase I. Moreover, Yang et al.[30] showed that rutin was more powerful in the inhibition of lipid peroxidation than ascorbic acid. At the concentration of 0.5 mg/ml, rutin and ascorbic acid inhibited lipid peroxidation by 68.8 and 26.2%, respectively.[30] Comparing our results from the present study with the previously published ones,[7] it appears that rutin is also more powerfull in the inhibition of DNase I (IC50 = 108.90 ± 9.73 μM) than ascorbic acid (IC50 = 330.74 ± 29.92 μM[7]).

Hypericum species possess significant antioxidant activity[36,37] which, in addition to their DNase I inhibitory property, make them significant dietary supplement in prevention and/or therapy of disease conditions induced by both oxidative and apoptotic DNA fragmentation. It is worth noticing that H. perforatum exhibited the most potent antioxidant activity,[36] as well as the most potent DNase I inhibition activity, among the investigated Hypericum species.

Conclusions

Water extracts of nine tested Hypericum species inhibited bovine pancreatic DNase I within the investigated concentrations (IC50 < 800 µg/ml), with H. perforatum being the most potent (IC50 = 391.26 ± 68.40 µg/ml). Rutin inhibited bovine pancreatic DNase I (IC50 = 108.90 ± 9.73 μM) in a non-competitive manner, and showed to be more potent DNase I inhibitor than crystal violet (IC50 = 362.95 ± 44.37 μM) used as a positive control. Its inhibition ability was further confirmed on rat liver DNase I (IC50 = 137.17 ± 16.65 μM). Among the tested Hypericum species, the highest flavonoid content, as well as the highest content of rutin was determined in H. perforatum (41.18 ± 0.40 mg/g plant material and 15.80 mg/g plant material, respectively). Due to the lack of DNase I inhibitor regarded as a "golden standard", as well as a relatively small number of known organic DNase I inhibitors, especially natural, our results may be of interest for the scientific community. Taking into account the importance of DNase I inhibition, more attention should be paid to consumption of rutin and H. perforatum in everyday human nutrition. Additionally, rutin consumption may be useful against HMGB1 release-related disorders, as well as in prevention of male infertility. Experimental Section Chemicals DNase I from bovine pancreas, DNA (sodium salt from calf thymus, type I, fibers), DMSO, perchloric acid, hypericin, hyperforin, quercetin, rutin, and (+)- catechin were purchased from Sigma-Aldrich. Sodium hydroxide, sodium nitrite, and aluminum chloride hexahydrate were purchased from Merck. Acetonitrile and water were purchased from Fisher Chemical (LC-MS and HPLC grade, respectively). Formic acid was from Carlo Erba. Crystal violet was purchased from Lach-Ner. Plant Material Table 1 provides relevant data on the tested plant species, voucher numbers in the Herbarium Moesicum Doljevac, Serbia (HMD), as well as the time and location of the plant material collection. Plant Material Extraction Extraction of plant material comprised of dried above-ground parts (leaves, stems and flowers, 1 g) was performed with boiling water (10 ml) for 10 minutes. Obtained water extracts were filtered and filled up to 10 ml. An aliquot of each extract was evaporated in order to estimate the amount of dry matter, while the remaining volume of the solution obtained by water extraction was adjusted to the concentration of 10.4 mg/ml. Total Flavonoid Content The total flavonoid content was measured by the aluminum chloride spectrophotometric method described by Zhishen et al.[38] and Yang et al.[39] Catechin was chosen as a standard and the results were expressed as milligram of catechin equivalents (CE) per gram of plant material (mg CE/g plant material). The levels of total flavonoid contents in samples were determined in triplicate (Table 1). Rutin Determination in Hypericum Extracts using UHPLC-DAD-HESI-MS/MS UHPLC-DAD-HESI-MS/MS analysis was performed by using a Thermo Scientific liquid chromatography system (UHPLC) composed of a quaternary pump with a degasser, a thermostated column compartment, an autosampler, and a diode array detector connected to LCQ Fleet Ion Trap Mass Spectrometer (Thermo Fisher Scientific, San Jose, California, USA) equipped with heated electrospray ionisation (HESI). Xcalibur (version 2.2 SP1.48) and LCQ Fleet (version 2.7.0.1103 SP1) software were used for instrument control, data acquisition and data analysis. Separations were performed on a Hypersil gold C18 column (50  2.1 mm, 1.9 µm) obtained from Thermo Fisher Scientific. UHPLC-DAD-HESI-MS/MS analysis was performed according to Kečkeš et al.[40] with slight modifications. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile. A linear gradient program at flow rate of 0.350 ml/min was used 0–2 min from 10 to 20% (B), 2–4.5 min from 20 to 90% (B), 4.5–4.8 min 90% (B), 4.8–4.9 min from 90 to 10% and 4.9–12.0 min 10% (B). The injection volume was 10 µl, and the column temperature was maintained at 25ºC. The separated compounds were detected at a wavelength of 230, 260, 320 and 360 nm, and each online spectrum was recorded within the range of 200–800 nm. The mass spectrometer was operated in negative mode. HESI-source parameters were as follows: source voltage 4.5 kV, capillary voltage –50 V, tube lens voltage –125.40 V, capillary temperature 300ºC, sheath and auxiliary gas flow (N2) 32 and 12 (arbitrary units), respectively. Identification of rutin was done according to the corresponding spectral characteristics: mass spectra, accurate mass, and characteristic fragmentation. After MS/MS fragmentation of [M–H]– ion at 609.13 m/z, that originates from rutin, two fragment ions were obtained; first at 151.00 m/z, representing deprotonated glycoside molecule, and second at 301.03 m/z, representing deprotonated [M–H]– aglycone. Content of rutin in water extracts of Hypericum species was additionally confirmed by standard of rutin. Determination of the exact rutin concentration in aqueous extracts was done using the standard calibration curve. Evaluation of DNase I Inhibition Inhibition of Commercial DNase I DNase I from bovine pancreas was employed for in vitro evaluation of enzyme inhibition, by spectrophotometric measurement of acid-soluble nucleotides formation at 260 nm, according to the previously published procedure.[7-10] All samples containing Hypericum water extracts were assayed for DNase I inhibitory activity at concentration of 800 μg/ml, and samples containing studied compounds were assayed for DNase I inhibitory activity at concentration of 200 μM. Those showing inhibition greater than 50% at these concentrations were tested in a broader concentration range to allow calculation of IC50 values. IC50 curves were generated using four concentrations of the studied extracts (800, 600, 400 and 200 μg/ml) or four concentrations of the studied compounds (200, 150, 100 and 50 μM). Inhibition of Rat Liver DNase I Adult female Wistar rat, weighing about 200 g and aged eight weeks, was obtained from the breeding colony of the Biomedical Research Centre, Faculty of Medicine, University of Nis. The animal was sacrificed after ketamine anesthesia injected intramuscularly at a dose of 90 mg/kg body weight. The liver was removed and cut in small pieces and homogenized in ice cold water, using laboratory homogenizer. The prepared 10% w/v homogenate was used for evaluation of enzyme inhibition by a procedure described by Ilić et al.[7] using rat liver homogenate instead of commercial bovine pancreatic DNase I. All experimental procedures were performed according to the Ethical Committee guidelines and rules for the protection of the welfare of experimental animals adopted by the Faculty of Medicine, University of Nis, according to the Animal Welfare Rules Republic of Serbia. The approval for the proposed animal experiment was obtained from the Animal Welfare Committee N° 21-4545-2/9 (Faculty of Medicine, University of Nis). Lineweaver-Burk Plots To determine the mode of DNase I inhibition by rutin, Lineweaver-Burk plot analysis was performed. This kinetic study was carried out in the absence and presence of the inhibitor with varying concentrations of DNA as the substrate. DNA concentration was varied (30, 50, 70, 100, 150, 250 and 400 µg/ml) at four series of fixed rutin concentrations (0, 50, 100 and 150 µM). Molecular Docking Studies Ligand Preparation The structures of molecules were built with ChemDraw Professional 16.0 (PerkinElmer Informatics, Inc.) and their geometries optimized with Chem3D 16.0 (PerkinElmer Informatics, Inc.) using MM2 force field until a minimum 0.100 Root Mean Square (RMS) gradient was reached. The optimized structure was refined with GAMESS interface using the semi-empirical AM1 method, QA optimization algorithm and Gasteiger Hückel charges for all atoms for 100 steps. FRED requires a set of input conformers for each ligand, which were generated with OMEGA (OMEGA version 2.5.1.4. OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com), with maximum number of conformations set to 100.[41,42] All the other options were left as default values. Receptor Preparation and Docking Protocol As no crystal structure with co-crystalized inhibitor is available, and since enzyme inhibition was assayed on bovine DNase, the crystal structures of bovine DNase I with N-acetylglucosamine and a DNA (PDB code: 2DNJ) was taken as the starting point.[43] All cofactors included were deleted to allow “blind” docking of the inhibitor. At beginning, “blind” docking was done inside a large grid box that surrounded all the enzyme with the volume of 146570 Å3, dimensions: 52,00 Å x 56,00 Å x 50,33 Å, and outer contour of 4804 Å2 using Make Receptor 3.0.1. 4.[44] After initial “blind” docking, a region in proximity of DNA-binding region (substrate-binding region) was used for further docking studies as majority of e-hits in blind docking were concentrated One grid box was created with volume of 31020 Å3, dimensions: 46,30 Å x 25,00 Å x 36,33 Å, and outer contour of 4804 Å2 using Make Receptor 3.0.1. The docking software FRED (OEDocking version 3.0.1. OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com) was used for docking studies with the default settings, and number of poses, which was set to 50.[44] The proposed five binding poses with the highest rank of the docked inhibitors were evaluated using final score and relative position to the native ligand. The graphical representations of the calculated binding poses were obtained using Discovery Studio Visualizer (version 16.1.0, Dassault Systèmes Biovia Corp). Acknowledgements The financial support of this work by Ministry of Education, Science and Technological Development of the Republic of Serbia (Grants No. OI 172044, TR 34012 and TR 31060), Slovenian Research Agency (Grant No. P1-0208) and Faculty of Medicine of the University of Nis (Internal project No. 4) is gratefully acknowledged. Author Contribution Statement A.S. created the experiment and article design. A.K. and J.L. performed plant material extraction. A.K., J.L. and G.K. evaluated the inhibition of commercial and rat liver DNase I. A.K. performed Lineweaver-Burk plot analysis. A.P., A.D. and S.S. determined total flavonoid and rutin content in Hypericum extracts. M.A. performed molecular docking studies. All authors participated in article writing and figures and tables preparation. References [1] Y. Higami, I. Shimokawa, ‘Apoptosis in the aging process’, Cell Tissue Res. 2000, 301, 125–132. [2] P. Anversa, T. Palackal, E. H. Sonnenblick, G. Olivetti, L. G. Meggs, J. M. Capasso, ‘Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart’, Circ. Res. 1990, 67, 871–885. [3] G. Olivetti, M. Melissari, J. M. Capasso, P. Anversa, ‘Cardiomyopathy of the aging human heart. Myocyte loss and reactive cellular hypertrophy’, Circ. Res. 1991, 68, 1560–1568. [4] L. Liu, G. Azhar, W. Gao, X. Zhang, J. Y. Wei, ‘Bcl-2 and Bax expression in adult rat hearts after coronary occlusion: age-associated differences’, Am. J. Physiol. Regul. Integr. Comp. Physiol. 1998, 275, R315–R322. [5] J. A. Nitahara, W. Cheng, Y. Liu, B. Li, A. Leri, P. Li, D. Mogul, S. R. Gambert, J. Kajstura, P. Anversa, ‘Intracellular calcium, DNase activity and myocyte apoptosis in aging Fischer 344 rats’, J. Mol. Cell. Cardiol. 1998, 30, 519–535. [6] A. Kolarevic, D. Yancheva, G. Kocic, A. Smelcerovic, ‘Deoxyribonuclease inhibitors’, Eur. J. Med. Chem. 2014, 88, 101–111. [7] B. S. Ilić, A. Kolarević, G. Kocić, A. Šmelcerović, ‘Ascorbic acid as DNase I inhibitor in prevention of male infertility’, Biochem. Biophys. Res. Commun. 2018, 498, 1073– 1077. [8] A. Kolarević, B. S. Ilić, N. Anastassova, A. T. Mavrova, D. Yancheva, G. Kocić, A. Šmelcerović, ‘Benzimidazoles as novel deoxyribonuclease I inhibitors’, J. Cell. Biochem. 2018, 119, 8937–8948. [9] A. T. Mavrova, S. Dimov, D. Yancheva, A. Kolarević, B. S. Ilić, G. Kocić, A. Šmelcerović, ‘Synthesis and DNase I inhibitory properties of some 5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines’, Bioorg. Chem. 2018, 80, 693–705. [10] A. Kolarević, B. S. Ilić, G. Kocić, Z. Džambaski, A. Šmelcerović, B. P. Bondžić, ‘Synthesis and DNase I inhibitory properties of some 4‐thiazolidinone derivatives’, J. Cell. Biochem. 2019, 120, 264–274. [11] K. Yazaki, T. Okuda, in ‘Medicinal and aromatic plants VI. Biotechnology in agriculture and forestry’, Ed. Y. P. S. Bajaj, 1994, Vol. 26, p. 167–178. [12] B. Šarić-Kundalić, C. Dobeš, V. Klatte-Asselmeyer, J. Saukel, ‘Ethnobotanical survey of traditionally used plants in human therapy of east, north and north-east Bosnia and Herzegovina’, J. Ethnopharmacol. 2011, 133, 1051–1076. [13] G. Stojanović, A. Đorđević, A. Šmelcerović, ‘Do other Hypericum species have medical potential as St. John's wort (Hypericum perforatum)?’, Curr. Med. Chem. 2013, 20, 2273–2295. [14] A. Singh, D. Jain, C. Tyagi, S. Singh, S. Kumar, I. K. Singh, ‘In silico prediction of active site and in vitro DNase and RNase activities of Helicoverpa-inducible pathogenesis related-4 protein from Cicer arietinum’, Int. J. Biol. Macromol. 2018, 113, 869–880. [15] C. Inocencio, D. Rivera, F. Alcaraz, F. A. Tomás-Barberán, ‘Flavonoid content of commercial capers (Capparis spinosa, C. sicula and C. orientalis) produced in mediterranean countries’, Eur. Food Res. Technol. 2000, 212, 70–74. [16] A. Romani, N. Mulinacci, P. Pinelli, F. F. Vincieri, A. Cimato, ‘Polyphenolic content in five Tuscany cultivars of Olea europaea L’, J. Agric. Food Chem. 1999, 47, 964–967. [17] B. D. Oomah, G. Mazza, ‘Flavonoids and antioxidative activities in buckwheat’, J. Agric. Food Chem. 1996, 44, 1746–1750. [18] D. P. Makris, J. T. Rossiter, ‘Domestic processing of onion bulbs (Allium cepa) and asparagus spears (Asparagus officinalis): effect on flavonol content and antioxidant status’, J. Agric. Food Chem. 2001, 49, 3216–3222. [19] K. R. Price, M. J. C. Rhodes, K. A. Barnes, ‘Flavonol glycoside content and composition of tea infusions made from commercially available teas and tea products’, J. Agric. Food Chem. 1998, 46, 2517–2522. [20] L. Wada, B. Ou, ‘Antioxidant activity and phenolic content of Oregon caneberries’, J. Agric. Food Chem. 2002, 50, 3495–3500. [21] K. R. Määttä, A. Kamal-Eldin, A. R. Törrönen, ‘High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection: Ribes species’, J. Agric. Food Chem. 2003, 51, 6736–6744. [22] J. L. Donovan, A. S. Meyer, A. L. Waterhouse, ‘Phenolic composition and antioxidant activity of prunes and prune juice (Prunus domestica)’, J. Agric. Food Chem. 1998, 46, 1247–1252. [23] H. Javed, M. M. Khan, A. Ahmad, K. Vaibhav, M. E. Ahmad, A. Khan, M. Ashafaq, F. Islam, M. S. Siddiqui, M. M. Safhi, F. Islam, ‘Rutin prevents cognitive impairments by ameliorating oxidative stress and neuroinflammation in rat model of sporadic dementia of Alzheimer type’, Neuroscience 2012, 210, 340–352. [24] A. Annapurna, C. S. Reddy, R. B. Akondi, S. R. C. Rao, ‘Cardioprotective actions of two bioflavonoids, quercetin and rutin, in experimental myocardial infarction in both normal and streptozotocin-induced type I diabetic rats’, J. Pharm. Pharmacol. 2009, 61, 1365–1374. [25] R. P. Webster, M. D. Gawde, R. K. Bhattacharya, ‘Protective effect of rutin, a flavonol glycoside, on the carcinogen-induced DNA damage and repair enzymes in rats’, Cancer Lett. 1996, 109, 185–191. [26] C. H. Jung, J. Y. Lee, C. H. Cho, C. J. Kim, ‘Anti-asthmatic action of quercetin and rutin in conscious guinea-pigs challenged with aerosolized ovalbumin’, Arch. Pharm. Res. 2007, 30, 1599–1607. [27] E. A. Ostrakhovitch, I. B. Afanas’ev, ‘Oxidative stress in rheumatoid arthritis leukocytes: suppression by rutin and other antioxidants and chelators’, Biochem. Pharmacol. 2001, 62, 743–746. [28] Q. H. Hu, C. Wang, J. M. Li, D. M. Zhang, L. D. Kong, ‘Allopurinol, rutin, and quercetin attenuate hyperuricemia and renal dysfunction in rats induced by fructose intake: renal organic ion transporter involvement’, Am. J. Physiol. Renal Physiol. 2009, 297, F1080–F1091. [29] R. A. Khan, M. R. Khan, S. Sahreen, ‘CCl4-induced hepatotoxicity: protective effect of rutin on p53, CYP2E1 and the antioxidative status in rat’, BMC Complement. Altern. Med. 2012, 12, 178. [30] J. Yang, J. Guo, J. Yuan, ‘In vitro antioxidant properties of rutin’, LWT - Food Sci. Technol. 2008, 41, 1060–1066. [31] C. F. L. Gonçalves, M. C. D. S. dos Santos, M. G. Ginabreda, R. S. Fortunato, D. P. de Carvalho, A. C. F. Ferreira, ‘Flavonoid rutin increases thyroid iodide uptake in rats’, PloS One 2013, 8, e73908. [32] Y. Yamada, T. Fujii, R. Ishijima, H. Tachibana, N. Yokoue, R. Takasawa, S. I. Tanuma, ‘DR396, an apoptotic DNase γ inhibitor, attenuates high mobility group box 1 release from apoptotic cells’, Bioorg. Med. Chem. 2011, 19, 168–171. [33] H. Yoo, S. K. Ku, Y. D. Baek, J. S. Bae, ‘Anti-inflammatory effects of rutin on HMGB1-induced inflammatory responses in vitro and in vivo’, Inflamm. Res. 2014, 63, 197– 206. [34] S. Bisht, M. Faiq, M. Tolahunase, R. Dada, ‘Oxidative stress and male infertility’, Nat. Rev. Urol. 2017, 14, 470–485. [35] E. Moretti, L. Mazzi, G. Terzuoli, C. Bonechi, F. Iacoponi, S. Martini, C. Rossi, G. Collodel, ‘Effect of quercetin, rutin, naringenin and epicatechin on lipid peroxidation induced in human sperm’, Reprod. Toxicol. 2012, 34, 651–657.
[36] N. Radulović, V. Stankov-Jovanović, G. Stojanović, A. Šmelcerović, M. Spiteller, Y. Asakawa, ‘Screening of in vitro antimicrobial and antioxidant activity of nine
Hypericum species from the Balkans’, Food Chem. 2007, 103, 15–21.
[37] D. Zheleva-Dimitrova, P. Nedialkov, G. Kitanov, ‘Radical scavenging and antioxidant activities of methanolic extracts from Hypericum species growing in Bulgaria’,
Pharmacogn. Mag. 2010, 6, 74–78.
[38] J. Zhishen, T. Mengcheng, W. Jianming, ‘The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals’, Food Chem. 1999, 64, 555–559.
[39] J. Yang, K. J. Meyers, J. van der Heide, R. H. Liu, ‘Varietal differences in phenol content and antioxidant and antiproliferative activities of onions’, J. Agric. Food Chem.
2004, 52, 6787–6793.
[40] S. Kečkeš, U. Gašić, T. Ćirković Veličković, D. Milojković-Opsenica, M. Natić, Ž. Tešić, ‘The determination of phenolic profiles of Serbian unifloral honeys using ultra- high-performance liquid chromatography/high resolution accurate mass spectrometry’, Food Chem. 2013, 138, 32–40.
[41] P. C. D. Hawkins, A. G. Skillman, G. L. Warren, B. A. Ellingson, M. T. Stahl, ‘Conformer generation with OMEGA: algorithm and validation using high quality structures from the Protein Databank and the Cambridge Structural Database’, J. Chem. Inf. Model. 2010, 50, 572–584.
[42] P. C. D. Hawkins, A. Nicholis, ‘Conformer generation with OMEGA: learning from the data set and the analysis of failures’, J. Chem. Inf. Model. 2012, 52, 2919–2936.
[43] A. Lahm, D. Suck, ‘DNase I-induced DNA conformation. 2 Å structure of a DNase I-octamer complex’, J. Mol. Biol. 1991, 222, 645–667.
[44] M. McGann, ‘FRED and HYBRID docking performance on standardized datasets’, J. Comput. Aided Mol. Des. 2012, 26, 897–906.