Quercetin - based rhodium(III) complex: Synthesis, characterization and diverse biological potentials

{"full_text": " Journal of Molecular Structure 1257 (2022) 132584\n\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Molecular Structure\n journal homepage: www.elsevier.com/locate/molstr\n\n\n\n\nQuercetin - based rhodium(III) complex: Synthesis, characterization\nand diverse biological potentials\nHeba A. Sahyon a, Fayez Althobaiti b, Abd El-Motaleb M. Ramadan a,\u2217, Ahmed M. Fathy c\na\n Chemistry Department, Faculty of Science, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt\nb\n Department of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia\nc\n Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt\n\n\n\n\na r t i c l e i n f o a b s t r a c t\n\nArticle history: Quercetin \u2013 based rhodium(III) complex, [RhIII L2 ClH2 O], L is quercetin, was synthesized via the direct\nReceived 13 November 2021 interaction between the polyphenolic natural product (3,3\u0003 ,4\u0003 ,5,7-pentahydroxyl\ufb02avone) and RhCl3 3H2 O.\nRevised 29 January 2022\n Characterization of the pure isolated metal complex by analytical, electrolytic conductance, TGA and spec-\nAccepted 4 February 2022\nAvailable online 5 February 2022\n troscopic techniques demonstrated the stoichiometric ratio 1:2 metal to ligand. The \ufb01nal structure of\n [RhIII L2 ClH2 O] was con\ufb01rmed using PXRD\u2013structural analysis by processing the XRD data of microcrys-\nKeywords: talline powder by the relevant computer program Expo 2014. The mimetic catalytic activity of superoxide\nQuercetin dismutase (SOD) was studied and the obtained results showed that [RhIII L2 ClH2 O] has mid activity com-\nRhodium(III) pared to other SOD mimics. By analogy with the work of the original-SOD and its functional models, a\nBiomimicking mechanism for SOD-mimicking catalytic activity of the newly synthesized rhodium(III) complex has been\nSOD\n proposed. The binding of the Rh(III) chelate to DNA was tested, and spectroscopic investigations indi-\nDNA binding\n cated the successful binding of the metal complex to DNA by the groove/electrostatic binding pattern\nCaspase9\nMMP9 with intrinsic binding constants Kb of 2.76 \u00d7 106 M\u22121 . In addition, the quercetin \u2013 based RhIII exhibits\n relatively higher DPPH scavenger power than the free quercetin. The in vitro data indicated the e\ufb03cient\n anti-proliferative activities of the current rhodium(III) complex compared with cis-platin with increased\n safety on normal cells. In addition, our data revealed that the studied RhIII complex could prevent cancer\n cell replication with increasing the p53 levels, inhibit both Bcl2 and MMP9, then activating caspase 9,\n which then cleaves caspase 3, leading to apoptosis triggering. Apoptosis activation led to cell cycle arrest\n at the pre-G1 phase and decreased Hela cell proliferation, which appeared in the decreased G2/M phase.\n Ongoing biological assays indicate that the current Rh(III) complex is a promising anti-proliferative agent\n with minimal toxicity to normal cells.\n \u00a9 2022 Elsevier B.V. All rights reserved.\n\n\n\n\n1. Introduction plexes with multiple ligands [15\u201317]. These rhodium(III) complexes\n were proven to have other antitumor pathways, such as binding\n Cisplatin was discovered in 1965 and is clinically considered to DNA mismatches [10,18\u201320] rather than disturbing the double\nthe most successful anticancer agent against several cancer types helix structure as cisplatin. In this context, the cancer cell\u2019s mito-\n[1\u20134]. Up till now, cisplatin was clinically used in limited cases chondria is aiming to produce apoptotic death by increasing their\nwith plenty of side effects, such as nephrotoxicity, cardiotoxic- free radical levels [21]. Recently, a RhIII - based complex was suc-\nity, and the appearance of drug resistance cancers [5\u20137]. As a cessfully prepared and established to induce malignant cell apop-\nresult of these toxicities, scientists have to innovate other non- tosis through induction of caspases with decreasing the Bcl2 ex-\nplatinum anticancer drugs with reduced side effects, such as pression through increased free radicals [13].\nruthenium [8,9] and rhodium [10\u201313]. Although hexa-coordinated Quercetin is a popular, edible Flavonoid found in vegetables and\nlow-spin rhodium(III) complexes with the electronic con\ufb01guration fruits with marked antioxidant and anticancer activities [22\u201329].\n(t2g 6 eg 0 ) is kinetically inert, some RhIII - based complexes have The structure of quercetin, with its phenolic OH groups, shows\nbeen shown to have signi\ufb01cant anticancer activities [12\u201314]. How- a remarkable ability to react with transition metal ions to form\never, rhodium(III) ion forms biologically active and stable com- stable metal chelates [30,31]. Recently, the ability of quercetin to\n form stable complexes with ruthenium, calcium, magnesium, zinc,\n \u2217\n iron, copper, and aluminum has been con\ufb01rmed [32\u201335]. Most\n Corresponding author.\n E-mail address: Ramadanss@hotmail.com (A.E.M. Ramadan).\n\n\n\nhttps://doi.org/10.1016/j.molstruc.2022.132584\n0022-2860/\u00a9 2022 Elsevier B.V. All rights reserved.\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\nquercetin-based metal complexes showed higher antioxidant and the treated/untreated Hela cells, and the DNA binding assay are\nanticancer activities than quercetin itself [32\u201335]. included in S1.\n The superoxide anion radical (O2 \u2022\u2013 ), a by-product from energy\nproduction processes by O2 , is the origin of the formation of the 2.3. Synthesis of quercetin \u2013 based rhodium(III) complex\nreactive oxygen species (ROS), which are extremely harmful to all\naerobic organisms. Native SOD-proteins including CuZn-SOD, Mn- Quercetin powder 0.002 mole was dissolved in a 50 mL al-\nSOD, Fe-SOD and Ni-SOD rid the cell of O2 \u2022\u2013 accumulation. The coholic solution containing 0.002 mole of NaOH. To this solution\ntherapeutic use of natural SOD proteins faced many complications, 0.001 mole of RhCl3 3H2 O dissolved in EtOH was added drop by\nsuch as cell wall permeability, immune problems, and reduced drop with stirring. The resulting mixture then was stirred at 25\nshelf life, which precluded their use as drugs. These concerns, com- \u00b0C for 30 min., during which the metal complex was formed in\nbined with the lack of clinical success with natural SOD-enzymes, a dissolved form. By slow evaporation of the reaction mixture a\nhave motivated efforts to develop synthetic alternatives such as dark red microcrystalline precipitate formed. The colored precipi-\ntransition metal complexes as functional models for SOD-proteins. tated residue was separated by \ufb01ltration and washed with small\nSeveral SOD-mimics have been synthesized with different transi- portions of a mixture of EtOH and diethyl ether (1:1) numerous\ntion metals such as copper, manganese, iron and nickel and most times and then was left to dry under anhydrous conditions for 1\nof them have demonstrated high e\ufb03ciency [36,37]. With regard to week.\nrhodium(III) complexes, there are no studies in the literature yet\nregarding their use as SOD-mimetics. 2.4. Biological examinations\n To our knowledge, the metal complex of rhodium(III) with\nquercetin has not yet been reported. The present work aims to 2.4.1. DPPH test\nsynthesize a quercetin \u2013 based Rh(III) complex to examine its in Serial dilutions (200\u201310 \u03bcg/mL) of both quercetin and quercetin\nvitro antioxidant and anticancer activities and compare it with \u2013 based RhIII complex were mixed with DPPH reagent, then in-\nfree quercetin. Superoxide dismutase-like activity, DNA binding, cubated and measured as mentioned in our pervious study [38].\ncell death mode and proposed anticancer mechanism of quercetin\u2013 Reduced absorbance values showed greater free radical scaveng-\nbased RhIII complex will be investigated. ing activity. All analyses were achieved in triplicate. To calculate\n the sample concentration which prevents the 50% from the color\n formed by the DPPH radical, a curve was plotted between the con-\n2. Experimental\n centration and color intensity and the IC50 value was calculated\n from it [39].\n2.1. Chemicals\n 2.4.2. In vitro anticancer activity of quercetin\u2013based rhodium(III)\n Dimethyl sulfoxide (DMSO), MTT [3-(4,5-dimethylthiazol-\n complex\n2-yl)\u22122,5-diphenyltetrazolium bromide], Nitroblue tetrazolium\n To identify the anticancer activity of the newly synthesized\n(NBT), NADH, phenazine methosulphate (PMS), 2,2-Diphenyl-1-\n quercetin-based rhodium(III) complex against human cancer cell\npicrylhydrazyl (DPPH), rhodium(III) chloride trihydrate and sodium\n proliferation, the MTT assay was done [40], as described in the\npyrophosphate decahydrate were purchased from Sigma-Aldrich,\n supplementary data S1.\nSt. Louis, MO, USA, Fetal Bovine serum and penicillin/streptomycin\nwere obtained from GIBCO, UK. Trypsin 0.25% (AMRESCO - USA).\n 2.4.3. Flowcytometric determination\nThe human cell lines; hepatocellular carcinoma (HepG-2), ep-\n 2.4.3.1. Annexin and Pi assay for apoptosis determination. The Hela\nitheliod carcinoma (Hela), mammary carcinoma (MCF-7), human\n cells treated with IC50 of quercetin- based rhodium(III) complex\nprostate cancer (PC-3), colorectal adenocarcinoma (Caco-2) and\n were suspended in cold PBS, centrifuged in a cooling centrifuge\nhuman lung \ufb01broblast (WI38) were obtained from American\n at 20 0 0 rpm for 30 min. The separated pellet was then trans-\nType Culture Collection (ATCC, NY, USA) via Holding company\n mitted into a sterilized plain tube. Finally, the annexin V kit (Cat.\nfor biological products and vaccines (VACSERA), Cairo, Egypt.\n No.556547 BD pharmingen FITC apoptosis Kit) was used to stain\nQuercetin dihydrate [(C15 H10 O7 2H2 O) 97% Lot.no. 10,181,203, Alfa\n the pellet, as reported in our previous study [38].\nCaesar Company, Germany], Dulbeco\u2019s Modi\ufb01ed Eagle\u2019s Medium\n(DMEM) (GIBCO, New York, USA; Cat.no.11995073), fetal bovine\n 2.4.3.2. Cell cycle examination. The Hela cells treated with\nserum (GIBCO, Grand Island, New York, USA;Cat.no.10099133),\n 9.40 \u03bcg/ml of the quercetin\u2013based-rhodium(III) complex was\nL-glutamine (Invitrogen, Grand Island, New York, USA; Cat.no.\n incubated in the CO2 incubator for 48 h. Then further steps was\n25,030,024), ethidium bromide and acridine orange (EB/AO)\n done to the treated/untreated cells as mentioned in our previous\n(Sigma-Aldrich, Deisenhofen, Germany). Annexin V kit (cat. No.\n work [9]. The cells were then examined using a BD FACSCal-\n556,547 BD Pharmingen FITC apoptosis kit) was purchased from\n ibur \ufb02ow cytometer (Becton Dickinson, Sunnyvale, CA, USA). The\nBecton, Dickinson and Company. Anti-rabbit antibodies (polyclonal\n treated and untreated Hela cells tubes were covered with dark\nIg) for anti-caspase-9 antibody ([2\u201322] ab69541), anti-MMP9 anti-\n foil and preserved for 12 h in dark place to be analysed by the\nbody (ab73734), Bcl2 anti-mouse (ab196495), \u03b2 -actin (monoclonal\n \ufb02ow-cytometer to examine the pre-G1 percentage.\nIg) (ab6276) were obtained from BD Abcam. The chemilumines-\ncent western blot ECL substrate reagent was obtained from Perkin\n 2.5. Analysis of p53 genes by real-time PCR\nElmer, Waltham, MA, USA. Qiagen RNA extraction kits, BioRad\nsyber green PCR MMX. All chemicals used in the current study are\n RNA was extracted from the Hela cells treated with the\nof the analytical grade and used without further puri\ufb01cation.\n quercetin\u2013based-rhodium(III) complex as well as untreated\n Hela cells using the RNeasy Mini Kit (Qiagen, Germantown,\n2.2. Characterization techniques MD, USA) according to the company\u2019s procedure. The ex-\n pression level of P53 gene (F:5\u0003 -AGAGTCTATAGGCCCACCCC-\n Characterization techniques were performed as described in the 3\u0003 & R:5\u0003 -GCTCGACGCTAGGATCTGAC-3\u0003 ), and housekeeping\nsupplementary materials S1. As well, the experimental details of gene; \u03b2 -actin (F: 5\u0003 -AGAGCTACGAGCTGCCTGAC-3\u0003 & R: 5\u0003 -\nsuperoxide dismutase biomimetic catalytic activity, SOD levels in AGCACTGTGTTGGCGTACAG-3\u0003 ) were assessed by real-time PCR.\n\n 2\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\nThen further steps were done as mentioned in our previous work\n[9]. qPCR was done by QuantiTect SYBR Green qPCR Master Mix\nin a Step One Plus real-time PCR system as follows: the adjusted\ntime for enzyme activation was 10 min at 95 \u00b0C followed by 40\ncycles of 15 s at 95 \u00b0C, 20 s at 55 \u00b0C, and 30 s at 72 \u00b0C for the\nampli\ufb01cation step. The \u03b2 -actin was used as a housekeeping gene\nto normalize the deviations in the p53 gene expression, and the\nresults were related to its mean critical threshold (CT) values by\nthe \u0003CT method.\n\n2.6. Immunohistochemical determination of Bcl2 protein\n\n Hela cells treated with the quercetin\u2013based-rhodium(III) com-\nplex, for 48 h, were harvested and centrifuged in cooling cen-\ntrifuges at 1700 rpm for 10 min. The supernatant was aspirated\n Fig. 1. Quercetin (3,3\u0003 ,4\u0003 ,5,7-pentahydroxyl\ufb02avone) structure.\ncarefully and the cell pellets were washed 3 times with sterile\nPBS. A volume of 50 \u03bcl of treated/untreated cells was aspirated\ncarefully, spread on clean, positive-charged glass slides, and re- There are \ufb01ve OH groups in the positions 3,3\u0003 ,4\u0003 ,5,7 in addition\nserved overnight in a cold place. After incubation, all slides were to a C=O group adjacent to each of the two hydroxyl groups 3\n\ufb01xed in methanol for 30 min. After \ufb01xation, the slides were im- and 5. These structural features allowed quercetin to form metal\nmersed in primary anti-Bcl2 antibody for 60 min at 24 \u00b0C. Then chelates with many transition metal ions such as ruthenium, cop-\nthe slides were dipped in PBS three times, and a suitable amount per, iron, cobalt, nickel, zinc, Cd and others [30,41\u201345]. However,\nof the anti-mouse IgG secondary antibody (EnVision + System several stoichiometric ratios have been reported, ranging from 1:1,\nHRP; Dako) was added and kept for half an hour at 24 \u00b0C. The 1:2, 2:1 to 3:1 for the ratio of quercetin to metal ion in the case of\ndi-aminobenzidine (Liquid DAB + Substrate Chromogen System; CdII , CuII , FeII and rare earth metals respectively [43\u201346].\nDako) commercial kit was used in visualization of the stained pro- For the current quercetin - based Rh(III) complex the analytical\ntein. Then, the Mayer\u2019s haematoxylin was used as counterstain. results as well as that of TGA showed that the molar ratio is 2:1\nLater, the slides were examined with a light microscope. The BCl2 (quercetin: RhIII ). The molecular formula of the pure isolated RhIII -\nprotein was quanti\ufb01ed by calculation of positive cells in no less based quercetin complex is [RhL2 Cl H2 O]. This complex shows ap-\nthan 10 high power \ufb01elds from 10 0 0 cells. preciable solubility in water, DMF and DMSO and partial solubility\n in alcohols such as methanol and ethanol. Electrical conductivity\n2.7. Protein expression of MMP9 and caspase 9 by western blot measurements in DMF solution (Table 1) demonstrated the non-\nanalysis electrolytic behavior of the newly synthesized quercetin - based\n Rh(III) complex [47]. This \ufb01nding con\ufb01rms the suggested molecular\n To detect the existence of MMP9 and caspase 9 proteins, the formula (Table 1) and indicates the participation of the chloride ion\ntreated/untreated Hela cell lysed samples were running in SDS gel as anionic ligand, in the coordination chromophore around Rh(III)\nelectrophoresis. After electrophoresis, each protein will be relo- ion.\ncated on polyvinylidene \ufb02uoride (PVDF) membranes (BioRad) then\nblocked with 5% nonfat dry milk. To image the MMP9 and caspase 3.2. Thermal analysis\n9 protein, speci\ufb01c primary antibody and anti-\u03b2 -actin antibody will\nattached to each protein making a protein-antibody complex. After Thermal analysis is one of the techniques that support the\nthat, a secondary antibody (EnVision + System HRP; Dako), con- results of elemental analysis and help determine the nature of\njugated with a speci\ufb01c enzyme, was added to be attached to that the hydration content accompanying the metal complex molecule.\ncomplex. Then, the enzyme substrate chemiluminescent Western Therefore, thermogravimetric analysis of the rhodium complex un-\nECL substrate was added to generates luminescence captured by der study was conducted to identify the nature of the water con-\nCCD camera-based imager (Chemi Doc imager, Biorad, USA), and tent and thermal behavior of RhIII - based quercetin complex. The\nthe bands intensities were then measured by Image Lab (Bio-Rad). thermogram of the current metal complex shows three phases of\nBy measuring the amount of luminescence, the quantity of the pro- the thermal decompositions (Supplementary materials S2).\ntein that reacted with the antibody can be calculated. The \ufb01rst phase occurs between 190 and 280 \u00b0C in several over-\n lapping steps and accompanied by a mass loss of 18.45% with\n3. Results and discussion DTGmax peaks at 200 \u00b0C. The observed mass loss is in an agree-\n ment with the theoretical value that corresponds to the volatiliza-\n3.1. General tion of the coordinated water molecule and chloride anion in ad-\n dition to the partial decomposition of the organic content. Since\n Quercetin (3,3\u0003 ,4\u0003 ,5,7-pentahydroxyl\ufb02avone) is the common the mass loss at this initial stage begins at a temperature of about\nname for the ligand used in the current study and is a natural 200 \u00b0C, the surface nature of the water content must be excluded\nproduct of plant origin. Its basic structure is shown in Fig. 1 in- and this \ufb01nding con\ufb01rms the coordination bonding of the water\ndicating the polynuclear and polyphenolic features. molecule.\n\n\n Table 1\n Molecular formula, physical properties and analytical data of quercetin - based Rh(III) complex.\n\n Found (calcd.)\n Complex Color \u0004M ( \u0005\u22121 cm2 mol\u22121 )\n %C %H %M\n\n [RhL2 Cl H2 O] Dark red 15.22 47.45 (47.59) 2.55 (2.64) 13.76 (13.60)\n\n\n\n 3\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n Table 2\n Thermal degradation of quercetin - based RhIII complex [RhL2 Cl H2 O].\n\n Complex Temperature \u00b0C DTGmax \u00b0C % Mass loss Found (calcd.) Species formed\n\n [RhL2 Cl H2 O] 190 \u2013 280 200 18.45(19.37) [RhL(2 \u2013 0.17%) ]\n 280 \u2013485 380, 450 25.25(26.19) [RhL(2 \u2013 0.35%) ]\n 485 \u2013 700 530 40.73(39.75) Rh2 O3\n\n\n Table 3\n Infrared spectra (cm\u22121 ) of quercetin and its RhIII complex.\n\n Compound \u03c5 (OH) \u03c5 (C = O) \u03c5 (C = C) \u03c5 (C-O-H) \u03c5 (C-O-C) \u03c5 (Rh-O) \u03c5 (Rh-Cl)\n Quercetin 3408 1664 1595 1410 1210 \u2013 \u2013\n [RhL2 Cl H2 O]. 3400 1620 1585 1390 1220 505 380\n\n\n\n\n The second stage of pyrolysis begins at 280, and ends at 485 \u00b0C,\nwith DTGmax peaks at 380 and 450 \u00b0C, indicating mass loss corre-\nsponding to the continuous volatilization of the organic content. At\nthis stage, both theoretical and practical values of mass loss agree\nin explaining the nature of the volatile part of the organic content,\nas is clear from the results in Table 2.\n This thermal decomposition event ends with the third and \ufb01nal\nstage, which sees the volatilization of the remaining portion of the\norganic ligand, leaving behind the metal oxide. The brown color\nof the residual indicates the trivalent state of rhodium in its oxide\nRh2 O3 and not the tetravalent state of rhodium in the black oxide,\nRhO2 [48]. This interpretation is based on the remarkable agree-\nment between the theoretical and experimental values (Table 2) of\nthe percentage of metal oxide remaining at the end of the pyrolysis\nprocess.\n It should be noted that the value of the rhodium content in\nthe remaining oxide (Rh2 O3 ) matches the value estimated by the\nelemental analysis of the suggested formula [RhL2 Cl H2 O].\n\n Fig. 2. The proposed structure of quercetin \u2013 based Rh(III) complex.\n3.3. Veri\ufb01cation of the coordination sites and bonding pattern\n\n To verify the coordination sites and bonding pattern of the cur-\nrent metal complex molecule, the infrared spectra of both the free\nquercetin and its rhodium(III) complex were measured and the re- support an OH at position 3 above position 5 in the formation of\nlated charts are given in S3. In this respect, it is necessary to iden- quercetin metal complexes [42,43,50]. This \ufb01nding is supported by\ntify the functional groups in the ligand molecule that are expected the observed decrease in the bond order of the two oxygen atoms\nto participate in the binding to the metal ion or to be affected groups, C=O and OH on site 3 due to coordination to the metal ion\nby the formation of the metal complex. In this context, the val- in the metal chelate which may give rise to a coupling of stretch-\nues of the wavenumbers characteristic of the vibrations of these ing of these two bonds. In the same context, the spectrum of the\nfunctional groups in both the free quercetin and its metal complex RhIII \u2013 quercetin based chelate shows two new bands at 1390 and\nwere determined as mentioned in Table 3. The data in Table 3, 1440 cm\u22121 which can be considered to be related to the symmet-\nrevealed that the characteristic stretching of the carbonyl group, ric and asymmetric stretching modes of (C\u2013O) group respectively\n\u03c5 (C = O), for the free quercetin appears at 1664 cm\u22121 was shifted at the chelation site [43].\nto lower wavenumber, 1620 cm\u22121 , in the spectrum of the metal The non-participation of ring oxygen in the formation of the\ncomplex. This \ufb01nding indicates involvement of the carbonyl oxy- metal complex is supported by the slight change in the frequency\ngen in coordination to RhIII ion [49]. of both \u03c5 (C\u2013O\u2013C) and \u03c5 (C=C) of ring II (Fig. 1). Alternatively,\n The question now is which of the hydroxyl groups, 3 or 5, ad- the new medium band appears at 505 cm\u22121 in the spectrum of\njacent to the carbonyl oxygen will participate in the bonding to quercetin - based RhIII complex is assigned to \u03c5 (Rh\u2013O) and con-\nthe metal ion. Since the bonding of the metal ion to the hydroxyl \ufb01rm complex formation [49]. Participation of the chloro anion\noxygen takes place with the deprotonated hydroxyl oxygen, so the (Cl\u00af) in the coordination chromophore of the newly synthesized\nacidity of the OH group will determine the hydroxyl group that rhodium(III) chelate is evidenced from the presence of the char-\nparticipates in the bonding to the metal ion. Referring to the struc- acteristic frequency of the bonding of the RhIII ion with the chlo-\nture of the quercetin molecule (Fig. 1), we \ufb01nd that the hydroxyl ride anion \u03c5 (Rh \u2013 Cl) at 380 cm\u22121 [49]. In the same respect, the\ngroup on site 3 is more acidic than that on site 5. This is due to presence of coordinated water was inferred from the broad band\nthe fact that, the hydroxyl group on site 3 is closer to the oxygen present at 3400 cm\u22121 in the spectrum of the metal complex that\nof the carbonyl group than the hydroxyl group on position 5. In did not appear in the free quercetin spectrum [49]. However, ther-\naddition, the ring to which the hydroxyl group is attached to the mogravimetric measurements indicated the coordination nature of\nposition 3 is heterogeneous due to the presence of the highly elec- the water content in the molecule of the present metal complex.\ntronegative oxygen atom. These structural features of the quercetin Based on the aforementioned results and discussions, the struc-\nmolecules in turn increase the degree of acidity of OH in posi- tural formula of the present quercetin \u2013 based RhIII complex has\ntion 3 over OH in position 5. However, previous relevant studies been proposed as given in Fig. 2.\n\n 4\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n Table 4\n Crystallographic data of quercetin \u2013 based RhIII complex.\n\n Empirical formula C32 H25 ClRhO15\n Formula weight 787.89\n T (K) 305\n \u03bb (A\u030a) 1.529040\n Crystal system Monoclinic\n Space group P2/m\n Centro symmetry Acentric\n Space Group Number 4\n Z 2\n Multiplicity 2\n Bravais Lattice P\n Lattice Symbol mP\n Unit cell dimensions:\n a (A\u030a), b (A\u030a), c (A\u030a) 5.881, 9.597, 3.063\n \u03b1 (\u00b0), \u03b2 (\u00b0), \u03b3 (\u00b0) 90.000, 95.122, 90.000\n Cell volume (A\u030a3 ) 172.161\n Volume per atom (A\u030a3 ) 1.757\n Calculated density (g/cm3 ) 5.57\n \u03b8 range for data collection (\u00b0) 30.000 \u2013 80.000\n Total re\ufb02ection 693\nFig. 3. Electronic absorption spectra of quercetin (1) and quercetin \u2013 based RhIII\n Rietveld results:\ncomplex (2) in DMF solution at room temperature. (For interpretation of the refer-\n Rp 18.726\nences to color in this \ufb01gure legend, the reader is referred to the web version of this\n Rwp 24.381\narticle.)\n R-Bragg 1.044\n R-F 1.240\n\n\n3.4. UV\u2013Vis spectral and magnetic investigations\n\n Rhodium(III) belongs to d6 \u2013 transition elements with 1 A1g Fig. 4 shows the PXRD spectrogram of quercetin \u2013 based RhIII\nground state. The hexa- coordinated Rh(III) center in an octahedral complex while Fig. 5 shows the degree of agreement between the\ncon\ufb01guration, is characterized by the low energy spin\u2013forbidden practical results and computer calculations.\ntransition 1 A1g \u2192 3 T1g (\u03c5 1 ) and the two spin \u2013 allowed transi- Table 4 summarizes the relevant crystallographic information\ntions 1 A1g \u21921 T1g , (\u03c5 2 ) and 1 A1g \u2192 1 T2g , (\u03c5 3 ) in an energy order of quercetin - based RhIII complex that crystallizes in crystal sys-\nof \u03c5 1 \u02c2 \u03c5 2 \u02c2 \u03c5 3 [51,52]. The room temperature spectrum in DMF tem monoclinic space group P2/m with cell parameters a = 5.881,\nsolution for the present quercetin - based RhIII complex exhibits b = 9.597 and c = 3.063 A\u030a. It is noticeable that a, b and c do not\nthree absorption bands (Fig. 3) at 740, 645, and 545 nm ascribed correspond exactly x, y and z because one of the three angles of\nto the electronic transitions, \u03c5 1 , \u03c5 2 and \u03c5 3 , in the octahedral stere- the monoclinic structure is unequal to 90\u00b0 (see Table 4).\nochemistry [52,53]. The energies of these electronic transitions are Fig. 6 illustrates the numbering drawing adopted for the opti-\nsimilar to other six-coordinated rhodium(III) complexes in an octa- mized coordination polyhedron in an octahedral con\ufb01guration and\nhedral geometry [52\u201356]. the hydrogen atoms have been omitted for clarity.\n To clarify the electronic properties of the studied compounds, The structural diagram in Fig. 6 indicates that the four oxy-\nthe spectra of free quercetin and its Rh(III) chelate were measured gen atoms O(2), O(7), O(8) and O(9) occupy the Basel plane of\nunder the same conditions and related spectra are given in Fig. 3. the octahedral geometry around RhIII - center. In the same respect,\nIn this regard, two strong absorption bands are observed at rela- the anionic ligand Cl(1) and the oxygen atom O(10) of the co-\ntively high-energy positions 255 and 350 nm in the case of un- ordinated water molecule are located on the two axial positions.\ncomplexed quercetin. These bands are associated with the \u03c0 \u2192 \u03c0 \u2217 The determination of the type of atoms occupying the vertical axis\naromatic transition of the cinnamoyl and benzoyl systems of the was based on the value of the bond between these two donor\nrings III and I respectively of the quercetin ligand (see Fig. 1). In sites Cl(1)-Rh(1)-O(10)= =171.27. This angle has the largest value\nthe spectrum of the quercetin \u2013 based Rh(III) complex, the elec- in comparison to the angles between the opposite sites in the Basel\ntronic transitions related to the quercetin component appear ap- plane O(2)-Rh(1)-O(8)= =170.74 and O(7)- Rh(1)-O(9)= =168.52\nproximately in the same energy regions as in the case of free (Table 5). Also, the bond distance between Rh(1) and the two axial\nquercetin. Cl(1) and O(10) donor sites is longer than that between Rh(1) and\n Magnetic susceptibility measurements at room temperature the four basal plane donor sites (Table 6). This noticeable elonga-\ndemonstrated the diamagnetic character of the magnetically di- tion of the axial bond distance indicates a tetragonal distortion in\nluted quercetin - based Rh(III) complex. This \ufb01nding indicates the the octahedron structure of the current quercetin \u2013 based Rh(III)\nlow-spin state (t2g 6 ) of the six coordinated Rh(III)-core with d2 sp3 complex.\nhybridization in an octahedral stereochemistry. The packing diagram of the unite cell which includes two\n molecules of metal complex is illustrated in Fig. 7.\n It is known that, metal complexes with coordination number\n3.5. PXRD-structural analysis six take the stereoscopic shape of an octahedron. The question\n arises about the perfection of this stereotype; is it an ideal oc-\n Nowadays, PXRD results processing technology with special- tahedron or a trigonal prism? The geometric index \u03c4 6 value was\nized computer software, such as Expo 2014, for structural analy- estimated from the relationship \u03c4 6 = =\u03b8 /60 and will decide the\nsis of metal complexes has become an alternative to X-ray struc- matter; with note that \u03b8 is the torsion angle between opposite\ntural analysis for a single crystal [36]. In this regard, the Rietveld- trigonometric faces in the octahedron. In the case that the \u03c4 6 is\nRe\ufb01nement methodology is used to reach a high degree of agree- equal to one, this indicates a perfect octahedron, while in the case\nment between the practical results and the computer processing of of the \u03c4 6 equal to zero the related structure is ideal trigonal prism\nthe X-ray spectroscopic data of the metal complex under study. [57,58]. The determined value of \u03c4 6 is 0.95 that con\ufb01rms the ideal-\n\n 5\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n\n\n Fig. 4. The microcrystalline XRD spectrum of quercetin \u2013 based RhIII complex.\n\n\n\n\nFig. 5. Good match between the experimental and calculated data for the microcrystalline XRD\u2013spectrum based on Rietveld Re\ufb01nement for quercetin \u2013 based RhIII complex.\n\n\n\n\n Fig. 6. Structural diagram of the optimum numbering of the octahedral con\ufb01guration for quercetin - based RhIII complex.\n\n\n\nity of the octahedral structure of the current quercetin\u2013based RhIII production source that reacts with NBT to form formazan as de-\ncomplex. scribed in reaction 1:\n\n3.6. Biomimicking SOD catalytic activity k\n NBT + O2 \u2022\u2212 \u2192 (F ) (1)\n The SOD like activity of present quercetin\u2013based RhIII complex\nwas evaluated spectrophotometrically by determining the percent- The rate of formazan formation is de\ufb01ned in relationship 2:\nage change in the optical density produced from the reduction of\nNBT to formazan (F) due to the presence of the metal complex. In\nthis assay, phenazine methosulphate (PMS) is a light-induced O2 \u2022\u2212 d[F]1 /dt=k[NBT][O2 \u2022\u2212 ] (2)\n\n 6\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n\n\n Fig. 7. Stereo view of the packing diagram of the unite cell which includes two molecules of metal complex.\n\n\n\n Table 5 In the same way, question\u2013based RhIII complex will act as\n The relevant bond angle (\u1db1) around Rh(III) \u2013 core of\n the native Mn+ -SOD and the inhibitory rate of formazan develop-\n [RhIII L2 Cl H2 O].\n ment after adding the current RhIII complex is assumed from the\n (\u03b8 = 57); Octahedral \u03c4 6 =0.95 Eq. (4):\n O2-Rh1-O7 86.39\n O2-Rh1-O8 170.74\n d[F]2 /dt=kc [RhIII complex][O2 \u2022\u2212 ] (4)\n O2-Rh1-O9 86.54\n O2-Rh1-Cl1 88.91\n O2-Rh1-O10 83.84 The catalytic rate constant kc (second order) is de\ufb01ned as\n O7-Rh1-O8 87.67 shown in the relationship 5:\n O7- Rh1-O9 168.52\n O7-Rh1-Cl1 92.04\n O7- Rh1-O10 82.69 d[F]1 / d[F]2 =\u089e1 /\u089e2 = 1 + kc [RhIII complex]/ k[NBT] (5)\n O8-Rh1-O9 98.15\n O8-Rh1-Cl1 98.4\n O8-Rh1-O10 88.36 where \u089e1 and \u089e2 are the relevant optical density at 560 nm in the\n O9-Rh1-Cl1 96.86 absence and in the presence of the RhIII complex respectively.\n O9-Rh1-O10 87.57 The concentration of the quercetin\u2013based RhIII complex (IC50 )\n Cl1-Rh1-O10 171.27\n at which NBT reduction is inhibited by 50% was determined graph-\n ically (Fig. 8) with knowing that NBT%= =(\u089e1 /\u089e1 \u2212 \u089e2 ) \u00d7 100.\n Table 6 Since the IC50 \u03b1 [NBT], thus kc = k[NBT]/IC50 ; where k, is\n The relevant bond distances around Rh(III) \u2013 5.94 \u00d7 104 M\u22121 s\u22121 and [NBT] is 30 \u03bcM [58].\n core of [RhIII L2 Cl H2 O]. The estimated values of IC50 and kc are 21.9 \u03bcM and\n Bond type Bond distance (\u01fa) 8.91 \u00d7 104 M\u22121 s\u22121 respectively indicating that the SOD mimetic\n Rh1 - O2 2.043\n catalytic activity of the quercetin \u2013 based RhIII complex is some-\n Rh1 - O7 2.025 what moderate and similar to related low-spin d6 metal complex\n Rh1- O8 1.807 (Table 7).\n Rh1 - O9 1.849 The mid value of the catalytic activity of the rhodium(III) com-\n Rh1 - Cl1 2.104\n plex in question could be ascribed to structural reasons. In this re-\n Rh1 - O10 2.065\n gard, the catalytic dismutation of O2 \u2022\u2212 will begin after the bond-\n ing of the superoxide anion radical to the RhIII center. Therefore,\n the spark of the catalytic cycle is the easy exchange of O2 \u2022\u2212 with\n The native SOD protein catalyzes disproportionation of O2 \u2022\u2212 as one of the six coordination sites of the octahedral structure. Al-\nshown in Eq. (3): though the complex molecule in question is coordinately saturated\n kc\n and does not have empty coordination sites for binding O2 \u2022\u2212 but\nMn+ \u2212 SOD + O2 \u2022\u2212 \u2192+ Mn+ \u2212 SOD + H2 O2 + O2 (3) it has two well-departing groups namely H2 O and Cl. In this con-\n 2H\n\n 7\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n\n\nFig. 8. Scheme of percentage reduction of NBT inhibition with increasing the con-\ncentration of quercetin-based RhIII complex.\n Fig. 9. Absorption spectra of quercetin-based RhIII complex (15 \u03bcM) in lack and ex-\n Table 7 istence of increasing amounts of CT-DNA (2 - 12 \u03bcM) in Tris\u2013HCl buffer (pH = 7.2).\n Comparison the biomimetic SOD activity of quercetin \u2013 based RhIII com-\n plex with others SOD mimics.\n\n Complex IC50 (\u03bcM) kc (M\u22121 s\u22121 ) Reference form the intermediate [(O2 \u2022\u2212 )-RhIII L2 H2 O]. Electron transfer from\n [RhIII L2 Cl H2 O] 21.9 8.91 \u00d7 104 This work O2 \u2022\u2212 to RhIII center follows this step which results in the reduc-\n [(CoL2 )2 O2 Cl2 ]Cl2 2H2 O 31.0 5.75 \u00d7 104 [36] tion of RhIII to RhII with release of a molecular oxygen molecule\n [CoLX LY ]Cl 87.0 3.82 \u00d7 104 [59] (O2 ). The second redox reaction occurs due to binding of the sec-\n [FeIII L2 Cl2 ]Cl H2 O 17.0 1.05 \u00d7 105 [36]\n FeIII TPAA 7.5 7.92 \u00d7 105 [60]\n ond O2 \u2022\u2212 to RhII center with spontaneous oxidation of RhII to RhIII\n [(MnIII L2 )2 O2 Cl2 ]Cl2 2H2 O 8.0 22.2 \u00d7 105 [36] and reduction of O2 \u2022\u2212 to O2 2\u2212 . Protonation of O2 2\u2212 before releas-\n [MnL8 ] 0.77 3.6 \u00d7 106 [58] ing is essential due to the high basicity of O2 2\u2212 . Therefore, in suc-\n [MnL9 ] 1.14 2.4 \u00d7 106 [58] cessive steps, O2 2\u2212 is protonated and liberated as H2 O2 and the\n [MnL10 ] 2.34 1.2 \u00d7 106 [58]\n catalyst returns to its primary form as shown in Scheme 1.\n MnSOD and FeSOD\u2217 \u2013 \u223c109 [61]\n \u2217\n Indicated native enzymes.\n\n 3.8. DNA binding study\ntext, the labile potency of the existing metal complex is the key\nthat tuning its catalytic potential. The interaction between DNA and metal complexes is com-\n According to Taube, the degree of liability or inertness of a tran- monly studied using electronic absorption spectroscopy. The DNA\nsition metal complex can be correlated with the D-electronic con- binding capacity of the current querectin\u2013based Rh(III) complex\n\ufb01guration of the metal ion [48]. For the octahedral structure, if a with calf thymus (CT)-DNA was evaluated by monitoring the spec-\ncomplex contains electrons in the anti-bonding orbitals eg \u2217 , the lig- troscopic changes of the electronic spectra of CT-DNA due to the\nands are expected to be relatively weakly bound and easily dis- interaction with the metal complex. However, interaction of CT-\nplaced; it is labile, while for a complex with empty eg \u2217 it is inert DNA with the metal complex leads to spectral changes which, by\n[48]. In the same context, if all the three- t2g levels are \ufb01lled ei- their characteristics, can determine the mode of interaction be-\nther singly or doubly, then the complex is inert kinetically. In any tween the metal complex and DNA. For example, at a \ufb01xed con-\ncase, when it comes to the reactivity of complexes, the phrases \"la- centration of a metal complex and a sequential rise in the concen-\nbile\" and \"inert\" are simply comparable. In general, complexes of tration of CT-DNA, the spectral change was detected leading to hy-\n(0.1 M) should be termed labile if they entirely react in less than perchromism or hyporchromism that may be associated with red\none minute, and inert if they take longer. shift or blue shift [62]. Hyporchromism indicates the intercalation\n However, the electronic con\ufb01guration of the present RhIII cen- mode, while hyperchromism occurs when the external contact pat-\nter in the low-spin octahedral geometry is t2g 6 eg 0 and thus it is tern (groove/electrostatic binding) is dominant [63,64].\nkinetically inert and this may explain its mid catalytic potential. For the current study, an absorbance titration experiment was\n Several studies concluded that the natural enzyme always re- performed using a \ufb01xed concentration of quercetin-based Rh(III)\nmains the highest in activity compared to its functional models complex to which increments of CT-DNA stock solution were added\nlisted in Table 7 as well as mentioned in the literature. and the obtained results are represented in Fig. 9.\n As shown in Fig. 9, the spectral changes for the quercetin-\n3.7. Proposed catalytic mechanism based RhIII complex at 250 nm due to \u03c0 -\u03c0 \u2217 intra-ligand transi-\n tion showed an increase in absorbance (hyperchromism) with a\n All previous studies demonstrated that the catalytic dismuta- noticeable red shift of about 4 nm, indicating the stability of the\ntion of O2 \u2022\u2212 by the M-SOD protein [61] or the SOD-mimics occurs DNA helix. This result indicated a surface binding to DNA [65,66],\nin a ping pong pattern [36,37,58]. Likewise the current quercetin- which leads to damage to the secondary structure of CT-DNA, as\nbased RhIII complex catalyzes the dismutation of O2 \u2022\u2212 in a ping- the phosphate group can provide the appropriate anchors to coor-\npong mechanism. In this regard, initiation the catalytic cycle begins dinate with the metal complex [67,68]. A similar behavior of such\nwith the exchange of bonding between O2 \u2022\u2212 and the anionic lig- kind of interaction of DNA with quercetin-iron has been already\nand (Cl\u2013 ) which leads to binding of O2 \u2022\u2212 to rhodium(III) center to reported [43].\n\n 8\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n Table 8\n Mean IC50 values (\u00b1 SEM) of quercetin and its RhIII complex against different\n cancer human cell lines as well as normal cell line (WI38) compared with cis-\n platin.\n\n Compounds Quercetin Quercetin \u2013 based RhIII Cisplatin\n\n HepG-2 129.28 \u00b1 13.5\u2217# 8.49 \u00b1 0.4# 4.50 \u00b1 0.12\n Hela 68.78 \u00b1 1.39\u2217# 9.40 \u00b1 0.46# 5.57 \u00b1 0.23\n MCF-7 61.72 \u00b1 3.29\u2217# 16.32 \u00b1 0.81# 4.17 \u00b1 0.12\n PC-3 91.92 \u00b1 7.32\u2217# 17.71\u00b1 0.87# 8.87 \u00b1 0.635\n Caco-2 80.92 \u00b1 9.04\u2217# 21.73 \u00b1 1.13# 12.49 \u00b1 0.64\n WI38 454.5 \u00b1 48.1\u2217# 40.58 \u00b1 2.8# 6.72 \u00b1 0.29\n \u2217\n p < 0.05 signi\ufb01cant compared with the quercetin \u2013 based RhIII complex.\n #\n p < 0.05 signi\ufb01cant compared with cisplatin.\n\n\n\n the results of the SOD-mimicking catalytic activity of the\n polyphenolic natural product quercetin and its rhodium(III)\n compound.\n\n 3.10. In vitro cytotoxic activity\n\n The cytotoxic effect of both quercetin and its rhodium(III) com-\n plex were determined, in triplicated manner, against HepG-2, Hela,\nFig. 10. Plot of [DNA]/(\u025ba -\u025bf ) versus [DNA] for the DNA binding assay of quercetin\n\u2013 based Rh(III) complex. [DNA] is the concentration of CT-DNA in base pairs, \u025ba MCF-7, PC-3, Caco-2 and WI38 as a normal cell line. Cisplatin was\ncorresponds to the extinction coe\ufb03cient observed (Aobs /[complex]). \u025bf is the extinc- used as standard anticancer drug in all cell lines. A concentration\ntion coe\ufb03cient of the free complex, \u025bb is extinction coe\ufb03cient of the complex fully scale from 100 to 1.56 \u03bcg/ml of quercetin and its rhodium(III)\nbound to CT-DNA, and kb is the intrinsic binding constant. complex and cisplatin were freshly prepared and every dilution\n was added into a well cultured with 0.5 \u00d7 105 cells, then in-\n In the same context, the intrinsic binding constants, Kb , of the cubated for 48 h. The cytotoxic effects of both quercetin and its\ncomplex was determined using the following equation [67]: rhodium(III) complex were assessed by MTT assay and the IC50s\n were calculated and compared with cisplatin IC50 .\n [DNA] [DNA] 1\n\u0002 \u0003=\u0002 \u0003+ \u0002 \u0003 (5) Fig. 12 presents different human cancer cells viabilities (%)\n \u03b5a\u2212 \u03b5 f \u03b5b\u2212 \u03b5 f kb \u03b5b\u2212 \u03b5 f that regularly reduced with increasing the quercetin \u2013 based RhIII\n The ratio of slope to intercept in the plot of [DNA]/(\u025ba -\u025bf ) ver- complex concentration which proven the anti-proliferative effect\nsus [DNA] (Fig. 10) gave the value of Kb . The calculated Kb value of of that complex in a concentration dependent manner. The IC50\nthe quercetin-based RhIII complex was found to be 2.76 \u00d7 106 M\u22121 values of the quercetin \u2013 based RhIII against the \ufb01ve cell lines\nwhich can be compared with the analogous quercetin-based ruthe- were signi\ufb01cantly reduced compared with uncomplexed quercetin\nnium complex with a Kb value of 3.05 \u00d7 103 M\u2212 1 [41,69]. (p \u2264 0.0 0 01, for all, Table 8) con\ufb01rming the improved anticancer\nSince quercetin is the common ligand for both ruthenium(III) and activity of the quercetin \u2013 based RhIII complex. Also, the IC50 val-\nrhodium(III) complexes, the marked variance in the binding abil- ues of quercetin \u2013 based RhIII complex were approximately two\nity with DNA is due to the type of metal ion. In the same context, folds increased on HePG-2, Hela and PC-3 cells compared with cis-\nthe value of the intrinsic binding constant, Kb , of the quercetin- platin indicating potent anticancer activities of the current Rh(III)\nbased Rh(III) complex is much higher than that of the quercetin- complex against those cell lines. Alternatively, the IC50 of quercetin\nbased Ru(III) complex and this \ufb01nding indicates that the Rh(III) \u2013 based RhIII complex against the normal cell line (WI38) was\nquercetin-based complex is relatively tightly bound to the DNA. signi\ufb01cantly increased compared with cisplatin indicating reduced\n toxicity to normal cell. These data indicated the e\ufb03cient anti-\n3.9. DPPH scavenging power proliferative activities of the quercetin \u2013 based RhIII complex com-\n pared with standard anticancer agent (cisplatin) with increased\n To con\ufb01rm the scavenging power of the rhodium(III) complex safety on normal cells.\nunder study to remove free radicals, an additional test of its abil-\nity for scavenging DPPH was performed. The ability of a compound 3.11. Quercetin \u2013 based RhIII complex apoptotic effect on Hela cell\nto neutralize a free radical is measured by DPPH\u2019s scavenging ac-\ntivity by forming a bond with the radical, resulting in an inert The percentages of apoptosis and necrosis were quantitatively\nproduct, or by providing hydrogen to the free radical. To mea- assessed in the Hela cells after treatment with quercetin \u2013 based\nsure that activity, the compound was reacted with the DPPH so- RhIII complex by the Annexin V/PI stain. Normal cells were de-\nlution and the disappearance of the DPPH color was related to signed for apoptotic cell death, but malignant cells had inhibited\nits scavenging activity [70]. Our data revealed a signi\ufb01cant de- that route. In apoptosis, nucleus fragmentation appears then the\ncrease (p = 0.047) in the DPPH IC50 value of the quercetin \u2013 based cell was shrank stimulating the phagocytic cell to engulf it [71].\nRhIII (23.53 \u00b1 3.56) compared with the DPPH IC50 value of the While, in necrosis the dysfunction of the plasma membrane ap-\nquercetin (36.99 \u00b1 3.13), which established the increased ROS scav- peared leading to cell swelling then suddenly exploded causing in-\nenger power of the quercetin \u2013 based RhIII than the free quercetin \ufb02ammation in a wide area [72]. Fig. 13 shows a signi\ufb01cant increase\n(Fig. 11). This superiority in the antioxidant activity of the exam- in the total apoptotic cell death (%) as sum of early and late apop-\nined rhodium(III) complex can be attributed to cooperative action tosis in the Hela cells treated with the IC50 of the synthesized RhIII\nbetween the rhodium(III) center and the two coordinated quercetin complex compared with untreated Hela cells (p \u2264 0.0 0 01). These\ncompartments of whole metal chelate molecule. results proposed that the quercetin \u2013 based RhIII complex may in-\n From that we can conclude that, the results obtained hibit the Hela cells proliferation through induction of apoptosis\nfrom the DPPH scavenging activity assay are consistent with (Fig. 13C). To endorse the hypothesis of apoptotic pathway; the cell\n\n 9\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n\n\n Fig. 11. DPPH inhibition percentage curve of quercetin \u2013 based RhIII complex and free quercetin.\n\n\n\n\n Fig. 12. Viability percentage of the human cancer cell lines after treatment with quercetin (Qu) or quercetin \u2013 based RhIII complex.\n\n\n\n\ncycle was assessed on the Hela cells treated with quercetin \u2013 based that the quercetin \u2013 based RhIII complex has prevented malig-\nRhIII complex. nant cells from entering another cell cycle by surface binding\n with DNA to avoid its replication (Fig. 13D), as observed from\n3.12. Cell cycle arrest prompted by quercetin \u2013 based RhIII complex the DNA binding study. By inhibiting DNA replication, the cell\n cycle stopped, and mitosis decreased, as observed in the deple-\n Division of the cells at certain phases in order to replicate is tion of G2/M percentage. To prove the apoptotic pathway of the\ncalled cell cycle. G1 (Gap 1) is the \ufb01rst phase to trigger the cell quercetin \u2013 based RhIII complex on Hela cells, the p53, MMP9\ncycle, then S and G2 phases and ends with mitosis (M phase). and caspase 9 gene expressions were detected and compared with\nThe Hela cells were treated with the IC50 of the quercetin \u2013 untreated cells.\nbased RhIII complex and incubated for 48 h, then the cell cy-\ncle was estimated by \ufb02ow cytometer using PI. The average per- 3.13. Quercetin \u2013 based RhIII complex effect on p53 genes and Bcl2\ncentage of cells in each phase was calculated. The propaga- protein\ntion in cell count (%) in the pre-G1 phase was associated with\nlate apoptotic death of malignant cells [73]. The arrest in the P53 is a protein that, when activated, can suppress any mu-\npre-G1 phase could be attributed to prevention of DNA replica- tation that happens in cells, so it is known as a tumor suppres-\ntion, stopping cell multiplication, leading to a decrease in G2/M sor gene. To suppress mutation, p53 can promote cell cycle ar-\nphase [39]. rest, inhibit DNA damage, and initiate apoptosis for malignant cells\n As shown in Fig. 13, the Hela cells treated with the present [74,75]. Cancer cells could diminish p53 gene activity to proliferate\nRhIII complex were arrested in the pre-G1 phase as a huge abnormally [76\u201378]. The current data indicated a markedly signi\ufb01-\npropagation of cells was detected compared with untreated Hela cant upregulation of p53 gene expression (p \u2264 0.0 0 01) in the Hela\ncells (p \u2264 0.0 0 01). On the other hand, a signi\ufb01cant decrease in cells treated with quercetin \u2013 based RhIII complex compared with\nG2/M percentage was observed in Hela cells treated with the untreated cells (Fig. 13A). P53 gene upregulation demonstrated\nquercetin \u2013 based RhIII complex compared with untreated cells that the quercetin \u2013 based RhIII complex may stop Hela cells pro-\n(p = 0.025). That ampli\ufb01cation in pre-G1 cell count indicated liferation through the p53 pathway. These data are complementary\n\n 10\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n\n\nFig. 13. The apoptotic/necrotic percentage in Hela cell treated with quercetin \u2013 based RhIII complex compared with untreated cells (A&B). Bar plots represented early, late\napoptosis and necrosis percentage (C). The cell cycle distribution in Hela cell treated with quercetin \u2013 based RhIII complex compared with untreated cells (D&E). Bar plots\nrepresented cell cycle distribution (%) (F) \u2217 p < 0.05 signi\ufb01cant compared with untreated cells.\n\n\n\n\nto our former results that the quercetin \u2013 based RhIII complex can hibit Bcl2 protein and trigger apoptotic cell death in cancer cells\ninduce Hela cells apoptosis and reduce mitosis in the G2/M phase [82].\nvia the p53activation. Our immunohistochemistry data revealed a signi\ufb01cant decrease\n Bcl2 (B-cell lymphoma 2) is a protein that plays an essential (p \u2264 0.0 0 01) in the Bcl2 protein in the Hela cells treated with\nrole in cancer progression by shutting down apoptosis [79,80]. Re- the quercetin \u2013 based RhIII complex compared with untreated cells\ncently, innovative chemotherapeutic agents have been planned to (Fig. 14B & C). That decrease in the Bcl2 protein could be evidence\ninhibit Bcl2 activity [81]. Also, the up-regulated p53 gene can in- of the apoptotic pathway through activating p53.\n\n 11\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n\n\nFig. 14. Bar plots presentation of RT-PCR analysis of the mean fold change of the p53 (A). Immunohistochemical analysis of Bcl2 gene in Hela cells treated with quercetin\n\u2013 based RhIII complex (C) compared with untreated cells (B). Bar plots and representative western blot of MMP9 and caspase 9 gene expression levels in Hela cells treated\nwith IC50 of quercetin \u2013 based RhIII compared with untreated cells (D&E). \u03b2 -actin was considered as standard gene control. \u2217 P < 0.05 signi\ufb01cant compared with untreated\nHela cells.\n\n\n\n3.14. Quercetin \u2013 based RhIII complex in\ufb02uence on MMP9 and ing the p53 levels. Increased p53 can inhibit both Bcl2 and MMP9,\ncaspase 9 proteins then activating caspase 9, which then cleaves caspase 3, leading to\n apoptosis triggering. Apoptosis activation led to cell cycle arrest at\n MMP9 is one of the matrix metalloproteinases that can dena- the pre-G1 phase and decreased Hela cell proliferation, which ap-\nture extracellular matrix components, leading to cell membrane peared in the decreased G2/M phase.\ndestruction. That destruction promoted malignant cell metastasis\n[83]. High levels of MMP9 were noticed in different types of can-\ncers [84\u201387]. 3.15. Antioxidant activity of the quercetin \u2013 based RhIII complex\n Caspases are protelytic enzymes that will be triggered at the\nend-stage of apoptosis to cleavage the DNA and split the cell mem- High cancer cells\u2019 proliferation is always accompanied with high\nbrane of the mutated cells [88]. Apoptotic cell death could be metabolic activities. High metabolic activities led to excess reac-\nidenti\ufb01ed by the activation of the caspases. Extrinsic and intrinsic tive oxygen species (ROS) [93]. Excess ROS have important role\napoptotic signaling pathways are the principal cell death pathways in cancer proliferation by starting DNA damage and controlling\n[89]. Both pathways end with caspase 8, 9 or 10 activation, trigger- metabolism [94]. In normal cells, the antioxidant system is there\ning the caspase3 cleavage [90,91]. Caspase3 consequantly degrades to consume the ROS produced by metabolism. From these antiox-\nDNA, split the membrane component and promotes the apoptotic idant enzymes is the superoxide dismutase (SOD), which removes\ndeath [92]. the superoxide radicals by converting them to H2 O2 [95,96]. Stim-\n Our western blot image presented a signi\ufb01cant decreased level ulation of high SOD production can lead to apoptotic cell death in\nof MMP9 protein (p = 0.026) with a signi\ufb01cant increase in caspase malignant cells [97].\n9 level (p = 0.009) in the Hela cells treated with the quercetin The DPPH assay improved the high scavenging activity of the\n\u2013 based RhIII complex compared with untreated cells (Fig 14D&E). quercetin \u2013 based RhIII complex with its high in vitro SOD like ac-\nThe decline in MMP9 level has proven the anti-proleferative and tivity. To con\ufb01rm the antioxidant activity inside cancer cells, the\nanti-metastasis effects of the quercetin \u2013 based RhIII complex SOD activity was evaluated in Hela cells treated with the quercetin\nagainst Hela cells. \u2013 based RhIII complex compared with untreated cells. Our data re-\n From our former data, we can hypothesize that the quercetin \u2013 vealed a markedly signi\ufb01cant increase in the SOD activity in Hela\nbased RhIII complex anti-proliferative effect is by binding to the cells treated with quercetin \u2013 based RhIII complex compared with\nDNA surface which prevent cancer cell replication with increas- untreated cells (Fig. 15). The increased SOD levels proved the an-\n\n 12\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\n investigations which indicated the successful binding of the metal-\n lic complex of quercetin to DNA by a groove/electrostatic binding\n pattern with intrinsic binding constants Kb 2.76 \u00d7 106 M\u22121 . The\n anti-proliferative activity of the quercetin \u2013 based RhIII complex\n was con\ufb01rmed by preventing replication of different types of can-\n cer cell lines. That anti-proliferative activities may be via the sur-\n face binding between the quercetin \u2013 based RhIII complex and the\n cancer cell\u2019s DNA. Also, the safety of the quercetin \u2013 based RhIII\n complex was con\ufb01rmed with its high IC50 on the normal cell line.\n Moreover, apoptosis activation was promoted by the quercetin \u2013\n based RhIII complex. The apoptotic pathway of the our complex\n was proven via increasing the p53 levels together with inhibiting\n both Bcl2 and MMP9, then activating caspase 9 which then cleaves\n caspase 3, leading to apoptosis triggering. Apoptosis activation led\n to cell cycle arrest at the pre-G1 phase that stops Hela cell pro-\n liferation, which appeared in the decreased G2/M phase. Another\n proposed anticancer pathway that the quercetin \u2013 based RhIII com-\nFig. 15. SOD activity in Hela cells treated with the IC50 of the quercetin \u2013 based plex has antioxidant activity appeared in high DPPH value, SOD-\nRhIII complex. \u2217 displays signi\ufb01cant difference (P = 0.001) compared with untreated like activity and elevated SOD levels in treated cells. That antioxi-\nHela cells. dant activity can remove the excess ROS from the cancer cell pre-\n venting DNA mutation and stopping the cancer cell\u2019s proliferation\n and stimulating apoptotic cell death.\ntioxidant activity of the quercetin \u2013 based RhIII complex inside the\n From the current data one can conclude that the quercetin \u2013\ncancer cells that can help in promoting apoptosis in Hela cells.\n based RhIII complex could be a promising anti-proliferative agent\n with low toxicity on normal cells, but in vivo studies must be done\n4. Conclusion\n to con\ufb01rm its safety on vital organs as well as its anticancer activ-\n ity.\n In conclusion, the polyphenolic 3,3\u0003 ,4\u0003 ,5,7-pentahydroxyl\ufb02avone\n(quercetin) forms a mononuclear metal complex with rhodium(III) Declaration of Competing Interest\nion in a molar ratio of 1:2 metal to ligand. The analytical and spec-\ntroscopic investigations used demonstrated the mode of bonding The authors declare that there are no con\ufb02icts of interest re-\nbetween quercetin and RhIII -center in the six-coordinated metal garding the publication of this research paper.\ncomplex that exists in a distorted octahedral geometry. The struc-\nture of the current quercetin \u2013 based RhIII complex was achieved Acknowledgements\nby processing the PXRD data by the relevant Expo 2014 computer\nprogram based on the Rietveld-Re\ufb01nement approach. The SOD- This work was funded by Taif University Researchers Support-\nlike activity of the present quercetin \u2013 based rhodium(III) complex ing Project number (TURSP-2020/222), Taif University, Taif, Saudi\nwas examined and the obtained results showed moderate activity Arabia\ncompared to other M-SOD mimics containing d6 -metals. By anal-\nogy with the mechanism of action of the natural enzyme and its Supplementary materials\nmimicry, a mechanism for the catalytic activity of the quercetin\u2013\nbased RhIII complex has been proposed (scheme 1). The ability of Supplementary material associated with this article can be\nthe Rh(III) chelate to bind to DNA was examined by spectroscopic found, in the online version, at doi:10.1016/j.molstruc.2022.132584.\n\n\n\n\n \u2022\u2212\n Scheme 1. Catalytic cycle of scavenging of O2 by quercetin\u2013based RhIII complex; [RhIII L2 ClH2 O] is represented by RhIII \u2013SOD.\n\n\n 13\n\fH.A. Sahyon, F. Althobaiti, A.E.M. Ramadan et al. Journal of Molecular Structure 1257 (2022) 132584\n\n\nReferences [27] M. Ezzati, B. Youse\ufb01, K. Velaei, A. Safa, A review on anti-cancer properties of\n Quercetin in breast cancer, Life Sci. (2020), doi:10.1016/j.lfs.2020.117463.\n [1] S.P. Dunuweera, R.M.G. Rajapakse, Discovery, chemistry, anticancer action and [28] L.T. Nguyen, Y.H. 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