The Isolation of Secondary Metabolites from Rheum Ribes L. and The

Food Chemistry 342 (2021) 128378

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Food Chemistry

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The isolation of secondary metabolites from Rheum ribes L. and the synthesis T of new semi-synthetic anthraquinones: Isolation, synthesis and biological activity ⁎ Ibrahim Halil Gecibeslera, , Faruk Dislib, Sinan Bayindirc, Mahmut Toprakc, Ali Riza Tufekcid, Ayse Sahin Yaglıoglug, Muhammed Altund, Alpaslan Kocake, Ibrahim Demirtasf, Sevki Ademd a Department of Occupational Health and Safety, Laboratory of Natural Product Research, Faculty of Health Sciences, Bingöl University, Bingöl, Turkey b Department of Physical Therapy and Rehabilitation, Faculty of Health Sciences, Bingöl University, Bingöl, Turkey c Department of Chemistry, Faculty of Science and Arts, Bingöl University, Bingöl, Turkey d Department of Chemistry, Faculty of Science and Arts, Çankiri Karatekin University, Cankiri, Turkey e Department of Biology, Faculty of Science and Arts, Bingöl University, Bingöl, Turkey f Department of Biochemistry, Faculty of Science and Arts, Igdır University, Igdır, Turkey g Department of Chemistry and Chemical Process Technology, Technical Sciences Vocational School, Amasya University, Amasya, Turkey

ARTICLE INFO ABSTRACT

Keywords: Rheum ribes L. (Rhubarb) is one of the most important edible medicinal plants in the Eastern Anatolia region and Rhubarb is called “Işkın” by local people. Resveratrol and 6-O-methylalaternin were isolated from the Rhubarb for the Food first time in addition to well-known secondary metabolites including emodin, aloe-emodin, β-sitosterol and Antioxidant α rutin. The new semi-synthetic anthraquinone derivatives with the N Fmoc-L-Lys and ethynyl group were syn- Anthraquinone thesized from the isolated anthraquinones emodin and aloe-emodin of Rhubarb to increase the bioactivities. Bioactivity α Aloe-emodin derivative with N Fmoc-L-Lys shows the highest inhibition values by 94.11 ± 0.12 and Human serum albumin Rheum ribes 82.38 ± 0.00% against HT-29 and HeLa cell lines, respectively, at 25 µg/mL. Further, modification of the aloe- α In silico emodin with both the ethynyl and the N Fmoc-L-Lys groups showed an antioxidant activity-enhancing effect. From molecular docking studies, the relative binding energies of the emodin and aloe-emodin derivatives to human serum albumin ranged from −7.30 and −10.62 kcal/mol.

1. Introduction Choowong, Supothina, & Pittayakhajonwut, 2012), neuroprotective and anti-inflammatory effects (Yang, Kim, Lee, & Song, 2018) and are Anthraquinones (9,10-dioxoantracenes) with a wide range of ap- active against multiple sclerosis (Szwed, 2013). plications constitute an important class of natural and synthetic com- R. ribes L. species from Polygonaceae family is very popular among pounds. Moreover, there is an increasing interest in developing new local people of Bingöl, Erzurum, Hakkari and Erzincan by names “Işkın, anthraquinone derivatives with biological activity. Anthraquinone-rich Uçgun and Işgın” is the only native Rheum species which grows wildly edible medicinal plants such as Rheum ribes, Rheum rhabarbarum, in the East and Southeast Anatolia (Öztürk, Aydoğmuş-Öztürk, Duru, & Frangula purshiana, Frangula alnus, Rubia tinctorum and Aloe vera have Topçu, 2007). Rheum species are medically important as they contain been used in folk medicine for a long time (Perassolo, Cardillo, Mugas, anthraquinone-derived compounds and R. ribes is also used as a source Montoya, Giulietti, & Talou, 2017; Çiçek, Ugolini, & Girreser, 2019; of several herbal medicines in the Middle East. Further, the plant is also Zhuang, Yu, Zhong, Liu, & Liu, 2019; Parvez, Al-Dosari, Alam, Rehman, used traditionally as laxative, expectorant, antihelmintic drug and for Alajmi, & Alqahtani, 2019). Anthraquinones show a broad spectrum of the treatment of rheumatic haemorrhoids, measles, smallpox, bile pharmacological activities, such as anti-HIV, cytotoxic and anti-plas- complications, stomach diseases, vomiting, diarrhea, hypertension, modial (Feilcke et al., 2019), antifungal and antibacterial obesity, ulcers and diabetes besides being used as a vegetable, snacks, (Mohamadzadeh, Zarei, & Vessal, 2020), antiviral (Zhang et al., 2016), appetizers and salad (Naqishbandi, Josefsen, Pedersen, & Jäger, 2009; antiplatelet and anticoagulant (Seo, Ngoc, Lee, Kim, & Jung, 2012), Abudayyak, 2019). In previous isolation studies, it has been reported antimalarial and anti-tuberculosis (Supong, Thawai, Suwanborirux, that the R. ribes has secondary metabolites including emodin, physcion,

⁎ Corresponding authors. E-mail address: [email protected] (I.H. Gecibesler). https://doi.org/10.1016/j.foodchem.2020.128378 Received 28 March 2020; Received in revised form 9 October 2020; Accepted 9 October 2020 Available online 15 October 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved. I.H. Gecibesler, et al. Food Chemistry 342 (2021) 128378

Fig. 1. Structure of secondary metabolites isolated from R. ribes (A) and semi-anthraquinones (B). chrysophanol, rhein, aloe-emodin, physcion-8-O-glucoside, aloee- 2. Materials and methods modin-8-O-glucoside, sennoside A, rhaponticin, quercetin, 5-deox- yquercetin, quercetin 3-O-rhamnoside, quercetin-3-O-galactoside, 2.1. Reagents quercetin-3-O-rutinoside, β-sitosterol-3β-glucopyranoside-6'-O-fatty acid esters, triacylglycerols, β-sitosterol, phytyl fatty acid esters, and Trifluoroacetic acid (TFA), copper (II) sulfate4 (CuSO ), potassium chlorophyllide A (Tosun & Kızılay, 2003; Ragasa et al., 2017). carbonate (K2CO3), sodium sulfate (Na2SO4), copper (I) iodide (CuI), α In the present study, the R. ribes, which has a very broad spectrum of N,N-diisopropylethylamine (DIPEA), hydrogen iodide (HI), N Fmoc-L- ε biological activity and is famous for its anthraquinones, was selected as Lys-N Boc, sterile dimethylsulfoxide (DMSO), 5-Bromo-2′-deoxyuridine a possible source of bioactive natural products and its anthraquinones (BrdU), sulfuric acid (H2SO4), sodium chloride (NaCl), human serum emodin (E) and aloe-emodin (AE) were selected as target precursors to albumin (HSA), sodium hydroxide (NaOH), ammonium hydroxide synthesize their new bioactive semi-synthetic derivatives. Further iso- (NH4OH), phosphoric acid (H3PO4), 1,1-diphenyl-2-picrylhydrazyl lation studies of R. ribes extract resulted in isolation of resveratrol and (DPPH), potassium hexacyanoferrate (III) (K3Fe(CN)6), trichloroacetic 6-O-methylalaternin, for the first time from R. ribes species, as well as acid (TCA), iron (III) chloride (FeCl3), iron (II) chloride (FeCl2), 3-(2- the isolation of four well-known secondary metabolites β-sitosterol, Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium aloe-emodin, emodin, and rutin (Fig. 1A). Following isolation of emo- salt hydrate (ferrozine), butylated hydroxyanisole (BHA), butylated dine and aloe-emodine from R. ribes we have designed and synthesized hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA) and α- new anthraquinones E-1 (9), AE-1 (10), FLE (11) and FLAE (12) con- tocopherol (Vit. E) were purchased from Sigma (St. Louis, MO, USA). taining L-lysine groups (Fig. 1B). Biological activity screening studies, All other chemicals including n-hexane (n-C6H12), chloroform (CHCl3), carried out during the current work, are i) anticancer activity assays dichloromethane (CH2Cl2), n-butanol (n-BuOH), methanol (CH3OH) evaluated against human prostate cancer (PC-3), human cervix cancer and ethyl acetate (EtOAc) were supplied in analytical purity. (HeLa) and human colon cancer (HT-29) cell lines, ii) antioxidant activities (DPPH free radical scavenging activity, ferrous chelating capacity, and ferric reducing power assays) and iii) interaction tests with 2.2. Plant material transport protein human serum albumin. The study showed that natural and semi-synthetic products obtained from R. ribes could be potentially The aerial part of R. ribes was collected from the rocky slopes of used in the development of new and promising natural products as food Bingöl Yelesen village at the altitude of 1970 m (38°52′07.8″N, additives. 40°19′26.7″ E) during flowering period. The plant materials were dried in dark place. Herbarium samples were prepared by Alpaslan Kocak and stored at Herbarium of Bingol University with the herbarium number of KOCAK-4003.

2 I.H. Gecibesler, et al. Food Chemistry 342 (2021) 128378

2.3. Extraction, isolation, synthesis and structure elucidation dissolving, each reaction flasks E-1 and AE-1 were mixed separately by adding CuI (1.01 mmol) and DIPEA (6.06 mmol), respectively. Then, α ε The aerial part of dried R. ribes (5 kg) was extracted with the N Fmoc-L-Lys-N N3 (3, 1.01 mmol) was added dropwise to the re- CH3OH:CH2Cl2 (1:1; v:v, 30 L, four consecutive times) and 523.8 g of action flask with a dropping funnel and stirred overnight at 61–62°C dry extract was obtained by evaporating the solvent using a rotary under reflux. After completion, the reaction mixture was cooled tola- evaporator. The dry extract (473.8 g) was suspended in distilled water boratory temperature and the reaction was completed by adding

(4L) and extracted with CHCl3 (1200 mL × 20), EtOAc (1200 mL × 9) NH4OH solution and extracted with chloroform (3 × 25 mL). The or- and n-BuOH (1200 mL × 6) in the given order of 216.3, 113.2 and ganic phase was dried over anhydrous Na2SO4, and the solvent was 74.2 g dry extracts were obtained, respectively. CHCl3, EtOAc and n- removed in vacuo. The compounds FLE and FLAE were purified from the BuOH extracts were individually subjected to silica gel (pore size: crude product mixtures FLE (521 mg) and FLAE (514 mg), respectively 40–63 µm) column chromatography (18 × 150 cm). During silica gel (Fig. S4B) on silica gel column (pore size: 20–40 µm; 25 g) and EtOAc:n- column chromatography of each extract, fractions were collected in C6H12 (1:1; v:v) as an eluent. 1 13 500 mL volume using gradients of n-C6H12:CH2Cl2 (0:100–100:0), The H-, C NMR and 2D-NMR spectra of the isolated natural CH2Cl2:EtOAc (0:100–100:0) and EtOAc:CH3OH (0:100–100:0), re- products and semisynthetic compounds were recorded at 298 K in spectively. As a result of column chromatography applications on DMSO‑d6 on a Agilent NMR 600 MHz (14.1 T) NMR spectrometer CHCl3, EtOAc and n-BuOH extracts, 267, 142 and 97 sub-fractions were (Agilent, Santa Clara, CA, USA) equipped with a 3 mm broadband probe 1 31 13 15 1 13 1 collected, respectively. The sub-fractions from the CHCl3, EtOAc and n- ( H, P, C, N) and with cooled H and C preamplifiers ( H at BuOH extracts were subjected to further isolation process using iso- 600 MHz and 13C at 150 MHz). Acquisition parameters included a 45 cratic high-pressure liquid chromatography system (HPLC, Buchi) hard pulse angle, a sweep width of 14 ppm, 1.7 s acquisition time, 0.1 s equipped with Sephadex LH 20 (pore size: 18–111 µm) and C18 reverse- pulse delay and continuous WALTZ-16 broadband 1H decoupling. Up to phase silica gel column (pore size: 20–40 µm; 5 × 60 cm). β-Sitosterol 2000 scans were collected per sample, corresponding to ~1 h of col-

(ST, 33 mg) was isolated from the sub-fractions 12–43 of CHCl3 extract lection time. 2D-NMR experiments were performed using standard by p-TLC using mobile solvent system n-C6H12:EtOAc (7:3; v:v). Emodin Agilent NMR programs. High-resolution mass spectrometric analysis (E, 29 mg) was isolated from the sub-fractions 61–66 of CHCl3 extract was carried out on an Agilent 1260 Infinity Series HPLC-TOF/MS (ESI/ by isocratic HPLC system at a flow of 2 mL/min using the mobile sol- MS) (Agilent, Santa Clara, CA, USA). Mass spectra were recorded in the vent system CH3OH:CHCl3 (35:65; v:v). Aloe-emodin (AE, 17 mg) was positive ion mode in the range of 100–2000 m/z, with a mass resolution isolated from the sub-fractions 122–188 of CHCl3 extract with silica gel of 20.000 and an acceleration voltage of 0.7 kV. column (230–400 mesh; 4 × 80 cm) and mobile phase n-C6H12:EtOAc The melting points of the compounds were determined by Stuart (100:0–0:100). Resveratrol (RE, 16 mg) was isolated from the sub- SMP50 Automatic Digital Melting Point Apparatus. The NMR spectra fractions 16–23 of n-BuOH extract with an isocratic HPLC combined with chemical shift values and the mass chromatograms of compounds with a C18 reverse-phase silica gel column (pore size: 20–40 µm; were given in the supplementary part (Figs. S6-S46). 5 × 60 cm) and H2O:CH3OH (100:0–0:100). 6-O-methylalaternin (AL, 23 mg) was isolated from the sub-fractions 47–56 of n-BuOH extract 2.4. Biological activity assays with isocratic HPLC system integrated with Sephadex LH-20 column (pore size: 18–111 µm; 5 cm × 60 cm). Rutin (RU, 39 mg) was isolated 2.4.1. Antiproliferative activity from the sub-fractions 59–63 obtained of n-BuOH extract using isocratic Antiproliferative activities were performed on human colon cancer HPLC system combined with C18 reverse-phase silica gel column (pore (HT-29), human cervix cancer (HeLa) and human prostate cancer (PC- size: 20–40 µm; 4 × 30 cm) and using H2O:CH3OH (100:0–0:100) 3) cell lines. The cancer cell lines were provided by Istanbul University (isolation chart can be seen in Fig. S1). Faculty of Pharmacy and were originally purchased from the American α ε Synthesis of the compounds N Fmoc-L-Lys-(N N3) (3): The compound Type Culture Collection (ATCC) with codes of HT-29 (ATCC® HTB- α ε N Fmoc-L-Lys-(N N3) was synthesized in the light of the literature 38™), HeLa (ATCC® CCL-2™) PC-3 (ATCC® CRL-1435™). The cancer cell (Sminia & Pedersen, 2012). The group Boc in the structure of N2–(((9H- lines stored in liquid nitrogen, were cultured in DMEM medium (10%; fluorene-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)lysine w/v, Dulbecco′s Modified - Eagle′s Medium) at 37 °C in an incubator α ε (N Fmoc-L-Lys-(N Boc), 1), which is a commercially available natural containing 95% moisture and 5% CO2 (Demirtas, Gecibesler, & amino acid derivative, was removed by reacting with TFA. The target Yaglioglu, 2013). All operations in anticancer activity experiments α ε amino acid derivative N Fmoc-L-Lys-(N N3) (3) was synthesized with were carried out in a sterile cabinet. Different concentrations of com- high efficiency over (((9H-fluorene-9-yl)methoxy)carbonyl)lysine pounds were pipetted into each well of the 96-well plate, except the α (N Fmoc-L-Lys, 2) in the reaction conditions specified in Fig. S2. 4.44 g control group, with a total fluid volume of 200 µL. Sterile DMSO was α ε of N Fmoc-L-Lys-N N3 (3) in (yield 84%) was obtained as yellow viscous added to the negative control wells instead of the test compounds, and liquid. the cells were incubated for 24 h. At the end of this period, the cell Synthesis of the anthraquinones E-1 (9) and AE-1 (10): Each of the proliferation experiment was carried out according to the manufac- compounds E (1.85 mmol) and AE (1.11 mmol) was dissolved in DMF turer's protocol as described below. All tests were done three times and (5 mL) in separate reaction flask with a magnetic stirrer at laboratory it repeated three times. Each of the 96 microplate wells was pipetted temperature. Thereafter, 3-bromoprop-1-yne (8, 1.85 and 1.11 mmol) with 20.000 cells and incubated for 24 h at 37 °C in a 5% CO2 in- and K2CO3 (4.60 and 2.76 mmol) were put into reaction flasks of E and cubator. At the end of 24 h, 20 µL of BrdU labelling solution was added AE separately. Then, the reaction mixture was stirred at 60–70 °C for to the wells and incubated for 4 h at 37 °C in a 5% CO2 incubator. The 5 h. The reaction flasks were cooled to laboratory temperature and labelling solution in the wells was removed by turning the plates’ up- extracted with EtOAc (3 × 25 mL). The organic phase was dried over side down. After the addition of 200 µL FixDenat, it was incubated for anhydrous Na2SO4 and its solvent was removed in vacuo. The com- 30 min at room temperature. After removing the FixDenat solution, pounds E-1 and AE-1 were purified from the crude product mixtures E-1 200 µL of the anti-BrdU-POD solution was added and incubated for (512 mg) and AE-1 (331 mg), respectively (Fig. S4A). Purification of the 90 min at room temperature. After removing the anti-BrdU-POD solu- products were performed using the silica gel column (25 g) and the tion, all wells were washed with wash solution (3 × 200 µL). Subse- mobile phase of EtOAc:n-C6H12 (1:1; v:v). quently, 100 µL of substrate solution was added to all wells and in- Synthesis of anthraquinones FLE (11) and FLAE (12): Each of the cubated for 30 min at room temperature. The absorbance at 450 and compounds E-1 (1.01 mmol) and AE-1 (1.01 mmol) were dissolved 650 nm was measured in the ELISA reader before and after adding separately in CHCl3 (20 mL) in two-necked reaction flasks. After 25 µL of 1.0 M H2SO4 solution to all wells.

3 I.H. Gecibesler, et al. Food Chemistry 342 (2021) 128378

2.4.2. Antioxidant activity the sketcher Marvin JS (version 16.4.18) was converted into SMILES by 2.4.2.1. DPPH free radical scavenging activity. The DPPH (1,1-diphenyl- JChem Web Services (version 14.9.29) installed on one of our servers 2-picrylhydrazyl) radical scavenging activity assay was performed (Ceylan, Erkan, Yaglioglu, Akdogan Uremis, & Koç, 2020). according to the method available in the literature (Gecibesler & Erdogan, 2019). Briefly, 0.5 mL of sample solutions were prepared in 2.5.2. Molecular docking studies different concentrations (5–200 µg/mL) and 2.5 mL DPPH radical The blind molecular docking works were implemented by utilizing solution (0.6 µM) was pipetted to test tubes. After pipetting, each the AutoDock-vina program at the SAMSON software platform (Trott & tube was mixed vigorously and incubated at the dark area for 60 min. Olson, 2010). The structure of the compounds was depicted at Mar- At the end of the incubation period, absorbance values were measured vinSketch, then converted into PDB format using Discovery Studio Vi- at 517 nm. DPPH radical scavenging activity was calculated with the sualiar 2020. The 3D crystal structure of HSA (pdb id:1AO6) was formula given below: downloaded from the protein data bank (https://www.rcsb.org/) (Sugio, Kashima, Mochizuki, Noda, & Kobayashi, 1999). At the DPPH radical scavenging activity(%)= [(ACSC A )/A ]× 100. SAMSON platform, water molecules were removed and hydrogen atoms In the equation, AC shows the absorbance value of the control and were added. The size of the grid box was set to 87 × 64 × 79 Å to cover AS indicates the absorbance value of the sample. all the active site residues having the centre of the grid at x = 27.0, y = 29.6, and z = 22.0. The minimum energy docked conformation 2.4.2.2. Ferric-ion reducing antioxidant activity. The ferric-ion reducing was determined at various conformations, and the optimal binding antioxidant activity was determined according to Gecibesler and energy conformation was selected for further analysis. The summarized Erdogan (2019). 2.5 mL of phosphate buffer (0.2 M, pH = 6.6) and 2D interaction diagrams were created using the Discovery Studio Vi- 2.5 mL of K3Fe(CN)6 (1.0%; w/v) were added to 1.0 mL of samples sualiar 2020 program. prepared in different concentrations in the test tubes. Then, the tubes were incubated at 50 °C for 20 min. 2.5 mL of TCA (10%; w/v) was 3. Results and discussion added to test tubes and centrifuged at 3000 rpm for 10 min. After centrifugation, 2.5 mL of supernatants was transferred into separate test 3.1. The characterization tubes and mixed with an equal volume of distilled water and 0.5 mL of FeCl3 (0.1%; w/v) solution. The absorbance of the samples was The emodin was isolated as pale orange amorphous powder. Its measured at 700 nm. molecular structure C15H10O5 (270.24 g/mol) was determined by the NMR spectra (Figs. S6-S11). The melting point (m.p.) was determined 2.4.2.3. Ferrous-ions chelating activity. The ferrous-ion chelating to be 255–256 °C compatible with previous study (Guo, Feng, Zhu, Ma, activity was carried out according to the method in the literature & Wang, 2011). Aloe-emodin was obtained as dark brown. Its molecular (Gecibesler & Erdogan, 2019). 0.5 mL of the samples prepared in structure C15H10O5 (270.24 g/mol was determined by the spectral data different concentrations (5–200 µg/mL) was added into the test tube (Figs. S12-S13). Its m.p. was 224–225 °C compared with the reference and 1.0 mL of distilled water and 0.05 mL of FeCl2 (2 mM) were added (Dai et al., 2014). The 6-O-methylalaternin was obtained as light orange into these test tubes. The reaction mixture in each test tube was amorphous solid. The molecular structure C16H12O6 (299.06 g/mol) homogeneously mixed with a vortex. After 30 s, 0.1 mL of ferrozine was examined by the spectral data (Figs. S14-S19) with the m.p. of solution (5 mM) was added to the tubes and incubated for 10 min at 295–296 °C in accordance with a study (Debbab et al., 2009). The room temperature. Absorbances of samples were measured at 562 nm molecular structure C14H12O3 (228.24 g/mol) of resveratrol was ap- after incubation. The ferrous-ion chelating activity was calculated using proved by 1D and 2D NMR spectra (Figs. S20-S25). Its physical ap- the formula given in DPPH radical scavenging assay. pearance was dark orange and the m.p. was 247–250 °C in accordance with another study (Lee, Kim, & Whang, 2017). The rutin was isolated

2.4.3. The binding capacity to HSA assays as yellow solid. The molecular structure C27H30O16 (610.52 g/mol) was The binding capacity of the samples to the human serum albumin determined by the NMR spectra (Figs. S26-S29) and its m.p. was (HSA) was evaluated by fluorimetric measurements (Toprak, 2016). 191–193 °C. The findings were found to be compatible with other study Briefly, HSA solutions were prepared in 0.05 M phosphate buffer(pH in the literature (Napolitano, Lankin, Chen, & Pauli, 2012). The β-si- 7.4) containing 0.1 M NaCl. 150 µL of each sample prepared in different tosterol was isolated in the form of a white amorphous powder. The concentrations was pipetted into test tubes. The solvent contained in molecular structure C29H50O (414.7 g/mol) was analyzed by the NMR the samples was removed under nitrogen flow. Then, 3 mL of HSA was spectra (Figs. S30-S35) and the m.p. 135–137 °C in accordance with the pipetted into the test tubes at a constant concentration of 5 µM. A reference study (Zhao et al., 2020). thermostat bath and quartzes (1 cm) were used for fluorescence mea- The compound 3 was obtained as yellow viscous liquid (4.44 g; surements. The fluorescence and synchronous fluorescence measure- 84%). The 1H NMR spectrum was given in Fig. S36. The compound E-1 ments of HSA were performed using a Perkin–Elmer (Model LS 55) was obtained as a pale yellow solid (364 mg; yield 63%) as following fluorescence spectrometers. The fluorescence spectra of HSA wereob- literature (Narender, Sukanya, Sharmab, & Bathula, 2013). The 1H tained at different liposome concentrations within the range of NMR and 13C NMR spectra were given in Figs. S37-S38. The compound 290–450 nm at an excitation wavelength of 280 nm. Fluorescence in- AE-1 was obtained as yellow solid (245 mg; yield 71%). The 1H NMR tensities were corrected for inner filter. and 13C NMR spectra were given in Figs. S39-S40. The compound FLE was obtained as a yellow solid (467 mg; yield 65%). The molecular + 2.5. In silico studies formula C39H34N4O9 ([M + H] m/z 703,2164; calculated + C39H34N4O9: [M + H] , m/z 703,2400) was found by mass spectrum 2.5.1. Physicochemical and pharmacokinetic properties for computational (Fig. S41). The 1H NMR and 13C NMR spectra were given in Figs. S42- methods S43. The compound FLAE obtained as an orange solid (428 mg; yield The Swiss ADME web tool used to estimate the compound proper- 61%). The m.p. greater than 400 °C and the molecular formula + ties including absorption, distribution, metabolism, elimination physi- (C39H34N4O9: [M + H] m/z 703.2344; calculated C39H34N4O9: cochemical and pharmacokinetic properties that make more efforts to [M + H]+, m/z 703.2400) was determined by the mass spectrum (Fig. support experimental studies. The program takes into account the six S44). The 13C NMR spectra was given in Figs. S45-S46. When the 1H most important physicochemical properties flexibility, lipophilicity, NMR spectrum of the compound FLE is examined, it can be seen that saturation, size, polarity and solubility. The molecule inputted through the total 34 proton signals in the structure of the molecule were

4 I.H. Gecibesler, et al. Food Chemistry 342 (2021) 128378 resonanced between 1 and 13 ppm. The peaks –COOH and –NH re- remarkable with the highest inhibition rate of 82.38 ± 0.14% at a sonating at 12.23 and 5.78 ppm, respectively are important indicators concentration of 25 µg/mL. This exceptional inhibition effect of the that confirm the structure of the compound FLE. On the other hand,17 FLAE compared to all other samples and anticancer agent 5-FU aliphatic proton signals (–CH, –CH2 and –CH3) in the FLE are also (73.7 ± 0.02%) at the same concentration, is worthy to be mentioned. compatible with the structure of the target molecule and confirm the In order to prevent proliferation in HeLa cancerous cells at the same structure of the FLE. Thirteen aromatic peaks (]CH) also appear to concentration, the inhibition percentage of other natural and semi- have resonance between 7.95 and 6.80 ppm. Three proton signals be- synthetic anthraquinones followed from large to small AE longing to the group –CH3 connected to the aromatic ring are found to (72.29 ± 0.01%), E-1 (34.99 ± 0.94%), E (28.66 ± 0.16%), AE-1 be resonant as singlet at 2.48 ppm. All these data confirm the structure (24.97 ± 0.09%), FLE (15.67 ± 0.02%). Similarly, the anthraquinone of the FLE. When the 13C NMR spectrum of the FLE is examined, a total FLAE showed the highest inhibition at concentration of 25 µg/mL of 24 olefinic carbon signals, including 15 quaternary and 9 aromatic against the HT-29 cell line. This important inhibition value of the FLAE –CH verifies the structure. There are six different carbon signals inthe (94.1 ± 0.12%) is worthy compared to the inhibition value of antic- aliphatic region. ancer agent 5-FU (90.09 ± 0.45%) at the same concentration. Like- wise the anthraquinone AE, the starting compound AE of the FLAE, 3.2. Biological activity showed the highest activity with an inhibition value of 91.9 ± 0.31% against HT-29 colon cancer cell line at a concentration of 25 µg/mL. Antiproliferative activities of the samples against the human cervix The samples generally showed significant increases in inhibition rates cancer (HeLa), colon cancer (HT-29) and prostate cancer (PC-3) cells with increased concentration against the human prostate cancer (PC-3) were carried out in the concentration ranges of 5, 25, 50 and 100 µg/ cell line (Fig. 2). Especially the anthraquinones E-1 (86.59 ± 0.2%), mL. Antiproliferative activities of R. ribes anthraquinones (E and AE), AE-1 (71.41 ± 0.52%), FLE (79.20 ± 0.17%) and FLAE semi-synthetic anthraquinones (E-1, AE-1 FLE and FLAE) and reference (77.3 ± 0.10%) showed a higher inhibition against PC-3 cells than 5- anticancer agent (5 FU) against HeLa cells were given in Fig. 2. The FU (53.2 ± 0.21%) at concentration of 25 µg/mL. anthraquinones FLAE (20.25 ± 0.00), E-1 (5.12 ± 0.28%), E The antioxidant activities of R. ribes secondary metabolites (E, AE, (9.33 ± 0.00%) and 5-FU (38.13 ± 0.20%) showed significant in- ST, AL, RU and RE), semi-synthetic anthraquinones (E-1, AE-1, FLE and hibitions at the lower concentration (5 µg/mL) against HeLa cell lines. FLAE) and reference antioxidants (BHA, BHT, Vit. E and EDTA) were Especially, the inhibition effect of the anthraquinone FLAE was evaluated for their antioxidant capacities by means of DPPH free radical scavenging activity, ferrous-ions chelating capacity and ferric-ions reducing capacity at the concentrations of 5, 10, 20, 40, 80, 100 and 200 μg/mL. The E, AE and AL showed slightly lower activities with values of 10.43 ± 1.19, 9.69 ± 0.01 and 12.4 ± 0.08%, respectively than metabolites RE and RU, while the metabolite ST did not show any DPPH radical scavenging activity. The semi-synthetic anthraquinones AE-1, FLE and FLAE showed a more moderate-higher activities with values of 13.56 ± 0.33, 14.91 ± 0.10, and 19.55 ± 0.30%, respectively compared to natural anthraquinones E, AE and AL. The ferric-ion reducing powers of the samples were assessed by colourimetric measurements of their reduction capacity of ferric ions (Fe3+) at different concentrations. Increased absorbance values at 700 nmin different concentration ranges indicate that the samples are capable to reduce Fe3+ ions to Fe2+ ions (Fig. S47). Therefore, the capacities of the samples to reduce ferric ions are also considered as an indicator for their antioxidant activity (Gecibesler, Behcet, Erdogan, & Askın, 2017). Except ST, the reducing antioxidant activities of samples generally increase with increasing concentration. When the capacity to reduce Fe3+ ions to Fe2+ ions of semi-synthetic anthraquinones was evaluated at the same concentration, it was observed that they showed a similar reducing activity trend in the range of 0.11 ± 0.00–0.18 ± 0.00 at 700 nm. The chelation of ferrous-ions (Fe2+) by antioxidants is considered as an indicator of antioxidant activity (Bilici et al., 2017). In this context, the capacities of the samples to chelate Fe2+ ions in different concentrations were determined by complex-based colourimetric measurements and compared with the standard compound EDTA. Among the samples, by comparing the chelating capacities of Fe2+ ions at 200 μg/mL concentrations, it was seen that isolated compounds showed the closest activity to EDTA (95.51 ± 0.35%). On the other hand, semi-synthetic anthraquinones E-1 (15.78 ± 0.32%), AE-1 (19.59 ± 0.30%), FLE (16.36 ± 0.23%) and FLAE (26.50 ± 0.70%) showed slightly higher activity than R. ribes anthraquinones E (14.52 ± 0.08%) and AE (13.02 ± 0.33%). HSA interactions with the semi-synthetic anthraquinones were screened using fluorescence spectroscopy. For this, increasing concentrations of the samples interacted in the fixed amount HSA solution in phosphate buffer (pH 7.4). After the interaction, fluorescence spectra of samples were recorded at 280 nm excitation. Fluorescence spectra Fig. 2. Antiproliferative activities of R. ribes secondary metabolites and semi- showing the interactions of semi-synthetic anthraquinones in increasing synthetic anthraquinones against HeLa, HT-29 and PC-3 cell lines. concentrations (1–50 µM) with HSA (5 µM) were given in Fig. 3. As

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Fig. 3. Fluorescence spectra of the anthraquinones E-1 (a), FLE (b) AE-1 (c) and FLAE (d) in different concentrations. seen in Fig. 3, HSA gave fluorescence peak at 347 nm. The semi-syn- differences in substitution, the FLAE had an activity enhancing effect. thetic anthraquinone E-1, which was added to the medium in increasing While compound AE had the highest activity (53.86 ± 0.01%) against concentration, affected the fluorescence intensity of the molecule HSA. PC-3 cells at a concentration of 5 µg/mL, the compound AE-1 formed by The fluorescence intensity was decreased in the beginning, but lateras the modification of the compound AE with the substituent ethynyl did the amount of E-1 increased, the emission of the HSA started to shift to not show inhibition. However, modification of the compound AE-1 with α the blue zone, a phenomena that depend on polarity. This shift was the N Fmoc-L-Lys group again increased inhibition against PC-3 cells by increased as the concentration of the semisynthetic anthraquinone E-1 23.64 ± 0.29%. The antioxidant activity, while the modification of the increased. So these data indicate that there is no spectral overlap be- compound E with the ethynyl group in DPPH radical showed a lowering tween HSA and E-1, and therefore there is no interaction in the form of activity, it was observed that the activity of the compound E-1 with the α binding between the two molecules. Similar findings were observed in N Fmoc-L-Lys group increased. However, modification of the com- α the semi-synthetic anthraquinones AE-1, FLE and FLAE. Further, these pound AE with both the ethynyl and the N Fmoc-L-Lys groups showed molecules did not interact with HSA but changed the polarity of the an activity-enhancing effect. Similarly, in the ferrous-ions chelating medium. This may be due to the low solubility of molecules in water activity experiment, the modification process with the related groups and their large molecular structures (Toprak & Arik, 2010). Since the showed an antioxidant activity enhancing effect. In the ferric-ion re- semi-synthetic anthraquinones did not interact in the phosphate buffer ducing antioxidant powers experiments, there was a moderate increase medium, they did not interact in the liposome medium. as a result of modification of the compound E with the ethynyl group, α Structure-activity relationship (SAR) studies have revealed that while the modification of the compound E-1 with the N Fmoc-L-Lys chemical modifications made with functional groups such as hydroxyls, group did not change the activity much. However, the modification of α alkyls, hydroxyl group of aliphatic esters, alkyl amines, ammonium the compound AE-1 with the N Fmoc-L-Lys group showed an anti- salts and halogens are also critical in different location of the skeleton oxidant activity-enhancing effect in terms of its reducing power(Fig of aloe-emodin and emodin. Modified compounds E-1, AE-1, FLE and S47). Modification of emodin with hydroxyl, quaternary ammonium, α FLAE with ethynyl and N Fmoc-L-Lys substituents were evaluated in iodine, ethyl amino and methyl groups has created very important SARs terms of structure–activity relationship (SAR). At 25 µg/mL con- that have antibacterial effect against methicillin resistant Staphylo- centration, FLAE compound had the highest capacity (94.11 ± 0.12%) coccus aureus and do not show cytotoxicity against non-cancer Vero to inhibit proliferation of HT-29 cell, while the FLE had the lowest in- cells (Chalothorn, Rukachaisirikul, Phongpaichit, Pannara, & Tansakul, hibition value with 17.81 ± 0.12%. Similarly, while the compound 2019). In a modification study with the bromine on the skeleton ofthe FLAE at the same concentration showed an enormous inhibition activity emodin, it was demonstrated by SAR study that the compound was a against the HeLa cells with value of 82.38 ± 0.00%, the FLE had the strong leader showing dose-dependent inhibition of the A549 lung lowest activity among the modified compounds (15.67 ± 0.00%). cancer cell line (Koerner et al., 2017). In a SAR study involving the

Unlike the FLE, the FLAE does not have a methyl group (–CH3) at po- quaternary ammonium salt derivatives of emodin, it was reported that sition 6, and also carries a methylene at position 3. Due to these lipophilicity and carbon chain length in the quaternary nitrogen atom

6 I.H. Gecibesler, et al. Food Chemistry 342 (2021) 128378

Fig. 4. Bioavailability radar of the anthraquinones E-1, FLE, AE-1 and FLAE. The pink area represents the optimal range for each properties (LIPO: Lipophilicity, SIZE: Molecular weight, POLAR: Total Polar Surface Area, INSOLU: Insolubility, INSATU: Insaturation, FLEX: Flexibility). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) are important factors that explain the bioactivities (Zheng et al., 2017). 3.3. In silico analysis Structure activity studies have been reported in which the derivatives of aloe-emodin, especially the thiosemicarbazide group, exhibit stronger Bioavailabilities of compounds E-1, AE-1, FLE and FLAE were de- tyrosinase inhibitory activity (Liu, Wu, & Chen, 2015). In a study in monstrated in Fig. 4. The pink area represents the optimal range for which aloe emodin derivatives were synthesized based on the SAR each properties (lipophilicity: LOGP between −0.7 and +5.0, size: MW study, antitumor activities were evaluated. Results of SAR studies have between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, shown that L-serine methyl ester, β-alanine ethyl ester and 3-(2-ami- solubility: log S not higher than 6, saturation: fraction of carbons in the noethyl) pyridine substituents were important for increased antitumor sp3 hybridization not less than 0.25, and flexibility: no more than 9 activities (Thimmegowda et al., 2015). Beyond the SAR studies men- rotatable bonds. According to the values, compound E-1 and AE-1 was tioned above, more studies are still needed to develop the best natural predicted orally bioavailable. The number of hydrogen bond acceptors food-based products. In order to increase the biological activity of (HBA) and donors (HBD) are within the Lipinski’s rules, n-ON < 10 natural products such as emodin and aloe-emodin, efforts to develop and n-OHNH < 5. The calculated logP must be smaller than 5. In our new bioactive products by modifying their structures with functional study the logP values were smaller than 5. The molecular weights of the substituents will increase the interest in foods of natural origin. compounds are at the range of 308.28 g/mol and 702.71 g/mol. The Blood-Brain Barrier (BBB) Score: 6-High, 0-Low. The BBB scores of compounds E-1, AE-1, FLE and FLAE was ranges from 0 to 3.04.

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Fig. 5. Affinity and view of docked conformation of compounds AE-1, E1, FLAE and FLEtoHSA.

Synthetic accessibility score of the compounds are from 1 (very easy) to energies of the compounds were calculated as − 7.83, −7.30, −10.10 10 (very difficult). Synthetic accessibility of all the compounds were and − 10.62 kcal/mol respectively for AE-1, E-1, FLAE and FLE. In between 3.00 and 5.76. Topological polar surface area (TPSA) must addition, the best Ki values were calculated as 1.80, 4.44, 0.03 and be < 70 Å2. TPSA values of compounds E-1, AE-1, FLAE and FLE were 0.01 µmol in the same order (Table S3). The binding results indicated bigger than 70 Å2. The solubility (log S) scale value ranges between that E-1, FLE, and FLAE are in close proximity to the HEME binding site, −10 (insoluble), −6 (poorly soluble), −4 (soluble), −2 (very soluble) and EA-1 is located at Sudlow’s site I (Fig. 6)(Ascenzi, Bocedi, Notari, and 0 (highly soluble). The solubilities of compounds were between Menegatti, & Fasano, 2005). Compounds FLE and FLAE are more pro- −7.35 and −3.50. The value ranges were soluble and poorly soluble. minent HSA binder than the other two compounds. The more negative the skin permeation (log Kp) the less skin permeant FLE exhibited the highest binding to HSA protein energy. It formed the molecule. For example, Diclofenac is a good topic anti-in- hydrogen bond interactions with Asp 108, Arg 415, His 146, Arg 117, flammatory with a predicted log Kp = −4.96 (cm/s), while ouabain and Gln 459. Results show that Lys 190, Tyr 138, Leu 182, Tyr 161, Leu has little chance to cross skin with a predicted log Kp = −10.94 (cm/ 115, Arg 197 and Ala 194 were critical residues in hydrophobic inter- s). The log Kp values of the compounds were between −6.95 and action of FLE with protein. FLE formed van der Waals interactions with −5.94 cm/s. According to Lipinski’s rule, E-1 and AE-1 could be a new Asn 458, Val 462, Leu 463, Pro 147, Asn 109, Pro 110, Leu 185, Glu bioactive molecule according to calculated data (Tables S1 and S2). 141, Met 123, Val 116, Arg 114, and Glu 425. Computational methods are widely used to evaluate the interaction As described in the 2D-interaction diagrams (Fig. 6), the optimum of biomolecules with small molecules. In order to explain the binding pose of AE-1 at HSA encircled by amino acids Arg 222, Tyr 150, His mechanism of the semi-synthetic anthraquinone derivatives with HSA, 242, (Hydrogen Bonds), Arg 218, Trp 214, Glu 153, His 283, Glu 188, a molecular docking study was carried out. From docking studies, 5 Glu 292, Ser 192, Phe 157, Gln 196, Val 241, Ser 287, lle 264, lle 290, possible free energy conformations were selected for HSA-compounds Leu 219, Phe 223, (van der Waals), Leu 260, Leu 238, Ala 291 (Hy- (Fig. 5), among them least binding free energies were preferred to drophobic interaction), Arg 257 (Unfavoroble), Ala 291, and Lys 195 create 2D interaction diagrams (Fig. 6). The relative binding to HSA (Carbon Hydrogen Bonds)

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Fig. 6. Molecular docking perspectives of HSA interacted with compounds AE-1, E1, FLAE and FLE using Discovery Studio.

The difference between experimental data and theoretical studies CRediT authorship contribution statement can be concluded from two reasons. The first is that the solubilities of the molecules are low (Toprak & Arik, 2010). The second is that pro- Ibrahim Halil Gecibesler: Writing - original draft, Investigation, teins display different behaviours in aqueous environments than X-ray Project administration, Formal analysis, Visualization, structures (Rudra, Dasmandal, Patra, Kundu, & Mahapatra, 2016). Our Conceptualization, Methodology. Faruk Disli: Investigation. Sinan experiment studies demonstrated that new anthraquinones interact Bayindir: Investigation, Writing - review & editing, Methodology. with HSA protein at low concentrations. The reason for not being af- Mahmut Toprak: Investigation, Writing - review & editing. Ali Riza fected in high concentration may be low solubility. In silico studies have Tufekci: Investigation. Ayse Sahin Yaglıoglu: Investigation. shown that molecules have a high potential for binding with HSA Muhammed Altun: Investigation. Alpaslan Kocak: Investigation. protein. Ibrahim Demirtas: Investigation, Writing - review & editing. Sevki Adem: Formal analysis, Writing - review & editing.

4. Conclusions Declaration of Competing Interest In summary, an effective semi-synthetic strategy has been devel- oped from the anthraquinones E and AE of native R. ribes to synthesize The authors declare that they have no known competing financial several anthraquinone derivatives E-1, AE-1, FLE and FLAE. Taking this interests or personal relationships that could have appeared to influ- strategy into account, four bioactive anthraquinone derivatives with ence the work reported in this paper. structural similarities were practically synthesized. Semi-synthesis of the anthraquinones E-1, AE-1, FLE and FLAE was obtained from anthraquinones E and AE with 2 steps for E-1 and AE-1 and 4 steps for FLE Acknowledgements and FLAE, a total yield of 63.0, 71.0, 18.0 and 16.8%, respectively. Besides, isolations of resveratrol (RE), and 6-O-methylalaternin (AL) This study was supported by the Republic of Turkey Ministry of from the R. ribes have been reported for the first time. Moreover, an- Agriculture and Forestry General Directorate of Agricultural Research tiproliferative and antioxidant activities of secondary metabolites and and Policies (Project No: TAGEM-16/AR-GE/52). As project re- new semi-synthetic anthraquinones and their interactions with HSA searchers, we thank the relevant institutions separately and offer our were compared. Among them, the anthraquinones E-1, AE-1, FLE and gratitude. FLAE exhibited higher antiproliferative activities against HT-29, PC-3 and HeLa cell lines, while these anthraquinones showed moderate to weak antioxidant activities and HSA interactions. Appendix A. Supplementary data

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2020.128378.

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