DSpace at VNU: Antitrypanosomal Activities and Mechanisms of Action of Novel Tetracyclic Iridoids from Morinda lucida Benth.

AAC Accepted Manuscript Posted Online March 2016 Antimicrob Agents Chemother doi:10.1128/AAC.01916-15 Copyright © 2016, American Society for Microbiology All Rights Reserved In-vitro Anti-trypanosomal Activities and Mechanisms of Action of Novel Tetracyclic Iridoids from Morinda lucida Benth Kwofie K D.1 ¶ , Tung N H.3 ¶ *, Suzuki-Ohashi M.1,2#, Amoa-Bosompem M.1, Adegle R.4, Sakyiamah M M.4, Ayertey F.4, Owusu K B-A.1, Tuffour I.1, Atchoglo P.1, Frempong K K.1, Anyan W K.1, Uto T.3, Morinaga O.3, Yamashita T.3, Aboagye F.4, Appiah A A 4, Appiah- Opong R.1, Nyarko A K.1, Yamaguchi Y.3, Edoh D.4, Koram K A.1, Yamaoka S.2, Boakye D A.1, Ohta N.2, Shoyama Y.3 and Ayi I.1 10 11 12 13 14 15 16 Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, P O Box LG 581, Legon, Ghana Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan Centre for Scientific Research into Plant Medicine, P O Box 73, Mampong - Akuapem, Ghana 17 18 Running title: Novel Anti-trypanosomal compounds from Morinda lucida 19 # Address correspondence to Mitsuko Suzuki-Ohashi (PhD) Email: mikkvip@tmd.ac.jp 20 ¶ 21 * These authors contributed equally to this work Present address: School of Medicine and Pharmacy, Vietnam National University, Hanoi 22 (VNU), 144 Xuan Thuy Str., Cau Giay, Hanoi, Vietnam 23 24 Abstract 25 Trypanosoma brucei parasites are a group of kinetoplastid protozoa which devastate 26 health and economic well-being of millions of people in Africa through the disease, Human 27 African Trypanosomiasis (HAT) New chemotherapy has been eagerly awaited due to severe 28 side effects and drug resistance issues plaguing current drugs Recently, there have been a lot of 29 emphases on the use of medicinal plants world-wide Morinda lucida Benth is one of the 30 popular medicinal plants widely distributed in Africa and several research groups have reported 31 on anti-protozoa activities of this plant In this study, we identified three novel tetracyclic 32 iridoids, Molucidin, ML-2-3 and ML-F52 from the CHCl3 fraction of M lucida leaves, 33 possessing activity against the GUTat 3.1 strain of T b brucei The IC50 value of Molucidin, 34 ML-2-3 and ML-F52 were 1.27 μM, 3.75 μM and 0.43 µM, respectively ML-2-3 and ML-F52 35 suppressed the expression of paraflagellum rod proteins, PFR-2 and caused cell cycle alteration, 36 which preceded apoptosis induction in bloodstream form of Trypanosoma parasites Novel 37 tetracyclic iridoids may be promising lead compounds for the development of new 38 chemotherapies of African trypanosomal infections in both humans and animals 39 40 Keywords 41 Kinetoplastids, Medicinal plants, Morinda lucida, tetracyclic iridoid, Human African 42 Trypanosomiasis, Trypanosoma brucei 43 Abbreviations 44 HAT - Human African trypanosomiasis 45 HR-ESI-MS – High-resolution electrospray ionisation mass spectrometry 46 NMR – Nuclear Magnetic Resonance 47 HMQC – Heteronuclear Multiple-Quantum Correlation 48 HMBC – Heteronuclear Multiple-Bond Correlation 49 NOESY – Nuclear Overhauser Effect Spectroscopy 50 IFA – Immunofluorescence Assay 51 DAPI – 4',6-diamidino-2-phenylindole 52 GAPDH – Glyceraldehyde 3-phosphate dehydrogenase 53 HPLC – High-Performance liquid chromatography 54 ECACC – European collection of cell cultures 55 EMEM – Eagle’s Minimum Essential Medium 56 FBS – Foetal Bovine Serum 57 MTT – 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide 58 SI – Selectivity Index 59 BSA – Bovine Serum Albumin 60 PBS – Phosphate Buffered Saline 61 NP-40 – Nonyl phenoxypolyethoxylethanol 62 63 64 65 66 67 68 Introduction 69 Human African trypanosomiasis (HAT), commonly known as sleeping sickness has remained a 70 serious health problem in many African countries with thousands of new infected cases annually 71 (1,2) Although millions of people are under threat of HAT in Africa, it is known as one of the 72 neglected diseases which lacks the necessary resources to bring new compounds to market for 73 possible drug development (3,4) HAT is caused by a protozoan parasites belonging to the genus 74 Trypanosoma, transmitted through the bites of tsetse flies In Africa, there are mainly two 75 species responsible for the disease; T brucei gambiense and T b rhodesiense T brucei 76 gambiense is responsible for about 98% of reported cases of sleeping sickness while T brucei 77 rhodesiense is 2% of reported cases (2) In 2012, 7216 cases were reported with emphasis on the 78 complexity of diagnosis, therefore the skilled personnel for case detection will be needed (2) 79 The current treatments for HAT are far from ideal (5) Chemotherapeutic agents against HAT 80 namely; suramin, pentamidine, melarsoprol and eflornithine (3,6–8) cause severe side effects (9), 81 requires lengthy parenteral administration and are unaffordable for most of the patients In 82 addition to those concerns, the increase in drug resistance urges the need for the discovery of 83 new chemotherapeutic agents against HAT.(10,11) 84 Recently there have been a lot of emphases on the use of medicinal plants world-wide (12–14) 85 Morinda lucida Benth (Rubiaceae), an evergreen medium-sized tree with dark-shiny leaves on 86 the upper surface, is one of the most popular medicinal plants widely distributed in Africa (15) 87 Phytochemical studies showed that M lucida is a natural resource rich in antraquinones like 88 oruwacin, 89 methylanthraquinone, 1,3-dihydroxyanthraquinone-2-carboxyaldehyde and many others (16–19) 90 It is used among traditional healers to treat fever, dysentery, abdominal colic and intestinal worm 91 infestation Several groups have reported on anti-protozoa activities of M lucida and some active oruwal, 3-hydroxyanthraquinone-2-carboxyaldehyde, 1,3-dihydroxy-2- 92 compounds isolated have been from it (20–25) Anthraquinones isolated from M lucida are 93 reported to have anti-leishmanial and anti-malarial activities (26) Three other compounds 94 purified from M lucida were also reported to have high activities against Plasmodium 95 falciparum (20,21) Although several groups have revealed anti-trypanosome activities of M 96 lucida crude extracts, the responsible compounds have not been isolated yet (27,28) 97 We previously reported on the anti-trypanosomal activity of the novel tetracyclic iridoid, 98 Molucidin (29) In the present study, we report in addition to Molucidin, the anti-trypanosomal 99 activities of two more novel tetracyclic iridoids namely; ML-2-3 and ML-F52, as well as other 100 known compounds (oruwalol, ursolic acid (30) and oleanolic acid ) isolated from the leaves of 101 M lucida In this study, we also report on the role of these compounds in apoptosis induction and 102 cell cycle alteration in trypanosome parasites It is also known that the Trypanosoma flagellum 103 plays a key role not only in motility but also in their morphology, growth and cell division In the 104 kinetoplastid flagellum, there is major protein known as the paraflagellar rod (PFR) which runs 105 adjacent to the canonical + axoneme structure The paraflagellar rod consists of protein 106 sub-units referred to as PFR-1 and PFR-2 (31–33) The important role of PFR-2 protein in 107 flagellum function was demonstrated when parasite mutants lacking PFR-2 protein exhibited 108 reduced swimming velocity and paralyzed phenotype hence reduction in survival rates (34,35) 109 PFR-2 protein appears to be a potential choice of target for the development of new 110 chemotherapy In this study we therefore report on the effect of the compounds on the expression 111 of PFR-2 protein and parasite morphology Activity and mechanistic results with novel 112 tetracyclic iridoids; Molucidin, ML-2-3 and ML-F52 suggest that they are promising lead 113 compounds for the development of new drugs against the kinetoplastid protozoans, Trypanosoma 114 brucei 115 Materials and Methods 116 117 Plant material and general procedures 118 This study involved the screening of several extracts from different parts of about 73 Ghanaian 119 medicinal plants, selected according to traditional knowledge, for anti-trypanosomal activity 120 Morinda lucida was found to have the strongest anti-trypanosomal activity among them 121 The leaves of M lucida were collected in Mampong, Ghana in 2012 and authenticated by one of 122 the authors (Y.S.) Voucher specimens have been deposited in the Department of Pharmacognosy, 123 Nagasaki International University, Japan and Centre for Scientific Research into Plant Medicine, 124 Ghana Plants material (crude extract) was screened in vitro against trypanosomes for 125 trypanocidal activity Extracts with activity were fractionated and the resulting fractions screened 126 in the same manner Fractions found to have anti-trypanosomal activity were further processed to 127 isolate compounds which were likewise screened for activity Compounds with high activities 128 were selected to establish their mechanism of action and their structures elucidated An 129 established 3-step screening system described elsewhere in this manuscript was employed 130 Optical rotations were obtained using a DIP-360 digital polarimeter (JASCO, Easton, USA) 131 NMR spectra were recorded on a JEOL ECX 400 NMR spectrometer (JEOL, Tokyo, Japan) 132 HR-ESI-TOFMS experiments utilized a JEOL AccuTOFTM LC 1100 mass spectrometer (JEOL, 133 Tokyo, Japan) Column chromatography was performed on silica gel 60 (230–400 mesh, 134 NacalaiTesque Inc., Kyoto, Japan) and YMC ODS-A gel (50 μm, YMC Co Ltd., Kyoto, Japan) 135 TLC was performed on Kieselgel 60 F254 (Merck, Damstadt, Germany) plates Spots were 136 visualized by spraying with 10% aqueous H2SO4 solution, followed by heating 137 138 139 Isolation of compounds 140 141 Air-dried and pulverized leaf sample of Morinda lucida (1100 g) was extracted with 50% 142 aqueous EtOH (2.0 L × times) at 40 oC under sonication After removal of solvent, the obtained 143 residue (203 g) was suspended in 1.0 L of water and successively partitioned with (1.0 L × 144 each) hexane, CHCl3, and EtOAc to obtain soluble fractions of hexane (2.1 g), CHCl3 (3.80 g), 145 and EtOAc (3.6 g) The CHCl3 fraction, the most active fraction against Trypanosoma, was 146 subjected to a silica gel column (45 × 350 mm) fractionation with hexane-EtOAc (2:1, v/v) as the 147 mobile phase to give seven sub-fractions (fr.1 ~ fr.7) Fr.1 (120 mg) was then rechromatographed 148 over a reversed-phase (RP) column (20 × 450 mm) with MeOH-H2O (10:1, v/v) to yield 149 compounds (white powder, 15 mg) and (white powder, 18 mg) Fr.2 (80 mg) was further 150 chromatographed over a RP column (20 × 450 mm) with MeOH-H2O (1:1, v/v) to obtain 151 compound (yellow solid, 30 mg) Similarly, fr.4 (140 mg) was loaded onto a RP column (20 × 152 450 mm) with MeOH-H2O (3:2, v/v) to yield compound (colorless crystal, 35 mg) 153 Subsequently, compound (colorless crystal, 50 mg) was purified from fr.6 (550 mg) by means 154 of a RP column (30 × 400 mm) with MeOH-H2O (3:5, v/v) followed by a silica gel column (20 × 155 350 mm) with CHCl3-MeOH (25:1, v/v) 156 157 Screening of compounds for anti-kinetoplastid activities 158 159 Trypanosome parasites 160 The GUTat 3.1 strain of the bloodstream form of T b brucei parasites was used in this study 161 Parasites were cultured in vitro according to the conditions established previously (36) Parasites 162 were used when they reached a confluent concentration of × 106 parasites/ml Estimation of 163 parasitemia was done with the Neubauer’s counting chamber Parasites were diluted to a 164 concentration of × 105 parasites/ml with HM1-9 medium and used for the various 165 experiments 166 167 In-vitro viability test for trypanosome parasites 168 The Alamar Blue assay (alarmaBlue® Assay, Life Technologies™, US) was carried out on 169 treated or untreated trypanosome parasites to ascertain their viability The assay was performed 170 in a 96-well plate following manufacturer’s instructions, with modification Briefly, 1.5 × 104 171 parasites were seeded with varied concentrations of plant material (extracts, fractions or 172 compounds) ranging from 0.78 µg/ml to 200 µg Final concentrations of ETOH and DMSO were 173 kept at less than 1% and 0.1%, respectively After incubation of parasites with or without plant 174 extracts or compounds for 24 h at 37 oC in 5% CO2, 10% Alamar Blue dye was added and 175 incubated another 24 h in darkness After a total of 48 h, the plate was read for absorbance at 540 176 nm using the TECAN Sunrise Wako Spectrophotometer Trend curve was drawn to obtain IC50 177 value of each plant materials (extracts, fractions and compounds) 178 179 180 Testing of compounds for cytotoxicity to mammalian cells 181 182 Cell cultures for cytotoxicity assay 183 The cytotoxicity of the compounds to mammalian cells were determined using four human 184 normal cell lines, namely, NB1RGB (skin fibroblast),, HF-19 (lung fibroblast), obtained from the 185 RIKEN Bio Resource Center Cell Bank (Japan), Chang Liver, and Hs888Lu (lung), obtained 186 from ECACC NB1RGB and HF-19 were maintained in Minimum essential medium-α (MEM9 187 α) Chang Liver and Hs 888Lu were grown in Eagle’s minimum essential medium (EMEM) and 188 RPMI1640, respectively All these media were supplemented with 10% FBS and 1% penicillin– 189 streptomycin and were then incubated at 37°C under 5% CO2 in fully humidified conditions 190 Cytotoxicity was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 191 bromide (MTT) assay The cells were treated with Molucidin, ML-2-3 or ML-F52 at 192 concentrations of 50 µM and below for 48 h Cells were plated at a density of 0.5 × 104 cells/well 193 into 96-well plates After 24 h incubation, cells were treated with various concentrations of each 194 of the purified compounds for 48 h Then, MTT solution was added to each well, and the cells 195 were incubated for another h The precipitated MTT-formazan product was dissolved in 0.04 N 196 HCl–isopropanol and the amount of formazan was measured at a wavelength of 595 nm by a 197 microplate reader (ImmunoMiniNJ-2300, Nihon InterMed, Tokyo, Japan) Cytotoxicity was 198 calculated as the percentage of live cells relative to the control culture The selectivity index (SI) 199 was expressed as the ratio of the IC50 value obtained for mammalian cells and the IC50 on 200 Trypanosome 201 202 FACS analysis for detection of apoptosis and cell cycle alteration 203 Trypanosoma cells were treated with either 6.25μM of Molucidin (about times of IC50), 6.25 204 μM of ML-2-3 (about times of IC50) or 0.78 μM of ML-F52 (about times of IC50) for 24 h 205 and then subjected to the nexin assay 206 Seeding and incubation of parasites with compounds were done under the same conditions as for 207 the Alamar Blue assay as described above without addition of the Alarmar Blue reagent In this 208 case, after 24 h incubation, Guava reagents for Nexin and cell cycle assays were added and each 209 assay was performed using Millipore guava easyCyte 5HT FACS machine according to the 210 manufacture’s instruction The Nexin assay and subsequent FACS analysis allowed for detection 211 of markers of apoptosis induction by the plant materials Similarly, the cell cycle assay and 10 602 603 43 Adesogan EK 1979 Oruwacin, a new irridoid ferulate from Morinda lucida Phytochemistry.18:175–6 604 605 606 607 44 Krohn K, Gehle D, Dey SK, Nahar N, Mosihuzzaman M, Sultana N, Sohrab MH, Stephens PJ, Pan JJ, Sasse F 2007 Prismatomerin, a new iridoid from Prismatomeris tetrandra Structure elucidation, determination of absolute configuration, and cytotoxicity J Nat Prod.;70(8):1339–43 608 609 610 45 Stephens PJ, Pan JJ, Devlin FJ, Cheeseman JR 2008 Determination of the absolute configurations of natural products using TDDFT optical rotation calculations: the iridoid oruwacin J Nat Prod.71(2):285–8 611 612 46 Shaha C 2006 Apoptosis in Leishmania species & its relevance to disease pathogenesis Indian J Med Res.123(3):233–44 613 614 47 Nguewa A, Fuertes MA, Cepeda V, Iborra S, Carrio J 2005 Pentamidine Is an Antiparasitic and Apoptotic Drug That Selectively Modifies Ubiquitin.2:1387–400 615 616 617 48 Welburn SC, Lillico S, Murphy NB 1999 Programmed cell death in procyclic form Trypanosoma brucei rhodesiense identification of differentially expressed genes during A induced death Mem Inst Oswaldo Cruz.94(2):229–34 618 619 620 49 Atawodi SE, Bulus T, Ibrahim S, Ameh DA, Nok AJ, Mamman M, Galadima M 2003 In vitro trypanocidal effect of methanolic extract of some Nigerian savannah plants African J Biotechnol ;2:317–21 621 622 623 50 Ralston KS, Lerner AG, Diener DR, Hill KL 2006 Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system Eukaryot Cell 5(4):696–711 624 625 28 626 Figure Legend 627 Table Anti-trypanosomal activities and cytotoxicities of three novel tetracyclic iridoids, 628 Molucidin, ML-2-3 and ML-F52 against four types of human fibroblast cell lines S.I values 629 were obtained with values of both anti-trypanosomal activities and cytotoxicity on each 630 compound, Molucidin (2),ML-2-3 (3) and ML-F52 (6) 631 632 Figure Chemical structures and activities of compounds isolated from M lucida 633 Structures of novel tetracyclic spirolactone iridoids, ML-2-2: Moludicin (2), ML-2-3 (3) and 634 ML-F52 (6), and known compounds, oruwalol (1), ursolic acid (4) and oleanolic acid (5), 635 isolated from Morinda lucida leaves with their respective IC50 values of 48 h incubation 636 637 Figure ML-2-3 and ML-F52 induced apoptotic cell death in Trypanosoma parasite cells 638 (a) Trypanosoma parasites incubated with varied concentrations of compounds ranging from 639 μM to 50 μM for 24 h at 37 oC and % CO2 were subjected to nexin assay Dot plots were 640 generated by flow cytometry Control represents negative control (untreated population) ML-2- 641 and ML-F52 induced strong apoptosis at minimum concentrations of 6.25 μM and 0.78 μM 642 respectively, whereas Molucidin showed no significant apoptotic induction (b) Percentages of 643 apoptotic parasites within the different compound-treated populations shown by a bar chart 644 645 Figure The effect of three novel compounds on parasite morphology and flagellum 646 formation (a) Immunohistochemistry results of Trypanosoma parasites incubated in the 647 presence or absence of either 5mM (4 x IC50) of Molucidin, 15mM(4 x IC50) of ML-2-3 or 648 0.43mM (IC50) of ML-F52 DAPI stained both nucleus (N) and kinetoplast (K) in parasite cells 649 Parasite flagellum was stained by FITC (Green) using anti-PFR-2 antibody ML-2-3 and ML- 650 F52 induced fragmented distorted nucleus which is indicated by arrowhead (H and K) but not 29 651 with Molucidin (E) The expression of PFR-2 was suppressed by ML-2-3 and ML-F52 (I and 652 L) In addition, ML-2-3 induced round shape cells having shorten flagellum (G) ML-F52 653 induced parasites that have two set of kinetoplasts and two set of flagellum in one parasite (K 654 and L) (b) The quantification analysis of PFR-2 protein in trypanosoma cells incubated with 655 either Molucidin, ML-2-3 or ML-F52 (concentrations are same with immunohistochemistry 656 study) by western blotting using anti-PFR-2 antibody ML-2-3 and ML-F52 but not Molucidin 657 suppressed the expression of PFR-2 proteins GAPDH proteins were detected as an internal 658 control 659 Figure PFR-2 suppression and cell cycle alteration preceded apoptosis event in ML-2-3- 660 treated Trypanosoma parasites (a) Time course nexin apoptosis assay using Trypanosoma 661 cells treated with 15µM of ML-2-3 Trypanosoma parasites were cultured in the presence of 662 15µM of ML-2-3 for different time periods (0, 0.5, 1.5, and 24 hours) and then percentages of 663 apoptotic and dead parasites were obtained using Flow cytometry The values are represented as 664 the mean of three different experiments (b) Time course western blot analysis on PFR-2 665 suppression using Trypanosoma cells treated with 15µM of ML-2-3 GAPDH was used as a 666 loading control (c) Time course cell cycle analysis using the same conditions as was done for 667 the time course western blot analysis with ML-2-3 treated Trypanosoma cells Percentages of 668 each phase of cells during cell cycle are shown as line graph for 24 h incubation 669 Figure In vivo efficacy test of three novel compounds, Molucidin, ML-2-3 and ML-F52 (a) 670 Parasitemia changes for 20 days post infection consecutive daily shots of 30mg/kg of each 671 compound were inoculated (ip) as well as vehicle-treated mice (Neg Control) ML-F52-treated 672 mice showed no parasitemia for 20 days (b) Survival rate curve for 20 days post infection ML- 673 F52 treated group showed 100% cure for 20 days post infection 674 30 675 676 677 678 679 31 680 Table 681 32 ... aromatic ring as above and the free carboxylic 326 function at C-14 of ML-2-3 featured by a relatively downfield shifted signal at δ 171.4 327 The relative configuration of ML-2-3 elucidated... 0% of late, 373 and 1.1% of early stages (Fig 2) ML-F52 showed the strongest induction of apoptosis with 14.2 374 % of early, and 2.3 % of late stages apoptotic cells at a very low concentration... Efficacy Of Methanolic Root And Leaf Extracts Of Morinda lucida J Nat Sci Res.;3(2):112–23 549 550 551 23 Obih PO, Makinde M, Laoye OJ 1985 Investigations of various extracts of Morinda lucida