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

AAC Accepted Manuscript Posted Online 7 March 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01916-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.

1

In-vitro Anti-trypanosomal Activities and Mechanisms of Action

2

of Novel Tetracyclic Iridoids from Morinda lucida Benth

3
4

Kwofie K. D.1 ¶ , Tung N. H.3 ¶ *, Suzuki-Ohashi M.1,2#, Amoa-Bosompem M.1, Adegle R.4,

5

Sakyiamah M. M.4, Ayertey F.4, Owusu K. B-A.1, Tuffour I.1, Atchoglo P.1, Frempong K. K.1,

6

Anyan W. K.1, Uto T.3, Morinaga O.3, Yamashita T.3, Aboagye F.4, Appiah A. A. 4, Appiah-

7

Opong R.1, Nyarko A. K.1, Yamaguchi Y.3, Edoh D.4, Koram K. A.1, Yamaoka S.2, Boakye D.

8

A.1, Ohta N.2, Shoyama Y.3 and Ayi I.1

9
10
11
12
13
14
15
16

1

Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of

Ghana, P. O. Box LG 581, Legon, Ghana
2

Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental

University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
3

Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten

Bosch, Sasebo, Nagasaki 859-3298, Japan.
4

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:

20

¶

21

*

These authors contributed equally to this work.

Present address: School of Medicine and Pharmacy, Vietnam National University, Hanoi
1


22

(VNU), 144 Xuan Thuy Str., Cau Giay, Hanoi, Vietnam

23

2

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

3


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
4


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,

5

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 3 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 9 + 2 axoneme structure. The paraflagellar rod consists of 2 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.

6


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

7


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 × 3 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 × 3

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 4 (white powder, 15 mg) and 5 (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 1 (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 3 (colorless crystal, 35 mg).

153

Subsequently, compound 2 (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 1 × 106 parasites/ml. Estimation of
8

163

parasitemia was done with the Neubauer’s counting chamber. Parasites were diluted to a

164

concentration of 3 × 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 4 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 5 times of IC50), 6.25

204

μM of ML-2-3 (about 2 times of IC50) or 0.78 μM of ML-F52 (about 2 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


212

subsequent FACS analysis allowed for detection of markers of cell cycle alteration by the plant

213

materials

214
215

Investigating effect of compounds on parasite morphology and

216

flagella function

217

To investigate the effect of the compounds on parasite morphology and their flagellum function,

218

immunohistochemistry using anti-paraflagellum rod proteins, α-PFR-2 antibody (37), was

219

performed with Molucidin-, ML-2-3- and ML-F52-treated trypanosome parasites. Briefly,

220

Parasites were incubated for 24 h under appropriate conditions (37oC, 5% CO2) with 5 µM (4

221

times of IC50) of Molucidin, 15 µM (4 times of IC50) of ML-2-3 and 0.43 µM (IC50) of ML-

222

F52. Parasites were then harvested after incubation with or without appropriate concentrations of

223

compounds, and fixed with 4% paraformaldehyde in 8-well chamber slides at room temperature

224

for 5 min. Washing steps were carried out with 500 μl of PBS twice and PBST (0.1% Triton X

225

100 in PBS) at room temperature for 5 min each. Blocking reagent (500 μl; 3% BSA in PBS)

226

was added and incubated for 30 min at room temperature. Primary and secondary antibody

227

incubation with parasites was done for 1 h each and DAPI (5 μg/ml DAPI in PBS) staining for

228

10 min. After washing steps as above, the slides were mounted using parmafluor mounting

229

reagent and covered with cover slips. The slides were observed under the Olympus fluorescent

230

microscope (Olympus BX53) to detect any phenotypic changes in T. b. brucei parasites.

231
232

PFR-2 protein expression analysis

233

Trypanosome parasites were incubated with or without compound in vitro and lysed with 0.5 %

234

NP-40 (38). Protein concentration of the lysate was determined using the Biorad Protein assay

235

reagents (BIO-RAD, USA). SDS-PAGE was run using INVITROGEN NUPAGE 12% Bis-Tris
11


236

Gel. The Proteins were blotted on a PVDF membrane (Immobilon P, MILLIPORE, USA), added

237

with mouse α-PFR-2 antibody, 1:500 dilution, and incubated at 4°C overnight. The membrane

238

was then incubated with anti-mouse HRP antibodies, 1:2000 dilution, for an hour at room

239

temperature.

240

MILLIPORE, USA) was added to the membrane in a ratio of 1:1. Detection was done using the

241

ATTO cooled CCD Camera System Ez-CaptureII (ATTO Corporation, Japan).

Chemiluminescence HRP Substrate A and B (Immobilon™ Western,

242
243

Time-course analysis for mechanisms of action by compounds

244

To investigate the sequence in which the events (apoptosis induction, PFR-2 suppression, and

245

cell cycle alteration) involved in mechanisms of action occur, we examined the time course for

246

the three events using ML-2-3-treated parasites. Trypanosoma brucei parasites were incubated

247

for 0, 0.5, 1.5, 3, 6 and 24 h with 15 μM of ML-2-3 and then subjected to both nexin assay and

248

western blot analysis using PFR-2 antibody. We further investigated the time course of cell cycle

249

alteration using ML-2-3-treated parasites as well at similar concentration and incubation periods.

250
251

Structural analysis and comparison of compounds

252

In addition to the comparison of its spectroscopic data with those of plumericin, prismatomerin,

253

and oruwacin, the relative configuration of ML-2-3 was then elucidated by NOESY experiment.

254
255

In vivo efficacy assay for active compounds

256

Six weeks old BALB/c female mice with an average weight of 20g were infected with 1 x 103

257

cells of T. b. brucei (TC-221 strain) and randomly grouped into four cages containing five mice

258

each. The first 3 groups were treated with 30 mg/kg body weight of Molucidin, ML-2-3 and ML-

259

F52 respectively 6 h post infection and continued daily afterwards for 5 consecutive days. The

260

last group received physiological saline containing less than 0.1% of DMSO as a vehicle control.
12


261

Parasitemia and weight were monitored daily until 20 days post infection. The experiments were

262

conducted in compliance with the internationally accepted principles for laboratory animal use

263

and care as contained in the Canadian Council on Animal Care guidelines on animal use protocol

264

review.

265

13


266

Results

267

Isolated compounds and their structures

268

Bioassay-guided column chromatography resulted in the isolation of six compounds

269

including three novel (Molucidin, ML-2-3 and ML-F52). The rest are, oruwalol (39) (1), ursolic

270

acid (4), and oleanolic acid (5)(40) (Fig. 1). The three novel componds, (Molucidin, ML-2-3 and

271

ML-F52) were found to share unique tetracyclic iridoids skelton. Chemical characteristics of the

272

respective compounds are as follows:

273

o
+
Molucidin: colorless crystal; [α]25
D -188.5 (c 1.0, CHCl3); HR-ESI-MS m/z: 399.1084 [M + H]

274

(calcd for C21H19O8, 399.1080); 1H-NMR (CDCl3, 400 MHz) δ: 3.58 (1H, dd, J = 10.0, 6.0 Hz,

275

H-9), 3.78 (3H, s, 14-COOCH3), 3.96 (3H, s, 3ʹ-OCH3), 4.05 (1H, dt, J = 10.0, 2.0 Hz, H-5),

276

5.22 (1H, s, H-10), 5.63 (1H, dd, J = 6.4, 2.4 Hz, H-7), 5.64 (1H, d, J = 5.6, H-1), 6.03 (1H, dd, J

277

= 6.4, 2.0 Hz, H-6), 6.99 (1H, d, J = 8.0 Hz, H-5ʹ), 7.26 (1H, dd, J = 8.0, 2.0 Hz, H-6ʹ), 7.43 (1H,

278

d, J = 2.0 Hz, H-2ʹ), 7.46 (1H, s, H-3), 7.78 (1H, s, H-13); and13C-NMR (CDCl3, 100 MHz) δ:

279

102.4 (C-1), 153.0 (C-3), 109.6 (C-4), 38.5 (C-5), 141.1 (C-6), 125.9 (C-7), 104.4 (C-8), 54.3 (C-

280

9), 82.2 (C-10), 120.1 (C-11), 170.0 (C-12), 144.9 (C-13), 166.7 (C-14), 51.7 (14-COOCH3),

281

126.5 (C-1ʹ), 112.4 (C-2ʹ), 149.1 (C-3ʹ), 147.0 (C-4ʹ), 115.1 (C-5ʹ), 125.9 (C-6ʹ), 56.0 (3ʹ-OCH3).

282

Molucidin has been described in our previous study (29).

283

o
Compound 3 (ML-2-3): colorless crystal; [α]25
(c 0.35, CHCl3);HR-ESI-MS m/z:
D -89.2

284

385.0925 [M + H]+ (calcd for C20H17O8, 385.0923); 1H-NMR (CDCl3,400 MHz) δ: 3.60 (1H, dd,

285

J = 10.0, 6.0 Hz, H-9), 3.95 (3H, s, 3ʹ-OCH3), 4.05 (1H, dt, J = 10.0, 2.0 Hz, H-5), 5.28 (1H, s,

286

H-10), 5.67 (1H, dd, J = 6.4, 2.4 Hz, H-7), 5.68 (1H, d, J = 5.6, H-1), 6.06 (1H, dd, J = 6.4, 2.0

287

Hz, H-6), 6.92 (1H, d, J = 8.0 Hz, H-5ʹ), 7.25 (1H, dd, J = 8.0, 2.0 Hz, H-6ʹ), 7.49 (1H, d, J = 2.0

288

Hz, H-2ʹ), 7.50 (1H, s, H-3), 7.75 (1H, s, H-13); and 13C-NMR (CDCl3, 100 MHz) δ: 103.6 (C-

289

1), 153.9 (C-3), 110.2 (C-4), 39.2 (C-5), 141.9 (C-6), 126.9 (C-7), 105.7 (C-8), 54.9 (C-9), 83.0
14


290

(C-10), 120.0 (C-11), 169.2 (C-12), 145.9 (C-13), 171.7 (C-14), 127.2 (C-1ʹ), 113.7 (C-2ʹ), 151.1

291

(C-3ʹ), 148.8 (C-4ʹ), 116.2 (C-5ʹ), 126.0 (C-6ʹ), 56.1 (3ʹ-OCH3).

292

The molecular formula of ML-2-3 (Compound 3) was defined as C20H17O8 on the basis of HR-

293

ESI-MS experiment. The 1H and 13C-NMR spectra of ML-2-3 showed two relatively downfield

294

CH signals at δ 103.6 (C-1) and δ 153.9 (C-3) correlated with H-1 at δ 5.68 (d, J = 5.6 Hz) and

295

H-3 at δ 7.49 (br s) in the HMQC spectrum, together with a quaternary carbon at δ 110.2 (C-4)

296

suggested an irridoid-like structure (41) In addition, the presence of a 1,3,4-trisubstituted

297

aromatic ring with a typical ABX coupling pattern [δ 7.49 (d, J = 2.0, H-2′), 6.92 (d, J = 8.0 Hz,

298

H-5′), and 7.25 (dd, J = 8.0, 2.0 Hz, H-6′)] in the 1H NMR spectrum, a carbonyl carbon at δ

299

169.2 (C-12), and two olefinic carbons at δ 120.0 (C-11) and 145.9 (C-13) proposed a

300

coumaroyl-like (C6-C3) moiety, to which link to the irridoid nucleus [30]. Furthermore, along

301

with a downfield quaternary carbon at δ 105.7 (C-8) and a CH group [δ 83.0 (C-10) and 5.28 (br

302

s, H-10)], the HMBC spectrum revealed the key correlations of H-1/C-10, H-10/C-12, H-10/C-

303

13, and H-13/C-10 indicated the connection of the C6-C3 moiety with the irridoid nucleus to form

304

a rigid spirolactone tetracyclic ring skeleton similar to plumericin (42), oruwacin (43) and

305

prismatomerin (44).

306

o
Compound 6 (ML-F52): white amorphous powder; [α]25
D -62 (c 0.33, CHCl3); HR-ESI-MS

307

m/z:413.1249 [M + H]+(calcd for C22H21O8, 413.1236); 1H-NMR (CDCl3, 400 MHz) δ: 1.31

308

(3H, t, J = 7.2 Hz, -OCH2CH3), 3.56(1H, dd, J = 9.6, 6.0 Hz, H-9), 3.96 (3H, s, 3ʹ-OCH3), 4.06

309

(1H, dt, J = 9.6, 2.0 Hz, H-5), 4.24 (2H, q, J = 3.6 Hz, -OCH2CH3), 5.22 (1H, s, H-10), 5.63 (1H,

310

dd, J = 6.4, 2.4 Hz, H-7), 5.64 (1H, d, J = 5.6, H-1), 6.03 (1H, dd, J = 6.4, 2.0 Hz, H-6), 7.00

311

(1H, d, J = 8.0 Hz, H-5ʹ), 7.25 (1H, dd, J = 8.0, 2.0 Hz, H-6ʹ), 7.43 (1H, d, J = 2.0 Hz, H-2ʹ),

312

7.46 (1H, s, H-3), 7.77 (1H, s, H-13); and 13C-NMR (CDCl3, 100 MHz) δ: 102.3 (C-1), 152.7 (C-

313

3), 109.8 (C-4), 38.5 (C-5), 141.1 (C-6), 126.4 (C-7), 104.4 (C-8), 54.3 (C-9), 82.2 (C-10), 120.2

314

(C-11), 170.0 (C-12), 144.8 (C-13), 166.3 (C-14), 60.5 (-OCH2CH3), 14.3 (-OCH2CH3), 126.4
15

315

(C-1ʹ), 112.4 (C-2ʹ), 149.1 (C-3ʹ), 147.0 (C-4ʹ), 115.1 (C-5ʹ), 126.0 (C-6ʹ), 56.0 (3ʹ-OCH3). The

316

structure of ML-F52 including stereochemistry was assigned by means of the NMR spectra and

317

optical rotation value.

318
319

Comparison of the compounds

320

NMR data of Molucidin was very similar to those of ML-2-3 (see below), the presence of a

321

methyl group, however, was evident from 13C NMR signal at δ 51.7 (OCH3) and 1H NMR signal

322

at δ 3.78 (s, OCH3) and the HR-ESI-MS showing a molecular ion peak at m/z 399.1084 [M + H]+

323

(calcd for C21H19O8, 399.1080).

324

NMR data of ML-2-3 was found to have close similarity with those reported of

325

prismatomerin except for the 1,3,4-trisubstituted 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 by NOESY experiment is as follows: The

328

NOESY spectrum of ML-2-3 revealed the cross-peaks of H-1/H-9 and H-5/H-9 indicating H-1, H-

329

5, and H-9 are cofacially oriented. Furthermore, the NOESY correlations of H-10 at δ 5.28 with

330

H-2′ at δ 7.47 and H-6′ at δ 7.25 and no observed NOESY interaction of H-10 with H-13

331

supported E-configuration of the C-11—C-13 double bond in ML-2-3. Based on these findings,

332

the relative configuration of ML-2-3 was determined to be similar to that of prismatomerin (44).

333

Recently, the absolute configuration of the spirolactone tetracyclic iridoids including plumericin,

334

oruwacin, and prismatomerin has been well assigned by the combination of NMR spectra and

335

optical rotation using computational calculation and experimental value (45). Subsequently, the

336

relative configuration of ML-2-3 defined the absolute configuration of its rigid spirolactone

337

tetracyclic skeleton as of either (1R,5S,8S,9S,10S) or (1S,5R,8R,9R,10R) and on the basis of the

338

o
negative optical rotation value { [α ] 25
D -89.2 (c 0.35, CHCl3)}, the absolute configuration of ML-2-

339

3 was then assigned as (1R,5S,8S,9S,10S).
16


340

The 1H and

13

341

Molucidin apart from the appearance of signals arising from an ethyl moiety [δ 4.24 (2H, q, J =

342

3.6 Hz, -OCH2CH3), 1.31 (3H, t, J = 7.2 Hz, -OCH2CH3); δ 60.5 (-OCH2CH3), 14.3 (-

343

OCH2CH3)] instead of the methyl group in Molucidin. This finding was further evident by the

344

HRMS result of a quasimolecular ion peak at m/z413.1249 [M + H]+(calcd for C22H21O8,

345

413.1236). The linkage of the ethyl group to C-14 was confirmed by an HMBC correlation

346

between the methylene signal at δ 4.24 (-OCH2CH3) and C-14 at δ 166.3.

C NMR spectra of ML-F52 (compound 6) closely resembled the data for

347
348

Anti-trypanosomal activities and cytotoxicity of isolated compounds

349

The three novel compounds, Molucidin (2), ML-2-3 (3) and ML-F52 (6) had anti-trypanosomal

350

activities with IC50 values of 1.27 μM, 3.75 μM and 0.43 μM, respectively. Two known

351

compounds, ursolic acid (4) and oleanolic acid (5) had moderate activities with IC50 of 15.37 μM

352

and 13.68 μM, respectively. Oruwalol (1) had no significant activity with 518 μM of IC50 (Fig.

353

1).

354

Cytotoxicity assay results (Table 1), showed Molucidin and ML-F52 to have relatively high

355

toxicity with IC50 values between 4.74 μM to 14.24 μM against all cell lines tested. On the other

356

hand, ML-2-3 did not show any cytotoxicity with 50 µM and below among all cell lines.

357

Regarding the selectivity index (SI) values, which represent how the compounds inhibit the

358

growth of the target organisms specifically ML-2-3 and ML-F52 but not Molucidin was more

359

than 10 for all the cell lines, suggesting that ML-2-3 and ML-F52 might be ideal lead

360

compounds compared with Molucidin for anti-typanosomal activity.

361

362

Mechanisms of trypanocidal acitvities for Molucidin, ML-2-3 and

363

ML-F52
17


364

Recently, apoptosis-like death mechanism in trypanosomatid parasites were found (46–48),

365

which could actually be exploited as a possible target to fight against trypanosomiasis. To

366

investigate if the novel compounds, Molucidin, ML-2-3 and ML-F52 involve apoptosis-like

367

cell death machinery in their anti-trypanosomal activities, we performed FACS Nexin assay

368

using trypanosome parasites treated with each compound for 24 hrs. ML-2-3-treated

369

Trypanosoma parasites showed significant apoptosis induction with 7.8 % of early, and 4.4% of

370

late stages apoptotic cells compared with untreated Trypanosoma parasites with 0.2% of early,

371

and 0% of late stages apoptotic cells (Fig. 2). On the other hand, even five times of IC50

372

concentration of Molucidin (6.25μM) showed no significant induction of apoptosis, 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 of 0.78 μM (Fig.

375

2). These findings demonstrated that ML-2-3 and ML-F52 but not Molucidin had apoptosis

376

induction activity against Trypanosoma parasites (Fig. 2).

377
378

Effect of compounds on parasite morphology and flagellum function

379

PFR-2, which is expressed in their paraflagellar rod plays a key role not only in their motility but

380

also in their cell cycle and proliferation. PFR-2 knockout in trypanosome parasites caused

381

incomplete cell division and resulted in aggregation of parasites (34,35). We also found a lot of

382

aggregated parasites (data not shown).We therefore investigated the involvement of PFR-2 as a

383

possible target candidate for the novel tetracyclic iridoids by immunohistochemistry using anti-

384

PFR-2 antibody as well as DAPI which stains parasite nucleus and kinetoplast. We observed

385

intact kinetoplast but totally disintegrated nuclei in stumpy-like parasites in Molucidin-treated

386

group (Fig. 3a, D-F) Flagellum however appeared to be normal with significant expression of

387

PFR-2 (Fig. 3a, D-F). ML-2-3 and ML-F52 on the other hand, induced fragmented nucleus with

388

normal kinetoplast. ML-2-3 induced typical short stumpy form whiles ML-F52 caused abnormal
18


389

cells which have two set of kinetoplasts and flagellum with fragmented nucleus (Fig. 3a, K and

390

L). Both ML-2-3 and ML-F2 treated cells appeared to have less expression of PFR-2 poteins in

391

their flagellums (Fig. 3a, G-L). We further ran quantitative western blotting using anti-PFR-2

392

antibody against parasites treated with Molucidine, ML-2-3 and ML-F52. The quantification of

393

PFR-2 protein clearly showed the suppression of PFR-2 expression by ML-2-3 and ML-F52 but

394

not with Molucidin (Fig. 3b).

395

population of parasites having two sets of Kinetoplast and two sets of flagellum in a cell.

Interestingly, in ML-F52-treated group, we found large

396

397

PFR-2 suppression and cell cycle alteration preceded apoptosis

398

induction in ML-2-3-treated parasites

399

We demonstrated that ML-2-3 and ML-F52 involved apoptosis-like cell death in their growth

400

suppression. Moreover, we found these two compounds inhibited PFR-2 expression in parasite

401

flagellum. The flagellum is also known to have a significant role in cell cycle and growth, and

402

PFR-2 is one of the responsible proteins for those activities. These findings led us to the

403

hypothesis that PFR-2 may be a possible target of ML-2-3 and ML-F52, resulting in cell cycle

404

alteration and apoptotic cell death. We therefore investigated the timings of all events, apoptosis

405

and PFR-2 suppression as well as cell cycle alteration. The time course analysis from 0.5 h to 24

406

h of ML-2-3-treated parasites was performed using Nexin assay, Western blotting with PFR-2

407

antibody and FACS cell cycle assay. ML-2-3-treated trypanosome parasites showed that

408

induction of both early and late stages of apoptosis occurred at 3 h and continued to 24 h of

409

exposure (Fig. 4a). On the other hand, western blot showed that PFR-2 protein expression was

410

significantly suppressed within 0.5 h of parasite exposure to ML-2-3 (Fig. 4b). Significant

411

changes in parasites’ cell cycle were also found to be induced within 0.5 h of parasites exposure

412

to ML-2-3, in which Sub G1 and G0/G1 phase cells increased from 37% to 60% and 35% to

413

46%, respectively; whereas G2/M phase cells decreased from 31% to 9% (Fig. 4c). These
19


414

changes however continued through to 24 h. S phase cells stayed stable. These results therefore

415

suggested that suppression of PFR-2 in flagellum and alteration in G0/G1 phase of cell cycle

416

preceded induction of apoptosis in ML-2-3-treated parasite cells.

417
418

Evaluation of mice in vivo efficacy for Molucidin, ML-2-3 and ML-

419

F52

420

Molucidin, ML-2-3 and ML-F52 were evaluated for in vivo efficacy using mice model. 6 weeks

421

female BALB/c mice (average of 20g body weight) (n = 5 per group) infected with 1 x 103 of T.

422

brucei (TC-221 strain) were administered intraperitoneally with 30mg/kg of each compound 6h

423

post infection and continued daily afterwards for 5 consecutive days. Results showed that 30

424

mg/kg of ML-F52 completely cleared trypanosome parasites and ensured the survival of mice

425

for 20 days post infection, while vehicle control mice died at day 9. ML-2-3 -treated mice also

426

died at 9 days post infection. Molucidin-treated mice were all dead by 7 days post infection. (Fig

427

5).

428
429
430
431

432

20


433

Discussion

434

The main aim of this study was to identify anti–trypanosomal compounds from the extracts of M.

435

lucida leaves, which is popularly used as a traditional medicine to treat parasitic diseases in West

436

Africa. Several groups had already reported that the leaves of Morinda lucida possessed anti-

437

trypanosomal properties (15,28,49), however, the responsible active components were yet to be

438

isolated. Hence novel compounds with trypanosomal activity isolated from this plant might be

439

good candidates for new chemotherapy for both sleeping sickness in humans and Nagana in

440

animals. We recently published one of novel tetracyclic iridoids, ML-2-2 as Molucidin which is

441

enantiomer of Oruwacin (29) .

442

In this study, two more active novel compounds, ML-2-3 and ML-F52, with three other known

443

compounds; oruwalol (1), ursolic acid (4) (30) and oleanolic acid (5) were identified together

444

with Molucidin from the extract of M. lucida leaves (Fig. 1). Molucidin, ML-2-3 and ML-F52

445

have novel tetracyclic iridoid skelton and their absolute configurations were determined as

446

(1R,5S,8S,9S,10S). The chemical structures revealed that their side chains have different

447

functional groups at C-4. ML-2-3 has a carboxylic acid while Molucidin and ML-F52 have a

448

methyl and ethyl ester functional groups, respectively (Fig. 1).

449

Molucidin, ML-2-3 and ML-F52 (; Fig. 1) had significant trypanocidal activities with 1.27 μM,

450

3.75 μM and 0.43µM, respectively. Cytotoxicity assays showed Molucidin to be more toxic than

451

ML-2-3 and ML-F52 in all the normal fibroblast cells tested (Table 1). SI values of three novel

452

compounds demonstrated ML-2-3 and ML-F52 to be more specific against trypanosome

453

parasites than Molucidin.

454

We also demonstrated that ML-2-3 and ML-F52 induced apoptosis in Trypanosoma cells

455

(Fig 2). This finding was supported by two other observations that ML-2-3 and ML-F52 caused

456

fragmented nuclei in DAPI stained parasite cells (Fig. 3a) and an increase of SubG1 phase
21


457

population in cell cycle analysis with ML-2-3-treated cells (Fig. 4c). Moreover, ML-2-3 and

458

ML-F52 suppressed the expression of PFR-2, (Fig 3b). It is known that flagellum plays a key

459

role not only in their motility but also cell cycle progression and cell division (29,30,47). Indeed,

460

we also demonstrated that cell cycle alteration occurred in ML-2-3-treated cells as well as PFR-2

461

suppression within 0.5 h of ML-2-3 treatment. Those events preceded an induction of apoptosis

462

that was observed within 3 h of incubation. These findings therefore suggest that ML-2-3 and

463

ML-F52 affect parasite flagellum formation potentially through suppression of PFR-2

464

expression, which may result in cell cycle disorder and eventually killing Trypanosoma parasite

465

by apoptosis-like death signal. A study in 2006 showed that PFR-2 knockdown induced

466

flagellum beat defect which eventually caused the incompletion of cytokinesis. As a result, PFR-

467

2 knockdown-parasites had double or triple flagella (50). Interestingly, in our experiments with

468

Trypanosoma cells, we noticed significant increase in parasites having two flagella in ML-F52-

469

treated cells (Fig. 3a, L).

470

Molucidin, on the other hand, showed neither apoptotic induction nor PFR-2 suppression

471

capabilities in Trypanosoma cells but caused complete nuclei disintegration as shown by DAPI

472

stain. Although three tetracyclic iridoids have activities in vitro, they may also have different

473

targets and mechanisms of action. Further mechanistic studies will be necessary to establish a

474

complete profile of actions of each compound, as well as to confirm the toxicity of Molucidin

475

and explore other beneficial scientific uses of this novel compound.

476

Mice in vivo efficacy test of Molucidin, ML-2-3 and ML-F52 against T. b. brucei parasites (Tc-

477

221 strain) showed that 5 consecutive daily shots of 30mg/kg ML-F52 showed complete

478

clearance of parasitemia, resulting in 100% cure for 20 days post infection (Fig. 5). Molucidin,

479

however, showed severe toxicity which eventually caused death at day 7. These results revealed

480

that ML-F52 might be the best lead compound for the development of new chemotherapy

481

against trypanosome.

22


482

In addition to the anti- trypanosomal activities observed, data from preliminary studies also

483

showed anti-Plasmodium activities of Molucidin, ML-2-3 and ML-F52 against P. falciparum in

484

vitro, while Molucidin also had significant efficacy against P. yoelli in preliminary in vivo

485

studies using BALB/c mice (manuscript in preparation).

486

Our current findings suggest that the novel tetracyclic iridoids; Molucidin, ML-2-3 and ML-

487

F52 may not only be active against T. brucei parasites but other protozoan parasites as well,

488

which therefore makes them promising lead compounds for new chemotherapies against

489

infections caused by protozoan parasites.

490

23


491

Acknowledgments

492

This study was supported by a Science and Technology Research Partnership for Sustainable

493

Development (SATREPS) Grant from Japan Science and Technology Agency (JST) and Japan

494

International Cooperation Agency (JICA) (2010-2014) and Japan Initiative for Global Research

495

Network on Infectious disease (J-GRID) Grant from Japan Agency for Medical Research and

496

Development (AMED) (2015-). We also thank Dr. Theresa Manful of the Biochemistry

497

Department

in

the

University

of

Ghana

24

for

the

gift

of

PFR-2

antibody.


498

References

499
500

1.

WHO. 2012 Trypanosomiasis, human African (sleeping sickness). Media Center, WHO
Fact Sheet.. p. 259. />
501
502

2.

CDC. 2012 Parasites - African Trypanosomiasis (also known as Sleeping Sickness). CDC
Home. />
503
504

3.

Barrett MP, Boykin DW, Brun R, Tidwell RR. 2007 Human African trypanosomiasis:
pharmacological re-engagement with a neglected disease. Br J Pharmacol. 152(8):1155–

505

506
507
508

4.

Blas E, Chitsulo L, Philippe MP, Engers HD, Jane F, Kayondo K, Kioy DW,
Kumaraswami V, Lazdin JK, Paul P, Oduola A, Ridley RG, Toure YT, Zicker F,
Morel CM. 2002 Strategic emphases for tropical diseases research : a TDR
perspective.10(10):435–40.

509
510

5.

Barrett MP, Croft SL. 2012 Management of trypanosomiasis and leishmaniasis. British
Medical Bulletin. p. 175–96.

511
512

6.

Bacchi CJ. 2009 Chemotherapy of human african trypanosomiasis. Interdiscip Perspect
Infect Dis 2009:195040.

513
514

7.

Simarro PP, Jannin J, Cattand P. 2008 Eliminating Human African Trypanosomiasis :
Where Do We Stand and What Comes Next ? PLoS Med.5(2):174–80.

515
516
517

8.

Sun T, Zhang Y. 2008 Pentamidine binds to tRNA through non-specific hydrophobic
interactions and inhibits aminoacylation and translation. Nucleic Acids Res 36(5):1654–
64.

518
519

9.

Wang CC. 1995 Molecular mechanisms and therapeutic approaches to the treatment of
African trypanosomiasis. Annu Rev Pharmacol Toxicol 35:93–127.

520
521

10.

Mpia B, Pe J. 2002 Combination of eflornithine and melarsoprol for melarsoprol-resistant
Gambian trypanosomiasis. Trop Med Int Health.7(9):775–9.

522
523

11.

Horn D. 2014 High throughput decodind of drug targets and drug resistance mechanisms
in African trypanosomes. Parasitology.141(1):77–82.

524
525

12.

Vijaya T, Maouli KC, Rao S. 2009 Phytoresources as Potential Therapeutic Agents for
Cancer Treatment and Prevention. J Glob Pharma Technol.1(1):4–18.

526
527

13.

Willcox ML, Gilbert B. Traditional Medicinal Plants for the Treatment and Prevention of
Human Parasitic Diseases. life Support Systems. p. 1–10.

528
529

14.

Sampath Kumar GV. 2014 An emphasis on global use of traditional medicinal system
and herbal hepatoprotective drugs. J Pharm Res.8(1):28–37.

25