An antiplasmodial depside from a nigerian lichen dirinaria
picta, epiphytic on the oil palm Elaeis Guineense

Un dépsido antiplasmodial de un liquen nigeriano dirinaria
picta, epífito en la palma de aceite Elaeis Guineense

Ozadheoghene E. Afieroho^1,*, Xavier Siwe Noundou², Rui WM. Krause², ]]> Michelle Isaacs³, Louise Olley⁴, Heinrich C. Hoppe³, Kio A. Abo^1
1Department of Pharmacognosy and Phytotherapy,
University of Port Harcourt, Nigeria
*Corresponding author:ozadheoqhene.afieroho@uniport.edu.nq
²Department of Chemistry, Rhodes University,
Grahamstown, 6140, South Africa
³Department of Biochemistry and Microbiology,
Rhodes University, Grahamstown, 6140, South Africa
⁴The Royal Botanic Garden, Edinburgh,
Scotland, United Kingdom ]]> Received 01 06 2018 Accepted 04 25 2018 Published 04 30 2018

Abstract

This study investigated the anti-plasmodial and cytotoxic potentials of the chloroform (LCE) and ethanol (LEE) extracts from the foliose lichen Dirinaria picta with the view of isolating anti-malarial drug lead compound(s). In vitro anti-plasmodial and cytotoxicity assays were done using the plasmodium lactate dehydrogenase assay and human HeLa cervica cell lines respectively. The structure of the isolated compound was elucidated using spectroscopic techniques. The LCE yielded a novel antiplasmodial depside 1 (antiplasmodial IC₅₀≈37 (μg/mL; cytotoxicity (IC₅₀ >100 μg/mL; Selectivity index >2.7) and an impure fraction LC2 (antiplasmodial IC₅₀ ≈ 79 μg/mL; cytotoxicity (IC₅₀ >100 μg/mL; Selectivity index <1.3). The LEE (antiplasmodial IC₅₀ ≈ 17 μg/mL; cytotoxicity (IC₅₀ ≈ 62 μg/mL; Selectivity index ≈ 3.7) showed a significantly (p < 0.05) better anti-plasmodial activity though more cytotoxic compared to depside 1 and LC2.The depside 1, LC2 and LEE were less cytotoxic compared to emetine (cytotoxicity (IC₅₀ = 0.02 μM ≈ 0.013 μg/mL) though not as active as the reference drugs chloroquine (antiplasmodial IC₅₀ = 0.031 μM ≈ 0.016 μg/mL). This is the first time report on the anti-malarial potential of Nigerian lichens and the isolation of a novel anti-plasmodial depside 1.

Keywords: Lichens, Depside, Anti-plasmodial, Cytotoxicity, Drug discovery.

RESUMEN

Este estudio investigo los potenciales anti-plasmodiales y citotoxicos de los extractos de cloroformo (LCE) y etanol (LEE) de liquenes foliosos Dirinaria picta con el objetivo de aislar el (los) compuesto (s) principal (es) del farmaco antipaludico. Los ensayos in vitro anti-plasmodial y de citotoxicidad se realizaron usando el ensayo de deshidrogenasa plasmodium lactato y las lineas celulares humanas HeLa cervica, respectivamente. La estructura del compuesto aislado se elucido usando tecnicas espectroscopicas. El LCE produjo un nuevo depsido antiplasmodial 1 (antiplasmodial IC50 ≈ 37 μg / ml; citotoxicidad (IC50 > 100 μg / ml; Indice de selectividad> 2,7) y una fraccion impura LC2 (antiplasmodial IC50≈ 79 μg / ml; citotoxicidad (IC50> 100) μg / mL; Indice de selectividad <1.3). El LEE (IC50 antiplasmodial ≈ 17 μg / mL; citotoxicidad (IC50 ≈ 62 μg / mL; Indice de selectividad ≈ 3.7) mostro una actividad antiplamodial significativamente mejor (p <0.05) aunque fue mas citotoxico en comparacion con depsido 1 y LC2. El depsido 1, LC2 y LEE fueron menos citotoxicos en comparacion con emetina (citotoxicidad (IC50 = 0.02 μM ≈ 0.013 μg / mL) aunque no tan activos como los medicamentos de referenda chloroquine (antiplasmodial IC50 = 0.031 μM = 0.016 μg / mL). Este es el primer informe sobre el potencial antipaludico de los liquenes nigerianos y el aislamiento de un nuevo depsido antiplamodial 1.

The global plague of malaria which has been worsened by the prevalence of drug resistant Plasmodium falciparum strains of the causative parasite, limited access to quality health facilities and high cost of orthodox drugs, remains a threat to children, expectant mothers and the poor people living in endemic regions especially of Sub-Saharan Africa[l]. In view of these obstacles to receiving effective treatment for malaria, the continued search for new anti-malarial agents that are relatively non-toxic and easily affordable is an imperative. Bioactive metabolites from the flora and fauna in nature are good leads in drug development [2]. Lichens are a symbiotic form of life consisting of an alga (photobiont) and a fungus (mycobiont). They are found growing on rocks and as epiphytes on trees. Their harsh niche conditions pre-disposed them towards secreting protective metabolites against different ecological and biological influences. Lichens substances (the secondary metabolites) includes: depsides, benzofuranoids, terpenoids, xanthones, and anthraquinones [3-4]. They have been reported to have anti-viral, anti-bacterial, anti-fungal, anti-protozoan, anti-herbivore, antioxidant, anti-tumor, anti-ulcerogenic, anti-nociceptive, anti-pyretic, and anti-inflammatory activities [3-4]. With respect to the biological activities of Nigerian lichens, few literatures on the antiviral activities of Nigerian lichens are documented [5-8]. As a follow-up to our interest in the isolation and characterization of bioactive metabolites from Nigerian mycoflora [9-12], this present study reports the antiplasmodial and cytotoxic activities of a novel depside 1 isolated from the chloroform extract of the lichen Dirinaria picta epiphytic on the oil palm tree Elaeis guineensis.

RESULTS AND DISCUSSION

Structural elucidation of compound 1

The structure of compound 1 (Figure 1) isolated from the LCE after chromatography separation was elucidated using nuclear magnetic resonance (ID and 2D), mass spectrometric, UV-visible and infra-red spectroscopic techniques. Compound 1 showed ¹H and ¹³C-NMR chemical shift signals similar to the reported lichen compound atranorin [13] except for the replacement of the aldehyde functional group in atranorin with a carboxylic acid group. This was confirmed from the DEPT-135 and HSQC experiments with the characteristic carbonyl carbon and proton signals of the aldehyde functional group evidently absent. This is a confirmation that the carbon-13 chemical shift signal at δC = 193.8 ppm for C₉ is quaternary, indicative of a carboxylic acid. Infact, the ¹H-NMR data ( see Table 3) of compound 1 has: two sp² hybridised methine (CH) protons ( one of which is the quartet at H₁₀; δH = 6.33 ppm, 1Hq J=0.6, and the other the singlet at H_6’; δH = 6.45 ppm, 1Hs ), one deshielded methyl proton singlet of a methoxyl (H₁₀; δH = 3.92 ppm, 3Hs) which is rationalised for the methyl ester moiety, in addition to three other less deshielded methyl protons signals rationalised for the two singlets at H₈ (δH = 2.47 ppm, 3Hs) and H₉' (δH = 2.02 ppm, 3Hs), and the doublet at H₁₁ (δH = 2.62 ppm, 3Hd, J=0.6). Five characteristic hydroxyl (OH) proton singlets of phenols were also evident at: 2-OH (δH = 12.44 ppm), 3-OH (δH = 10.58 ppm), 6-OH (δH = 12.49 ppm), 8-OH (δH = 9.90 ppm) and 3'-OH (δH = 11.90 ppm). The presence of the methyl allylic enol residue was unambiguously assigned at the position para to the carboxylic acid moiety from the long range (²J_C,H, ³J_C,H, ⁴J_C,H,) HMBC experiments rationalised in Table 1 for H₁₀ and H₁₁. A further confirmatory evidence was seen in that the doublet protons of this allylic methyl signals (H₁₁; δH = 2.62 ppm, 3Hd, J=0.6), were observed from COSY experiment (see Table 1) to be vicinally coupled to the olefinic proton quartet (H₁₀; δH = 6.33 ppm, 1Hq J=0.6). In all, a total of twenty-one (21) carbon signals were rationalised from the ¹³C-NMR data (see Table 3) for fifteen quartenary (in the region 102- 194 ppm, three of which have been assigned to the carbonyl group of the carboxylic acid moiety at C₉ and that of the ester moieties at C₇ and C₇'), two methine, and four methyl. One of the methyl is due to the methoxy group of the methyl ester moiety which is rationalised for the deshielded δC signal at 52.4 ppm for C₁₀'. The structure of compound 1 was thus confirmed to be a 4-(1-hydroxylprop-1-en-1-yl) atranorin-1-carboxylic acid derivative with the systemic name: 2,3,6-trihydroxy-5-((3-hydroxy-4-(methoxycarbonyl)-2,5-dimethylphenoxy)carbonyl)-4-(1-hydroxyprop-1-en-1-yl)benzoic acid.

Its molecular formula of C₂₁H₁₀O₁₁, corresponded to the molecular mass of 448 g / mol as seen from its ESI (Positive mode)-Mass spectrum: [m/z (rel. int)]: 449(2) [M+H]⁺, 373 (100) [M-75]. The base peak at m/z 373 is rationalised from cleavages at the olefinic bond of the methyl allylic group and loss of the carboxylic acid moiety.

The novel antiplasmodial depside compound 1 (antiplasmodial IC₅₀ ≈ 37 μg/mL) was significantly (p < 0.05) less active compared to the standard drug chloroquine (antiplasmodial IC₅₀ = 0.03 μM ≈ 0.016 μg/mL) and the other impure lichen fractions LEE (antiplasmodial IC₅₀ ≈17 μg/mL) and LC2 (antiplasmodial IC₅₀ ≈ 79 μg/mL). The trend in the dose response pLDH activity (Figure 2 and Table 2) revealed that a marked inhibition of the pLDH activity was evident as from 25 μg/mL for the compound 1 and LEE. This was however not so for the LC2 where a similar trend was not evident until 100 μg/mL. Generally, the trend in pLDH activity was observed to be of the order: Chloroquine > LEE> Compound 1 > LC2. The plasmodium parasite lactate dehydrogenase (pLDH) is the last enzyme in the parasite glycolytic pathway and is produced by both the sexual and asexual stages of parasites, as well as the mature gametocytes of all human Plasmodium species [14-16]. Due to its dependence on anaerobic glucose metabolism, the pLDH plays an important role in catalysing energy production in the parasite [16-17]. Its associated activity is reported to disappear within 24 hours of effective malaria treatment [17] thus the pLDH antigen is considered a specific marker for the presence of viable plasmodium in blood.

Generally, a marked onset of cytotoxicity was observed for the test lichen samples after 25 μg/mL. At 100 μg/mL, the trend in cytotoxicity (Figure 3 and Table 2) expressed as % cell viability was of the order: LEE (14.003 %) > Compound 1 (55.405%) > LC2 (71.037%). Their IC₅₀ compared to the reference drug emetine indicated relative lower cytotoxicity with a trend: emetine (IC₅₀ = 0.013 μg/mL) > LEE (IC₅₀ = 62.38 μg/mL) > Compound 1(IC₅₀>100 μg/mL) > LC2 (IC₅₀ > 100 μg/mL). Report by the United States National Cancer Institute regards plant extract with cytotoxic IC₅₀ of 20 μg/mL or lower as being highly cytotoxic [18-20]. Those with IC₅₀ greater than 100 μg/mL are regarded to be of low to non toxicity [18]. The observed low cytotoxicity of the isolated lichen compound 1, and the impure lichen samples LEE and LC2 is suggestive that their anti-plasmodial activity may not necessarily be due to general cytotoxicity of the extract. This is an indication of their potential as non-toxic agents for drug development. Their trend in selectivity index was observed to be LEE (SI = 3.7) > Compound 1 (SI >2.7) > LC2 (SI >1.3). Selectivity index is a measure of how a drug substance is toxic to the parasite cells compared to the host (mammalian) cells. Higher SI values is therefore indicative of better selectivity [13].

EXPERIMENTAL

Lichen species

Dirinaria picta (thalli) epiphytic on oil palm tree Elaeis guineense were collected from the premises of the University park (Abuja campus, 4.9018°N, 6.9205°E) of the University of Port Harcourt, Port Harcourt, Rivers State, Nigeria.

Materials

All solvents used were of analytical grade (Sigma-Aldrich, Germany) or previously distilled. The spectra of ID and 2D NMR were recorded on a Brucker Avance NMR spectrometer of 300 MHz in the Chemistry Department of the Rhodes University, Grahamstown, South Africa using CDCl₃ as solvent. The Infra-red (IR) spectra were recorded on 1600 ATI Matson Genesis series FTIR™ spectrometer. Mass spectra were recorded on ESI positive mode on a mass spectrometer equipped Waters Synapt G2. The chromatographic separation was performed using normal phase silica gel (Mesh size 230-400) for column chromatography. The purity confirmation was done using Thin Layer Chromatography (TLC) using Iodine fume as developer, and by melting point determination.

Extraction and isolation

The dried pulverised lichen (60 g) was cold macerated for 72 hours with chloroform and ethanol in succession with fresh replacement of solvent at 24 hours interval to obtain the chloroform extract (LCE) and ethanol extract (LEE) used in this study after concentration using a rotary evaporator. The lichen chloroform extract LCE (3.2 g) was dissolved in chloroform and pre-adsorbed on silica gel in the ratio of 1:1 w/w to form a homogenous paste which was allowed to air dry in a fume cupboard. The mixture was loaded on a chromatography column (internal diameter 4.1 cm and packed with normal phase silica gel mesh 230-400 to a height of 30 cm). The column was eluted isocratically with chloroform (1200 mL) based on prior analytical Thin Layer Chromatography (TLC) evaluation. The eluted fractions were collected in 50 ml portion. From fractions 5-10 eluted with chloroform was isolated compound 1 after re-crystalisation from acetone. Its purity was confirmed from TLC and melting point determination. Fractions 13-17 were pooled together based on TLC as fraction LC2

Compound 1 ( a 4-(1-hydroxylprop-1-en-1-yl)atranorin-1-carboxylic acid derivative with the systemic name: systemic name: 2,3,6-trihydroxy-5-((3-hydroxy-4-(methoxycarbonyl)-2,5-dimethylphenoxy)carbonyl)-4-(1-hydroxyprop-1-en-1-yl)benzoic acid), molecular formula: C₂₁H₁₀O₁₁, yield: 124 mg, white amorphous solid; Melting point: 103-108 °C; Freely soluble in chloroform, dichloromethane; acetone, methanol and DMSO; Molecular Weight: 448 g / mol; UV in MeOH λ_max1 281 nm and λ_max2 340 nm; IR spectrum [frequency, v, cm^-1]: 2925 C-H str aliphatic, 1650 (C=O), 1581, 1451_def (C=C) 1106, 1077, 1031 (C-0 ester, carboxylic acid,), 935, 862, 728 (out-of-plane CH deformation of olefin and aromatic systems); ¹H and ¹³C NMR (Tables 1 and 3); ESI (Positive mode)-Mass spectrum: [m/z (rel. int)]: 449(2) [M+H]⁺, 373 (100)[M-75]

Plasmodium falciparum growth inhibition assay

This was done using the LEE, LC2 and the isolated compound 1. Briefly, the P. falciparum (3D7 strain) parasites were maintained in a medium composed of RPMI 1640 supplemented with 2 mM L-glutamine, 25 mM Hepes (buffered between a pH of 7.2 and 7.4), 5%(w/v) Albumax II, 20 mM glucose, 0.65 mM hypoxanthine, 60 μg/mL gentamicin sulfate and 2-4% (v/v) human red blood cells, in an atmosphere containing a mixture of O₂, CO₂, N₂ (5:5:90 v/v/v). For the growth inhibition assays, parasite cultures were adjusted to 2% parasitaemia and 1% haematocrit (final) and incubated for 48 hours, after addition of the test samples [(final test concentrations range of 0.006104 -100 μg/mL prepared in duplicate following a 4-fold serial dilutions approach in 96-well plates (200 μL culture/well; two wells per test sample dilution)]. After the incubation period, the levels of parasite in the wells were determined by colorimetric determination of parasite lactate dehydrogenase activity[21]. Chloroquine (eight final test concentration within the range 0.00001 - 100 μM) prepared following a 10-fold serial dilutions approach in 96-well plates was used asstandard anti-malarial drug for comparison. The Abs 620 values in test sample's and standard drug (chloroquine) wells were converted to percentage parasite viability relative to wells containing untreated parasite cultures. The median pLDH inhibition concentration (IC₅₀) values were derived from graphs of mean % parasite viability against Log (test sample concentration) using the non-linear regression function of Microsoft Excel 2007 software.

Mammalian cell growth inhibition assay

This was done using the LEE, LC2 and the isolated compound 1. Briefly mammalian HeLa cells were plated in 96-well plates at 2x1O⁴ cell per well in 150 μL the culture medium. The culture medium was prepared from Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5mM L-glutamine, 10% (v/v) fetal bovine serum and antibiotics (penicillin/streptopmycin/amphotericin B). After an overnight incubation in a 5% CO₂ humidified incubator, the various concentration (0.006104 -100 μg/mL) of the test samples prepared following a 4-fold serial dilutions approach in 96-well plates were added to the cultures (duplicate wells; 200 μL final culture volume) and incubation continued for an additional 48 hours. The viability of cells in individual wells was assessed by adding 20 (xL of resazurin toxicology reagent (Sigma-Aldrich) per well and measuring fluorescence intensity (exc. 560 nm/em. 590 nm) in a Spectramax M3 plate reader after an incubation of 2 hours. Fluorescence readings in experimental wells were converted to % cell viability relative to control wells containing untreated cells and used to obtain the dose-response plots of mean % cell viability against log (test sample concentration) using the non-linear regression function of Microsoft Excel 2007 software with the median inhibition concentration IC₅₀ values derived from the plot by extrapolation. Emetine various concentration (0.000005 -50 μM) prepared following a 10-fold serial dilutions approach in 96-well plates was used as standard drug for comparison.

CONCLUSIONS

This study is reporting for the first time, the anti-malarial potential of the Nigerian lichen Dirinaria picta epiphytic on E. guineense as well as the isolation and structural elucidation of the novel depside 1 -a methylallyl enol carboxylic acid derivative of atranorin as an anti-malarial drug lead compound. Further work is ongoing to isolate the constituents in LEE as well as LC2 and to evaluate them for anti-plasmodial activity.

ACKNOWLEDGEMENT

AOE gratefully acknowledged financial support from the Royal Society of Chemistry JWT Jones travelling Fellowship award of 28^th August 2015 to visit Rhodes University, Grahamstown, South Africa where the spectroscopy and bioassay components of this project was done, and Dr Andre Aptroot of the ABL Herbarium The Netherlands for assisting in the authentication of the lichen species used in this study. The bioassay component of the project was funded by the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package. XSN is grateful for a Rhodes University Postdoctoral Fellowship.

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