Journal of Ethnopharmacology 169 (2015) 176–182

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Clerodane diterpenes from Polyalthia longifolia (Sonn) Thw. var. pendula: Potential antimalarial agents for drug resistant Plasmodium falciparum infection Stephen Y. Gbedema a,c,n, Marcel T. Bayor a, Kofi Annan b, Colin W. Wright c a Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana b Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana c School of Pharmacy, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK

art ic l e i nf o

a b s t r a c t

Article history: Received 16 February 2015 Received in revised form 9 April 2015 Accepted 13 April 2015 Available online 23 April 2015

Background: Plasmodium falciparum drug resistance is a major public health challenge in sub-Sahara Africa. Many people are now resorting to the use of herbs in managing malaria due to the increasing treatment failures with the conventional drugs. In this study the ethanolic extract of Polyalthia longifolia (Sonn) Thw. var. pendula, a variety fondly used in folklore medicine in Ghana was investigated for potential antimalarial drug development. Method: The ethanolic extract of P. longifolia (Sonn) Thw. var. pendula stem bark was screened against the multidrug resistant, K1 strain of P. falciparum by the parasite lactate dehydrogenase (pLDH) assay and a good antiplasmodial activity (IC50 22.04 7 4.23 mg/ml) was observed which led to further chromatographic analysis in search for actives. Results: Bioassay guided fractionation of the extract yielded; three clerodane diterpenes [16-hydroxycleroda-3,13-dien-16,15-olide (1), 16-oxocleroda-3,13E-dien-15-oic acid (2) and 3,16-dihydroxycleroda-4(18),13 (14)Z-dien-15,16-olide (3)], a steroid [beta-stigmasterol (4)] and two alkaloids [darienine (5) and stepholidine (6)]. While compounds 4, 5 and 6 exhibited weak antiplasmodial activity (IC50 22–105 mg/ml), the clerodane diterpenes exhibited significantly potent (po0.005) blood schizonticidal activity (IC50: 3–6 mg/ml). This is the first report of the antiplasmodial activity of compounds 2 and 3. In combination assay with chloroquine, compounds 1, 2, 3 and 5 antagonized the antiplasmodial activity of chloroquine while 4 and 6 demonstrated a synergistic action. Conclusion: The potent antiplasmodial activity of the extract of P. longifolia (Sonn) Thw. var. pendula and compounds therein strongly suggests its usefulness as an antimalarial agent and supports its inclusion or exploitation in formulations of herbal remedies for malaria in Ghana. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Malaria Synergistic action Parasite lactate dehydrogenase Herbal remedies

1. Introduction Malaria is an important tropical infectious disease of concern, ranking high among the WHO listing of ‘Infectious diseases of poverty’. It remains a major cause of morbidity and mortality world-wide; exerting a huge negative impact on population health and economic development in countries where it is endemic (WHO, 2011; Sachs and Malaney, 2002). The WHO report indicated 216 million cases of malaria globally with 655,000 deaths in 2010. n Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. E-mail address: [email protected] (S.Y. Gbedema).

http://dx.doi.org/10.1016/j.jep.2015.04.014 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

Out of this, 174 million cases with 596,000 malaria deaths occurred in sub-Sahara Africa (Schwartz et al., 2012; WHO, 2012). Malaria therefore, remains a major public health challenge and a significant economic burden in Africa. Plasmodium falciparum, is established as the commonest malaria parasite in West Africa, transmitted by the female Anopheles mosquito, which injects the sporozoites into humans during probing for a blood meal. The sporozoites are rapidly taken up into the liver where they replicate and develop into merozoites. The merozoites when released into the bloodstream, quickly invade erythrocytes to begin the erythrocytic cycle. In high parasitaemia, the parasites can become sequestered within the blood capillaries of major internal organs like the brain, resulting

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of Pharmacognosy, KNUST. The material was sun-dried and milled into a coarse powder for extraction. 2.2. Extraction and isolation of compounds

Fig. 1. Polyalthia longifolia (Sonn) Thw. var. pendula.

in cerebral malaria in high-risk groups, particularly children and pregnant women. Currently there are no ideal options for the eradication of the disease in sub-Sahara Africa. Vector control and the use of chemotherapeutic drugs are the main measures available for control of the disease since there is no licensed malaria vaccine. Prevention of malaria related morbidity and mortality therefore depends largely on early treatment of the infection. However, there are many limitations associated with current therapy. Very few health facilities exist in poor endemic countries and are mainly located in the urban centers, hardly accessible to the most susceptible and vulnerable rural populace. The cost of treatment is also not affordable to a large majority, resulting in the influx and distribution of cheap, fake and sub-standard antimalarial drugs (Newton et al., 2006) with widespread treatment failures and the development of resistance by P. falciparum to most of these agents (Wellems, 2002; Riddley, 2002; Jambou et al., 2005). As such, the reliance on herbs as first line of treatment for malaria among the poor and the rural populace is still high in Ghana and the subregion. Polyalthia longifolia (Sonn) Thwaites variety pendula (family Annonaceae) is native to India and now widely cultivated in the tropical and subtropical regions of Asia and Africa. It is an evergreen, tall (up to 12 m) and slender tree that grows symmetrically and produces fresh and shining green foliage (Fig. 1). Its peculiar dropping branching habit gives the plant a handsome narrow columnar shape. These features encourage its use as a hedge around buildings for beauty, shade and as a wind break. This makes it readily available and is used in many traditional systems of medicine for the treatment of various ailments notably malaria (Ichino et al., 2006; Verma et al., 2008). However, apart from a report by Annan et al. (2015) which investigated the use of this plant species in the treatment of chloroquine-sensitive malaria, its effectiveness in the treatment of multidrug-resistant P. falciparum infection, a major public health challenge in Ghana, is yet to be ascertained. As such, the current study sought to investigate and evaluate the antiplasmodial activity of extracts, fractions and isolates of P. longifolia (Sonn) Thw. var. pendula growing in Ghana, against the multidrug-resistant P. falciparum, and this we now report.

2. Method 2.1. Plant material The stem bark of P. longifolia (Sonn) Thw. var. pendula was collected in June 2011 from Tikrom near Kumasi, Ghana. The plant was identified and authenticated by Dr. G.H. Sam of the Department of Herbal Medicine, KNUST, Kumasi. A voucher specimen (PL-01/11/031) was archived in the herbarium of the Department

One kilogram (1.0 kg) of the powdered material was macerated in 5 l of 70% ethanol at room temperature for 3 days and filtered. The marc was extracted 2 more times with fresh menstruum. The filtrates were pooled, concentrated to a syrupy mass at 40 1C in a rotary evaporator (Buchi, Swaziland) and then dried in vacuo at room temperature (yield 2.28% w/w). Twenty-one grams (21.0 g) of the extract was fractionated by a flash column chromatography (Still, 2002; Still et al., 1978) on 50 g of silica gel 60G (average particle size 5–40 mm; Merck) by sequential elution with 1.50 L each of n-hexane, dichloromethane, ethyl acetate and methanol– ethyl acetate (1: 9). The ethyl acetate fraction (7.25 g) which exhibited the highest level of antiplasmodial activity (Table 2) was further fractionated repetitively on column chromatography using n-hexane, graded with quantities of ethyl acetate (n-hexane: ethyl acetate 100:0, 99:1, 98:2, 96:4 up to 0:100) followed by analytical and preparative TLC, leading to the isolation of compounds 1 (2.7 g), 2 (0.03 g) and 3 (0.014 g). The dichloromethane and methanol–ethyl acetate (1: 9) fractions were also similarly fractionated to yield compounds 4 and 5, and 6, respectively. The isolated compounds were spectroscopically analyzed and subsequently identified as; 16hydroxycleroda-3,13-dien-16,15-olide (1), 16-oxocleroda-3,13(14) E-dien-15-oic acid (2) and 3,16-dihydroxycleroda-4(18),13(14)Zdien-15,16-olide (3), beta-stigmasterol (4), darienine (5) and L-stepholidine (6). 2.3. Parasite culture and maintenance Multidrug resistant P. falciparum (K1 strain), obtained from Prof. D.C. Warhurt of the London School of Hygiene and Tropical Medicine, UK, was maintained in continuous culture with stock stored in liquid nitrogen. Culture media was made up of RPMI 1640 medium (SigmaAldrich, St. Louis, MO, UK) supplemented with D-glucose (2.0 g/l), NaHCO3 (2.3 g/l), TES buffer (2-[[1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl]amino]ethanesulfonic acid) (9.2 g/l) and gentamicin (0.04 g/l) (Sutar et al., 1992). Serum was prepared in our laboratory from human A þ blood purchased from Yorkshire Blood Transfusion Centre, Leeds, UK. Malaria parasites were maintained in A þ erythrocytes in culture medium supplemented with A þ serum according to the methods of Trager and Jensen (1976) and Fairlamb et al. (1985). Parasitaemia estimation as well as parasite visualization was done using light microscopy of Giemsa-stained slides under oil immersion. 2.4. Determination of in vitro antiplasmodial activity Drug sensitivity assays were carried out in 96-well flat bottomed microtitre plates as described by Desjardins et al. (1979) with some modifications (Addae-Kyereme et al., 2001). The crude extracts were dissolved in dimethyl sulfoxide (200 mL) and prediluted with culture medium to make a final concentration of 1000 mg/ml. All the drug solutions used were freshly prepared and sterilized by passing through a 0.2 mm syringe filter. Artemether and chloroquine diphosphate were used as reference drugs. Ring stage infected erythrocytes (50 ml per well with 2.5% hematocrit and 1.5% parasitaemia) were incubated in duplicates with two-fold serial dilutions of each drug for 48 h. Parasitaemia was measured using the parasite lactate dehydrogenase (pLDH) assay (Makler and Hinrichs, 1993; Malik et al., 2004).

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O

O

O

O

O

O OH

OH

OH

HO

(2)

(1)

(3)

O

H

HO

H3CO

H

H

OCH3

(4)

HO

N

(5)

H3CO N

HO H OCH3

(6)

2.5. Parasite lactate dehydrogenase (pLDH) assay Nitroblue tetrazolium (NBT) (1 mg/ml) and phenazine ethosulphate (PES) (0.05 mg/ml) solutions were mixed in a ratio of 20:1 and kept in an aluminum foil-covered container. APAD reagent was also prepared by dissolving 3-acetyl pyridine adenine dinucleotide (APAD) (0.74 g/1), lithium lactate (19.2 g/l), diaphorase (0.1 g/l) and triton X 100 (2 ml/1) in tris buffer (pH 9.2). All chemicals were purchased from Sigma-Aldrich Inc., St. Louis, MO, UK and all reagents used were freshly prepared. The APAD reagent (50 ml) and NBT/PES solution (50 ml) were added to each well and mixed thoroughly thereby initiating the lactate dehydrogenase reaction. Color development of the LDH plate was monitored colorimetrically at 650 nm with the aid of a plate reader (MRX Dynatech Laboratories, California, USA) after half an hour of incubation in the dark at 37 1C. Three independent experiments were conducted in each case and by means of Microsoft Excel software (Microsoft Inc.) IC50 values were calculated from log dose-response curves. 2.6. Drug combination assay Various dilutions of chloroquine in combinations with each of the isolated compounds were prepared in fixed ratios (Table 1) as

OH

Table 1 Fixed ratio combinations of chloroquine and isolated compounds. Solution Chloroquine: compound ratio

Chloroquine (10 lg/ml)

Compound 1–6 (10 lg/ml)

0 ml 2.0 ml 4.0 ml 6.0 ml 8.0 ml 10.0 ml

10.0 ml 8.0 ml 6.0 ml 4.0 ml 2.0 ml 0 ml

Chloroquine Compound 1–6 1 2 3 4 5 6

0 1 2 3 4 5

5 4 3 2 1 0

described by Fivelman et al. (2004). Parasitized red blood cells were added to the various drug combination solutions which were serially diluted with complete RPMI 1640 medium in flat-bottom 96-well microtiter plates. The antiplasmodial activity was evaluated against the K1 strain of P. falciparum using the pLDH method. Verapamil was used as a reference chemo-sensitizing agent. Three independent experiments were conducted and isobolic curves were plotted from the fractional IC50 (FIC) values obtained.

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3. Results and discussion Plants have played a dominant role in the discovery of antimalarials as exemplified by quinine and artemisinin. The anticipation that new compounds or leads for even more effective antimalarials against the fast spreading multidrug resistant P. falciparum strains could still emerge from plant sources cannot be overemphasized. In pursuance of this objective, this study sought to investigate the antiplasmodial activity of extracts of the stem back of P. longifolia (Sonn) Thw. var. pendula, a species widely used locally for treating malaria in Ghana. In accordance with the criteria adopted by Gessler et al. (1994) and Omoregie and Sisodia (2012) in classifying antiplasmodially active extracts, the ethanolic extract of the species was ‘very active’ (IC50 r25 mg/ml) against the multidrug resistant K1 strain of P. falciparum (Table 2). These initial results appeared to lend some scientific support to the use of P. longifolia (Sonn) Thw. var. pendula in treating malaria and encouraged further investigations. The sequentially fractionated portions of the extract, in n-hexane, dichloromethane, ethyl acetate, and methanol–ethyl acetate (1:9), also exhibited potent antiplasmodial activity (IC50 10.07–47.80 mg/ml). These results are consistent with reports (in the literature) of potent antiplasmodial activity associated with extracts of P. longifolia (Gbedema, 2014) and other species of Polyalthia: extracts of Polyalthia avecta roots and Polyalthia viridis stem bark have been indicated to be active against the multidrug resistant K1 strain of P. falciparum (IC50 20 mg/ml and 10 mg/ml, respectively) (Kanokmedhakul et al., 2005; Kanokmedhakul et al., 2003; Ichino et al., 2006). The aqueous, methanolic and hexane fractions of Polyalthia oliveri stem bark extract were reported to have exhibited potent activity (IC50 0.05–8.09 mg/ml) against the Cameroonian field isolates of P. falciparum (Kemgne et al., 2012). Similarly, the hexane extract of Polyalthia debilis was reported as active (IC50 10–100 mg/ml) against the chloroquine resistant strains of P. faciparum (T9.94) (Prachayasittikul et al., 2009). However, the results of this current study appear to be the first report of antiplasmodial activity of extracts of P. longifolia (Sonn) Thw. var. pendula stem bark against the multidrug resistant malaria parasite. Spectroscopic analysis of compounds isolated from this extract led to the characterization and identification of three of them as the clerodane diterpenes [16-hydroxycleroda-3,13(14)-dien-16,15olide (1); 16-oxocleroda-3,13(14)E-dien-15-oic acid (2); and 3,16dihydroxycleroda-4(18),13(14) Z-dien-15,16-olide (3)] (Phadnis et al., 1988; Hara et al., 1995). The rest were; a steroid [betastigmasterol (4)] (Habib et al., 2007; Woldeyes et al., 2012) and two alkaloids [darienine (5) and L-stepholidine (6)] (Arango et al.,

179

1987; Tavares et al., 2005). These compounds exhibited varying degrees of antiplasmodial activity against the blood schizonts of the multidrug resistant K1 strain of P. falciparum (Table 2). According to Basco et al. (1994) and Batista et al. (2009) antiplasmodial activity of compounds can be classified as ‘very good’ (IC50 1–20 mM); ‘good’ (IC50 20–100 mM), ‘weak’ (IC50 100– 200 mM) and ‘inactive’ (IC50 4200 mM). Based on this criteria, compounds 1, 2 and 3 (the clerodane diterpenes) exhibited ‘very good’ antiplasmodial activity (IC50 9.59–18.41 mM) (Table 2) that lent further credence to the use of P. longifolia (Sonn) Thw. var. pendula as an antimalarial agent. Annan et al. (2015) have recently reported the antiplasmodial activity (IC50 4.5–213.8 mM) of six clerodane diterpenes (16-hydroxycleroda-3,13(14)-dien-15-oic acid; bisclerodane imide; cleroda-3-ene,pyrole-15,16-dione; cleroda-3ene,pyrolidine-15,16-dione; cleroda-3,13(14)-dien-15,16-diamide; and cleroda-3-ene-15,16-diamide) isolated from this species of Polyalthia against chloroquine-sensitive 3D7 strain of P. falciparum. Ichino et al. (2006) also reported antiplasmodial activity (IC50 11.32 mM) of 16-hydroxycleroda-3,13(14)-dien-16,15-olide (1) which was isolated from the ethanol extract of P. viridis leaves. Some other bioactivities, reported of compound 1 together with 2 and 3, are anti-tumor, antimicrobial, antileishmanial, anti-inflammatory, antidyslipidemic and antioxidant properties (Ma et al., 1994; Hagiwara et al., 1995; Rashid et al., 1996; Sashidhara et al., 2009; Katkar et al., 2010). This is however, the first report of the antiplasmodial activity of compounds 2 and 3, with compound 2 (16-oxocleroda-3,13(14)Edien-15-oic acid) being the most active (IC50 9.59 mM). Other clerodane diterpenes have been reported to display varying degrees of antiplasmodial activity. Gomphostenin and gomphostenin-A isolated from Gomphostemma niveum (Lamiaceae) exhibited antiplasmodial activity (IC50 114.90 and 9.80 mM, respectively) against MRC-02 strain of P. falciparum (Sathe and Kaushik, 2010). Casearlucin A, casamembrol A and laetiaprocerine A–D, all of which were isolated from Laetia procera (Flacourtiaceae) stem bark, reportedly showed ‘good to moderate’ antiplasmodial activity (IC50 0.54–27.5 mM) against both F32 and FCb1 strains of the parasite (Jullian et al., 2005). Others are the caseargrewiins A–D, isolated from Casearia grewiifolia (Flacourtiaceae) with potent antiplasmodial activity (IC50 3.6–7.9 mM) against K1 strain of P. falciparum (Kanokmedhakul et al., 2005). Antiplasmodial activity has, as well, been reported of various other classes of terpenes. The kaurane diterpene ent-kaur-16-en19-oic acid, isolated from Schefflera umbellifera (Araliaceae) exhibited antiplasmodial activity (IC50 106.50 mM) against chloroquinesusceptible strain D10 of P. falciparum (Mthembu et al., 2010). Diterpenes, (þ )-8,11,13-totaratriene-12,13-diol and (þ )-8,11,13-

Table 2 Antiplasmodial activity of extracts and isolates of P. longifolia (Sonn) Thw. var. pendula stem bark against K1 strain of P. falciparum. Extracts/compounds/drug

IC50 7 SD (lg/ml)

Ethanolic extract n-Hexane fraction Dichloromethane fraction Ethyl acetate fraction Methanol–ethyl acetate (1: 9) fraction 16-Hydroxycleroda-3,13-dien-16,15-olide (1) 16-Oxocleroda-3,13(14)E-dien-15-oic acid (2) 3,16-Dihydroxycleroda-4(18),13(14)Z-dien-15,16-olide (3) Beta-Stigmasterol (4) Darienine (5) L-Stepholidine (6) Artemether Chloroquine diphosphate SD ¼standard deviation, (n ¼3).

22.047 4.23 47.80 7 1.33 22.917 0.76 10.077 0.18 26.067 0.97 5.337 0.70 3.05 7 0.19 6.157 0.65 63.36 7 9.39 22.05 7 1.47 104.337 8.95

(lM)

(16.767 2.20) (9.59 7 0.60) (18.41 71.95) (153.79 7 22.79) (81.377 5.42) (319.05 7 27.37) (0.0623 7 0.057) (0.4127 0.015)

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1.5 1 0.5 0

(Chanda et al., 2012), extracts from the plant have rather displayed potent gastroprotective action against ethanol/HCl induced gastric mucosa damage (Chanda et al., 2011). Research has shown that significantly less chloroquine accumulation occurs in chloroquine-resistant P. falciparum as compared to the sensitive parasites (Wellems and Plowe, 2001; Henry et al., 2006) and that could be responsible for the treatment failures associated with chloroquine. Chloroquine is more available, inexpensive and affordable; hence the development of an agent that can increase its accumulation in drug resistant parasites (Pradines et al., 2002) may enable the reuse of this drug in regions with chloroquine-resistant malaria and help reduce their malaria burden. In our study, the isobolic curves constructed for the various combination solutions depicted the interactions of chloroquine with the isolates (Fig. 2). The IC50s of Chloroquine alone, isolated compounds or reference drugs alone were normalized to one unit. In each combination, IC50 was plotted as a fraction of each agent alone IC50 (FIC). Concave and convex isobolic curves respectively, reveal synergistic and antagonistic effects of chloroquine and isolated compounds or reference drugs. The straight line is the additive effect.

FIC compound 2

FIC compound 1

abietatrien-12-ol, isolated from Harpagophytum procumbens (devil's claw) also displayed excellent antiplasmodial activity (IC50 o1 mM) against K1 and D10 (chloroquine-sensitive) strains of P. falciparum (Clarkson et al., 2003). Another terpenoid compound that has revolutionised the treatment of multidrug resistant P. falciparum infection worldwide is artemisinin, isolated from Artemisia annua. Compound 1, 2 and 3 could therefore be useful leads for the discovery of other more effective antimalarial chemotherapeutic drugs. The safety of P. longifolia (Sonn) Thw. var. pendula as a medicinal plant in humans can be predicted from the long history of oral use in many traditional settings across the globe and a number of cytotoxic studies conducted on the phytochemical compounds and extracts obtained from it. Compound 1, 2, 3 and other clerodane diterpenes isolated from this plant species have shown potent anti-tumor activity against a number of human cancer cell lines but no significant cytotoxic effects were observed in normal human cells including macrophages (Chang et al., 2006; Lee et al., 2009; Sashidhara et al., 2010; Misra et al., 2010). Also, while acute oral toxicity study of the methanol extract did not show any significant toxic effect in the wistar albino rats used

0

0.5

1

1

0.5

0

0

1

0.5

0

0

0.5

0.5

0

1

0

FIC compound 6

FIC compound 5

0.5

0.5

0.5

1

1

0.5

0 0

0.5 FIC Chloroquine

FIC Chloroquine

FIC Verapamil

1

0.5

0 0

0.5 FIC Chloroquine

1

FIC Chloroquine

1

0

1

1

FIC Chloroquine

0

0.5 FIC Chloroquine

FIC compound 4

FIC compound 3

FIC Chloroquine

1

Fig. 2. Isobolic graphs of chloroquine diphosphate in combination with isolated compounds.

1

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Compounds 4, 5 and 6 did not show any appreciable antiplasmodial activity against the drug resistant K1 strain of P. falciparum; but 4 and 6 appeared to have synergized the action of chloroquine diphosphate against the parasite. Some structural modifications of these two compounds may improve their synergistic actions. Compounds 1, 2, 3 and 5 however, antagonized the action of chloroquine. This is also the first report of chloroquine combination studies of the isolated compounds. Though in vitro studies do not necessarily correlate with human in vivo studies, these results seem to indicate some clinical consequences of concomitant administration of phytomedicines containing these compounds and some conventional drugs. While co-administration that leads to synergy may result in toxic reactions, antagonism will cause therapeutic failures (Fasinu et al., 2012); and must be avoided when using remedies of P. longifolia in treating malaria.

4. Conclusion The stem bark extract of P. longifolia (Sonn) Thw. var. pendula together with three compounds (1, 2 and 3) isolated therein had displayed potent antiplasmodial activity that strongly supports its use in phytomedicines for treating malaria in Ghana. The active compounds (1, 2, 3 and 5) however, antagonized the action of chloroquine against K1 strain of P. falciparum. Meanwhile, the antiplasmodially inactive compounds (4 and 6), apparently synergized the action of chloroquine diphosphate against the malaria parasite. A serious clinical concern of this study is a potential herbdrug interaction that may result from concurrent administration of P. longifolia phytomedicines and conventional drugs used especially in the treatment of malaria. Acknowledgments The authors are grateful to the Commonwealth Scholarship Commission, United Kingdom, and the Government of Ghana for providing funds for the study (Scholarship number: GHCN-201122). We are also grateful to Prof. D.C. Warhurt (London School of Hygiene and Tropical Medicine, UK) for providing us with the malaria parasites.

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