ISOLATION AND CHARACTERISATION OF SECONDARY METABOLITES WITH ANTIPLASMODIAL ACTIVITY IN SELECTED MEDICINAL PLANTS OBTAINED FROM NIGER STATE, NIGERIA

ABSTRACT

Malaria continues to be a global burden as the efficacy of most anti-malarial drugs have been compromised by the evolution of resistant parasites. Plants present unlimited sources of novel substances with many therapeutic potentials. This study was designed to obtain bioactive compounds with antiplasmodial potential and high safety margin from selected medicinal plants that could be useful against both sensitive and resistant parasites. Eight medicinal plants; namely Agelanthus dodoneifolius, Securidaca longepedunculata, Neocarya macrophylla, Merremia hederacea, Zanthoxylum zanthoxyloides, Leptadenia hastata, Polycarpea linearfolia  and  Lophira  alata collected in  Niger State,  Nigeria,  extracted in methanol were subjected to phytochemical screening and acute toxicity study using standard methods. The plant extracts were screened for antiplasmodial activity in vitro against chloroquine-sensitive  (CQS)  strain,  NF54  and  chloroquine-resistant  strain  (CQR)  K1  of Plasmodium falciparum while the in vivo activity was tested against CQS(NPK) strain of Plasmodium berghei at 100, 200 and 400 mg/kg bw.  Subchronic toxicological screening of the most active extracts (P. linearfolia and L. hastata) was carried out at 200 mg/kg bw orally for 28 days in rats. Biochemical and haematological parameters were monitored while histopathological examination of the liver, kidney, heart and spleen of test animal and control groups were also undertaken. The most active extracts in vivo were subjected to bioassay guided fractionation. 1H NMR, 13C NMR and HPLC-ESI/MS were used to characterize active compounds from the potent fractions.    The plant extracts contained a variety of phytochemicals, except phytosteroids that was absent in A. dodoneifolius. Acute toxicity study revealed P. linearfolia was safest with LD50 value of 4500 mg/kg bw. Z. zanthoxyloides recorded highest in vitro inhibition against CQS and CQR with IC50 of 1.07 and 1.31 µg/ml respectively.  In the in vivo activity of the tested extracts, the parasite density of L. hastata (576 parasites/µl) and P. linearfolia (473 parasites/µl) at 400 mg/kg bw were comparable to the standard control (431 parasites/µl). Mean survival time of L. hastata and P. linearfolia treated groups were also comparable to the standard in the range of 31-35 days. Biochemical parameters of the rats for the 2 extracts administered subchronically revealed a significant (p<0.05) increase in creatinine, bilirubin and HDL concentrations when compared to the control. However, the LDL concentrations of the treated groups decreased significantly (p<0.05). Serum glucose concentration decreased while AST activity increased significantly (p<0.05) for P. linearfolia treated group when compared to the control. RBC count, haemoglobin concentration and haematocrit of P. linearfolia treated group decreased significantly(p<0.05) compared to the control. White blood cell indices, of L. hastata group increased significantly(p<0.05) compared  to the control. Histopathological result showed normal cell architecture of all the organs analysed except the spleen that showed hyperplastic lymphoid follicles in L. hastata treated group.  The six fractions of L. hastata: Lh1, Lh2 and Lh3 had IC50 value higher than10 µg/ml, while Lh4A, Lh4B and Lh5 had IC50 values of 4.24,8.50 and 7.24 µg/ml. All the six fractions of P. linearfolia, had IC50 higher 10 µg/ml except Tk3 with a value of 2.40 µg/ml.  Administration of fractions Lh3 of L. hastata and Tk3 of P. linearfolia resulted in complete parasite clearance on day 14 post infection. Spectral analysis identified two antiplasmodial compounds: 2,2‘ –methylene bis(6-tert-butyl-4-methylphenol) and Sclerinone A in P. linearfolia extract, and 2,2‘ –methylene bis(6-tert-butyl-4- methylphenol) in L. hastata. These antiplasmodial agents could serve as templates for the synthesis of new antimalarial drugs operating synergistically against both CQ sensitive and resistant strains of Plasmodium falciparum.

CHAPTER ONE

1.0       INTRODUCTION

1.1       Background to the Study

Malaria is an infection caused by Plasmodium protozoan parasite, a major human health threat with almost half of the world’s population at risk (Asrar et al., 2014, WHO, 2015). Malaria is a typical example of a disease that influences the efficiency of individuals, families and the society as a whole (Naghibi et al.,2013). The global incidence of malaria cases in 2015 is 214 million, with African continent being most susceptible (WHO, 2015). The global malaria mortality recorded in 2015 is 438, 000. Africa region has the highest (90%) mortality rate, followed by south East region (7%) and Eastern Mediterranean region (2%) with having the least mortality rate. Nigeria and Democratic Republic of Congo carries 35 % of these deaths burden (WHO, 2016).

The human malaria is caused by six Plasmodium genus species, P. malariae, P. falciparum, P. vivax, P. ovale, P. knowlesi and P. simium. The later is solely found in the Brazilian Atlantic Forest (Grigg and Snounou, 2017). P. falciparum is the most virulent specie and important cause of morbidity and mortality (Akkawi et al., 2014). Female Anopheles mosquitoes are the vectors for transmission of malaria parasites. (David-Bosne et al.,2013). These Plasmodium parasites are transmitted by over 70 species of Anopheles mosquitoes (Thota and Yerra, 2016).

Clinical manifestation of malaria include fever, protraction and anaemia. Acute forms of the disease can result in delirium, metabolic acidosis, multi organ system failure, coma and death eventully if untreated (Fidock et al.,2004). Proinflammatory cytokines are released as part of the pathogenesis of acute malaria (Aguiar et al.,2012a) and these cytokines contribute to the suppression of erythropoiesis. There are many anti- inflammatory and analgesics drugs in the market use in modern treatment but they are not devoid of adverse drug effects (Osadebe and Okoye, 2003). Thus, new drugs with less detrimental effects are needed and warranting their search.

The problem of mosquito vector resistance to insecticides and parasite resistance, especially Plasmodium falciparum to the commonly available antimalaria drugs has resulted in recrudescence of malaria (Naghibi et al.,2013). Therefore, new drugs need to be studied and produced from plants that have not yet been exposed to drug pressure in order to triumph over the issue of emerging resistance and putting into cognisance the safety and affordability of the new drug. Floral diversity offers the hope for such novel agents Medicinal  plants  have  been  used  in  the  treatment  of  parasitic  diseases  since  time immemorial.  Plants like Cinchona succiruba (Rubiaceae) have history for the treatment of malaria infection. Numerous compounds isolated from rich natural resources form various structures for optimization to obtain improved therapeutics (Vangapandu  et al.,2007). Some of such components include alkaloids, flavonoids and terpenoids. Alkaloids are physiologically active nitrogenous base secondary metabolites found in plants,  fungi,  bacteria  and  marine  organism  (Kaur  et  al.,2009).  Literature  search indicated that a host of antiplasmodial alkaloids have been derived from African flora, ranging from indole alkaloids, amides, cryptolepines and many yet to be identified (Onguine-Amoe et al.,2013). Several flavonoids from medicinal plants, as well as from dietary sources have been found to possess in vitro and in vivo antiplasmodial actvity against both sensitive- and resistant- strains of P. falciparum. Examples of flavonoids having  antimalarial  activity are  acacetin,  genistein,  baicaclein,  kaempferol,  chrysin, hesperetin, quercetin, isoquercetin, luteolin, naringenin and myricetin, just to name a few  with  such  potency  (Rudrapal  and  Chetia,  2017).  Terpene  is  the  second  non- nitrogenous secondary metabolite apart from flavononids, having numerous reports of its efficacy as antiplasmodial agent (Hussein and El-Anssary, 2018). The common ones are artemisinin and its derivatives (examples include artemether, artesunate, arteether and B-dihydroartemisinin) that has come as an alternative to Chloroquine in malaria treatment (Brown, 2010). Similarly, nerolidol, balsaminoside B, farnesol, karavilagenin C, limonene, and the karavoates B and D are also terpenes that have exhibited greater in vivo and in vitro antimalarial activity against strains of P. berghei and P. falciparum respectively (Grabriel  et al.,2016  and  Batisa  et  al.,2009).  Many of these bioactive compounds are yet to be discovered. The use of such compounds as antiplasmodial agents can only be enchanced when empirical evidences exist for their efficacy and toxicity profile. They could also be used as template for the synthesis of more active, less toxic drug derivatives because drug efficacy, pharmacology and toxicity are important parameters in the selection of compounds for development.

1.2       Statement of the Research Problem

Despite intensive efforts to control malaria, its devastating heatlh effects remain unabated. Ninety five percent of global malarial burden is in sub-Saharan Africa. Most vulnerable group are pregnant women and children under the age of five. The numbers of  easily  available,  affordable  and  effective  antimalarial  drugs  are  few.  Viable multistage vaccines for the disease are unavailable. The situation is further compounded by the spread and intensification of drug resistant malaria parasites. The morbidity and mortality from the diseases are on upward trend. WHO recent estimates put annual global  malaria  infection  at  350-500  million,  with  about  1-2  million  fatalities.  The disease   accounts   for   over   100,000   deaths,   60%   outpatient   visits   and   30% hospitalizations in Nigeria (WHO, 2016).

The economic impacts of malaria infection in endemic countries cause significant loses in gross domestic product annually. In Africa 12 billion dollars is lost due to malaria infection every year and the disease also consumes forty percent of all public health expenditure.   (Zelefack   et   al.,2012).   Malaria   is   a   major   obstacle   to   economic development and a cause of poverty in areas of it‘s prevalence. In Africa, 89 % of malaria control schemes and commodities are financed by global programs and 11 % by local governments (Nkumama et al.,2017).

1.3       Justification for the Study

Antimalaria drugs such as sulphadoxine-pyrimethamine, artemisinin and chloroquine have  been  compromised  by  drug  resistant  P.  falciparum.  Fixed  dose  ACT  drug regimens are also being threatened by treatment failure. Sourcing for effective new drugs especially from plant sources is thus justifiable. Quinine, the original antimalarial alkaloid  was  isolated  from  Cinchona  ledgeriana  and  Artermisinin  from  Artemisia annua, hence plants may provide yet another active antimalaria drug. The threat from malaria thus necessitates screening of medicinal plants with reputation in the treatment of the disease for leads that can be developed into new generation of cures. Hence, there is need to source and advance the course for new drugs development from medicinal plants having folklore background in the malaria treatment with the aim of surpassing the problem of emerging resistance, such drugs should be safe and affordable. The selected plants are claimed by traditional healers to be of importance in the management of malaria.

Herbal medicaments have gotten global application, acceptability and efficacy which have predispose millions of people to the fundamental but rarely reported problems of herbal toxicity and mis-adventuring. Most herbal treatments are in the forms of crude or partially purified extracts which are often dispensed over a period of time with the chances of accruing organ toxicity and even death among users (Yuan et al.,2016). The need for in vitro and in vivo toxicological profiling of such new drugs as an integral component in their development is imperative (Jigam et al.,2012). Such routine evaluations include acute toxicity studies which are critical as they provide a rough idea about the nature of the medicinal extract in addition to determining safe doses for clinical use. Sub-chronic and chronic studies are necessary in the elucidation of target organs of toxicity and demonstration of dose-response relationships.

1.4       Aim and Objectives of the Study

1.4.1    Aim

The  aim  of  this  study  is  to  isolate  and  characterize  secondary  metabolite  with antiplasmodial potential from selected medicinal plants in Niger State, Nigeria.

1.4.2    Objectives

The objectives of this study are to:

i.      determine the percentage yield of the crude extracts from selected medicinal plants

ii.      determine  the  qualitative  and  quantitative  phytochemical  constituents  of  the crude plant extracts

iii.     determine acute toxicity of the crude extracts.

iv.      screen crude plant extracts for antiplasmodial activity

v.      determine subchronic toxicological effects of the most active extracts

vi.      fractionate the most active extract and test for their antiplasmodial activity.

vii.     determine the spectral characteristics of the most active fraction

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