Evaluation of the antimalarial properties and safety profile of a polyherbal product (NEFANG)
Background: The emerging resistance of Plasmodium species to currently available antimalarials remains a public health concern and necessitates the need to develop new, effective, safe and affordable drugs. Compounds with antioxidant, anti-inflammatory and antipyretic activities are believed to modulate plasmodial infection. Natural products constitute and remain a reliable and alternative source. Nefang is a polyherbal preparation composed of Mangifera indica (bark-MiB and leaf-MiL) and leaves of Psidium guajava (Pg), Carica papaya (Cp), Cymbopogon citratus (Cc), Citrus sinensis (Cs) and Ocimum gratissimum (Og). It is used traditionally for the treatment of malaria in the South West Region of Cameroon. There are no documented studies on the efficacy and safety of this product. OBJECTIVES: The present study aimed at evaluating the efficacy and safety of Nefang and its constituents, for antiplasmodial and related biological activities useful for the treatment of malaria, using in vitro and in vivo techniques. METHODS: An ethnopharmacological survey was carried out to document the folkloric information on formulation and use of Nefang. Preliminary phytochemical screening of the constituent plants was carried out. Cytotoxicity was determined by the Resazurin Fluorimetric Cell Viability Assay method using two human cell lines (Hep G2 and U2OS). The in vitro antiplasmodial activity of the extracts was analyzed on CQ-sensitive (3D7) and multi-drug resistant (Dd2) strains of Plasmodium falciparum using the SYBR-Green 1 fluorescence-based method. Interactions studies involving the different solvent extracts were further carried out using a variable potency ratio drug/extract combination approach and isobologram analysis. In vivo antiplasmodial activities of Nefang and its active components, during early and established infection as well as the prophylactic activity were investigated in rodent models infected with P. berghei and P. chabaudi chabaudi, using the Peter’s 4-day suppressive activity, Rane’s curative tests and repository activity methods respectively. In vivo toxicity of Nefang and its constituents was assessed by the single-dose (acute) and repeated dose (sub-acute and sub-chronic) toxicity testing on rodent models. Evaluation of the constituent plants for their antioxidant, antipyretic, anti-inflammatory and antinociceptive antioxidant activities were determined using the radical scavenging activity, total phenolic content estimation and ferric reducing antioxidant power (FRAP) assay methods while in vivo activity of Nefang was evaluated in carbon tetrachloride-induced oxidative stressed wistar rats. Antipyretic activities were determined using the D-amphetamine and Brewer’s Yeast induced pyrexia methods. Anti-inflammatory activities were determined using the carrageenan-induced rat paw edema method while antinociceptive activities were determined using the tail pressure, tail flick and hot plate methods. RESULTS: The ethnopharmacological survey study revealed that the respondents had a good knowledge of malaria and its causes. They could also identify the constituent plants and recognized the need for Good Agricultural and Cultivation Practice (GACP) in the harvesting process. Five different formulations used for the preparation of Nefang were described: MiB: MiL: Pg: Cp: Cc: Cs: Og (w/w) - (i) 4:2:2:1:1:1:1 (ii) 3:1:1:1:1:1:1 (iii) 2:2:1:1:1:1:1 (iv) 2:1:1:1:1:1:1 (v) 1:1:1:1:1:1:1. Administration was by oral or rectal (enema) routes. Preliminary phytochemical screening of the constituent plant extracts of Nefang, revealed the presence of flavonoids, phenols, triterpenes and sterols in all extracts. Saponins were present in all except Cc, tannins in all except Cp and Cc while alkaloids were found only in MiB and Og. Cytotoxicity testing of Nefang and the solvent extracts revealed no significant toxicity of the extracts relative to their antiplasmodial activities (Selectivity Index>20). The derived EC50 values (3D7/Dd2, µg/mL) of the extracts were as follows: Nefang-96.96/55.08; MiB65.33/34.58, MiL-82.56/40.04, Pg-47.02/25.79, Cp-1188/317.5, Cc-723.3/141, Cs-184.4/105.1, and Og-778.5/118.9. Synergism was obtained with the following pairs: MiB/Pg (Combination Index=0.351), MiL/Pg (0.358), MiB/Cs (0.366), MiL/Cs (0.482), Pg/Cs (0.483), and Cs/Og (0.414) when analyzed at equipotency ratios. This was further confirmed at variable potency combination ratios and by isobologram analysis. Evaluation of the in vivo antiplasmodial activities revealed that Nefang and MiB/Pg exhibited significant (p<0.05) suppressive, prophylactic and curative activities when compared to the control with percentage suppression of parasitemia (P. berghei/P.c. chabaudi) of 82.9/86.3 and 79.5/81.2 respectively. Chemotherapeutic effects of Nefang ranged from 61.2 – 86.1% with prevented loss of body weight and reduction in body temperature in experimental animals. Evaluation of the in vivo toxicity revealed that no observed adverse effect levels (NOAEL) for all extracts were > 5000 mgkg-1 bwt. In the sub-acute and sub-chronic toxicity testing, plasma biochemical analysis revealed slightly significant (p<0.05) increase in some renal (blood urea nitrogen) and hepatic (transaminases) parameters after administration of Cp, Cs and Cc ethanol extracts. Aqueous extracts exhibited hypolipidemic and hypoglycemic effects in experimental rats. No significant hematological adverse effects were observed. Evaluation of biological activities: In vitro antioxidant assays revealed that Pg, MiL and MiB exhibited greatest radical scavenging activities (>90% inhibition). Their total phenolic contents ranged from 61.7 - 67.2 mg catechine equivalent/g of extract. In vivo activity of Nefang showed significant (p<0.05) increase in superoxide dismutase and decrease in malondialdehyde. Best percentage suppression of pyrexia (amphetamine/brewer’s yeast; p<0.05) was exhibited by Cc (95/97) followed by Og (85/94), MiL (90/89), MiB (88/84) and Cs (82/89), comparable to paracetamol (100/95). Anti-inflammatory studies revealed paw edema inhibition (%) as follows (p<0.05): Indomethacin (47), MiL (40), Cp (30), MiB (28) and Og (22), suggesting best activity by MiL. Antinociceptive studies revealed significant (p<0.01) pain inhibition (%) as follows: Paracetamol (97), Og (113), MiL (108), Pg (84) and MiB (88). Og and MiL exhibited the best activities. CONCLUSION: This study supports the traditional use of Nefang for the treatment of malaria and gives information on its formulation, folk use, efficacy and safety. The demonstrated in vitro and in vivo antiplasmodial activities suggest that Nefang has a promising antimalarial activity. The in vivo findings showed that Nefang is relatively safe for oral administration at doses tested. In addition to its antimalarial activity, the constituents of Nefang contain biologically active compounds with antioxidant, analgesic, antipyretic and anti-inflammatory activities whose synergism with the antiplasmodial activity could complement its antimalarial effects. Furthermore, suitable combinations of the constituent plants of Nefang with better activities were revealed, suggesting that re-formulation of Nefang could yield optimum activity, exploitable towards a rational antimalarial phytotherapeutic drug discovery.