DOI: 10.14457/tu.the.2015.159
title: Antimalarial activity, toxicity, pharmacokinetics and metabolic drug interaction of Plumbago Indica linn.

description: Malaria is widespread in tropical and subtropical regions.  Throughout the history of mankind, this highly infectious disease has been one of the major causes of human illness and death.  Plasmodium falciparum is the most virulent and widespread infectious malarial species in tropical and subtropical countries due to the resistance of the parasite to most of the available antimalarial drugs.  As antimalarial drug resistance compromises the effective treatment of the disease, there is a pressing need for ongoing drug discovery research.  Natural products including medicinal plants may offer relatively cheap alternative treatment opportunities for malaria patients.  The crude ethanolic extract of Plumbago indica Linn. has been shown to possess good to moderate antimalarial activity (class III antimalarial activity) in our previous in vitro screening.  Among the 32 plants investigated, Plumbago indica Linn. showed the most promising activity against both K1 chloroquineresistant (IC50 3 µg/ml) and 3D7 chloroquine-sensitive (IC50 6.2 µg/ml) clones, with highest selectivity (SI = 44.7 and 21.6, respectively).  Plumbagin is the major active constituent in several plants including Plumbago indica Linn. (root).  This compound has been shown to exhibit a wide spectrum of pharmacological activities such as activities against malaria, leishmania and trypanosome parasites, as well as against virus, cancers, and bacteria. Plumbagin exhibited promising antimalarial activity with in vitro IC50 (concentration that inhibits enzyme activity by 50%) against 3D7 chloroquine-sensitive Plasmodium falciparum and K1 chloroquine-resistant Plasmodium falciparum clones of 580 (270-640) and 370 (270-490) nM, respectively.  In vivo antimalarial activity was investigated in Plasmodium berghei infected mouse model.  Chloroquine exhibited the most potent antimalarial activity in mice infected with Plasmodium berghei ANKA strain with respect to its activity on the reduction of parasitaemia on day 4 and the prolongation of survival time.  Plumbagin at the dose of 25 mg/kg body weight given for 4 days produced moderate antimalarial activity with regards to its inhibitory activity on the reduction of parasitaemia and the prolongation of survival time.Toxicity testing in ICR mice indicated maximum oral dose at the dose levels up to 100 (single oral dose) and 25 (daily doses for 28 days) mg/kg body weight for acute and subacute toxicity, respectively whereas toxicity testing in wistar rats indicated maximum oral dose at the dose levels up to 150 (single oral dose) and 25 (daily doses for 28 days) mg/kg body weight for acute and subacute toxicity, respectively.  Fifty percent lethal dose (LD50) of plumbagin in wistar rats was 250 mg/kg body weight.  Permeation (Papp) of plumbagin (2-8 µM) for the apical to basolateral and basolateral to apical directions were 10.29-15.96×10-6 and 7.40-9.02×10-6 cm/s, respectively, with the efflux ratios of 0.57-0.73.  Plumbagin was not either a substrate or inhibitor of P-glycoprotein (p-gp).  It did not interfere with the P-glycoprotein-mediated R123 transport across Caco-2 cell monolayer, as well as the function of P-glycoprotein and the expression of MDR-1 (multidrug resistance 1) mRNA.  Results suggest moderate permeability of plumbagin across the Caco-2 cell monolayer in both directions.  The transport mechanism is likely to be a passive transport.  The propensity to inhibit CYP-mediated hepatic metabolism (CYP1A2, CYP2C19, CYP2D6 and CYP3A4) of the five Thai medicinal plants with promising activities against malaria (Table 1) using human liver microsomes were investigated.  Results showed that all plants exhibited varying inhibitory potencies on various CYP isoforms.  With regard to the inhibitory activity of each plant extract on the four CYP isoforms, PI and DM showed potent inhibitory activities on most CYP isoforms (CYP2C19, CYP2D6, and CYP3A4, respectively) while DL showed potent inhibitory activities on the two CYP isoforms (CYP1A2 and CYP3A4 respectively).  Moreover, plumbagin showed significant inhibitory effects on all cytochrome P450 (CYP) isoforms under investigation, but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation.  The IC50 values (mean±SD) of plumbagin and nootkatone (selective inhibitor) for CYP2C19 were 0.78±0.01 and 27.31±0.66 µM, respectively.  The inhibitory activities on CYP1A2-mediated phenacetin O-deethylation and CYP3A4-mediated nifedipine oxidation were moderate.  The IC50 values of plumbagin and α-naphthoflavone (selective inhibitor) for CYP1A2 were 1.39±0.01 and 0.02±0.36 µM, respectively.  The corresponding IC50 values of plumbagin and ketoconazole (selective inhibitor) for CYP3A4 were 2.37±0.10 and 0.18±0.06 µM, respectively.  Furthermore, Plumbagin did not induce hepatic drug metabolizing enzymes, especially CYP1A2 and CYP3A11 of treated mice with plumbagin (6.25, 12.50 and 25.00 mg/kg body weight) for 28 days, and that plumbagin exhibited a little inhibitory effect on CYP1A2 but did not reach the level of significance.  The pharmacokinetic study in wistar rats showed that the average (±) half-life (T1/2) and time to reach maximum blood concentration (Tmax) of plumbagin were 9.98±1.63 and 5.00±0.00 h, respectively after oral administration.  Maximum blood concentration (Cmax) was 0.46±0.08 (µg/ml).  The mean residence time (MRT) of plumbagin was 13.67±1.62 h.  In addition, the area under the time curve (AUC0-inf) was 6.72±0.74 (µg•h/ml) whereas (AUC0-48h) was 6.47±0.62 (µg•h/ml).  On the other hand, systemic clearance (CL) and volume of distribution (Vd) of plumbagin were 7.51±0.84 (l/h) and 0.11±0.01 (l), respectively.  Comparison between blood kinetics of healthy mice and P. berghei-infected mice following intravenous injection of 99mTc-plumbagin complex demonstrated the mean residence time (MRT) of the labelled complex in healthy mice (3.16 h) was significantly longer than P. berghei-infected mice (2.21 h) (p&lt;0.001).  In addition, the area under the time curve (AUC0-inf) was significantly higher in healthy (13.54 %ID/g*h) compared with P. berghei-infected (7.85 %ID/g*h) mice (p&lt;0.001).  On the other hand, systemic clearance (CL) and volume of distribution (Vd) of 99mTc-plumbagin were significantly higher in P. berghei-infected (12.76 l/h vs 41.95 ml) compared with healthy (7.39 l/h vs 29.56 ml) mice (p&lt;0.001).  Plumbagin was rapidly cleared from blood circulation and major routes of excretion were renal, hepatobiliary and pulmonary routes.  Malaria disease state influenced the pharmacokinetics and disposition of plumbagin in animal model.Results suggest that clinical relevance of the interference of human drug metabolizing enzymes should be aware of for further development scheme of plumbagin as antimalarial drug when used in combination with other antimalarial drugs which are metabolized by these CYP isoforms.  Preparation of modified formulation is required to improve its systemic bioavailability.  Increase in the dose of plumbagin, together with improvement of its oral bioavailability may be required if the compound will be selected as a candidate compound for treatment of malaria.