Crude methanolic extracts were successfully extracted from fresh leaves and stems of the species A. paniculata and P. pellucida. With crude extracts in hand, their ability to suppress parasitemia and inhibit parasite growth were evaluated. Following the incubation of synchronized Pf D-10 with the AP-PP extract at various ratios (80:20 to 20:80) in DMSO, CQ (1 µM) and 0.2% DMSO, a thin blood smear stained with Giemsa was prepared. Parasitemia, parasite growth and inhibition rates were determined microscopically.
The chemosuppression of parasitemia against the malarial parasite Pf D-10 was evaluated (Figure 3a). The parasitemia rates decreased when Pf D-10 were treated with the AP-PP extract. At a ratio of 80: 20 to 40:60, the antiplasmodial activity of the AP-PP extract were limited (ca. 2.8 to 3.0 %). Interestingly, parasitemia rate decreased when the ratio of the PP extract was increased. Parasitemia rates at ratio of 30: 70 and 20:80 exhibited significant activity (p < 0.05). Results of the in vitro test in Figure 1b indicate that the growth malarial parasite Pf D-10 (chloroquine-sensitive) was suppressed by the AP-PP extract. The AP-PP extract exhibited higher parasite growth of Pf D-10 at a ratio of 80: 20 to 40:60, while at a ratio of 20:80 exhibited a significantly killing effect of 1.5 % (p < 0.05). The results in Figure 3c indicate that parasite Pf D-10 was inhibited by the AP-PP extract. Although there is no significant inhibition of Pf D-10 at ratio of 80:20 to 40:60, but the AP-PP extract at higher ratio of the PP extract (20:80) exhibited significant inhibition effect of 50% (p < 0.05). As expected, there was good correlation among the increased ratio of the PP extract and inhibition effects. The antiplasmodial activity of the AP-PP extract was increased in a ratio dependent manner. Furthermore, we determined the IC50 values of the AP-PP extract at a ratio of 20:80. The extract was tested at a concentration of 0.01, 0.1, 1, 10 and 100 µg/mL and evaluated the inhibition rates after 48 h of incubation. An analysis of inhibition–response curves were used to determine the IC50 value. The AP-PP extract at a ratio of 20:80 was considered to be marginally potent on the basis of an in vitro antiplasmodial activity of plant extract against Plasmodium falcifarum strain with an IC50 value of 62.01 µg/mL [20].
Since the increased ratio of the PP extract exhibited significant activity in contrast to the increased ratio of the AP extract, it is essential to evaluate the PP extract to facilitate the discovery of their biological function and mode of action. We next evaluated the potential of the PP extract alone against parasite Pf D-10. The antiplasmodial activity of the PP extract was performed at a concentration ranging from 0.01 to 100 µg/mL. As shown in Fig. 2a, the PP extract possessed promising antiplasmodial activity against Pf D-10. At the dilute concentration of 0.01 µg/mL, the antiplasmodial activity of the PP extracts were limited (ca. 4.5%). Parasitemia rates were approximately 3.7%, 3.4%, and 2.4% at a concentration of 0.1, 1 and 10 µg/mL, respectively. Interestingly, at the highest concentration (100 µg/mL) the PP extract exhibited a significantly higher activity in reducing parasetimia of 1.2%. The results in Fig. 2a indicate the inhibition of the Pf D-10 induced by the PP extract. The PP extract exhibited low suppression of Pf D-10 at the lowest concentration (0.01 µg/mL), while at a concentration of 10 and 100 µg/mL exhibited significant inhibition effect of 51% and 92%, respectively (p < 0.01 and p < 0.001). DMSO was used as a negative control showed no antiplasmodial activity, but CQ as an antimalarial reference was more active than the test samples. The results, recorded in Fig. 2c, suggested that the PP extract displayed promising antiplasmodial activity on the basis of plant extract with IC50 value of 4.0 µg/mL (IC50 of a promising extract should be less than 10 µg/mL) [20].
A comparison with the results of Mishra et al., the in vitro antiplasmodial activity of P. pellucida L. Kunth possessed a significantly higher activity (IC50, 4.0 µg/mL) then A. paniculata (Burm. f.) Nees (IC50, 7.2 µg/mL). These results clearly show that the increased ratio of the PP extract in the combination of the AP-PP extract provoke better antiplasmodial activity on the basis in vitro assay. Although, andrographolide is a major well known bioactive antimalarial compound from A. paniculata, however the phytochemical constituents from P. pellucida L. Kunth may provide stronger antiplasmodial activity. Therefore it is important to isolate and identify the compound(s) to facilitate the discovery of their biological function. Unlike the PP extract, the low responses of the AP and AP-PP extracts are presumably due to compound cytotoxicity rather than specific activity against the parasite itself or being negatively influenced by poor pharmacokinetics.
We next evaluated the potential of the PP extract in BALB/c albino mice infected with Pb ANKA. The in vivo efficacy of the PP extract was evaluated following the procedure described by Peter (a four-day suppressive test) [19]. BALB/c albino mice were inoculated with Pb ANKA intraperitoneally. After the fourth days of treatment with 0.5% CMC-Na as a negative control, chloroquine diphosphate as a positive control and the PP extract at a daily dose of 1, 10 and 100 mg/kg/body, Giemsa-stained thin blood smears were prepared on the fifth day for each mice. The parasitemia and parasite growth suppression were determined microscopically (Fig. 3a). The survival of mice was carefully recorded until day 21. The PP extract was considered to be partially active on the basis of an in vivo antiplasmodial activity with an ED50 value of 12.86 mg/kg/ body weight (Fig. 3b). Clinically available drug quinine against Pb ANKA has an [ED50] of 34 mg/kg/day and slow clearance [21]. The results in Fig. 3c indicate that at a daily dose of 1, 10 and 100 mg/kg/body after the fourth day treatment, the PP extract exhibited a chemosuppression of parasitemia against Pb ANKA in mice. At the lowest dose (1 mg/kg/body), intraperitoneally administration of the PP extract led to 4.4% of parasitemia in contrast to untreated mice (6.4%). Namely, 31% of parasites were killed induced by the PP extract. The rate of parasitemia reduced as the concentration of the PP extract was increased. The parasitemia rates decreased to approximately 3.2% and 2.4% at the PP extract concentrations of 10 and 100 mg/kg/body, respectively. Furthermore, the chemosuppression of parasitemia on day 0 to day 4 was also evaluated (Fig. 3d). Parasitemia rates of untreated mice were approximately 1.0% (day 0), 3.2% (day 1), 4.6% (day 2), 5.7% (day 3) and 7.2% (day 4), indicating that the number of parasites in the blood was consistently increasing after infection on day 0. At a daily dose of 1, 10 and 100 mg/kg/body, the PP extract exhibited a significant chemosuppressive effect against Pb ANKA in contrast to untreated mice. The parasite growth inhibition in mice was evaluated (Fig. 3e). The PP extract administered intraperitoneally in mice at a daily dose of 1, 10 and 100 mg/kg/body suppressed Pb ANKA in a dose dependent manner. Suppression rates were approximately 64% (100 mg/kg/body), 50% (10 mg/kg/body) and 31% (1 mg/kg/body), respectively (untreated mice defined as 0%). These results clearly demonstrated the parasite killing induced by the PP extract. Survival of infected mice were also increased due to the treatment of the PP extract. Untreated mice succumbed to death after 11 days of infection with Pb ANKA, while mice treated with 1, 10, 100 mg/kg/body of the PP extract died on days 13, 16 and 19 after treatment, respectively. A positive control groups treated with chloroquine diphosphate at a daily dose of 25 mg/kg/body all survived up to 21 days, indicating that the infected mice were completely cured of Pb ANKA. This is consistent with the results of Fang et al.[22]
Although many therapeutic antimalaria have been reported, insufficient efficacy has been a critical issue in treating malaria. Also, antimalarial drug resistance is a major hurdle. Peptides [23, 24] and amino acid-malaria drug conjugated [25–27] were reported for their antiplasmodial activity, however the chemical synthesis of hydrophobic peptide was very challenging [28, 29]. Tropical plants are well known as source of bioactive compounds, thus they are potential for the development of a new class antimalarial agent. Although, the use of the species A. paniculata and their combination with P. pellucida L. Kunth was practically used to treat malaria disease, however there is less information about the beneficial of the species P. pellucida alone for treating malaria disease in humans. Herein we report the potential of species P. pellucida from Indonesia as a source of new antimalaria compounds. This is the first report of the potential of P. pellucida as a source of antiplasmodial agent and we have shown its promising efficacy in an in vivo mouse model. Our results suggest a novel P. pellucida that has the potential as a source of antimalarial agent. We plan to do an in vivo test with the combination of the PP extract and an antimalarial drug artesunate [30] to improve the efficacy of drug action. In addition, we plan to isolate and analyze bioactive metabolites from the species P. pellucida that may be provide more efficacious in antiplasmodial activity and hope to contribute to the development of a new class of antimalarial agent.