Antiplasmodial Potential of Indonesian Medicinal Plants


 Background: Species A. paniculata (Burm. f.) Nees known as″ Sambiloto ″ and P. pellucida L. Kunth known as″ Suruhan ″ are mainly distributed in Indonesia and their combination was used as a traditional medicine for treating malaria diseases. However, no information appears to have evaluated the antiplasmodial potential of the two plants. This research aimed to evaluate the antiplasmodial activity of the two plants and the species P. pellucida L. Kunth alone as a source of antiplasmodial agent. Methods: In vitro test of the AP-PP and PP extracts against Pf D-10 (chloroquine-sensitive) were performed as described by Desjardins et al. An in vivo test of the PP extract in mice infected with Pb ANKA was performed using Peters´ 4-day suppressive test. Parasitemia, growth and inhibition rates were determined via Giemsa-stained smear of blood and analyzed microscopically. Survival was followed up until day 21 post-infection.Results: The increased ratio of the PP extract (20:80) exhibited significant antiplasmodial in contrast to the high ratio of the AP extract (IC50, 62.01 mg/mL). Further evaluation of the PP extract alone displayed better antiplasmodial activity with an IC50 value of 4.0 mg/mL. Furthermore, an in vivo test of the PP extract in BALB/c albino mice infected with Pb ANKA exhibited a significant chemosuppressive effect in a dose-dependent manner.Conclusion: The increased ratio of the PP extract exhibited a major contribution for their activity. The PP extract alone showed better antiplasmodial activity than the AP extract and their combination. An in vivo test confirmed the efficacy of the PP extract in mouse model.


Introduction
Malaria is a serious global health problem caused by infection of the species Plasmodium transmitted to humans by female Anopheles mosquitoes [1]. This disease continues to be a world health issue because of the lack of preventative measures [1][2][3] as well as the increasing resistance to many of the existing antimalarial drugs such as quinine, chloroquine, amodiaquine, me oquine, piperaquine, lumefantrine, primaquine, artemisinin derivatives and artemisinin-based combination treatments with artesunateamodiaquine, artesunate-me oquine, artesunate-sulfadoxine-pyrimethamine and artemether-lumefantrine [4]. Currently, there are no commercially available malaria vaccine [1], despite many decades of intense research efforts. Thus, chemotherapeutics using combination drugs appears to be the best option [5] and there is an urgent need to discover new antimalarial compounds. Medicinal plants have commonly been used as a source for new active compounds [6]. For example, Artemisinin, an antimalarial drug which was discovered from Artemisia annua L., by You-You Tu in China in the early 1970s [7]. Tropical plants are known to be a rich reservoir of bioactive metabolites, thus they are potential sources of new antimalarial drugs [8].
The family Acanthaceae is widely grown in tropical and subtropical regions [9]. Traditionally, this plant has been used to treat acute diarrhea, cough, common cold, in ammation, boils, skin eruptions, and seasonal fever [10]. Phytochemical screening has revealed the presence of a variety of secondary metabolites with important pharmacological activities [11]. Species A. paniculata known as″ Sambiloto ″ is mainly distributed from the northeast to the south of Indonesia and often consumed as a traditional medicine [12]. Although the antiplasmodial activity of this plant against Plasmodium falciparum strain have been reported [13], but traditional communities believed their combination with P. pellucida would be bene cial for the better antimalarial treatment [14,15]. Because of P. pellucida use against malaria, we evaluated the activity of combination and different extracts against Pf D-10 and the potential of P. pellucida plant as a source of antimalarial agent.

Plant extraction
Dried leaves and stems of P. pellucida (2 kg) and A. paniculata (2 Kg) were macerated with methanol (12 L) at RT for 2 days. After ltrating, the ltrate was evaporated in vacuo to give the crude PP extract (211 g) and AP extract (278 g), respectively.

Parasite synchronization and maintenance
Malarial parasite Pf D-10 strain (chloroquine-sensitive) was obtained from the University of Tokyo, Japan.

In vitro antiplasmodial activity
The crude AP-PP extract was prepared at 20 mg/mL stock solutions in DMSO. Chloroquine diphosphate (Sigma, Burlington, MA, USA) stock solution as an antimalarial reference was prepared in water (Milli-Q grade) at 1 mM, while 0.2% DMSO was used as a negative control. Except for CQ, the nal solution contained 0.2% DMSO. An in vitro test was performed as described by Desjardins et al [16]. A suspension of 200 µL of synchronized parasites (0.5% parasitemia and 4% hematocrit) were incubated with various ratios of the AP-PP extract ranging from 20:80 to 80:20 in DMSO, CQ ( nal concentration of 1 µM) and 0.2% DMSO under the same conditions mentioned before. After incubation, a thin blood smear stained with Giemsa was prepared [17,18]. In vitro test of the PP extract was performed at various concentration of 0.01 to 100 µg/mL. Parasitemia, parasite growth and inhibition rates were determined microscopically by calculating the number of infected erythrocytes from 500 erythrocytes. An analysis of dose-response curves was used to determine IC 50 values as the mean of three experiments (n = 3).

Experimental animals and parasites
Thirty (male) BALB/c albino mice aged 6-8 weeks and weighing 25-28 g were used in this study, provided by the Animal Experimental Development Unit in Gadjah Mada University, Yogyakarta, Indonesia. The mice were maintained RT, with food and water given ad libitum at the Animal Laboratory in the Institute of Tropical Disease, Universitas Airlangga. The parasite Pb ANKA strain was obtained from the Eijkman Institute for Molecular Biology, Jakarta, Indonesia. The parasite had been maintained at the Institute of Tropical Disease, Universitas Airlangga by a combination of passages in male BALB/c mice and cryoscopic storage.

Experimental design and treatment of mice
An in vivo antiplasmodial activity of the crude PP extract in mice infected with Pb ANKA was performed as described by Peter [19] with minor modi cations. Thirty mice (BALB/c albino) were inoculated with the parasite Pb ANKA strain (10 6 erythrocytes parasitized) intraperitoneally. The volume of inoculum was 200 µL. Mice were divided into ve groups of six mice each (three experimental and two control groups). Three experimental groups were treated with the crude PP extract at a concentration of 1, 10 and 100 mg/kg/body in 0.5% CMC-Na (Total volume of 200 µL). A negative control group was treated with 0.5% CMC-Na and a positive control group was treated with chloroquine diphosphate (25 mg/kg/body) intraperitoneally once a day for four days (day-1 to day-4). The animals stopped receiving treatments after 4 days of treatments. On the fth day, thin blood smears from the tail of the mice were prepared on a slide. After drying and xing with methanol, the slide was stained with 15% Giemsa-stained solution [17,18] for 10 min. The slide was nally rinsed with water and dried at RT. The percentage of parasitemia and parasites growth suppression were determined via Giemsa-stained smear of blood and analyzed microscopically. Survival was followed up until day 21 post-infection.

Results And Discussions
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 signi cant 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 signi cantly 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 signi cant 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 signi cant 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 IC 50 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 IC 50 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 IC 50 value of 62.01 µg/mL [20].
Since the increased ratio of the PP extract exhibited signi cant 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 signi cantly 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 signi cant 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 IC 50 value of 4.0 µg/mL (IC 50 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 signi cantly higher activity (IC 50 , 4.0 µg/mL) then A. paniculata (Burm. f.) Nees (IC 50 , 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 speci c activity against the parasite itself or being negatively in uenced by poor pharmacokinetics.
We next evaluated the potential of the PP extract in BALB/c albino mice infected with Pb ANKA. The in vivo e cacy 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 fth 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 ED 50 value of 12.86 mg/kg/ body weight (Fig. 3b). Clinically available drug quinine against Pb ANKA has an [ED 50 ] 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 signi cant 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 de ned 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, insu cient e cacy has been a critical issue in treating malaria. Also, antimalarial drug resistance is a major hurdle. Peptides [23,24] and amino acidmalaria drug conjugated [25][26][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 bene cial 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 rst report of the potential of P. pellucida as a source of antiplasmodial agent and we have shown its promising e cacy 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 e cacy of drug action. In addition, we plan to isolate and analyze bioactive metabolites from the species P. pellucida that may be provide more e cacious in antiplasmodial activity and hope to contribute to the development of a new class of antimalarial agent.

Conclusions
In summary, the combination of the species A. paniculata and P. pellucida has been assessed in vitro antiplasmodial activity against Pf D-10. At a ratio of 20:80 exhibited signi cant antiplasmodial activity. In vitro test of the PP extract alone showed a signi cantly higher antiplasmodial activity in contrast to the AP extract alone and their combinations. In vivo analysis of the PP extract at a daily dose of 1, 10 and 100 mg/kg/body showed signi cant chemosuppressive effect in mice infected with parasite Pb ANKA. The raised concentrations of the PP extract exhibited dose-dependent manner.  extract. An IC50 value was calculated using GraphPad Prism software. The results in Figure 2a indicate parasitemia rates of the PP extract at a concentration of 0.01 g/mL (black), 0.1 g/mL (white), 1 g/mL