Anti-plasmodial effect of plant extracts from Picrolemma huberi and Picramnia latifolia

The compound leaves and stems of the P. huberi and P. latifolia plants were collected at the Joaquín Antonio Uribe botanical garden in Medellín, Antioquia, Colombia. A botanical sample of the evaluated species is deposited in JAUM (Herbarium of the Botanical Garden of Medellín), P. huberi under the code Tobon Juan Pablo 2392 and P. latifolia under the code 65508. The plant material was dried at room temperature for 7 days. The raw extracts were prepared by grinding the plant material in an industrial mill with 5 mm particles, obtaining 255.29 g of compound leaves and 135.59 g of stems from P. huberi, and 125.66 g of stems and 166.93 g of compound leaves from P. latifolia.

The extractions were carried out following the protocols previously proposed [15, 16]. Briefly, the ground plant material was sectioned; P. huberi was extracted by percolation of leaves/petiole/rachis, leaves, petiole/rachis and cortex using solvents of different polarity (ethanol and hexane). P. latifolia was extracted by percolation of bark/leaves and bark/petiole using ethanol; each percolation was performed for 72 h, with drying intervals of 24 h between each solvent. Subsequently, each extract was filtered with Whatman paper and concentrated to dryness under vacuum using the Heidolph Laborota 4001/g3 rotary evaporator (Sigma-Aldrich) [15, 16].

A second collection of Picrolemma huberi plant material was made, found on the Guada road, at the Guada estate (1662 meters above sea level) in the town of Amalfi, Antioquia, Colombia (coordinates of the place: 6°52′006″ N 75°8′49,9″). The plant material (Cortex) was subjected to the same extraction procedure described above [15, 16].

Finally, a 10 mg/mL stock solution was prepared from each of the individual plant extractions by diluting them in 100% dimethyl sulfoxide (DMSO). These stock solutions were then used in the subsequent biological evaluations.

Two strains with different resistance phenotypes of P. falciparum [17], strain 3D7 and strain FCR3 were used. For both strains, the parasite cultures were incubated at 37 °C in RPMI 1640 culture medium supplemented with 10% inactivated fetal bovine serum, 25 mM Hepes, 0.2 mM Hypoxanthine, 5% Sodium Bicarbonate, and 25 μg/mL of gentamicin sulfate (G1397 SIGMA) in the presence of 5% CO₂, 5% O₂ and 90% N₂ [18].

Several concentrations of each extract (from 100 to 0.02 µg/mL and DMSO: 0.99% for the highest concentration) were evaluated in the 3D7 and FCR3 strains; diphosphate salt of chloroquine (≥ 98%, SIGMA C6628), evaluated in a range of 1.03 μg/mL to 0.00118 μg/mL, was used as a treatment control in each assay [14]. Culture medium in the absence of the extract was used as a growth control. A suspension of parasitized red blood cells with a haematocrit of 2%, a total parasitaemia of 1%, and with a predominance > 80% of young trophozoite stage, were prepared. The cultures with the treatments were incubated at 37 °C for 48 h in the presence of 5% CO₂, 5% O₂ and 90% N₂ [14]. After 48 h of incubation, 50 µL of 0.4% SYBR Green was added to each well and incubated for 10 min, to intercalate the fluorochrome into the DNA of the parasites [19]; after 10 min incubation, fluorescence emission detection was performed using the BD AccuriTM C6 flow cytometer, measured at 485 nm excitation and 530 nm emission [19]. Each concentration was evaluated in duplicate, and 2 or more independent assays were performed. To find the inhibitory concentration 50% (IC₅₀), the percentages of parasitaemia were analyzed using a non-linear slope-dependent regression with GraphPad Prism™ version 5.01. An extract was considered highly active with an IC₅₀ < 5 μg/mL, promising extracts with an IC₅₀ of 6–15 μg/mL, extracts with moderate activity with a IC₅₀ of 16–30 μg/mL, extracts with low activity with an IC₅₀ of 31–50 μg/mL, and inactive extracts if the IC₅₀ was > 50 μg/mL [20].

HepG2 cells (ATCC HB-8065™) incubated at 37 °C with 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% inactivated FBS, 2 nM l-glutamine and 1× Pen/strep amphotericin B were used [21]. 200,000 cells/mL were seeded in 96-well flat bottom plates (COSTAR 3599) (20,000 cells/well) [22, 23]. Seven serial concentrations of each extract (from 200 to 0.15 µg/mL in DMSO: 1.98% for the highest concentration) were evaluated in duplicate in two independent assays. For each assay, cells cultured under the same conditions in the absence of the extract were used as a growth control. FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) was used as a cytotoxicity control.

The cell viability reading was performed following the enzymatic micro-method MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) described by Mosmann in 1983 [24]. Production of formazan was measured on a Multiskan Spectrum Thermo scientific spectrophotometer, measured at 570 nm excitation and 595 nm emission. The data obtained were analysed by non-linear regression with the GraphPad Prism™ version 5.01 program, to determine the cytotoxic concentration of each extract that reduced cell viability by 50% (CC₅₀). An extract was considered very toxic with CC₅₀ < 10 µg/mL, moderately toxic with CC₅₀ 11–30 µg/mL, slightly toxic with CC₅₀ 31–50 µg/mL, and potentially non-toxic with CC₅₀ > 50 µg/mL [21].

The haemolytic effect of the extracts was evaluated in human A+ erythrocytes at a concentration of 40% in isotonic sodium phosphate buffer (10 mM, pH 7.4) [25]. Seven serial concentrations starting at 10 µg/mL for P. latifolia and 0.5 µg/mL for P. huberi were evaluated in two independent trials. Erythrocytes treated with isotonic sodium phosphate buffer solution were used as a baseline haemolysis control, and 1% Tween 20 was used as the maximum haemolysis control. The erythrocyte suspension with the treatments was incubated at 37 °C for 1 h and the amount of haemoglobin released was determined on a Multiskan Spectrum Thermo scientific spectrophotometer at 540 nm [25]. The percentage of haemolysis was calculated with the following formula: % haemolysis = (treatment of each extract in isotonic solution)/(treatment in tween − isotonic solution) × (100) [25].

The FCR3 strain parasite cultures were synchronized in the ring stage by lysis with 5% d-sorbitol (Sigma-Aldrich), with a haematocrit of 2% and a parasitaemia of 1% [26]. For the determination of the anti-plasmodial activity, each of the extracts was evaluated during several maturation periods, every 4 h, and in triplicate [26]. The parasite solution (200 µL) was added to Flat-bottomed 96 well plates (COSTAR 3599). A dilution of the ethanolic extracts (50 µL) were added at time 0 in the first three columns. The dishes were gently shaken and incubated at 37 °C in the presence of 5% CO₂, 5% O₂ and 90% N₂. After the incubation period (4 h), the supernatant from the first three columns was removed and the parasitized erythrocytes from these wells were washed with RPMI-1640 medium; the whole dish was then centrifuged at 839 g for 10 min and 200 µL RPMI-1640 medium was added to each well of the first three columns. After this, the dilutions of the ethanolic extracts were placed in the remaining three columns and the dish was incubated for 4 more hours; after this time, the wells were washed with RPMI-1640 medium and this process was repeated every 4 h until the maturation period (of 48 h or more per cycle) was completed. After 48 h of incubation, 50 µL of 0.4% SYBR Green was added to each well and incubated for 10 min to intercalate the fluorochrome into the parasitic DNA and to detect the fluorescence emission [19]. After the 10 min incubation, fluorescence emission detection was performed using the BD AccuriTM C6 flow cytometer, measured at 485 nm excitation and 530 nm emission [19]. Finally, the data obtained were analysed by non-linear regression using the GraphPad Prism™ software version 5.01, to find the inhibitory concentration 50% (IC₅₀) [21].

The in vitro antiplasmodial activity of 10 extracts of P. latifolia and P. huberi plants was evaluated in FCR3 and 3D7 strains of P. falciparum; the results are described in detail in Table 1. Chloroquine was used as control, with an IC₅₀ of 0.0057 µg/mL for the strain 3D7 (chloroquine sensitive phenotype) and IC₅₀ of 0.064 µg/mL for strain FCR3 (chloroquine resistant phenotype).

In the FCR3 strain, the extracts from P. latifolia species presented IC_50s from 1.33 to 7.57 µg/mL, and were considered as highly active extracts [20]. The IC_50s for P. huberi were from 0.04 to 39.33 µg/mL, of which the bark extract dissolved in ethanol:water (90:10) (with hexane degreasing pretreatment) and the bark extract dissolved in ethanol:water (90:10) without the pretreatment, showed high antiplasmodial activity with IC_50s of 0.04 and 0.05 µg/mL, respectively. The extract obtained from the P. huberi plant that was collected in the town of Amalfi, shows an IC₅₀ of 0.18 µg/mL, maintaining the highly active extract classification.

For the 3D7 strain, inhibitory concentrations from 0.07 to 1.21 µg/mL were observed for P. latifolia extracts. Extracts from P. huberi showed a range of IC_50s, from 0.08 to 156.9 µg/mL.

Regarding the evaluation of in vitro toxicity of P. latifolia extracts in HepG2 cells, the CC₅₀ of the bark/petiole ethanolic extract was 3.5 µg/mL and the CC₅₀ of the ethanolic extract of bark/leaves was 146.6 µg/mL. On the other hand, the toxicity classifications for P. huberi extracts ranged from very toxic (CC₅₀ = 0.38 µg/mL) to potentially nontoxic (CC₅₀ = 143.7 µg/mL) [21].

The leaves of the two species of plants tested were not toxic in HepG2 cells. The CC₅₀ of the P. latifolia bark/leaves ethanolic extract was 146.6 µg/mL. Similarly, the CC₅₀ of the hexane extract from P. huberi leaves/petiole/rachis and the hexanic acid extract from the P. huberi leaves alone, was 142.5 and 143.7 µg/mL, respectively. In contrast, some degree of toxicity was observed in the extracts obtained from bark, petiole and rachis, where no leaves were present, from both plant species regardless of the solvent used; with CC_50s < 50 µg/mL (slightly toxic extracts, moderately toxic and very toxic).

The selectivity index (SI) of extracts of P. latifolia and P. huberi were assessed and demonstrate specific antiplasmodial activity rather than toxicity in HepG2 cells, since most of the indices were ≥ 2. The extracts with an SI < 2, such as the hexane extract from bark, would not be good candidates for the treatment of malaria. Overall, the hexane extracts induced more toxicity when compared to the ethanolic extracts.

The haemolytic capacity of P. latifolia and P. huberi extracts was evaluated in healthy red blood cells. The ethanolic extract of P. latifolia evaluated at a concentration of 10 µg/mL (8.5× IC₅₀) showed a haemolysis percentage of 1.35%, while the ethanolic extract of P. huberi evaluated at a concentration of 0.5 µg/mL (10× IC₅₀) showed a haemolysis percentage of 0.43%. Thus, these ethanolic extracts do not have haemolytic capacity in healthy red blood cells when compared with the total haemolysis control (1% Tween 20). The cytotoxic activity of all the plant extracts evaluated in human erythrocytes is shown in Table 2.

The concentrations of P. latifolia extracts that inhibited parasite growth by 50% at 20 and 28 h was 0.3 and 0.7 µg/mL, respectively. During the life cycle of P. falciparum, 20 and 28 h correspond to the young and mature trophozoite stage of development [32]. Growth of parasites was inhibited by 50% between 20 and 36 h when they were exposed to plant extract concentrations of 0.05–0.1 µg/mL from the P. huberi species. This time period (20–36 h) covers all the asexual intraerythrocytic stages of parasite development, from juvenile trophozoites to schizonts (Fig. 1).

For P. latifolia, the MTD was established at 2000 mg/kg body weight. This indicates that the ethanolic extract from the plant was not toxic for Balb/c mice at 2000 mg/kg weight in oral treatments. For P. huberi, the MTD was established below 205 mg/kg body weight.

Blood samples were taken to evaluate blood chemistries and blood chemical parameters to determine possible alterations in the liver function of the 3 mice under treatment. Mild leukocytosis was found with a value of 11.3 × 10³/µL (reference range in mice is 5.0–8.93 × 10³/µL) with predominance of neutrophilia and mild thrombocytosis with a value of 672.00 × 10³ µL (reference range in mice: 200–590 × 10³ µL) in mice treated with P. latifolia at 2000 mg/kg (recommended maximum concentration for evaluations). Increased aspartate transaminase enzyme (AST) values were observed in the blood chemistry analysed: 17.8 μ/L, 3 times above the reference values (reference range in mice: 0.0–5.0 μ/L), indicating possible alterations in the liver function in mice treated with P. latifolia at 2000 mg/kg and with P. huberi at 61.03 mg/kg. Since the maximum tolerable concentration recommended by the 2008 OECD guidelines was used, these alterations suggest that the mice exhibited basic immunological reactions to foreign agents. The Alanine transaminase enzyme (ALT), creatinine and total bilirubin were not altered. There were no changes in animal weight or signs of pain when touched [27].

Samples of liver, kidney and spleen tissue were taken from mice treated with the MTD of the ethanolic plant extracts from P. latifolia and P. huberi for pathology studies. In mice treated with the P. latifolia ethanolic extract, acute splenitis or spleen inflammation was observed, characterized by an increase in organ size. A slight abnormal increase in liver size and an increase in the size of the hepatocyte nucleus or moderate to severe karyomegaly were observed. No tissue damage was observed in the kidney. In mice treated with the P. huberi ethanolic extract, a slight increase in hepatocyte nucleus size, binucleation, congestion, and macrophages in the liver were observed. Additionally, a slight abnormal increase in kidney size or hyperplasia, and mild congestion in the spleen were observed. Further studies are needed to identify the importance of these lesions in the functioning of the organs.

Table 3 shows the anti-malarial activity of each extract using the in vivo mouse model. Parasitaemia is expressed as the mean percentage of parasitized red blood cells for each treatment group compared to the vehicle control group ± the standard deviation (± SD). Inhibition of parasitaemia is expressed as the proportion of total red blood cells analysed without parasites (mean percentage ± SD) from the mice treated with plant extracts or chloroquine. There was a near elimination of 100% in the positive parasitaemia elimination controls. For mice treated with an extract of Cinchona officinalis (positive control group for parasitaemia removal by a quinine-containing plant extract), parasitaemia inhibition rates of 96.3 ± 4,3% were observed, and for mice treated with chloroquine, inhibition was 99.7 ± 1.5% (Table 3, Fig. 2b).

Regarding the specific treatments, parasitaemia was inhibited by 52.1 ± 3.4% (effective dose 50) in the mice treated with the 1000 mg/kg dose of P. latifolia ethanolic extract (Fig. 2b), and classified as an extract with good to moderate activity [33]. Statistical analysis of the means with a Tukey post hoc test revealed significant differences between the parasitaemias observed during the three-day follow-up treatment with the 1000 mg/kg dose, when compared to the DMSO vehicle control treatment (P < 0.05), (Fig. 2a).

On the other hand, in the evaluations carried out with P. huberi, parasitaemia inhibition averages of up to 93 ± 32,9% were determined at the 150 mg/kg dose (Table 3, Fig. 2b). Thus, according to Rasoanaivo et al. [32] the P. huberi extract was classified as a very good extract or one with good activity. Statistical analysis of the means with a Tukey post hoc test revealed significant differences (P < 0.05) between the parasitaemias observed during the 3 day follow-up in the mice treated with the 150 mg/kg dose of P. huberi extract, relative to the DMSO vehicle control group (Fig. 2a).

The pathology results from the mice treated with the ethanolic extract of P. latifolia were the following: parasitic hepatitis characterized by hyperplasia, vacuolar degeneration, karyomegaly, and with presence of malaria pigment in the liver; mild acute congestive splenitis characterized by congestion with the presence of malaria pigment in the spleen; mild renal congestion characterized by vacuolar degeneration with presence of malarial pigment in the kidney; and finally, alterations in the liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST) were observed. For mice treated with the ethanolic extract of P. huberi, the following pathologies were observed: mild vacuolar degeneration and congestion in the liver; mild reactive hyperplasia and mild apoptosis in the spleen; nephrosis and mild congestion in the kidney; and alterations in the liver enzymes ALT and AST.