Anti-Plasmodium falciparum Activity of Extracts from 10 Cameroonian Medicinal Plants

Background: In the midst of transient victories by way of insecticides against mosquitoes or drugs against malaria, the most serious form of malaria, caused by Plasmodium falciparum, continues to be a major public health problem. The emergence of drug-resistant malaria parasites facilitated by fake medications or the use of single drugs has worsened the situation, thereby emphasizing the need for a continued search for potent, safe, and affordable new antimalarial treatments. In line with this need, we have investigated the antiplasmodial activity of 66 different extracts prepared from 10 different medicinal plants that are native to Cameroon. Methods: Extracts were evaluated for their capacity to inhibit the growth of the chloroquine-sensitive (Pf3D7) and resistant (PfINDO) strains of P. falciparum using the SYBR green fluorescence method. The cytotoxicity of promising extracts against human embryonic kidney cells (HEK293T) mammalian cells was assessed by MTT assay. Results: The antiplasmodial activity (50% inhibitory concentration, IC50) of plant extracts ranged from 1.90 to >100 μg/mL against the two strains. Six extracts exhibited good activity against both Pf3D7 and PfINDO strains, including cold water, water decoction, and ethyl acetate extracts of leaves of Drypetes principum (Müll.Arg.) Hutch. (IC503D7/INDO = 4.91/6.64 μg/mL, 5.49/5.98 μg/mL, and 6.49/7.10 μg/mL respectively), water decoction extract of leaves of Terminalia catappa L. (IC503D7/INDO = 6.41/8.10 μg/mL), and water decoction extracts of leaves and bark of Terminalia mantaly H.Perrier (IC503D7/INDO = 2.49/1.90 μg/mL and 3.70/2.80 μg/mL respectively). These promising extracts showed no cytotoxicity against HEK293T up to 200 μg/mL, giving selectivity indices (SIs) in the range of >31.20–80.32. Conclusions: While providing credence to the use of D. principum, T. catappa, and T. mantaly in the traditional treatment of malaria, the results achieved set the stage for isolation and identification of active principles and ancillary molecules that may provide us with new drugs or drug combinations to fight against drug-resistant malaria.


Introduction
Malaria is one of the world's most severe and deadly infectious diseases, and primarily affects the most disadvantaged populations. In fact, approximately 216 million cases of malaria Gastroenteritis, hypertension, diabetes, oral and skin conditions, oral and genital candidiasis [17,27] Leaf Tml D 27.70 Bark Tmb D 23.10 # The % yield (w/w) of extraction was calculated from the weight of extract relative to 100 g of starting plant material. Annona senegalensis (As) (Asb WEt : Hydroethanol extract of bark of As; Asb W : Aqueous maceration extract of bark of As; Asb D : Decoction extract of bark of As; Asb Et : Ethanol extract of bark of As; Astw WEt : Hydroethanol extract of twigs of As; Astw W : Aqueous maceration extract of twigs of As; Astw D : Decoction extract of twigs of As; Astw Et : Ethanol extract of twigs of As; Asst WEt : Hydroethanol extract of stems of As; Asst W : Aqueous maceration extract of stems of As; Asst D : Decoction extract of stems of As; Asst Et : Ethanol extract of stems of As; Asl WEt : Hydroethanol extract of leaves of As; Asl W : Aqueous maceration extract of leaves of As; Asl Et : Ethanol extract of leaves of As; Asl D : Decoction extract of leaves of As  [32] with some modifications [25]. Parasites were cultured in fresh O +ve human erythrocytes suspended at 4% (v/v) hematocrit in complete RPMI 1640 medium (16.20 g/L RPMI 1640 (Sigma, Munich, Germany) containing 25 mM HEPES, 11.11 mM glucose, 0.20% sodium bicarbonate (Sigma, Munich, Germany), 0.50% Albumax I (Gibco, Waltham, MA, USA), 45 µg/mL hypoxanthine (Sigma, Munich, Germany) and 50 µg/mL gentamicin (Gibco, Waltham, MA, USA) and incubated at 37 • C in an atmosphere of 5% O 2 , 5% CO 2 , and 90% N 2 . The spent medium was replaced with fresh complete medium every day to propagate the culture. Giemsa-stained blood smears were examined microscopically under oil immersion to monitor cell-cycle transition and parasitemia.

In Vitro Anti-Plasmodial Assay
Plant extracts were assessed for in vitro antiplasmodial activity using the SYBR green I-based fluorescence assay set up as described by Smilkstein et al. [33]. Crude extracts were prepared at 25 mg/mL in dimethyl sulfoxide (DMSO), while the chloroquine (Sigma-Aldrich, New Delhi, India) stock solution used as standard drug was prepared in water (Milli-Q grade) at 1 mM. All stock solutions were then diluted in 96-well, round-bottom, tissue culture-grade plates (Corning, New York, USA) with fresh RPMI 1640 culture medium to achieve the required concentrations for testing. In all cases, except for chloroquine (positive control), the final solution contained 0.4 % DMSO, which was found to be non-toxic to the parasite. Extracts were tested at concentrations ranging from 0.10 to 100 µg/mL, and chloroquine was used at 1 µM. All tests were performed in triplicate.
Briefly, 100 µL of sorbitol-synchronized parasites [34] were incubated under normal culture conditions (37 • C, 5% CO 2 , 5% O 2 , 90% N 2 ) at 1% parasitemia and 2% hematocrit in flat-bottomed, 96-well plates (Corning, Corning, NY, USA) in the absence or presence of increasing concentrations of crude extracts for 48 h. Chloroquine (Sigma-Aldrich, New Delhi, India) was used as positive control, while 0.4% (v/v) DMSO was used as the negative control. Following incubation, 100 µL of SYBR green I lysis buffer (Tris (20 mM, pH 7.5), EDTA (5 mM), saponin (0.008%, w/v), and Triton X-100 (0.08%, v/v)) was added to each well and mixed gently twice, and incubated in dark at 37 • C for 1 h. Fluorescence was then measured with a Victor fluorescence multi-well plate reader (Perkin Elmer, Waltham, MA, USA) with excitation and emission wavelength bands centered at 485 and 530 nm, respectively. The fluorescence counts were plotted against drug concentration and the 50% inhibitory concentration (IC 50 ) was determined by analysis of dose-response curves using the IC Estimator-version 1.2 software (http://www.antimalarial-icestimator.net/MethodIntro.htm) (Free Software Foundation, Boston, MA, USA). Resistance indices (RIs) were calculated as IC 50 Pf INDO/IC 50 Pf 3D7. Results were validated microscopically by examination of Giemsa-stained smears of extract-treated/untreated parasite cultures.

Cytotoxicity Study of the Selected Extracts Using MTT Assay
The cytotoxic effect of antiplasmodial extracts was assessed using the MTT assay [35], targeting human embryonic kidney cells (HEK239T cells) cultured in complete medium containing 13.5 g/L DMEM (Gibco, Waltham, MA USA), 10% fetal bovine serum (Gibco, Waltham, MA USA), 0.21% sodium bicarbonate (Sigma-Aldrich, New Delhi, India) and 50 µg/mL gentamicin (Gibco, Waltham, MA, USA). Essentially, HEK239T cells at 104 cells/200 µL/well were seeded into 96-well flat-bottomed tissue culture plates (Corning, Corning, NY, USA) in complete medium. Then, 50 µL of serially diluted extracts solutions (≤200 µg/mL) were added after 24 h of seeding and the samples incubated for 48 h in a humidified atmosphere at 37 • C and 5% CO 2 . DMSO at final concentrations (v/v) of 0.4% and 10% were used as negative (100% growth) and positive (0% growth) controls respectively. Twenty microliters of a stock solution of MTT (5 mg/mL in 1× phosphate-buffered saline) were added to each well, gently mixed, and incubated for additional 4 h. After spinning the plate at 1500 rpm for 5 min, the supernatant was carefully removed and 100 µL of 10% DMSO (v/v) was added. Formazan formation was read on a microtiter plate reader (Versa Max Microplate Reader, Molecular Devices, San Jose, CA, USA) at 570 nm. The 50% cytotoxic concentrations (CC 50 ) of extracts were determined by analysis of dose response curves (Graphpad prism 5.0, GraphPad, La Jolla, CA, USA). Selectivity indices (CC 50 /IC 50 ) were calculated for each extract.

Results and Discussion
Medicinal plants have and will always play a vital role in the management of community health and the discovery of novel chemotherapeutic agents since they are rich repositories of a wide range of metabolites that have promise against diverse diseases. Therefore, collection of plants based on the ethnomedical knowledge is still an attractive starting point for drugs discovery. In the present study, 10 medicinal plants from Cameroon were evaluated for their antiplasmodial activity against chloroquine (CQ)-sensitive P. falciparum 3D7 and CQ-resistant P. falciparum INDO strains.

Results and Discussion
Medicinal plants have and will always play a vital role in the management of community health and the discovery of novel chemotherapeutic agents since they are rich repositories of a wide range of metabolites that have promise against diverse diseases. Therefore, collection of plants based on the ethnomedical knowledge is still an attractive starting point for drugs discovery. In the present study, 10 medicinal plants from Cameroon were evaluated for their antiplasmodial activity against chloroquine (CQ)-sensitive P. falciparum 3D7 and CQ-resistant P. falciparum INDO strains.
The extraction yields as indicated in table 1 varied from 1.10% to 29.17%, depending on the plant part and solvent of extraction. The highest yields were obtained with the aqueous maceration extract of fruit of Ficus benjamina (Fbfr W : 29.17%), followed by decoction extract of leaves of T. mantaly (Tml D : 27.70%) and aqueous maceration extract of leaves of F. benjamina (Fbl W : 26.71%). The results of the in vitro evaluation of the potential of the 66 extracts from medicinal plants to inhibit the growth of the Pf3D7 and PfINDO strains are presented in Figure 1 and summarized in Table 2. The antiplasmodial activity of plant extracts ranged from 1.90 to >100 μg/mL against the two strains. Bagavan et al. [36] have classified the antiplasmodial activity of plant extracts as good (IC50 < 10 μg/mL), moderate (IC50 > 10 to <25 μg/mL), and weak (IC50 > 25 μg/mL). Therefore, out of the 66 extracts tested, six showed good activity (IC50 = 2.49-6.49 μg/mL), 10 exhibited moderate activity (IC50 = 12.41-25.08 μg/mL), while 50 displayed weak (IC50 > 25 μg/mL) antiplasmodial activity against the malaria parasites ( Figure 1A,B). All extracts tested were nearly equipotent against both sensitive and resistant strains of the malaria parasite.
The most active extracts were the water maceration and decoction, and ethyl acetate extracts of leaves of D. principum (IC503D7/INDO = 4.91/6.64, 5.49/5.98, and 6.49/7.10 μg/mL respectively), water decoction extract of leaves of T. catappa (IC503D7/INDO = 6.41/8.10 μg/mL) and water decoction extracts of leaves and bark of T. mantaly (IC503D7/INDO = 2.49/1.90 and 3.70/2.80 μg/mL, respectively). As shown in Table 2, the promising extracts listed above exhibited no cytotoxicity on HEK293T at up to 200 μg/mL, giving selectivity indices (SI) in the range of >31.20-80.32.  The antiplasmodial activity of Drypetes principum against both Pf3D7 and Pf INDO strains is being reported for the first time. For this plant and considering the criteria of Bagavan et al. [36], the water maceration (Dpst w ) and decoction (Dpst D ) extracts of stems showed no activity (IC50 > 100 µ g/mL), contrasting with the water maceration and decoction extracts of the twig (Dptw W ; Dptw D ) that exerted weak activity, the ethyl acetate extracts of twigs and stems (Dptw E ; Dpst E ) that exhibited moderate activity and finally the promising extracts (water maceration of the leaf: Dpl W -IC50Pf3D7/INDO = 4.91/6.64 µ g/mL; ethyl acetate extract of the leaf: Dpl E -IC50Pf3D7/INDO = 5.49/5.98 µ g/mL; and the decoction of the leaf: Dpl D -IC50Pf3D7/INDO= 6.49/7.10 µ g/mL) that showed good activity (IC50 < 10 µ g/mL). Extracts from the leaves were promising given their potent activities in both polar water extracts and less polar ethyl acetate extract. Unlike leaves, the activities associated with extracts from twigs were solvent-dependent, ranging from moderate when using ethyl acetate to weak with aqueous extracts. The ethyl acetate extracts of twigs and stems were more active than their respective water extracts counterparts, suggesting that their antiplasmodial metabolites are more soluble in ethyl acetate than water. However, as stated earlier, independently of the solvent used, extracts from leaves exhibited good activities, indicating that the leaves of D. principum may contain various classes of antiplasmodial metabolites that are soluble in water (whether macerated or whether decocted) and in ethyl acetate, or the same metabolites may dissolve in both solvents due to their amphipathic nature.
Moreover, decoction extracts from the leaves of T. catappa and leaves and bark of T. mantaly exhibited very promising activity against Pf 3D7 (IC50 = 2.49-6.41 µ g/mL) and PfINDO (IC50 = 1.90-8.10 µ g/mL). Earlier, Abiodun et al. [24] reported that hexane, ethyl acetate and methanol extracts of leaves of T. catappa showed potent antipasmodial activity against PfK1(IC50 = 3.05-10.10 µ g/mL) and PfNF54 (IC50= 6.68-21.93 µ g/mL) with hexane extract being less potent (IC50 = 10.10-21.93 µ g/mL). A previous study by Mbouna et al. [27] showed that water and methanol extracts of leaves, stem bark, and roots of T. mantaly displayed very good activity against Pf3D7 (IC50 = 1.03-5.09 µ g/mL) and PfINDO (IC50 = 0.26-7.01 µ g/mL). Equally, in vitro antiplasmodial activity of other species of the genus Terminalia has been previously reported [37,38]. Thus, extracts from stem bark of Terminalia avicennoides (IC50 = 10.99-14.76 µ g/mL (Pf3D7) and 9.31-12.56 µ g/mL (PfK1) and isolated compounds including ellagic acid (IC50 = 12.14 and 11.20 μg/mL), flavogallonic acid (IC50 = 8.89 and 8.35 µ g/mL), punicalagin (IC50 = 9.42 and 8.79µ g/mL), castalagin (IC50 = 10.57 and 9.63 µ g/mL) and The antiplasmodial activity of Drypetes principum against both Pf 3D7 and Pf INDO strains is being reported for the first time. For this plant and considering the criteria of Bagavan et al. [36], the water maceration (Dpst w ) and decoction (Dpst D ) extracts of stems showed no activity (IC 50 > 100 µg/mL), contrasting with the water maceration and decoction extracts of the twig (Dptw W ; Dptw D ) that exerted weak activity, the ethyl acetate extracts of twigs and stems (Dptw E ; Dpst E ) that exhibited moderate activity and finally the promising extracts (water maceration of the leaf: Dpl W -IC 50 Pf3D7/INDO = 4.91/6.64 µg/mL; ethyl acetate extract of the leaf: Dpl E -IC 50 Pf3D7/INDO = 5.49/5.98 µg/mL; and the decoction of the leaf: Dpl D -IC 50 Pf3D7/INDO= 6.49/7.10 µg/mL) that showed good activity (IC 50 < 10 µg/mL). Extracts from the leaves were promising given their potent activities in both polar water extracts and less polar ethyl acetate extract. Unlike leaves, the activities associated with extracts from twigs were solvent-dependent, ranging from moderate when using ethyl acetate to weak with aqueous extracts. The ethyl acetate extracts of twigs and stems were more active than their respective water extracts counterparts, suggesting that their antiplasmodial metabolites are more soluble in ethyl acetate than water. However, as stated earlier, independently of the solvent used, extracts from leaves exhibited good activities, indicating that the leaves of D. principum may contain various classes of antiplasmodial metabolites that are soluble in water (whether macerated or whether decocted) and in ethyl acetate, or the same metabolites may dissolve in both solvents due to their amphipathic nature.

Discussion
Ethyl acetate extracts of stems and twigs of Alchornea lacifolia (Alst E , Altw E , respectively) displayed moderate antiplasmodial activity (IC 50 Pf 3D7/INDO ranging 12.44-16.64 µg/mL) against both P. falciparum strains, whereas the corresponding aqueous extracts were weakly active or inactive (>25 to >100 µg/mL). Moreover, leaf and trunk extracts displayed weak antiplasmodial activity to inactivity against the sensitive and resistant P. falciparum strains. Okokon et al. [26] have recently reported that ethyl acetate extract of roots of A. lacifolia exhibits weak activity against both strains of P. falciparum with IC 50 values of 38.44 µg/mL (Pf 3D7) and 40.17 µg/mL (Pf INDO) which suggests that stems and twigs may be preferred over roots and leaves of A. lacifolia as sources of antiplasmodial metabolites.
Activity of extracts from A. senegalensis varied considerably depending of extract type and parasite strain, but none of them exhibited good antiplasmodial potency (IC 50 < 10µg/mL). However, moderate activity was recorded for the ethanol and hydroethanol extracts of bark (Asb Et , Asb Wet ) and hydroethanol extracts of stems and leaves (Asst Wet , Asl Wet ) with IC 50 ranging 13.16-25.08 µg/mL against both P. falciparum strains. Of note, the maceration extract of A. senegalensis bark (Asb W ) showed moderate activity against the sensitive Pf 3D7 strain but was rather inactive against the resistant INDO strain (IC 50 > 100 µg/mL). Wele et al. [21] have recently reported moderate to weak antiplasmodial activity of ethanol extracts from leaves of A. senegalensis against Pf 3D7 and Pf Dd2 (IC 50 = 23.93 and 29.47 µg/mL respectively). However, leaf ethanolic crude extract of A. senegalensis growing in the Democratic Republic of Congo was reported to exhibit weak activity against P. falciparum FcM29 (IC 50 = 32.52 µg/mL) [39], corroborating the findings of Ndjonka et al. [40] who reported weak antiplasmodial activity (IC 50 = 94.80 µg/mL) of ethanol extract of leaves of A. senegalensis collected in Cameroon. In comparison to literature data, our findings suggest that hydro-ethanol may be the best solvent for extraction of antiplasmodial compounds from A. senegalensis leaves, twigs, and stems, while ethanol could be more appropriate for extraction of active compounds from the bark.
Water and hydroethanol extracts of leaves, stems, and fruit as well as decoction extracts of Ficus benjamina were investigated against P. falciparum strains. Overall, the results showed that only the water maceration extract of leaves (Fbl W ) could exhibit moderate (IC 50 =12.41 µg/mL) to weak (IC 50 = 26.35 µg/mL) activity against Pf 3D7 and Pf INDO, respectively. A weak activity was also recorded against Pf INDO for the hydroethanol extract of stems (Fbst Wet ) with IC 50 of 52.91 µg/mL, but this extract showed to be inactive against the sensitive P. falciparum 3D7 strain (>100 µg/mL). In contrast to the present findings, Hayat et al. [22] reported that hydroethanol and petroleum ether leaf extracts of F. benjamina exhibited weak (IC 50 = 31.80 µg/mL) and moderate (IC 50 = 14.50 µg/mL) antiplasmodial effects against Pf 3D7. Extracts of the other investigated species of Ficus genus (F. exasperata) exhibited mostly weak antiplasmodial activity (IC 50 > 25 µg/mL) against both P. falciparum strains.
The extracts of Senna alata were mostly inactive, with the exception of the leaf ethanol and decoction extracts (Cal Et , Cal D ) that showed weak antiplasmodial activity against both strains (IC 50 > 31.36 µg/mL) and the twig ethanol extract that also weakly inhibited the resistant Pf INDO (IC 50 = 37.06 µg/mL) strain. Our findings corroborate those of Zirihi et al. [41] who recorded no antiplasmodial activity at concentrations up to 50 µg/mL in the leaf ethanol extract of Senna alata. Besides, Kayembe et al. [23] reported promising antiplasmodial activity (IC 50 = 12.50 µg/mL) in the seed ethanol extract of C. alata. In addition, Kaushik et al. [25] reported antiplasmodial activity in the ethyl acetate extract of C. alata against both CQ-sensitive 3D7 (IC 50 = 18.00 µg/mL) and CQ-resistant INDO (IC 50 = 20.00 µg/mL) strains. This suggests that for this plant, ethyl acetate might be a better solvent for extraction of promising metabolites from the leaves as compared to ethanol and water.
This study also shows that the ethanol and methanol extracts of Occimum gratissimum have weak antiplasmodial activity (IC 50 > 25 µg/mL) against both 3D7 and INDO strains. Abiodun et al. [19] also reported comparable activity profile (IC 50 = 36.71 µg/mL) of methanol extract of O. gratissimum leaves against the chloroquine-sensitive Pf NF54 strain. The same authors further reported very good activity from ethyl acetate extract of leaves of O. gratissimum against Pf K1 (IC 50 = 1.80 µg/mL) and Pf NF54 (IC 50 = 3.61 µg/mL) respectively [24]. This activity variation indicates that instead of alcohols like ethanol and methanol, ethyl acetate should be the solvent of choice for extracting potent antipasmodial compounds from the leaves and roots of O. gratissimum. The results achieved also indicated that the decoction extract of Cananga odorata flower was inactive (IC 50 > 100 µg/mL) against both Pf 3D7 and Pf INDO. Similar work undertaken by Nyugen-Poupin et al. [42], but targeting leaves instead, and using cyclohexane as extractant showed a moderate activity against Pf FcB1 strain with IC 50 = 12.50 µg/mL. Overall, reported differences in the antiplasmodial activity of plant extracts may result from the influence of many factors such as time and site of plant collection, maturity of plant parts, intra-species variations, part investigated, edaphic substrate, climate, methods used for extraction, type of bioassay, parasite strain etc. [43].
The resistance index (RI) which indicates the inhibitory potential of a drug against both sensitive and resistant strains of P. faciparum was determined for each extract using IC 50 values against Pf 3D7 and Pf INDO strains. The RI ranged from 0.43 to >6.91. Given that extracts with RI ≤ 1 might be considered promising against both sensitive and resistant parasite strains, 16  Selectivity index (SI), defined as the ratio of CC 50 HEK293T to IC 50 P. falciparum was also determined. The higher the SI, the more promising is the extract due to its selective action on malaria parasites. Eleven out of the 14 selected plant extracts that were evaluated for cytotoxicity displayed strong selectivity (SI > 10.58) for P. falciparum. The highest SI values were obtained for the decoction extract of leaves and bark of T. mantaly (SI > 80.32). Overall, water, ethyl acetate, and decoction extracts of leaves of D. principum, and decoction extracts of leaves of T. catappa and T. mantaly are considered of interest since they display high antiplasmodial activity (IC 50 = 1.90-8.10 µg/mL) with high selectivity indices (SI > 31.20) against both P. falciparum 3D7 and INDO strains.

Conclusions
The need to continue searching for new antimalarial molecules is driven by the continuous spread of multi-drug resistant malaria parasites. The present study has found that the leaves of D. principum, and T. catappa and bark of T. mantaly possess significant antiplasmodial activities, with good selectivity against chloroquine-sensitive and -resistant strains of P. falciparum. These findings confirm the use of much of these plants in the treatment of malaria and related symptoms. Further studies on these extracts, including bioassay-guided fractionation, are likely to yield new antimalarial compounds and ancillary molecules which could be developed as alternative drug combination therapies against malaria.

Acknowledgments:
The authors gratefully acknowledge support from the National Herbarium of Cameroon for plant identification, and support from the Seeding Labs' Instrumental Access Grant (SL2012-2) to Boyom F.F.

Conflicts of Interest:
The authors declare no conflict of interest.