Antimalarial Studies and Phytochemical Profiling of Laportea aestuans (L.) Chew Extractives

Abstract

Background and objectives

This study validated the antimalarial efficacy of Laportea aestuans extract using chemosuppressive and curative models, identified its most active fraction, and performed chemical profiling of the active fraction.

Methodology

The aerial part of L. aestuans (LA) was extracted with 80% methanol and assayed for its chemosuppressive and curative antimalarial activities through the oral administration of the extract (100, 200, 400 and 800 mg/kg) to groups consisting of five mice each. The extract was thereafter suspended in water and partitioned with dichloromethane to afford the dichloromethane (DLA) and aqueous (ALA) fractions. The fractions (10, 20, 40, and 80 mg/kg) were thereafter tested for their antimalarial activity followed by GC-MS analysis of the most active fraction.

Results

The toxicity level was found to be above 2000 mg/kg. The activity recorded for the four-day chemosuppressive antimalarial tests showed a dose-dependent activity up to 400 mg/kg. The activity was significantly lower than that of chloroquine. The antimalarial activity of the extract at the lowest dose of 100 mg/kg was 42%. In the curative test, the extract of LA gave a dose-dependent activity that was significantly higher than the negative control. The DLA and ALA fractions gave a comparable curative activity across all doses. At 10 mg/kg, ALA and DLA gave percentage clearance of 81.25 ± 1.84 and 87.45 ± 1.35, respectively, which were significantly higher than that of the negative control (0.00 ± 0.00) and significantly lower than that of the positive control (94.93 ± 0.26). The chemical profiling revealed the presence of non-polar and medium-polar constituents in the dichloromethane fraction.

Conclusion

The good antimalarial activity elicited by LA extractives supports its usage in ethnomedicine as an antimalarial agent.

Keywords

antimalarial chemo suppressive curative GC-MS profiling

Introduction

Parasitic diseases are most prevalent in developing countries, particularly those situated in warm and humid areas of tropical, subtropical, and temperate regions. Seven of the 10 globally important tropical diseases are parasitic diseases, including malaria, schistosomiasis, lymphatic filariasis, onchocerciasis, leishmaniasis, African trypanosomiasis and Chagas disease.^1–3 Malaria is a disease in humans caused by Plasmodium parasites, which is transmitted to humans via bite from infected female Anopheles mosquitoes.⁴ Mild symptoms include fever, chills and headache while severe symptoms include fatigue, confusion, seizures, and difficulty breathing.⁵ Infants, children below 5 years, pregnant women, travelers, and people with HIV or AIDS are at higher risk of severe infection.^6,7

The latest World Malaria report indicates a slight increase in malaria cases, with 249 million reported in 2022 compared to 244 million in 2021.⁷ The WHO African Region bears the overwhelming majority of the global malaria burden, with an estimated 94% of all malaria cases and 95% of deaths occurring there in 2022. Children under 5 years of age accounted for about 78% of all malaria deaths in the region.⁷

Malaria is preventable by using insecticide-treated nets or taking prophylactics. Treatment typically involves antimalarial medications.⁸ However, resistance to these medications has emerged as a major challenge over time.⁹ A prime example is the emergence of partial artemisinin resistance, which has significantly threatened global malaria control efforts in the past decade.¹⁰ While new studies explore the development of malarial vaccines,^11,12 there remains a pressing need to investigate natural sources for the discovery of more effective antimalarial lead molecules with minimal side effects.

Plant-derived medicines have been an integral aspect of traditional health care in many regions of the world for thousands of years. There is a rising interest in them as sources for the treatment of diseases.^13,14 Medicinal plants biosynthesize numerous secondary metabolites that are sources of bioactive organic compounds, which help defend against infection or disease.¹⁵ These substances may also act as attractants, insecticides, antifeedants, oviposition inhibitors, repellents, growth inhibitors, and juvenile hormone analogs.¹⁶ Medicinal plants have shown potential use in the treatment of malaria. Scientific studies and traditional evidence have helped establish the antimalarial activities of African medicinal plants, including Fagara zanthoxyloides, Monodora myristica, Piliostigma thonningii, Cassia nigricans, Dracaena manni, Stachytarpheta cayennensis, and Newbouldia laevis.¹⁷

Phytochemicals like benzophenanthridine alkaloids, sesquiterpenes, triterpenoid, saponins, glycoside/glucoside, and pheophytins are some of the identified active compounds isolated from these medicinal plants.¹⁷ There are also predictions of the mechanism of action of some of these plant metabolites. For example, androstan-17-one,3-ethyl-3-hydroxy-, (5 alpha) identified in the fruits of Piper betle has been predicted to interact with plasmepsin 1 and 2, which are employed in the degradation of hemoglobin upon infection by Plasmodium falciparum.¹⁸ Since medicinal plants have shown potential as a veritable source of antimalarial compounds, this study evaluates the antimalarial properties of Laportea aestuans, which is traditionally used in the treatment of malaria.

Laportea aestuans (Urticaceae), the West Indian Nettle, is a little-branched, annual plant native to tropical regions. It is now widely spread throughout both the western hemisphere and eastern hemisphere tropics and subtropics.¹⁸ It is usually 1 metre tall, up to 3 metres. The stem is fleshy, becoming slightly woody at the base, and the whole plant is densely covered with stinging hairs up to 1 mm long.¹⁹ It is widely used in the management of various diseases in African traditional medicine. The pulped whole plant is eaten or the plant sap is drunk as an anthelmintic and for the treatment of hernias. Externally, it is also applied in the treatment of fevers in children, malaria (personal communication), oedema, and ulcers. The sap, often mixed with palm oil or kaolin, is applied to abscesses and wounds; on the head of children to close the fontanel; on the abdomen to ease childbirth; and on the gums to relieve toothache.²⁰ A macerated fresh leaf of L. aestuans is used to massage the body for the treatment of intercostal pain and stitches on the side. Slightly scorched or smoked leaves are applied to burns and used against migraine.¹⁸ A leaf decoction of the plant is applied on swellings; instilled in the eye to treat minor eye infections; and as an embrocation to strengthen rachitic children and to relieve fever.¹⁹

Extract of L. aestuans is reported to elicit antioxidant properties, while its essential oil whose constituent is mainly methyl salicylate exhibits significant antioxidant and antimicrobial activities (Oloyede, 2011).¹⁹ Other studies reported the phytochemical, toxicity, antimicrobial, and antioxidant screening of extracts obtained from the plant.^19–21 Despite the rich pharmacological studies performed on the plant, its antimalarial activity is yet to be investigated. Therefore, this study uses chemosuppressive and curative models to examine its antimalarial efficacy. In addition, the study performed chemical profiling for the identification of compounds present in their active fractions.

Methodology

Plant Collection, Extraction, and Partitioning

The aerial parts of L. aestuans were collected from the Obafemi Awolowo University (OAU) campus. The plant material was identified and authenticated at the Forestry Research Institute of Nigeria, Ibadan, Nigeria, with the voucher number FHI 11035. Three hundred grams (300 g) of fresh-weight plant material was crushed and extracted with 80% methanol for 48 h. The extract was then filtered and concentrated to yield 3 g of residue. The residue was suspended in water and partitioned with dichloromethane, affording 1.2 g of a dichloromethane fraction and 1.8 g of an aqueous fraction.

Antimalarial Studies

Animals

Healthy Swiss albino mice, both male and female (nonpregnant), weighing between 18 to 24 g, were procured from the Animal House, Faculty of Basic Medical Sciences, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria. The mice were housed in aluminum cages with wood shavings as bedding material and were offered water freely and Growers’ mash as food, maintaining under a 12-h light/dark cycle. The animal experimental protocol received approval from the Health Research and Ethics Committee of the Institute of Public Health, Obafemi Awolowo University, Ile-Ife, Nigeria. All procedures adhered to the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication, No. 83-123, revised 1985). Also, the study was approved by the Postgraduate Committee of Obafemi Awolowo University with registration number: SCP17/18/H/1272. Acclimatization was ensured for a minimum of seven days before the commencement of experiments, and the mice were randomly assigned to groups of five for the study.

Parasite

Plasmodium berghei strain NK65, susceptible to Chloroquine, was kindly donated by Professor O. G. Ademowo of the Institute of Advanced Medical Research and Training (IMRAT), University College Hospital, Ibadan. This strain was employed for evaluating the in vivo chemosuppressive and curative antimalarial activities. The parasite strain was preserved by successively transferring blood from infected mice to uninfected mice. The donor mouse was euthanized, and blood was collected via cardiac puncture into a heparinized bottle to prepare the inoculum. This inoculum was then diluted with a normal saline solution to ensure that 0.2 mL of the inoculum contained 1.0 × 10⁷ parasitized red blood cells.

in Vivo Antimalarial Assays

The efficacy of the extract in suppressing and treating malaria was assessed by orally administering varying doses (100 mg/kg, 200 mg/kg, 400 mg/kg, and 800 mg/kg) to mice, alongside chloroquine (CQ) at 10 mg/kg for comparison, while a control group received normal saline. Each group comprised five mice. Administration commenced two hours after infection and continued daily for 3 days in the suppression model. In the treatment model, administration began on the third day post-infection and continued daily for 5 days. Prior to administering the extracts or standard drug, the body temperature of each mouse was measured using a digital clinical thermometer inserted into the rectum.

In both models, parasitemia levels were evaluated for each mouse on Day 4 (D4) and daily thereafter in the suppression and treatment models, respectively. This assessment involved cell counting in 5 fields of view on a microscope slide with a thin blood smear obtained from the tail of each mouse. The smears were fixed with methanol and stained with Giemsa. The average parasitemia in each group was calculated to determine the percentage of suppression and treatment efficacy of the extract using the formula:

(A - B) / A \times 100,

where A and B represent the mean parasitemia in the negative control and the test groups, respectively. The suppressive activity of the extract was determined by assessing the percentage reduction of parasitemia in the treated groups compared to the untreated infected group.

Survival Times

All the animals used for this study were observed for 28 days for possible mortality, while the survival time and percentage of survivors were calculated using the formula below^22,23:

\begin{aligned} % S u r v i v a l = & ((N u m b e r o f a n i m a l s (D a y 0) \\ - N u m b e r o f A n i m a l s (D a y 7)) \times 100) \\ \div N u m b e r o f A n i m a l s (D a y 0) . \end{aligned}

Statistical Analysis

Values were expressed as mean ± SEM and analyzed statistically using One-way Analysis of Variance (ANOVA) followed by Student Newmann Keul's post-hoc for comparisons to determine the source of significant difference for all values. Values of P < 0.05 were of statistical significance.

GC-MS Profiling

The dichloromethane fraction of L. aestuans underwent GC-MS analysis utilizing a Varian 3800/4000 gas chromatograph mass spectrometer equipped with an Agilent fused silica capillary CP-Sil 5 CB column (30 m × 0.25 mm i.d.). This system was linked to a Varian 4000 mass spectrometer operating in Electron Impact (EI) mode (70 eV; m/z 30-1000 amu; source temperature 230 °C and quadruple temperature 150 °C). The initial temperature was held at 150 °C and then gradually increased to a maximum of 300 °C. A 1 μL sample was injected in split mode (10:1). Nitrogen gas was employed as the carrier gas at a flow rate of 0.8 mL/min, and the total runtime was set at 40 min.

For the identification of phytochemical components, the National Institute of Standards and Technology MS library (NIST-MS library 2009) was utilized, comparing the obtained spectrum via GC–MS to identify compounds present in the samples. Retention indices (RI) were determined relative to a homologous series of n-alkanes (C6–C28) on the CP-Sil 5 CB column under identical chromatographic conditions. The relative concentrations of components were determined through peak area normalization, with no response factors calculated. All samples and their replicates were injected as a single batch in a random order to distinguish technical variations from biological ones. Moreover, the pooled samples prepared were employed as quality controls (QCs) and injected at regular intervals during the analytical run. This approach facilitated the generation of a dataset for assessing repeatability.

Results

Acute Oral Toxicity

Extracts that mice can tolerate at doses of up to 5000 mg/kg without exhibiting any signs of toxicity can be considered non-toxic and safe.^23–27 Regarding the aqueous methanol extract of L. aestuans, doses of up to 5000 mg/kg did not result in mortality or manifest any adverse effects such as skin changes, aggressiveness, diarrhea, restlessness, dizziness, weakness, or refusal to consume food or water. Therefore, it can be inferred that the tested extract is devoid of toxicity.²⁸ Similar findings were also reported in studies involving Plumeria alba and Azadirachta indica.^25,28

Evaluation of Chemosuppressive Effect of LA Extract

The chemosuppressive effect of LA extract varied from 42% to 67% across doses ranging from 100 to 800 mg/kg. Comparatively, chloroquine demonstrated a higher chemosuppressive effect of 80%, which was significantly greater (p < 0.05) than that of the LA extract at all tested doses. Furthermore, the mean survival time of mice treated with LA extract ranged from 11.00 ± 0.32 to 13.00 ± 0.00 days across different doses. In contrast, mice treated with chloroquine exhibited a mean survival time of 26.40 ± 1.60 days, while those in the negative control group survived for 6.60 ± 0.75 days (Table 1).

Table 1.

Antiplasmodial Activity of LA Leaf Extract in Mice Infected Plasmodium Berghei in 4-day Chemosuppressive Test.

Test Doses/Substance (mg/kg)	% Parasitaemia	% Suppression	Mean Survival Time (in Days)
NC	5.30 ± 0.18^d	0.00 ± 0.00^a	6.60 ± 0.75^a
100	3.07 ± 0.17^c	42.20 ± 1.31^b	11.00 ± 0.32^b
200	2.06 ± 0.04^b	60.99 ± 0.63^c	11.80 ± 0.20^b
400	1.73 ± 0.02^b	67.17 ± 0.96^d	11.00 ± 0.95^b
800	2.15 ± 0.13^b	59.19 ± 3.37^c	13.00 ± 0.00^b
CQ	1.04 ± 0.04^a	80.31 ± 1.14^e	26.40 ± 1.60^c
ED₅₀		328.10 ± 2.42
ED₉₀		613.61 ± 15.53

Keys: Data show the mean ± SEM, n = 5. NC (negative control): Tween 80 in normal saline; Chloroquine (10 mg/kg) = positive control. Only values with different superscripts (a, b, c, d or e) within columns are significantly different (p < 0.05, one-way analysis of variance followed by the Student–Newman–Keul's post hoc test).

Evaluation of the Curative Effect of LA Extract

The findings from the analysis of the curative potential of LA extract at doses of 100, 200, and 400 mg/kg revealed a decrease in percentage parasitemia from 8.04–8.72% on day 1 to 1.76–1.92% on day 5. Additionally, the curative effects observed for doses of 800 mg/kg and chloroquine (5 mg/kg) on day 5 resulted in a reduction of percentage parasitemia to 1.19% and 0.9%, respectively (Table 2).

Table 2.

Percentage Parasitaemia in an in Vivo Antimalarial Activities (Curative) Test of the Methanol Extract of the LA at Different Doses at DAY 0 to 5.

Doses (mg/kg)			% Parasitaemia
EXTRACTS/ DRUG	DOSES (mg/kg)	DAY 1	DAY 3	DAY 5
NC	0	8.04 ± 0.06^a	9.73 ± 0.18^d	12.26 ± 0.21^c
LA	100	8.72 ± 0.08^a	6.13 ± 0.67^c	1.92 ± 0.22^b
	200	8.43 ± 0.09^a	3.64 ± 0.17^b	1.83 ± 0.08^b
	400	8.10 ± 0.24^a	2.07 ± 0.09^a	1.76 ± 0.09^b
	800	8.12 ± 0.27^a	1.98 ± 0.04^a	1.19 ± 0.10^a
CQ	10	8.02 ± 0.07^a	1.93 ± 0.74^a	0.90 ± 0.03^a

Keys: LA = Data show the mean ± SEM, n = 5, NC = Negative Control (Tween 80 in normal saline); CQ = Chloroquine (10 mg/kg). Only values with different superscripts (a, b, c, d, e, f or g) within columns are significantly different (p < 0.05, one-way analysis of variance followed by the Student–Newman–Keuls’ post hoc test).

Percentage Clearance and Survival Time of LA Extract in Mice

On day 5, LA demonstrated percentage clearances of 84.28%, 85.04%, 85.65%, and 90.26% at doses of 100, 200, 400, and 800 mg/kg, respectively, in comparison to the 0.00% observed in the negative control group. In contrast, chloroquine exhibited a clearance of 92.66% (Table 3). The antiplasmodial efficacy of LA peaked at a dose of 400 mg/kg. The mean survival times, recorded in days, for the various doses, negative control, and chloroquine were 18.20, 21.40, 24.40, 26.80, 6.00, and 28.00, respectively (Table 3). The calculated ED₅₀ and ED₉₀ for the extract were 210.15 mg/kg and 399.21 mg/kg, respectively (Table 3).

Table 3.

Percentage Clearance in an in Vivo Antimalarial Activities (Curative) Test of the Methanol Extract of the LA at Different Doses at DAY 0 to 4.

Doses (mg/kg)			% Clearance		Mean Survival Time (in Days)
EXTRACTS/ DRUG	DOSES (mg/kg)	DAY 1	DAY 3	DAY 5	Mean Survival Time (in Days)
NC	0	0.00 ± 0.00^a	0.00 ± 0.00^a	0.00 ± 0.00^a	6.00 ± 0.55^a
LA	100	−8.47 ± 1.41^a	36.93 ± 6.95^b	84.28 ± 1.85^b	18.20 ± 0.58^b
	200	−4.84 ± 1.13^a	62.43 ± 2.28^c	85.04 ± 0.66^b	21.40 ± 1.21^c
	400	−0.83 ± 3.73^a	78.67 ± 1.16^d	85.65 ± 0.63^b	24.40 ± 1.29^d
	800	−0.98 ± 2.88^a	79.64 ± 0.68^d	90.26 ± 0.89^c	26.80 ± 0.58^d,e
CQ	10	0.22 ± 1.51^a	80.11 ± 0.43^d	92.66 ± 0.19^c	28.00 ± 0.00^e
ED₅₀		907.65 ± 450.89	289.34 ± 12.79	210.15 ± 1.86
ED₅₀		1360.83 ± 788.87	581.67 ± 16.91	399.21 ± 4.56

Evaluation of Curative Effect of LA Solvent Fractions

The percentage parasitemia induced by the solvent fractions of LA on day 5 at various doses of ALA10, ALA20, ALA40, ALA80, DLA10, DLA20, DLA40, and DLA80 mg/kg were 2.65, 1.83, 1.49, 3.15, 1.77, 1.24, 0.91, and 2.36%, respectively, compared to the negative control's parasitemia level of 14.27%. In contrast, chloroquine resulted in a parasitemia level of 0.72% (Table 4).

Table 4.

Percentage Parasitaemia in an in Vivo Antimalarial Activities (Curative) Test of the Solvent Fractions of LA at Different Doses at DAY 1 to 5.

Doses (mg/kg)			% Parasitaemia
EXTRACTS/ DRUG	DOSES (mg/kg)	DAY 1	DAY 3	DAY 5
NC	0	9.20 ± 0.17^b	11.08 ± 0.15ⁱ	14.27 ± 0.41^h
ALA	10	8.08 ± 0.33^a	2.154 ± 0.11^f	2.65 ± 0.18^f
	20	7.82 ± 0.41^a	1.87 ± 0.04^d	1.83 ± 0.06^d
	40	8.54 ± 0.20^a,b	2.10 ± 0.08^e	1.49 ± 0.07^b
	80	8.14 ± 0.26^a,b	2.74 ± 0.10^h	3.15 ± 0.11^g
DLA	10	8.50 ± 0.26^a,b	1.46 ± 0.03^b	1.77 ± 0.14^c
	20	8.37 ± 0.06^a,b	1.58 ± 0.03^b	1.24 ± 0.02^a
	40	8.26 ± 0.02^a,b	1.15 ± 0.05^a	0.91 ± 0.03^a
	80	7.99 ± 0.18^a	2.22 ± 0.04^g	2.36 ± 0.10^e
CQ	10	8.32 ± 0.13^a,b	1.73 ± 0.05^c	0.72 ± 0.02^a

Keys: DLA = Dichloromethane fraction; ALA = Aqueous phase. Data show the mean ± SEM, n = 5, NC = Negative Control (Tween 80 in normal saline); CQ = Chloroquine (10 mg/kg). Only values with different superscripts (a, b, c, d, e, f, g, h or i) within columns are significantly different (p < 0.05, one-way analysis of variance followed by the Student–Newman–Keuls’ post hoc test).

Percentage Clearance of LA Solvent Fractions in Mice

At day 5, the solvent fractions of LA showed varying percentages of clearance at different doses of ALA10, ALA20, ALA40, ALA80, DLA10, DLA20, DLA40, and DLA80 mg/kg, with values of 81.25%, 87.07%, 89.60%, 77.93%, 87.45%, 91.28%, 93.62%, and 83.30%, respectively, in comparison to the 0.00% clearance observed in the negative control. Chloroquine, in contrast, exhibited a clearance of 94.93% (Table 5). The calculated ED₅₀ and ED₉₀ values for ALA and DLA at day 5 were 23.14; 39.18 and 21.94; 37.20 mg/kg, respectively (Table 6).

Table 5.

Percentage Clearance in an in Vivo Antimalarial Activities (Curative) Test of the Solvent Fractions of LA at Different Doses at DAY 1 to 5.

Doses (mg/kg)			% Clearance
EXTRACTS/ DRUG	DOSES (mg/kg)	DAY 1	DAY 3	DAY 5
NC	0	0.00 ± 0.00^a	0.00 ± 0.00^a	0.00 ± 0.00^a
ALA	10	12.16 ± 3.12^a,b	77.11 ± 0.98^c	81.25 ± 1.84^b
	20	14.74 ± 5.33^b	83.09 ± 0.45^b	87.07 ± 0.77^b
	40	7.00 ± 2.97^a,b	81.01 ± 0.70^a	89.60 ± 0.17^b
	80	11.28 ± 3.80^a,b	75.23 ± 0.93^a	77.93 ± 0.18^a
DLA	10	7.39 ± 2.54^a,b	86.82 ± 0.31^c	87.45 ± 1.35^b
	20	8.82 ± 2.10^a,b	85.76 ± 0.34^b	91.28 ± 0.09^b
	40	10.11 ± 1.70^a,b	89.59 ± 0.44^a	93.62 ± 0.02^b
	80	12.91 ± 3.00^a,b	79.96 ± 0.45^a	83.30 ± 1.17^a
CQ	10	9.49 ± 1.30^a,b	84.34 ± 0.51^a	94.93 ± 0.26^a

Keys: DLA = Dichloromethane fraction; ALA = Aqueous phase. Data show the mean ± SEM, n = 5, NC = Negative Control (Tween 80 in normal saline); CQ = Chloroquine (10 mg/kg). Only values with different superscripts (a, b, c, d, e, f or g) within columns are significantly different (p < 0.05, one-way analysis of variance followed by the Student–Newman–Keuls’ post hoc test).

Table 6.

Comparative ED₅₀ and ED₉₀ in an in Vivo Antimalarial Activities (Curative) Test of the Solvent Fractions of LA at Different Doses at DAY 1 to 5.

DAYS	Effective Doses (ED₅₀ and ED₉₀)
	ALA		DLA
	ED₅₀	ED₉₀	ED₅₀	ED₉₀
DAY 1	68.37 ± 25.50	109.09 ± 50.03	273.03 ± 73.64	496.17 ± 132.24
DAY 3	24.22 ± 0.20	41.62 ± 0.27	22.82 ± 0.10	38.41 ± 0.15
DAY 5	23.14 ± 0.06	39.18 ± 0.52	21.94 ± 0.21	37.20 ± 0.19

Keys: DLA = Dichloromethane fraction; ALA = Aqueous phase. Data show the mean ± SEM, n = 5.

Chemical Profiling of DCM Fraction of L. aestuans

The results of the GC–MS analysis for the dichloromethane fraction are outlined in Table 7, revealing the presence of 23 compounds. Notably, four of these compounds collectively accounted for 54% of the total compounds. These prominent constituents include 9,12-Octadecadienoic acid (Z,Z)- (19.06%), 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- (18.66%), and tetradecanoic acid (16.28%). The dichloromethane fraction of the LA extract contained a variety of compounds categorized as esters, fatty acids, and terpenes (see Table 7). Emeka et al ²⁹ previously documented the effectiveness of 9,12-Octadecadienoic acid (Z,Z)- in managing hyperlipidemia and atherosclerosis.

Table 7.

Phytoconstituents Identified from the Dichloromethane Fraction of L. aestuans.

Peak	RT	Compound Detected	Mol. Formula	MW	Peak Area (%)	Comp (wt%)	Fragmentation pattern (m/z)
1	2.51	3,4-Hexanediol, 2,5-dimethyl-	C₈H₁₈O₂	146	2.43	2.54	41, 73, 146^a
2	7.50	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-	C₆H₈O₄	144	1.18	2.05	43, 101, 144^a
3	9.81	6-Octen-1-ol, 3,7-dimethyl-	C₁₀H₂₀O	156	0.78	1.23	41, 69, 156^a
4	12.00	Dodecane	C₁₂H₂₆	170	0.98	1.65
5	13.50	Cyclohexaneethanol	C₈H₁₆O	128	0.41	1.00	43, 57, 170^a
6	14.25	Mono(2-ethylhexyl) phthalate	C₁₆H₂₂O₄	278	0.42	1.43	41, 70, 278^a
7	14.76	2,6-Octadien-1-ol, 3,7-dimethyl-, acetate	C₁₂H₂₀O₂	196	1.29	2.82	43, 69, 196^a
8	15.70	9,12-Octadecadienoic acid (Z,Z)-	C₁₈H₃₂O₂	270	0.43	1.38	41, 67, 270^a
9	18.92	9-Hexadecenoic acid, methyl ester, (Z)-	C₁₇H₃₂O₂	268	2.54	1.00	60, 73, 268^a
10	21.50	Dodecanoic acid, methyl ester	C₁₃H₂₆O₂	214	1.72	2.37	41, 74, 214^a
11	23.00	Pentadecanoic acid,13-methyl ester	C₁₆H₃₂O₂	256	0.86	0.87	43, 74, 256^a
12	24.71	Dodecanoic acid	C₁₂H₂₄O₂	200	0.85	3.42	43, 73, 200^a
13	25.50	Benzoic acid, 4-hydroxy-3,5-dimethoxy-	C₉H₁₀O₅	198	1.49	0.76	43, 73, 180^a
14	26.75	9,12-Octadecadienoic acid (Z,Z)-	C₁₈H₃₂O₂	280	20.65	19.06	41, 67, 280^a
15	28.00	9-Octadecenoic acid, methyl ester	C₁₉H₃₆O₂	296	1.30	0.65	41, 55, 296^a
16	28.92	9,12,15-Octadecatrienoic acid, (Z,Z,Z)-	C₁₈H₃₀O₂	278	19.74	18.66	43, 79, 278^a
17	29.03	Tetradecanoic acid	C₁₄H₂₈O₂	228	17.17	16.28	41, 73, 228^a
18	29.70	Methyl tetradecanoate	C₁₅H₃₀O₂	242	3.07	0.96	43, 74, 242^a
19	31.18	Methyl stearate	C₁₉H₃₈O₂	294	2.59	2.21	41, 74, 294^a
20	31.50	Oleic Acid	C₁₈H₃₄O₂	282	8.58	8.24	41, 72, 337^a
21	34.25	13-docosenoic acid	C₂₂H₄₂O₂	338	2.64	2.18	43, 74, 338^a
22	40.69	Eicosanoic acid, methyl ester	C₂₁H₄₂O₂	336	2.56	3.00	43, 74, 336^a
23	42.18	Tetracosanoic acid, methyl ester	C₂₅H₅₀O₂	382	5.33	5.75	43, 74, 382^a

Molecular ion fragment.

Discussion

The endemic nature of malaria and the resistance of the parasite to current synthetic antimalarial drugs has led to the continuous search for new molecules to replace them.³⁰ As a result of malaria's prevalence, several in vivo, in vitro, and in silico investigations have been reported, justifying the ethnomedical use of medicinal plants as antimalarial remedies.^31–33 These studies have gone beyond mere justification, with previous reports describing the antimalarial compounds found in leaves, roots, and stem bark of some antimalarial plants.^25,34 Notably, some of these compounds are non-polar extracts obtained using non-polar solvents.^33,35

In our search for new antimalarial leads, we considered extracts of LA due to its relevant traditional applications and reported pharmacological activities. Traditional medicine considers LA extracts safe with no adverse effects. This study confirmed the LA extract's low toxicity, with a level exceeding 2000 mg/kg. This aligns with a previous report where the LA extract showed no toxicity even at a dosage of 5000 mg/kg.¹⁹

We then tested the extract's antimalarial effects using chemosuppressive, prophylactic, and curative models at doses of 100, 200, 400, and 800 mg/kg. The four-day chemosuppressive tests yielded a percentage chemosuppression significantly higher than the negative control. The observed activity was dose-dependent up to 400 mg/kg, but significantly lower than that of chloroquine (positive control). At the lowest dose (100 mg/kg), the extract's activity (42%) fell short of the optimal 50% threshold. Nigussie and Wale³⁶ define a very good in vivo antimalarial activity as one that suppresses malaria by ≥50% at 100 mg/kg. Interestingly, we observed no corresponding increase in chemosuppressive activity at doses exceeding 400 mg/kg.

Studies have shown that at a higher dosage, some plant metabolites exhibit increased biological effects with increasing doses, then plateau or reverse at higher doses, a phenomenon known as biphasic dose response. The biphasic dose-response displayed by the LA extract in the chemosuppression test could be due to the saturation of the binding sites where its constituents bind.^37,38 A similar result was reported by Alehegn et al³⁹ in their study on the antimalarial activity of crude extract of the leaves of Bersama abyssinica Fresen. The survival time (in days) showed that the extract was able to prolong the life of the animals significantly more than that of the negative control but significantly less than chloroquine. Only at 800 mg/kg did the extract prolong the life of the mice more than 12 days.

In the curative antimalarial assay, the methanol extract of LA gave a dose-dependent activity that was significantly higher than the negative control. The activity elicited by the LA extract at the highest dose of 800 mg/kg was comparable to that of chloroquine (positive control). This suggests that further purification of the compound might yield even more active compounds if the activity is not due to synergistic action. The LA extract at all doses extended the lifespan of the mice to more than triple that of the negative control group (more than 12 days). The result showed that increasing the dose of the LA extract significantly increased the survival time of the mice compared to the negative control (6.00 ± 0.55). The extract also showed a comparable survival time (26.80 ± 0.58 days) to that of chloroquine (28.00 ± 0.00) at the highest dose of 800 mg/kg. Extracts are considered active if they can extend the survival of the animal beyond 12 days.⁴⁰ Also, Faloye et al⁴¹ reported that the survival time for treated mice in antiplasmodial studies which doubled that of the negative control, was evidence of good antiplasmodial activities of the extract. Even at the lowest dose of 100 mg/kg, the LA extract was able to triple the survival time of the animals relative to negative control and therefore could be considered as an indication of good antiplasmodial activity.

The median effective doses (ED₅₀ and ED₉₀) of the extracts were determined to establish a basis for comparison of the antimalarial activities between the crude extracts and subsequent partitioned fractions.³⁶ Median effective doses have been used to compare the activities of different formulation ratios and partitioned fractions of MAMA Decoction.³⁵ Previous studies have reported that a lower ED₅₀ and ED₉₀ indicate higher activity.³⁷

Comparing the ED₅₀ and ED₉₀ in the chemosuppressive and curative models, LA exhibited a lower ED₅₀ (210.15 ± 1.86) in the curative model than in the chemosuppressive model (328.10 ± 2.42). This suggests that LA elicits a better curative effect than a suppressive effect. Therefore, the curative model was chosen for further investigation on the solvent fractions of LA.

In the curative antimalarial studies of the partitioned fractions, the solvent fractions (ALA and DLA) displayed comparable curative activity across all doses. This suggests that the antimalarial potency of the plant is distributed equally between the polar and non-polar solvents. It also suggests that the active phytochemical constituents can be extracted from both solvents or that the antimalarial effect is produced by more than one phytochemical constituent.⁴²

At the lowest dose (10 mg/kg), ALA and DLA produced significantly higher percentage clearances (81.25 ± 1.84 and 87.45 ± 1.35, respectively) compared to the negative control (0.00 ± 0.00) but significantly lower than the positive control (94.93 ± 0.26) at the same dose. The ED₅₀ and ED₉₀ values of ALA and DLA were 23.14 ± 0.06; 39.18 ± 0.52 and 21.94 ± 0.21; 37.20 ± 0.19, respectively. This indicates that a larger portion of the antimalarial constituents of LA reside in the non-polar fraction (DLA). Previous studies have shown that active antiplasmodial compounds of some antimalarial plants reside in the non-polar fractions.^25,42

The ED₅₀ and ED₉₀ of chloroquine were reported to be 2.19 ± 0.10 and 4.29 ± 0.10, respectively,^26,43 which are significantly better than the values reported for DLA (21.94 ± 0.21; 37.20 ± 0.19). However, it is projected that purification of the dichloromethane fraction would further improve its activity. Further purification of DLA might yield compounds comparable to or even better than chloroquine in their antiplasmodial activity.

Limitations of the Study

The study focused on evaluating the antimalarial activity of the extract and fractions of L. aestuans. To identify the chemical constituent responsible for the most active fraction's antimalarial activity and elucidate its possible mechanism of action, molecular docking and molecular dynamics simulation could be performed. This would further elucidate the possible mechanism of action of the identified phytochemical.

Conclusion

This study employed chemosuppressive and curative models to evaluate the antimalarial potency of L. aestuans, with the curative model identifying the most active fraction. The curative model yielded considerably higher antiplasmodial activity compared to the chemosuppressive model in infected mice. The dichloromethane fraction exhibited the strongest antiplasmodial activity. However, both solvent fractions tested elicited good antimalarial activity, suggesting that the chemical constituents distributed between the non-polar and polar fractions largely contribute to the antimalarial efficacy of L. aestuans. The chemical profiling revealed the presence of non-polar and medium-polar constituents in the dichloromethane fraction.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

This study was approved by the Postgraduate Ethical Committee of Obafemi Awolowo University, Ile-Ife, Nigeria, with registration number: SCP17/18/H/1272.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Statement of Human and Animal Right

All procedures in this study were conducted in accordance with the Animal Care approved protocols of the Postgraduate Ethical Committee of Obafemi Awolowo University, Ile-Ife, Nigeria.

Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Kolade Olatubosun Faloye

Joseph A. Aladesanmi

Samuel A. Oguntimehin

References

Madsen

Stauffer Jr

. Aquaculture of animal Species: their eukaryotic parasites and the control of parasitic infections. Biology (Basel) 2024;13(1):41.

Caraballo

Zakzuk

Lee

, et al. Particularities of allergy in the tropics. World Allergy Organization Journal 2016;9(3):20.

Wang

. Introduction to parasitic disease. In: Radiology of parasitic diseases: a practical approach. World Health Organization; 2023:3‐3.3. Malaria. 2016.

Paton

Childs

Itoe

, et al. Exposing anopheles mosquitoes to antimalarials blocks plasmodium parasite transmission. Nature 2019;567(7747):239‐243.

Warrell

. Clinical features of malaria. In: Essential malariology. 4th ed. CRC Press; 2017:191‐205.

Centers for Disease Control and Prevention (CDC), Parasites. August 2023 Review.

World Health Organization. World Malaria Report 2022 Global report.

Daily

. Malaria 2017: update on the clinical literature and management. Curr Infect Dis Rep 2017;19(8):1‐7.

Achan

Mwesigwa

Edwin

D’alessandro

. Malaria medicines to address drug resistance and support malaria elimination efforts. Expert Rev Clin Pharmacol 2018;11(1):61‐70

10.

Ippolito

Moser

Kabuya

J-BB

, et al. Antimalarial drug resistance and implications for the WHO global technical strategy. Curr Epidemiol Rep 2021;8(2):46‐62.

11.

Amelia

Iyori

Emran

, et al. Down–selecting circumsporozoite protein–based malaria vaccine: a comparison of malaria sporozoite challenge model. Parasite Immunol 2019;41(5):12624.

12.

Yusuf

Yoshii

Iyori

, et al. A viral-vectored multi-stage malaria vaccine regimen with protective and transmission-blocking efficacies. Front Immunol 2019;10(5):478977.

13.

Mohana

Satish

Raveesha

. Antibacterial evaluation of some plant extracts against some human pathogenic bacteria. Adv Biol Res (Rennes) 2008;2(3–4):49‐55.

14.

Ajayi

Akintola

. Evaluation of antibacterial activity of some medicinal plants on common enteric food-borne pathogens. Afr J Microbiol Res 2010;4(4):314‐316.

15.

Bello

Ayoola

Obembe

Akinwunmi

. Antidiabetic and toxicity studies of the extract of four Nigerian medicinal plants. European J Med Plants 2022;33(11):32‐45.

16.

Berestetskiy

. The chemical ecology approach to reveal fungal metabolites for arthropod pest management. Microorganisms 2021;9(7):1379.

17.

Ungogo

Ebiloma

Ichoron

, et al. A review of the antimalarial, antitrypanosomal, and antileishmanial activities of natural compounds isolated from Nigerian flora. Front Chem 2020;8:617448.

18.

Tallei

Kepel

Alorabi

, et al. Appraisal of Bioactive Compounds of Betel Fruit as Antimalarial Agents by Targeting Plasmepsin 1 and 2: A Computational Approach. Pharmaceuticals 2021;14(12):1285.

19.

Ashidi

Lawal

. Analgesic and anti-inflammatory potential of the ethanol extract of leaves of Laportea aestuans (L.) chew in albino wistar rats. Afr J Sci Nat 2017;5(2):46‐54.

20.

Oloyede

Oyelola

. Chrysen-2-ol derivative from west Indian wood nettle Laportea aestuans (l.) chew inhibits oxidation and microbial growth in vitro. EXCLI J 2013;12(7):894.

21.

Oguntimehin

Oriola

Obuotor

Aladesanmi

. Bioassay guided phytochemistry of aerial part of Laportea Aestuans (l.) chew (Urticaceae). Niger J Nat Prod Med 2017;23(1):6‐12.

22.

Morrison

Twumasi

. Comparative studies on the in vitro antioxidant properties of methanolic and hydro-ethanolic leafy extracts from eight edible leafy vegetables of Ghana. Afr J Biotechnol 2010;9(32):5177‐5184.

23.

Adetunji

Olugbami

Adegoke

, et al. Reno-Hepatoprotective and antidiabetic properties of methanol leaf extract of Laportea Aestuans in Wistar rats. J Evid Based Integr Med 2021;26(3):2515690X211017464.

24.

Peters

. Drug resistance in Plasmodium berghei Vinca and Lips 19481 chloroquine resistance. Exp Parasitol 1965;17(1):80‐89.

25.

Ryley

Peters

. The antimalarial activity of some quinolone esters. Ann Trop Med Parasitol 1965;17(1):80‐89.

26.

Adesida

Odediran

Elujoba

. Investigation on the antimalarial properties of Plumeria alba linn (Apocynaceae) cultivated in Nigeria Nig. J Nat Prod and Med 2021;25(2):34‐42.

27.

Lagarto Parra

Menéndez Soto-del Valle

Piloto Ferrer

, et al. Antidiabetic, hypolipidemic, antioxidant and anti-inflammatory effects of Momordica charantia L. Foliage extract. J Pharm Pharmacogn 2021;9(4):537‐548.

28.

Lorke

. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54(4):275‐287.

29.

Emeka

Charity

Augustine

. Evaluation of biologically active constituents of ethanol extracts of H. verticillata leave, L. aestuans leave and seeds of L. aestuans. J Med Plant 2022;10(2):196‐203.

30.

Wells

TNC

Alonso

Gutteridge

. New medicines to improve control and contribute to the eradication of malaria. Nat Rev Drug Discovery 2009;8(11):879‐891.

31.

Hidayati

Widyawaruyanti

Ilmi

, et al. Antimalarial activity of flavonoid compound isolated from leaves of artocarpus altilis. Pharmacogn J 2020;12(4):835‐842.

32.

Abdelmohsen

Bayoumi

SAL

Mohamed

, et al. Naturally occurring phenylethanoids and phenylpropanoids: antimalarial potential. RSC Adv 2023;13(38):26804‐26811.

33.

Adeyoju

Ajayi

Adepiti

Elujoba

. Comparative in vivo antimalarial activities of aqueous and methanol extracts of MAMA powder-A herbal antimalarial preparation. J Ethnopharmacol 2022;283(3):114686.

34.

Jha

Sit

. Extraction of bioactive compounds from plant materials using combination of various novel methods: a review. Trends Food Sci Technol 2022;119(5):579‐591.

35.

Odediran

Elujoba

Adebajo

. Influence of formulation ratio of the plant components on the antimalarial properties of MAMA decoction. Parasitol Res 2014;113(5):1977‐1984.

36.

Nigussie

Wale

. Medicinal plants used in traditional treatment of malaria in Ethiopia: a review of ethnomedicine, anti-malarial and toxicity studies. Malar J 2022;21(1):262.

37.

Odediran

Akosile

Bamigboye

. In vivo chemosuppressive activities of combinations of four Nigerian ethnomedicinal antimalarial ferns. GSC Biol Pharm Sci 2022;20(1):145‐158.

38.

Mulisa

Girma

Tesema

, et al. Evaluation of in vivo antimalarial activities of leaves of Moringa oleifera against Plasmodium berghei in mice. Jundishapur J Nat Pharm Prod 2018;13(1):1–5.

39.

Alehegn

Yesuf

Birru

. Antimalarial activity of crude extract and solvent fractions of the leaves of Bersama abyssinica fresen.(Melianthaceae) against Plasmodium berghei infection in Swiss albino mice. Evidence-Based Complementary Altern Med 2020;6(6):1–14.

40.

Ajaiyeoba

Ashidi

Abiodun

, et al. Antimalarial ethnobotany: in vitro. Antiplasmodial activity of seven plants identified in the Nigerian middle belt. Pharm Biol 2005;42(8):588‐591.

41.

Faloye

Adesida

Oguntimehin

, et al. LC-MS analysis, computational investigation, and antimalarial studies of Azadirachta indica fruit. Bioinf Biol Insights 2023;17(1):11779322231154966.

42.

Jodynis-Liebert

Kujawska

. Biphasic dose-response induced by phytochemicals: experimental evidence. J Clin Med 2020;9(3):718.

43.

Adebajo

Odediran

Aliyu

, et al. In vivo antiplasmodial potentials of the combinations of four Nigerian antimalarial plants. Molecules 2014;19(9):13136‐13146.