Antitrypanosomal activity of Argemone mexicana extract and fractions in the animal model of Trypanosoma brucei brucei infection
-
Ifeoma L. Chukwu
,
Malachy C. Ugwu
Abstract
Background
This study investigated the antitrypanosomal activity of Argemone mexicana extract and fractions in the animal model of Trypanosoma brucei brucei infection.
Methods
The whole plant was cold-macerated with methanol. The liquid–liquid partitioning of the extract with n-hexane, ethyl acetate, butanol, and water produced various fractions of the extract. Infection was established by the inoculation of T. brucei brucei-infected red blood cells in the animals. Treatment with the extract and fractions was done orally for 5 days postinfection at 200 and 400 mg/kg doses. Diminazene aceturate 3.5 mg/kg and 5 mL/kg 10% Tween 80 served as standard and vehicle control, respectively. Parasite load, packed cell volume (PCV), animal body weight, and survival as well as serum liver function enzymes’ activities were also assessed.
Results
The extract and the n-hexane fraction showed the presence of all the tested phytocompounds except tannins and cardiac glycosides, respectively. The extract showed a reduction in parasitemia while the order of activity for the fractions was n-hexane ≫ water ≫ butanol ≫ ethyl acetate. The n-hexane fraction produced complete protection against parasite-induced mortality just like the reference standard and a higher increase in PCV compared with the reference standard. The extract, n-hexane, and water fractions showed protection against infection-induced liver damage with a significant (P < 0.05) difference when compared to the vehicle control group.
Conclusion
A. mexicana showed antitrypanosomal activity which may be attributed to the presence of phytocompounds particularly saponins, which were present in the extract and fractions that showed antitrypanosomal activity but absent in fractions that showed no or weak antitrypanosomal activity.
Abbreviations
- RBC
-
red blood cell
- HAT
-
human African trypanosomiasis
- NITR
-
Nigerian Institute for Trypanosomiasis Research
- BW
-
body weight
- AST
-
aspartate aminotransferase
- ALP
-
alanine aminotransferase
- PCV
-
packed cell volume
- ABC
-
ATP-binding cassette
- TNF-α
-
tumor necrosis factor-alpha
1 Introduction
Human African trypanosomiasis (HAT) otherwise called sleeping sickness is a parasitic disease that is caused by the species of the genus Trypanosoma, which affects humans, as well as domestic and wild animals. The infection and its associated disease are restricted to sub-Saharan Africa where the vector (tsetse flies) and its reservoir hosts co-exist [1].
Trypanosomiasis has an overwhelming impact on the poorest countries in the world. Available estimates for HAT indicate that 60 million people are at risk in sub-Saharan Africa [2]. Irrespective of the millions of people under threat of HAT, only about 10% are screened for trypanosomiasis and, of these, only a small proportion are treated annually (Black & Seed, 2018). It is estimated that 300,000–500,000 people live with sleeping sickness, and in some areas, the incidence of the disease exceeds that of acquired immune deficiency syndrome [3,4].
The search for vaccination against African trypanosomiasis remains elusive and the current control strategy relies on the use of trypanocidal drugs. Inadequate drugs for treatment, high cost of synthetic drugs coupled with adverse side effects, and rapid development of resistance are some of the limiting factors in the fight against trypanosomiasis. New chemotherapy has been eagerly awaited; however, HAT being one of the neglected tropical diseases presents an unattractive market for drug development investment. The lack of the necessary resources to bring new compounds to market for possible drug development coupled with the fact that the population affected by the disease cannot afford expensive drugs underscores the continuing need for the discovery of alternative, cheap, and effective drugs [5].
Historically, medicinal plants have served as sources of new pharmaceutical products and inexpensive starting materials for the synthesis of many known drugs. Medicinal plants have also continued to serve as preferred primary health care in many rural communities and resource-constrained settings. This is due to a number of reasons including accessibility, affordability, and effectiveness.
Argemone mexicana L., known as Ghamoya belongs to class: Magnoliopsida Dicotyledons; subclass: Magnoliidae; order: Papaverales; family: Papaveraceae, is a plant that is widely distributed in many tropical and subtropical countries including West Africa [6]. The plant has been used as a cure for leprosy, skin diseases, inflammation, and bilious fevers [7]. Extracts of different parts of this plant are used in chronic skin diseases and also as emetic, expectorant, demulcent, and diuretic; the seeds and seed oil are employed as a remedy for dysentery, ulcers, asthma, and other intestinal affections [8,9,10]. Furthermore, various parts of A. mexicana plants have been shown to have activity against malaria and leishmaniasis, caused by Plasmodium falciparum and Leishmania donovani, respectively [11]. Other species of Agemone like Agemone ochroleuca have been reported to show the profound activity against malaria and Chagas disease caused by P. falciparum and T. cruzi [12]. A. mexicana has also been used in ethnomedicine for the treatment of other protozoan parasitic infections like trypanosomiasis; however, little has been done scientifically to authenticate these claims. This study investigated the trypanocidal activity of A. mexicana extract and fractions in the animal model of Trypanosoma brucei brucei infection.
2 Materials and methods
2.1 Collection of plants
Whole plant of A. mexicana (without flowers) was collected from Sagamu, Nigeria (6.8322°N, 3.6319°E; 46 km to Lagos), on the morning of July 16, 2021, and authenticated by a trained taxonomist Mr Nwafor Felix of the Department of Pharmacognosy and Environmental Medicine, University of Nigeria, Nsukka. A voucher specimen (No. PCG 474/A/034) was deposited at the Herbarium of Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Agulu. The plant materials were washed with clean water, air-dried, and pulverized into coarse powder.
2.2 Extraction and fractionation
The pulverized plant (900 g) was cold-macerated in methanol using a 1:10 solid-to-solvent ratio and the resulting solution concentrated to dryness in vacuo using the rotary evaporator at 40°C. Two-thirds of the extract was subjected to the liquid–liquid partition successively with n-hexane, ethyl acetate, and butanol to obtain fractions soluble in these solvents. The percentage yield of the extract and fractions was calculated relative to the powder and extract, respectively. The extraction and fractionation solvents were products of JHD, China.
2.3 Experimental animals
Twelve-week-old male Wistar rats were used for the study. The animals were reared at the Animal House of the Nigerian Institute for Trypanosomiasis Research (NITR), Asaba, Nigeria. All animals were fed standard feed (Vital Animal Feed, Nigeria) and water ad libitum. All animals will be kept under standard conditions. Before the experiments, the blood sample of the animals was screened for trypanosomes by wet film preparations to ensure that they were parasite free and uninfected prior to the commencement of the study. The animals were housed in standard laboratory conditions. At the end of the experiment, the animals were euthanized by the overdose of sodium pentobarbital through intraperitoneal injection.
-
Ethical approval: The research related to animal use has been complied with the Principles of Laboratory Animal Care (National Institute of Health Publication No. 86.23, revised 1985) and as described in the animal experimental protocol reviewed and approved by the Institutional Animal Care and Use Committee (NAU/FPS/PHAT/065-91).
2.4 Test organisms
Federe strain of trypnosoma isolates (T. brucei brucei) was obtained from stabilates maintained at the NITR, Vom, Plateau State, Nigeria, and confirmed at the NITR, Asaba, Nigeria, before use. The parasites were maintained in the Animal House of Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Agulu, by serial passage in rats until required.
2.5 Phytochemical analysis
The presence of secondary metabolites in the extract and fractions were determined using standard methods as described by Odoh et al. [13].
2.6 Acute toxicity studies on non-infected animals
Acute oral toxicity was done using Lorke’s method as described by Agyigra et al. [14]. The acute toxicity of the extract was determined in mice using the method of Lorke (1983). The tests were done in phases. The first phase (determination of the toxic range) involves three groups of three mice that received 10, 100, and 1,000 mg/kg orally. The treated mice were observed for 24 h for mortality. The death pattern in the first phase determined the doses used for the second phase. Because there were no deaths recorded in the first phase, a fresh batch of four mice received 1,600, 2,900, 3,900 and 5,000 mg/kg of the extract each. The treated animals were observed for lethality or signs of acute intoxication for 24 h. The LD50 was estimated as the geometric mean of the highest nonlethal dose and the least toxic dose.
2.7 Experimental design
2.7.1 Inoculation of rats with T. brucei brucei
Blood obtained from a donor was diluted with dextrose saline to serve as the inoculum [15]. The rats were inoculated through intraperitoneal injection of 0.2 mL of the inoculum containing 1 × 106 parasites/mL [16]. After the inoculation, animals were examined daily by microscopic examination of blood film obtained from the tail vein. After the establishment of parasitemia, animals were grouped into 13 groups of 6 rats as follows:
Group 1: uninfected untreated control.
Group 2: infected and treated with a standard drug (diminazeneaceturate, berenil 3.5 mg/kg).
Group 3: vehicle control infected treated with 5 mL/kg 10% Tween 80 (vehicle).
Group 4: infected and treated with 200 mg/kg body weight (BW) of plant methanolic extract.
Group 5: infected and treated with 400 mg/kg BW of plant methanolic extract.
Group 6: infected and treated with 200 mg/kg of the n-hexane fraction.
Group 7: infected and treated with 400 mg/kg of the n-hexane fraction.
Group 8: infected and treated with 200 mg/kg of the ethyl acetate fraction.
Group 9: infected and treated with 400 mg/kg of the ethyl acetate fraction.
Group 10: infected and treated with 200 mg/kg of the butanol fraction.
Group 11: infected and treated with 400 mg/kg of the butanol fraction.
Group 12: infected and treated with 400 mg/kg of the water fraction.
Group 13: infected and treated with 200 mg/kg of the water fraction.
Dose of extracts was selected based on the results of acute toxicity. All treatments were initiated postinfection (day 0) once daily through oral route and parasitemia monitored after 5 days treatment by microscopic examination.
2.7.2 Determination of mean survival time of parasite-infected animals
Mortality was monitored daily from the first day of treatment following the establishment of parasitemia to the last day of treatment. Percentage survival for each group was calculated as follows:
2.7.3 Experimental parameters
The BWs of each rat were determined by weighing the rats with a sensitive weighing balance. Packed cell volume (PCV) was determined using the microhematocrit method.
The enzyme activities were determined using commercial reagent kits (Randox Laboratories Limited, UK). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined according to the method of Reitman and Frankel [17], while the alkaline phosphatase (ALP) was determined according to the method of King and Amstrong [18].
2.8 Statistical analyses
The data obtained were expressed as mean + SD. Data analyses were done using the Kruskal–Wallis analysis of variance test. The differences between the treatment groups were determined by multiple comparisons of mean ranks for all groups. In all cases, a probability error of less than 0.05 was selected as the criterion for statistical significance.
3 Results
3.1 Yield of plant extract/fraction and phytochemical analysis
The plant extract exhibited a poor yield of 5.3% (Table 1). A greater portion of the extract was soluble in butanol solvent while the least partitioning of the constituents of the extract was recorded for the ethyl acetate and water fractions. The extract contained phytocompounds like alkaloids, flavonoids, terpenoids, steroids, and cardiac glycosides while tannins were absent. The n-hexane fraction also contained all the tested phytocompounds except cardiac glycosides. The ethyl acetate and butanol fractions do not contain tannins and saponins (Table 1).
Total phenolic content, yield, and phytocompounds present in the extract and fractions
| Phytocompounds | Extract | Ethyl acetate F. | Butanol F. | n-Hexane F. | Water F. |
|---|---|---|---|---|---|
| Alkaloids | + | + | + | + | + |
| Tannins | − | − | − | + | − |
| Flavonoids | + | + | + | + | + |
| Steroids | + | + | + | + | − |
| Terpenoids | + | + | + | + | + |
| Saponins | + | − | − | + | + |
| Cardiac glycosides | + | + | − | − | + |
| Total phenolic content (mgGAE/g) | 58 ± 1.01 | 102 ± 1.06 | 84 ± 1.12 | 26 ± 1.04 | 76 ± 1.12 |
| Yield (%) | 5.3a | 18.5b | 35.4b | 24.6b | 18.5b |
+ means present; − means absent.
aCalculated from 900 g of pulverized powder.
bCalculated from 26 g of extract.
3.2 Acute toxicity of the extract on non-infected animals
No mortality was recorded following single oral doses of the extract up to 5,000 mg/kg. There was also a lack of obvious signs of toxicity in all the groups that received different doses of the extract. The median oral lethal dose of the extract was estimated to be above 5,000 mg/kg.
3.3 Effect of the extract and fraction on parasitemia and survival of T. brucei brucei-infected animals
The extract, n-hexane, water, and butanol fractions showed reduction in parasitemia. The reference treatment (diminazene aceturate) exhibited complete clearance of the parasite from the blood. The extract though unable to produce complete eradication of the parasite showed a significant (P < 0.05) reduction in parasitemia compared with the vehicle-treated control group (Figure 1). The n-hexane and water fractions at 200 and 400 mg/kg also showed a significant (P < 0.05) reduction in parasitemia compared to the vehicle control group. Although slight differences existed in the response to 200 and 400 mg/kg treatment, these differences in efficacy between both doses were not statistically significant (P > 0.05). Ethyl acetate fraction exhibited a total lack of antitrypanosomal activity while the activity produced by butanol fraction was not significantly (P > 0.05) different from the parasite-infected control group.
Effect of the extract and fractions on the parasitemia of T. brucei brucei-infected animals. (a) 200 mg/kg extract and fraction effect on mean parasitemia; (b) 200 mg/kg extract and fraction effect on mean changes in parasitemia; (c) 400 mg/kg extract and fraction effect on mean parasitemia; and (d) 400 mg/kg extract and fraction effect on mean changes in parasitemia. *P < 0.05 compared to the vehicle-treated control group.
For the survival rate following treatment, the reference drug showed complete protection against parasite-induced mortality (Figure 2). The same complete protection was also recorded for both doses of n-hexane fraction and 400 mg/kg of water fraction. The survival rate exhibited by the butanol fraction was the same as the untreated control group. Although the ethyl acetate fraction did not show anti-trypanosomal activity, it offered 20% protection against parasite-induced mortality compared to untreated vehicle control group.
Effect of the extract and fractions on percentage survival of T. brucei brucei-infected animals.
3.4 Effect of the extract and fraction on BW of T. brucei brucei-infected animals
Just like the uninfected group, the reference drug-treated group showed a significant (P < 0.05) increase in BW compared with the infected control group (Figure 3). Similarly, the butanol fraction-treated group at both 200 and 400 mg/kg showed a significant (P < 0.05) increase in BW compared with the infected control group. Although the extract, n-hexane, and water fractions produced a significant (P < 0.05) reduction in parasitemia, they also exhibited a significant reduction in BW when compared with the infected control group (Figure 3).
Effect of extract and fractions on BW of T. brucei brucei-infected animals. (a) 200 mg/kg extract and fraction effect on BW; (b) 200 mg/kg extract and fraction effect on mean changes in BW; (c) 400 mg/kg extract and fraction effect on BW; and (d) 400 mg/kg extract and fraction effect on mean changes in BW. *P < 0.05 compared to the vehicle-infected control group (significant increase) and # P < 0.05 compared to the vehicle-infected control group (significant decrease).
3.5 Effect of the extract and fraction on PCV of T. brucei brucei-infected animals
The extract and water fraction produced a significant (P < 0.05) increase in PCV just like diminazene aceturate. Higher increase in PCV was recorded for n-hexane fraction compared to the reference drug (diminazene aceturate) (Figure 4). Just like the infected control group, there was reduction in PCV of the ethyl acetate and butanol treated groups. However, these reductions were significantly (P < 0.05) less than the infected control group.
Effect of extract and fraction on PCV of T. brucei brucei-infected animals. (a) 200 mg/kg extract and fraction effect on PCV; (b) 200 mg/kg extract and fraction effect on percentage changes in PCV; (c) 400 mg/kg extract and fraction effect on PCV; and (d) 400 mg/kg extract and fraction effect on percentage changes in PCV. *P < 0.05 compared to the vehicle-treated control group.
3.6 Effect of the extract and fraction on liver function enzymes
T. brucei brucei infection produced a significant (P < 0.05) increase in liver function enzymes when compared to the infected control group. The extract, n-hexane, and water fraction showed protection against infection-induced liver damage as shown by a significant (P < 0.05) reduction in the serum liver function enzymes. Just as was recorded in the parasitemia response to treatment, 200 and 400 mg/kg doses exhibited no significant differences in there effects. Thus, it is possible that a lower dose would also be effective. The ethyl acetate and butanol fractions showed no protection against the parasite-induced elevation in serum liver function enzymes (Figures 5–7). The serum liver function enzyme concentrations recorded for these fraction-treated groups were significantly (P < 0.05) higher than the infected control group. A similar pattern of activity was recorded for the extract and fractions across the three liver function enzymes evaluated.
Effect of the extract and fractions on serum the ALT activity of T. brucei brucei-infected animals. (a) 200 mg/kg extract and fraction effect on ALT activity; (b) 200 mg/kg extract and fraction effect on mean changes in ALT activity; (c) 400 mg/kg extract and fraction effect on ALT activity; and (d) 400 mg/kg extract and fraction effect on mean changes in ALT activity. *P < 0.05 compared to the vehicle-infected control group (significant decrease) and # P < 0.05 compared to the vehicle-infected control group (significant increase).
Effect of extract and fractions on serum AST activity of T. brucei brucei-infected animals. (a) 200 mg/kg extract and fraction effect on AST activity; (b) 200 mg/kg extract and fraction effect on mean changes in AST activity; (c) 400 mg/kg extract and fraction effect on AST activity; and (d) 400 mg/kg extract and fraction effect on mean changes in AST activity. *P < 0.05 compared to the vehicle-infected control group (significant decrease) and # P < 0.05 compared to the vehicle-infected control group (significant increase).
Effect of extract and fractions on serum ALP activity of T. brucei brucei-infected animals. (a) 200 mg/kg extract and fraction effect on ALP activity; (b) 200 mg/kg extract and fraction effect on mean changes in ALP activity; (c) 400 mg/kg extract and fraction effect on ALP activity; and (d) 400 mg/kg extract and fraction effect on mean changes in ALP activity. *P < 0.05 compared to the vehicle-infected control group (significant decrease) and # P < 0.05 compared to the vehicle-infected control group (significant increase).
4 Discussion
Microbial resistance to the few conventional antitrypanosomal drugs, lack of effective vaccines due to antigenic variation exhibited by these parasites, and the adverse effects associated with the existing antitrypanosomal drugs have encouraged the exploration of natural products to feed the pipeline of drug developments for trypanosomiasis control and elimination [19]. Wistar rat murine model was used in the evaluation of the antitrypanosomal activity of A. mexicana. The choice of this animal model was based on its reported usefulness in the investigation of HAT disease-associated pathologies as well as its usefulness in the identification of new potential drug targets, new drug therapies, and potential drug toxicity [20,21]. Despite the fact that this model features some definite but unavoidable limitations like; differences in genetic background between human and animal host, also differences in pathogenesis between the human and animal infecting subspecies. These limitations have been argued to be outweighed by the advantages of the model in the understanding of different aspects of the physiopathology of HAT and for the development of new diagnostic tools and drugs [22].
A. mexicana showed the presence of secondary metabolites with a wide variety of structural classes that have been previously reported for their pharmacological activities against T. brucei brucei. Structural-based design analysis has revealed that compounds possessing a number of hydrogen bond accepting and/or donating groups like flavonoids show extensive interaction with trypanosomal glycolytic enzyme – glyceraldehydes-3-phosphate dehydrogenase as well as interfering with the activities of trypanosome alternative oxidase [23,24]. These inhibitory activities have been linked to the trypanocidal effect due to the total block of the energy production of the bloodstream forms of this parasite [25]. The presence of flavonoids in the extract of A. mexicana may have contributed to its antitrypanosomal activity. However, the presence of this same phytocompound in the ethyl acetate fraction of the extract could not reproduce the antitrypanosomal activity seen in the extract. This observation creates some doubt in attributing the activities of the extract to the presence of flavonoids. The process of fractionation of the extract may have possibly affected the interactions of phytocompounds that may have been working synergistically in the extract to produce the antitrypanosomal effect. Similarly, the molecular size, number of hydroxyl groups, and the substitution pattern of the hydroxyl groups in flavonoid skeleton play an important role in their antiparasitic potentials against T. brucei brucei [26]. The process of fractionation may have affected these structural features in the flavonoids that partitioned into the ethyl acetate fraction or the fraction may have selectively accumulated flavonoids not possessing the antitrypanosomal activity. Other pharmacokinetic influences have also been documented to affect the biological activities of flavonoids. Their bioavailability, metabolism, and elimination are dependent on the subtype of flavonoids and on the type of glycosides they contain [27]. These effects may also explain the lack of antitrypanosomal activity recorded for the ethyl acetate fraction that is rich in flavonoids.
Apart from flavonoids, several alkaloids of plant origin have been documented as lead phytocompounds in drug development for trypanosomiasis [28]. They have been associated with the intercalation of DNA, induction of apoptosis, inhibition of the microtubule assembly, as well as protein synthesis as mechanisms of their anti-trypanosomal action [29]. The presence of this phytocompound in the extract and its accumulation in the most active fraction (n-hexane fraction) in moderately high concentration could account for the significant antitrypanosomal activities of the extract and n-hexane fraction of A. mexicana.
Similarly, the antitrypanosomal and antimicrobial activities of saponins, steroids, and terpenoids, which were detected in the most active fraction, have been previously demonstrated [30]. ATP-binding cassette (ABC) transport proteins at T. brucei brucei plasma membranes account for the major drug resistance mechanism of this parasite [31]. The upregulation of these ABC transporters, which include p-glycoprotein and multi-drug resistance-associated transporters, results in the energy-dependent extrusion of anti-trypanosomal compounds, pro-drugs, or active metabolites [29]. A combination of alkaloids with steroidal saponins has been documented to result in the synergistic potentiation of trypanocidal activity [27]. Steroidal saponins (genins) increase the uptake of polar compounds and also serve as an inhibitor of ABC transporters, which are active in trypanosomes [32,33]. By inhibiting efflux system and thus increasing the internal concentration of alkaloids, their combination with alkaloids has been shown to enhance their individual trypanocidal activity [27]. The steroids present in the n-hexane fraction may have occurred as genins, which could have given this fraction edge over other fractions in terms of antitrypanosomal activity. Similarly, the saponins present in the extract and active fractions may have acted singly or in synergism with other phytocompounds.
Treatment with the extract and fraction not only reduced parasitemia but also the outcome of parasite infection as animals treated with these extract and fraction demonstrated a decrease in parasitemia that is associated with prolonged host survival. The reduction of parasitemia and prolongation of the lifespan of infected animals can be associated with the trypano-suppressive effect of the individual class of phytocompounds and/or the synergistic effect of each class of phytocompounds in the extract and fractions.
Measurement of PCV, which is an index of anemia, gives a reliable indication of the disease status and productive performance of trypanosome-infected animals [34]. Anemia associated with trypanosomiasis is a consistent and significant finding in both human and animal studies [35]. This established consequence of trypanosome infection is consistent with our result on reduced PCV following T. brucei brucei infection of the experimental animals.
Host and parasite-derived factors have been demonstrated to contribute to acute anemia associated with trypanosomal infection [33]. Red blood cells (RBCs) from infected animals have been shown to exhibit enhanced osmotic fragility and altered fatty acid membrane composition compared with RBCs from non-infected animals [36]. Host-derived factors such as tumor necrosis factor-alpha (TNF-α) have been suggested to be a driving force for the observed changes in RBC fragility, given that it was shown that it can decrease the RBC half-life and thereby fuel RBC senescence [37]. The importance of host-derived factors such as TNF-α in acute anemia development was further substantiated by the observation that T. brucei-infected TNF-α-deficient animals exhibited greatly reduced acute anemia levels compared with control animals [33].
Besides TNF-α produced by activated myeloid cells, NO was found to be an important factor affecting T. brucei-associated acute anemia development [38,39]. Inhibitors of NO synthase enzymes have been documented to alleviate acute anemia associated with parasitemia [36,37]. In line with these documented influences of inflammatory mediators in acute anemia associated with T. brucei infection, it is expected that anti-inflammatory phytocompounds that downregulate these inflammatory processes could alleviate anemia development associated with host response to T. brucei infection. Steroids among other phytocompounds are known for their effectiveness as anti-inflammatory agents [40]. Of particular importance is their repression of inducible nitric oxide synthase enzyme and cytokine gene transcription associated with inflammation [41]. The presence of steroids in the extract and its partitioned in the most active fraction could have contributed to improved PCV and survival rate of animals treated with these extract and fraction.
Parasite-derived factors, such as extracellular vesicles in the case of T. brucei infection, have been shown to contribute to the modification of RBCs and their subsequent elimination [33]. The fusion of these released extracellular vesicles causes changes in the physical properties of the RBC membrane, which enhances erythrophagocytosis and thereby fuels anemia development [42]. Reduction in parasitemia and its consequent reduction in parasite-derived factors and improved PCV may be attributed to the presence of steroid and terpenoid phytocompounds.
Mechanical injury to erythrocytes can also mediate anemia associated with parasite-derived factors [43]. The lashing action of the powerful locomotory flagella and microtubule-reinforced bodies of the parasite during parasitemia may induce mechanical injury to erythrocytes leading to their enhanced elimination and anemia [43]. Part of the established mechanisms of antitrypanosomal actions of alkaloid is the inhibition of microtubule assembly [44]. The presence of this phytocompound in the extract and its distribution in the fractions could have influenced the differential activity of the n-hexane, ethyl acetate, butanol, and water fractions.
Specific organ damage during trypanosomiasis is one of the major contributing factors to the disease pathology [45]. Unlike most other trypanosome genus members, T. brucei is particularly tissue invasive, causing cellular infiltration into the parenchyma of most organs [43]. The tissue colonization process is usually accompanied by a marked inflammatory response driven by macrophages, plasma cells, and lymphocytes [46]. Oxidative stress and the production of proinflammatory cytokines are other processes associated with trypanosomal hepatocellular inflammation [47]. A. mexicana has been reported for its antioxidant and anti-inflammatory activities [47]. It could be possible that the extract and fraction of A. mexicana through radical scavenging and anti-inflammatory activity of its phytocompounds could have limited pathogenicity by reducing the recruitment and activation of inflammatory cells. Histopathological analysis was not done because, during the period of this study, our tissue processing unit (histology unit) was not functional due to some technical issues.
The acute toxicity of A. mexicana whole plant showed no motility up to 5,000 mg/kg; this is in alignment with the studies reported on the leave and root of A. mexicana; these studies revealed these parts to show no mortality at the highest tested doses of 1,000 and 5,000 mg/kg, respectively [48,49]. However, poisoning of the seed oil of A. mexicana has been reported by several studies. A study by Verma et al. [50] reported mustard seed oil adulterated with A. mexicana seed oil to cause poisoning known as “epidemic dropsy” in many parts of India and some other countries like Mauritius, Fiji Islands, Madagascar, and South Africa since the mustard seed oil is a popular cooking medium in these areas. Also, single exposure of Argemone oil even at low doses has been reported to produce genotoxic effects in mice [51,52]. The toxicity of Argemone is reported to be primarily due to the alkaloid known as sanguinarine, which is said to be 2.5 times more toxic than other toxic alkaloid di-hydrosanguinarine, although both are inter-convertible by simple oxidation and reduction process [50]. The concentrations of sanguinarine and hydrosanguinarine in the oil were reported to be 5 and 87%, respectively [50]. The lack of mortality observed in this present study as well as reported in the aerial part and root of this plant suggests that the toxic alkaloids, sanguinarine and hydrosanguinarine, reported in the seed oil may either be absent in these parts or be present in very minute concentrations.
5 Conclusion
The extract of A. mexicana showed antitrypanosomal activity which may be attributed to the presence of phytocompounds particularly saponins, which were present in the extract and fractions that showed antitrypanosomal activity but absent in fractions that showed no or weak antitrypanosomal activity.
The contributions of the individual phytocompounds to the antitrypanosomal activity of A. mexicana were not determined in this study. Data from this study suggest that saponins are responsible for the activity shown by the extract and active fractions; however, whether these saponins are acting singly or in combination with other phytocompounds is unknown; thus, the isolation and evaluation of these phytocompounds to determine their activity both singly and in combination is currently in progress in our laboratory. Also, further studies on the toxicity of A. mexicana whole plant to ascertain the safety of this plant part are also ongoing in our laboratory.
Following the discovery of the active phytochemicals, the toxicity profile and other preclinical studies, such as the pharmacokinetics profile. The results of these assays will inform the subsequent steps for usage in clinical trials for use in people.
Acknowledgments
The authors wish to acknowledge Nnamdi Azikiwe University Awka, Anambra State, the host of this research work.
-
Funding information: The authors state no funding involved.
-
Author contributions: I.L.C.: Conceptualization, Methodology; A.A.A.: Data curation, Writing-Original draft preparation; I.S.M.: Visualization, Investigation; M.C.U.: Supervision, Validation; I.R.I.: Supervision, Writing-Reviewing; U.U.O.: Editing, software.
-
Conflict of interest: The authors state no conflict of interest.
-
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
[1] Franco JR, Simarro PP, Diarra A, Jannin JG. Epidemiology of human African trypanosomiasis. Clin Epidemiol. 2014;6:257–75. 10.2147/CLEP.S39728.Search in Google Scholar PubMed PubMed Central
[2] WHO. Trypanosomiasis, human African (sleeping sickness); 2021. https://www.who.int/news-room/fact-sheets/detail/trypanosomiasis-human-african-(sleeping-sickness) (accessed 14 October 2021).Search in Google Scholar
[3] Kagira JM, Maina N, Njenga J, Karanja SM, Karori SM, Ngotho JM. Prevalence and types of coinfections in sleeping sickness patients in Kenya (2000/2009). J Trop Med. 2011;2011:248914. 10.1155/2011/248914.Search in Google Scholar PubMed PubMed Central
[4] Hnaide HA. African trypanosomiasis (sleeping sickness); 2019. https://emedicine.medscape.com/article/228613-overview (accessed 16 February 2022).Search in Google Scholar
[5] Field MC, Horn D, Fairlamb AH, Ferguson MA, Gray DW, Read KD, et al. Anti-trypanosomatid drug discovery: An ongoing challenge and a continuing need. Nat Rev Microbiol. 2017;15:217–31. 10.1038/nrmicro.2016.193.Search in Google Scholar PubMed PubMed Central
[6] Rahangdale GK, Indurwade NH, Chauragade S, Katre SG, Goplani AK, Shelke SB, et al. Study of psoriasis treatment using traditional medicine. IJCRT. 2020;8:2320–882.Search in Google Scholar
[7] Ali H, Sabiha S, Sadequl I, Shahina BR, Meherun N, Nurul I. Repellent activity of Argemone mexicana L. extracts against Aphis gossypii Glover and Tribolium castaneum (Hbst.) adults. J Pharmacog Phytochem. 2017;6:466–9.Search in Google Scholar
[8] Brahmachari G, Gorai D, Roy R. Argemone mexicana: Chemical and pharmacological aspects. Rev Bras Farmacogn. 2013;23:559–75. 10.1590/S0102-695X2013005000021.Search in Google Scholar
[9] Sharanappa R, Plant GMVidyasagar. Profile, phytochemistry and pharmacology of argemone mexicana Linn. A review. Int J Pharm Pharm Sci. 2014;6:45–53.Search in Google Scholar
[10] Magaji MB, Haruna Y, Muhammad A. Phytochemical screening and antioxidant activity of Argemone mexicana leaf methanol extract. Ann Pharmacol Pharm. 2019;4:1167.Search in Google Scholar
[11] Calderón AI, Angela I, Calderón LI, Ortega-Barría RE, Solís PN, Zacchino S, et al. Screening of Latin American plants for antiparasitic activities against malaria, Chagas disease, and leishmaniasis. Pharm Bio. 2010;48:545–53. 10.3109/13880200903193344.Search in Google Scholar PubMed
[12] Abdel-Sattar E, Maes L, Mahmoud Salama M. In vitro activities of plant extracts from Saudi Arabia against malaria, leishmaniasis, sleeping sickness and Chagas disease. Phytother Res. 2010;24:1322–8. 10.1002/ptr.3108.Search in Google Scholar PubMed
[13] Odoh UE, Obi PE, Ezea CC, Anwuchaepe AU. Phytochemical methods in plant analysis. First ed. Nigeria: Pascal Communications; 2019.Search in Google Scholar
[14] Agyigra IA, Ejiofor JI, Magaji MG. Acute and subchronic toxicity evaluation of methanol stem-bark extract of Ximenia americana Linn (Olacaceae) in Wistar rats. Bull Fac Pharm Cairo Univ. 2017;55:263–7.10.1016/j.bfopcu.2017.08.004Search in Google Scholar
[15] Herbert WJ, Lumsden WH. Trypanosoma brucei: A rapid “matching” method for estimating the host’s parasitemia. Exp Parasitol. 1976;40:427–31.10.1016/0014-4894(76)90110-7Search in Google Scholar PubMed
[16] Ayawa NG, Ramon-Yusuf SB, Wada YA, Oniye SJ, Shehu DM. Toxicity study and anti-trypanosomal activities of aqueous and methanol whole plant extracts of Brillantaisia owariensis on Trypanosoma brucei-induced infection in BALB/c mice. Clin Phytosci. 2021;7:39. 10.1186/s40816-021-00267-3S.Search in Google Scholar
[17] Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am J Clin Path. 1957;28:56–63.10.1093/ajcp/28.1.56Search in Google Scholar PubMed
[18] King EJ, Amstrong AR. A convenient method for determining serum and bile phosphatase activity. J Clin Path. 1954;7(4):322–6.Search in Google Scholar
[19] Yabu Y, Yoshida A, Suzuki T, Nihei C, Kawai K, Minagawa N, et al. The efficacy of ascofuranone in a consecutive treatment on Trypanosoma brucei brucei in mice. Parasitol Int. 2003;52:155–64.10.1016/S1383-5769(03)00012-6Search in Google Scholar PubMed
[20] Adebiyi OE, Omobowale TO, Abatam MO. Neurocognitive domains of neuropathological damages in experimental infection with Trypanosoma brucei brucei in Wistar rats. Heliyon. 2021;7:e08260.10.1016/j.heliyon.2021.e08260Search in Google Scholar PubMed PubMed Central
[21] Jolayemi KO, Mamman M, Sanic D, Okoronkwo MO, Amaye. J. In vitro and in vivo changes observed in Trypanosoma brucei brucei infected rats treated with artesunate and/or diminazene acefurate. Sokoto J Vet Sci. 2020;18:211–20.10.4314/sokjvs.v18i4.5Search in Google Scholar
[22] Kennedy PG. Animal models of human African Trypanosomiasis – very useful or tool far removed. Trans R Soc Trop Med Hyg. 2007;101:1061–2. 10.1016/j.trstmh.2007.05.001.Search in Google Scholar PubMed
[23] Mamoon-Ur R, Ali S, Alamzeb M, Igoli J, Clements C, Shah SQ, et al. Phytochemical and antitrypanosomal investigation of the fractions and compounds isolated from Artemisia elegantissima. Pharm Bio. 2014;52:983–7. 10.3109/13880209.2013.874534.Search in Google Scholar PubMed
[24] Ott R, Chibale K, Anderson S, Chipeleme A, Chaudhuri M, Guerrah A, et al. Novel inhibitors of the trypanosome alternative oxidase inhibit Trypanosoma brucei brucei growth and respiration. Acta Trop. 2006;100:172–84.10.1016/j.actatropica.2006.10.005Search in Google Scholar PubMed
[25] Tasdemir D, Kaiser M, Brun R, Yardley V, Schmidt TJ, Tosun F, et al. Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: In vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies. Antimicrobial Agents and Chemotherapy. 2006;50:1352–64. 10.1128/AAC.50.4.1352-1364.2006.Search in Google Scholar PubMed PubMed Central
[26] Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: An overview. Sci World J. 2013;2013:162750. 10.1155/2013/162750.Search in Google Scholar PubMed PubMed Central
[27] Merschjohann K, Sporer F, Steverding D, Wink M. In vitro effect of alkaloids on bloodstream forms of Trypanosoma brucei and T. congolense. Planta Med. 2001;67:623–7. 10.1055/s-2001-17351.Search in Google Scholar PubMed
[28] Krstin S, Peixoto HS, Wink M. Combinations of alkaloids affecting different molecular targets with the saponindigitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei. Antimicrob Agents Chemother. 2015;59:7011–7. 10.1128/AAC.01315-15.Search in Google Scholar PubMed PubMed Central
[29] Atawodi SE, Ogunbusola F. Evaluation of anti-trypanosomal properties of four extracts of leaves, stem and root barks of Prosopis africana in laboratory animals. Biokem. 2009;21:101–8. 10.4314/biokem.v21i2.56478.Search in Google Scholar
[30] Baker N, De Koning HP, Maser P, Horn D. Drug resistance in African trypanosomiasis: The melarsoprol and pentamidine story. Trends Parasitol. 2013;29:1–16. 1016/j.pt.2012.12.005.Search in Google Scholar
[31] Rautio J, Humphreys JE, Webster LO, Balakrishnan A, Keogh JP, Kunta JR, et al. In vitro p-glycoprotein inhibition assay for assessment of clinical drug interaction potential of new drug candidates: A recommendation for probe substrates. Drug Metab Dispos. 2006;34:786–92. 10.1124/dmd.105.008615.Search in Google Scholar PubMed
[32] Eid SY, El-Readi MZ, Eldin EE, Fatani SH, Wink M. Influence of combinations of digitonin with selected phenolics, terpenoids and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells. Phytomed. 2013;21:47–61. 10.1016/j.phymed.2013.07.019.Search in Google Scholar PubMed
[33] Mbewe NJ, Namangala B, Sitali L, Vorster I, Michelo C. Prevalence of pathogenic trypanosomes in anaemic cattle from trypanosomosis challenged areas of Itezhi-tezhi district in central Zambia. Parasit Vectors. 2015;8:638. 10.1186/s13071-015-1260-0.Search in Google Scholar PubMed PubMed Central
[34] Stijlemans B, De Baetselier P, Magez S, Van Ginderachter JA, De Trez C. African Trypanosomiasis-associated anemia: The contribution of the interplay between parasites and the mononuclear phagocyte system. Front Immunol. 2018;9:218. 10.3389/fimmu.2018.00218.Search in Google Scholar PubMed PubMed Central
[35] Cnops J, De Trez C, Stijlemans B, Keirsse J, Kauffmann F, Barkhuizen M. NK-, NKT- and CD8-derived IFNγ drives myeloid cell activation and erythrophagocytosis, resulting in trypanosomosis-associated acute anemia. PLoS Pathog. 2015;11:e1004964. 10.1371/journal.ppat.1004964.Search in Google Scholar PubMed PubMed Central
[36] Moldawer LL, Marano MA, Wei H, Fong Y, Silen ML, Kuo G. Cachectin/tumor necrosis factor-alpha alters red blood cell kinetics and induces anemia in vivo. FASEB J. 1989;3:1637–43. 10.1096/fasebj.3.5.2784116.Search in Google Scholar PubMed
[37] Mabbott N, Sternberg J. Bone marrow nitric oxide production and development of anemia in Trypanosoma brucei-infected mice. Infect Immun. 1995;63:1563–6.10.1128/iai.63.4.1563-1566.1995Search in Google Scholar PubMed PubMed Central
[38] Mabbott NA, Coulson PS, Smythies LE, Wilson RA, Sternberg JM. African trypanosome infections in mice that lack the interferon-gamma receptor gene: Nitric oxide-dependent and -independent suppression of T-cell proliferative responses and the development of anaemia. Immunol. 1998;94:476–80. 10.1046/j.1365-2567.1998.00541.x.Search in Google Scholar PubMed PubMed Central
[39] Patel SS, Savjani JK. Systematic review of plant steroids as potential anti-inflammatory agents: Current status and future perspectives. J Phytopharmacol. 2015;4:121–5.10.31254/phyto.2015.4212Search in Google Scholar
[40] Barnes PJ. How corticosteroids control inflammation: Quintiles prize lecture 2005. Br J Pharmacol. 2006;148:245–54. 10.1038/sj.bjp.0706736; Szempruch J, Sykes SE, Kieft R, Dennison L, Becker AC, Gartrell A. Extracellular vesicles from Trypanosoma brucei mediate virulence factor transfer and cause host anemia. Cell. 2016;164:246–57. 10.1016/j.cell.2015.11.051.Search in Google Scholar PubMed PubMed Central
[41] Mbaya A, Kumshe H, Nwosu CO. The mechanisms of anaemia in trypanosomosis: A review, anemia, Donald S. Silverberg, London, UK: IntechOpen; 2012. 10.5772/29530.Search in Google Scholar
[42] Wink M, Schimmer O. Molecular modes of action of defensive secondary metabolites, In Annual plant reviews, volume 39: Functions and biotechnology of plant secondary metabolites. Second ed. Hoboken, New Jersey: Blackwell Publishing Ltd; 2010.10.1002/9781444318876Search in Google Scholar
[43] Kariuki C, Kagira JM, Mwadime V, Ngotho M. Virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates in a Swiss white mouse model. Afr J Lab Med. 2015;4(2015):137–45. 10.4102/ajlm.v4i1.137.Search in Google Scholar
[44] Vincendeau P, Bouteille B. Immunology and immunopathology of African trypanosomiasis. An Acad Bras Cienc. 2006;78:645–65. 10.1590/S0001-37652006000400004.Search in Google Scholar
[45] Penas FN, Cevey AC, Siffo S, Mirkin GA, Goren NB. Hepatic injury associated with Trypanosomacruzi infection is attenuated by treatment with 15-deoxy-Δ prostaglandin J2. Exp Parasitol 170. 2016;170:100–8. 10.1016/j.exppara.2016.09.015.Search in Google Scholar PubMed
[46] Srivastava A, Srivastava M. Fungi toxic effect of some medicinal plants (on some fruit pathogens). Philippine J Sci. 1998;127:181–7.Search in Google Scholar
[47] Prabhakaran D, Rajeshkanna A, Senthamilselvi MM. Antioxidant and Anti-inflammatory activities of the flower extracts of Argemone mexicana L. IJPSR. 2020;11(1):323–30. 10.26452/ijrps.v11i1.1824.Search in Google Scholar
[48] Mergu P, Venugopal SP. Preliminary phytochemical analysis and oral acute toxicity study of the root of Argemone mexicana Linn. IJRDPL. 2016;5(2):2010–7.Search in Google Scholar
[49] Sourabie TS, Ouedraogo N, Sawadogo WR, Nikiema JB, Guissou IP, Nacoulma OG. Biological evaluation of anti-inflammatory and analgesic activities of Agremone mexicana Linn. (Papaveraceae) aqueous leaf extract. IJPSR. 2012;3(9):451–8.Search in Google Scholar
[50] Verma SK, Devb G, Tyagia AK, Goomberc S, Jain GV. Argemone mexicana poisoning: Autopsy findings of two cases. Forensic Sci Int. 2001;115(2001):135–41.10.1016/S0379-0738(00)00322-4Search in Google Scholar PubMed
[51] Ansari KM, Chauhan KS, Dhawan A, Khanna SK, Mukul DA. Unequivocal evidence of genotoxic potential of Argemone oil in mice. Int J Cancer. 2004;112:890–5.10.1002/ijc.20319Search in Google Scholar PubMed
[52] Ghosh I, Mukherjee A. Argemone oil induces genotoxicity in mice. Drug Chem Toxicol. 2016;39:407–11. 10.3109/01480545.2015.1135339.Search in Google Scholar PubMed
© 2022 the author(s), published by De Gruyter
This work is licensed under the Creative Commons Attribution 4.0 International License.
Articles in the same Issue
- Research Articles
- Antitrypanosomal activity of Argemone mexicana extract and fractions in the animal model of Trypanosoma brucei brucei infection
- Domestic dogs as a threat to sloths in Costa Rica: A clinical case report and review of the problem
- Review Article
- Brucella transmission from domestic and wild animals to dromedary camel: Diagnostic methods and zoonotic threats – A review
- Communication
- Surgical management and outcome of a laryngeal myxosarcoma in a dog
Articles in the same Issue
- Research Articles
- Antitrypanosomal activity of Argemone mexicana extract and fractions in the animal model of Trypanosoma brucei brucei infection
- Domestic dogs as a threat to sloths in Costa Rica: A clinical case report and review of the problem
- Review Article
- Brucella transmission from domestic and wild animals to dromedary camel: Diagnostic methods and zoonotic threats – A review
- Communication
- Surgical management and outcome of a laryngeal myxosarcoma in a dog
