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Most dangerous and prevalent form of malaria is caused by the Plasmodium falciparum mediated malaria and poses the greatest threat to the humans. Emergence of multi drug resistant parasite hindered the prevention of malaria burden worldwide. This study is mainly focused on the erythrocytic membrane stabilisation using regionally available medicinal plant extracts and its corelation with the oxidative stress generated during the intracellular erythrocytic stages development of Plasmodia. The results disclosed that antioxidant potential of the medicinal plants can diminish the reactive oxygen species generation leads to restrict the plasmodial invasion into erythrocytes ultimately decreases the parasitic load. Hence, the evidence of the effective phytochemicals present in the selected medicinal plants can be the promising anti-plasmodial drug candidates as a future perspective.
Department of Biochemistry, School of Sciences, Gujarat University, India
Department of Zoology, BMTC, and Human Genetics, School of Sciences, Gujarat University, India
Dhara Jani
Department of Zoology, BMTC, and Human Genetics, School of Sciences, Gujarat University, India
Linz-Bouy George
Department of Zoology, BMTC, and Human Genetics, School of Sciences, Gujarat University, India
Hyacinth Highland
Department of Zoology, BMTC, and Human Genetics, School of Sciences, Gujarat University, India
*Address all correspondence to: urjajoshi@gujaratuniversity.ac.in
1. Introduction
The burden of malaria is still felt worldwide and caused by Apicomplexa parasite Plasmodium spp., in which, Plasmodium falciparum (P. falciparum) primarily causes severe malaria and remains the leading cause of morbidity and mortality worldwide. According to World Health Organisation (WHO), 241 million cases of malaria and 627,000 deaths were reported globally in 2022 [1] in which the WHO African Region has consistently reported a significant portion of the worldwide malaria burden. Moreover, Malaria cases in the WHO South-East Asia Region dropped significantly and made up roughly 2% (5 million cases in 2020) of all malaria cases worldwide [1]. Still, more than 4,00,000 people around the world killed due to malaria every year [2, 3]. It mostly occurs due to the delay in treating P. falciparum caused malaria. Plasmodia requires at least two hosts a) Female Anopheles mosquitoes – for sexual cycle b) Human beings – for asexual cycle, to accomplish its life cycle. It infects in form of sporozoites via female Anopheles mosquitoes’ bites enters the human being, within 60 mins it invades hepatocytes via circulatory system [4]. Eventually sporozoites matures and complete its pre erythrocytic stages into 6–15 days, which is clinically remains silent, and leave the liver in form of merozoites and enters the circulation to invade erythrocytes [4]. During erythrocytic stages, merozoites develop into (trophozoites – ring stages – schizonts - merozoites) in <48 hrs and on the time of maturation thousand of merozoites leave that erythrocyte to infect new erythrocytes. Bursting of erythrocytes into circulation onset the progression of symptoms including chills, fever, headache, etc. associated with malaria [5]. Severe malaria includes the series of complications include cytoadherence, and sequestration found in Plasmodium falciparum infected erythrocytes (Pf-iEs) leads to unregulated inflammatory processes, sequestration, coma, severe anaemia, multiple organ dysfunctionality and cerebral malaria like complications in vital organs [5]. In addition, mortality rates in P. falciparum caused malaria remained elevated mainly in pregnant women, young children >5 years, etc. [6]. Although the availability of wide spectrum of anti-malarial drugs in the markets, emergence of drug resistant of malaria parasite especially of P. falciparum has created an urgent demand for newer, more efficacious, anti-plasmodial agents, with minimal side effects. The search for a safe, efficient treatment agent for the control and management of this dreadful disease may perhaps now have an answer thanks to several plant extracts and formulations that are highly concentrated in powerful phytochemicals. According to estimates from the WHO, 80% of people rely on herbal remedies as their primary form of healthcare. Around 21,000 plant species have the potential to be used as medical plants, according to the WHO [7]. The prophylactic anti-malarial medication derived from medicinal plants is one possible source since certain secondary plant compounds have a significant potential for cell-cell and cell-molecular interactions [8]. Natural products have been essential in the discovery and development of new medications for a long time since plant primary and secondary metabolites have significant biological functions [9].
Here, two medicinal plants:
Lantana camara L. (L. camara), generally known as wild or red sage has been extensively studied for its phytochemical composition in last few decades and belongs to the Verbenaceae family [10]. It is used in the treatment of cancer, chicken pox, measles, asthma, ulcers, swellings, high blood pressure, rheumatism, and other conditions due to its active phytochemicals and components [11, 12]. The plant is traditionally used to treat tetanus, epilepsy, diarrhoea, gastropathy, and metabolic process infections and disorders (cough, cold, and bronchitis) [12]. It is also used as a diaphoretic, carminative, antispasmodic agent, tonic, and antiemetic. Infusions of the leaves are used for bilious fever, eczema, and eruptions. Pulverised leaves are used for cuts, wounds, ulcers, and swellings. Protozoal infections, rheumatism, skin rashes, dermatitis, eczema, mycotic infections, tract infections, grippe, and T.B. are all treated with the foundation [13].
Calotropis procera L (C. procera) is a medium-sized tree or spreading shrub belong to the family Apocynaceae. It can be found in areas with excessively drained soil where yearly precipitation can reach up to 2000 mm and in arid habitats where rainfall is restricted to 150 to 1000 mm, as well it found in typical habitats including roadside sand dunes, seashore dunes, and heavily populated urban areas [14]. C. procera possess a wide range of pharmacological activity. Plant part such as root bark, stem bark, leaf, flower, and latex and their extracts, fraction, and isolated compound proved larvicidal, anticancer, acaricidal, schizonticidal, antibacterial, anthelmintic, insecticidal, anti-inflammatory and anti-diarrheal special effects [15, 16]. Numerous cardenolides, alkaloids, flavonoids, sterols, organic carbonates, norditerpenic esters, cysteine protease procerain and other chemicals have made this plant a popular subject of study for many years. Although, due to the existence of cardenolides, plants have the potential to be poisonous (cardiac glycosides). Cardenolides were discovered to be most abundant in the latex [17]. According to research, the leaf of this plant consists of cardenolides 162 mg/g at dry weight and 2 mg/g [18].
Recent research indicates that during malarial infections, the concentration of reactive oxygen species (such as superoxide and hydroxyl radicals) rises [19], due to haemoglobin digestion, parasite metabolism and host defense mechanism of the Pf-iEs. Reactive oxygen species plays a crucial role in various physiological processes for both host and parasite, as well as overproduction of intracellular ROS attack and damage lipids, proteins, nucleic acids, and integrity of cell membrane, which affects the survival of the cell and induce, the gradual apoptosis by suppressing the specific gene expressions [20]. To maintain the intracellular redox homeostasis parasite activates the machinery includes enzymes like iron-superoxide synthetase (Fe-SOD), glutathione-S-transferase (GST), glutathione synthetase (GS), γ-glutamylcysteine synthetase (γ-GCS), thioredoxin reductase (TrxR), and peroxiredoxins (nPrx) but, it lacks the catalase and glutathione peroxidase [21]. Therefore, it is understood that oxidative stress has a significant clinical and pathological role in malaria infection [22]. In addition, oxidative stress factor is also an effective therapeutic tool for example quinolines and artemisinin act chiefly via the production of ROS but, resistance development against Quinoline and Artemisinin- Based Combination Therapies (ACTs) in Southeast Asia and various regions of Africa over a long-term usage adds more complications in treating P. falciparum infected patients [21].
The goal of the current study is to evaluate the antioxidant activity and potential anti-plasmodial activity of the hydro-alcoholic extract of L. camara and C. procera leaves as well as to investigate how it affects the stabilisation of the erythrocyte membrane and in reducing the oxidative damage that this parasite onslaught caused in erythrocytes.
Traditional antimalarial drugs use Reactive Oxygen Species (ROS), which eventually causes their parasiticidal effects. However, the antimalarial activity mediated by ROS is something that Plasmodium’s evolutionary dynamism actively works to displace [21]. In light of this, current studies have focused on developing natural medicines to reduce Plasmodium infections. The inclusion of several secondary phytochemicals endows L. camara L. and C. procera L. plant extracts with strong therapeutic capabilities.
To perform the phytochemical extraction from the selected plants (L. camara and C. procera), the following method has been implemented.
2.1 Plant collection and extraction
Leaves of L. camara and C. procera were collected from the Gujarat University campus, Ahmedabad, Gujarat. Samples of plant material were authenticated by the Botany Department, Gujarat University, Ahmedabad, India. The plant material was thoroughly cleansed with distilled water, shade dried at ambient temperatures (27–37°C), then manually powdered using a commercial electrical stainless-steel blender. For defattation step, powder was treated with petroleum ether and continuously stirred on a magnetic stirrer for 48 hours. Using a Soxhlet extractor, 20 grammes of defatted powdered plant material were extracted for 72 hours with 200 ml of solvent (hydro-alcoholic: 70:30). At low pressure, the diluted crude solvent that had accumulated in the flask was concentrated. The yield that was obtained after drying was stored at 4°C until use [23].
2.2 In vitro cultivation of chloroquine (CQ)-sensitive strain 3D7 of Plasmodium falciparum
It is obtained from National Institute Malaria Research (NIMR), New Delhi. Erythrocytic stages were developed to determine the correlation between the anti-plasmodial efficacy of the plant extracts and the reactive oxygen species generated. Asynchronized P. falciparum culture was maintained according to the method described by Trager and Jensen [24] with minor modifications.
2.3 Determination of antioxidant activity of the plant extracts by DPPH assay
Both the plant extracts were assessed by the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) to determine its antioxidant activity by scavenging the free radicles as per the method reported by Gyamfi et al. [25]. EC50 value of the plant extract was evaluated by the dose response curve using Microsoft Excel.
2.4 In vitro study of erythrocyte membrane stabilisation and % inhibition of parasite entry
In many underdeveloped nations today, medicinal plants represent an important part of the traditional healthcare infrastructure. The bioactive chemicals found in these medicinal plants have been used to create several medications since antiquity [26, 27]. This study also suggests that the extract may alter the erythrocyte membrane in a way that renders red blood cells hostile and incompatible as parasite host cells or that it may prevent the parasite from entering the erythrocyte even when extract is not present in the growing media. L. camara and C. procera extracts are rich in phenolic and flavonoid components [12, 28].
Erythrocyte membrane stabilisation efficacy of the two plant extracts were assessed using the haemolysis method mentioned by Jansen et al. [29] and Linz-Buoy et al. [28]. In which, control O+ve erythrocytes were resuspended in 10% PBS (v/v) and incubated with crude plant extracts consisting of the range (1000 μg/ml with serial dilution up to 7.18 μg/ml concentration) under agitation for 24 hours at room temperature, this mixture then subjected to centrifugation for 5 minutes at 10,000 x g followed by the absorbance of the supernatants was measured at 550 nm with a microplate reader.
2.4.1 In vitro % inhibition of parasite entry into RBCs
In vitro percent inhibition of parasite entry into RBCs was calculated as mentioned in Linz-Buoy et al., [12]. For each concentration, growth inhibition was calculated as a percentage of the number of schizonts compared to five untreated controls. Dose-response curves were used to get the mean IC50 values (percentage of schizonts vs. logarithm of drug concentration).
2.5 Oxidative stress parameters
By preventing the emergence of new free radical species, stopping radical chain reactions, changing existing free radicals into less dangerous molecules, and repairing oxidative damage, antioxidants work through protective processes at various levels within cells [30].
2.5.1 Cellular sample preparation
Intracellular ROS: Cells were harvested after 48 hours of incubation at 37°C. Cultured cells were harvested and centrifuged to remove the culture media at 500 g for 10 minutes at 4°C. The pelleted cells were haemolysed in four times volume of ice-cold injection water/1x RBC lysis buffer and centrifuged again at 4°C. This cell lysate was then used to measure intracellular ROS by Metta et al. [31] with minor modifications.
2.6 Parameters
2.6.1 Lipid peroxidation (LPO)
The method of Okhawa et al. [32] was used to measure the quantities of thiobarbutiric acid reactive species (TBARS) in Control erythrocytes, Pf-iEs, and Pre-treated Control erythrocytes with the plant extract.
2.6.2 Superoxide dismutase (SOD)
The NADH-phenazinemethosulphate-nitroblue tetrazolium formazon is the foundation of the superoxide dismutase (SOD) test. The activity of SOD in Control erythrocytes, Pf-iEs, and Pre-treated Control erythrocytes with the plant extracts by the method of [33].
2.6.3 Catalase
As hydrogen peroxide is broken down by the enzyme catalase, its UV absorbance at 240 nm can be used in this method to evaluate enzyme activity. The method of Sinha [34] was employed to determine the activity of catalase enzyme in control erythrocytes, Pf-iEs, and Pre-treated Control erythrocytes with the plant extracts. By decrease in absorbance, the activity of an enzyme can be calculated.
2.6.4 Reduced glutathione (GSH)
The method outlined by Ellaman et al. [35] was used to measure the reduced glutathione (GSH) level. When GSH reacts with DTNB (5, 5′-dithiobis nitro benzoic acid), a yellow compound formed. Control erythrocytes, Pf-iEs, and Pre-treated Control erythrocytes with the plant extracts were taken as samples.
2.6.5 Glutathione peroxidase (GPx)
The Rotruck et al. [36] method was used to measure the glutathione peroxidase (GPx) enzyme activity in control erythrocytes, Pf-iEs, and pre-treated control erythrocytes A cytosolic enzyme called GPx catalyses the conversion of peroxide radicals to alcohols and oxygen as well as the conversion of hydrogen peroxide to water and oxygen.
2.7 %Inhibition of growth proliferation (MTT) assay
3- (4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) Assay is based on the capacity of Mitochondria succinate dehydrogenase dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into an insoluble, coloured formazan product which is measured spectrophotometrically at 540 nm [37, 38]. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability/cytotoxicity of the cells.
In vitro confirmations of the toxic effect of the phytocomponents have measured on the HeLa cell lines with 6 serial dilutions from 250 to 0.0156 µg/ml. The formazan crystals were formed, following the reduction of MTT by metabolically active (viable) cells.
2.8 Haemoglobin (Hb) determination
Haemoglobin determination was carried out using a Sahli’s hemoglobinometer with standard colour comparator and the final value was recorded as g/dl Haemoglobin content [39]. The amount of Hb of control, Pf-iEs and pre-treated erythrocytes is given inFigure 8.
3.1 Antioxidant activity of the plant extracts by DPPH assay
Antioxidant activity demonstrates the plant extract’s ability to scavenge Reactive Oxygen Species (ROS) in a dose-dependent manner. The antioxidant potency of two plant extracts is revealed in this study. Ascorbic acid is used as the standard here (Figure 1).
Figure 1.
Bar graph presentation of antioxidant activity of two selected plant’s hydro-alcoholic extracts, compared with the standard ascorbic acid. Values are Mean ± S.E. n = 6. (* p<0.01, ** p < 0.001, *** p <0.0001).
As antioxidants are required to protect cells from oxidative stress, the L. camara and Calotropis Procera extracts tested in this study were found to be high in antioxidants L camara showed the higher antioxidant activity compared to C. procera plant extract, with 85.23% at the highest concentration of 250 g/ml. Linz-Buoy et al. [28] demonstrated in vitro that this effective antioxidant capability is beneficial in this control.
3.2 In vitro erythrocyte membrane stabilisation and % inhibition of parasite entry
The % erythrocyte membrane stabilisation assay with haemolysis (osmotic fragility test) is shown in Figure 2. The property of erythrocyte membrane stabilisation is performed in dose dependent manner i.e., from the highest concentration (250 μg/ml to 7.81 μg/ml).
Figure 2.
The % erythrocyte membrane stabilization with haemolytic assay reported via bar graph presentation of selected hydroalcoholic extracts of the two plants (L. camara and C. procera).
Only 2.48 percent of the erythrocytes were hemolyzed when treated with the hydroalcoholic extract of L. camara at the highest concentration (250 g/ml) used in the study. Pretreatment with C. procera hydroalcoholic extracts yielded 7.66 percent at the concentration (250 g/ml).
In the pretreatment of control erythrocytes, haemolysis increases as the crude extract concentration decreases.
3.2.1 % Inhibition of parasite entry
The experimental results of this study showed that pre-treatment of the hydroalcoholic extracts of selected two plants showed anti-plasmodial efficacy for an asynchronized culture of P. falciparum MRC2 and RKL-9 (Tables 1 and 2).
MRC-2
Concentration
1.95 μg/ml
3.91 μg/ml
7.81 μg/ml
15.63 μg/ml
31.25 μg/ml
62.5 μg/ml
125 μg/ml
250 μg/ml
% Inhibition
L. camara (HA)
41.5 ± 0.09
43 ± 0.12
48 ± 0.05
51.5 ± 0.07
63 ± 0.28
75.5 ± 0.16
86 ± 0.04
86.5 ± 0.21
C. procera (HA)
26.02 ± 0.23
30.09 ± 0.47
35.65 ± 0.59
45.65 ± 0.63
59.1 ± 1.21
68.23 ± 0.08
81 ± 0.45
83.45 ± 0.23
Table 1.
% Inhibition of entry of P. falciparum MRC-2 strain in pre-treated erythrocytes with hydro-alcoholic extract of L. camara and C. procera. This shows the hydro-alcoholic extract of L. camara and C. procera were assessed for their anti-plasmodial activity. Here, asynchronized culture of P. falciparum MRC-2 were subjected to pre-treated control erythrocytes for 24 hours.
RKL-9
Concentration
1.95 μg/ml
3.91 μg/ml
7.81 μg/ml
15.63 μg/ml
31.25 μg/ml
62.5 μg/ml
125 μg/ml
250 μg/ml
% Inhibition
L. camara (HA)
26.02 ± 0.23
30.09 ± 0.47
35.65 ± 0.59
45.65 ± 0.63
59.1 ± 1.21
68.23 ± 0.08
81 ± 0.45
83.45 ± 0.23
C. procera (HA)
29.5 ± 1.09
35.0 ± 0.19
40.0 ± 0.06
50.0 ± 0.11
52.5 ± 0.21
65.0 ± 0.19
70 ± 0.05
73.5 ± 0.17
Table 2.
% Inhibition of entry of P. falciparum RKL-9 strain in pre-treated erythrocytes with hydro-alcoholic extract of L. camara and C. procera. This shows the hydro-alcoholic extract of L. camara and C. procera were assessed for their anti-plasmodial activity. Here, asynchronized culture of P. falciparum RKL-9 were subjected to pre-treated control erythrocytes for 24 hours.
Among these two plants L. camara is found more effective in killing 50% of malaria parasites in the appropriate course of incubation time (24 hours) of the asynchronized parasite of strain MRC-2 and RKL-9 according to the experimental evidence obtained in our study. Among the two plants L. camara reported highest antioxidant activity and thus same result reflected in the % erythrocyte membrane stabilisation assays and proves its potent anti-plasmodial activity.
3.3 Oxidative stress parameters
3.3.1 Lipid peroxidation (LPO)
When Pf-iEs were compared to control erythrocytes, lipid peroxidation was significantly increased (Figure 3). The results also revealed that, when compared to the negative control, pre-treated erythrocytes with the plant extracts had significantly lower levels of lipid peroxidation, as evidenced by higher MDA values.
Figure 3.
Showing the lipid peroxidation (TBARS) level in control, infected and pre-treated erythrocytes with the hydroalcoholic extracts of L. camara and C. procera. extract. Values are mean ± S.E. # (×104 nmoles of MDA/100mg cells wt/60min).
The results also indicated that there was a significant reduction (** p < 0.001) in lipid peroxidation as indicated by the elevated MDA values, for the control erythrocytes when compared to negative control, were treated with both the extracts when subjected to Pf-MRC2 and Pf-RKL9 for 24 hours.
3.3.2 Superoxide dismutase (SOD)
While Pf-iEs were compared to control erythrocytes, the activity of superoxide dismutase was found to be significantly lower (p < 0.001). Once control erythrocytes were pre-treated with hydroalcoholic extracts of L. camara and C. procera and then subjected to Plasmodium infection (MRC-2 and RKL-9) for 24 hours, SOD activity increased significantly (Figure 4).
Figure 4.
SOD enzyme activity in control and Pf-iEs, compared with the pre-treated control erythrocytes with the hydroalcoholic extracts of L. camara and C. procera. The unit of SOD is SOD (units/mg protein). Values are mean ± S.E. ** p < 0.001; * p < 0.01.
3.3.3 Catalase
The results obtained show that catalase activity significantly decreases with Plasmodium infection but, it is significantly improved with the use of plant extracts (Figure 5).
Figure 5.
Shows the catalase enzyme activity in control erythrocytes, PfMRC-2 and PfRKL-9 infected and treated control erythrocytes with the hydroalcoholic extracts of L. camara and C. procera. Values are mean ± S.E. Unit of catalase activity measured: mmol of H2O2 consumed/min/mg protein) ** p < 0.001; * p < 0.01.
3.3.4 Reduced glutathione (GSH)
The study revealed a significant (p < 0.01) increase in reduced glutathione level in the Pf-iEs. After pre-treatment with the hydroalcoholic extracts of both the plants, decrease in the GSH level was observed (Figure 6).
Figure 6.
Shows the reduced Glutathione content differs during the Plasmodium infection as it increases compared to control erythrocytes but, when pre-treated with hydroalcoholic extracts, decreases the glutathione level. Among two plants L. camara hydroalcoholic extract shows most effective decline. Values are mean ± S.E. * p < 0.01. The unit of GSH measurement (µg/100mg cells weight).
3.3.5 Glutathione peroxidase (GPx)
The study revealed that the Plasmodium infection resulted in a considerable decline in GPx activity. Following the application of the plant extracts via pre-treatment to control erythrocytes, there were noticeable increase seen in the Glutathione peroxidase activity (Figure 7).
Figure 7.
Showing GPx enzyme activity in control erythrocytes, Pf-iEs and pre-treated erythrocytes with hydroalcoholic extracts of L. camara and C. procera. Values are mean ± S.E. ** p < 0.001.
3.4 % Inhibition of growth proliferation (MTT) assay
The result decreases of % growth proliferation after pre-treatment with both extracts has tabulated in Table 3.
Concentration of the crude extract of the plants (μg/ml)
7.81 μg/ml
15.63 μg/ml
31.25 μg/ml
62.5 μg/ml
125 μg/ml
250 μg/ml
L. camara (HA)
98.12 ± 0.51
96.90 ± 1.24
97.56 ± 0.90
97.88 ± 0.43
97.79 ± 1.09
98.46 ± 0.90
C. procera (HA)
98.08 ± 0.54
97.53 ± 0.77
97.08 ± 1.15
97.17 ± 0.41
97.24 ± 0.67
96.86 ± 1.2
Table 3.
Showing the results of MTT assay in which % growth inhibition of extracts of both L. camara and C. procera on the HeLa cells. In vitro confirmation of the toxic effect of the phytocomponents has measured on the HeLa cell lines with 6 dilutions from 250 to 7.81 μg/ml. The formazan crystals were formed, following the reduction of MTT by metabolically active (viable) cells.
3.5 Haemoglobin (Hb) determination
The highly significant decrease of Hb content has been observed in the infected erythrocytes (iEs). Compared with the control erythrocytes much significant changes in the amount of Hb has not been observed in the pre-treated erythrocytes (Figure 8).
Figure 8.
Showing the haemoglobin content in control, Pf-iEs and pre-treated erythrocytes with plant extracts. Values are Mean ± S.E. *p<0.01 **p<0.001.
Membrane stabilising profiles of various extracts of L. camara on bovine red blood cells exposed to both heat and hypotonic induced lysis were reported previously [40]. Earlier studies have shown that various herbal drugs can stabilise the red blood cell membrane [41]. The mode of action of the extracts could relate to binding to the erythrocyte membranes with subsequent alteration of the surface charges of the cells. This might have prevented physical interaction with aggregating agents or promote dispersal by mutual repulsion of like charges which are involved in the haemolysis of red blood cells. It has been reported that certain saponins and flavonoids exerted profound stabilising effect on lysosomal membrane both in vivo and in vitro, while tannins and saponins possess ability to bind cations, there by stabilising erythrocyte membrane and other biological macro molecules [41]. It is not surprising that P. falciparum develops resistance to antimalarial medications whose mode of action is dependent on the production of ROS in a shorter amount of time. This shows a common pathway of resistance to the K13 propeller gene mutation, which is supported by the development of artemisinin resistance to novel endoperoxide-based hybrid molecules [42].
The ROS-managing machinery of the parasite could be disrupted to preserve and improve the actions of the antimalarials, ensuring the ongoing relevance of ROS-producing antimalarials [21]. Moreover, aromatic and therapeutic plants make up much of India’s natural resources. According to reports, phenolic and flavonoid compounds function as antioxidants to exert anti-allergic, anti-inflammatory, antidiabetic, antimicrobial, antiviral, antithrombotic, and vasodilatory effects. As a result, they may prevent diseases like cancer, cardio-vascular disease, cataract, eye disorders, and Alzheimer’s [43, 44].
Free radicals are vital to many metabolic processes and play a crucial role in aerobic metabolism and life. Reactive oxygen species (ROS) have been implicated in mediating oxidative damage to macromolecules such lipids, proteins, and DNA. Antioxidants protect cells at multiple levels by inhibiting the formation of free radical species, interfering with radical chain reactions, converting existing free radicals into less harmful molecules, and repairing oxidative damage [30]. Flavonoids and phenolic compounds are abundant in L. camara extracts [28]. Flavonoids and tannins are most likely responsible for the free radical scavenging effect. Plant phenolic compounds also serve as primary antioxidants.
Becker et al. [45] have shown an increase in the lipid peroxidation of Plasmodium infected RBCs. Moreover Erel et al. [46] have demonstrated that plasmodia succeed in accumulating free radical scavenging enzymes within their own cells but deplete them in red blood cells of the host. Polyphenols (flavonoids) have been known to effectively restrict free radical induced peroxidation of lipid. According to [47] in addition to their protein binding and direct scavenging activity, these potent antioxidants interact with membrane lipids and prevent the access of deleterious molecules across the cell membrane. Thus, the infected erythrocytes, treated with the extracts showed decreased lipid peroxidation, due to the potent antioxidant activity of these extracts.
The current study found a highly significant decrease in the erythrocyte antioxidant machinery, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), indicating the presence of a high amount of ROS. Plasmodium erythrocytic stages are subjected to a variety of oxidative stress-inducing events, such as haemoglobin metabolism [48]. P. falciparum generates H2O2 within RBCs during haemoglobin degradation [45]. Furthermore, the Fenton reaction exposes intra-erythrocytic parasite stages to increased ROS formation [49, 50]. P. falciparum-infected erythrocytes produced significantly more hydroxyl (OH) radicals and H2O2 than uninfected erythrocytes, according to Atamna and Ginsburg [51]. Plasmodium infection was found to cause a significant increase in lipid peroxidation in red blood cells; however, when treated with a hydro-alcoholic extract of L. camara, a significant decrease in LPO in infected RBCs was obtained.
SOD activity in plasmodium-infected RBCs was significantly lower than in normal RBCs. Several other studies have found a decrease in SOD activities in erythrocytes in malaria patients [52, 53], which supports the findings. This supports its role as an antioxidant, where levels decreased to counteract oxidative stress. The main characteristics of these changes are changes in erythrocyte GSH content, lipid peroxidation levels, and oxidative stress enzymes like SOD, CAT, and GPx. L. camara and C. procera extracts were found to be effective enough to overcome this change and return the cell to normalcy as a result, the L. camara and C. procera extracts exhibit enormous potential and promise in controlling Plasmodium ingress into the host erythrocytes and further reducing the subsequent oxidative stress.
Herbal products are well thought-out to be symbols of safeguard in comparison to the synthetic product that are regarded as unsafe to human life and environment. Phytochemical and pharmacological studies are conducted on L. camara and C. procera. Based on the results obtained in the study, it can be inferred that the leaves of L. camara are rich sources of lot of secondary metabolites/phytocomponents which can be used as a prophylactic drug against malaria.
Finally, the current investigation’s experimental work was focused on using membrane stabilisation to assess the anti-plasmodial activity of L. camara hydro-alcoholic crude extract. This extract did show evidence of potent phytochemicals with erythrocyte membrane stabilising activity. Furthermore, this study found that L. camara has better anti-plasmodial activity than C. procera against MRC2 and RKL9 Plasmodium falciparum strains, in contrast to synthetic products, which frequently have side effects and are dangerous to human life. This plant extract’s potent antioxidant phytochemicals are both effective and relatively safe.
Both extracts of L. camara leaves had shown positive results on the effect on erythrocyte membrane stabilisation. They could inhibit the entry of parasites, also show effective antioxidant property, and has no toxic effect due to certain unknown compounds in the crude extracts on the normal cells which is proved by the MTT assay.
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Written By
Urja Joshi, Dhara Jani, Linz-Bouy George and Hyacinth Highland
Submitted: 21 July 2022Reviewed: 28 July 2022Published: 13 October 2022
Open access peer-reviewed chapter
