Evaluation of the Bioactivities of Rumex crispus L. Leaves and Root Extracts Using Toxicity, Antimicrobial, and Antiparasitic Assays

Traditional folks in different parts of the world use Rumex crispus L. for the treatment of microbial infections, malaria, and sleeping sickness in the form of decoction or tincture. In the search for a natural alternative remedy, this study aimed to evaluate the antimicrobial, antitrypanosomal, and antiplasmodial efficacy and the toxicity of R. crispus extracts. Antimicrobial potency of the extracts was evaluated using the agar dilution method to determine the minimum inhibitory concentration (MIC). The antitrypanosomal activity of the extracts was evaluated with the Trypanosoma brucei brucei model while the antimalaria potency was tested using Plasmodium falciparum 3D7 strain. Toxicity was then tested with brine shrimp assay and cytotoxicity (HeLa cells). The acetone extract of the root (RT-ACE) reveals the highest antimicrobial potency with the lowest MIC value of <1.562 mg/mL for all bacteria strains and also showed high potent against fungi. RT-ACE (IC50: 13 μg/mL) and methanol extract of the leaf (LF-MEE; IC50: 15 μg/mL) show a strong inhibition of P. falciparum. The ethanol extract of the root (RT-ETE: IC50: 9.7 μg/mL) reveals the highest inhibition of T.b. brucei parasite. RT-ETE and RT-ACE were found to have the highest toxicity in brine shrimp lethality assay (BSLA) and cytotoxicity which correlates in the two assays. This research revealed Rumex crispus has potency against microorganisms, Trypanosoma, and Plasmodium and could be a potential source for the treatment of these diseases.


Introduction
Medicinal plants have been used for ages in several ways for antimicrobial, hypoglycemic, antihelminthic, antiulcerogenic, hepatoprotective, antipyretic, analgesic, insecticidal, piscicide, pesticide, pharmaceutical, and cosmetic purposes [1]. e biological activities and efficacy of medicinal plants have been tested in traditional medicine and in several fields of science [2]. eir potency and curative potentials were documented independently across the globe suggesting that plants could be a potential source of novel drugs [2][3][4]. ere has been a rapid increase in the use of medicinal plants as a curative prototype, and new products are emanating from these natural products into the commercial market daily. ese products are believed by the consumers to be safer and effective than the chemotherapeutic conventional drugs [4,5]. It was estimated that about 61% of newly developed drugs between 1981 and 2002 have their source from plants and are successful in the treatment of cancer and several other diseases [2,6]. Many medicinal plants have been screened for antimicrobial, antiplasmodial, and antitrypanosomal activities, and some of these plants showed prospect in the treatment of these infections [3,7,8]. In most cases, the medicinal properties of a plant are a function of its phytochemical constituents [9]. Drugs. ere have been reports of multiple drug resistance (MDR) in the human pathogenic microbial organisms due to indiscriminate use of commercially available drugs [4]. e capacity of diseases to drug resistance is the clinical determinants of the pathogenesis of a particular pathogen. For instance, there have been reports of higher resistance to a group of antibiotics in an area where it is regularly misused [10,11]. Multiple drug-resistant strains of pathogens and parasites are economically important, and these pose a huge threat to the public health system. is calls for an immediate intervention with new drugs that use a different mechanism of action in the treatment of these diseases. On the other hand, there are several scientific evidences to the success of medicinal plants in treating diseases; hence, plants could be a novel source that will combat drug resistance [5].

Resistance of Microorganisms to
Plants are known to produce complex bioactive organic compounds that possess several bioactivities. e compounds are secreted in various parts of the plants such as seeds, fruits, flowers, leaves, roots, stems, bark and some plant secretions (isothiocyanates, plant pigment, plant gum, phytoalexins, essential oils, and hydrolytic enzymes) [12]. e bioactive constituent in medicinal plants has shown tremendous health benefits. For instance, the major antibacterial bioactive compounds in plants are anthraquinones and flavonoids which are active against several human pathogenic bacteria [13]. Phenolic compounds, flavonoids, and artemisinin are active against malaria causing parasite, Plasmodium falciparum [14]. It was also reported that flavonoids and glycosides in plants inhibit the growth of African sleeping sickness (Trypanosoma brucei) parasites [15]. e increase in the occurrence and sustenance of drug-resistant pathogens to drugs has drawn the attention of scientists and pharmaceutical communities towards harnessing the potentials of plant-derived substances used successfully in different countries by traditional medical practitioners [16].

Tests for Toxicity, Antimicrobial, and Antiparasite.
ere is a rapid increase in interest and demand for medicinal plants in various parts of the world because they are considered natural, safer, and cheaper [17]. On the other hand, there is a general concern in the scientific communities on the safety, efficacy, and pharmacokinetics of medicinal plants, irrespective of their wide acceptance and usage. e possible side effects of plant products cannot be ruled out regardless of their adoption as a novel source of drugs [18,19]. e source of the toxicity of plants may be due to the contaminants in the environment or from the plant-secreted phytochemicals [20,21]. Different biological models have been tested via in vitro and in vivo assays to qualify and quantify the potential toxicity of extracts of medicinal plants. However, recent studies employed the use of preliminary toxicity assessment tools using brine shrimp species: Artemia franciscana, Artemia salina, amnocephalus platyurus, and Artemia urmiana [22][23][24], which is considered a useful tool to evaluate the potential toxicity of plant extracts. Cytotoxic screening using HeLa human cervix carcinoma cells is an advanced tool to screen substances inducing apoptosis. In addition, a comparative toxicity study of plants extract could be tested using in vivo study. e result, however, showed quite a good correlation in the mean value of in vitro study lethal concentration (LC 50 ) and the acute oral lethal dose (LD 50 ) of in vivo study [23,25].
Rumex crispus L. (curled dock) is commonly used by traditional healers for treatment of various diseases and correction of disorders such as gastrointestinal tract disorders, antihelminthic diseases, anti-inflammatory, and arthritis, and it is also used as laxative, antipyretic, antioxidant, and antimicrobial [5,26]. More so, there are reports of the antimicrobial [27][28][29], antitrypanosomal [30], and antiplasmodial [31] properties and toxicity of R. crispus which were evaluated in this research. is is necessary to validate the claim of traditional healer practitioners in Eastern Cape, South Africa, and any other part of the world that use R. crispus for the treatment of these ailments.

Organic Extraction.
e leaves and roots of Rumex crispus were collected from a fallow damp field, University of Fort Hare (32°47′1.23″S, 26°51′9.85″E), in the summer of 2017. It was identified, and a sample of the specimen was placed in Giffen herbarium (Idr-Med-2017/03), University of Fort Hare. e leaves and roots of the plant specimen were carefully sorted, and the infected ones were removed. e specimen was rinsed, dried in an oven at 30°C until a fixed weight, and then pulverized with a mechanical grinder (Polymix PX-MFC90D, Switzerland). e pulverized sample was stored in an airtight container, in a refrigerator at 4°C until use for extraction.
Crude extraction of R. crispus was done using standard procedures. e choice of solvent for the extraction process is an imperative factor that must be considered during the antimicrobial and toxicity tests. e extracting potentials of different solvents differ; therefore, extraction was done using solvents of low reactivity: a technically graded acetone, methanol, ethanol, and distilled water. e extractant (solvent) and the plant materials used were in the ratio of 1 : 10 (g/mL). e dried root (RT) and the aerial part (LF) of the plant sample (100 g) were macerated with the extractant solvents (1000 mL) at room temperature. e crude extract of methanol (MEE), ethanol (ETE), and acetone (ACE) were concentrated under reduced pressure by using a rotatory evaporator, considering the boiling point of the respective solvent, and then dried in a desiccator at a mild temperature (40°C). e filtrate of water samples (WAE) was chilled at − 40°C with a shell refrigerant (PolyScience AD15R-40-A12E, USA) and freeze-dried (Savant vapour trap, RV-T41404, USA) for 48 h. e plant crude extracts were stored in sterile bottles and refrigerated at 2 to 4°C until further use.

Determination of Minimum Inhibitory Concentrations
(MICs) of the Extracts. Minimum inhibitory concentrations (MICs) were used to determine the lowest concentration of the extracts that will inhibit visible microbial growth after an incubation period of 24 h (bacteria) and 48 h (fungi). Usually, MICs are diagnostic laboratory tools used to confirm microbial resistance but are often used in research laboratories to determine the in vitro activity of antimicrobials and its MIC breakpoints [32]. ese strains of bacteria were collected from the Department of Microbiology, University of Fort Hare, South Africa. e concentration of the plant extracts that inhibits the microbial growth after 24 h of incubation was determined by MIC assay as described [33] with modification. e agar dilution method was used for the MIC to determine the antibacterial efficiency of R. crispus, and the resultant efficacy was compared with a commercial antibiotic drug (erythromycin). e collected strains of bacteria were subcultured for 24 h at 37°C in a sterilized Mueller-Hinton Agar (Oxoid ™ ), following the manufacturer preparation instructions. e bacteria were harvested using 5 mL of sterilized saline solution, and the inoculum was adjusted to 10 6 CFU/mL by comparison with 0.5 McFarland standard {0.5 mL of 0.048 M BaCl 2 (1.17% w/v BaCl 2 ·2H 2 O) + 99.5 mL of 0.18 M H 2 SO 4 (1% v/v)} between 106 and 108 CFU/mL and then the inoculum was applied to the agar prepared in sterilized Petri plates [13]. A dilution range of 1.562-25 mg/mL was prepared for the plant extracts, and concentration range of 0.5-8 μg/mL of erythromycin was used for the positive control. e plant's extracts were dissolved in 10% dimethylsulphoxide (DMSO) (except aqueous extracts) and distilled water and then purified by filtering through Millipore filter paper. Mueller-Hinton agar was prepared following the manufacturer's instructions; a volume of 20 mL of the agar was measured in Erlenmeyer flask and autoclaved. ereafter, 1 mL of the prepared extract was added, swirled, and poured into sterile disposable Petri dishes. e agar was allowed to solidify at room temperature. e standardized inoculum was diluted in sterile saline solution and Mueller-Hinton broth in ratio 1 : 10, to attain 0.5 McFarland standard between 106 and 108 CFU/mL. An aliquot volume of 0.1 mL of the inoculum was pipetted and transferred to the agar medium, and the plates were incubated for 24 h at 37°C.

Antifungal Screening.
e fungi Trichophyton tonsurans ATCC 28942, Trichosporon mucoides ATCC 201382, Penicillium aurantiogriseum ATCC 16025, Penicillium chrysogenum ATCC 10106, Candida glabrata ATCC 66032, and Candida albicans ATCC 32077 were collected from the Department of Botany, University of Fort Hare, and subcultured on Sabouraud Dextrose Agar (Oxoid ™ ), at 30°C for 48 h. e antifungal assay was carried out according to the method described by Lass-Flörl et al. [34], with modification. e medium (Sabouraud Dextrose Agar) was prepared following the manufacturer's instructions, and 5 mg/100 mL of chloramphenicol was added to the agar media before autoclaving to inhibit bacteria growth. e suspensions of the inoculum were transferred into a sterile saline solution from the subculture, by carefully collecting the conidia with a sterile cotton swab. e suspension was homogenized for 15 s with a vortex (2,000 rpm), and the inoculum was diluted in Mueller-Hinton broth in ratio 1 : 10, to obtain a final test inoculum load range between 1.0 × 10 6 and 5.0 × 10 6 CFU/ mL. e adjusted working inoculum was plated on solidified sterilized Sabouraud Dextrose Agar plates with plant extract's dilution range of 0.625-10 mg/mL, and the control agar plates was diluted at the range of 1-16 μg/mL of nystatin; all plates were incubated for 48 h at 30°C.

Screening of R. crispus against Plasmodium falciparum.
e malaria parasites (Plasmodium falciparum, sensitive strain; 3D7) were used in blood-stage culture to test the antimalarial efficacy of R. crispus extracts through in vitro studies, following the method described by Murugan et al. [7]. e parasites were preserved in RPMI 1640 medium containing 2 mML glutamine and 25 mM Hepes (Lonza). e medium was further supplemented with 5% Albumax II, 20 mM glucose, 0.65 mM hypoxanthine, 60 μg/mL gentamycin, and 2-4% hematocrite human red blood cells. e parasites were cultured at 37°C under an atmosphere condition of 5% CO 2 , 5% O 2 , and 90% N 2 in sealed T75 culture flasks. e infected erythrocytes were transferred into a fresh medium to propagate the culture, and parasitaemia (the concentration of parasites in the culture) was measured by light microscopy of Giemsa-stained thin blood smears.
In the experiment, the tested extracts were prepared in 0.4% dimethyl sulfoxide (DMSO). en, the parasiticidal potency of tested extracts was done by single concentration screening. e single concentration screening was conducted by adding 25 μg/mL (extracts) to parasite cultures in 96-well, clear plates and incubated for 48 hours in a CO 2 incubator at 37°C. After 48 hours, 20 μL of culture was removed from each well and combined with 125 μL of a mixture of Malstat and NBT/PES solutions in a fresh 96-well plate. ese solutions were used to measure the activity of the parasite lactate dehydrogenase (pLDH) enzyme in the cultures. A purple product is formed when pLDH is present [8], and this product is quantified in a Spectramax M3 microplate reader at the absorbance of 620 nm. For each extract concentration, percentage parasite viability and pLDH activity in extract-treated wells relative to untreated controls were calculated. e extracts were tested in triplicates, and standard deviations (SD) were derived. For comparative purposes, chloroquine (a standard antimalarial drug) was used as a standard (IC 50 values ranging from 0.01 to 0.05 μM).

Antitrypanosomal Activity of R. crispus Extracts.
e subspecies responsible for sleeping sickness in Nagana, Trypanosoma brucei brucei (T.b. brucei), is infective to humans and is commonly used as a drug screening model in the laboratory. is research adopted the T.b. brucei model as described in [35], with modification. e blood-stage forms of T.b. brucei (Strain 427) were grown in Iscove's Modified Dulbecco's Medium (IMDM) enhanced with 10% fetal bovine serum, and the culture was incubated in 5% CO 2 , at 37°C.

Evidence-Based Complementary and Alternative Medicine
For primary screening of extracts' trypanocidal activity, a single concentration of 50 μg/mL in duplicate was prepared from the extract stock in IMDM and added to the culture of T.b. brucei in a 96-well plate. e mixture was incubated for 48 h, and the number of survived parasites was determined by adding a resazurin-based reagent which would be reduced to resorufin by living cells. Resorufin is a fluorescent compound (Excitation560/Emission590); hence, absorbance was measured in a multiwell fluorescence plate reader (Spectramax M3) to determine viable cells. e percentage parasite viability of each extract was determined by comparing the resorufin fluorescence in treated wells in relation to untreated controls. e resultant was then plotted against log (extract concentration), and the IC 50 from the resulting dose response in nonlinear regression was obtained. e percentage parasite viability and response to extracts were then compared with pentamidine (a drug for trypanosomiasis) as a positive control.

Toxicity Test.
e toxicity of plant was tested by exposing the eggs and larvae of Artemia salina to the plant extracts and comparing their LC 50 values with toxic compound (potassium dichromate: K 2 CrO 7 ), suitable as a positive control for this test. Further toxicity was done by testing extracts on HeLa cells (human cervix adenocarcinoma). All experiments were carried out in accordance with the guidelines for the Care and Use of Laboratory Animals. e research methodology was approved by the University of Fort Hare, Animal Ethical Committee (reference number: AFO121SIDR01).

Brine Shrimp Hatchability Assay (BSHA).
e BSHA was carried out as previously described by Otunola et al. [36], with modification. e eggs of Artemia salina (brine shrimp) were obtained from aquaculture outlet in East London, South Africa. e eggs of Artemia salina was hatched at optimum condition, in seawater (salinity 38 ppt and pH 8.5), collected in East London, South Africa (32°59 S 27°52 E). e concentration of the extracts and a standard drug (K 2 CrO 7 ) were graded (0.0625, 0.125, 0.25, 0.5, and 1.0 mg/mL) by serial dilution in 30 mL of filtered seawater, in sterile Petri dishes. Each concentration of extracts and positive and negative control were tested in triplicate. Ten eggs of brine shrimp were introduced into the Petri dishes. e hatched nauplii were counted against the ratio of the number of eggs introduced at the beginning of the experiment at 12 h interval, and the level of toxicity was evaluated using probit regression ( Figure 1).

Brine Shrimp Lethality Assay (BSLA).
In the study, BSLA was carried out adopting the method as described in [37], with slight modifications. e brine shrimp eggs were incubated at the optimum condition in a glass jar for 48 h. A count of 10 nauplii with an inverted microscope was pipetted into different Petri dishes with a graded concentration of extracts, potassium dichromate, and control seawater.
e lethality of larvae was observed with an inverted microscope at an interval of 12 h, and the total lethality was computed using the Standardized Lethality Index (SLI). e condition of the exposed larvae was evaluated with SLI, as used by Agrafioti et al. [38]. Based on the index, each experimental nauplius was ranked from 0 to 3, to measure the intensity of the response. In the scale; responses 0: normal nauplii, responses 1: nauplii were knocked down but were able to swim for short intervals, responses 2: nauplii were knocked down and unable to swim but with visible movement, and responses 3: nauplii were dead (Table 1, Figures 2-4). us, the lethality index was calculated with the equation: where N 0 : number of nauplii at rank 0; N 1 : number of nauplii at rank 1; N 2 : number of nauplii at rank 2; N 3 : number of nauplii at rank 3; N t : total number of nauplii; N ob : number of observations (three days, six observations); Max w : maximum lethality coefficient; and lethality coefficients:

Cytotoxicity
Assay. e cytotoxicity of the extracts of R. crispus was estimated using HeLa (human cervix adenocarcinoma) cells as described by Larayetan et al. [8]. e stock solutions were prepared in 1% DMSO to attain a final concentration of 50 μg/mL. e HeLa cells were incubated at a fixed concentration (50 μg/ml) of the extracts in 96-well plates for 48 h. e viability of the cells after treatment with extracts was determined by adding resazurin-based reagent and reading resorufin fluorescence in a microplate reader (Spectramax M3). e results were expressed as percentage viability in resorufin fluorescence-treated extracts per wells relative to the untreated wells (controls). e cytotoxicity of the extracts was done in duplicate, and the positive control, standard drug (emetine), was used for comparative reference.

Statistical Analysis.
e mean of the triplicates of the results for the assay was expressed as mean ± SD. e data were analyzed statistically by one-way analysis of variance, and differences between data were extrapolated by Fisher's Least Significant Difference (LSD) test, where it is applicable. e relationship between the response to the stimulus of the independent variables (concentration) and the quantal (dead or alive, all or none) was analyzed by probit regression to estimate the LC 50 and LC 99 using statistical software (SPSS).

Antimicrobial Activity of Plant Extracts.
e antibacterial and antifungal potency of the extracts is given in Tables 2  and 3. e result shows that all extracts of R. crispus have a varying degree of antimicrobial activities with a variation in the level of potency. e concentration effect of the plant extracts and standard drug is reported in Table 2. e MIC was confirmed by the absence of the growth of tested bacterial strains on the surface of the agar which shows the potential bactericidal activity of the plant's extracts against the tested bacteria. e acetone extract of the root (RT-ACE) registered the highest antimicrobial potency with the lowest MIC value of <1.562 mg/mL, in all the experimental strains of bacteria. Also, the ethanol extract of the root (RT-ETE) and methanol extract of the root (RT-MEE) show high inhibitory capacity of the bacteria. On the contrary, the MIC of water extract of the root (RT-WAE), water extract of the leaf (LF-WAE), and acetone extract of the leaf (LF-ACE) was low. e methanol extract of the leaf (LF-MEE) and ethanol extract of the leaf (LF-ETE) showed a moderate but an undulating MIC on the bacterial strains. e mean MIC value of R. crispus root extracts proved to have significant effects against bacterial strains, more than the leaf extracts, which aligns with previous research as reported by Mostafa et al. [33], on how extracts of the underground parts of many plants could be more active than the aerial parts.

Antifungal Activity of Plant Extracts.
Antifungal susceptibility testing remains less developed compared to antibacterial testing. However, it is an adoption of antibacterial testing. e knowledge of the mechanism of fungal resistance to drugs through the MIC method has been valuable in identifying and measuring resistance in in vitro systems [34]. In the study, the endpoint was read visually with a viewing mirror and the growth level (MIC) was recorded (Table 3). e highest level of MDR was recorded in    C. glabrata for all the extracts (>10 mg/mL) as recorded in Table 3. High resistance was also recorded in P. chrysogenum and C. albicans across the extracts and the positive control. It was established by Lass-Flörl et al. [34] that some fungi (mold) are considered resistant to some antifungal drugs (itraconazole) at MICs ≥8 μg/mL but susceptible to MICs of 0.125-0.5 μg/ml. e MICs of the extracts against tested fungal extracts showed varying concentrations as shown in Table 3. e overall MIC of leaf extracts showed a higher antifungal potency, contrary to the lesser MIC of leaf in the antibacterial test. is may be due to the slight differences in the phytochemical composition of the leaf and root of R. crispus.

Antiplasmodial Activity of R. crispus Extracts.
e solvent extracts of R. crispus were subjected to an in vitro screening of malaria-causing parasite (P. falciparum strain     chloroquine were evaluated: 13 μg/mL, 15 μg/mL, and 10 μM, respectively, as shown in Figures 6 and 7.

Antitrypanosomal Activity of R. crispus
Extracts. e potential of R. crispus extracts was tested on T.b. brucei parasite, causative agents of African sleeping sickness. e ethanol extract of the root of R. crispus (RT-ETE) has the highest potency among the extracts, acting against the parasite and reducing the viability to 43.27 ± 2.92% as shown in Figure 8. RT-ETE was thereafter put forward for further IC 50 screening, and the outcome was compared to the IC 50 of pentamidine (standard control): IC 50 : 9.7 μg/mL and IC 50 : 0.017 μM, respectively. e IC 50 was obtained from the doseresponse curve extrapolated by nonlinear regression using percentage viabilities plotted against logarithm (Figures 9  and 10).

Brine Shrimp Hatchability.
e percentage inhibition of the hatchability of the eggs of Artemia salina after 72 h exposure to the plant extracts and standard drug at optimum incubation conditions is shown in Figure 1. e entire tested extract composite shows some degree of inhibition of the rate of hatching of A. salina eggs compared to the control. e highest percentage of successfully hatched eggs into larvae in the least concentration used (0.0625 mg/mL) was observed in the extracts RT-ETE (57%), RT-WAE (58%), LF-ETE (54.67%), and LF-WAE (56.67%). is could be due to the polarity of the extraction solvents (ethanol and water), which brings about the fair inhibition of hatchability of the brine shrimp eggs. e hatching success of the cysts in RT-ACE has no significant difference, compared to the positive control (K 2 CrO 7 ). At the concentration 0.125 mg/mL of the tested samples, there was an overall more successful hatch of the cyst with the highest percentage in the extract of RT- MEE (70%), while the least was in RT-ACE (12.67%). At the highest concentration of 1 mg/mL, the toxicity of the extracts expressed their potency as postulated. e extracts of the root of R. crispus showed higher toxicity compared to the leaf extracts, and hence, the success of hatchability was very low (Figure 1). e overall percentage hatchability of A. salina cyst against the extracts is shown in Figure 2. e water extracts of the root (72.4%) and leaf (72.2%) are not significantly different; they have the highest hatchability and hence less toxic compared to other extracts. e acetone extract of the root (RT-ACE) has the lowest percentage hatchability, and it is not significantly different from the standard drug (K 2 CrO 7 ) with an inhibition percentage of 16.4% and 21.8%, respectively ( Figure 2).

Brine Shrimp Lethality.
e exposure of A. salina to toxins could have led to an elevation of glutathione S-transferase (sGST) activity in the phase II detoxication system of the larva. e depletion of glutathione levels and intracellular thiols, which act against oxidative stress in this organism, by these toxins leads to the death of the larva [39]. In most cases, the difference between toxins and nontoxic is dose or concentration. e lethal concentration (LC 50 and LC 99 ) of the extracts and the standard toxic drug was evaluated with probit regression and recorded as shown in Table 1. In the study, the level of toxicity of the extracts and the control in descending order is as follows:  Table 1). e overall percentage mortality of all concentrations of the extracts of R. crispus in 72 h, as shown in Figure 6, indicated that there was no significant difference between the toxicity of the extracts of RT-MEE (43%), RT-ACE (42.8%), RT-WAE (45.6%), and LF-MEE (45.8%). e highest toxicity among the extracts was RT-ETE (58.6%), while K 2 CrO 7 has the mortality of 89.2% as the control. e acetone extract of the leaf (LF-ACE) has the lowest mortality of 21.6% among all tested samples contrary to the acetone extract of the root (RT-ACE). e percentage sensitivity of the larva to extracts per each concentration used was tested and the effect of the various concentrations of the plant extracts used on the mortality of the larvae is shown in Figures 3 and 4. At the lowest concentration (0.0625 mg/mL) of the root extracts followed by 0.125 mg/mL, the toxicity of the extracts and the control drug was not fully expressed. e percentage lethality of all the extracts at these concentrations (0.0625 mg/ mL and 0.125 mg/mL) as shown in Figure 3 was low and less than 50% of the shrimp were knocked down. At the concentration 1 mg/mL, there is no significant difference (P < 0.05) between the toxicity of RT-MEE (67.3%), RT-ETE (72.6%), and K 2 CrO 7 (75.3%), which is highly toxic. However, the toxicity of the aqueous extract (RT-WAE) was mild at the mortality of 48% of nauplii ( Figure 11). In Figure 4, the concentrations of the leaf extracts used range from 0.25 to 4 mg/mL, which is slightly higher than the concentration used for the root extracts due to its less toxicity as confirmed by the preliminary lethal concentration test done.
ere is no significant difference between the mortality of shrimp larva in the concentrations 0.25-2 mg/ mL of the leaf extracts of R. crispus. e toxicity of the leaf extract was fully expressed at the concentration of 4 mg/mL. LF-MEE has the highest percentage mortality of 50%, followed by LF-WAE (42.6%) while the LF-ACE has the lowest mortality of 12%. e toxicity of LF-ACE is the least compared to other extracts of the leaf, contrary to the toxicity of RT-ACE, which has the highest toxicity in the root extract (Figures 3 and 4).

Cytotoxicity Activity.
e extracts were tested against HeLa (human cervix adenocarcinoma) cells at a concentration of 50 μg/mL. e result ( Figure 12) revealed all extracts are not cytotoxic except RT-ETE (46.55 ± 4.84%) and RT-ACE (37.31 ± 5.69%) which reduce the viability of HeLa cells to below 50%.

Discussion
e potential of phytomedicine for developing antimicrobial and antiparasitic drugs seems rewarding because most plants that have been tested against microbes and parasites show enormous therapeutic outcome with presumed fewer side effects but these potentials are yet untapped [40,41]. e genus Rumex has been reported and verified scientifically to be effective against a broad range of microorganisms [28,29,41,42] and parasites [43]. e verification of the antimicrobial and antiparasitic potential of R. crispus extracts in this research shows that it could be a potential source for new antibacterial, antifungal, antitrypanosomal, and antiplasmodial drugs.
is study revealed extracts of Rumex crispus were effective against bacterial growth at different levels of concentrations and there were significant positive correlations between extract concentration and antimicrobial activity through the test for the MIC of bacteria ( Table 2) (Table 3). In this research, the acetone extract of the root shows a tremendous inhibition power against bacterial growth at the concentration of <1.562 mg/mL compared to other extracts. However, the overall antibacterial potency of the extracts of the root was significantly higher than the extracts of the leaf, which support the previous research done by Orbán-Gyapai et al. [29]. Contrary to the antibacterial potency of the leaf extracts of R. crispus, the overall MIC of the extracts shows a higher antifungal potency compared to the root extracts. Furthermore, specific in vivo studies are recommended to determine the efficacy of these extracts in the treatment of antimicrobial infections. e antiplasmodial activity of R. crispus extracts was tested and compared with the standard antimalarial drug (chloroquine). Generally, a compound is considered inactive against Plasmodium when it shows an IC 50 > 200 μM, low activity when IC 50 is 100-200 μM, moderately active when IC 50 is 20-100 μM, good activity when IC 50 is 1-20 μM, and excellent or potent antiplasmodial activity when IC 50 is <1 μM [44]. For natural products or extracts in which the molar mass is difficult to determine, they are categorized as inactive, moderate, good, and excellent antiplasmodials if the IC 50 values were >5 μg/mL, 1-5 μg/mL, 0.1-1 μg/mL, and <0.1 μg/mL, respectively [45]. RT-ACE and LF-MEE have the highest potency against the Plasmodium parasite with an IC 50 of 13 μg/mL and 15 μg/mL, respectively. However, these two extracts brought about reduction in plasmodium lactate dehydrogenase (pLDH) activity to less than 50% as shown in Figure 1.
e antitrypanosomal activity of the extracts of R. crispus was examined using Trypanosoma brucei assay. It was found that only RT-ETE reduced the viability of T.b. brucei below 50%; hence, RT-ETE was put forward for IC 50 screening and the IC 50 value was found to be 9.7 ug/mL. Larayetan et al. [8] confirmed the IC 50 value of compounds on Trypanosoma ≤20 μg/mL are considered good, IC 50 between 20 and 60 μg/ mL are considered moderate, while IC 50 > 100 μg/mL is termed not active. e test for toxicity using brine shrimp assay is a viable tool for preliminary evaluation of the toxicity of samples, especially plant extracts [46,47]. e toxicity of R. crispus was evaluated using hatchability of cysts and lethality of the larvae of Artemia salina with varying concentrations. In Table 1 and Figures 2-4, 10, shows the lethal concentration (LC) and mean toxicity of the root of R. crispus are almost twice as that of the leaf. is could be due to the active phytochemical content of the root which was confirmed to be roughly more than in the leaf [5]. e aqueous extracts also show a mean value of lesser toxicity compared to other solvent extracts except for aqueous extracts of the root that show slightly higher toxicity; hence, the aqueous extracts are presumed to be less toxic when compared to other solvent extracts.
Bastos et al. [48] interpreted the toxicity of brine shrimp in accordance with Meyer toxicity index that LC 50 values > 1 mg/mL are considered nontoxic and LC 50 values ≥ 0.5 mg/mL but LC 50 values <1 mg/mL are considered mild toxic, while LC 50 values< 0.5 mg/mL are considered highly toxic [48]. Nevertheless, it was found in this study that RT- ETE has the lowest LC 50 value (LC 50 0.275 mg/mL) followed by the LC 50 value of RT-ACE (LC 50 0.363 mg/mL). e response of the larva to LF-ETE (LC 50 4.658 mg/mL), LF-MEE (LC 50 2.734 mg/mL), LF-WAE (LC 50 2.990 mg/mL), and LF-ACE (LC 50 1.824 mg/mL) thus reveal the extracts are nontoxic, according to Bastos et al. [48].
To further ascertain the toxicity level of R. crispus extracts, cytotoxicity of the extracts was conducted using HeLa (human cervix adenocarcinoma) cells at a concentration of 50 μg/mL. e study shows that RT-MEE, RT-WAE, LF-MEE, LF-ETE, LF-ACE, and LF-WAE did not reduce the percentage viability of HeLa cells below 50%; hence, samples did not cause significant cytotoxic effects, which denote their safety when used as drugs. On the contrary, RT-ETE and RT-ACE reveal some degree of toxicity and reduce the viability of HeLa cells to 46.55 ± 4.84% and 37.31 ± 5.69%, respectively ( Figure 12). However, it is worthy of note that there is a correlation between the outcome of brine shrimp lethality assay (BSLA) and cytotoxicity assay using HeLa cells; both assays revealed that RT-ETE and RT-ACE are most toxic among the extracts.

Conclusion
Based on the results of the research, the extracts of R. crispus have therapeutic potentials against some microbes (fungal and bacteria) and parasites (Trypanosoma and Plasmodium). It was confirmed that the antimicrobial activity of the extracts does not dependent on Gram-negative or Grampositive bacteria and hypha or single-celled-form fungi. e level of toxicity and potency of the extracts correlate with the inhibition of the viability of HeLa cells and Artemia salina. e results, therefore, further confirms the use of Rumex crispus by traditional healers and recognized the potential of medicinal plants as the prime source of novel compounds in the search of new treatment for African sleeping sickness, malaria, and microbial infections.