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Article

Phytochemical Analysis and Amoebicidal Evaluation of Different Agave Species

by
Ana Laura Rodríguez-Zapata
1,
Jorge Ivan Mora-Frias
1,
Miguel Angel Briano-Elias
2,
Armando Pérez-Centeno
3,
Lucia Barrientos-Ramírez
2,
Ramón Reynoso-Orozco
4,
Mario Nava-Villalba
1 and
Araceli Castillo-Romero
1,*
1
Departamento de Microbiología y Patología, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Calle Sierra Mojada 950, Independencia Oriente, Guadalajara 44340, Jalisco, Mexico
2
Departamento de Madera, Celulosa y Papel, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de Guadalajara, Cam. Ramón Padilla Sánchez 440, Las Agujas 45221, Jalisco, Mexico
3
Departamento de Física, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de Guadalajara, Blvd. Marcelino García Barragán 1421, esq. Calzada Olímpica, Guadalajara 44430, Jalisco, Mexico
4
Departamento de Biología Celular y Molecular, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Camino Ramón Padilla Sánchez No. 2100 Nextipac, Zapopan 45200, Jalisco, Mexico
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(5), 1905; https://doi.org/10.3390/app14051905
Submission received: 30 January 2024 / Revised: 21 February 2024 / Accepted: 23 February 2024 / Published: 26 February 2024
(This article belongs to the Special Issue Novel Interventions for Control of Human Pathogens)
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Abstract

:
Amoebiasis, a disease caused by the protozoan Entamoeba histolytica, represents a serious public health problem, mainly in developing countries. The first line of therapy for amoebiasis treatment is metronidazole (MTZ); however, clinical isolates of E. histolytica with resistance to MTZ and varying sensitivity to other antiamoebic drugs threaten the effectiveness of the prevention and treatment of this parasitic infection. Natural products stand out as a promising strategy to develop new, safe and more effective alternatives. In this study, we determined and compared the phytochemical profiles of Agave tequilana, Agave angustifolia, Agave rhodacantha, and Agave maximiliana and described their cytotoxic effect on E. histolytica trophozoites. The results show that the four Agaves kill E. histolytica in a species–time–dose-dependent manner. A morphologic analysis of the treated parasites showed evident morphological alterations suggestive of programmed cell death with nuclear alterations; it also highlighted the presence of rounded cells with protuberances/perforations in the membrane and cells that appeared to have exploded. The overall activity of Agave ethanolic extracts in E. histolytica can help provide new strategies to advance alternative treatments against amoebiasis.

1. Introduction

Human amoebiasis represents a severe public health problem worldwide; millions of people are infected, resulting in over 100,000 deaths yearly. Entamoeba histolytica, the causative agent of amoebiasis, is associated most commonly with intestinal infections but can also present extraintestinal manifestations leading to an amoebic liver abscess; in rare cases, it affects the lungs, heart, and brain [1]. Immunocompromised persons may develop the most severe symptoms and higher case fatality rates due to invasive amoebiasis. In areas with poor sanitation and high-risk hygiene behaviors, its prevalence remains as high as 40% [2]. In Mexico, this parasitosis is classified as one of the main causes of diarrheal disease, with high prevalence and mortality rates; incidences over 70% have been related to geographical, socioeconomic, and environmental factors [3]. There are multiple pharmacological treatments to control amoebiasis which are classified as luminal, systemic, or mixed amoebicides; nitroimidazoles are first-line drugs, in particular, metronidazole (MTZ) is the most widely used standard therapy for invasive amoebiasis. However, the indiscriminate use of amoebicidal compounds has stimulated an increasing presence of strains resistant to these antibiotics and significantly high therapeutic failure rates [4,5,6]. Currently, plants and natural products continue to be important sources of novel bioactive compounds [7,8,9,10]. Several studies have identified, quantified, and classified bioactive compounds, such as flavonoids, saponins, terpenes, and steroids, with potential uses as antioxidant, anti-inflammatory, and antimicrobial compounds [11,12,13,14,15,16]. The genus Agave consists of approximately 200 species; 150 are in Mexico, with 119 endemics [17,18,19]. This plant has been used principally to produce distilled alcoholic beverages, representing an economic benefit of billions of dollars yearly [20,21,22]. However, during production, a considerable amount of waste is generated, including the plant’s spiky leaves [21,22,23]. Due to the presence of secondary metabolites such as phenols, flavonoids, phytosterols, and saponins in Agave crude leaf extracts with well-known biological activities, they have been associated with several benefits for human health, curing and preventing many diseases including infectious diseases and cancer [24,25,26,27,28,29,30,31]. In particular, as antiparasitics, extracts of Agave americana have been reported to exhibit significant leishmanicidal activities; 0.05 mg/mL was enough to eliminate promastigotes and axenic amastigotes after 24 h of treatment [32]. In addition, other studies reported by Botura et al. (2011, 2013) proved that an extract from Agave sisalana has effective activity against the eggs, larvae, and adult worms of gastrointestinal nematodes in in vitro assays [33,34]. Guerra JO et al. (2008) reported cytostatic and growth inhibition activity in steroidal saponins from the plant Agave brittoniana against Trichomonas vaginalis [35]. Other species, such as Agave tequilana and Agave angustifolia, have been highlighted principally for their therapeutic benefits as antibacterial compounds [36,37]. Even though there have been entirely positive results in biological assays, Agave extracts have not been widely evaluated against the most relevant intestinal protozoa. A first report by Quintanilla-Licea et al. (2020) showed 69% growth inhibition against Entamoeba histolytica trophozoites by extracts of Agave lechugilla Torr [38]. In this study, we determined and compared the phytochemical profiles of Agave tequilana, Agave angustifolia, Agave rhodacantha, and Agave maximiliana and their cytotoxic effect on E. histolytica trophozoites. Our investigation supports that Agave crude extract compounds are good candidates for the discovery of the untapped potential of the agro-wastes of the tequila industry in the development of novel amoebicidal alternatives.

2. Materials and Methods

2.1. Harvesting of the Raw Material and Ethanolic Extraction

Leaves of Agave tequilana Weber, Agave angustifolia Haw, Agave rhodacantha Trel, and Agave maximiliana Baker were collected during the period of October to November 2020 at the “Gorupa” property, from 4.5 Km S of Chiquilistlán, and 0.5 km from the “El Agostadero” crossroads, respectively. The collected samples were washed, and once dry, a 60 g sample of dried leaves was cut, placed in flasks containing 600 mL of ethanol, and incubated at 150 rpm for 48 h at room temperature (TA). Subsequently, the extracts were filtered through Whatman paper no.2 to remove fibers. The collected ethanolic extracts were concentrated under vacuum in a rotary evaporator (R-300, BÜCHI Labortechnik AG, Swiss) at 40 °C to yield a 100 mL volume. The samples were frozen for 48 h at −20 °C. The samples were subsequently dried by freeze-drying for 48 h (−36 °C, Scientz-10N Freeze Dryer, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China). The powders collected were stored at 4 °C until use.

2.2. UV–Vis

Absorbance spectra were obtained using a UV–Vis spectrophotometer (Genesys 10UV, Thermo Fisher Scientific, Waltham, MA, USA), scanning wavelengths from 300 to 800 nm with a 1 nm step. For the measurements, 3 mL of each Agave extract was poured into a quartz cell. The acquired data were analyzed using OriginLab software (version OriginPro 2023b).

2.3. Phytochemical Screening

Standard extraction and screening procedures were used to determine the phytochemical components of A. tequilana, A. maximiliana, A. angustifolia, and A. rhodacantha.

2.3.1. Test for Flavonoids

For the identification of flavonoids, the Shinoda test [39,40] was used, in which 1–2 mg of crude extract was dissolved in 1 mL of ethanol in a test tube and a few drops of concentrated hydrochloric acid and two magnesium filings were added; if the solution turned bright red, the test was positive.

2.3.2. Test for Sugars

For the identification of sugars, the Anthrone test [41] was used. This technique consisted of placing 1 mL of the sample in a test tube before dissolving the sample in water. Subsequently, 1–2 mg of Anthrone reagent and about 4 drops of sulfuric acid were added. The test is positive when a blue-green ring appears at the interface.

2.3.3. Test for Sterols and Triterpenes

For the identification of sterols and triterpenes, the Lieberman–Burchard test [39] was used; the reagent was prepared by mixing 1 mL of acetic anhydride, 1 mL of chloroform, and five drops of sulfuric acid. A couple of drops of the reagent were added to l–2 mg of the sample dissolved in chloroform. The appearance of any coloration within an hour indicates a positive test for steroids and triterpenes, particularly when they have a high degree of unsaturation.

2.3.4. Test for Saponins

For the identification of saponins, 2 mg of sample was dissolved with l.5 mL of water and stirred manually for three minutes to observe the formation of foam [42,43].
The overall results were expressed by cross-testing, with (−) negative, (+) weak positive, (++) moderate positive, and (+++) strong positive results.

2.4. Culture and Maintenance of E. histolytica

The trophozoites of E. histolytica (HM1-IMSS strain) were grown axenically in TYI-S-33 medium at a pH of 6.8 supplemented with 15% bovine serum, penicillin, and streptomycin 1% (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C [44]. The culture tubes were monitored via microscopic examination, and subcultures were made twice weekly.

2.5. In Vitro Susceptibility Assay

To evaluate the effect of the ethanolic agave extracts on the growth of E. histolytica, 15,000 trophozoites/mL were grown at 37 °C for 24, 48, and 72 h in the presence of 0, 100, 300, or 600 μg/mL of A. tequilana, A. maximiliana, A. angustifolia, or A. rhodacantha. Untreated cells and the extracts’ diluent, 0.4% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), were used as negative controls, and 1.4 μg/mL of MTZ (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control. After the incubation periods, trophozoites were collected by chilling the cell culture tubes on ice for 20 min, and they were counted in a Neubauer chamber. The percentage of parasite growth inhibition was determined in relation to the DMSO control, which was considered to represent 100% parasite growth.

2.6. Cell Viability Assay

To determine the effect of the Agave extracts on the viability of trophozoites, an exclusion staining method with trypan blue was used. From cultures exposed to DMSO, 0, 100, 300, or 600 μg/mL of A. tequilana, A. angustifolia, A. rhodacantha, and A. maximiliana, a volume of 10 μL of culture was mixed with 10 μL of trypan blue (0.4% GibcoBRL, Grant Island, FL, USA) for 24 and 48 h. The total number of parasites, including those in samples which excluded the dye, was counted in a Neubauer chamber, and cell viability was expressed as the percentage of viable cells: [100 × (living cells)/(dead cells + living cells)].

2.7. Bright-Field Microscopy

For a microscopy analysis, Agave- or DMSO-treated trophozoites were washed and adhered to poly-L-lysine pretreated coverslips. Then, the adhered parasites were fixed with 4% paraformaldehyde for 30 min. Finally, the coverslips were washed in phosphate-buffered saline (PBS) and mounted on glass slides using ProLong Gold mounting medium containing DAPI (ProLong Gold, Thermo Fisher Scientific, USA). The cells were analyzed under an Eclipse Ts2 microscope (Nikon Instruments Inc., Melville, NY, USA)

2.8. Statistical Analysis

All the results obtained were the average of three independent experiments, each in triplicate. All data are expressed as mean ± standard deviation values. Data were statistically analyzed using a one-way ANOVA (GraphPad Prism version 6.01 for Windows, GraphPad Software, La Jolla, CA, USA), and p values ≤ 0.05 were considered significantly different.

3. Results and Discussion

3.1. Ethanolic Extracts of Agave tequilana, Agave angustifolia, Agave rhodacantha, and Agave maximiliana UV–Vis Analysis and Phytochemical Screening

Several authors have proved that during phytochemical extraction, solvents have a significant impact on the level of polyphenols extracted [45]. In general, tannins, polyphenols, polyacetylenes, flavonols, terpenoids, sterols, and alkaloids can be readily extracted with ethanol [46,47,48]. In this study, a UV–Vis spectroscopy analysis revealed six weak and strong, intense bands ranging in wavelength from 400 to 700 nm (Figure 1); as reported by other authors, these bands reveal the presence of alkaloids, flavonoids, terpenes, steroids, saponins, tannins, and coumarins [45,48,49,50,51,52]. The profile showed the peaks at 409–414 nm, 499–506 nm, 535–539 nm, 558–561 nm, 607–614 nm, and 666 nm. The higher absorbance values were obtained from A. tequilana, followed by A. angustifolia (Table 1). The fact that the Agave species had absorbance peaks of unequal intensity is not surprising to us since the phytochemical constituents of plants vary among species; also, the concentration depends on the metabolite type, the characteristics of the plant, the tissue, the stage of development, and climatic factors [53,54,55,56,57]. The phytochemical screening of the Agave extracts confirmed the presence of flavonoids, terpenes, steroids, saponins, tannins, and coumarins, the contents of which varied considerably among the Agave species. The concentration of terpenes was high (+++) in the four species analyzed, while tannins were moderately (++) present. Only A. angustifolia and A. rhodacanta presented high concentrations of flavonoids (+++). A. maximiliana was found to be the species with the lowest concentrations of flavonoids, steroids, and coumarins (Table 2).

3.2. Ethanolic Extracts of Agave Leaves Inhibit the Growth of E. histolytica Trophozoites

The in vitro amoebicidal activity of ethanolic extracts of the species Agave tequilana, Agave angustifolia, Agave rhodacantha, and Agave maximiliana was evaluated. Our results showed that all the Agave species studied in the present investigation showed promising amoebicidal activity (Figure 2). The negative control (DMSO 0.4%) did not exhibit any significant differences compared with untreated cells. Remarkably, the highest amoebicidal activity was observed with A. tequilana; at 72 h, with concentrations of 300 and 600 µg/mL, a more than 90% decrease in the trophozoite count was observed compared to the negative controls (Figure 2D), with IC50 = 193 µg/mL (Table 3). With A. angustifolia, a growth inhibitory effect of more than 60% was seen only at 600 µg/mL during the entire exposure period (Figure 2F–H), with IC50 = 364 µg/mL (Table 3). The extracts with moderate activity were A. rodhacantha and A. maximiliana (Figure 2I–P); large and undefined IC50 values were set at 1220 and 1824 µg/mL, respectively, for purposes of illustration (Table 3). Our results correspond with those reported by Quintanilla-Licea et al. (2014), who showed the amoebicidal activity of A. lechuguilla [38]; however, our results showed that for E. histolytica, higher doses are necessary to achieve a reduction of more than 90%. In addition, we confirmed that other species of the Agave genus present varying degrees of amoebicidal activity. The phytochemical analysis (Table 2), showing differences in the presence and relative abundance of some metabolites, could explain the different efficacies of the Agave extracts when killing E. histolytica. In addition, in this research study, there was a positive correlation between the higher absorbance values of A. tequilana and A. angustifolia (Figure 1 and Table 1) and the higher amoebicidal properties observed for these species. Chromatographic separation, chromatography–mass spectrometry, and a UV–Vis analysis confirmed the abundant presence of quercetin, kaempferol, gallic acid, β-sitosterols, and steroidal saponins in A. tequilana and A. angustifolia [36,58,59,60,61]. This variety of compounds has been associated with numerous medicinal uses and biological activities. In E. histolytica, studies by Moises Martines-Castillo et al. (2018) demonstrated the amoebicidal activity of epicatechin, kaempferol, and quercetin [62]. Similar amoebicidal activity has been reported by Arrieta et al. (2001) for β-sitosterol and β-sitosterol glucoside phenolic [63]. In the latter work, the authors described that pure compounds were less active than complete plant extracts, suggesting an important synergist activity with the presence of several metabolites. On the other hand, MTZ is considered the drug of choice for treating amoebiasis; in this study, at 24 h, 600 µg/mL of A. angustifolia showed greater efficacy than MTZ (Figure 3A). After 48 h, A. tequilana and A. angustifolia were demonstrated to be more effective than MTZ (Figure 3B,C). Even though Agave extracts showed higher amoebicidal activity in comparison to MTZ, the plants use natural toxins as a defense mechanism. Additional studies are necessary to establish the toxicity or adverse health effects of Agave extracts for their use as amoebicidal alternatives.

3.3. Agave Extracts Reduced the Cell Viability of E. histolytica Trophozoites

The percentage of viable parasites was measured after 48 h of incubation with Agave extracts using the trypan blue exclusion test. Interestingly, the extracts that showed less amoebicidal activity turned out to be from the species with the highest presence of blue-stained cells (nonviable cells) (Figure 4I). With A. maximiliana, at 24 h, only 73%, 61%, and 52% were viable at 100, 300, and 600 µg/mL, respectively (Figure 4E). A. maximiliana was the species with the highest concentration of saponins (Table 2). Saponins have properties that facilitate the formation of insoluble complexes with components of the cell membrane (steroids, proteins, and phospholipids, especially cholesterol), which contributes to the permeability of the cell membrane, leading to the formation of pores, leading to a massive release of cytoplasmic content and the formation of apoptotic bodies, which eventually induces the death of the parasite [64,65]. In other Agave extracts, it has been reported that saponins from A. lophantha and A. brittoniana at concentrations of 500, 100, and 10 µg/mL decreased the viability of T. vaginalis, G. lamblia, and E. histolytica [35,66]. The DMSO-treated cells did not exhibit any significant difference compared with the untreated cells.

3.4. Agave Extracts Induce Morphological Alterations in E. histolytica Trophozoites

The effect of the Agave extracts on trophozoite morphology was evaluated using bright-field microscopy. All concentrations of the Agave extracts stimulated a wide variety of morphological alterations compared to the control trophozoites. No differences were found in the morphology of untreated and DMSO-treated trophozoites; the cells showed a distinctive pleomorphic form and the presence of pseudopods (Figure 5A,B). After Agave exposure, there was a prevalence of detached trophozoites with a rounded form; there were parasites with apparent protuberances in the membrane, and cell lysis and DAPI staining revealed apparent changes in the diameter of nuclei (Figure 5). With A. tequilana and A. angustifolia, more than 60% of cells showed damage; ruptured trophozoites and cell debris were observed (Figure 5C,D). With A. maximilana, 80% of the parasite population presented a granular surface, and an accumulation of filopodial protrusions prevailed (Figure 4G,H). Phytochemical screening indicated a greater presence of flavonoids and steroids in A. tequilana, A. angustifolia, and A. rhodacantha (Table 2), which could be responsible for the drastic morphological alterations observed due to these extracts. Several studies support the amoebicidal activity of flavonoids, and (−)-epicatechin and kaempferol have been related to growth inhibition, decreased cell viability, and nuclear and morphological alterations in E. histolytica trophozoites. Damage to cytoskeletal structures with changes in essential mechanisms such as adhesion, cytolysis, and nuclear alterations followed by programmed cell death are the principal findings [67,68]. In addition, it has been demonstrated that the motility of E. histolytica depends on the sustained instability of the intracellular hydrostatic pressure that drives the cyclic generation and healing of membrane blebs [69]. Furthermore, actin filaments constitute the physical backbone of membrane protrusions [70]. The prevalence of the filopodial protrusions that A. maximiliana caused in trophozoites could be related to alterations in intracellular hydrostatic pressure and the actin cytoskeleton. Our results suggest that the trophozoites’ deaths occurred principally due to a loss of membrane integrity, changes within the nucleus, and cytoskeletal alterations; biochemical studies are necessary to obtain evidence of programmed cell death in Entamoeba histolytica due to Agave extracts.

4. Conclusions

The results obtained in this study demonstrate that the ethanolic leaf extracts of Agave tequilana, Agave angustifolia, Agave rhodacantha, and Agave maximiliana present different efficacies of amoebicidal activity. This is the first study focused on identifying structural damage in E. histolytica due to Agave extracts. The result of this study supports the traditional claim that the plants are antiparasitic alternatives, and efforts are currently under way to identify the bioactive component(s) of the Agave species and their cytotoxicity and selectivity.

Author Contributions

Conceptualization, R.R.-O. and A.C.-R.; data curation, A.L.R.-Z., L.B.-R. and M.N.-V.; formal analysis, A.C.-R.; funding acquisition, A.C.-R.; investigation, A.L.R.-Z., J.I.M.-F., M.A.B.-E., A.P.-C. and A.C.-R.; methodology, A.L.R.-Z., J.I.M.-F., M.A.B.-E., A.P.-C., L.B.-R. and A.C.-R.; project administration, A.C.-R.; resources, L.B.-R. and A.C.-R.; software, A.L.R.-Z. and A.P.-C.; supervision, A.C.-R.; validation, A.P.-C., L.B.-R., R.R.-O., M.N.-V. and A.C.-R.; visualization, A.L.R.-Z.; writing—original draft, A.L.R.-Z., L.B.-R. and A.C.-R.; writing—review and editing, A.P.-C., L.B.-R., R.R.-O., M.N.-V. and A.C.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by Consejo Estatal de Ciencia y Tecnología de Jalisco (Coecytjal, Fodecijal 2019-8129).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

Ana Laura Rodríguez-Zapata thanks CONACYT for the undergraduate scholarship (761449) received. Miguel Angel Briano-Elias thanks COECYTJAL for the scholarship received.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 01905 g001
Figure 1. UV–Vis absorbance spectra analysis of the ethanolic extracts of A. angustifolia, A. maximiliana, A. rhodacantha, and A. tequilana from 300 to 800 nm.
Figure 1. UV–Vis absorbance spectra analysis of the ethanolic extracts of A. angustifolia, A. maximiliana, A. rhodacantha, and A. tequilana from 300 to 800 nm.
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Figure 2. Extracts of Agave inhibit E. histolytica growth. Growth inhibition curves of A. tequilana (A), A. angustifolia (E), A. rhodacantha (I), and A. maximiliana (M). Percentages of inhibition at 24 h of A. tequilana (B), A. angustifolia (F), A. rhodacantha (J), and A. maximiliana (N). Percentages of inhibition at 48 h of A. tequilana (C), A. angustifolia (G), A. rhodacantha (K), and A. maximiliana (O). Percentages of inhibition at 72 h of A. tequilana (D), A. angustifolia (H), A. rhodacantha (L), and A. maximiliana (P). The boxes represent the 25–75th percentiles, and the medians are indicated. The whiskers show the range. Data are representative of at least three independent experiments and represented as mean ± SEM values. * p ≤ 0.05, *** p ≤ 0.0005, and **** p ≤ 0.0001 compared to the control group (DMSO-treated cells).
Figure 2. Extracts of Agave inhibit E. histolytica growth. Growth inhibition curves of A. tequilana (A), A. angustifolia (E), A. rhodacantha (I), and A. maximiliana (M). Percentages of inhibition at 24 h of A. tequilana (B), A. angustifolia (F), A. rhodacantha (J), and A. maximiliana (N). Percentages of inhibition at 48 h of A. tequilana (C), A. angustifolia (G), A. rhodacantha (K), and A. maximiliana (O). Percentages of inhibition at 72 h of A. tequilana (D), A. angustifolia (H), A. rhodacantha (L), and A. maximiliana (P). The boxes represent the 25–75th percentiles, and the medians are indicated. The whiskers show the range. Data are representative of at least three independent experiments and represented as mean ± SEM values. * p ≤ 0.05, *** p ≤ 0.0005, and **** p ≤ 0.0001 compared to the control group (DMSO-treated cells).
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Figure 3. Comparison of growth inhibition rates of Agave extracts (600 µg/mL) and MTZ (1.4 µg/mL) at 24 h (A), 48 h (B), and 72 h (C). The boxes represent the 25–75th percentiles, and the medians are indicated. The whiskers show the range. Error bars represent the standard deviation (n = 3 experiments performed in triplicate; * p ≤ 0.05, ** p ≤ 0.005, and **** p ≤ 0.0001).
Figure 3. Comparison of growth inhibition rates of Agave extracts (600 µg/mL) and MTZ (1.4 µg/mL) at 24 h (A), 48 h (B), and 72 h (C). The boxes represent the 25–75th percentiles, and the medians are indicated. The whiskers show the range. Error bars represent the standard deviation (n = 3 experiments performed in triplicate; * p ≤ 0.05, ** p ≤ 0.005, and **** p ≤ 0.0001).
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Figure 4. Percentage inhibition of viability due to the effect of Agave ethanolic extracts in E. histolytica. A. angustifolia at 24 h (A) and 48 h (B), A. rhodacantha at 24 h (C) and 48 h (D), A. maximiliana at 24 h (E) and 48 h (F), and A. Tequilana at 24 h (G) and 48 h (H). Inverted optical microscope (Nikon Eclipse Ts2) pictures of trypan-blue-stained trophozoites after DMSO or Agave extract treatment (600 µg/mL, 48 h) (I). The boxes represent the 25–75th percentiles, and the medians are indicated. The whiskers show the range. Results were obtained from three experiments independently using the triplicate SD. **** p ≤ 0.0001. Bar = 100 µm.
Figure 4. Percentage inhibition of viability due to the effect of Agave ethanolic extracts in E. histolytica. A. angustifolia at 24 h (A) and 48 h (B), A. rhodacantha at 24 h (C) and 48 h (D), A. maximiliana at 24 h (E) and 48 h (F), and A. Tequilana at 24 h (G) and 48 h (H). Inverted optical microscope (Nikon Eclipse Ts2) pictures of trypan-blue-stained trophozoites after DMSO or Agave extract treatment (600 µg/mL, 48 h) (I). The boxes represent the 25–75th percentiles, and the medians are indicated. The whiskers show the range. Results were obtained from three experiments independently using the triplicate SD. **** p ≤ 0.0001. Bar = 100 µm.
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Figure 5. Inverted optical microscope (Nikon Eclipse Ts2) pictures of trophozoites treated with ethanolic extracts of Agave. Untreated trophozoites (A) and those treated with 0.4% DMSO (B). A. Tequilana (C), A. angustifolia (D,E), A. rhodachanta (F), and A. maximiliana (G,H). White arrows indicate alterations in nuclei. Black arrows indicate filopodial protrusions. Bar = 50 µm.
Figure 5. Inverted optical microscope (Nikon Eclipse Ts2) pictures of trophozoites treated with ethanolic extracts of Agave. Untreated trophozoites (A) and those treated with 0.4% DMSO (B). A. Tequilana (C), A. angustifolia (D,E), A. rhodachanta (F), and A. maximiliana (G,H). White arrows indicate alterations in nuclei. Black arrows indicate filopodial protrusions. Bar = 50 µm.
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Table 1. UV–Vis absorbance peak intensity values of the ethanolic extracts of A. angustifolia, A. maximiliana, A. rhodacantha, and A. tequilana..
Table 1. UV–Vis absorbance peak intensity values of the ethanolic extracts of A. angustifolia, A. maximiliana, A. rhodacantha, and A. tequilana..
Ethanolic Extracts
A. angustifoliaPeak123456
Wavelength (nm)666610560535505409
Absorbance
(arb. u) 		0.2300.0440.0260.0430.0640.575
A. maximilianaPeak123456
Wavelength (nm)666611558537503412
Absorbance
(arb. u) 		0.0900.0240.0180.0200.0440.185
A. rhodacanthaPeak123456
Wavelength (nm)666614561539499414
Absorbance
(arb. u) 		0.0890.0160.0070.0150.0300.141
A. tequilanaPeak123456
Wavelength (nm)666607561537506409
Absorbance
(arb. u) 		0.3780.1120.0790.1050.1190.930
Table 2. Screening of ethanolic Agave extracts.
Table 2. Screening of ethanolic Agave extracts.
PhytochemicalA. tequilanaA. angustifoliaA. rhodachantaA. maximiliana
Flavonoids++++++++++
Terpenes++++++++++++
Steroids+++++++++++
Saponins+++++++++
Tannins++++++++
Coumarins+++++++++
++ Moderate concentration; +++ High concentration.
Table 3. IC50 value of Agave extracts on E. histolytica growth after 72 h of incubation.
Table 3. IC50 value of Agave extracts on E. histolytica growth after 72 h of incubation.
Ethanolic ExtractIC50 µg/mL *
A. tequilana193.47 ± 22.16
A. angustifolia363.54 ± 10.41
A. maximiliana1219.93 ± 18.05
A. rhodanactha1824.10 ± 7.7772
* Data are expressed as mean concentration ± SD (n = 3) values.
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Rodríguez-Zapata, A.L.; Mora-Frias, J.I.; Briano-Elias, M.A.; Pérez-Centeno, A.; Barrientos-Ramírez, L.; Reynoso-Orozco, R.; Nava-Villalba, M.; Castillo-Romero, A. Phytochemical Analysis and Amoebicidal Evaluation of Different Agave Species. Appl. Sci. 2024, 14, 1905. https://doi.org/10.3390/app14051905

AMA Style

Rodríguez-Zapata AL, Mora-Frias JI, Briano-Elias MA, Pérez-Centeno A, Barrientos-Ramírez L, Reynoso-Orozco R, Nava-Villalba M, Castillo-Romero A. Phytochemical Analysis and Amoebicidal Evaluation of Different Agave Species. Applied Sciences. 2024; 14(5):1905. https://doi.org/10.3390/app14051905

Chicago/Turabian Style

Rodríguez-Zapata, Ana Laura, Jorge Ivan Mora-Frias, Miguel Angel Briano-Elias, Armando Pérez-Centeno, Lucia Barrientos-Ramírez, Ramón Reynoso-Orozco, Mario Nava-Villalba, and Araceli Castillo-Romero. 2024. "Phytochemical Analysis and Amoebicidal Evaluation of Different Agave Species" Applied Sciences 14, no. 5: 1905. https://doi.org/10.3390/app14051905

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