Phytotoxic and Cytotoxic Effects, Antioxidant Potentials, and Phytochemical Constituents of Stevia rebaudiana Leaves

Stevia (Stevia rebaudiana), recognized for its low-calorie, sugar-free attributes, and various health benefits, has potential applications beyond human consumption, particularly in agriculture. This study explored the potential uses of Stevia in both agricultural and healthcare contexts by examining its plant-inhibitory, cytotoxic, and antioxidant effects. The methanolic extract of Stevia leaves was fractionated into hexane, ethyl acetate, chloroform, and water fractions. These fractions were then subjected to the bioassay analyses above and underwent identification of their chemical constituents. The results indicated that the ethyl acetate fraction demonstrated significant inhibitory effects on weed germination and growth of Beggars tick (Bidens frondosa) (100% inhibition at 1000 ppm of dose). This fraction also exhibited the highest antioxidant activity, total phenolic, and total flavonoid contents (IC50 DPPH = 18.67 μg/mL, 103.50 mg GAE/g fraction, and 410.16 mg QE/g fraction, respectively). In contrast, the chloroform fraction showed the highest cytotoxic effect (LC50 = 700.01 ppm) in the brine shrimp (Artemia salina) mortality evaluation. Pearson's correlation analysis revealed a positive correlation among plant inhibitory effects, antioxidant potentials, and phenolic/flavonoid contents of Stevia. FTIR spectra confirmed the presence of phenols and nonpolar components in the ethyl acetate and chloroform fractions. In addition, GC-MS analysis successfully identified Stevia's key constituents, including tetracontane, hexadecane, hexadecanoic acid, methyl ester in the ethyl acetate fraction, and spiro [4.5] decan-7-one and 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one in the chloroform fraction. This study underscores the potential of S. rebaudiana as a source of natural antioxidants and herbicides, offering valuable insights into its diverse applications in agriculture.


Introduction
Stevia, scientifcally known as (Stevia rebaudiana Bertoni), is an annual plant native to Latin America and is classifed under the Asteraceae family.Tis plant is popular as a low-sugar calorie source.Te leaf of this plant contains various steviol glycosides, such as isosteviol, steviolbioside, stevioside, and rebaudioside (A-F), which are known for their sweet favor.Tis plant is used as a sugar substitute in the production of sauces, jams, and dental products [1].Using Stevia at a concentration of 5% compared to sugar achieves the same level of sweetness [2].
Despite its widespread use for sweeteners and medicinal purposes, limited studies have focused on Stevia's phytotoxic and cytotoxic efects on some indicator species.A previous study investigated the cytotoxicity of Stevia against brine shrimp (Artemia salina) and found its methanol extract exhibited a lethal concentration (LC 50 ) value of 66.78 mg/ mL [10].Another research has reported the cytotoxic properties of this herb against various cell lines, such as SKBR3, MCF-7, and MDA-MB-231.Te inhibitory efects of this plant on crops have been reported in some indicator plants such as Allium cepa [11], Cajanus cajan, Arachis hypogea, Glycine max, Cicer arietinum, Sorghum vulgare [12], Vigna mungo, and Triticum aestivum [13].
Furthermore, several studies have explored the antioxidant properties of Stevia rebaudiana leaf.In a study conducted by Ruiz et al. [14], the antioxidant activity of two Mexican varieties of Stevia, Criolla and Morita, showed comparable DPPH free radical-scavenging activity, ranging from 86.4% to 84.3%.Tis activity was also refected in its Trolox equivalent antioxidant capacity, ranging from 18.5 to 623.7 mM/mg.Escutia -Lopez et al. [15] investigated the antioxidant potential of aqueous Stevia leaf extract, demonstrating a signifcant inhibition of DPPH free radicals ranging from 60% to 72.37% at a concentration of 10 mg/ mL.In addition, Rao et al. [16] observed a DPPH-scavenging activity of 52.46% for Stevia leaf powder at a dose of 100 μg/ mL.Tese studies collectively highlight the substantial antioxidant activity associated with Stevia rebaudiana leaf.
Research investigating the impact of diferent polarity fractions on Stevia's inhibitory efects against the growth and germination of an invasive weed like Bidens frondosa remains scarce.To the best of our knowledge, studies exploring how varying extraction solvent polarities infuence Stevia's ability to inhibit the germination and growth of Bidens frondosa have not been reported.Tis research gap highlights the need for our current study, which aims to evaluate the inhibitory efects of Stevia against weed using diferent polarities of extraction solvents.By examining these phytotoxic efects, we seek to enhance our understanding of the potential use of Stevia as a natural herbicide and optimize its utilization of Stevia beyond human consumption.In addition, this study assessed the antioxidant and cytotoxic properties of this plant using the same fractions, providing insights into both its weed-inhibitory efects and antioxidant activity.Tis comprehensive investigation contributes valuable knowledge for agricultural and pharmaceutical applications, particularly in weed management and the development of natural product derived from Stevia.

Plant Fractionation.
A total of 7000 g of Stevia leaves was redried at 60 °C in the oven and pulverized into powder.Ten, an amount of 500 g of dried leaves powder of S. rebaudiana was dispersed in 1000 mL of methanol and stored at room temperature for fve days.After a fltration process, the fltrate of Stevia extract was evaporated by using a rotary evaporator (Buchi Rotavapor ® Rotary Evaporator R3, B ÜCHI Labortechnik AG, Switzerland) at 40 °C and 337 mbar.Ten, a total of 44.67g of the methanol extract was fractionated using diferent polar solvents following low to high polarity to obtain 6.17, 0.90, 0.86, and 9.83 g of hexane, chloroform, ethyl acetate, and water fractions, respectively.

Total Phenolic Contents (TPCs).
Te Folin-Ciocalteu (FC) solution was utilized for assessing the total phenolic levels of the extracts following the study conducted by Minh et al. [17].A volume of sample (20 μL) with a concentration of 1000 μg/mL was combined with 100 μL FC reagent (10%).Ten, an 80 μL of sodium carbonate (5%) was added after giving the mixture a short twirl in a vortex.Te mixture was incubated in a dark room for thirty minutes.Te absorbance of the mixture was measured by utilizing a microplate reader (Multiskan GO, Termo Fisher Scientifc, Tokyo, Japan) at 750 nm wavelength.Te quantities of phenolics per gram of fraction were expressed in milligrams of gallic acid equivalent (GAE).

Total Flavonoid Contents (TFCs).
Te procedure reported by Tuyen et al. [18] was employed to elucidate the total favonoid contents of samples.A volume of 100 μL containing 1000 μg/mL of sample or 10-200 μg/mL quercetin was mixed with 100 μL of aluminum chloride (2%).Te mixture was left to incubate for 15 minutes at ambient conditions.Ten, a microplate reader (Multiskan GO, Termo Fisher Scientifc, Tokyo, Japan) was used to read the absorbance at 430 nm of wavelength.Te total favonoid contents were quantifed as milligrams of quercetin (QE) equivalent per gram of sample.
2.5.DPPH Radical-Scavenging Activity.Te method reported by Andriana et al. [19] was modifed to assess the antioxidant activity of Stevia by measuring the DPPH scavenging activity of the samples compared to quercetin as a positive control.A total volume of 20 μL quercetin (1-20 μg/mL) or sample (100-3000 μg/mL) in methanol was combined with 80 μL of a 0.5 mM DPPH solution.Te mixture was subsequently left to incubate for 30 minutes in the dark at ambient conditions.Te absorbances of samples were measured by using a microplate reader (Multiskan GO, Termo Fisher Scientifc, Tokyo, Japan) at a 517 nm wavelength.Te percentage of DPPH-scavenging activity was calculated using the following equation: where A c was the absorbance of DPPH without a sample, and A s was the absorbance of DPPH with a sample.Te antioxidant activities of all samples were expressed as inhibition concentration (IC 50 ) at μg/mL.Te test solution (300 μL) was pipetted to each well, which had been prepared with two flter papers, and was allowed to evaporate for an hour.Subsequently, a volume of 300 μL of distilled water was added to each well.Te inhibitory germination, shoot, and root lengths were evaluated at fve days.Te plant inhibitory potentials of Stevia against L. sativa and B. frondosa were expressed as an inhibitory percentage of germination, shoot, and root size over control.

Brine Shrimp Lethality Assay (BSLA).
Te toxicity efects of S. rebaudiana fractions were assessed by the brine shrimp lethality assay following the previous studies [21,22].A total of 1 mg of each sample was dispersed in dimethyl-sulfoxide (DMSO), and the serial dilution method was used to create solutions with various concentrations using simulated seawater (0-1000 μg/mL).Te mixtures were then transferred to a 12-well plate with 10 live brine shrimp larvae in 300 μL of artifcial seawater.Te plates were examined after 24 hours to count the Artemia nauplii that had persisted on each plate.In this bioassay, the absence of regulated forward movement throughout the prescribed observation period of 30 seconds served as the criterion for determining mortality.Te data were utilized to determine the mortality rate of brine shrimp larvae for every concentration level and the control group.Carbofuran was utilized as a positive control due to its potent toxicity and accessibility [23].

Identifcation of Chemical Functional Groups of Stevia by
Fourier-Transform Infrared Spectroscopy (FTIR).Te chemical functional groups in S. rebaudiana were determined by FTIR analysis as outlined in a prior technique [24] to complement the GC-MS analysis.Te samples were placed right away into an FTIR system (Bruker A7.8) and scanned from 490 to 4000 cm −1 and then every specimen was examined three times.Te spectra were ATR and displayed as the percentage of transmittance.

Identifcation of Chemical Constituents of Stevia by GC-MS.
Te chemical constituents of Stevia fractions were determined utilizing a GC-MS system (Agilent 7890B/MSD 5977 A, Agilent Technology, Inc., California, United States) according to the previous report [25].A 1 μL sample was introduced into the HP-5MS column, an Agilent column with 30 meters long, 250 μm in diameter internally, and 0.25 μm thick.Te split-less helium induction was employed, with GC oven temperature set to 250 °C, the pressure at 7.0699 pounds per square inch, and the overall fow rate at 104 mL per minute.Te starting temperature was 40 °C, maintained for one minute.Te temperature rate was set to increase at 10 °C per minute to a maximum temperature of 325 °C with a four-minute hold interval.Te scanned mass range was 122 amu-1021 amu.Ions were generated via electron ionization (EI), and subsequently, positive ions were gathered in preparation for analysis via mass spectrometry (MS).Utilizing the Agilent ChemStation software, which integrated the NISTmass spectral library for data peak processing, the GC-MS system was managed.
2.10.Statistical Analysis.MetaboAnalyst 6.0 software (https://metaboanalyst.ca/) was utilized to analyze the data using the one-way analysis of variance (ANOVA) method.Afterward, to determine the signifcant diference between the control and treatment groups, a Tukey's test with a 95% confdence interval (p < 0.05) was conducted.Each assay was conducted three times to ensure accuracy and reliability.Te data were expressed in the form of standard deviations and mean values.Pearson's correlation evaluation was conducted to determine the relationship between the total phenolic and total favonoid contents of S. rebaudiana and its antioxidant, cytotoxicity, and phytotoxicity potentials.  1.Total phenolic contents showed a signifcantly positive correlation with L. sativa and B. frondosa growth and total favonoid contents.Tese results suggested that the inhibitory efects of Stevia on L. sativa and B. frondosa tend to increase with rising TPC and TFC values.In contrast, TPC and TFC negatively correlated to the IC 50 value of DPPH, which means they were proportional to the antioxidant activity of S. rebaudiana.Furthermore, total phenolic and favonoid contents have a negligible correlation to brine shrimp lethality.

Identifcation of the Chemical Functional Groups by FTIR.
Te spectra of Fourier transform infrared spectroscopy (FTIR) of fractions from S. rebaudiana are displayed in Figure 2. Tese spectra revealed peak characteristics associated with various chemical functional groups in S. rebaudiana fractions.Te water fraction exhibited indicative peaks of O-H stretching (3294.05cm −1 ), C-H stretching (2927.62 cm −1 ), C�O stretching (1720.31cm −1 ), and aromatic compounds (1593.02and 817.72 cm −1 ).Te ethyl acetate fraction exhibited similar peaks, emphasizing the presence of alcohol or phenol groups (3251.62 cm −1 ), alkanes (2927.62 cm −1 ), esters (1161.02cm −1 ), and aromatic compounds (1600.74,1512.02, and 810.01 cm −1 ).Te chloroform fraction possessed similar features to the ethyl acetate extract, with the addition of peaks indicating C�O stretching and chloroalkane groups (1242.02cm −1 ).Te hexane fraction showed a low intensity of O-H (3344.20)but was rich in alkanes (2919 and 2850 cm −1 ), carbonyl (C�O) stretch (1704), indicating the presence of aldehydes, ketones, or carboxylic acids.Te hexane fraction also showed C-H bending vibration (1461) and C-O (carbon-oxygen), which is associated with alkanes, ethers, esters, or alcohols [26].A combined FTIR with other analytical techniques is required to identify specifc chemical components in the samples.

Discussion
In this study, various fractions of S. rebaudiana including chloroform, hexane, water, and ethyl acetate were evaluated for their plant inhibitory, antioxidant, and cytotoxicity potentials.We found the ethyl acetate fraction exhibited the most potent radical scavenging efect against DPPH free radicals (IC 50 � 18.67 ± 0.61 μg/mL) as well as TPC (103.50 ± 0.18 mg GAE/g fraction) and TFC contents (410.16 ± 0.47 mg QE/g fraction).Tese fndings are in line with some previous studies that evaluated ethyl acetate extracts in diferent species, such as Coix lacryma-jobi and

Scientifca
Tridax procumbens [25,27].Generally, ethyl acetate extract contains semipolar compounds, including phenolic and favonoids, which possess antioxidant properties [28].Several studies also reported the correlation between TPC and TFC to antioxidant and plant inhibitory efects in some species, for example, Anacyclus pyrethrum, Salvia ofcinalis, and Ambrosia trifda [29][30][31].Tese phenolic compounds might be responsible for the antioxidant activity of Stevia.Moreover, several studies have reported that phenolic compounds inhibit free radicals through mechanisms such as hydrogen transfer or single electron transfer via proton transfer [32].Given the presence of phenolic compounds in the ethyl acetate fraction of Stevia, it is likely that its free radical-scavenging activity involves one or more of the following mechanisms: hydrogen atom transfer, single electron transfer, sequential proton loss electron transfer, and transition metal chelation [33].However, further studies are needed to better understand which mechanisms contribute to the antioxidant activity of Stevia.
In line with the antioxidant activity, the ethyl acetate fraction of S. rebaudiana reached the highest inhibitory activity in the growth and germination of L. sativa and B. frondosa (Table 2).At the 1000 μg/mL dose, this fraction suppressed the germination and growth of B. frondosa.It also inhibited L. sativa root and shoot lengths (IC 50 shoot and root lengths � 872.78 and 6 Scientifca 764.20 μg/mL, respectively) (data not shown).Tis inhibitory is comparable to some previous studies that reported that the ethyl acetate extract gave the most inhibitory efect on some indicator species such as Raphanus sativus and Echinochloa crus-galli compared to hexane, chloroform, and water [27,28].Te ethyl acetate extract was reported to possess some phytotoxic allelochemicals, such as phenolic acids.For example, the ethyl acetate fraction from Tridax procumbens contains phenolic acids such as benzoic acid, vanillin, ferulic acid, and ellagic acid [34].Tese phytochemicals have been reported to inhibit plant growth through several mechanisms, including disrupting photosynthesis and respiration, altering membrane permeability, inhibiting cell division, decreasing enzyme function and activity, and suppressing protein synthesis [35].Te current study highlights that the ethyl acetate fraction of Stevia exhibited signifcantly higher levels of total phenolic and favonoid contents compared to other samples.Given the presence of these bioactive compounds, it is possible that similar mechanisms may contribute to the phytotoxic efects of Stevia.However, further analyses are needed to determine the specifc modes of action of these phytochemicals in Stevia's phytotoxic activity.
In contrast with antioxidant and plant inhibitory assays, chloroform gave the most cytotoxic efect in the brine shrimp lethality assay in comparison with hexane, ethyl acetate, and water fractions.Some terpenoid compounds such as thymol, p-cymene, c-terpinene, and carvacrol were reported strong toxicity in the Artemia lethality assay.Tese compounds induce a dose-dependent inhibition of cell proliferation, disrupt cellular processes, induce apoptosis, or interfere with cell membrane integrity [36].Tis was in line with the result of GC-MS analysis that revealed chloroform fraction component consisted of benzofurans and terpenoids.

Conclusions
In this study, we found that the ethyl acetate fraction of S. rebaudiana exhibited the most potent inhibitory efects on germination and growth of B. frondosa and L. sativa, alongside signifcant antioxidant activity and high total favonoid and phenolic contents.We also revealed that the inhibitory efect on DPPH free radical scavenging, plant germination, and growth were proportional to the total phenolic and favonoid contents of S. rebaudiana.Conversely, the chloroform fraction demonstrated the highest level of cytotoxicity in the brine shrimp lethality test.FTIR spectra indicated the presence of phenolic and nonpolar groups in ethyl acetate and chloroform fractions, consistent with the GC-MS, which identifed several major components in the Stevia fractions.Tese fndings suggest that Stevia has potential as a natural source of antioxidative agents and herbicides, highlighting its potential use in various sectors, including agriculture and pharmaceuticals.However, this study only evaluated Stevia's phytotoxic, cytotoxic, and antioxidant properties in a controlled laboratory environment and was limited to a few indicator species.To fully understand Stevia's potential as a natural antioxidant and herbicide and to elucidate its mechanisms of action, further research is required.Tis includes conducting larger-scale studies such as feld trials to assess its herbicidal efects and in vivo assays to evaluate its cytotoxicity and antioxidant activity.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Table 1
Even at lower concentrations, the inhibitory efects of ethyl acetate fraction remain signifcant.Tese results show that diferent fractions of Stevia exhibit varying levels of plant inhibitory activity on the tested plant species.262.28 ppm), ethyl acetate (1061.45± 46.16 ppm), and water (3391.78± 388.76 ppm).Tis fraction, at a concentration of 1000 ppm, demonstrated a mortality rate of 62.50 ± 17.68%, suggesting moderate toxicity.Similarly, the hexane extract at a concentration of 1000 ppm exhibited a mortality rate of 55.00 ± 21.21%, indicating moderate toxicity.In contrast, the water extract exhibits lower toxicity.At a concentration of 1000 ppm, it displayed a mortality rate of 15.00 ± 0.00%.Tese fndings reveal the varying degrees of toxicity of the diferent fractions and reference compounds.Te hexane, chloroform, and ethyl acetate fractions of Stevia demonstrate moderate toxicity with varied mortality rates and LC 50 values.Te water fraction exhibited the least cytotoxic efect, whereas carbofuran showed the highest.3.4.Correlation among TPC, TFC, Antioxidant, Cytotoxicity, and Phytotoxicity Potentials of S. rebaudiana.Pearson's correlations of total phenolic, favonoid, DPPH radicalscavenging activity, brine shrimp toxicity, and plant inhibitory potentials of S. rebaudiana are illustrated in Figure displays the antioxidant potentials, TPC, and TFC of diferent fractions of S. rebaudiana.Te DPPH free radicals and3.2.Plant Inhibitory Potential of S. rebaudiana Fractions.Te inhibitory efects of S. rebaudiana on plant germination and growth were evaluated against B. frondosa and L. sativa.Table2presents the results of plant inhibitory activity of S. rebaudiana against L. sativa and B. frondosa.Te ethyl acetate fraction demonstrated a strong inhibitory activity across all parameters, followed by chloroform, water, and hexane fractions.At the dose of 1000 ppm, it showed the highest inhibition rates on L. sativa germination (46.67 ± 11.55%), root height (56.28 ± 6.21%), and shoot length (61.64 ± 5.90%).Tis fraction also completely inhibits the germination, root height, and shoot length of B. frondosa.

Table 3 :
Brine shrimp lethality test of diferent fractions of S. rebaudiana.
bcdeData are presented as the mean ± standard deviation.According to Tukey's test, values denoted by distinct letters imply statistically signifcant variations (p < 0.05).* � positive control.

Table 4 :
Figure 2: Te spectra characteristic peaks associated with various functional groups in S. rebaudiana extracts by FTIR.Te phytochemical constituents of diferent fractions of S. rebaudiana.