Bioactivity of Fractions and Pure Compounds from Jatropha cordata (Ortega) Müll. Arg. Bark Extracts

Medicines for chronic inflammation can cause gastric ulcers and hepatic and renal issues. An alternative treatment for chronic inflammation is that of natural bioactive compounds, which present low side effects. Extracts of Jatropha cordata (Ortega) Müll. Arg. have been evaluated for their cytotoxicity and anti-inflammatory activity; however, testing pure compounds would be of greater interest. Campesteryl palmitate, n-heptyl ferulate, palmitic acid, and a mixture of sterols, i.e., brassicasterol, campesterol, β-sitosterol, and stigmasterol, were obtained from an ethyl acetate extract from J. cordata (Ortega) Müll. Arg. bark using column chromatography. The toxicity and in vitro anti-inflammatory activities were evaluated using RAW 264.7 murine macrophage cells. None of the products assessed exhibited toxicity. The sterol mixture exhibited greater anti-inflammatory activity than the positive control, and nitric oxide (NO) inhibition percentages were 37.97% and 41.68% at 22.5 μg/mL and 30 μg/mL, respectively. In addition, n-heptyl ferulate decreased NO by 30.61% at 30 μg/mL, while campesteryl palmitate did not show anti-inflammatory activity greater than the positive control. The mixture and n-heptyl ferulate showed NO inhibition; hence, we may conclude that these compounds have anti-inflammatory potential. Additionally, further research and clinical trials are needed to fully explore the therapeutic potential of these bioactive compounds and their efficacy in treating chronic inflammation.


Introduction
Certain forms of cancer, cardiovascular and cerebrovascular diseases, diabetes, and several other diseases have been strongly linked to chronic inflammatory processes [1][2][3][4].Until now, the main treatments for these processes include the use of synthetic medicines, whose prolonged consumption may lead to undesirable side effects such as gastric ulcers, The four steroids present in this mixture have the same steroidal skeleton and only differ in the side chain, i.e., through the presence of a methyl group for campesterol and an ethyl group for β-sitosterol in C-24, and through the presence of a double bond at C-22 for brassicasterol and at C-23 for stigmasterol in the side chain.By comparing the NMR data of the mixture with those described in the literature for the individual compounds, In addition, n-heptyl ferulate was identified by comparing its NMR data with those described in [20].Its 13 C NMR spectrum, and the HSQC spectrum, led to the identification of 17 carbon resonances, which were assigned to two CH 3 , six CH 2 , five CH, and four quaternary carbon atoms.Among the carbon resonances, the signals at δ C 167.5 (C-9), 144.8 (C-7), and 115.8 (C-8) were assigned to the α, β-unsaturated carboxyl; and the six aromatic carbon signals at δ C 127.2 (C-1), 109.39 (CH-2), 146.8 (C-3), 148.0 (C-4), 114.9 (CH-5), and 123.1 (CH-6) correspond to the trisubstituted benzene ring; the signal at δ C 56.9 is ascribable to a methoxyl group.Also, this spectrum reveals the presence of a C7 saturated hydrocarbon chain, evidenced by the seven signals at δ C 64.70 (C-1 ), 32.0 (C-6 ), 29.6 (C-2 ), 29.4 (C-4 ), 28.8 (C-3 ), 22.7 (C-5 ), and 14.2 (C-7 ).The 1 H-NMR spectrum reveals signals for an AB olefinic spin system at δ H 7.59 (d, J = 15.9Hz, H-7) and 6.27 (d, J = 15.9Hz, H-8), and one aromatic ABX spin system at δ H 7. 06 (dd, J = 8.1, 1.8 Hz, H-6), 7.02 (d, J = 1.8 Hz, H-2), and 6.9 (d, J = 8.1 Hz, H-5), indicating a 1,3,4 trisubstituted benzene ring.In addition, signals for a methoxyl group (δ H 3.91, s) and one OH group (δ H 5.85, sbr) were also observed.The aliphatic side chain was evidenced by the signals at δ H 4.17 (2H, t, J = 6.7 Hz, H-1 ), 1.67 (2H, q, H-2 ), 1.36 (8H, m, H-3 -H-6 ), and 0.86 (3H, t, J = 6.8 Hz, H-7 ).Furthermore, these signals were verified by proton-carbon correlations in the HMBC spectrum.Therefore, compound 2 was determined as n-hepthyl ferulate based on the abovementioned experimental results and previous reports from the literature [20] (Figure 1).Experimentally, n-heptyl ferulate was obtained as yellow crystals, and its molecular weight was determined by the positive FAB/MS ion peak at m/z = 315 [M + Na] + .Figures A3-A6, in Appendix A, correspond to the spectra 1 H-NMR, 13 C-NMR, HSQC, and HMBC of n-heptyl ferulate, respectively.
The four steroids present in this mixture have the same steroidal skeleton and only differ in the side chain, i.e., through the presence of a methyl group for campesterol and an ethyl group for β-sitosterol in C-24, and through the presence of a double bond at C-22 for brassicasterol and at C-23 for stigmasterol in the side chain.By comparing the NMR data of the mixture with those described in the literature for the individual compounds, and the C-H correlations of the HSQC and HMBC spectra, it was possible to identify the signals corresponding to each one, as is described in the experimental section (Figure 1).Figures A7 and A8, in Appendix A, correspond to the 1 H-NMR spectrum of the mixture of sterols (brassicasterol, campesterol, β-sitosterol, and stigmasterol) and the 13 C-NMR spectrum of sterols (brassicasterol, campesterol, β-sitosterol, and stigmasterol).

Cell Cytotoxicity
The cytotoxicity of fractions F-1 and F-2 at concentrations of 3.125, 6.25, 12.5, 25, and 50 µg/m was assessed for their effect on viability in murine RAW 264.7 macrophage cells.Lipopolysaccharides (LPS) and lipopolysaccharides + indomethacin (LPS + Indo) were used as negative (C−) and positive (C+) controls, respectively.The corresponding analysis of variance (Table 1) showed that concentration was the only significant factor, Plants 2023, 12, 3780 5 of 24 and all concentration levels were equal and significantly different from the negative control (Table 2); hence, both fractions did not show cell cytotoxicity at the concentrations used in the experiment (Figure 2).used as negative (C−) and positive (C+) controls, respectively.The corresponding of variance (Table 1) showed that concentration was the only significant factor, concentration levels were equal and significantly different from the negative con ble 2); hence, both fractions did not show cell cytotoxicity at the concentrations the experiment (Figure 2).Similar results occurred for subfractions SF-1 and SF-2, at 3. 75, 7.5, 15, 22.5, and 30 µg/mL, as shown in Tables 3 and 4 and Figure 3.No subfractions at any concentrations resulted in cytotoxic in murine RAW 264.7 macrophage cells.resulted in cytotoxic in murine RAW 264.7 macrophage cells.The analysis of variance showed that the compounds (campesteryl palmitate heptyl ferulate, C-2, and the steroid mixture) and concentration were statistically cant (Table 5).Tukey's test found C-2 to be different from C-1 and the mixture (T Neither concentration resulted in cell cytotoxic compared to the negative control ( ble 7). Figure 4 shows profiles for the two compounds and the mixture with re concentration and confirms the above interpretation.The results showed no mac toxicity of compounds or the mixture at any of the studied concentrations.The analysis of variance showed that the compounds (campesteryl palmitate, C-1, n-heptyl ferulate, C-2, and the steroid mixture) and concentration were statistically significant (Table 5).Tukey's test found C-2 to be different from C-1 and the mixture (Table 6).Neither concentration resulted in cell cytotoxic compared to the negative control (C−) (Table 7).Figure 4 shows profiles for the two compounds and the mixture with respect to concentration and confirms the above interpretation.The results showed no macrophage toxicity of compounds or the mixture at any of the studied concentrations.The potential anti-inflammatory activity of fractions, subfractions, and compou was assessed using the effects on NO inhibition in murine RAW 264.7 macrophage stimulated with LPS.

Nitric Oxide (NO) Inhibition
The potential anti-inflammatory activity of fractions, subfractions, and compounds was assessed using the effects on NO inhibition in murine RAW 264.7 macrophage cells stimulated with LPS.
The inhibition of NO from fractions F-1 and F-2 was studied at concentrations of 3.125, 6.25, 12.5, 25, and 50 µg/mL.The analysis of variance (Table 8) shows that concentration was the only significant factor (p-value < 0.05).Tukey's test showed that none of the concentrations were statistically different of C+, and all were better than C− (Table 9). Figure 5 shows that only F-2 at a concentration of 50 µg/mL was better than C+.The inhibition of NO from fractions F-1 and F-2 was studied at concentration 3.125, 6.25, 12.5, 25, and 50 µg/mL.The analysis of variance (Table 8) shows that con tration was the only significant factor (p-value < 0.05).Tukey's test showed that non the concentrations were statistically different of C+, and all were better than C− (Tab Figure 5 shows that only F-2 at a concentration of 50 µg/mL was better than C+.The effects of subfractions and their concentrations on NO inhibition are show the following.The analysis of variance (Table 10) shows that only the concentration statistically significant (p < 0.05).Tukey's test (Table 11) found that concentrations fro The effects of subfractions and their concentrations on NO inhibition are shown in the following.The analysis of variance (Table 10) shows that only the concentration was statistically significant (p < 0.05).Tukey's test (Table 11) found that concentrations from 15 to 30 µg/mL had comparable effects to C+.In fact, Figure 6 shows that SF-1 and SF-2 were comparable to C+ at 30 µg/mL.Finally, in the case of compounds, both factors were statistically signifi (Table 12).According to Tukey's test, campesteryl palmitate and the stero hibited greater NO inhibition than n-heptyl ferulate (Table 13, Table 14).H the steroid mixture at concentrations of 22.5 and 30 µg/mL produced bett C+ (Figure 7).Finally, in the case of compounds, both factors were statistically significant (p < 0.05) (Table 12).According to Tukey's test, campesteryl palmitate and the steroid mixture exhibited greater NO inhibition than n-heptyl ferulate (Tables 13 and 14).However, only the steroid mixture at concentrations of 22.5 and 30 µg/mL produced better results than C+ (Figure 7).

Discussion
The anti-inflammatory ethyl acetate extract of Jatropha cordata (Ortega) Müll.Arg. was separated using the NO inhibition bioassay, and an active SF-2 was obtained.This fraction was mainly composed of steroids (70%).SF-2 was further purified using column chromatography to yield campesteryl palmitate, n-heptyl ferulate, and a mixture of four sterols, identified as brassicasterol, campesterol, β-sitosterol, and stigmasterol (Figure 1) based on their spectroscopic characteristics and a comparison of the spectroscopic data with those in the literature.A quantitative analysis showed that the percentages of brassicasterol, campesterol, β-sitosterol, and stigmasterol in the SF were 8.74%, 22.8%, 18.30%, and

Discussion
The anti-inflammatory ethyl acetate extract of Jatropha cordata (Ortega) Müll.Arg. was separated using the NO inhibition bioassay, and an active SF-2 was obtained.This fraction was mainly composed of steroids (70%).SF-2 was further purified using column chromatography to yield campesteryl palmitate, n-heptyl ferulate, and a mixture of four sterols, identified as brassicasterol, campesterol, β-sitosterol, and stigmasterol (Figure 1) based on their spectroscopic characteristics and a comparison of the spectroscopic data with those in the literature.A quantitative analysis showed that the percentages of brassicasterol, campesterol, β-sitosterol, and stigmasterol in the SF were 8.74%, 22.8%, 18.30%, and 29.20%, respectively.
Plant sterols are naturally occurring bioactive compounds in plant materials [21,22].They are highly present in lipid-rich plant foods such as nuts, seeds, legumes, and olive oil and have been shown to elicit a broad range of pharmacological activities, such as antiallergy, antitumor [23], antimalarial, antiobesity, antimicrobial [24], antidepressant [25], antinociceptive [26], and antileishmanial activities [27], cardiovascular protection [28], and antiaging and hepatoprotective activities [29].All plant species have their characteristic phytosterol (PS) composition, with more than 250 PS being recognized so far [30].Campesterol, β-sitosterol, and stigmasterol are the most common plant-derived sterols in the human diet.All contain a core skeleton of cholesterol but possess a different side chain.β-sitosterol and stigmasterol have an ethyl group at C-24, whereas campesterol has a methyl group.Stigmasterol has a double bond at C-22/C-23, and sitosterol has a saturated side chain.The compounds brassicasterol and D-7-avenasterol are minor constituents [31].
The compound campesteryl palmitate did not inhibit NO production at the concentrations evaluated, although this compound has been reported as a constituent of a wide variety of plants and nutraceuticals [32].Reports of its biological activities were not discovered during our research.Furthermore, Compound 1 did not inhibit NO production at the concentrations evaluated compared to fractions and subfractions of J. cordata (Ortega) Müll.Arg., which showed significant activity.
The compound n-heptyl ferulate displayed an important anti-inflammatory effect since it diminished the concentration of NO by 30.61% at 30 µg/mL.This compound is an ester derivative of ferulic acid, which is a hydroxycinnamic acid widely distributed in cereals, fruits, vegetables, and beverages [33,34].In these foods, Compound 2 is found in its free form and as ester derivatives, displaying a wide range of biological activities, including anticancer, antibacterial, anticarcinogenic, and anti-inflammatory activity [35,36].Esterification has shown some advantages over precursor compounds [37][38][39][40].In this context, the compound n-heptyl ferulate has been previously reported as a natural product from Jatropha podagrica [20], but no activity was assessed.This is the first report to focus on the anti-inflammatory activity of n-heptyl ferulate.
Another pure compound isolated and identified was palmitic acid, which has already been widely reported in the literature with pharmacological activities such as antiviral [41,42], anti-inflammatory [41,43], analgesic [41,44], and lipid metabolism-regulating activities [41,45].Regarding its anticancer activity, several authors have reported that palmitic acid induces cell cycle arrest [41,46] and promotes apoptosis of human neuroblastoma cells [41,47] and breast cancer cells [41,48].In addition, palmitic acid can inhibit hepatoma cell proliferation by changing membrane fluidity and blocking glucose metabolism [41,49].Cantrell et al. [50] demonstrated that palmitic acid obtained from Jatropha curcas L. at a concentration of 25 nmol/cm 2 acts as a repellent against Aedes aegypti (L.) mosquitoes (Diptera: Culicidae).Othman et al. [51] reported that palmitic acid obtained from fractions of n-hexane extracts of J. curcas root presented anti-inflammatory activity in RAW 264.7 cells at an effective concentration of 1 mg/mL.Aati et al. [8], studying palmitic acid obtained from J. pelargoniifolia root essential oil, reported anti-inflammatory, antipyretic, anticonceptive, analgesic, and antioxidant activity.Regarding antimicrobial activity, Shaaban et al. [44] reported that palmitic acid has an effect against S. aureus, P. aeruginosa, K. pneumoniae, Lalthanpuii, and Lalchhandama.Shaaban et al. [52] mentioned that the antimicrobial effect of palmitic acid is due to its structure, shape, the length of its carbon chains, and the presence, number, position, and orientation of double bonds.As for its anticancer activity, Zhu et al. [41] tested palmitic acid in human prostate cancer cell lines PC3 and DU145 at 0.1, 1, 1, 5, 10, 25, and 50 µM concentrations, concluding that it has anticancer activity in prostate cancer by arresting G1 phase and suppressing tumor metastasis regulatory proteins.The underlying mechanism of these effects could be attributed to the inhibition of the PI3K/Akt pathway.Diverse biological activities of palmitic acid have been reported by [53,54] who, beginning with a methanolic extract of Chrozophora tinctoria (L.) from the family Euphorbiaceae, at concentrations of 1000, 500, 250, and 125 µg/mL, reported antioxidant, nematicidal, pesticidal, hypocholesterolemic, nematicidal, hemolytic, and 5-alpha reductase inhibitory activities.
The compounds that constitute the mixture of free and esterified sterols are brassicasterol, campesterol, β-sitosterol, and stigmasterol.Brassicasterol has been reported for its antiaging activity under oxidative stress and decreased ROS and MDA levels [55] and against Alzheimer's disease, which is attributed to its bioactivity against amyloid beta and tau receptors [56].Similarly, antiherpes simplex type I and antituberculosis activities attributed to the inhibition of vital enzymes involved in HSV-1 replication and Mtb cell wall biosynthesis have been reported [57], as have the inhibitory properties of human angiotensin-converting enzyme [58].Kuwabara et al. [59] reported the accumulation of cholesterol precursors (latosterol, 7-dehydrocholesterol, and desmosterol) and their decrease by altering mRNA and biosynthesis protein levels, increasing sterol 8,7-isomerase (EBP) enzymes, and decreasing DHCR7 and 24-dehydrocholesterol reductase (DHCR24).
Brassicasterol has been reported for its anticancer activity in prostate cancer, which was attributed to AKT and AR dual-targeting signaling [60], and in bladder cancer through its androgen receptor (AR) antagonist action and AR (androgen receptor) expressionreducing effect in bladder epithelial cells [61].Brassicasterol has also demonstrated activity in hepatocellular carcinoma [58] through the suppression of the AKT signaling pathway.
As for the compound stigmasterol, Viswanatham et al.
In addition, mixtures of the above compounds have been shown to have diverse biological activities.Dumandan et al. [66] showed that the mixture of brassicasterol, campesterol, and stigmasterol presented antimicrobial activity.Prabhakar et al. [67] found that the mixture of brassicasterol, campesterol, and β-sitosterol presented activity against androgenic alopecia as they were potential inhibitors of 5α-reductase1.Abou-Hussein et al. [68] determined that the mixture of brassicasterol and campesterol manifested antiinflammatory activity in vivo.Akintayo [69] and Sekandí et al. [70] reported that a mixture of campesterol, β-sitosterol, and stigmasterol exhibited antioxidant, antimicrobial, and sunscreen activities.Hérnandez-Hérnandez et al. [71] reported that the mixture of βsitosterol and stigmasterol had antioxidant, antimicrobial, and antifungal activities.Finally, Mahrous et al. [72] found anti-inflammatory activity, attributing it to the decrease in NO, prostaglandin PGE2, TNF-α, and PKC levels by 19, 29.35, 16.9, and 47.83%, respectively.

GC-MS Analysis
The components present in the acetylated steroidal mixture were analyzed with GC-MS using an HP Agilent Technologies 6890 gas chromatograph equipped with an MSD 5973 quadrupole mass detector (HP Agilent, Wilmington, DE, USA) and an HP-5MS capillary column (length: 30 m; inner diameter: 0.25 mm; film thickness: 0.25 M).A constant flow of carrier helium was adjusted to the column at 1 mL per minute.The inlet temperature was set at 250 • C, while the oven temperature was initially maintained at 40 • C for 1 min and increased to 280 • C at 10 • C/min intervals.The mass spectrometer started in positive electron impact mode with an ionization energy of 70 eV.Detection was performed in selective ion monitoring (SIM) mode, and peaks were identified and quantified using target ions.Compounds were identified by comparing their mass spectra with the NIST library version 1.7a.Relative percentages were determined by integrating the peaks using the GC ChemStation software, version C.00.01.The composition was reported as a percentage of the total peak area.

In Vitro Anti-Inflammatory Activity
The in vitro anti-inflammatory evaluation of J. cordata (Ortega) Müll.Arg.bark fractions, subfractions, and pure compounds was performed with a murine macrophage cell model RAW 264.7 (Tib-71TM ATCC).The inflammatory process was induced using lipopolysaccharides produced by Escherichia coli (LPS), applying the fractions, subfractions, and pure compounds as inhibitory treatments of proinflammatory cytokines.The stages of the evaluation are detailed below.

Cell Culture and Cytotoxicity Assay
A murine macrophage cell line RAW 264.7 was cultured in Dulbecco's modified Eagle's medium in an F-12 nutrient mixture (DMEM/F12 medium) supplemented with 10% heat-inactivated fetal bovine serum (FBS).Cells were maintained in a humidified atmosphere containing 5% CO 2 at 37 • C and subcultured by scraping and seeding in 25 cm 2 flasks.To assess cell viability, 2 × 10 4 cells/well in 200 µL of medium were seeded into a 96-well plate and incubated for 24 h.Subsequently, cells were treated with fractions, subfractions, and pure compounds at various concentrations (3.125, 6.25, 12.5, 25, and 50 µg/mL for fractions and subfractions; 3.75, 7.5, 15, 22.5, and 30 µg/mL for pure compounds) using DMSO as vehicle (0.21%, v/v), indomethacin (30 µg/mL) as positive control, and untreated cells as negative control.After 2 h, inflammation was induced with lipopolysaccharide (LPS) at a concentration of 4 µg/mL (for wells with extracts, vehicle, indomethacin, and 100% stimulus control) as a proinflammatory stimulus and without LPS (negative control), incubating for 22 h.
Percent cell viability (%CV) was calculated using the following equation: where a S = the absorbance of the sample and a LPS = the average LPS absorbance.

Nitric Oxide (NO) Inhibition
After cell viability determination, the cell-free supernatants were collected and used for nitric oxide (NO) quantification.Nitrite ion (NO 2 − ), the stable final product of NO, is an indicator of NO production in cell-free supernatants and was measured according to the Griess reaction.A volume of 50 µL of supernatant from each extract was mixed with 100 µL of Griess reagent (50 µL of 1% sulfanilamide and 50 µL of 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid) for 10 min at room temperature.The optical density of the mixture, at 540 nm (OD 540 ), was measured with a microplate reader and the concentration of nitrite in the samples prepared in fresh culture medium was calculated using a NaNO 2 standard curve [73,74].
(2) The corrected absorbance, (a c ), was calculated for each fraction and subfraction at concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 µg/mL, and for pure compounds at concentrations of 0, 3.75, 7.5, 15, 22.5, and 30 µg/mL, using the difference where a s = the absorbance of the sample and a 0 NaNO 2 = the average absorbance at zero concentration of the NaNO 2 curve.
(3) The concentration of NaNO 2 (µM) present in each of the fractions, subfractions, and pure compounds was determined using the following equation: where c µM NaNO 2 = the micromolar concentration of sodium nitrate.

Figure 2 .
Figure 2. Mean percentage profiles of cell viability in RAW 264.7 murine macrophages, st with LPS and treated with fractions of various concentrations.

Figure 2 .
Figure 2. Mean percentage profiles of cell viability in RAW 264.7 murine macrophages, stimulated with LPS and treated with fractions of various concentrations.

Figure 3 .
Figure 3. Mean percentage profiles of cell viability in RAW 264.7 murine macrophages, st with LPS and treated with subfractions of various concentrations.

Figure 3 .
Figure 3. Mean percentage profiles of cell viability in RAW 264.7 murine macrophages, stimulated with LPS and treated with subfractions of various concentrations.

Figure 4 .
Figure 4. Mean percentage profiles of cell viability in RAW 264.7 murine macrophages, stimu with LPS and treated with pure compounds and the steroid mixture at various concentrations and LPS + Indo (Indomethacin) were the positive (C+) and the negative (C−) controls, respectiv

Figure 4 .
Figure 4. Mean percentage profiles of cell viability in RAW 264.7 murine macrophages, stimulated with LPS and treated with pure compounds and the steroid mixture at various concentrations.LPS and LPS + Indo (Indomethacin) were the positive (C+) and the negative (C−) controls, respectively.

Figure 5 .
Figure 5. Mean percentage profiles of NO inhibition in RAW 264.7 murine macrophages, stimu with LPS and treated with fractions.LPS and Indo (Indomethacin) were the positive (C−) an negative (C+) controls, respectively.

Figure 5 .
Figure 5. Mean percentage profiles of NO inhibition in RAW 264.7 murine macrophages, stimulated with LPS and treated with fractions.LPS and Indo (Indomethacin) were the positive (C−) and the negative (C+) controls, respectively.

Figure 6 .
Figure 6.Mean percentage profiles of NO inhibition in RAW 264.7 murine macroph with LPS and treated with subfractions at various concentrations.LPS and Indo were the positive (C−) and the negative (C+) controls, respectively.

Figure 6 .
Figure 6.Mean percentage profiles of NO inhibition in RAW 264.7 murine macrophages, stimulated with LPS and treated with subfractions at various concentrations.LPS and Indo (Indomethacin) were the positive (C−) and the negative (C+) controls, respectively.

Figure 7 .
Figure 7. Mean percentage profiles of NO inhibition in RAW 264.7 murine macrophages, stimulated with LPS and treated with pure compounds and the mixture at various concentrations.LPS and Indo (Indomethacin) were the positive (C−) and the negative (C+) controls, respectively.

Figure 7 .
Figure 7. Mean percentage profiles of NO inhibition in RAW 264.7 murine macrophages, stimulated with LPS and treated with pure compounds and the mixture at various concentrations.LPS and Indo (Indomethacin) were the positive (C−) and the negative (C+) controls, respectively.

Table 1 .
ANOVA of fractions and concentrations for cell viability.

Table 2 .
Comparisons of cell viability means for concentrations using Tukey's test.
Means that do not share a letter are significantly different (p < 0.05).

Table 1 .
ANOVA of fractions and concentrations for cell viability.
p-value < 0.05 was considered statistically significant.

Table 2 .
Comparisons of cell viability means for concentrations using Tukey's test.
Means that do not share a letter are significantly different (p < 0.05).

Table 3 .
ANOVA of subfractions and concentrations for cell viability.

Table 4 .
Comparisons of cell viability means for concentrations using Tukey's test.
Means that do not share a letter are significantly different (p < 0.05).

Table 3 .
ANOVA of subfractions and concentrations for cell viability.

Table 4 .
Comparisons of cell viability means for concentrations using Tukey's test.
Means that do not share a letter are significantly different (p < 0.05).

Table 5 .
ANOVA of compounds and concentrations for cell viability.
p-value < 0.05 was considered statistically significant.

Table 6 .
Comparisons of cell viability means for compounds using Tukey's test.
Means that do not share a letter are significantly different (p < 0.05).

Table 7 .
Comparisons of cell viability means for concentrations using Tukey's Test.
Means that do not share a letter are significantly different (p < 0.05).

Table 5 .
ANOVA of compounds and concentrations for cell viability.
p-value < 0.05 was considered statistically significant.

Table 6 .
Comparisons of cell viability means for compounds using Tukey's test.

Table 7 .
Comparisons of cell viability means for concentrations using Tukey's Test.
Means that do not share a letter are significantly different (p < 0.05).

Table 8 .
ANOVA of fractions and concentrations for NO inhibition.
p-value < 0.05 was considered statistically significant.

Table 9 .
Comparisons of NO inhibition means for concentrations using Tukey's test.
Means that do not share a letter are significantly different (p < 0.05).Plants 2023, 12, x FOR PEER REVIEW 8

Table 8 .
ANOVA of fractions and concentrations for NO inhibition.
p-value < 0.05 was considered statistically significant.

Table 9 .
Comparisons of NO inhibition means for concentrations using Tukey's test.
Means that do not share a letter are significantly different (p < 0.05).

Table 10 .
ANOVA of subfractions and concentrations for NO inhibition.

Table 11 .
Comparisons of NO Inhibition Means for Concentrations using Tukey's test.
Means that do not share a letter are significantly different.Plants 2023, 12, x FOR PEER REVIEW

Table 10 .
ANOVA of subfractions and concentrations for NO inhibition.

Table 11 .
Comparisons of NO Inhibition Means for Concentrations using Tukey's t Means that do not share a letter are significantly different.

Table 12 .
ANOVA for compounds and mixture NO inhibition.
Means that do not share a letter are significantly different.
Means that do not share a letter are significantly different.
Means that do not share a letter are significantly different.