INTRODUCTION:

The search for biologically active compounds in plants that present antioxidant or anticancer activity generates great interest and has been the objective of various researches worldwide for several decades. Although there are many efforts to develop drugs and treatments to combat cancer, some of them with adverse side effects, this disease still represents the principal causes of death in the world;^1,2 that is why new compounds of natural origin that could help combat it are constantly being sought.

A549 cells, derived from a human lung adenocarcinoma, are a fundamental tool in the research of respiratory diseases and lung cancer, and have allowed the investigation of the molecular parameters of lung cancer and the assessment of the efficacy of antitumor treatments, in addition, due to their characteristics, they are an ideal and commonly used model for these trials.³ HeLa cells, derived from a human cervical carcinoma, represent one of the most broadly employed cell types in biomedical research,⁴ they have served as a valuable model for cytotoxicity studies due to their high capacity to divide rapidly, maintain their viability in culture conditions, they are compatible with colorimetric assays and are very useful in anticancer drug research. The MCF7 cells that come from a type of breast cancer, have allowed scientists to study how tumors work, how they react to certain drugs and how cancer can be tackled more effectively; These cells have estrogen receptors, so they respond to hormonal treatments, and their ability to grow in three-dimensional cultures, better imitating what happens in the human body, makes them an ideal model for researching more personalized and specific treatments for this type of cancer.⁵ Fibroblasts are fundamental cells in connective tissue, they help in wound healing processes and are responsible for synthesizing and maintaining the extracellular matrix and collagen; these cells are relevant in inflammatory processes and in the progression of certain diseases, such as fibrosis and cancer.⁶

On the other hand, the search for compounds with antioxidant activity also generates great interest because this property allows neutralizing the effect of free radicals and reactive oxygen species (ROS) or nitrogen species (RNS) that are present in our body and that when not properly controlled, can favor the attack on the cell membrane, proteins and on the DNA, triggering multiple neurogenerative, inflammatory, mutation-related and other diseases,^7-9 so in some cases antioxidant activity and anticancer activity may be related.^10-13 The DPPH and ABTS procedures have been frequently employed to calculate the antioxidant potential of the specimens obtained from plants. They are considered popular, simple, rapid and economical methods and are based on the change in absorbance of ions and radicals at certain wavelengths (515-517nm for DPPH and 732-744nm for ABTS) due to electron transfer causing the reduction of the oxidizing species causing a color change.^14-15

The species Tripogandra serrulata is part of the more than 600 plants that belong to the Commelinaceae family, which is a taxonomic family with a wide pantropical distribution and is characterized by containing species with a great diversity of chemical components and a great variety of biological and medicinal properties, highlighting the antimicrobial, anticancer, antioxidant, anti-inflammatory, antihyperglycemic, antifebrile properties, among others.^16-23 Currently, there are very few bibliographic records of research on the chemical compound or the biological and curative characteristics of the T. serrulata species, so the current research proposes the assessment of the cytotoxic and antioxidant activity, as well as the preliminary phytochemical study, of the excerpt and some portions obtained from the leaves and stems of this plant collected in the department of Arauca (Colombia).

MATERIALS AND METHODS:

Plant material collection:

The leaves and stems of Tripogandra serrulata were harvested at kilometer 9 on Caño Limón road in the city of Arauca (7°00′59″N, 70°44′40″W; altitude 127m a.s.l.) in Colombia. A sample of this species is in the Orinocense Herbarium of the Universidad Nacional de Colombia, Orinoquia Campus (HORI) under the code HORI3292.

Extraction and Fractionation:

The leaves and stems were dried at 45°C for 72hours, then this material was pulverized and subjected to maceration with ethanol (96%) for 72 hours. The solvent was filtered and removed under reduced pressure, obtaining a dark-colored semi-solid residue. A part of this extract was used for fractionation by the modified Kupchan method,²⁴ using dichloromethane (FO), butanol (FB) and water (FW) as solvents. The yields of the extract and fractions are calculated with equation 1.

Weight of the sample obtained (g)

% Yield = ------------------------------------ x 100 Eq.---(1)

Weight of the initial material (g)

Phytochemical Inspection:

The ethanolic excerpt (EE) was analyzed for alkaloids presence, reducing sugars, steroids, terpenes, coumarins, aglycones, flavonoids, cardiotonic glycosides, polyphenols and tannins, saponins, anthocyanins, leucoanthocyanins and anthraquinones.^25-29 The fractions obtained (FO, FB and FW) were analyzed by NMR (¹H y HSQC) to detect characteristic signals of the different types of metabolites present in them.

Cytotoxic Potential:

The cytotoxic potential of the ethanolic extract (EE) and fractions (FO, FB and FW) was evaluated on HeLa cells (human cervical carcinoma), MCF7 (breast cancer), A549 (human lung adenocarcinoma) and fibroblasts. The procedure described by Muñoz et al. (2018) was used.³⁰ Each of the samples evaluated was dissolved in DMSO (Dimethyl sulfoxide), due to its ability to dissolve a wide range of organic compounds without significantly affecting its cell viability at low concentrations (generally ≤ 1% v/v in the final assay). 8 stem solutions for each sample at 25000μg/ml concentration were made. The stock solutions were stored at -20°C to minimize degradation or loss of chemical activity. From the mother solutions, a series of dilutions in the culture medium (Dulbecco's Modified Eagle Medium – DMEM with 10% fetal bovine serum and antibiotics) were prepared to reach the desired final concentrations for the cytotoxicity assay (0, 5, 10, 25, 50, 100, and 200μg/ml).

· HeLa cell growing (human cervical carcinoma), MCF7 (breast cancer), A549 (human lung adenocarcinoma) and fibroblasts

They were cultured under controlled conditions in T75 boxes to ensure optimal growth and consistent cell viability throughout the experiment. DMEM high-glucose (4.5g/l) culture medium supplemented with 10% Fetal Bovine Serum (FBS) and an antibiotic mixture (penicillin 100U/ml, streptomycin 100µg/ml) was used. Cells were cultured at 37°C, in a cell culture incubator with a 5% CO₂ atmosphere and a relative humidity close to 95%. The culture means was replaced every 2-3 days to ensure a constant supply of nutrients and the removal of metabolic products that could accumulate and affect cell growth. Cell confluence was regularly monitored under an inverted microscope. The optimal confluence for the cytotoxicity assay was maintained between 70-80%.

· MTT Assay Procedure:

When cells reached 70-80% confluence in the T75 box, they were trypsinized using a 0.25% trypsin-EDTA solution. Adherent cells were dissociated from the substrate, trypsin was neutralized with an FBS supplemented medium, and cells were harvested by centrifugation and cell viability was assessed before each assay using the Trypan Blue exclusion method, ensuring that the cells used had a viability greater than 95%. These cells were seeded in culture plates (96 wells for the MTT assay) at a density of 10,000 cells per microwell and grown for 24hours at 37°C with 5% CO₂ to allow its adhesion and uniform growth.

After the initial incubation, the culture medium was withdrawn from each well and was substituted with 100 µl of fresh medium having the different configurations of the 8 fractions (0, 5, 10, 25, 50, 100, and 200µg/ml) to cover a broad spectrum of cytotoxic activity. The cells were incubated with the samples (extract and fractions) for 24hours, where the appropriate interaction between the samples and the cells was allowed, ensuring a detectable cellular response. An MTT solution at a configuration of 5mg/ml in PBS buffer (sodium phosphate and sodium chloride, pH 7.4) was made immediately before each assay to ensure its stability. After the incubation period with the samples, 10µl of the MTT solution was mixed to each well (final concentration of 0.5mg/ml). The plates were placed back in the incubator and kept at 37°C with 5% CO₂ for 3 hours, time required for the viable cells to reduce the MTT to purple formazan crystals. At the end of the MTT inoculation, the growth medium was carefully discarded from the trays to remove excess MTT and other cellular debris, leaving only the formazan crystals at the bottom of each well. 100µl of DMSO was mixed to each plate to dissolve the formazan crystals.

The plates were gently agitated in the dark for 10-15 minutes at ambient temperature (or until the crystals were completely dissolved) to ensure uniform solubilization of the formazan. The absorbance of each plate was measured at a UV of 570nm using a microplate reader. This absorbance peak corresponds to the formazan, whose purplish color is in proportion to the number of visible cells.

The absorbency of each well was determined at 570 using a microplate lector. The wells containing only the solvent (0μg/ml sample concentration) served as a negative control, and their absorbance represented the 100% cell viability. The percentage of cell viability for each sample concentration was calculated using equation 2:

Cell viability = -------- x 100 % eq…………(2)

Where: AT= Treatment Absorbance (average absorbance reading of wells treated with a specific concentration of the sample). AC= Control Absorbance (average absorbance reading of wells containing negative control)

· Statistical Analisis:

The GraphPad Prism (Version 9) statistical software for statistical analysis and generation of graphs and dose-response curves, and IC₅₀ calculations was used. The IC₅₀ (concentration required to reduce cell viability to 50%) was estimated for each compound using the percentages of viability using a sigmoid curve using a non-linear fit. Bar graphs were employed to represent the percent of cell viability in each concentration for each compound; error lines indicate the standard error of the average.

Antioxidant Potential:

· 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay:

The procedure outlined by Perez-Carrascal et al. (2016) was used.³¹ A solution of the DPPH radical in ethanol (54 mM in ethanol) was prepared previously (at least 12 hours beforehand), which was kept refrigerated and protected from light until use. Initially, a diluted DPPH solution is prepared (from the 54 mM solution) with an adjusted absorbency of 0.300±0.05 at λ=517nm (using the GENESYS 50 spectrophotometer). The inhibitory capacity of this radical presented by the ethanolic extract was evaluated at different concentrations in a range between 20 and 100μg/ml (dissolved in DMSO). For which, 20μl of a stock solution of the extract were taken and diluted with 1980μl of the diluted DPPH solution, to reach the concentration of interest (20 - 100μg/ml). In addition, a blank treatment was used, made by mixing 20μl of the extract solution along with 1980μl of ethanol, this was done in order to subtract the interference that may occur due to the color of the extract solutions. A reference treatment was prepared by mixing 20μl of DMSO and 1980μl of the diluted DPPH solution, in order to subtract any interference from the DMSO solvent on the radical. All treatments were stored in the dark for 30 minutes and absorbency was taken at 517nm. With the data obtained the % inhibition was calculated using equation 3, and the median inhibitory concentration (IC₅₀) was then determined. A calibration curve with Trolox was used to express the antioxidant capability as Trolox equivalence (TEAC, µmol Trolox/g extract).

% Inhibition = [1- (As-Ab)/ Ar] x 100 eq------(3)

Where As= sample absorbance; Ab= blank absorbance; Ar= reference absorbance

· 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay

The procedure outlined by Perez-Carrascal et al. (2016) was performed.³¹ The procedure is similar to that described for the DPPH assay. The ABTS radical solution was prepared (at least 12hours before) by mixing 9900μl of an aqueous ABTS solution (3.5mM) with 100μl of a potassium persulfate S₂O₈K₂ solution (125mM). This radical solution was diluted with phosphate buffer (NaH₂PO₄•H₂O/Na₂HPO₄•2H₂O, pH 7.4) to an absorbency of 0.700±0.05 at 732nm (using the GENESYS 50 spectrophotometer). The antioxidant potential of the extract was tested over a range of concentrations between 10 and 30μg/ml. The blank treatment was prepared using 20μl of the excerpt solution together with 1980μl of phosphate buffer. The reference treatment was prepared by mixing 20μl of DMSO and 1980μl of the diluted ABTS solution. Likewise, the treatments were left in the darkness for 30 min and the absorbance data were transformed employing equation 3.

Determination of Total Phenol Content:

The total phenol content was calculated employing the Folin-Ciocalteu reagent, using the method outlined by Kaur y Kapoor (2002),³² with some modifications. Aqueous stock solutions of gallic acid and the ethanolic excerpt were obtained. From the mother solution of gallic acid, various solutions at concentrations between 1-5µg/ml were prepared. The corresponding volume of the stock solution of gallic acid (or the extract) was combined with 500µl of Folin-Ciocalteu reagent solution (2N). After 5 minutes, 1250µl of a Na₂CO₃ solution (20%) was added, adjusting the final volume to 2000µl with distilled water. The mixture was left to stand for 1 hour at ambient temperature. After incubation, the absorbency of the mixtures was determined at 760nm by means of the GENESYS 50 spectrophotometer. The value of the total phenol concentration was obtained from the regression equation of the calibration curve (y= 0.1217x+0.0257 with R²= 0.9985) and was stated as mg of gallic acid/g of excerpt.

Total flavonoids Determination:

The total flavonoid concentration was obtained employing the AlCl₃ technique with the methodology used by Madhvi y colaboradores (2020).³³ Stock solutions of quercetin (in ethanol) and the extract were prepared, in order to work in a range of concentrations between 20 and 100µg/ml. 1500µl of AlCl₃ at 2% (in ethanol) was mixed to 500µl of the quercetin (or excerpt) solutions. After 60 minutes, the absorbency was taken at 420nm at ambient temperature with the GENESYS 50 spectrophotometer. The value of the total flavonoid concentration was obtained from the regression equation of the calibration curve (y= 0.0035x + 2.9319 with R²= 0.9816), which was expressed as mg of quercetin/g of excerpt.

RESULTS AND DISCUSSION:

Extraction, fractionation and phytochemical analysis:

The ethanolic excerpt (EE) of the leaves and stems of T. serrulata was obtained with an extraction efficiency of 4.3%. A part of this extract was subjected to fractionation by the Kupchan method to obtain three fractions of different polarities using dichloromethane (FO, 36.9% efficiency), n-butanol (FB, 5.3% efficiency) and water (FW, 7.1%) as solvents, indicating a greater presence of low and medium polarity compounds in the EE extract due to the high affinity of the compounds with dichloromethane. The EE Phytochemical analysis and the NMR spectra analysis (¹H and HSQC) of FO and FB fractions (Figure 1) suggest the presence of a variety of compounds present, such as reducing sugars, aglycones, cardiotonic glycosides, steroidal compounds, saponins, as well as the absence or very low concentration of alkaloids, coumarins, polyphenols and tannins, anthocyanins, leucoanthocyanins and anthraquinones, terpenes and flavonoids.

In the spectrum of the FO fraction (figure 1a) the presence of a group of signals between 0.8 and 2.9ppm is observed, which correspond to protons that couple with saturated carbons in the region between 12 and 41 ppm, which may correspond to compounds of lipid nature; a set of signals between 3.0 and 3.8ppm is also observed on saturated carbons between 10 and 21ppm. A set of signals between 3.8 and 4.7ppm is observed on carbons between 52 and 67ppm, which could correspond to protons and carbons of unsaturations or close to hydroxyl groups; between 4.8 and 5.6 protons appear on carbons 122 and 132ppm, which corresponds to the region of unsaturated carbons of aromatic compounds or alkenes. Low intensity signals are observed between 6.1 and 6.3ppm that couple with one or more carbons above 65ppm. Finally, some sets of low intensity signals are observed between 7.8 and 8.0ppm, between 8.4 and 8.7 ppm, between 9.5 and 9.9ppm in the aromatic proton region, coupled with carbons between 90 and 130ppm. In the spectrum of the FB fraction (figure 1b) a set of signals between 3.0 and 4.0ppm of protons is observed that are found on carbons emerging in the region between 50 and 65ppm and could be accredited to the presence of reductant sugars considering the results of the phytochemical analysis performed. Very low intensity signals are observed between 1.1 and 1.3ppm that couple with carbons above 28 and 30ppm, which, due to the polarity of the fraction, can be attributed to amino acid-type compounds. In the spectrum (NMR-¹H) of the FW fraction no signals of interest were observed, only that of the solvent used (D₂O) stands out with great intensity.

Figure 1: NMR-¹H spectra of the FO (a) and FB (b) fractions of T. serrulate

Cytotoxic potential:

The Cytotoxicity assays performed on various tumor cell lines revealed significant differences in cytotoxicity indices for each of the samples evaluated. These variations are represented in terms of IC₅₀ figures, which represent the amount of the sample necessary to suppress cell growth by 50%. The values obtained for each cell line and sample analyzed are presented in Table 1, considering that an IC₅₀ value < 20μg/mL suggests a potent cytotoxic activity; an IC₅₀ in the range of approximately 20 to 80μg/mL suggests a moderate cytotoxic activity; the values approximately between 81 and 150μg/mL suggest low cytotoxicity; and an IC₅₀ higher than these values indicate little or no cytotoxicity.

Table 1: IC₅₀ values (μg/ml) of cytotoxicity assays for T. serrulata extract and fractions

	IC₅₀(μg/ml)
Sample	MCF7	HeLA	A549	Fibroblast
EE	>200	136,9	>200	>200
FO	163,3	44,95	80,01	40,61
FB	>200	>200	>200	>200
FW	>200	>200	>200	>200

The FB and FW fractions of T. serrulata showed high IC₅₀ values (all greater than 200μg/mL) in the cell lines analyzed, suggesting no cytotoxicity under the experimental conditions used. The EE showed low or no toxicity, although HeLa cells showed sensitivity to this sample, which could be related to specific characteristics of this cell line, such as its molecular profile or proliferation rates, also taking into account that the EE showed no toxicity in normal fibroblasts indicating a certain degree of selectivity, so it is possible to suggest that this extract may be safe for cancer cells, although it is necessary to continue carrying out trials with this extract and, if possible, isolate the compound or compounds responsible for this cytotoxic activity, carry out structural modifications and structure-activity relationship studies or combinations with other agents to improve their efficacy, so that they can be considered in the development of antitumor therapies. The FO fragment was the one with the most activity against the cell types evaluated, especially against HeLa cells and fibroblasts; these results suggest a higher toxicity towards normal cells, so its use in antitumor treatments would be limited since it could cause adverse effects in non-tumor tissues. It is then necessary to balance antitumor effectiveness with safety for healthy cells if additional studies are to be carried out with this fraction, considering the isolation of the compound or compounds responsible for the activity, considering structural modifications and structure-activity relationship studies to evaluate its potential use in antitumor therapies, due to its effectiveness against cell lines.

Antioxidant Potential:

The antioxidant potential was tested for the EE extract of T. serrulata. In the DPPH assay, a concentration range between 20 and 100μg/ml was used and the inhibition percentages were between 39 and 100%. For the ABTS assay, a scale of concentration between 10 and 30μg/ml was used with inhibition percentages between 36 and 59%. In both methods, a significant increase (P value <0.05) of the antioxidant potential was observed with increasing concentration. The mean inhibitory concentration (IC₅₀) values for each assay, as well as the Trolox equivalent value (TEAC) for DPPH and ABTS are shown in Table 2.

The percentage of inhibition calculated for each extract concentration was represented against the concentration to achieve the IC₅₀ figure, which is understood as the quantity of antioxidant compound needed to eliminate 50% of free radicals found in the test system. This IC₅₀value is in an inverse variation to the antioxidant capacity, low IC₅₀ values mean a higher antioxidant potential.³³ The ethanolic extract of the leaves and stems of T. Serrulata presents high antioxidant activity against DPPH radical and against the ABTS radical cation.

Total Phenolic and Flavonoid Content Estimation:

The value determined for the total phenolic content equivalents to gallic acid was 49.0±0.01mg AGE/g extract and can be considered to be within a medium range for gallic acid equivalents (AGE) values in plant extracts.³⁴ The content of total flavonoids as quercetin equivalents was rather low 2.0±0.01mg QE/g excerpt. These results are in accordance with those found in the phytochemical analysis in which the results for the presence of flavonoids did not show an intense or very evident coloration, thus indicating a very low concentration of this type of compounds. In addition, the very low intensity signals in the RMN-¹H spectrum in the region of the aromatic protons in the FO and FB fractions (Figure 1), suggest the possible presence of phenolic concentrations of the T. serrulata leaves and stems, but in a very low amount.

In some studies, carried out with plants of the Commelinaceae family, the occurrence of phenolic concentrations and these species potent antioxidant activity have been recorded.^22,23,35,36 The results for T. serrulata obtained in this study of the antioxidant potential are in accordance with the results for other plants of this same family. It is possible to propose future research to obtain the extract of leaves and stems using other solvents (for example a hydroalcoholic mixture) or another extraction method and to be able to extract the phenolic compounds in a higher concentration, determine their identity, evaluate the antioxidant potential with these and other methods and compare the results obtained with those presented in this research.

CONCLUSION:

The ethanolic extract EE and the fractions obtained from the species T. serrulata show the presence of a variety of secondary metabolites, characteristic of many plants of the Commelinaceae family, as well as an excellent antioxidant property of the EE excerpt against the DPPH radical and against the ABTS radical cation and a moderate content of total phenols. The results obtained in these antioxidant tests allow proposing the species T. serrulata as a natural source of antioxidants, although it is necessary to continue the studies with other antioxidant tests to have more complete information on this property of pharmacological interest. In the anticancer tests, only the FO sample of T. serrulata showed a moderate cytotoxic activity profile, although with greater toxicity towards non-tumor cells. The IC₅₀ values found show the effect of the FO fraction against cell lines, although this effectiveness could be exploited as long as additional studies are carried out to improve the selectivity against cancer cells and the safety against normal cells. These results represent the first study of this type for the species T. serrulata collected in the Colombian Orinoquia.

CONFLICT OF INTEREST:

To the best of our knowledge, the authors do not have any kind of conflict of interest in relation to this research.

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Received on 22.04.2025 Revised on 03.06.2025

Accepted on 09.07.2025 Published on 24.07.2025

Available online from July 28, 2025

Res. J. Pharmacognosy and Phytochem. 2025; 17(3):197-204.

DOI: 10.52711/0975-4385.2025.00032

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

DPPH			ABTS
EE IC₅₀ (μg/ml)	TROLOX IC₅₀ (μg/ml)	TEAC (μmol Trolox/g extract)	EE IC₅₀ (μg/ml)	TROLOX IC₅₀ (μg/ml)	TEAC (μmol Trolox/g extract)
28.98±0.04	2.67±0.06	551.83	19.25±0.03	3.05±0.10	633.74