Cytotoxicity of Nine Medicinal Plants from San Basilio de Palenque (Colombia) on HepG2 Cells

The utilization of plants with medicinal properties is deeply rooted in the traditional knowledge of diverse human populations. This study aims to investigate the cytotoxicity of nine plants commonly used by communities in San Basilio de Palenque, Bolivar (Colombia), for managing inflammation-related illnesses. Hydroethanolic extracts from various plant parts such as roots, stems, barks, or leaves were prepared through a process involving drying, powdering, and maceration in an ethanol–water (7:3) solution. The extracts were subsequently freeze-dried and dissolved in DMSO for the bioassays. Cytotoxicity against the human hepatoma HepG2 cell line was assessed using the MTT assay, with extract concentrations ranging from 0 to 500 µg/mL and treatment durations of 24 and 48 h. The total phenolic content of the nine extracts varied from 96.7 to 167.6 mg GAE/g DT. Among them, eight hydroethanolic extracts from Jatropha gossypiifolia L., Piper peltatum L., Malachra alceifolia, Verbesina turbacensis, Ricinus communis, Desmodium incanum, and Dolichandra unguis-cati showed low toxicity (IC50 > 500 µg/mL, 24 h) against HepG2 cells. On the other hand, the extracts of Aristolochia odoratissima L. (IC50 = 95.7 µg/mL) and Picramnia latifolia (IC50 = 128.9 µg/mL) demonstrated the highest cytotoxicity against the HepG2 cell line, displaying a modest selectivity index when compared to the HEKn cell line after 48 h of treatment. These findings suggest that medicinal plants from San Basilio de Palenque, particularly Picramnia latifolia and Aristolochia odoratissima, have potential activity against cancer cells, highlighting their potential for further research and development.


Introduction
Medicinal plants serve as valuable sources of new drugs globally [1]. In Europe, over 1300 medicinal plant species are utilized, with approximately 90% derived from wild resources [2]. In the United States, a significant number of major prescription drugs are based on natural sources. The World Health Organization (WHO) reports that around 80% of the population in developing countries relies on traditional medicine for their healthcare needs [3]. The utilization of medicinal plants is rapidly increasing worldwide. However, due to overexploitation, habitat destruction, and the growing human population's consumption of plants, approximately 15,000 species are currently threatened with extinction, and nearly 20% of their wild resources have already been depleted [4,5]. The American continent, particularly South America, is recognized for its vast cultural and biological wealth, hosting one of the largest areas of biological diversity globally. Approximately 40% of the continent's biodiversity is concentrated in South America, particularly in its tropical forested regions [6].
The tropical forests of Colombia boast remarkable biodiversity of flora and fauna, thanks to their abundant rainfall, which is the highest among all the regions in the Americas. This climatic condition, characterized by a bimodal regime, directly supports diverse The DPPH free radical scavenging activity of P. latifolia is depicted in Figure 1. At a concentration of 4 mg/mL, the extract demonstrated a scavenging activity of 84.1%. The IC 50 value for P. latifolia extract was determined to be 0.95 mg/mL. The antioxidant assay yielded values ranging from 20 to 476.6 µM Trolox. ratissima L. and P. latifolia Tul. stems against the cytotoxicity induced by zearalenone (α-ZEL) and APAP on HepG2 cells.

Total Phenolic, Flavonoid, Tannin Contents, and DPPH Free Radical Scavenging Activity
The quantification of total phenolic, flavonoid, and tannin contents, as well as DPPH free radical scavenging activity, is presented in Table 1. The phenolic contents of the hydroethanolic extracts ranged from 96.7 to 169.4 mg GAE/g DT, with the highest concentration observed in A. odoratisimma (167.6 mg GAE/g DT). The total flavonoid content varied from 6.9 to 95.1 mg RE/g DT, while P. latifolia exhibited the highest levels of condensed tannins (73.5 mg CE/g DT). Among the extracts, D. incanum (Sw.) displayed the highest DPPH free radical scavenging activity, with an IC50 value of 0.04 mg/mL. The DPPH free radical scavenging activity of P. latifolia is depicted in Figure 1. At a concentration of 4 mg/mL, the extract demonstrated a scavenging activity of 84.1%. The IC50 value for P. latifolia extract was determined to be 0.95 mg/mL. The antioxidant assay yielded values ranging from 20 to 476.6 µM Trolox.

Screening of Cytotoxic Activity of Hydroethanolic Extracts
The cytotoxicity of the examined plant extracts on HepG2 cells is depicted in Figure 2. HepG2 cells exposed to the P. latifolia extract at concentrations ranging from 32 to 500 µg/mL showed a significant concentration-dependent reduction in cell viability, particularly after 48 h (IC 50 = 17.2 µg/mL). In contrast, the other extracts exhibited lower cytotoxicity at both exposure times, with IC 50 values exceeding 500 µg/mL.

Screening of Cytotoxic Activity of Hydroethanolic Extracts
The cytotoxicity of the examined plant extracts on HepG2 cells is depicted in Figure  2. HepG2 cells exposed to the P. latifolia extract at concentrations ranging from 32 to 500 µg/mL showed a significant concentration-dependent reduction in cell viability, particularly after 48 hours (IC50 = 17.2 µg/mL). In contrast, the other extracts exhibited lower cytotoxicity at both exposure times, with IC50 values exceeding 500 µg/mL.

Cell viability (%)
Plants 2023, 12, x FOR PEER REVIEW 5 of 13 The IC50 values after 48 hours of exposure were found to be low for the extracts of P. latifolia (IC50: 17.2 µg/mL) and A. odoratissima (IC50: 54.2 µg/mL). The results on the cytotoxicity of noncancerous cells (HEKn) exposed to A. odoratissima and P. latifolia extract and the selectivity index (SI) are shown in Figure 3 and Table 2. These extracts demonstrated lower cytotoxicity towards HEKn cells compared to HepG2 cells, with IC50 values of 38.9 µg/mL and 54.2 µg/mL for P. latifolia and A. odoratissima, respectively, after 48 h of exposure. Notably, the P. latifolia extract exhibited a more pronounced cytotoxic effect on cancer cells compared to non-cancer cells (SI: 2.3, 48 h). Furthermore, both extracts displayed The IC 50 values after 48 h of exposure were found to be low for the extracts of P. latifolia (IC 50 : 17.2 µg/mL) and A. odoratissima (IC 50 : 54.2 µg/mL). The results on the cytotoxicity of noncancerous cells (HEKn) exposed to A. odoratissima and P. latifolia extract and the selectivity index (SI) are shown in Figure 3 and Table 2. These extracts demonstrated lower cytotoxicity towards HEKn cells compared to HepG2 cells, with IC 50 values of 38.9 µg/mL and 54.2 µg/mL for P. latifolia and A. odoratissima, respectively, after 48 h of exposure. Notably, the P. latifolia extract exhibited a more pronounced cytotoxic effect on cancer cells compared to non-cancer cells (SI: 2.3, 48 h). Furthermore, both extracts displayed moderate selectivity, as depicted in Figure 3. * Significant difference (p < 0.05) when compared to the control group (C).
The IC50 values after 48 hours of exposure were found to be low for the extracts of P. latifolia (IC50: 17.2 µg/mL) and A. odoratissima (IC50: 54.2 µg/mL). The results on the cytotoxicity of noncancerous cells (HEKn) exposed to A. odoratissima and P. latifolia extract and the selectivity index (SI) are shown in Figure 3 and Table 2. These extracts demonstrated lower cytotoxicity towards HEKn cells compared to HepG2 cells, with IC50 values of 38.9 µg/mL and 54.2 µg/mL for P. latifolia and A. odoratissima, respectively, after 48 h of exposure. Notably, the P. latifolia extract exhibited a more pronounced cytotoxic effect on cancer cells compared to non-cancer cells (SI: 2.3, 48 h). Furthermore, both extracts displayed moderate selectivity, as depicted in Figure 3.

Cytoprotective effects of P. latifolia against α-ZEL and APAP
The protective effect of P. latifolia on α-ZEL and APAP after 24 hours of exposure is depicted in Figure 4. The results demonstrated that simultaneous treatment of HepG2 cells with α-ZEL (12.5 and 25 µM) or APAP (12.5 and 25 mM) along with the extract (8 µg/mL), significantly (p < 0.05) preserved cell viability, with cytoprotection ranging from

Cytoprotective Effects of P. latifolia against α-ZEL and APAP
The protective effect of P. latifolia on α-ZEL and APAP after 24 h of exposure is depicted in Figure 4. The results demonstrated that simultaneous treatment of HepG2 cells with α-ZEL (12.5 and 25 µM) or APAP (12.5 and 25 mM) along with the extract (8 µg/mL), significantly (p < 0.05) preserved cell viability, with cytoprotection ranging from 8% to 13% and 21% to 8%, respectively. Additionally, it was observed that the IC 50 remained similar under these conditions. Plants 2023, 12, x FOR PEER REVIEW 6 of 13 8% to 13% and 21% to 8%, respectively. Additionally, it was observed that the IC50 remained similar under these conditions.  . Cytoprotective effects of P. latifolia extract on HepG2 cells exposed to α-ZEL and APAP after treatment for 24 h. * Significant difference (p < 0.05) compared to the group treated with α-ZEL or APAP.

ALT and AST Activities
The results of ALT and AST activities in HepG2 cells exposed to hydroalcoholic extracts from P. latifolia and A. odoratissima are presented in Figure 5. No statistically significant differences were observed in the mean ALT or AST activities induced by the extracts of the two plants when compared to the control cells. Moreover, it was found that both α-ZEL and β-ZEL exhibited high cytotoxicy. . Cytoprotective effects of P. latifolia extract on HepG2 cells exposed to α-ZEL and APAP after treatment for 24 h. * Significant difference (p < 0.05) compared to the group treated with α-ZEL or APAP.

ALT and AST Activities
The results of ALT and AST activities in HepG2 cells exposed to hydroalcoholic extracts from P. latifolia and A. odoratissima are presented in Figure 5. No statistically significant differences were observed in the mean ALT or AST activities induced by the extracts of the two plants when compared to the control cells. Moreover, it was found that both α-ZEL and β-ZEL exhibited high cytotoxicy.

Discussion
Medicinal plants hold significant importance as therapeutic agents in folk medicine, especially in developing countries. In this study, semi-structured interviews and open discussions were conducted with six plant connoisseurs to gather detailed information on 100 plant species used in traditional medicine in San Basilio de Palenque, Colombia. From this extensive list, nine plant species were selected based on their wide usage in traditional medicine for various diseases. These nine species were then screened for cytotoxicity on HepG2 cells. Among the tested species, hydroethanolic extracts from the stems of A. odoratissima and P. latifolia were specifically evaluated for their potential anticancer activity using the normal liver epithelial cell line (HEKn) as a representative of normal human cells, to assess the toxicity of both extracts Plants are known for their rich content of biologically active secondary metabolites, which play a positive role in human health by preventing and treating various diseases. Phenolic compounds, including flavonoids, are among the key compounds with beneficial properties such as antioxidant, anti-carcinogenic, cardioprotective, immune system support, anti-inflammatory, skin protective, and antibacterial activities [11]. Flavonoids, being the most abundant phenolics in plants, possess diverse bioactivities attributed to their Figure 5. Effect of plant extracts, α-ZEL, and β-ZEL on ALT (left) and AST (right) levels measured in culture media of unexposed control cells and exposed cells. * Significant difference (p < 0.05) when compared to the negative control group (C-). α-ZEL and β-ZEL were used as positive controls.

Discussion
Medicinal plants hold significant importance as therapeutic agents in folk medicine, especially in developing countries. In this study, semi-structured interviews and open discussions were conducted with six plant connoisseurs to gather detailed information on 100 plant species used in traditional medicine in San Basilio de Palenque, Colombia. From this extensive list, nine plant species were selected based on their wide usage in traditional medicine for various diseases. These nine species were then screened for cytotoxicity on HepG2 cells. Among the tested species, hydroethanolic extracts from the stems of A. odoratissima and P. latifolia were specifically evaluated for their potential anticancer activity using the normal liver epithelial cell line (HEKn) as a representative of normal human cells, to assess the toxicity of both extracts Plants are known for their rich content of biologically active secondary metabolites, which play a positive role in human health by preventing and treating various diseases.
Phenolic compounds, including flavonoids, are among the key compounds with beneficial properties such as antioxidant, anti-carcinogenic, cardioprotective, immune system support, anti-inflammatory, skin protective, and antibacterial activities [11]. Flavonoids, being the most abundant phenolics in plants, possess diverse bioactivities attributed to their potent antioxidant potential [11]. The results of this study revealed high levels of antioxidant compounds, specifically phenolics and flavonoids, in the hydroethanolic extracts. The highest concentrations of total phenols (>150 mg GAE/g DT) were found in A. odoratissima, J. gossypiifolia L., P. peltatum L., D. incanum (Sw.), and D. unguis-cati (L.) L.G. Lohmann. Regarding flavonoids, concentrations exceeding 30 mg RE/g DT were detected in P. latifolia, D. incanum (Sw.), and A. odoratissima. These findings align with a study conducted by Mariyammal et al. [12], which reported 3.2-fold lower phenolic content in the hydroalcoholic leaf extract of Aristolochia tagala compared to the species of the genus Aristolochia evaluated in this work (52.58 ± 0.06 mg GAE/g vs. 167.6 ± 1.3 mg GAE/g).
The screening of cytotoxic activity revealed that A. odoratissima and P. latifolia hydroethanolic extracts exhibited a high cytotoxic effect on HepG2 cells, with IC 50 values of 54.2 µg/mL and 17.2 µg/mL, respectively, after 48 h of exposure. Previous studies have reported higher IC 50 values (>200 µg/mL) for A. odoratissima methanolic extract on human kidney (HK-2) cells [13]. In San Basilio de Palenque, various connoisseurs have highlighted the usage of Aristolochia species in treating several diseases, including diabetes, cancer, inflammation, snakebites, viral and bacterial infections, and stomach problems, among others. These medicinal plants from the Aristolochia genus are not only utilized in America but also in Asian countries [12]. In Colombia, A. odoratissima is commonly known as 'capitana' [14]. In San Basilio de Palenque, it is primarily used for reducing inflammation-related pain and as an antidote against venomous animals, which aligns with the properties reported by Montiel-Ruiz et al. [15]. It was somewhat unexpected to find that the extract of D. incanum presented a very low IC 50 (0.04 mg/mL) in relation to the total flavonoid content, which is within the same order of magnitude as the other extracts. However, this behavior could be due to several factors. First is the presence of one or several substances that can interfere with the DPPH assay, as has been documented for pigments [16], but this probably was not the case, as the extract had very little color. Second, substances different from flavonoids, such as alkaloids bearing free phenolic groups, can also exert antiradical activities [17].
Picramnia latifolia Tul. (Picramniaceae) is a plant species found in tropical regions. Extracts derived from this plant have been reported to possess anti-plasmodial effects [18]. Moreover, the P. latifolia extract exhibited a high selectivity index (>2), which could be attributed to its content of flavonoids and phenols. Interestingly, co-treatment with the P. latifolia extract and α-ZEL demonstrated a cytoprotective effect on HepG2 cells. It is worth noting that the evaluated extracts did not induce significant changes in the activity of liver damage marker enzymes (ALT and AST) when compared to the control group (untreated cells), suggesting the potential safety of both extracts for phytotherapeutic treatments.
The current uses and reported secondary metabolites for studied plants are presented in Table 3. Table 3. Medicinal applications of plants and their reported chemical constituents.

J. gossypiifolia
Anti-inflammatory, anti-hemorrhagic, edematogenic, and antimicrobial. Latex used to treat wounds and bites of snakes.

Species Current Uses Reported Secondary Metabolites References
V. turbacensis Anticryptococcal.

Plant Material
The study began with semistructured interviews and open discussions involving six plant connoisseurs who provided detailed information on 100 plant species commonly used in traditional medicine. From this extensive list, nine species were selected based on their widespread use in treating various diseases. Plant samples, including roots, stems, bark, or leaves, were collected from different areas in San Basilio de Palenque between August and September 2022. These plant specimens were then identified and deposited at the Colombian National Herbarium, with voucher specimens available (refer to Table 4). Additionally, the detailed information obtained from the interviews and discussions is provided in Table S1.

Extraction
The plant materials were air dried in the laboratory at room temperature (22-24 • C) and subsequently cut and powdered at the Ecotoxicology Laboratory, University of Cartagena (Cartagena, Colombia). The dried plant material was ground, and 200 g of the powdered material was subjected to extraction for 48 h using a mixture of ethanol (99.9%) and MilliQ water in a 70:30 ratio (v/v). After 24 h, the extract was filtered using Whatman paper, and the plant material was then subjected to another 24 h of hydroalcoholic extraction. The combined solutions were rotary evaporated to a volume of 40 mL and subsequently freeze-dried. Finally, the extracts were stored at −20 • C until further use [33].

Total Phenol Content
The total phenolic content was assayed according to Sánchez-Gutiérrez et al. [34] with modifications. Briefly, an aliquot of diluted hydroethanolic extract (0.25 mL) was added to 0.25 mL of distilled water and 0.125 mL of Folin-Ciocalteu reagent. The mixture was shaken and allowed to stand for 5 min before the addition of 0.375 mL of 20% (w/v) Na 2 CO 3 . After incubation in the dark for 2 h, absorbance at 760 nm was read versus a prepared blank using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The total phenol content of the hydroethanolic extract was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry tissue (DT) (mg GAE/g DT) from a calibration curve with gallic acid. All samples were analyzed in three replicates.

Total Flavonoid Content
The measurement of total flavonoids was performed using the AlCl 3 method, following the procedure described by Sánchez-Gutiérrez et al. [34] with slight modifications. In brief, a diluted hydroethanolic extract (0.4 mL) or a standard solution of rutin was mixed with AlCl 3 ·6H 2 O solution (2%, 0.4 mL) and allowed to react for 3 min. The absorbance of the resulting mixture was measured at 415 nm against a blank using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The total flavonoid content was calculated as milligrams of rutin equivalents per gram of dry tissue (mgRE/g DT) using a calibration curve constructed with rutin. All samples were analyzed in triplicate.

Total Condensed Tannins
The evaluation of condensed tannins was conducted using the vanillin-H 2 SO 4 method, following the methodology described by Sánchez-Gutiérrez et al. [34]. Briefly, diluted hydroethanolic extract (0.25 mL) was mixed with 1% (w/v) vanillin solution in methanol (0.25 mL) and 25% (v/v) H 2 SO 4 (0.25 mL). The mixture was allowed to stand for 15 min, and the absorbance was measured at 500 nm against a methanol blank using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The total condensed tannin content was expressed as milligrams of (+) catechin equivalents per gram of dry tissue (mg CE/g DT). All samples were analyzed in triplicate.

DPPH• Free Radical Scavenging Assay
The free radical scavenging activity was determined using a standard kit (Bioquochem, Llanera, Asturias, Spain) following previously described methods by Alvarez-Ortega et al. [35]. A 100,000 µg/mL solution of the hydroethanolic extract in Milli-Q water was prepared. To assess the scavenging activity, 200 µL of DPPH• reagent was mixed with 20 µL of the extract (with gradually increased concentrations ranging from 0.125 to 6 mg/mL) and incubated at room temperature for 1 h. After incubation, the absorbance was measured at 517 nm using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A TroloxR standard curve was utilized to determine the TEAC (Trolox Equivalent Antioxidant Capacity) of the tested extract concentrations. The concentration of the extract solution that caused 50% inhibition of the DPPH radical (IC 50 ) was determined from the nonlinear regression plot of the inhibition percentage against the concentrations.

MTT Assay
The cell lines were seeded in 96-well plates at a density of 2 × 10 4 cells per well and allowed to adhere for 24 h. The HepG2 cells were then treated with the extract at concentrations ranging from 500 to 3.9 µg/mL (1:2 dilutions) for 24 h. Subsequently, MTT solution was added to each well at a concentration of 3 mg/mL, and the plates were further incubated at 37 • C for 4 h. After incubation, 0.2 mL of DMSO was added to dissolve the formazan crystals, and the absorbance was measured at 620 nm using a spectrophotometric microplate reader (Varioskan™ LUX, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell viability was determined by comparing the absorbance of treated cells to that of untreated cells. Three independent experiments were performed with four replicates each. Furthermore, the cytotoxicity of P. latifolia and A. odoratissima extracts on primary HEKn cells was assessed. The selectivity index (SI) was obtained calculated as the ratio of the IC 50 value of noncancer cells to the IC 50 value of cancer cells.

Cytoprotective Effects of P. latifolia and A. odoratissima Extracts against ZEN Metabolites
To assess the cytoprotective effect of P. latifolia and A. odoratissima extracts against ZEN metabolite-induced toxicity, a concentration of 8 µg/mL was selected for both extracts, which showed no significant cytotoxicity. The experiments consisted of treating the cells with a fixed concentration of P. latifolia extract (8 µg/mL) in combination with various concentrations of α-ZEL or β-ZEL (ranging from 0.4 to 100 µM, with 1:2 dilutions). The plates were then incubated for 24 h and 48 h at 37 • C in a 5% CO 2 atmosphere, and cell viability was assessed using the MTT assay. Three independent experiments were conducted, with four replicates for each condition.

Biochemical Analysis
The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured according to established methods [37,38]. The absorbance at 535 nm was measured using a Bio-systems BTS-350 spectrophotometer (Barcelona, Spain). The concentrations of AST and ALT were expressed as units per liter (U/L).

Statistical Analysis
The results are presented as the mean ± standard error of the mean (X ± SEM). The normality of the data was assessed using the Shapiro-Wilk test. Multiple data comparisons were performed using ANOVA, followed by Dunnett's test. The IC 50 values for each treatment were determined using nonlinear sigmoid curve fitting. Statistical significance was defined as p < 0.05. All statistical analyses were conducted using GraphPad Prism 8.0 software.

Conclusions
The biota of San Basilio de Palenque presents a promising source of anti-inflammatory plant species rich in flavonoids, phenolics, and tannins. P. latifolia shows potential for isolating natural products with anticancer properties, while the extract of A. odoratissima holds promise as an immunomodulator plant and a potential candidate for further investigations in developing natural compounds with immunomodulatory and anticancer properties. However, further studies are required to fully explore and understand the potential benefits of these plants.