Chemical composition and in vitro bioactivities of hydroalcoholic extracts from Turnera pumilea, Hyptis lantanifolia, and Ageratina popayanensis cultivated in Colombia

Abstract

Colombia’s strategic location, encompassing diverse climates and ecosystems, enables abundant flora. This environment offers bioprospecting opportunities, emphasizing sustainable plant use. Our main objective was to expand the knowledge of the chemical, pharmacological, and cosmetic potential of three cultivated species, namely, T. pumilea, H. lantanifolia, and A. popayanensis. UHPLC-ESI-Orbitrap-HRMS and quantification of total flavonoid, phenolic, and condensed tannin contents were employed to elucidate the chemical composition. The antioxidant activity and photoprotective capacity were evaluated by spectrophotometry. Calu-1 and HepG2 cells were used to assess cytotoxicity by MTT assay, and early intracellular ROS production was evaluated using an H₂-DCFDA probe. The results demonstrate the richness of the chemical composition of the studied species, identifying compounds with therapeutic properties, including gallic acid, p-coumaric acid, caffeic acid, rosmarinic acid, chlorogenic acid, apigenin, and rutin, which are recognized as antioxidant and ultraviolet protective agents, supporting their uses in traditional medicine. The cytotoxicity evaluation on Calu-1 and HepG2 cells revealed the antiproliferative effects of these plants, especially the T. pumilea extract, on Calu-1 cells, and the possible influence of ROS generation, which could explain the observed effects. This research underlines the need for thorough evaluations of toxicological effects and the characterization of the mechanism of action involved to support the results obtained.

Introduction

Colombia ranks second in South America in botanical diversity, consolidating itself as a megadiverse country¹ that remains largely underexplored². Its strategic geographical position, which encompasses a wide variety of climates and the coexistence of diverse ecosystems, plays a crucial role in the richness and abundance of its flora³. This privileged environment opens significant opportunities for bioprospecting studies, highlighting the need to promote the sustainable use of plant resources.

However, in the Colombian territory, unsustainable agricultural practices, climate change, and sociocultural factors that induce population displacements threaten the conservation of species and ancestral knowledge about their medicinal properties. This situation puts at risk not only biodiversity, but also cultural heritage linked to native flora⁴. Faced with this panorama, the search for economic development based on biodiversity promotes recognition of plants’ potential and the adoption of sustainable agricultural practices, to preserve the vegetable wealth of the country and guarantee its long-term responsible use⁵. Thus, the conservation and management of natural resources are presented as fundamental pillars to maintain the biological diversity that characterizes Colombia and to ensure that future generations can benefit from its multiple uses, both in traditional medicine and in scientific and technological innovation. Research initiatives in this field facilitate the discovery of valuable bioactive compounds and emphasize the urgent need for responsible management of natural resources (https://bio-reto-xxi.uis.edu.co/ accessed March 10, 2024).

Within this context, the chemical analysis and biological activities identified in extracts from grown plants play a fundamental role in the valorisation of biodiversity, serving as a basis for the sustainable development of products intended for human consumption, such as cosmetics, cosmeceuticals, and pharmaceuticals^{6, 7, 8–9}. To this end, three plant species cultivated in Bucaramanga, Colombia: Turnera pumilea, Hyptis lantanifolia, and Ageratina popayanensis, have been selected based on their notable chemical and biological characteristics, which hold potential relevance for the cosmetic and pharmaceutical industries.

Turnera pumilea L. is one of 140 accepted species of the genus Turnera in Plants of the World Online of Royal Botanic Gardens [https://powo.science.kew.org/ accessed March 10, 2024], sometimes identified as part of the Turneraceae family. However, the Angiosperm Phylogeny Group (APG) III belongs to the Passifloraceae family¹⁰so the APG III guidelines will be considered in this research. The common name of this species is “Damiana” or “Yellow Damiana”. It is described as an annual subshrub that grows mainly in the seasonally dry tropical biome, with a native distribution area ranging from Mexico to Tropical America. Species of this genus have a wide range of traditional uses, including abortive properties, and the treatment of anemia, diabetes, respiratory tract, gastrointestinal, and skin disorders¹⁰.

Hyptis lantanifolia Poit. (Lamiaceae) is a species popularly known as “Santa Maria Bush” or “Coquito de Monte” that grows in neotropical regions with ample distribution in Colombia and Mexico [https://www.gbif.org/es/species/2926810 accessed March 16, 2024]. Species within this genus have been utilized in traditional medicine for treating viral processes, infections, fever, cramps, and disorders in the liver, gastrointestinal tract, and skin¹¹. In recent years, scientific evaluation of the genus Hyptis has revealed its potential as a hormone modulator, and anti-inflammatory, anticancer, antimicrobial, insecticidal, molluskicidal, and acaricidal agent¹². The water extract obtained from the whole plant of H. lantanifolia has shown potent inhibition of human immunodeficiency virus-reverse transcriptase (HIV-RT); hence, its anti-HIV effects are presently under investigation¹³.

Ageratina popayanensis (Hieron.) R.M.King & H.Rob. The so-called “Chilco Negro” or “Amargoso” is a species of the Asteraceae family native to Colombia with distribution in the northern region of the Andes, including Venezuela [https://www.gbif.org/es/species/5400619 accessed March 16, 2024]. Different studies on extracts and phytocompounds identified in species of this genus have revealed a wide spectrum of bioactivities, including anti-inflammatory, antiviral, antimicrobial (relief of onychomycosis), antiprotozoal, antioxidant, antidiabetic, antinociceptive, antifeedant, larvicidal, acaricidal, and wound-healing activities¹⁴.

This research aims to generate valuable insights into the hydroalcoholic extracts of T. pumilea, H. lantanifolia, and A. popayanensis, focusing on their chemical composition and potential applications in the pharmaceutical and skincare industries. Key areas of investigation include the in vitro antioxidant capacity, photoprotective effects, cytotoxicity against human cancer cell lines (Calu-1 and HepG2), and early reactive oxygen species (ROS) generation. These findings will contribute to understanding the traditional uses and the sustainability of Colombia’s rich botanical resources.

Materials and methods

Vegetal material

Aerial parts of T. pumilea L. (Passifloraceae), H. lantanifolia Poit (Lamiaceae), and A. popayanensis (Hieron) R. King & H. Rob (Asteraceae) were obtained from the garden of the National Center for Agroindustrialization of Aromatic and Medicinal Tropical Plant Species (CENIVAM) at the Industrial University of Santander (UIS, Bucaramanga, Colombia). Botanical identification was carried out at the UIS Herbarium (national registry RNC:030) under the supervision of the curator, Prof. Dr. Andrés Felipe Castaño González. The process was based on morphological comparison with reference specimens and the use of standard taxonomic keys for the families Passifloraceae, Lamiaceae, and Asteraceae. The corresponding exsiccatae and voucher specimens were deposited at the UIS Herbarium under the following accession codes: T. pumilea (UIS 21972), H. lantanifolia (UIS 21974), and A. popayanensis (UIS 22040).

Hydroalcoholic extracts

The aerial parts of the plants (100 g), either fresh before distillation or residual biomass was dried and ground, were combined with a 70% aqueous ethanol solution (2 L) and placed in an ultrasonic bath (Elmasonic S15H, Singen, Germany) at 50 °C for 1 h. Afterward, the extracts were filtered, concentrated using a Heidolph rotary evaporator (Hei-VAP, Advantage HL, Chicago, IL, USA), and then lyophilized (VirTis AdVantage Plus, SP Scientific, Gardiner, NY, USA). The final products were stored at 4 °C, in the dark. Solvent extractions were carried out in triplicate.

Sample preparation for UHPLC-ESI-Orbitrap-HRMS analysis

The samples were weighed (0.1 mg) and dissolved in a mobile phase of methanol (MeOH): water 50:50 with 0.1% formic acid (FA) and 5 mM ammonium formate (1 mL) to achieve a final concentration of 100 mg/L. Phenolic compounds were analyzed using an ultra-high performance liquid chromatography system on a VanquishTM instrument (Thermo Scientific, Waltham, MA, USA), equipped with a thermostatically controlled column compartment (40 °C). Chromatographic separation was performed on a Zorbax Eclipse XDB C18 column (Sigma Aldrich, St. Louis, MO, USA) 50 mm L × 2.1 mm I.D. with a particle size of 1.8 μm. The flow rate of the mobile phase, containing (A) water (0.1% FA + 5 mM FA) and (B) MeOH (0.1% FA + 5 448 mM FA), was 300 µL/min. The initial gradient conditions were 100% A, which was linearly changed to 100% B over 8 min, held for 4 min, returned to 100% A in 1 min, and held for 3 min. The injection volume was 2 µL. The liquid chromatograph was connected to a Q Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) with a heated electrospray ionization source (HESI-II). C-Trap ions were transferred to a high-energy collisional dissociation (HCD) cell and fragmented with collision energies ranging from 10 to 70 eV. Data were acquired and analyzed using Thermo Scientific™ Dionex™ Chromatography Data System (CDS) software, version 7.2, and Thermo Xcalibur 3.1 software (Thermo Scientific, San Jose, CA, USA). Compound identification was performed by comparing retention times (tR), exact masses, isotopic patterns, and MS spectra with those of standard substances, and by consulting spectral databases [e.g., HMDB (Wishart, D.S., Guo, A., Oler, E. & Wang, F. HMDB 5.0: The human metabolome database for 2022. Nucleic Acids Research. 2022. Available on-line: www.hmdb.ca(open in a new window) and MassBank (MassBank. Available on-line: www.massbank.eu/MassBank/(open in a new window)] and relevant literature. The confidence level for each compound was assigned according to the Metabolomics Standards Initiative (MSI) and is indicated in the table footnotes as follows: (a) confirmed identification using authentic standards (MSI Level 1); (b) tentative identification based on literature reports for species of the genera Turnera, Hyptis, and Ageratina (MSI Level 2); and (c) tentative annotation based solely on exact mass and isotopic pattern (MSI Level 3).

Total flavonoid, phenolic, and tannin contents

For the quantification of total flavonoid, phenolic, and condensed tannin contents the methodology of Sánchez-Gutiérrez et al.¹⁵ was followed. Absorbance measurements were acquired using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The total flavonoid content was quantified by constructing a standard curve of the flavonoid rutin (A_{415 nm} = 0.0089x + 0.0017, R² = 0.9994) at five concentrations ranging from 0.002 to 0.0315 mg/mL. Flavonoid levels were expressed as milligrams of rutin equivalents per gram of dry tissue (mg RE/g DT), with the extract concentration for analysis set at 1.0 mg/mL.

Similarly, the total phenolic content was determined using a gallic acid standard curve (A_{760 nm} = 0.0046x + 0.044, R² = 0.9966) established from seven concentrations ranging from 0.003 to 0.200 mg/mL. The phenolic content was reported as milligrams of gallic acid equivalents per gram of dry tissue (mg AGE/g DT), with the extract concentration for analysis set at 1.0 mg/mL.

The total condensed tannin content was assessed through a (+) catechin standard curve (A_{500 nm} = 0.0037x + 0.1181, R² = 0.9873) derived from eight concentrations ranging from 0.0078 to 1.00 mg/mL. Condensed tannin levels were expressed as milligrams of (+) catechin equivalents per gram of dry tissue (mg CE/g DT), with the extract concentration for analysis set at 1.0 mg/mL.

Antioxidant activity

The DPPH assay, a widely recognized and used method in antioxidant research, was used to determine the antioxidant capacity of the extracts^4,16,17. This assay allows estimating the efficacy of antioxidant compounds in neutralizing superoxide anion (O₂•−), hydroxyl (•OH), alkoxyl (RO•), and peroxyl radicals (ROO•). These molecules can cause cellular damage and contribute to the development of various pathologies associated with the generation of ROS, such as cancer¹⁸ and skin disorders⁸.

The principle of the DPPH assay is based on the reduction of the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl), which presents an intense purple color, to a non-radical form, which progressively fades upon interaction with antioxidants. The antioxidant capacity evaluated in this assay reflects a mixed mechanism involving both electron transfer and hydrogen atom transfer. The reaction rate of the DPPH radical depends largely on the steric accessibility of the radical site, which influences the efficiency with which antioxidants can donate electrons or hydrogen atoms to neutralize it¹⁹.

This study employed the kit from Bioquochem (Llanera, Asturias, Spain) to determine the DPPH radical scavenging activity¹⁷. Each extract was tested in duplicate at five different concentrations (1:2 dilution), ranging from 31.23 to 500 µg/mL. The extract (20 µL) was subsequently reacted with DPPH solution (200 µL) in the dark at room temperature for 30 min. A Trolox standard curve was prepared at the various concentrations (500, 400, 300, 200, 100, and 0 µg/mL). The absorbance data at 517 nm were acquired with a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The scavenging DPPH radical percentage (%) for each standard point was obtained with the following formula: % inhibition = [1 − (Abs _Sn/Abs _S1)] × 100, where Abs _S1 is the DPPH•+ radical absorption without inhibition and Abs _Sn is the DPPH•+ radical absorption of the corresponding standard. The Trolox equivalent antioxidant capacity (TEAC) of the tested concentrations of extracts was subsequently calculated via the following formula: TEAC (µM) = % inhibition − intercept/slope.

Photoprotective capacity

Based on its wavelength, the ultraviolet (UV) radiation emitted by sunlight is classified into three categories: UVA (320–400 nm), UVB (280–320 nm), and UVC (100–280 nm). While UVC rays are largely absorbed by the ozone layer and do not reach the Earth’s surface, approximately 95% and 5% of UVA and UVB rays penetrate the atmosphere, respectively. These UV radiations pose a significant health risk when overexposed, as they can trigger various conditions, such as photoaging, immune system suppression, and tumor development. Currently, the use of plant extracts as natural sunscreens is being explored to counteract these harmful effects. Plant extracts are an interesting source of bioactive compounds (flavonoids and phenolic compounds) that exhibit a remarkable capacity to absorb radiation in the wavelength range between 280 and 400 nm, corresponding to the UVB and UVA regions of the UV spectrum. This property makes them valuable ingredients for incorporation into cosmetic and cosmeceutical formulations, where they act as natural filters that contribute to skin protection. In addition to their absorption capacity, these compounds possess antioxidant activities that help neutralize free radicals generated by sun exposure^20,21.

In this study, the photoprotective capacity of the extracts was evaluated by spectrophotometry with a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA)⁷. The absorbance data at different wavelengths and optical path lengths were recorded to determine the sun protection factor (290–320 nm; 5 nm), critical wavelength (290–400 nm; 1 nm), UVA/UVB ratio (290–400 nm; 5 nm), and erythema transmission/pigmentation transmission (292–372 nm; 5 nm). The UV protection criteria used were those described previously^3,7.

Sun protection factor (SPF)

SPF values were obtained by applying the Mansur equation²²:: where CF is the correction factor (equal to 10), EE (λ) × I(λ) are constants at each wavelength obtained from the correlation between EE (erythematous effect of radiation of wavelength λ) and I (the solar intensity at wavelength λ) (Supplementary Table S1), and Abs is the absorbance of the solution at wavelength λ.

Critical wavelength (λc)

λc is the wavelength at which 90% of the area under the absorbance curve is found, considering the integral of the absorption spectrum (290 nm to 400 nm)²³. This value was determined via the following equation: where A is the absorbance at wavelength λ^3,7.

UVA/UVB ratio

To determine the UVA/UVB ratio, the average UVA absorbance was related to the average UVB absorbance using the following equation:

, where A(λ) is the effective absorbance related to the transmittance of the sunscreen²³.

Percentages of erythema transmission and pigmentation transmission

The ability to induce redness (erythema transmission) or sunspots on the skin (pigmentation transmission), was determined from the transmission (T), to then apply the following Eq²⁴.:

1. , where Fe (erythema flux) is a constant that corresponds to wavelengths ranging from 292 to 338 nm (Supplementary Table S2).

2. where Fp (pigmentation flux) is a constant that corresponds to wavelengths ranging from 322 to 372 nm (Supplementary Table S2).

Cytotoxicity and early intracellular ROS production

In this study, the MTT assay was employed to evaluate the in vitro cytotoxicity of plant extracts by measuring cell viability. MTT (3-(4, 5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide), a water-soluble tetrazolium salt, is selectively absorbed by metabolically active cells and enzymatically reduced by mitochondrial succinate dehydrogenase to form insoluble purple formazan crystals. These crystals are subsequently dissolved using an organic solvent, typically dimethyl sulfoxide (DMSO), and quantified spectrophotometrically. The intensity of the purple color directly correlates with the number of viable cells, providing a reliable indicator of cellular metabolic activity²⁵.

The human lung epidermoid carcinoma cells (Calu-1) and human hepatocellular carcinoma cell line (HepG2) obtained from the American Type Tissue Culture Collection (ATCC, USA) and ScienCell (Carlsbad, CA, USA) were cultured in Eagle’s minimum essential medium (EMEM; Quality Biological, Gaithersburg, MD, USA) supplemented with 10% FBS (Biowest, Riverside, MO, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MI, USA), and maintained in a humidified atmosphere with 5% CO₂ at 37°C⁴.

The cells were seeded in 96-well plates at a density of 2 × 10⁴ cells per well and incubated at 37 °C in 5% CO₂ for 24 h. The cells were then treated with the extract at concentrations ranging from 3.9 to 500 µg/mL for 24 h. Subsequently, MTT solution was added to each well at a concentration of 5 mg/mL, and the plates were further incubated at 37 °C for 4 h. After incubation, 200 µL of DMSO (Panreac Applichem^®, Barcelona, Spain) was added to dissolve the formazan crystals in each well, and the absorbance was measured at 570 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 for each treatment⁵.

Finally, the percentages of cell viability were normalized and calculated with the following equation: cell viability (%) = (OD_test/OD_control)×100, where OD is the optical density or absorbance of the test or control, respectively.

To quantify the early ROS production of the three extracts, hydrogen peroxide (H₂O₂) at a concentration of 200 µM was used as a positive control, which resulted in high intracellular ROS production at each recorded time interval (up to 120 min)³. In this study, the results revealed that the hydroalcoholic extracts from T. pumilea, H. lantanifolia, and A. popayanensis evaluated in HepG2 and Calu-1 cells showed no significant differences in the levels of ROS detected compared with those in the control.

Data analysis

Data were obtained in triplicate (n = 3) for the total content of flavonoids, phenolics, and condensed tannins, as well as for the photoprotective activity, cytotoxicity, and early intracellular ROS, and in duplicate (n = 2) for antioxidant activity. The results are expressed as the mean ± SD (standard deviation). The normality of the data obtained from the cell viability assays employing Calu-1 and HepG2 cell lines was assessed using the Shapiro-Wilk test. Significant differences between experimental groups were evaluated using a one-way analysis of variance (ANOVA), followed by Dunnett’s test. Each treatment’s mean inhibitory concentration at 50% (IC₅₀ values) was calculated using a non-linear sigmoid curve fitting with a four-parameter logistic model. For each IC₅₀ estimate, 95% confidence intervals were reported. This fit was performed on the cell proliferation data versus the logarithm of the concentration of the compound. The results were analyzed using GraphPad Prism 8.0.

Results

Hydroalcoholic extracts of the plant species T. pumilea, H. lantanifolia, and A. popayanensis were analyzed by UHPLC-ESI+/−-Orbitrap-MS. The extracted ion currents (EICs), presented in Fig. 1A and C, allowed the precise detection and quantification of the specific ions present in each extract, filtered in SIM mode to identify both protonated [M + H] + and deprotonated [M − H] − species. The analysis facilitated the chemical characterization of the compounds in the evaluated extracts. The initial identification was based on comparing exact masses and isotopic patterns, allowing the assignment of molecular formulas. These data were subsequently compared with information available in scientific literature, especially in studies related to species of the genera Turnera, Hyptis, and Ageratina, providing taxonomic and chemical support for the assignments made. Finally, the identification was confirmed by comparing retention times (tR) and mass spectra with reference standards, ensuring the correct assignment of key metabolites. Furthermore, a high-energy collision dissociation (HCD) cell was used to generate characteristic fragments that further elucidated the structure of the detected compounds. The complete results of this analysis are summarized in Tables 1 and 2, and 3.

Fig. 1 [Images not available. See PDF.]

Extracted ion currents (EICs), obtained using UHPLC-ESI^+/−-Orbitrap-MS, operated in SIM mode to filter the protonated [M + H]⁺ or deprotonated [M − H]⁻ molecules of substances present in the hydroalcoholic extract of (A) T. pumilea, (B) H. lantanifolia, and (C) A. popayanensis.

Table 1. Exact masses of protonated [M + H]⁺ and deprotonated [M – H]^– molecules, identified by UHPLC/ESI-Q-Orbitrap-HRMS, in the hydroalcoholic extract of T. pumilea.

Peak N° Figure 1A	Compound	tR (min)	Formula	Exp. masses		Δppm	HCD, eV	Product-ions	Formula	m/z I(%)	Ref.
				[M-H]–	[M + H]+
1	Gallic acid a	1.96	C7H6O5	169.01324	-	0.56	10	[(M-H)-CO2]-	C6H5O3	125.02327 (76.52)	26
2	Apigenin trihexoside b, c	4.51	C33H40O20	-	757.2169	2.26	10	[(M + H)-C6H10O5]+ [(M + H)-C6H10O5-C4H8O4]+ [(M + H)−2C6H10O5]+ [(M + H)−2C6H10O5-C4H8O4]+ [(M + H)−3C6H10O5]+	C27H31O15 C23H23O11 C21H21O10 C17H13O6 C15H11O5	595.16528 (21.18) 475.12283 (5.48) 433.11224 (4.07) 313.06985 (1.28) 271.05936 (2.26)	27
3	p-Coumaric acid a	4.72	C9H8O3	163.03894	-	0.18	10	[(M-H)-CO2]-	C8H7O	119.04900 (100.00)	26
4	Luteolin hexoside-deoxy-hexoside b, c (Isomer 1)	4.91	C27H30O15	-	595.1655	0.47	20	[(M + H)-C6H10O4]+ [(M + H)-C6H10O4 -H2O]+ [(M + H)-C6H10O4 −2H2O]+ [(M + H)-C6H10O4 -C2H8O4]+ [(M + H)-C6H10O4 -C4H8O4]+ [(M + H)-C6H10O4 -C6H10O5]+	C21H21O11 C21H19O10 C21H17O9 C19H13O7 C17H13O7 C15H11O6	449.10727 (100.00) 431.09674 (14.75) 413.08630 (3.68) 353.06516 (1.52) 329.06503 (8.08) 287.05472 (4.11)	28
5	Apigenin di-hexoside b, c	5.06	C27H30O15	-	595.1647	1.8	20	[(M + H)-C4H8O4]+ [(M + H)- C6H10O5]+ [(M + H)- C6H10O5 -H2O]+ [(M + H)- C6H10O5 −2H2O]+ [(M + H)- C6H10O5 -C2H8O4]+ [(M + H) - C6H10O5 -C4H8O4]+ [(M + H)-C6H10O5 -C4H8O4-H2O]+ [(M + H)- C6H10O5 -C6H10O5]+	C23H23O11 C21H21O10 C21H19O9 C21H17O8 C19H13O6 C17H13O6 C17H11O5 C15H11O5	475.12283 (7.68) 433.11234 (100.00) 415.10184 (16.15) 397.09137 (6.38) 337.07019 (5.91) 313.07013 (29.48) 295.05966 (4.53) 271.05972 (17.59)	29
6	Luteolin O-hexoside-deoxy-hexoside b, c (Isomer 2)	5.22	C27H30O15	-	595.166	0.45	10	(M + H)-C6H10O4]+ [(M + H)-C6H10O4 -C6H10O5]+	C21H21O11 C15H11O6	449.10724 (20.74) 287.05432 (4.09)	30
7	Apigenin 7- glucoside a	5.59	C21H20O10	-	433.1125	1.08	10	[(M + H)- C6H10O5]+	C15H11O5	271.05954 (68.41)	31
8	Luteolin-O-hexoside b, c (Isomer 1)	5.68	C21H20O11	447.09259	-	1.55	10	[(M-H)-C6H10O5]- [(M-H)-C6H10O5 -C7H4O4]-	C15H9O6 C8H5O2	285.03998 (100) 133.02837 (0.62)	30
9	Luteolin-O-hexoside b, c (Isomer 2)	5.91	C21H20O11	447.09253	-	1.69	10	[(M-H)-C6H10O5]- [(M-H)- C6H10O5 -C7H4O4]-	C15H9O6 C8H5O2	285.03992 (20.48) 133.02742 (0.16)	30
10	Luteolin p-coumaroyl-O-hexoside b, c (Isomer 1)	6.33	C30H26O13	593.12933	-	0.61	20	[(M-H)- C9H6O2]- [(M-H)- C9H6O2-H2O]- [(M-H)- C9H6O2-C6H10O5]- [(M-H)-C9H6O2-C6H10O5-C7H4O4]−	C21H19O11 C21H17O10 C15H9O6 C8H5O2	447.09274 (28.45) 429.08221 (2.63) 285.04010 (100) 133.02844 (0.50)	27
11	Apigenin p-coumaroyl-O-hexoside b, c (Isomer 1)	6.54	C30H26O12	-	579.1489	1.32	10	[(M + H)- C9H6O2]+ [(M + H)- C9H6O2-C6H10O5]+	C21H21O10 C15H11O5	433.11481 (0.13) 271.05966 (100.00)	32
12	Apigenin p-coumaroyl-O-hexoside b, c (Isomer 2)	6.66	C30H26O12	-	579.1492	0.92	10	[(M + H)- C9H6O2]+ [(M + H)- C9H6O2-C6H10O5]+	C21H21O10 C15H11O5	433.11337 (0.35) 271.05984 (56.11)	32
13	Apigenin a	6.70	C15H10O5	-	271.0599	0.83	50	[(M + H)-C8H6O]+ [(M + H)-C7H4O4]+	C7H5O4 C8H7O	153.01822 (11.85) 119.04932 (4.91)	33
14	Chrysoeriol b, c	6.73	C16H12O6	-	301.0703	1.07	50	[(M + H)-CH3]+• [(M + H)-CH3 -CO]+• [(M + H)-C9H8O2]+	C15H10O6 C14H10O5 C7H5O4	286.04691 (100.00) 258.05206 (36.14) 153.01819 (1.05)	33
15	Luteolin p-coumaroyl-O-hexoside b, c (Isómero 2)	6.75	C30H26O13	593.12952	-	0.92	20	[(M-H)-C9H6O2]- [(M-H)-C9H6O2 -H2O]- [(M-H)-C9H6O2 -C6H10O5]- [(M-H)-C9H6O2 -C6H10O5 -C7H4O4]-	C21H19O11 C21H17O10 C15H9O6 C8H5O2	447.09277 (14.34) 429.08209 (33.67) 285.04007 (100.00) 133.02838 (0.52)	27
16	Luteolin p-coumaroyl-O-hexoside a, b (Isomer 3)	6.91	C30H26O13	593.12939	-	1.13	20	[(M-H)-C9H6O2]- [(M-H)-C9H6O2-H2O]- [(M-H)- C9H6O2 -C6H10O5]- [(M-H)-C9H6O2 -C6H10O5 -C7H4O4]-	C21H19O11 C21H17O10 C15H9O6 C8H5O2	447.09271 (27.65) 429.08209 (51.09) 285.04007 (100.00) 133.02852 (0.62)	27
17	Cirsimaritine a	7.00	C17H14O6	-	315.0859	1.39	30	[(M + H)-CH3]+• [(M + H)-CH3-H2O]+• [(M + H)-CH3-H2O-CO]+• [(M + H)-CH3-H2O-2CO]+•	C16H12O6 C16H10O5 C15H10O4 C14H10O3	300.06250 (15.39) 282.05191 (16.05) 254.05701 (3.12) 226.06227 (0.14)	33

Identification criteria: ^a Confirmatory identification by comparison of retention times (t_R) and mass spectra with those of reference substances: gallic acid (≥ 97%), p-coumaric acid (≥ 98%), apigenin-7-glucoside (≥ 90%), apigenin (≥ 95%), and cirsimaritin (≥ 98%). ^b Tentative identification on the basis of data from the scientific literature on species of the genus Turnera. ^c Tentative identification on the basis of the study of exact masses and isotopic ratios. HCD, high-energy collision dissociation cell.

Table 2. Exact masses of protonated [M + H]⁺ and deprotonated [M–H]^– molecules, identified by UHPLC/ESI-Q-Orbitrap-HRMS, in the hydroalcoholic extract of H. lantanifolia.

Peak N° Figure 1B	Compound	tR (min)	Formula	Exp. masses		Δppm	HCD, eV	Product-ions	Formula	m/z I(%)	Ref.
				[M-H]–	[M + H]+
1	Dihydroxyphenyllactic acid c	2.83	C9H10O5	197.04445	-	0.53	30	[(M-H)-H2O]− [(M-H)-CH2O2]− [(M-H)-H2O-CO2]− [(M-H)-CH2O2-CO]−	C9H7O4 C8H7O3 C8H7O2 C7H7O2	179.03427 (100) 151.03928 (4) 135.04420 (83) 123.04417 (71)	34
2	Caffeic acid a	4.19	C9H8O4	179.03423	-	0.34	10	[(M-H)-CO2]−	C8H7O2	135.04417 (100)	35
3	Apigenin C-diglucoside b, c	4.46	C27H30O15	-	595.16574	1.08	30	[(M + H)-H2O]+ [(M + H)−2H2O]+ [(M + H)−3H2O]+ [(M + H)−4H2O]+ [(M + H)-H2O -C4H8O4]+ [(M + H)−2H2O-C5H10O5]+ [(M + H)−3H2O-C6H10O5]+ [(M + H)−2C5H10O5]+	C27H29O14 C27H27O13 C27H25O12 C27H23O11 C23H21O10 C22H17O8 C21H15O7 C17H11O5	577.15381 (40) 559.14343 (37) 541.13293 (27) 523.12244 (18) 457.11192 (100) 409.09052 (19) 379.08005 (10) 295.05930 (11)	35
4	Rosmarinic acid a	5.46	C18H16O8	359.07614	-	1.1	30	[(M-H)-C9H6O3]- [(M-H)-C9H8O4]- [(M-H)-C9H8O4-H2O]- [(M-H)-C9H8O4-CO2]-	C9H9O5 C9H7O4 C9H5O3 C8H7O2	197.04492 (15) 179.03427 (23) 161.02353 (100) 135.04416 (15)	36
5	Isorhamnetin rutinoside b, c	5.8	C28H32O16	623.16066	-	1.14	40	[(M-H)-C12H20O9]- [(M-H)-C12H20O9-CH3]-• [(M-H)-C12H20O9-CH3-CHO]- [(M-H)-C12H20O9-CH3-C8H5O3]−	C16H11O7 C15H8O7 C14H7O6 C7H3O4	315.05078 (100) 300.02701 (11) 271.02471 (3) 151.00256 (1)	37
6	Derived from hydroxycinnamic acid c	6.22	C21H26O9	421.14931	-	0.99	10	[(M-H)-H2O]- [(M-H)−2H2O]- [(M-H)-C6H8O3]- [(M-H)-C6H8O3-C6H10O2]- [(M-H)-C6H8O3-C6H10O2-H2O]-	C21H23O8 C21H21O7 C15H17O6 C9H7O4 C9H5O3	403.13971 (100) 385.12939 (13) 293.10309 (9) 179.03421 (18) 161.02350 (19)	-
7	Jaceidin b, c	6.43	C18H16O8	-	361.09179	0.79	30	[(M + H)- CH3]+• [(M + H)−2 CH3]+ [(M + H)- CH3-H2O]+•	C17H14O8 C16H11O8 C17H12O7	346.06732 (12) 331.04425 (3) 328.05737 (17)	37
8	Sarotrhin c	6.72	C18H16O8	359.07614	-	0.94	30	[(M-H)- CH3]-• [(M-H)−2 CH3]- [(M-H)−3 CH3]-• [(M-H)−3 CH3-CO]-• [(M-H)−3 CH3−2CO]-•	C17H12O8 C16H9O8 C15H6O8 C14H6O7 C13H6O6	344.05347 (12) 329.03012 (100) 314.00677 (48) 286.01175 (53) 258.01688 (6)	38
9	Cirsimaritine a	7.02	C17H14O6	313.07172	-	1.05	30	[(M-H)-CH3]−• [(M-H)−2CH3]− [(M-H)−2CH3-CO]− [(M-H)−2CH3−2CO]− [(M-H)- 2CH3-CO-C6H4O]−	C16H10O6 C15H7O6 C14H7O5 C13H7O4 C8H3O4	298.04794 (61) 283.02478 (100) 255.02980 (28) 227.03462 (8) 163.00285 (17)	39
10	Casticine b, c	7.11	C19H18O8	-	375.10744	0.88	30	[(M + H)-CH3]+• [(M + H)−2CH3]+ [(M + H)-CH3-H2O]+•	C18H16O8 C17H13O8 C18H14O7	360.08295 (15) 345.05991 (7) 342.07297 (17)	37
11	Brickellin c	7.38	C20H20O9	-	405.11716	2.1	40	[(M + H)-CH3]+• [(M + H)−2CH3]+ [(M + H)−2CH3-H2O]+ [(M + H)−2CH3-H2O-CO]+	C19H18O9 C18H15O9 C18H13O8 C17H13O7	390.09409 (1) 375.07059 (43) 357.06009 (57) 329.06512 (100)	34

Identification criteria: ^a Confirmatory identification by comparison of retention times (t_R) and mass spectra with those of reference substances: caffeic acid (≥ 98%), rosmarinic acid (≥ 97%), and cirsimaritin (≥ 98%). ^b Tentative identification on the basis of data from the scientific literature on species of the genus Hyptis. ^c Tentative identification on the basis of the study of exact masses and isotopic ratios.

Table 3. Exact masses of protonated [M + H]⁺ and deprotonated [M – H]^– molecules, identified by UHPLC/ESI-Q-Orbitrap-HRMS, in the hydroalcoholic extract of A. popayanensis.

Peak N° Figure 1C	Compound	tR (min)	Formula	Exp. masses		Δppm	HCD, eV	Product-ions	Formula	m/z I(%)	Ref.
				[M-H]–	[M + H]+
1	Caffeoyl quinic acid b, c	3.24	C16H18O9	353.08755	-	0.79	20	[(M-H)-C9H6O3]− [(M-H)-C7H10O5]− [(M-H)-C7H10O5-H2O]− [(M-H)-C7H10O5-CO2]−	C7H11O6 C9H7O4 C9H5O3 C8H7O2	191.05533 (100) 179.03416 (71) 161.02348 (5) 135.04411 (24)	40
2	Chlorogenic acid a	3.79	C16H18O9	353.08749	-	2.22	20	[(M-H)-C9H6O3]−	C7H11O6	191.05533 (100)	41
3	Caffeoyl quinic acid isomer b, c	3.79	C32H36O18	707.18219	-	0.99	10	[(M-H)-C16H18O9]− [(M-H)-C16H18O9-C9H6O3]−	C16H17O9 C7H11O6	353.08752 (45) 191.05533 (100)	40
4	Caffeic acid a	4.13	C9H8O4	179.03421	-	1.81	10	[(M-H)-CO2]−	C8H7O2	135.04413 (100)	-
5	p-Coumaric acid a	4.74	C9H7O3	163.03897	-	1.40	10	[(M-H)-CO2]−	C8H7O	119.04920 (100)	40
6	3,4-dicaffeoylquinic acid a	5.05	C25H24O12	515.11908	-	1.31	20	[(M-H)- C9H6O3]- [(M-H)- C9H6O3-H2O]− [(M-H)−2C9H6O3]- [(M-H)-C16H16O8]- [(M-H)−2C9H6O3-H2O]− [(M-H)-C16H16O8-CO2]-	C16H17O9 C16H15O8 C7H11O6 C9H7O4 C7H9O5 C8H7O2	353.08762 (100) 335.07718 (10) 191.05539 (31) 179.03418 (33) 173.04471 (26) 135.04407 (6)	41
7	Rutin a	5.37	C27H30O16	-	611.15936	2.12	10	[(M + H)-C6H10O4]+ [(M + H)-C6H10O4-C6H10O5]+	C21H21O12 C15H11O7	465.10172 (11) 303.04922 (100)	42
8	Dicaffeoylquinic acid isomer b, c	5.39	C25H24O12	515.11896	-	1.07	20	[(M-H)-C9H6O3]− [(M-H)−2C9H6O3]− [(M-H)-C16H16O8]− [(M-H)−2 C9H6O3-H2O]− [(M-H)-C16H16O8-CO2]−	C16H17O9 C7H11O6 C9H7O4 C7H9O5 C8H7O2	353.08759 (100) 191.05533 (7) 179.03416 (22) 173.04471 (33) 135.04404 (4)	40
9	Kaempferol 3-rutinoside a	5.73	C27H30O15	-	595.16528	1.83	30	[(M + H)-C6H10O4]+ [(M + H)- C6H10O4- C6H10O5]+	C21H19O11 C15H9O6	447.09277 (10) 285.03998 (100)	42

Identification criteria: ^a Confirmatory identification by comparison of retention times (t_R) and mass spectra with those of reference substances: chlorogenic acid (≥ 99%), caffeic acid (≥ 98%), p-coumaric acid (≥ 98%), 3,4-dicaffeoylquinic acid (≥ 95%), rutin (≥ 94%), and kaempferol-3-rutinoside (≥ 98%). ^b Tentative identification on the basis of data from the scientific literature on species of the genus Ageratina. ^c Tentative identification on the basis of the study of exact masses and isotopic ratios. HCD, high-energy collision dissociation cell.

Total flavonoid, phenolic, and condensed tannin contents of hydroalcoholic extracts

The total contents of flavonoids, phenolics, and condensed tannins in the hydroalcoholic extracts of the three plant species evaluated are presented in Table 4. The hydroalcoholic extract of H. lantanifolia exhibited the highest flavonoid content (175.9 ± 12.4 mg RE/g DT), whereas A. popayanensis had the lowest (108.8 ± 0.7 mg RE/g DT). Turnera pumilea showed the highest phenolic content (238.3 ± 1.3 mg GAE/g DT), with H. lantanifolia displaying the lowest phenolic content (191.3 ± 0.5 mg AGE/g DT). In terms of condensed tannins, Ageratina popayanensis had the highest content (43.0 ± 0.2 mg CE/g DT), whereas H. lantanifolia had the lowest (22.0 ± 0.7 mg CE/g DT).

Table 4. Quantification of flavonoids, phenolics, and condensed tannins in hydroalcoholic extracts, along with the Estimation of their free radical-scavenging and antioxidant activities.

Hydroalcoholic extract	Flavonoidsa (mg RE/g DT)	Phenolicsb (mg GAE/g DT)	Condensed tanninsc (mg CE/g DT)	Scavenging radical (%)	TEACd
T. pumilea	171.2 ± 2.3	238.3 ± 1.3	28.7 ± 0.5	18	105
H. lantanifolia	175.9 ± 2.4	191.3 ± 0.5	22.0 ± 0.7	22	93
A. popayanensis	108.8 ± 0.7	198.0 ± 0.8	43.0 ± 0.2	44	226

(a) A₄₁₅ = 0.0085x + 0.0084, R² = 0.9993, rutin curve obtained at concentrations of 0.002–0.125 mg/mL. The total flavonoid content was expressed as milligrams of rutin equivalents per gram of dry tissue (mg RE/g DT). (b) A_{760 nm}=0.0046x + 0.044, R² = 0.9966, gallic acid curve obtained at concentrations of 0.003–0.200 mg/mL. Total phenolic content was expressed as milligrams of gallic acid equivalents per gram of dry tissue (mg GAE/g DT). (c) A_{500 nm}=0.0037x + 0.1181, R²=0.9873, catechin curve obtained at concentrations of 0.0078–1.00 mg/mL. Condensed tannin content was expressed as milligrams of catechin equivalents per gram of dry tissue (mg CE/g DT). Data are the mean ± SEM (n = 3). (d) Evaluation at the concentration of 125 µg/mL. b. TEAC (trolox equivalent antioxidant capacity), y = 0.1753x − 0.9145. R² = 0.9941. The data are presented as the mean ± SEM (n = 2).

DPPH radical scavenging activity

The antioxidant activities of the three hydroalcoholic extracts measured using the assay of free radical scavenging (DPPH) are presented in Table 4. Ageratina popayanensis showed the highest TEAC value (226), indicating strong antioxidant activity, followed by T. pumilea (105) and H. lantanifolia (93). For the scavenging of DPPH radicals, Ageratina popayanensis exhibited the highest percentage (52%), while T. pumilea and H. lantanifolia showed values of 18% and 22%, respectively. The IC₅₀ values (concentrations required for 50% inhibition) for DPPH scavenging were > 500 µg/mL for T. pumilea (Supplementary Table S3), 496 µg/mL for H. lantanifolia (Supplementary Table S4), and 313 µg/mL for A. popayanensis (Supplementary Table S5), indicating that A. popayanensis had the most potent DPPH scavenging activity among the three extracts.

Photoprotective capacity of hydroalcoholic extracts

The in vitro photoprotective capacity of hydroalcoholic extracts from T. pumilea, H. lantanifolia, and A. popayanensis is summarized in Table 5. The sun protection factor (SPF) values were 41.9 ± 0.1, 38.6 ± 0.7, and 38.2 ± 0.2, respectively, indicating their potential as sunscreens. The extracts also provided long-wavelength UVA protection, with λc values of 385.2 ± 0.1 nm, 380.1 ± 0.2 nm, and 392.4 ± 0.1 nm. The UVA/UVB ratios were 1.6 ± 0.2, 1.5 ± 0.2, and 1.1 ± 0.2, respectively, with A. popayanensis being the most effective at providing maximum protection. The extracts also exhibited low erythema and pigmentation transmission percentages, indicating their potential for quick tan prevention and further supporting their use as sunscreen agents. To validate the results, the photoprotective activity of two sunscreens (commercial products claiming to have SPF 50+) was determined using a 1:1 ratio of sunscreen and absolute ethanol (Supplementary Table S6).

Table 5. In vitro photoprotective properties of hydroalcoholic extracts from T. pumilea, H. lantanifolia, and A. popayanensis.

Extract	T. pumilea	H. lantanifolia	A. popayanensis	Photoprotective potential
SPFa	41.9 ± 0.1	38.6 ± 0.7	38.2 ± 0.2	Sunscreen
λc (nm)	385.2 ± 0.1	380.1 ± 0.2	392.4 ± 0.1	Long-wavelength UVA-protection
UVA/UVB	1.6 ± 0.2	1.5 ± 0.2	1.1 ± 0.2	Maximum protector (****)
Erythema transmission (%)	12.5 ± 0.1	15.4 ± 0.1	15.8 ± 0.1	Quick tan
Pigmentation transmission (%)	17.9 ± 0.1	23.6 ± 0.1	22.9 ± 0.1	Sunscreen

a. Values at a concentration of 0.75 mg/mL.

Cytotoxicity and early intracellular ROS in human cancer cell lines

The antiproliferative effects of the extracts were evaluated in the Calu-1 and HepG2 cell lines, and the results are presented in Fig. 2; Table 6. The A. popayanensis extract did not exhibit significant cytotoxicity in either studied cell lines. The IC₅₀ values were 444 µg/mL for Calu-1 cells and 387 µg/mL for HepG2 cells. On the other hand, the IC₅₀ values of 201 µg/mL and 216 µg/mL obtained in HepG2 cells indicated that both the T. pumilea and H. lantanifolia extracts did not exert a relevant antiproliferative effect. However, a more notable antiproliferative effect was observed in Calu-1 cells, with IC₅₀ values of 97 µg/mL for T. pumilea and 116 µg/mL for H. lantanifolia. The IC₁₀ and IC₂₅ values are shown in Supplementary Tables S7-S10.

The evaluation of early intracellular ROS generation (Fig. 3) revealed that, compared with the positive control (H₂O₂, 200 µM), the extracts did not induce the production of ROS at the concentration evaluated (15.63 µg/mL). However, when comparing the levels of ROS generated by the different extracts, it was evident that after 45 min of exposure, the T. pumilea extract slightly increased the production of intracellular ROS in Calu-1 cells.

Fig. 2 [Images not available. See PDF.]

Cytotoxicity in Calu-1 and HepG2 cells exposed to hydroalcoholic extracts from T. pumilea, H. lantanifolia, and A. popayanensis for 24 h. *. There was significant difference in viability compared with that of the control group (C-) (p < 0.05). Data are the mean ± SEM (n = 3).

Table 6. Cytotoxic potential (IC₅₀) of hydroalcoholic extracts from T. pumilea, H. lantanifolia, and A. popayanensis on Calu-1 cells and HepG2 cells for 24 h.

Cell line		Extracts evaluated
		T. pumilea	H. lantanifolia	A. popayanensis
Calu-1	IC50 (µg/mL)	97	116	444
	R2	0.9869	0.8086	0.9737
	p value	< 0.0001	0.0147	< 0.0001
	95% confidence intervals	77.6 to 116.4	92.8 to 139.2	355.2 to 532.8
HepG2	IC50 (µg/mL)	201	216	387
	R2	0.9877	0.9571	0.9165
	p value	< 0.0001	0.0001	0.0002
	95% confidence intervals	160.8 to 241.2	172.8 to 259.2	309.6 to 464.4

Fig. 3 [Images not available. See PDF.]

Early intracellular ROS levels in hydroalcoholic extracts of T. pumilea, H. lantanifolia, and A. popayanensis at a concentration of 15.63 µg/mL on Calu-1 (A) and HepG2 (B).

Discussion

The hydroalcoholic extract obtained from the aerial parts of T. pumilea underwent UHPLC-ESI-Orbitrap-HRMS analysis, which led to the identification of various phenolic compounds, flavones, and their glycosylated derivatives. A robust analytical strategy was employed, combining the use of reference substances, literature reports, and spectral databases to achieve a high level of confidence in compound identification. According to the Metabolomics Standards Initiative, five compounds were confirmed using commercial reference standards: gallic acid (≥ 97%), p-coumaric acid (≥ 98%), apigenin-7-glucoside (≥ 90%), apigenin (≥ 95%), and cirsimaritin (≥ 98%). Additional flavones and glycosides were tentatively identified based on literature reports for the genus Turnera and MS spectral interpretation.

To establish the differences and similarities between T. pumilea and its related species, a literature review was conducted. This review led to the identification of T. diffusa as the most prominent species in terms of chemical characterization within the genus, facilitating comparative analysis of the species and enhancing our understanding of their flavonoids and phenolic profiles. The compounds identified include apigenin, pinocembrin, velutin, gonzalitosin I, apigenin 7-O-glucoside, apigenin 7-O-(6”-O-p-E-coumaroyl)-glucoside, apigenin 8-C-(2-O-rhamnosyl)-glucoside, luteolin 8-C-(2-O-rhamnosyl)-glucoside, luteolin 8-C-(2-O-rhamnosyl)-quinovoside, diosmetin 8-C-(2-O-rhamnosyl)-glucoside, chrysoeriol 7-O-glucoside, and a range of other flavonoids and glycoside derivatives^{43, 44, 45–46}. Additionally, phenolics, phenolic acids, and maltol derivatives such as arbutin, ellagic acid, 4-O-β-D-glucopyranosyl-p-coumaric acid, and maltol 3-O-glucoside have been identified from various sources^46,47.

Other interesting compounds reported in T. pumilea (not identified in our study) are cyanogenic glycosides, which constitute a structurally less diverse yet significant class of natural products⁴⁸. Spencer et al.⁴⁹identified four cyanogenic glycosides derived from cyclopentenyl glycine: deidaclin, volkenin, tetraphyllin A, and tetraphyllin B. These are defense agents against herbivores and play crucial roles in the early stages of plant development by releasing toxic hydrogen cyanide upon tissue damage⁴⁸.

According to UHPLC-ESI-Orbitrap-HRMS analysis of compounds in T. pumilea, gallic acid and p-coumaric acid have emerged as noteworthy natural products recognized for their anti-inflammatory and anti-cancer properties^50,51and antimelanogenic effects⁵⁰respectively. Apigenin, a significant flavone in cosmetology, has properties that contribute to the effective treatment of skin inflammatory conditions, UV-induced skin damage, skin aging, and vitiligo^52,53along with its anticancer^53,54and antiviral attributes⁵⁵. Chrysoeriol is recognized for its diverse effects including anti-inflammatory, anti-osteoporosis, antibacterial, antifungal, neuroprotective, and anti-cancer properties⁵⁶. Cirsimaritin, another flavone, demonstrates antioxidant, antimicrobial, antiparasitic, anticancer, antidiabetic, and anti-inflammatory effects⁵⁷. Apigenin 7-glucoside has been associated with skin anti-inflammatory activity in rats⁵⁸antimicrobial effects against Staphylococcus aureus and Enterococcus faecalis, as well as inhibitory effects on tyrosinase (TYR-), Acetylcholinesterase (AChE-), and butyrylcholinesterase (BuChE-)⁵⁹.

A review of T. diffusa highlights the relevance of this species in popular medicine, as it is considered an important therapeutic agent for the ancient Mayan civilization⁶⁰. In this review, basic information on the in vitro and in vivo biological activities of the extracts, essential oils, and phytocompounds studied by different authors is summarized and presented as antioxidant, antiphotoaging, antimicrobial, cytotoxic, anti-inflammatory, hepatoprotective, nephroprotective, testicular protective, neuroprotective, hypoglycemic/antidiabetic/antihyperglycemic, anti-obesity, analgesic, antidepressant and anxiolytic, aphrodisiac, potent activator of the pregnane-xenobiotic and aryl-hydrocarbon receptors, anti-glycosylation, monoamine oxidases inhibitor, phosphodiesterase 5 inhibitor, anti-aromatase and estrogenic, skin penetration enhancement, and smooth muscle relaxant.

The hydroalcoholic extract of T. pumilea evaluated in our study showed the ability to neutralize DPPH radicals, although with moderate efficacy, given that the concentration required to achieve 50% inhibition (IC₅₀) exceeded 500 µg/mL. At a concentration of 500 µg/mL, the extract achieved 39% radical scavenging, which, although lower than the IC₅₀ threshold, is consistent with the activity reported in previous studies. In particular, Wong-Paz et al.⁶¹ observed greater antioxidant capacity (77.9–79.7%) at a higher concentration (1 mg/mL), suggesting an expected dose-response relationship for this type of extract. However, it is important to note that variability in antioxidant activity can be influenced by factors such as the extraction method, the specific chemical composition of the extract, and the total concentration of phenolic compounds present. In this regard, Wong-Paz et al.⁶¹ reported a phenolic content of 101.5 mg GAE/g with an EC₅₀ of 305.4 µg/mL, while Tsaltaki et al.⁶² indicated a maximum phenolic content of 183.7 mg GAE/g DW. Our study recorded a phenolic content of 238.3 mg GAE/g DW at 1 mg/mL, exceeding those previously reported and could reflect differences in the plant source, extraction conditions, or analytical methods used. This disparity between phenolic content and antioxidant capacity suggests that the quantity, quality, composition, and synergy of the phenolic compounds present in the extract influence this activity. Therefore, although the T. pumilea extract demonstrates antioxidant potential, the efficacy observed in the DPPH assay indicates that its activity is moderate compared to other extracts with similar or lower phenolic profiles.

This study presents the in vitro photoprotective efficacy of T. pumilea at a concentration of 0.75 mg/mL. The findings revealed that the extract displayed an SPF of 41.9 and a λc of 385.2 nm, indicative of its broad-spectrum photoprotective capabilities. These results complement the research conducted by Wong-Paz et al.⁶¹who investigated the anti-photoaging effects of T. pumilea in HaCaT and HDF cells exposed to UVB radiation. Their study demonstrated the ability of the hydroalcoholic extract to regulate key pathways involved in photoaging, including MMP-1/procollagen type 1, TGF-β1/Smad, and MAPK pathways⁶¹.

We evaluated the cytotoxic potential and early intracellular ROS of the hydroalcoholic extract derived from the leaves of T. pumilea against HepG2 (liver cancer) and Calu-1 (lung cancer) cell lines. Our findings revealed that the extract exhibited cytotoxic activity in both cell lines, with IC₅₀ values of 201 µg/mL and 97 µg/mL for HepG2 and Calu-1 cells, respectively. The results indicate that the extract was more potent against Calu-1 cells than against HepG2 cells under the tested conditions. The literature concerning the cytotoxicity of T. pumilea extracts in HepG2 and Calu-1 cells is limited. However, we found two relevant studies that employed different extraction methods. A previous study used aqueous extracts obtained from the leaves and reported a CC₅₀ value of 43.87 µg/mL⁶³. Another study utilized essential oils extracted from the plants and reported CC₅₀ values ranging from 186.5 to 199.3 µg/mL⁶⁴. Interestingly, these latter results are comparable to our observations using the hydroalcoholic extract. Furthermore, no early intracellular ROS production due to oxidative stress induced by the extract was detected at a concentration of 15.63 µg/mL in HepG2 cells; however, a slight increase in the generation of reactive species was observed in Calu-1 cells. Previous studies have shown that this lung carcinoma cell line responds to various treatments with a significant increase in intracellular ROS, which is associated with the induction of cell death and apoptosis, through the modulation of pathways related to cancer growth, survival, and angiogenesis⁶⁵. Furthermore, the sensitivity of Calu-1 to cytotoxic agents, such as ABT-263, has been reported to correlate with intracellular ROS levels, suggesting a regulatory role for these species in the response to antitumor treatments⁶⁶. In this context, moderate ROS generation may act as a physiological signal that modulates oxidative stress and apoptosis pathways, indicating a possible early activation of oxidative stress mechanisms that could contribute to the cytotoxicity of the T. pumilea extract⁶⁷.

Within the genus Hyptis, the most studied species have been H. verticillata, H. radicans, and H. multibracteata. The limited bibliography about H. lantanifolia leads us to highlight the potent inhibitory activity of the cytopathic effect induced by HIV in cultured cells employing the aqueous extract obtained from its aerial parts, as well as its activity in essential enzymes (HIV-reverse transcriptase and HIV protease enzymes)^13,68.

Studies conducted on the genus Hyptis have shown its potential in Brazilian folk medicine¹¹including its anti-inflammatory, antimicrobial, antisecretory properties, anticancer, hepatoprotective, antiviral, acaricidal, insecticidal, molluskicide, and hormonal modulating activities. In addition, many chemical components have been identified, including lignans, triterpenes, diterpenes, sesquiterpenes, monoterpenes, flavonoids, polyphenols, and alkaloids^11,12.

UHPLC-ESI-Orbitrap-HRMS analysis of the hydroalcoholic extract of H. lantanifolia leaves led of the identification of caffeic acid (≥ 98%), rosmarinic acid (≥ 97%), and cirsimaritin (≥ 98%) with reference substances. Other flavonoids such as vicenin 2 (apigenin 6,8-C-diglucoside), jaceidin, casticin, sarothrin, isorhamnetin rutinoside, and brickellin were tentatively identified (Table 2). Among of the mentioned compounds, caffeic acid and rosmarinic acid have been previously identified in species within this genus and are considered major constituents of the Lamiaceae family¹¹.

Molecules identified in the H. lantanifolia leaves recognized for their antioxidant, antimicrobial, and anticancer potential. In addition, caffeic acid affects diabetes, atherosclerosis, and Alzheimer’s disease⁶⁹. Danshensu properties are remarkable in cardiovascular disease, cerebral lesions, and different health conditions, including tumorigenesis and pancreatitis⁷⁰. Rosmarinic acid affects diabetes, inflammatory and neurodegenerative disorders, and liver diseases⁷¹. Vicenin 2 prevents Helicobacter pylori-associated infection and protects the gastric cells⁷²; jaceidin affects Ehrlich’s ascites carcinoma, attenuating tumor progression⁷³; cirsimaritin has remarkable antidiabetic, antiparasitic, and anti-inflammatory effects⁵⁷; casticin ameliorates scopolamine-induced cognitive dysfunction⁷⁴ and protects against inflammation in human osteoarthritis chondrocytes⁷⁵isorhamnetin rutinoside induces the apoptosis of human myelogenous erythroleukemia cells⁷⁶; and brickellin reduces both the formation of advanced glycation end products (AGEs) and oxidative damage to the proteins⁷⁷.

The antibacterial activity of different species of the genus Hyptis has been evaluated⁹¹¹. However, H. lantanifolia is highlighted as an antiretroviral agent since the water extract of the aerial parts of this species acts as a potent inhibitor of HIV-RT, with an IC₅₀ value of 7 µg/mL¹³. In our study, the results of the quantitative analysis of the polyphenols in the hydroalcoholic extract of H. lantanifolia leaves revealed the following total phenolic content: 191.3 mg GAE/g DT > flavonoids: 175.9 mg RE/g DT > condensed tannins: 22.0 mg CE/g DT. Furthermore, the extract showed antioxidant activity, with an IC₅₀ value of 496 µg/mL and sunscreen properties, with an SPF of 38.6 and a λc of 380.1 nm. Additionally, the assessment of their cytotoxic potential revealed that the ability of Calu-1 cells (non-small cell lung cancer) to inhibit proliferation was greater than that of HepG2 cells (human hepatoma), with IC₅₀ values of 116 µg/mL and 216 µg/mL, respectively.

Chemical analysis of the evaluated extract of A. popayanensis revealed the presence of phenolic acids (caffeoyl quinic acid, chlorogenic acid), hydroxycinnamic acid (p-coumaric acid), phenylpropanoids (3,4-di-caffeoylquinic acid and its isomer), a trihydroxy flavone (kaempferol 3-O-rutinoside), and rutin, a glycoside that combines quercetin (flavonol) and rutinoside (Table 3). Some of the identified compounds in this extract are recognized as antioxidant agents, including caffeoyl quinic acid⁷⁸3,4-di-caffeoylquinic acid⁷⁹and chlorogenic acid, which are also anti-inflammatory, antimicrobial, hypoglycemic, immunomodulatory, anticardiovascular, and antimutagenic⁸⁰. P-coumaric acid is effective in treating inflammation, diabetes, cardiovascular, and nervous system diseases. Furthermore, p-coumaric acid has shown a positive effect on the prevention and treatment of diseases by inhibiting oxidative stress and exerting anti-aging effects on the skin⁸¹. Kaempferol 3-O-rutinoside acts on different targets and is useful in wound healing⁸². The activities of rutin highlight this glycoside compound as an antimicrobial, antifungal, and antiallergic agent⁸³.

In the evaluation of the three plant species, the hydroalcoholic extract derived from A. popayanensis leaves presented a notably higher concentration of condensed tannins (43.0 ± 0.2 mg CE/g DT). Additionally, this extract exhibited superior scavenging activity against DPPH radicals, with a lower IC₅₀ value of 313 µg/mL and a scavenging rate of 44%. Furthermore, it had minor inhibitory effects on the proliferation of Calu-1 and HepG2 cells.

The cellular permeability of the compounds identified in A. popayanensis has not been extensively studied in cell lines such as Calu-1 or HepG2. However, scientific literature provides relevant data on the permeability of these compounds in Caco-2 cells, a widely used model for simulating the human intestinal barrier^{84, 85, 86, 87, 88, 89–90}.

These studies demonstrate that some compounds have low apparent permeability, such as chlorogenic acid, caffeoylquinic acid isomers, and 3,4-dicaffeoylquinic acid. Other compounds, including caffeoylquinic acid, caffeic acid, dicaffeoylquinic acid, and rutin, show moderate permeability, while p-coumaric acid exhibits high permeability. It is important to note that the biological effects of these compounds do not depend exclusively on their cellular permeability^91,92.

For instance, 3,4-dicaffeoylquinic acid can exert relevant actions through interactions on the cell surface or through its high affinity for plasma proteins, which modulates its bioavailability and pharmacological activity. Furthermore, the same compound (rutin) can exhibit different levels of permeability depending on the cell type and the transport mechanisms involved. In certain cell lines, rutin can demonstrate high permeability mediated by transporters such as P-glycoprotein (P-gp) and the multidrug resistance proteins MRP2 and MRP3, while in Caco-2 cells, the permeability of the same compound may be moderate due to differences in the expression and activity of these transporters. A study of the extract’s permeability in Calu-1 and HepG2 cells will lead to a general permeability profile and allow analysis of the results compared with the existing scientific literature.

Finally, T. pumilea, H. lantanifolia, and A. popayanensis did not exhibit early intracellular ROS production compared with the positive control (H₂O₂, 200 µM). However, among these species, T. pumilea demonstrated intracellular ROS generation in Calu-1 cells, which could be linked to the observed cytotoxic properties, as reflected by an IC₅₀ value of 97 µg/mL.

Conclusions

The hydroalcoholic extracts of T. pumilea, H. lantanifolia, and A. popayanensis, which belong to the Passifloraceae, Lamiaceae, and Asteraceae families, respectively, were cultivated, collected, and processed following standardized procedures⁴. The chemical composition of the studied extracts was characterized using UHPLC-ESI-Orbitrap-HRMS analysis. Their activities, including antioxidant capacity, photoprotective effects, cytotoxicity in human cancer cells, and early reactive oxygen species (ROS) production, were assessed through in vitro assays. Collectively, these results highlight the potential of the three evaluated species as promising candidates for antioxidant, photoprotective, and anti-aging applications, particularly in protecting against UV-induced skin damage. Notably, the antiproliferative effects of T. pumilea on non-small cell lung cancer cells underscore its anticancer potential. Further research is needed to elucidate the underlying mechanisms and optimize the use of these botanical extracts in skin care formulations and pharmaceutical products.

Future directions include the evaluation of the antioxidant capacity of the extracts using complementary methods such as ferric reducing antioxidant power (FRAP) and oxygen free radical absorbance capacity (ORAC), which will further demonstrate their applicability in the cosmetic, pharmaceutical, and food industries. Furthermore, the inclusion of normal and cancerous human cell lines, as well as in vivo studies, is crucial to identify potential adverse effects and ensure therapeutic safety and efficacy. The moderate ROS generation induced by T. pumilea extract in Calu-1 cells suggests a controlled pro-oxidant mechanism that triggers oxidative stress and apoptosis. Future studies should focus on the regulation of these pathways and their impact on cell viability to clarify the role of ROS in the observed cytotoxic effects.

Author contributions

Conceptualization, K.C.-G. and P.Q.-R.; methodology, E.E.-S., K.C.-G., J.O.-V., and P.Q.-R.; formal analysis, P.Q.-R., K.C.-G. and J.O.-V.; investigation, J.O.-V., and E.E.-S., and P.Q.-R.; resources, K.C.-G. and J.O.-V.; data curation, P.Q.-R.; writing—original draft preparation, P.Q.-R.; writing—review and editing, P.Q.-R., K.C.-G. and J.O.-V.; supervision, K.C.-G. and J.O.-V.; project administration E.E.-S. and J.O.-V.; funding acquisition, E.E.-S. and J.O.-V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science, Technology and Innovation (Minciencias), the Ministry of Education, the Ministry of Industry, Commerce and Tourism, and ICETEX, Programme Ecosistema Científico–Colombia Científica, from the Francisco José de Caldas Fund, Grant RC-FP44842-212-2018; The Ministry of Environment and Sustainable Development of Colombia supported the Universidad Industrial de Santander through access permits to genetic resources and derivatives for bioprospecting (Contract No. 270–2019); and Minciencias - Programa Orquídeas, mujeres en la ciencia: agentes para la paz (Call No. 935–2023) through the Francisco José de Caldas Fund and from the municipality of Zambrano Bolivar, (Grant RC 112721-262-2023); and University of Cartagena (Resolution No.02-2024).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare that there are no conflicts of interest.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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