1 Anti-inflammatory, antinociceptive, antioxidant, and antimicrobial activities of 1 hydroalcoholic extracts of Witheringia solanacea L’Hér 2 Actividad antiinflamatoria, antinociceptiva, antioxidante y antimicrobiana de 3 extractos hidroalcohólicos de Witheringia solanacea L’Hér 4 5 Running title: Pharmacological activities of Witheringia solanacea 6 7 Cristina Herrera1*, Fabián Delgado-Rodríguez1, Navilla Apú1, Verónica Madrigal-Gamboa2, Marta Porras1 8 9 1Pharmaceutical Research Institute (INIFAR), School of Pharmacy, University of Costa Rica, 11501-2060 San José, Costa Rica. 10 2School of Pharmacy, University of Costa Rica, 11501-2060 San José, Costa Rica. 11 *E-mail address: cristina.herrera@ucr.ac.cr 12 13 *Corresponding author address: 14 Pharmaceutical Research Institute (INIFAR), School of Pharmacy, University of Costa Rica, 11501-2060 San José, Costa Rica. 15 Tel: +506 25118330 16 42 number of pages, 9 figures, and 2 tables 17 Institutional e-mail of the authors: 18 Author Institutional e-mail (mandatory) Other e-mail (gmail, yahoo, etc.) ORCID (0000-1111-2222-3333) Cristina Herrera cristina.herrera@ucr.ac.cr cristina.herreraarias@gmail.com 0000-0001-5555-2302 Fabián Delgado- Rodríguez fabian.delgadorodriguez@ucr.ac.cr 0000-0002-8268-7162 Navilla Apú navilla.apu@ucr.ac.cr naviapu21@gmail.com 0000-0002-6848-7252 Verónica Madrigal-Gamboa veronica.madrigalgamboa@ucr.ac.cr vrmadrigal@gmail.com 0000-0003-3340-555X Marta Porras marta.porras@ucr.ac.cr 0000-0001-5626-1005 mailto:cristina.herrera@ucr.ac.cr mailto:fabian.delgadorodriguez@ucr.ac.cr https://orcid.org/0000-0002-8268-7162 mailto:NAVILLA.APU@ucr.ac.cr https://orcid.org/0000-0002-6848-7252 mailto:VERONICA.MADRIGALGAMBOA@ucr.ac.cr https://orcid.org/0000-0003-3340-555X mailto:marta.porras@ucr.ac.cr https://orcid.org/0000-0001-5626-1005 2 Contribution Details 19 20 Contribution Herrera C Delgado- Rodríguez F Apú N Madrigal- Gamboa V Porras M Concepts or Ideas X X Design X X Definition of intellectual content X X Literature search X X Experimental studies X X X X X Data acquisition X X X X Data analysis X X Statistical analysis X X Manuscript preparation X X Manuscript editing X X X X X Manuscript review X X X X X 21 ABBREVIATIONS: DPPH: 2,2-diphenyl-1-picrylhydrazyl assay; DW: dry weight; E%: percentage of edema; 22 EDTAE: ethylenediaminetetraacetic acid equivalents; F1: fruit extract from Mora; F2: fruit extract from Puerto 23 Jiménez; FICA: ferrous iron chelating activity assay; FRAP: ferric reducing antioxidant power assay; GAE: gallic 24 acid equivalents; HAT: hydrogen atom transfer; MIC: minimum inhibitory concentration; ORAC: oxygen radical 25 absorbance capacity assay; QE: quercetin equivalents; SET: single electron transfer; SL1: stem and leaf extract from 26 Mora; SL2: stem and leaf extract from Puerto Jiménez; TE: Trolox equivalents; TFC: total flavonoid content; TPC: 27 total phenolic content. 28 29 3 ABSTRACT 30 Context: Witheringia solanacea has traditionally been used in Latin American medicine for its anti-inflammatory and antimicrobial 31 properties, as well as for general pain management. However, few pharmacological studies have been conducted to validate these 32 traditional uses. 33 Aims: To determine the chemical composition and evaluate selected pharmacological activities of W. solanacea extracts using 34 various experimental models. 35 Methods: Hydroalcoholic extracts from fruit and aerial parts of W. solanacea were analyzed by high-performance thin layer 36 chromatography to detect secondary metabolites. Qualitative phytochemical screening and quantification of total phenolic and 37 flavonoid contents were performed. The antioxidant and antibacterial activities of the extracts were evaluated in vitro. 38 Additionally, acute oral toxicity, analgesic, and anti-inflammatory activities of the aerial-part extract were assessed in rats. 39 Results: Phytochemical analysis confirmed the presence of phenolics, flavonoids, coumarins, terpenoids, triterpenes, steroids, and 40 alkaloids. Fruit extracts exhibited higher antioxidant activity than aerial-part extracts. The extracts showed only limited 41 antibacterial activity, with effects observed only against E. faecalis (MIC ≈ 5 mg/mL). The aerial-part extract was classified as non-42 toxic (LD₅₀ > 2000 mg/kg). In vivo, this extract produced significant analgesic effects in the tail-flick model and significantly 43 reduced carrageenan-induced paw edema and leukocyte infiltration, with effects comparable to those of indomethacin. 44 Conclusions: W. solanacea aerial part extract exhibits analgesic and anti-inflammatory properties that support its traditional use 45 for pain and inflammation, although only limited antibacterial activity was observed. 46 47 Keywords: anti-inflammatory; antimicrobial; antinociceptive; antioxidant; oral toxicity; Witheringia solanacea. 48 49 RESUMEN 50 Contexto: La planta Witheringia solanacea ha sido utilizada tradicionalmente en la medicina latinoamericana por sus propiedades 51 antiinflamatorias y antimicrobianas, así como para el tratamiento del dolor en general. Sin embargo, pocos estudios 52 farmacológicos han validado estos usos tradicionales. 53 Objetivos: Determinar la composición química y evaluar algunas actividades farmacológicas de los extractos de W. solanacea 54 mediante diversos modelos experimentales. 55 Métodos: Se analizaron extractos hidroalcohólicos de frutos y partes aéreas de W. solanacea mediante cromatografía de capa fina 56 de alta resolución para detectar metabolitos secundarios. Además, se realizaron pruebas fitoquímicas cualitativas y determinación 57 del contenido de compuestos fenólicos y flavonoides totales. Los extractos fueron evaluados para determinar su actividad 58 antioxidante y antibacteriana in vitro. También se evaluó la toxicidad oral aguda y las actividades analgésicas y antiinflamatorias 59 del extracto de partes aéreas en ratas. 60 Resultados: El análisis fitoquímico confirmó la presencia de compuestos fenólicos, flavonoides, cumarinas, terpenoides, 61 triterpenos, esteroides y alcaloides. Los extractos de frutos mostraron una mayor actividad antioxidante que los extractos de 62 partes aéreas. Por otro lado, los extractos presentaron una débil actividad antibacteriana, observándose efectos únicamente contra 63 4 E. faecalis (CMI ≈ 5 mg/mL). El extracto de partes aéreas se clasificó como no tóxico (DL₅₀ > 2000 mg/kg). In vivo, este extracto 64 produjo efectos analgésicos significativos en el modelo de retiro de la cola y redujo significativamente el edema plantar inducido 65 por carragenina y la infiltración leucocitaria, con efectos comparables a los de la indometacina. 66 Conclusiones: El extracto de partes aéreas de W. solanacea presenta propiedades analgésicas y antiinflamatorias que respaldan su 67 uso tradicional para el tratamiento del dolor y la inflamación; sin embargo, se observó únicamente una actividad antibacteriana 68 limitada. 69 70 Palabras claves: antiinflamatorio; antimicrobiano; antinociceptivo; antioxidante; toxicidad oral; Witheringia solanacea 71 5 INTRODUCTION 72 Witheringia solanacea L’Hér is a small shrub (1-4 m high) that belongs to the Solanaceae 73 family. This species is widely distributed from southern Mexico to Bolivia in South America, 74 through Central America and the Caribbean islands. The plant is typically found between 0 75 and 2000 m above sea level (Pequeno et al., 2017). 76 W. solanacea has several medicinal ethnobotanical applications in Mesoamerica and South 77 America. For example, in Mexico and Nicaragua, this plant is used to treat skin illnesses such 78 as acne (Jacobo-Herrera et al., 2006; Coe, 2008). In Costa Rica, W. solanacea is used to treat 79 infections; the fruit is eaten to alleviate headaches and stomachaches, and the roots are used 80 to treat diabetes (García et al., 2006). In Panama, the whole plant and fruit are used for general 81 body pain, skin diseases, hypertension, and as an anthelmintic (Gupta et al., 1993; Caballero-82 George et al., 2001). In Ecuador, the plant is widely used by several indigenous tribes; infusions 83 and the juice from the leaves and fruit are employed to treat headaches, inflammation, skin 84 infections, stomachaches, diarrhea, bronchitis and tuberculosis (Ballesteros et al., 2016). 85 Despite the extensive use of this plant in traditional Latin American medicine, few 86 pharmacological studies have confirmed the traditional use of W. solanacea. Some studies have 87 investigated the hypoglycemic activity (Herrera et al., 2011; Pequeno et al., 2021) and the 88 activity against malaria and leishmaniasis (Chinchilla et al., 2012; Chinchilla-Carmona et al., 89 2014) of different extracts and parts of the plant. 90 Moreover, the phytochemistry of W. solanacea is not well known, but it has been related to 91 Witheringia coccoloboides, from which seven physalins have been isolated. Physalins are 92 steroidal lactones commonly present in the Solanaceae family and have a variety of biological 93 activities, such as antitumor, antibacterial, and anti-inflammatory properties (Wu et al., 2021; 94 Meira et al., 2022). Physalins B, D, and F have been isolated from the leaves of W. solanacea. 95 6 Physalins B and F have demonstrated anti-inflammatory effects in in vitro models using cell 96 cultures (Jacobo-Herrera et al., 2006). 97 To date, no studies have assessed the anti-inflammatory and analgesic activities of W. 98 solanacea in animal models, which could provide additional biologically relevant evidence 99 supporting the ethnobotanical applications of this plant. Furthermore, to the best of our 100 knowledge, no previous information has been reported regarding the evaluation of the 101 antibacterial effect of W. solanacea extracts, despite its traditional use for treating infections. 102 Given the limited pharmacological data available on W. solanacea, this study evaluated its 103 antimicrobial, anti-inflammatory, and analgesic activities using hydroalcoholic extracts of 104 fruit and aerial parts from two regions of Costa Rica. Acute oral toxicity, antioxidant capacity, 105 and phytochemical profiles were also determined. 106 MATERIAL AND METHODS 107 Collection of plant material 108 Aerial parts of W. solanacea were collected in February 2021 in two different locations of 109 Costa Rica: Puerto Jiménez, Puntarenas province (South Pacific Region) (8o33´14´´ N, 110 83o23´56´´ W) and Mora, San José province (Central Region) (9o54´ 52.9´´ N, 84o16´50.6´´ W). 111 Taxonomic identification was performed by an expert botanist from the Nacional University 112 of Costa Rica. A voucher specimen of the species was deposited in the “Juvenal Valerio 113 Rodríguez” Herbarium (Nacional University of Costa Rica) under the acquisition numbers 114 JVR5362 and JVR5363, respectively. 115 Preparation of extracts 116 Combined stems and leaves of W. solanacea were shade-dried and ground to a particle size 117 of 2 mm using a blade mill (Wiley, United States). Ripe fruits were frozen at -80 °C and freeze-118 dried using a LABCONCO Freezone 6 system (United States). The dried fruit was milled with 119 7 a food processor and sieved to a maximum particle size of 2 mm. Each plant material was 120 macerated separately in a 1:10 ratio with 80%(v/v) ethanol for two weeks. The extract was 121 decanted, and the plant residue was subjected to a second one-week maceration under the 122 same conditions. The resulting extract was decanted and combined with the first extract. The 123 pooled extract was filtered through a 0.45 µm PVDF membrane and ethanol was removed by 124 evaporation at 40 °C (Büchi R-100, Switzerland). The remaining aqueous phase was freeze-125 dried to obtain the dried extracts. Four extracts were obtained, corresponding to: fruit from 126 Mora (F1), fruit from Puerto Jiménez (F2), stems and leaves from Mora (SL1), and stems and 127 leaves from Puerto Jiménez (SL2). Extraction yields were calculated, and the extracts were 128 stored at -20 °C for further analyses. 129 Chemical characterization of the extracts 130 Extracts solutions (10 mg/mL) were prepared in a 1:1 methanol-water mixture, and 25 µL 131 were applied onto HPTLC-grade silica gel 60 F254 plates (20 x 10 cm; Millipore, Germany). 132 Samples were sprayed as 8 mm x 1 mm bands using the ATS4 autosampler (CAMAG, 133 Switzerland) at 150 nL/s, with nitrogen as the propellant gas. The first band was positioned 134 20 mm from the left edge of the plate, and the solvent front was set at 80 mm from the bottom. 135 After application, the plates were dried for 30 seconds using the ADC2 automatic developing 136 chamber (CAMAG, Switzerland). The chamber was then saturated with the mobile phase. 137 Following saturation, the plates were developed and dried for 5 minutes. Chromatographic 138 development was conducted at room temperature (24 ± 3 °C) and relative humidity (51 ± 3 %). 139 Chromatograms were visualized under visible light or UV light (254 or 366 nm) using a TLC 140 visualizer (CAMAG, Switzerland). 141 Conditions for the detection of phenolic compounds by HPTLC 142 For the detection of highly polar phenolic compounds (e.g. glycosidic phenolics), a single 143 development system was employed using a mobile phase composed of butan-2-ol: n-butanol, 144 8 ethyl acetate, and formic acid (60:40:15:10). The development chamber was saturated for 20 145 minutes prior to use. For low-polarity phenolic compounds (e.g. non-glycosidic phenolics), a 146 two-step development system was applied. Plates were first developed with a mobile phase 147 of toluene, ethyl acetate, and formic acid (60:45:3), followed by a second elution using toluene, 148 ethyl acetate, n-hexane, and formic acid (60:30:10:3). The chamber was saturated for 10 minutes 149 before each elution step, and plates were dried in the ADC2 development chamber prior to 150 the second development. After development, plates were derivatized using potassium 151 ferricyanide and ferric chloride reagents (detection of phenolics), aluminum chloride reagent 152 (detection of flavonoids) or potassium hydroxide solution (detection of coumarins, flavonoids, 153 and quinones). All reagents were prepared and applied according to Lock de Ugaz (1994). 154 Conditions for the detection of terpenoids by HPTLC 155 Terpenoids were detected using a mobile phase of chloroform, methanol, and water 156 (100:40:5) after 20 minutes of chamber saturation. Following development, plates were 157 derivatized with either anisaldehyde/sulfuric acid or Liebermann-Burchard reagent, both 158 prepared and applied according to Lock de Ugaz (1994). 159 Conditions for the detection of alkaloids by HPTLC 160 Alkaloids were detected using a mobile phase of chloroform, methanol, and ammonium 161 hydroxide (47.5:47.5:5) after 20 minutes of chamber saturation. Following development, plates 162 were derivatized with Dragendorff’s reagent, as described by Lock de Ugaz (1994). 163 Additional qualitative tests 164 Additionally, the extracts underwent preliminary phytochemical screening to identify 165 secondary metabolite groups, including carbohydrates, reducing sugars, amino acids and 166 peptides, saponins, tannins, and cardiac glycosides, following standard protocols (Lock de 167 Ugaz, 1994; Tiwari et al., 2011). 168 9 Determination of Total Phenolic Content (TPC) 169 TPC of the extracts were determined in triplicate using the 96-well Folin-Ciocalteu method 170 described by Bobo-García et al. (2015). Results were expressed as the mean concentration of 171 gallic acid equivalents (GAE) (mg GAE/g dry weight (DW)) ± standard error of the mean 172 (SEM). 173 Determination of Total Flavonoid Content (TFC) 174 TFC of extracts was measured in triplicate using a modified aluminum chloride method 175 based on Magalhães et al. (2012). Quercetin was used as the standard at a concentration range 176 of 20–60 µg/mL. TFC values were expressed as mean quercetin equivalents (QE) (mg QE/g 177 DW) ± SEM. 178 Evaluation of antioxidant activity 179 Oxygen Radical Absorbance Capacity (ORAC) assay 180 ORAC values were determined following the method of Kenny et al. (2015), with minor 181 modifications. Fluorescence decay curves of standard and samples were recorded over 1 hour. 182 A Trolox calibration curve was constructed within a linearity range of 20-60 µM. Each sample 183 was analyzed in triplicate, and antioxidant capacity was expressed as mean Trolox equivalents 184 (TE) (µmol TE/g DW) ± SEM. 185 Ferric Reducing Antioxidant Power (FRAP) assay 186 FRAP values were measured using a microplate-adapted version of the method described 187 by Işıl et al. (2010). Trolox standards (20-100 µM) were prepared in methanol. Extract 188 concentrations were adjusted to fall within the linear range of the calibration curve. Aliquots 189 of 20 µL of Trolox or extract solutions were dispensed into microplate wells, followed by the 190 addition of 130 µL of 30 mM HCl, 25 µL of 1.2% (w/v) potassium ferricyanide, and 25 µL of 191 10 0.1% (w/v) iron (III) chloride hexahydrate. Methanol was used as the blank for the standard 192 instead of Trolox, while distilled water replaced potassium ferricyanide in the extract blanks 193 to correct for intrinsic absorbance. Plates were incubated at room temperature, and the 194 absorbance was measured at 700 nm using a Synergy HT microplate reader (BioTek 195 Instruments, USA). Each assay was performed in triplicate, and results were reported as mean 196 TE (µmol TE/g DW) ± SEM. 197 2,2- Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay 198 The concentration of extract required to reduce 50% of DPPH radicals (IC50) was 199 determined using a microplate assay method adapted from Kenny et al. (2015). The IC50 values 200 were obtained by interpolation from dose-response curves fitted with the Hill equation. Each 201 determination was conducted in triplicate, and antioxidant activity was reported as mean IC50 202 (µg/mL) ± SEM. 203 Ferrous Iron Chelating Activity (FICA) assay 204 FICA was assessed following the procedure described by Santos et al. (2017), with minor 205 modifications. A 0.3 mM iron (II) chloride solution was used as iron (II) source, and the EDTA 206 standard curve ranged from 25-100 µM. Each assay was performed in triplicate, and results 207 were expressed as mean EDTA equivalents (EDTAE) (µmol EDTAE/g DW) ± SEM. 208 Evaluation of antibacterial activity 209 Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), Enterococcus 210 faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC BAA-211 2452), Klebsiella pneumoniae (ATCC 10031), and Salmonella enterica subsp. enterica serovar 212 Typhimurium (known as S. typhimurium) (ATCC 14028) were employed as test 213 microorganisms. The minimum inhibitory concentration (MIC) of the extracts was determined 214 using the broth microdilution method, following the guidelines established by the European 215 11 Committee for Antimicrobial Susceptibility Testing (EUCAST, 2003) of the European Society 216 of Clinical Microbiology and Infectious Diseases (ESCMID). Extracts were tested at a 217 maximum concentration of 10 mg/mL. Ciprofloxacin hydrochloride was used as the positive 218 control for E. coli and E. faecalis, while ceftriaxone disodium salt was used for the remaining 219 strains. Dimethyl sulfoxide (DMSO) was employed as the solvent for the extracts, at a final 220 concentration not exceeding 1% (v/v) in the culture broth. For antibiotic controls, the culture 221 broth was used without any added solvent. 222 Experimental animals 223 Sprague–Dawley rats (11-12 weeks old) were obtained from the Biological Testing 224 Laboratory (LEBi), University of Costa Rica. All experimental protocols were approved by the 225 Institutional Committee for the Care and Use of Laboratory Animals (CICUA 038-2020) and 226 complied with the International Guiding Principles for Biomedical Research Involving 227 Animals (CIOMS). Animals were maintained under standard conditions (22 ± 2 ◦C, light/dark 228 cycles of 12 h) and ad libitum access to food and water. 229 Due to limited fruit availability, only the stem and leaf extract (SL2) was used for in vivo 230 tests. SL2 was selected over SL1 because of its higher total phenolic content and antioxidant 231 activity, suggesting a greater presence of bioactive compounds. 232 Acute oral toxicity test 233 The acute oral toxicity of SL2 was evaluated according to OECD guideline 423. A single 234 oral dose of 2000 mg/kg (dissolved in distilled water) was administered to six female Sprague-235 Dawley rats (11 weeks old, 260 ± 15 g) fasted overnight. A control group received distilled 236 water. Animals were observed at 30, 60, 120, 180, and 240 minutes after treatment and daily 237 for 14 days. Clinical signs (e.g. tremors, convulsions, dehydration, salivation, diarrhea, 238 lethargy), as well as respiratory, circulatory, autonomic, and central nervous system function, 239 12 were monitored. Body weight was recorded every two days. At the end of the observation 240 period, animals were euthanized (150 mg/kg i.p. pentobarbital overdose), and necropsies 241 were performed to assess macroscopic changes in target organs. 242 Evaluation of antinociceptive activity by the tail flick test 243 Antinociceptive effects were assessed using the tail flick test described by D’Amour and 244 Smith (1941), with a Tail Flick Unit-Thermal Stimulation device (Ugo Basile, model 7360). Male 245 Sprague–Dawley rats (12 weeks old, 350 ± 20 g) were randomized into four groups (n = 8) and 246 treated i.p. with SL2 extract dissolved in saline solution (500 or 1000 mg/kg), morphine (5 247 mg/kg) as positive control, or saline solution (1 mL) as negative control. Tail withdrawal 248 latency was measured 10 minutes before (pre-treatment latency), and at 30, 60, 90, and 120 249 minutes after administration of treatments (post-treatment latency). All measurements were 250 performed at the midpoint of the distal third of the tail. A 20 s cut-off time was applied to 251 prevent tissue damage. Results were expressed as percentage of antinociception (A%) 252 according to the following formula: A% = (post-treatment latency – pre-treatment latency) / (cut-253 off time – pre-treatment latency) x 100. 254 Evaluation of anti-inflammatory activity by the carrageenan-induced paw edema model 255 The anti-inflammatory effect was assessed according to the method described by Winter et 256 al. (1962), with minor modifications. Male Sprague–Dawley rats (11 weeks old, 300 ± 20 g) 257 were randomized into four groups (n = 8) and treated i.p. with SL2 extract dissolved in saline 258 solution (250 or 500 mg/kg), indomethacin (50 mg/kg) as positive control, or saline (1 mL) as 259 negative control. After one hour, 0.1 mL 1% (w/v) of carrageenan was injected subplantarly 260 into the right hind paw, and 0.1 mL of saline into the left paw. Paw thickness was measured 261 in duplicate at 1, 2, 4, 6, and 24 hours using a digital caliper (Kroeplin, model C330). Results 262 were expressed as percentage of edema (E%) according to the following formula: E% = (right 263 paw size – left paw size) / (left paw size) x 100. 264 13 Histology of paw tissue 265 At the end of the anti-inflammatory evaluation, animals were euthanized by decapitation. 266 Subplantar skin samples were fixed in 10% formalin-PBS for 48 h, processed, embedded in 267 paraffin, sectioned (5 µm), and stained with hematoxylin–eosin. Histopathological evaluation 268 focused on tissue congestion and leukocyte infiltration. 269 Statistical analysis 270 Results were expressed as means ± SEM. Data from antioxidant activity assays were 271 analyzed by one-way ANOVA followed by Tukey’s post hoc tests and Pearson´s correlation. In 272 vivo results were analyzed using two-way repeated measures ANOVA followed by Dunnett´s 273 multiple comparisons test. A p value < 0.05 was considered statistically significant. Analyses 274 were performed using the GraphPad Prism® version 8. 275 RESULTS 276 Chemical characterization of the extracts 277 Data from the detection of different groups of metabolites in the ethanolic extracts obtained 278 from W. solanacea are summarized in Table 1. HPTLC chromatograms are shown in Figs. 1 to 279 4. Fruit extracts contained a more diverse array of phenolic compounds compared to extracts 280 from stems and leaves (Fig. 1A). Moreover, most of the phenolics present in the extracts were 281 highly polar. The flavonoids present in F1 and F2 consisted of a mixture of polar and non-282 polar compounds, with F1 containing the greatest number of such compounds (Fig. 1B and 283 Fig. 2B). Coumarins were only detected in SL1 and SL2 under low-polarity HPTLC 284 development conditions; however, under high-polarity elution conditions, coumarins were 285 also found in F2 but not in F1 (Fig. 1C and Fig. 2C). 286 287 14 Table 1. Results of qualitative tests for the detection of metabolite groups in W. solanacea extracts. 288 Detected metabolites Extract F1 F2 SL1 SL2 Metabolites detected by HPTLC Phenolics + + + + Flavonoids + + + + Quinones - - - - Coumarins - + + + Terpenoids + + + + Triterpenes and Steroids + + + + Alkaloids + + - - Metabolites detected by qualitative phytochemical screening Carbohydrates (Molisch test) + + + + Carbohydrates (Benedict test) + + + + Amino acids and peptides (Ninhydrin test) + + + + Amino acids and peptides (Biuret test) + + + + Saponins (Foam test) - - - - Tannins (Protein precipitation test) - - - - 15 Cardiac glycosides (Kedde test) - - - - Cardiac glycosides (Keller-Killiani test) - - - - +: Positive result; -: Negative result. F1: Fruit extract from Mora; F2: Fruit extract from Puerto Jiménez; SL1: Stem and leaf extract from Mora; SL2: Stem and leaf extract from Puerto Jiménez. 289 Figure 1. Chromatograms showing the detection of polar phenolic compounds in W. solanacea extracts. A) General phenolics (derivatization with potassium ferricyanide and ferric chloride reagent). B) Flavonoids (derivatization with aluminum chloride reagent). C) Coumarins, flavonoids, and quinones (derivatization with potassium hydroxide reagent). 16 Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; UV: chromatogram under UV light (366 nm); Vis: chromatogram observed under visible light. Major positive changes are highlighted with yellow or black boxes. 290 Figure 2. Chromatograms showing the detection of non-polar phenolic compounds in W. solanacea extracts. A) General phenolics (derivatization with potassium ferricyanide and ferric chloride reagent). B) Flavonoids (derivatization with aluminum chloride reagent). C) Coumarins, flavonoids, and quinones (derivatization with potassium hydroxide reagent). Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; UV: chromatogram under UV 17 light (366 nm); Vis: chromatogram observed under visible light. Major positive changes are highlighted with yellow or black boxes. Extracts were also rich in terpenoids (Fig. 3). Differences were observed between extracts 291 of the same plant material collected from different regions; moreover, F1 and SL1 showed a 292 more diverse terpenoid profile compared to F2 and SL2, respectively. The extracts were 293 particularly abundant in triterpenoid and steroidal compounds (Fig. 3B). The fruit extracts also 294 showed the presence of alkaloids, with F1 and F2 exhibiting a similar profile for this group of 295 metabolites (Fig. 4). However, SL1 and SL2 were negative for the presence of alkaloids. 296 18 Figure 3. Chromatograms showing the detection of terpenoids in W. solanacea extracts. A) General terpenoids (derivatization with anisaldehyde/sulfuric acid reagent). B) Steroids and triterpenoids (derivatization Liebermann-Burchard reagent). Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; UV: chromatogram under UV light (366 nm); Vis: chromatogram observed under visible light. Major positive changes are highlighted with yellow or black boxes. 297 Figure 4. Chromatograms showing the detection of alkaloids in W. solanacea extracts by derivatization with Dragendorff reagent. Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; Vis: chromatogram observed under visible light. Major positive changes are highlighted with black boxes. Determination of TPC, TFC and antioxidant activity 298 The results of the TPC and TFC assays are presented in Fig. 5A and Fig. 5B. Fruit extracts 299 showed higher levels of TPC and TFC compared to stem and leaf extracts. F1 had the highest 300 TPC value (59.1 ± 0.9 mg GAE/g), whereas F2 showed the highest TFC level (127.7 ± 5 mg 301 QE/g). 302 19 Figure 5. Determination of TPC, TFC, and antioxidant activity of W. solanacea extracts. A) TPC values. B) TFC values. C) ORAC values. D) FRAP values. E) IC50 DPPH values. F) FICA values. Different capital letters above the bars indicate significant differences (p < 0.05). Results are expressed as mean ± SEM (n = 3). DPPH: 2,2-diphenyl-1-picrylhydrazyl assay; DW: dry weight; EDTAE: ethylenediaminetetraacetic acid equivalents; F1: fruit extract from Mora; F2: fruit extract from Puerto Jiménez; FICA: ferrous iron chelating activity assay; FRAP: ferric reducing antioxidant power assay; GAE: gallic acid equivalents; ORAC: oxygen radical absorbance capacity assay; QE: quercetin equivalents; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; TE: Trolox equivalents; TFC: total flavonoid content; TPC: total phenolic content. Regarding the antioxidant activity assays, fruit extracts exhibited the strongest antioxidant 303 activity in ORAC, FRAP, and DPPH assays (Fig. 5C, Fig. 5D, and Fig. 5E). F1 was the most 304 potent extract in these assays, with ORAC, FRAP, and DPPH IC50 values of 1796 ± 63 µmol 305 TE/g, 472.7 ± 5.4 µmol TE/g, and 51.6 ± 2.5 µg/mL, respectively. In the FICA assay (Fig. 5F), 306 F2 and SL2 were the most active, with FICA values of 104.6 ± 0.5 µmol EDTAE/g and 103.1 ± 307 0.7 µmol EDTAE/g, respectively. 308 20 On the other hand, Fig. 6 indicates a significant correlation between TPC and TFC values 309 and the antioxidant activities evaluated by ORAC, DPPH and FRAP assays. However, the 310 results from the FICA assay did not show a correlation with either TPC or TFC. 311 Figure 6. Pearson’s correlation matrix. A) Correlation coefficients (r). B) p-values matrix. DPPH: 2,2-diphenyl-1-picrylhydrazyl assay; FICA: ferrous iron chelating activity assay; FRAP: ferric reducing antioxidant power assay; ORAC: oxygen radical absorbance capacity assay; TFC: total flavonoid content; TPC: total phenolic content. Determination of antibacterial activity 312 21 Table 2 summarizes the results from the broth microdilution assay. SL2 was the only active 313 extract, with a MIC value of 5 mg/mL against E. faecalis, which is classified as exhibiting mild 314 antibacterial activity based on the criteria reported by Bussmann et al. (2010). 315 22 Table 2. Antibacterial activity of W. solanacea extracts against selected bacterial strains. 316 Bacteria Minimum Inhibitory Concentration F1 (mg/mL) F2 (mg/mL) SL1 (mg/mL) SL2 (mg/mL) Control* (µg/mL) S. aureus (ATCC 6538) >5 >5 >5 >5 4 S. epidermidis (ATCC 12228) >5 >5 >5 >5 2 E. faecalis (ATCC 29212) >5 >5 >5 5 0.5 P. aeruginosa (ATCC 15442) >5 >5 >5 >5 32 E. coli (ATCC BAA-2452) >5 >5 >5 >5 0.03 K. pneumoniae (ATCC 10031) >5 >5 >5 >5 0.03 S. typhimurium (ATCC 14028) >5 >5 >5 >5 0.06 *Control: ciprofloxacin chloride for E. coli and E. faecalis, or ceftriaxone disodium salt for other bacteria. F1: fruit extract from Mora; F2: fruit extract from Puerto Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez. 317 23 Acute toxicity test 318 The acute toxicity test revealed that oral administration of SL2 extract at a single dose of 319 2000 mg/kg did not cause mortality in the rats during the 14 days of the assay. Since the 320 median lethal dose (LD50) is greater than 2000 mg/kg, this extract is classified as non-toxic 321 according to OECD guideline 423. Moreover, the extract did not cause significant changes in 322 behavior, general appearance, or any signs of toxicity during 14 days following 323 administration. Body weight gain in both control and treated groups was similar. Necropsy 324 conducted 14 days post-administration revealed no significant macroscopic alterations in the 325 target organs. 326 Antinociceptive activity 327 The results of the antinociceptive activity of the SL2 extract are presented in Fig. 7. The tail-328 flick test showed a significant (p = 0.0334) increase in baseline latency time (i.e., the time 329 required to withdraw the tail) 30 min after the treatment with 1000 mg/kg of SL2 extract 330 compared to the control group. No significant effect was observed at later time points or with 331 the 500 mg/kg dose. As expected, morphine (5 mg/kg) caused a significant increase in latency 332 time at 30, 60, and 90 minutes post-treatment compared to the control (p = 0.0005, p = 0.0009, 333 and p = 0.0119, respectively). Administration of the vehicle did not significantly affect the 334 nociceptive threshold. 335 24 Figure 7. Antinociceptive effect of the stem and leaf extract from Puerto Jiménez (SL2) in the tail flick test in rats. Results are presented as the mean ± SEM of the percentage of antinociception (n = 8). Statistical significance was determined by two-way repeated measures ANOVA followed by Dunnett´s multiple comparisons test. *p < 0.05, ***p < 0.001 compared to control (saline solution). Anti-inflammatory activity 336 The results of the anti-inflammatory activity of the SL2 extract are shown in Fig. 8. Injection 337 of carrageenan into the hind paw tissue of rats pretreated with vehicle (control group) caused 338 a progressive increase in paw edema that persisted for 24 hours, with a maximum effect at 4 339 and 6 hours. Pretreatment with 500 mg/kg of SL2 extract significantly reduced the edema 340 induced by carrageenan at 2 and 4 hours post-injection compared to the control group (p = 341 0.0209 and p= 0.0043, respectively). No significant reduction was observed at 1, 6 or 24 hours, 342 although a trend toward decreased edema was noted across all time points, similar in 343 magnitude to the indomethacin group. Pretreatment with 250 mg/kg of SL2 extract 344 significantly (p = 0.0222) reduced edema only at 4 hours post-injection. No significant 345 reduction in inflammation was observed at 1, 2, 6, or 24 hours. Indomethacin (50 mg/kg) 346 25 produced a significant reduction in paw edema at 2 and 4 hours post-injection as compared to 347 the control group (p = 0.0229 and p = 0.0143, respectively). 348 Figure 8. Anti-inflammatory effect of the stem and leaf extract from Puerto Jiménez (SL2) extract in carrageenan-induced paw edema in rats. Results are presented as the mean ± SEM of the percentage of edema (right hind paw) relative to non-inflamed control (left hind paw) (n = 8). Statistical significance was determined by two-way repeated measures ANOVA followed by Dunnett´s multiple comparison test. *p < 0.05 compared to control (saline solution). The histological biopsies of the rat paw 24 hours after carrageenan injection are presented 349 in Fig. 9. Paw tissue from the control group pretreated with vehicle (Fig. 9A) showed typical 350 signs of acute inflammation, including leukocyte infiltration and congestion, compared to 351 normal paw tissue (Fig. 9B). Pretreatment with indomethacin decreased paw swelling and 352 leukocyte infiltration (Fig. 9E). Pretreatment with 500 mg/kg of SL2 extract similarly reduced 353 swelling and leukocyte infiltration to a comparable extent (Fig. 9C). Paw tissue from animals 354 pretreated with 250 mg/kg of SL2 extract showed more leukocyte infiltration and congestion 355 than the group treated with the higher dose (Fig. 9D). 356 26 Figure 9. Paw tissue dissected 24 hours after injection of A) carrageenan or B) saline solution in control groups; C) 500 mg/kg, D) 250 mg/kg of stem and leaf extract from Puerto Jiménez (SL2); and E) 50 mg/kg of indomethacin. Arrows indicate leukocyte infiltration. Arrowheads indicate congestion and swelling in the tissue. Hematoxylin–eosin staining. Scale bar = 50 µm. DISCUSSION 357 In Latin American countries, W. solanacea has traditionally been used for various purposes, 358 including as an anti-inflammatory and antimicrobial agent, and for general pain relief. 359 Ethnobotanical reports describe the use of different parts of the plant – such as fruits, stems, 360 and leaves - prepared using diverse methods (Gupta et al., 1993; Caballero-George et al., 2001; 361 García et al., 2006; Jacobo-Herrera et al., 2006; Coe, 2008; Ballesteros et al., 2016). Given the 362 27 diversity in traditional preparations, ethanolic extracts of each plant material were prepared 363 for chemical characterization and biological evaluation. 364 The primary finding from chemical characterization was the variation among extracts from 365 different plant parts and collection sites. Phytochemical screening and HPTLC analysis 366 revealed the presence of phenolics, flavonoids, coumarins, terpenoids, alkaloids, triterpenes, 367 and steroids. Variation in phytochemical profiles between plant parts is typical in medicinal 368 plants (Ak et al., 2020). Furthermore, chemical differences related to collection sites have been 369 reported in other species (Camara et al., 2021). F1 and SL1 were collected in Costa Rica´s 370 Central Valley (cooler, moderate rainfall), while F2 and SL2 were from the South Pacific 371 Region (hotter, more humid). Environmental factors such as altitude, temperature, solar 372 radiation, and precipitation are known to influence the metabolite profiles (Liu et al., 2016). 373 Genotypic variation may also contribute to the observed differences (Camara et al., 2021). 374 These aspects warrant further in-depth investigation. 375 The extracts were rich in triterpenoid and steroidal compounds, consistent with previous 376 reports of these metabolite groups in species of the Witheringia genus, including W. solanacea 377 (Pequeno et al., 2017). Previous studies have isolated physalins —steroidal lactones— from 378 this species (Jacobo-Herrera et al., 2006). 379 As expected for Solanaceae species, fruit extracts were positive for alkaloids. Several 380 alkaloids from this family exhibit anti-inflammatory activity (Xia et al., 2021). Thus, identifying 381 the alkaloids in W. solanacea fruit and evaluating their anti-inflammatory potential is a 382 promising research direction. Unfortunately, this analysis could not be conducted due to 383 limited fruit availability. 384 Consistent with qualitative assays, in vitro analyses revealed differences in TPC and TFC 385 values, and antioxidant activity among extracts from different regions, likely due to 386 28 environmental variation. Fruit extracts showed higher TPC and TFC values, and stronger 387 antioxidant activities compared to stem and leaf extracts. Similar differences in TPC, TFC, and 388 antioxidant activity among plant parts have been reported in related species, such as Withania 389 somnifera (Alam et al., 2012; Sahoo et al., 2024) and Physalis peruviana (Ertürk et al., 2017), 390 although their extracts exhibited lower values than those of W. solanacea in the present study. 391 The antioxidant capacity was evaluated using assays based on different chemical 392 mechanisms. Phenolic compounds and flavonoids are known to stabilize free radicals and to 393 chelate transition metals involved in Fenton and Haber-Weiss reactions (Perron and 394 Brumaghim, 2009). A positive correlation was observed between TPC/TFC and antioxidant 395 activities in the ORAC, DPPH, and FRAP assays. In contrast, no such correlation was found 396 with FICA results, possibly due to differences in compounds responsible for metal chelation. 397 This hypothesis is supported by the observed variations in phenolic composition among the 398 extracts. 399 Antibacterial activity was assessed due to traditional use of W. solanacea for treating 400 infections (García et al., 2006; Jacobo-Herrera et al., 2006). Only the stem and leaf extract of W. 401 solanacea from Puerto Jiménez showed mild activity against E. faecalis, a pathogen involved in 402 external and internal infections. Although limited, these findings are noteworthy due to the 403 lack of prior antibacterial studies, despite its traditional use. 404 In vivo studies were conducted only with the stem and leaf extract due to limited fruit 405 availability. SL2 was selected based on higher TPC and antioxidant activity than SL1, 406 suggesting greater concentration of active metabolites. 407 Toxicological evaluation is essential for medicinal plant research. Thus, the acute oral 408 toxicity of SL2 was assessed prior to its pharmacological evaluation. Results indicated no acute 409 toxicity at doses up to 2000 mg/kg. This is a relevant finding, given the lack of previous 410 29 toxicological studies for W. solanacea or related species. These results are consistent with 411 previous studies conducted with extracts from plants of the Solanaceae family, in which many 412 species have been shown to exhibit low acute oral toxicity in animal models at doses of up to 413 5000 mg/kg (Parra et al., 2001; Epoh et al., 2019; Moussaoui et al., 2020). 414 Pain is a common symptom associated with numerous medical conditions. In Latin 415 America, W. solanacea is traditionally used to treat headaches and general pain (Gupta et al., 416 1993; Caballero-George et al., 2001; García et al., 2006); although no pharmacological studies 417 had been reported prior to this work. The tail flick test is a well-established method for 418 assessing analgesic properties, measuring the latency of the tail flick reflex following thermal 419 stimulation, which activates pain responses at spinal and/or supraspinal levels (Barrot, 2012). 420 Although the SL2 extract of W. solanacea produced an analgesic effect only during the first 30 421 minutes of the test, this finding is noteworthy considering its traditional use for pain relief and 422 the absence of previous pharmacological evidence. 423 The phenolic-rich profile of W. solanacea may explain its analgesic potential, as such 424 compounds possess antinociceptive activity (Sun and Shahrajabian, 2023). Additionally, some 425 physalins—compounds identified in W. solanacea—have been reported to possess analgesic 426 properties (Wu et al., 2021; Jacobo-Herrera et al., 2006). These components may contribute to 427 the observed analgesic effects of the plant. However, further studies should be conducted 428 using additional nociception and pain models to confirm this activity and to identify the 429 specific active metabolites involved. 430 The carrageenan-induced paw edema model, which mimics acute inflammation, was used 431 to assess anti-inflammatory activity. This model involves two phases: an early phase involving 432 the release of mediators such as histamine, serotonin, and bradykinin, and a later phase 433 characterized by increased cyclooxygenase (COX) and nitric oxide synthase activity, leading 434 to edema and leukocyte infiltration (McKim et al., 2016). The SL2 extract reduced edema at 2 435 30 and 4 hours and decreased leukocyte infiltration at 24 hours, similar to indomethacin, 436 suggesting inhibition of pro-inflammatory enzymes. These findings indicate a modulatory 437 effect on acute inflammation; however, further mechanistic studies are needed. 438 Previous research demonstrated that physalins B and F (but not D) from W. solanacea 439 modulate NF-κB in cell models (Jacobo-Herrera et al., 2006). Other studies have confirmed the 440 anti-inflammatory effects of physalins through suppression of cytokines (Meira et al., 2022). 441 However, in vivo evidence of anti-inflammatory activity for W. solanacea was previously 442 lacking. The present findings provide novel in vivo confirmation that supports its traditional 443 medicinal use. 444 Finally, antioxidant polyphenols may contribute to the observed anti-inflammatory effects 445 (Roy et al., 2022). Phenolic acids and flavonoids have been shown to mitigate oxidative stress 446 in inflammation models, including carrageenan-induced paw edema, in which oxidative stress 447 is a key component (Albarakati, 2022). Thus, in addition to physalins, polyphenols are likely 448 contributors to the anti-inflammatory activity of W. solanacea. Future studies could include 449 fractionation of the active extract to isolate specific anti-inflammatory or analgesic 450 compounds, and assessment of their effects on inflammatory mediators (e.g. COX, cytokines) 451 to elucidate mechanisms. 452 CONCLUSION 453 Extracts from the aerial parts of W. solanacea demonstrated anti-inflammatory and 454 antinociceptive effects with no observed acute toxicity. These results support the traditional 455 use of the plant for treating inflammation and pain. In contrast, antibacterial activity was 456 weak. The observed anti-inflammatory and analgesic effects may be attributed to antioxidant 457 phenolics, flavonoids, and known physalins present in the extracts. W. solanacea therefore 458 represents a promising source of compounds for managing inflammation and pain. 459 31 CONFLICT OF INTEREST 460 The authors declare that they have no competing financial interests that could influence the results reported in 461 this paper. 462 ACKNOWLEDGMENT 463 The authors would like to thank Jorge Poveda and Minor Carranza from the “Juvenal Valerio Rodríguez” 464 Herbarium (Universidad Nacional, Costa Rica), and German Madrigal from the Pharmaceutical Research 465 Institute (INIFAR, for its acronym in Spanish), University of Costa Rica, for their help in the collection and 466 identification of the plant. The authors also thank the technical staff and students from the School of Pharmacy, 467 University of Costa Rica, and from the Pharmaceutical Research Institute, University of Costa Rica, for their help 468 in some parts of the experiments. This work was supported by the University of Costa Rica (grant number 817-469 C1-096). 470 DECLARATION OF GENERATIVE AI IN THE WRITING PROCESS 471 During the preparation of this work, the authors used ChatGPT in order to check grammar, spelling, and clarity. 472 After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the 473 published article. 474 REFERENCES 475 Ak G, Zengin G, Sinan KI, Mahomoodally MF, Picot-Allain MCN, Cakir O, Bensari S, Yilmaz MA, Gallo M, 476 Montesano D (2020) A comparative bio-evaluation and chemical profiles of Calendula officinalis L. extracts 477 prepared via different extraction techniques. 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Phytochemistry 191: 112925. https://doi.org/10.1016/j.phytochem.2021.112925. 590 Xia W, Kolli AR, Koshibu K, Martin F, Kondylis A, Kuczaj A, Tan WT, Yeo YS, Tan G, Teng C, Woon K, Schneider 591 T, Talikka M, Phillips BW, Vanscheeuwijck P, Peitsch MC, Hoeng J (2021) In vivo profiling of a natural alkaloid, 592 anatabine, in rodents: pharmacokinetics and anti-inflammatory efficacy. J Nat Prod 84: 1012-1021. 593 https://doi.org/10.1021/acs.jnatprod.0c01044. 594 https://doi.org/10.51929/jms.38.52.2021 https://doi.org/doi.1007/978-3-030-78160-6_20 https://doi.org/10.1016/j.foodchem.2016.07.091 https://doi.org/10.3390/molecules28041845 https://doi.org/10.3181/00379727-111-27849 https://doi.org/10.1016/j.phytochem.2021.112925 https://doi.org/10.1021/acs.jnatprod.0c01044 36 Legends of figures 595 Figure 1. Chromatograms showing the detection of polar phenolic compounds in W. solanacea extracts. A) 596 General phenolics (derivatization with potassium ferricyanide and ferric chloride reagent). B) Flavonoids 597 (derivatization with aluminum chloride reagent). C) Coumarins, flavonoids, and quinones (derivatization with 598 potassium hydroxide reagent). 599 Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto 600 Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; UV: chromatogram under UV 601 light (366 nm); Vis: chromatogram observed under visible light. Major positive changes are highlighted with yellow or black 602 boxes. 603 604 Figure 2. Chromatograms showing the detection of non-polar phenolic compounds in W. solanacea extracts. A) 605 General phenolics (derivatization with potassium ferricyanide and ferric chloride reagent). B) Flavonoids 606 (derivatization with aluminum chloride reagent). C) Coumarins, flavonoids, and quinones (derivatization with 607 potassium hydroxide reagent). 608 Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto 609 Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; UV: chromatogram under UV 610 light (366 nm); Vis: chromatogram observed under visible light. Major positive changes are highlighted with yellow or black 611 boxes. 612 613 Figure 3. Chromatograms showing the detection of terpenoids in W. solanacea extracts. A) General terpenoids 614 (derivatization with anisaldehyde/sulfuric acid reagent). B) Steroids and triterpenoids (derivatization 615 Liebermann-Burchard reagent). 616 Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto 617 Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; UV: chromatogram under UV 618 light (366 nm); Vis: chromatogram observed under visible light. Major positive changes are highlighted with yellow or black 619 boxes. 620 621 Figure 4. Chromatograms showing the detection of alkaloids in W. solanacea extracts by derivatization with 622 Dragendorff reagent. 623 Dr: observation after derivatization; Dv: observation after development; F1: fruit extract from Mora; F2: fruit extract from Puerto 624 Jiménez; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; Vis: chromatogram observed 625 under visible light. Major positive changes are highlighted with black boxes. 626 627 37 Figure 5. Determination of TPC, TFC, and antioxidant activity of W. solanacea extracts. A) TPC values. B) TFC 628 values. C) ORAC values. D) FRAP values. E) IC50 DPPH values. F) FICA values. 629 Different capital letters above the bars indicate significant differences (p < 0.05). Results are expressed as mean ± SEM (n = 3). 630 DPPH: 2,2-diphenyl-1-picrylhydrazyl assay; DW: dry weight; EDTAE: ethylenediaminetetraacetic acid equivalents; F1: fruit 631 extract from Mora; F2: fruit extract from Puerto Jiménez; FICA: ferrous iron chelating activity assay; FRAP: ferric reducing 632 antioxidant power assay; GAE: gallic acid equivalents; ORAC: oxygen radical absorbance capacity assay; QE: quercetin 633 equivalents; SL1: stem and leaf extract from Mora; SL2: stem and leaf extract from Puerto Jiménez; TE: Trolox equivalents; TFC: 634 total flavonoid content; TPC: total phenolic content. 635 636 Figure 6. Pearson’s correlation matrix. A) Correlation coefficients (r). B) p-values matrix. 637 DPPH: 2,2-diphenyl-1-picrylhydrazyl assay; FICA: ferrous iron chelating activity assay; FRAP: ferric reducing antioxidant power 638 assay; ORAC: oxygen radical absorbance capacity assay; TFC: total flavonoid content; TPC: total phenolic content. 639 640 Figure 7. Antinociceptive effect of the stem and leaf extract from Puerto Jiménez (SL2) in the tail flick test in rats. 641 Results are presented as the mean ± SEM of the percentage of antinociception (n = 8). Statistical significance was determined by 642 two-way repeated measures ANOVA followed by Dunnett´s multiple comparisons test. *p < 0.05, ***p < 0.001 compared to control 643 (saline solution). 644 645 Figure 8. Anti-inflammatory effect of the stem and leaf extract from Puerto Jiménez (SL2) extract in carrageenan-646 induced paw edema in rats. 647 Results are presented as the mean ± SEM of the percentage of edema (right hind paw) relative to non-inflamed control (left hind 648 paw) (n = 8). Statistical significance was determined by two-way repeated measures ANOVA followed by Dunnett´s multiple 649 comparison test. *p < 0.05 compared to control (saline solution). 650 651 Figure 9. Paw tissue dissected 24 hours after injection of A) carrageenan or B) saline solution in control groups; C) 652 500 mg/kg, D) 250 mg/kg of stem and leaf extract from Puerto Jiménez (SL2); and E) 50 mg/kg of indomethacin. 653 Arrows indicate leukocyte infiltration. Arrowheads indicate congestion and swelling in the tissue. Hematoxylin–eosin staining. 654 Scale bar = 50 µm. 655