Golden berry fruit modulates inflammation in LPS-stimulated RAW 264.7 macrophages and the DSS-induced acute colitis model

Golden berry fruit modulates inflammation in LPS-stimulated RAW 264.7 macrophages and the DSS-induced acute colitis model Jenny Castro a,b , Guillermo Lopez-Lluch c , Juan Carlos Rodríguez d , Rocío de la Puerta e , Lía Barrios f , Rub´ en Salas a , Luis Franco a,* a Biological Evaluation of Promising Substances Group, Universidad de Cartagena, Cartagena, Colombia b Faculty of Chemistry and Pharmacy, Universidad del Atl´ antico, Barranquilla 081007, Colombia c Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Sevilla, Spain d Departamento de Fisiología, Anatomía y Biología Celular, Universidad Pablo de Olavide, Sevilla, Spain e Department of Pharmacology, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain f Histopathology Research Group, Universidad de Cartagena, Cartagena, Colombia ARTICLE INFO Keywords: Physalis peruviana L Golden berry Dietary supplements RAW 264.7 Inflammatory bowel disease Dextran sodium sulphate-induced acute colitis ABSTRACT Physalis peruviana L. (golden berry) fruit is attractive for its many health benefits, associated with the presence of anti-inflammatory and antioxidant compounds. High fruit consumption has been associated with a lower risk of developing chronic non-communicable diseases. This work evaluated the anti-inflammatory potential of ten fruits cultivated in Colombia, determining their capacity to inhibit ⋅NO production in LPS-activated RAW 264.7 macrophages. The most active extracts were evaluated for their effect on the production of IL-1β, IL-6, TNFα , and PGE2. Golden berry was the most active extract, so its immunomodulatory effect was evaluated in a model of DSS-induced colitis in BALB/c mice. Dietary supplementation with golden berry attenuates the pathological symptoms of colitis. This effect may be associated with the inhibition of neutrophil infiltration and impacts on the production of IL-6, IL-1β, IL-10, TNFα , and ROS. Our results suggest that consistently consuming golden berries could benefit intestinal inflammation. 1. Introduction Suboptimal nutrition ranks first among lifestyle-related NCD risk factors worldwide and has been identified as the most important preventable NCD risk factor. In recent decades, as part of globalization, western lifestyles that include unhealthy dietary patterns have been adopted worldwide; this trend is known as the “nutrition transition” and is characterized by the replacement of a diet traditionally rich in fruits and vegetables with a diet high in calories from simple carbohydrates and animal fats. This has increased the incidence of NCDs worldwide (Astrup, Dyerberg, Selleck, & Stender, 2008; Beaglehole & Yach, 2003; Kimokoti & Millen, 2016). Although the association between inflammation and chronic diseases is widely recognized, the causality and extent to which inflammation contributes to and acts as a risk factor for disease development remain unresolved (Minihane et al., 2015). Noncommunicable diseases are non-infectious and long-lasting (more than three to six months); most of which can only be treated but not cured, and usually develop slowly and asymptomatically but result in devastating complications, leading to premature death and poor quality of life (Meetoo, 2008; Senthilkumar & Kim, 2013). These diseases are the leading cause of mortality globally. It is estimated that 70 % of all deaths worldwide are attributable to noncommunicable diseases (Holt, Kaviani, Sheth, & van Driel, 2018; Ruby, Knight, Perel, Blanchet, & Roberts, 2015). These diseases have traditionally been high in developed countries due to unhealthy diets and physical inactivity (Wagner & Brath, 2012). However, a rapid increase in these diseases has been observed in lowand middle-income countries, with significant adverse social, economic, and health effects (Alwan et al., 2010). In recognition of the impact of a healthy diet on preventing the onset of NCDs, the World Health Organization (WHO) has issued expert dietary recommendations to reduce the risk of NCDs, including consuming a significant amount of vegetables and fruits (Kimokoti & Millen, 2016). Fruits are a food group of great importance in human nutrition because they are a significant source of vitamins, minerals, and dietary fiber (Palafox- * Corresponding author at: Biological Evaluation of Promising Substances Group, Facultad de Ciencias Farmac´ euticas, Universidad de Cartagena, Zaragocilla Campus, Calle 50 No. 24–120, Cartagena 130014, Colombia. E-mail address: [email protected] (L. Franco). Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff https://doi.org/10.1016/j.jff.2025.106665 Received 28 December 2023; Received in revised form 12 December 2024; Accepted 2 January 2025 Journal of Functional Foods 125 (2025) 106665 Available online 8 January 2025 1756-4646/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/bync/4.0/ ). Carlos, Ayala-Zavala, & Gonz´ alez-Aguilar, 2011). High dietary fruit consumption has been associated with a lower risk of chronic and degenerative diseases, while low fruit consumption is among the top ten risk factors most implicated in overall mortality (Li et al., 2016; Slavin & Lloyd, 2012). This relationship is supported by studies showing that regular consumption of fruits in the diet can modulate chronic inflammation, which has been recognized as a pathophysiological mechanism underlying the onset of many NCDs (Joseph, Edirisinghe, & BurtonFreeman, 2016). Fruits grown in Colombia such as yellow pitaya (Hylocereus megalanthus), banana passion (Passiflora cumbalensis), purple passion (Passiflora edulis), golden berry (Physalis peruviana), tamarillo (Solanum betaceum), lulo (Solanum quitoense), soursop (Annona muricata), kalipatti sapota (Manilkara zapota), sapote (Pouteria sapota), and sour guava (Psidium friedrichsthalianum), and are commonly consumed in Colombia and are widely used to treat various chronic non-communicable diseases (Mejia et al., 2020). Among these stands out the cape gooseberry (Physalis peruviana), which is appreciated nationally and internationally not only for its attractive color and flavor but also for its multiple beneficial health properties, being used by various cultures to treat digestive disorders such as indigestion and heartburn, as well as to strengthen the immune system and prevent respiratory diseases (Carrillo-Perdomo, Aller, Cruz-Quintana, Giampieri, & Alvarez-Suarez, 2015; Franco, Matiz, Calle, Pinz´ on, & Ospina, 2007; Navarro-Hoyos et al., 2022; Pinto et al., 2009). This has aroused great interest in the scientific community, leading to multiple studies that have identified bioactive compounds in this fruit, such as β-carotene, lycopene, lutein, quercetin, rutin, peruviosas, campesterol, β-sitosterol, stigmasterol, avenasterol, lupeol, linalool, and several withanolides (Kasali et al., 2021; Mier-Giraldo, Díaz-Barrera, Delgado-Murcia, Valero-Valdivieso, & C´ aez-Ramírez, 2017). These compounds have antioxidant and antiinflammatory properties that could contribute to the prevention and treatment of diseases involving chronic inflammation, such as cardiovascular, respiratory, and neurodegenerative diseases, cancer, diabetes mellitus, and inflammatory bowel disease (Pan, Lai, & Ho, 2010). This work evaluated the in vitro anti-inflammatory potential of ten ethanolic extracts of fruits grown in Colombia, determining their capacity to inhibit nitric oxide (⋅NO) production in LPS-activated RAW 264.7 macrophages. In addition, the effects of the active extracts on the production of IL-1β, IL-6, TNFα , and PGE-2 in LPS-activated RAW 264.7 macrophages were determined, as it was hypothesized that the fruit inhibiting the highest number of inflammatory mediators could have an immunomodulatory effect when regularly included in the diet. To corroborate this hypothesis, the golden berry extract, which proved to be the most active, was evaluated in the dextran sulfate sodium (DSS)- induced IBD model in BALB/c mice, which simulates the prolonged and exacerbated chronic inflammatory process that characterizes the inflammatory bowel disease. 2. Materials and methods 2.1. Reagents Penicillin-streptomycin, trypan blue, lipopolysaccharide from Escherichia coli (LPS), sodium nitrite, N-[1,1-naphthyl] ethylenediamine dihydrochloride, sulfanilamide, TEMED, 2 ′ ,7 ′ -dichlorofluorescein diacetate (DCFH-DA), phosphate buffer saline (PBS) tablets, O-Dianisidine, ethylenediaminetetraacetic acid (EDTA), hydrogen peroxide (H 2 O 2 ), indomethacin, dexamethasone, rofecoxib, hematoxylin and eosin, were purchased from Sigma Aldrich (St Louis, MO). Ethanol, dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle Medium (DMEM), and Fetal Bovine Serum (FBS) were obtained from Thermo Fisher Scientific (Pittsburgh, PA). Acrylamide/bisacrylamide, Tris HCL, Bradford assay, precision plus protein standards dual color, Tris/Glycine/SDS buffer, Tris/Glycine buffer, Blotting-grade blocker were obtained from Bio-Rad. Bromide of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), hexadecyl-trimethylammonium bromide (HTAB), were obtained from Calbiochem® (San Diego, CA). Macrophages RAW 264.7 were acquired from the American Type Culture Collection (Manassas, VA). Phosphoric acid was obtained from JT Baker (Phillipsburg, NJ). ELISA kits from eBiosciences. Western HRP substrate was obtained from Millipore and TPER tissue protein extraction reagent from Thermo Scientific. 2.2. Fruit samples and determination of physicochemical parameters Fruits included in this study were obtained from Fusagasuga (Cundinamarca) and Turbana (Bolivar) and involved yellow pitaya (Hylocereus megalanthus), banana passion (Passiflora cumbalensis), purple passion (Passiflora edulis), golden berry (Physalis peruviana), tamarillo (Solanum betaceum), lulo (Solanum quitoense), soursop (Annona muricata), kalipatti sapota (Manilkara zapota), sapote (Pouteria sapota) and sour guava (Psidium friedrichsthalianum). Taxonomic identification was carried out at the Herbarium of the Universidad de Antioquia, Medellín, Colombia; one specimen of each species was kept at that institution. 2.3. Preparation of extracts Fruits (1000 g each) were washed with tap water, cut into small pieces, homogenized in a professional blender (Ninja BN751), frozen at −80 ◦C and lyophilized (Labconco FreeZone 2.5, Kansas City, MO, USA). The powder was extracted in covered containers for 3 days with three volumes of ethanol (96 %) at room temperature, frequently shaking; this procedure was repeated until exhausted. The extract was filtered and concentrated in a rotary evaporator (Buchi R100, Switzerland) at a controlled temperature (38–40 ◦C) and reduced pressure to remove ethanol. Information on the fruits chosen for the study and yields are presented in Table 1. 2.4. Cell culture experiments Murine macrophage cell line RAW 264.7 (ATCC® TIB-71™ Rockville, MD, USA) was maintained in DMEM supplemented with 10 % heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere of 5 % CO 2 at 37 ◦C. For the in vitro assays, the extracts were dissolved in DMSO, ensuring that the final concentration of DMSO in the culture medium was less than 1 %. Table 1 Information on fruits included in the study. Fruits Part used Extract yields (%) Voucher number Yellow pitaya Hylocereus megalanthus pulps and seed 30,08 199,083 Banana passion Passiflora cumbalensis pulps 81.41 199,085 Purple passion Passiflora edulis pulps 84,44 199,081 Golden berry Physalis peruviana Whole fruit 44,70 199,086 Tamarillo Solanum betaceum pulps 42,87 199,080 Lulo Solanum quitoense Pulps and seed 60,0 199,084 Soursop Annona muricata pulps 57,91 201,951 Kalipatti sapota Manilkara zapota pulps 49,95 201,955 Sapote Pouteria sapota pulps 36,21 201,953 Sour guava Psidium friedrichsthalianum pulps 52,66 201,952 Extract yields: (g of extracts obtained/ g of freeze-dried fruit)*100. J. Castro et al. Journal of Functional Foods 125 (2025) 106665 2 2.4.1. Assessment of cell viability The toxicity of fruit extracts on RAW 264.7 macrophages was assessed by the colorimetric MTT method (Scudiero et al., 1988). RAW 264.7 macrophages were seeded in sterile 96-well plates (2 ×10 4 cells/ well) and incubated at 37 ◦C for 48 h. The medium was removed, and the cells were washed with PBS and treated for 30 min with different concentrations of fruit extracts (2000–500 μ g/mL) dissolved in the medium, after which they were activated with LPS (1 μ g/mL) and incubated again for 24 h at 37 ◦C, cells were washed with PBS, and 100 μ L of MTT solution (0.25 mg/mL) was added to each well of the plate. The plates were incubated at 37 ◦C for 4 h in a CO 2 incubator. Finally, the supernatant was removed, 100 μ L of DMSO was added to dissolve the formazan crystals, and the absorbance was determined at 550 nm on a Multiskan EX microplate reader (Thermo Scientific, Waltham, MA, USA). In each trial, a group of cells not exposed to the extracts was included as a negative control, and a group exposed to Triton X-100 (20 %) as a maximally toxic control. Viability was calculated as a percentage, considering the negative control as 100 % viability. The concentrations used to evaluate the fruit extracts were defined in terms of their solubility in DMSO (2000 μ g/mL was the maximum concentration at which all fruit extracts could be solubilized in DMSO). 2.4.2. Determination of nitric oxide (NO) To determine the effect of fruit extracts on the release of nitric oxide, RAW 264.7 macrophages were seeded in sterile 96-well plates (2 ×10 4 cells/well) and incubated at 37 ◦C for 48 h. The medium was removed, and the cells were washed with PBS and treated for 30 min with the fruit extracts (2000, 1000 and 500 μ g/mL), after which they were activated with LPS (1 μ g/mL) and incubated again for 24 h at 37 ◦C. After this time, 70 μ L of the supernatants were removed, and NO released as its end product (nitrite) was measured by the Griess method (Green et al., 1982). Supernatants were mixed with equal volumes of Griess reagent (1 % sulfanilamide in 3 % phosphoric acid and 0.1 % naphthyl ethylenediamine dihydrochloride), and the mixture was incubated at room temperature for 5 min. The absorbance was measured at 550 nm with a Multiskan EX microplate reader (Thermo Scientific, Waltham, MA, USA). Nitrite concentration was determined using a standard curve prepared with sodium nitrite (1–200 μ M) dissolved in PBS. The concentrations of the extracts under study were selected based on the viability assessment results observed in the MTT assay (Table S1). A control group of unstimulated LPS, a group treated with LPS alone, and a group treated with 1400 W, used as a positive control for inhibition of nitric oxide production, were included in each trial. 2.4.3. Cytokine and prostaglandin E2 (PGE2) production RAW 264.7 macrophages were seeded in 24-well plates (2 ×10 5 cells/well) and incubated for 48 h. Cells were pretreated with the fruit extracts (2000, 1000, and 500 μ g/mL) for 30 min and incubated for 24 h with LPS (1 μ g/mL). After incubation, the medium was collected. Quantification of TNFα , IL-6, and IL-1β secreted into the culture medium was performed with commercial enzyme-linked immunosorbent assay (ELISA) kits purchased from eBioscience, following the manufacturer’s protocol. PGE2 production was measured with a commercial competitive ELISA kit (Enzo®), following the manufacturer’s instructions. A non-stimulated LPS control group and a group treated with LPS alone were included in each assay. 2.4.4. Western blot analysis Briefly, RAW 264.7 macrophages previously treated with various concentrations of the fruit extracts were collected and dissolved in RIPA lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors. Protein concentration was determined by Bradford assay (Bio-Rad). The exact amount of protein samples (80 μ g) was separated on a 10 % sodium dodecyl sulfate-polyacrylamide gel and electrically transferred to nitrocellulose membranes. The membranes were then blocked with 5 % (w/v) milk powder and washed in TBST buffer, incubated with a specific primary antibody for two hours at room temperature, and then incubated with a horseradish peroxidaseconjugated secondary antibody for one hour at room temperature. Immunoreactive bands were detected by being exposed to X-ray film, and their densities were quantified using Carestream Molecular Imaging Software. Western blot data were quantified to determine differences between treatment groups. 2.5. In vivo animal experiments 2.5.1. Animals Sixto eight-week-old BALB/c female mice were obtained from the Instituto Nacional de Salud (Bogot´ a, Colombia). Animals were housed in filtered-capped polycarbonate cages and kept in a controlled environment (22 ±3 ◦C, 65–75 % humidity, under a 12 h light/darkness cycle) with access to food and water ad libitum. All the experiments were designed and conducted in accordance with local and international regulations (EU Directive 2010/63/EU) and approved by the Committee of Ethics in Research of the University of Cartagena (Minutes No. 74 of June 5th, 2014). For the in vivo assay, the golden berry extract was dissolved in ethanol (96 %) for incorporation into the animal feed; this solvent was finally removed during the feed preparation process. 2.5.2. Acute toxicity study The acute oral toxicity study was performed following the protocol of Castro, Ocampo, & Franco, 2015 (Castro et al., 2015) with modifications. Briefly, BALB/c mice (n =6) fasted overnight with water ad libitum, and the golden berry extract was administered orally at 2000 mg/ kg. Mortality and general behavioral changes were observed for three days. For histological analysis, liver and kidney samples were preserved in buffered formalin, stained with hematoxylin and eosin, and analyzed by a blinded pathologist employing light microscopy (Olympus BX41, Tokyo, Japan). 2.5.3. Diets and chronic toxicity study Sixto eight-week-old BALB/c mice (n =6) were fed chow supplemented with golden berry extract (0.15 % or 0.3 %) for 30 days. These percentages were selected based on the acute toxicity study and considering that the amount of extract used may be reasonably achievable in a human population. For 30 days, general behavioral changes associated with alertness, such as passivity, irritability, and nervousness, or changes related to motor activity, such as mobility, tactile response, and response to pain, were monitored, and liver and kidney samples were preserved in buffered formalin and stained with hematoxylin and eosin for histological analysis by a blinded pathologist. 2.5.4. Dextran sulfate sodium (DSS)-induced acute colitis Colitis was induced by employing the method described by Kim, Shajib, Manocha, & Khan, 2012, with some modifications (Kim et al., 2012). Briefly, mice were randomly divided into five groups (n =7): control group, DSS alone, DSS +GB (food supplemented with 0.15 and 0.3 % of golden berry extract), and GB alone. All study groups of animals were fed for 30 days with commercial mouse food (Rodent LabDiet 5010). The control and DSS groups received the non-supplemented diet, while the DSS +GB and GB groups received the diet supplemented with GB extract. For the last nine days, the DSS groups were exposed to 3 % DSS in drinking water ad libitum. During the induction of colitis with DSS, mice were weighed daily, and rectal bleeding and diarrhea were assessed. An overdose of anesthesia will painlessly euthanize those animals that meet the termination criteria defined in the project. The disease activity index (DAI) was determined according to the parameters described by Li et al., 2014 (Li et al., 2014). On day 30 of the test, the mice were sacrificed, and the colon was removed, cleaned, weighed, and length measured. A sample of colon tissue was preserved in buffered formalin and stained with hematoxylin and eosin or periodic acid-Schiff (PAS) for histopathological analysis by a blinded pathologist. The rest of J. Castro et al. Journal of Functional Foods 125 (2025) 106665 3 the colon was kept to determine ROS level, MPO activity, and cytokine levels. 2.5.5. Measurement of tissue cytokine level Colonic tissue was homogenized in Tissue Protein Extraction Reagent (T-PER) with a protease inhibitor cocktail (Roche) at 4 ◦C using the TissueRuptor® (Qiagen, Haan, Germany). The samples were centrifuged at 12.000 rpm at 4 ◦C, and IL-1β, IL-6, IL-10, and TNFα levels were determined in the supernatants using a commercial enzymelinked immunosorbent assay (ELISA) kits (eBioscience), following the manufacturer’s protocol. Results were expressed as picograms of cytokine per milligram of protein (pg/mg), quantified by the Bradford method using a standard commercial kit (Biorad 500–0206). 2.5.6. Measurement of tissue ROS level ROS levels in the colon of mice were determined using the technique described by (Song et al., 2008). Colon biopsies were frozen in liquid nitrogen and vigorously homogenized in 0.1 M phosphate buffer (pH 7.4), and their homogenate was incubated with DCF-DA (20 μ M) for 30 min. Fluorescence intensity was measured in a Fluoroskan Ascent 96well plate reader (Thermo Scientific, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. 2.5.7. MPO activity Enzyme activity was determined according to the technique described by Castro, Rivera, & Franco, 2019 (Castro et al., 2019), with modifications. Briefly, colon tissue was homogenized in phosphate buffer (pH: 7.4) at 4 ◦C using TissueRuptor® (Qiagen, Haan, Germany). The samples were centrifuged at 10.000 rpm at 4 ◦C; the pellet obtained was suspended in a solution of 0.5 % HTAB and 0.3 % EDTA in phosphate buffer (pH 6.0). The homogenate obtained was subjected to two rapid freeze-thaw cycles, sonicated for 10 s, and finally centrifuged for 10 min at 10.000 rpm at 4 ◦C. The recovered supernatant was used to assess MPO activity; 50 μ L of the supernatant was mixed with 50 μ L of ODianisidine (0.067 %), 50 μ L of 0.5 % HTAB solution, and 50 μ L of hydrogen peroxide (H 2 O 2 0.003 %). OD 450 was determined using a Multiskan Go microplate reader (Thermo Scientific, Waltham, MA, USA). Results were expressed as units of MPO per milligram of protein; one unit of activity is defined as the amount of enzyme capable of degrading 1 μ mol of hydrogen peroxide in one minute at 25 ◦C. 2.6. Statistical analysis Results are expressed as the mean ±standard error of the mean (SEM) of two (in vivo assay) or three (in vitro assay) independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Values of p <0.05 were considered significant. 3. Results 3.1. Effect of fruit extracts on NO, IL-1β, IL-6, TNFα and PGE-2 production in LPS-activated RAW 264.7 macrophages The results of the evaluation of the effect of ethanolic extracts on NO production in RAW 264.7 macrophages stimulated with LPS (1 μ g/mL) reveal that the extracts of purple passion, yellow pitaya, tamarillo, and golden berry were the most active (Fig. 1). Fig. 2 shows the effect of active extracts on the production of IL-6, IL-1β, TNFα , and PGE-2 in RAW 264.7 macrophages stimulated with LPS (1 μ g/mL) for 24 h. It can be observed that except for the purple passion extract, all the others significantly decreased IL-6 and IL-1β production, with the golden berry extract being the most active (Fig. 2A–B). At the same time, TNFα and PGE2 were significantly inhibited by all the extracts tested; again, the golden berry extract showed high inhibitory activity of these mediators (Fig. 2C–D). These results allowed us to identify the golden berry extract as having the best anti-inflammatory potential. Therefore, we decided to evaluate its effect on the NF-kB transcription factor and its immunomodulatory effect in the DSS-induced IBD model in mice. 3.2. Inhibition of NF-kB-dependent LPS activation As shown in Fig. 3, the level of IκB α protein in the cytoplasm decreased by LPS treatment, indicating the degradation of IκB α , while in Fig. 1. Effect of the extracts on NO production in RAW 264.7 macrophages stimulated with LPS (1 μ g/mL) for 24 h. 1400 W (10 μ M) was used as a positive control. Results represent the mean ±standard error of the mean. (*p <0.01, **p <0.001 and ****p <0.00001 statistically significant compared to the LPS-treated group). J. Castro et al. Journal of Functional Foods 125 (2025) 106665 4 the golden berry extract-treated groups, the levels of IκB protein in the cytoplasm were maintained, showing a protective effect of NF-κB activation in a concentration-dependent manner. In addition, we examined the effect of golden berry extract on NF-κB activation, determining NFκB p65. As can be seen, stimulation with LPS caused translocation of p65 from the cytosol to the nucleus, while treatment with golden berry Fig. 2. Effect of purple passion, yellow pitaya, tamarillo, and golden berry extracts on IL-6, IL-1β, TNFα and PGE-2 production in RAW 264.7 macrophages stimulated with LPS (1 μ g/mL) for 24 h. 1400 W (10 μ M), Dexamethasone (20 μ M), and Rofecoxib (10 μ M) were used as positive control. Results represent the mean ±standard error of the mean. (*p <0.01, **p <0.001, ***p <0.0001 and ****p <0.00001 statistically significant compared to the LPS-treated group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 3. Inhibition of nuclear transcription factor NF-κB by golden berry extracts in RAW 264.7 macrophages stimulated with LPS (1 μ g/mL) for 24 h. The data shown are representative of three independent experiments. J. Castro et al. Journal of Functional Foods 125 (2025) 106665 5 extracts reduced this translocation (Fig. 3). 3.3. Anti-inflammatory effect of golden berries in mice with inflammatory bowel disease Considering the anti-inflammatory background of golden berry and the results obtained in this study, where golden berry extract showed the best anti-inflammatory potential by significantly inhibiting the production of NO, IL 6, IL-1β, TNF α , and PGE2 in LPS stimulated macrophages, we continue to evaluate the immunomodulatory effect of a diet supplemented with golden berry extract in mice with DSS-induced inflammatory bowel disease. Initially, an acute toxicity study was performed, which showed that the administration of a dose of 2000 mg/kg of the gold berry extract did not produce signs of toxicity during the 72 h after administration, and the histological analysis did not show alterations at hepatic or renal level. Similarly, no toxicity signs were detected in the chronic toxicity study, where animals were fed a diet supplemented with golden berry extract (0.15 % and 0.3 %) for 30 days and checked daily. In addition, the histopathological analysis showed that the diet supplemented with golden berry extract (0.15 % and 0.3 %) did not produce liver or kidney damage Fig. S1. Inflammatory bowel disease is characterized by weight loss, so this parameter was considered an important marker of the disease. The animals were weighed daily during the 30-day test period. Animals consuming the golden berry extract-supplied diet were observed to have a lower weight loss than the disease control group (DSS) (Fig. 4A). In addition, the disease activity index was determined, corresponding to a score calculated based on weight variation, fecal consistency, and blood in the feces. Coherently, the DSS group obtained the highest score (Fig. 4B). On day 30 of the trial, animals were sacrificed. A macroscopic analysis of the colon was performed, measuring its length and calculating the weight/length ratio indicative of intestinal mucosa damage. As expected, the healthy control had a normal appearance, and the length of the colon tissue was greater than that of the dextran sulfate sodium (DSS)-induced colitis group. Groups given the feed supplemented with golden berry extract (0.15 and 0.3 %) had a greater colonic length than those given only DSS (Fig. 4 C and D). Regarding the results of the weight/length ratio, an increase in this ratio was observed in the group with dextran sulfate sodium (DSS)-induced colitis due to the thickening and shortening of the tissue as a consequence of damage to the intestinal mucosa and submucosa caused by this agent; while in the groups treated with the golden berry extract, the index was lower, indicating an improvement in the intestinal inflammatory process (Fig. 4E). The findings of the histological study are consistent with the results of the macroscopic analysis. The healthy group and the group that only received the 0.30 % supplemented diet were in normal condition and without cellular alterations. In contrast, the IBD-induced groups showed ulceration, edema, fibrosis, inflammation, neutrophil infiltration, and increased thickness of the submucosal and muscular layers. However, restoration of colon tissue with decreased neutrophil infiltration was observed in groups treated with food supplemented with golden berry extract (0.15 % and 0.30 %) (Fig. 5A). Inflammatory bowel disease may be accompanied by a decrease in goblet cells, which contribute significantly to the proper functioning of the intestine by producing antimicrobial proteins, cytokines, and mucus (Choi et al., 2022; Gustafsson & Johansson, 2022). PAS staining allows the identification of goblet cells by staining the mucin granules of these cells purplish red (Zugibe, 1970). Evaluation of PAS-stained tissue shows that the group treated with the food supplemented with golden berry extract (0.15 % and 0.30 %) has a lower goblet cell depletion than the DSS group. On the other hand, the group that only received 0.3 % supplemented feed did not show significant differences from the healthy group, indicating that the extract is not responsible for the depletion of goblet cells (Fig. 5B). A decrease in MPO enzyme activity was observed, indicating a decrease in neutrophil infiltration into colonic tissue, which was accompanied by a reduction of levels of the reactive oxygen species (ROS) (Fig. 6 A and B). Furthermore, significant inhibition of levels of the proinflammatory cytokines IL-1β and IL-6 was observed in the colonic tissue of the groups with dietary supplementation with a golden berry extract (Fig. 6 C and F), as well as a downward trend in the levels of TNFα and rising trend in the levels of the anti-inflammatory cytokine IL-10 (Fig. 6 D and E). The simultaneous effect of golden berry extract on all these mediators constitutes an immunomodulatory effect with a Fig. 4. Golden berry dietary supplementation attenuates pathological symptoms of dextran sulfate sodium (DSS) induced acute colitis. BALB/c mice were fed with golden berry (0.15 y 0.30 %) for 30 days and exposed to 3 % DSS in drinking water during the last 10 days. A: Body weight changes following the DSS colitis induction. B: Disease activity index. C and D: Macroscopic analysis (measuring the length of the colon). E: Weight/length ratio of the colon. The results represent the mean ±SEM. [n =10]. (*p < 0,01 and ****p < 0.00001 statistically significant compared with DSS group). J. Castro et al. Journal of Functional Foods 125 (2025) 106665 6 significant impact on colonic tissue restoration in mice with DSSinduced inflammatory bowel disease. 4. Discussion In recent years, fruits have attracted increasing interest among researchers. This interest is related to the fact that fruits may potentially prevent and treat some chronic diseases (Li et al., 2016; Slavin & Lloyd, 2012). Experimental studies have shown that modulating the inflammatory response through fruit intake produces positive health outcomes (Pan et al., 2010; Zhu, Du, & Xu, 2018). Nitric oxide was selected as an inflammatory biomarker to perform the initial screening of the anti-inflammatory potential of the ten fruit extracts to be evaluated. Although ⋅NO is not one of the common inflammatory biomarkers, it is an important inflammatory mediator, as inhibition of iNOS activity or down-regulation of iNOS expression is desirable to reduce the extent of the inflammatory response (Zhu et al., 2018). Moreover, it can be easily quantified at a meager cost, making it a suitable method for screening plant species for metabolites with antiinflammatory activity. Therefore, we evaluated the in vitro antiinflammatory potential of the ten fruit extracts by assessing their ability to inhibit the production of inflammatory mediator nitric oxide in LPS-activated RAW 264.7 macrophages, which allowed us to initially identify that purple passion, yellow pitaya, tamarillo, and golden berry extracts were the most active. To further investigate the anti-inflammatory potential of the four Fig. 5. Representative images of the histological study (A) Hematoxylin and eosin staining (B) Periodic acid-Schiff [PAS] staining. Fig. 6. Effect of dietary supplementation with golden berry extract on myeloperoxidase enzyme [MPO] activity, reactive oxygen species (ROS) production, and the IL-1β, TNFα , IL-10, and -IL-6 levels in the colonic tissue. Results represent mean ±standard error of the mean [SEM] [n =10]. (**p <0.001, ***p <0.0001 and ****p <0.00001 statistically significant compared to the DSS group). J. Castro et al. Journal of Functional Foods 125 (2025) 106665 7 active extracts, clinically important inflammatory biomarkers that could be affected by regular fruit consumption and exert effects on inflammatory processes characteristic of chronic non-communicable diseases were selected. Human clinical studies in different populations and age groups show an association between fruit intake and reduced levels of common inflammatory biomarkers, such as CRP, IL-1β, IL-6, TNFα or PGE-2 (Calder et al., 2011; Calder et al., 2017; Wu & Schauss, 2012; Zhu et al., 2018). On the other hand, there is an association between the development and progression of chronic non-communicable diseases such as neurological disorders, cancer, diabetes, obesity, and inflammatory bowel disease, with increased levels of the inflammatory biomarkers MCP-1, CRP, TNFα , IL-6, IL-1β, NO, and PGE2 (Pan et al., 2010). Except for CRP, these inflammatory biomarkers are mainly secreted by macrophages, so identifying substances that can inhibit the production of these inflammatory mediators in these cells is a good starting point for the prevention and treatment of many chronic diseases. In this regard, we subsequently determined the effect of the four active extracts on the inflammatory biomarkers IL-1β, IL-6, TNFα , and PGE-2. Identifying the golden berry extract as the most active corresponds with results presented in other studies in which golden berry extracts have shown immunomodulatory effects on the expression of inflammatory biomarkers in human cervical cancer cells (HeLa), murine fibroblasts (L929) and liver (Mier-Giraldo et al., 2017; Pino-de la Fuente et al., 2020). The anti-inflammatory activity that some fruits may be due to bioactive substances present in these foods, such as phenolic compounds, saponins, alkaloids, polyunsaturated fatty acids, and terpenoids, among others; these compounds can inhibit pathways related to inflammation (Adefegha, 2018; Calder et al., 2017; Prasad, Sung, & Aggarwal, 2012; Szostak, Cybulska, Kłosiewicz-Latoszek, & SzostakWęgierek, 2013; Zhu et al., 2018) Modulating the inflammatory response by fruit phytochemicals has been linked to inhibiting several transcription factors, including NF-κB (Iddir et al., 2020; Oveissi et al., 2019). The nuclear transcription factor (NF-κB) is an essential molecular regulator of innate and adaptive immune systems and is ubiquitous in the cytoplasm. The presence of exogenous stimuli such as bacterial infections and gut microflora can stimulate the TLR4 receptor, which in turn activates a phosphorylation cascade leading to the activation of TAK1, which binds to the IKK complex via ubiquitin chains, allowing it to phosphorylate and activate IKKβ. The IKK complex phosphorylates the IκB α protein that acts as an inhibitor of NF-κB, which undergoes proteolytic degradation by the proteasome, allowing NF-κB to translocate to the nucleus. Classical NF-kB is a heterodimer consisting of a p50 and a p65 subunit. Transactivator domains at the C-terminal end of p65 make it a potent activator of gene expression from kB sites. Once in the nucleus, it binds to promoters that induce the coordinated expression of many genes encoding cytokines, chemokines, enzymes and adhesion molecules involved in the amplification and maintenance of the inflammatory response (Guo et al., 2024; Kawasaki & Kawai, 2014; Ma et al., 2024; Schottelius & Baldwin Jr, 1999). Thus, inhibition of the transcription factor NF-κB is often considered a valuable strategy for treating inflammatory disorders. This pathway represents an important and attractive therapeutic target for compounds that selectively interfere with it (Niu et al., 2015; Yoon & Baek, 2005). To determine whether the inhibitory effect of golden berry extract on the production of NO, IL6, IL-1β, TNFα , and PGE2 was related to the inhibition of the transcription factor NF-κB, we evaluated the protective effects of this extract on the degradation of IκB α in LPS-stimulated macrophages. As the results show, golden berry extract prevents the degradation of IκB α in a concentration-dependent manner. It maintains the levels of this protein in the cytoplasm, consequently avoiding the phosphorylation and translocation to the nucleus of the NF-κB factor. This decreases the production of proinflammatory mediators (Fig. S2). These results are in agreement with a previous study in which compounds isolated from golden berries were shown to significantly inhibit NF-κB transcription factor activity in HEK 293 / NF-kB-Luc cells (Chang, Sang-Ngern, Pezzuto, & Ma, 2016). A model of inflammatory bowel disease was selected to determine in vivo the anti-inflammatory potential of golden berry extract. Among the various chronic diseases with inflammation, this pathology is characterized by developing a severe inflammatory response, which has been well studied and described during the last decades (Garavaglia et al., 2024; Yue et al., 2024). In an attempt to understand the pathogenesis of IBD and progress in the search for new treatments, various animal models have been developed to simulate this disease. The dextran sulfate sodium (DSS) model is one of the most widely used; it is a reproducible model that morphologically and symptomatically resembles ulcerative colitis, one of the most common forms of inflammatory bowel disease. It is by far the most popular murine model of inflammatory bowel disease because it is easy to develop due to its wide availability and low cost (Fredin et al., 2008; Katsandegwaza, Horsnell, & Smith, 2022; Kiesler, Fuss, & Strober, 2015). The importance of diet in preventing and developing IBD lies in the fact that diet not only directly affects intestinal inflammation by regulating inflammatory mediators but can also influence epigenetic modifications and intestinal microbiota (Spooren et al., 2013); moreover, some food products, including fruits, can regulate the immune system response and modify intestinal inflammation (Bernstein, 2017; Salaritabar et al., 2017). The fruit of the species Physalis peruviana L., commonly known as golden berry or cape gooseberry, is used in traditional medicine to reduce blood sugar levels, prevent cataract formation, and treat intestinal problems such as ulcers and diarrhea (RodríguezEcheverry, 2010) in addition many studies are also found in the literature on the various activities of this fruit, including reports of antiinflammatory activity (Chang et al., 2016; Hassan, Serag, Qadir, & Ramadan, 2017; Mier-Giraldo et al., 2017). However, no reports relate the effect of fruit consumption to the anti-inflammatory effects of IBD. An approach to this is the work done by Moya et al., where they elaborated on an extract obtained by in vitro digestion that simulates the oral, gastric, and intestinal phases of human digestion and evaluated the effect on the expression of inflammatory markers in an in vitro inflammation model of Caco-2 intestinal epithelium-like cells (Moya, Mirada, Rivera, & Arredondo, 2024). Given the anti-inflammatory background of gold berry extract and the complex role of nutrition in the etiology of inflammatory bowel disease (IBD) (Gentschew & Ferguson, 2012; Hou, Abraham, & El-Serag, 2011), we evaluated the immunomodulatory effect of a diet supplemented with golden berry extract in mice with DSSinduced inflammatory bowel disease in BALB/c mice, with this study the first report that demonstrates this activity. These results suggest that regular consumption of the fruit or a functional food based on it could benefit intestinal inflammation. They establish a starting point for future studies in animal models exploring the effect on the intestinal microbiota and other signaling pathways involved in the inflammatory response, such as inflammasome activation and the MAPK pathway, as well as in clinical studies in patients with IBD, intending to improve their quality of life. 5. Conclusion Dietary supplementation with golden berry (Physalis peruviana) fruit extract showed an immunomodulatory effect in the DSS-induced inflammatory bowel disease model in BALB/c mice, attenuates the pathological symptoms of DSS-induced acute colitis, decreasing the infiltration of polymorphonuclear neutrophils into the tissue and reactive oxygen species, and modulating the levels of important mediators of the inflammatory process. Our results suggest that consistent consumption of a functional food based on golden berries in the diet could benefit intestinal inflammation. Further studies are required to identify the components responsible for the activity, its impact on the microbiota, and the immunomodulatory effects it could have at the systemic level. Supplementary data to this article can be found online at https://doi. J. Castro et al. Journal of Functional Foods 125 (2025) 106665 8 org/10.1016/j.jff.2025.106665. Ethics statement The animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the recommendations of the European Union regarding animal experimentation (Directive of the European Council 2010/63/EU). The protocol was approved by the Committee of Ethics in Research of the University of Cartagena (Minutes No. 74 of June 5th, 2014). All efforts were made to minimize animal suffering. CRediT authorship contribution statement Jenny Castro: Writing – original draft, Visualization, Methodology, Investigation. Guillermo Lopez-Lluch: Writing – review & editing, Supervision, Methodology. Juan Carlos Rodríguez: Writing – review & editing, Validation, Methodology. Rocío de la Puerta: Writing – review & editing, Supervision, Resources. Lía Barrios: Methodology, Investigation. Rub´ en Salas: Writing – review & editing, Methodology, Investigation, Funding acquisition. Luis Franco: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgments This work was supported by Colombia’s Ministry of Science Technology and Innovation (Mincienciasgrant 689-2014) and the Universidad de Cartagena (grant 025 -2019). The authors also thank Daneiva Caro, Nely Mejia, Yuri Palacio, Jaime Salgado, and Daniela Amarís for collaborating during experiments. Graphical abstract created with BioRender.com. References Adefegha, S. A. (2018). Functional foods and nutraceuticals as dietary intervention in chronic diseases; novel perspectives for health promotion and disease prevention. Journal of Dietary Supplements, 15(6), 977–1009. Alwan, A., MacLean, D. R., Riley, L. M., d’Espaignet, E. T., Mathers, C. D., Stevens, G. A., & Bettcher, D. (2010). Monitoring and surveillance of chronic non-communicable diseases: Progress and capacity in high-burden countries. The Lancet, 376(9755), 1861–1868. Astrup, A., Dyerberg, J., Selleck, M., & Stender, S. (2008). 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