Acute Effect of a Single Dose of Tomato Sofrito on Plasmatic Inflammatory Biomarkers in Healthy Men

Sofrito is a Mediterranean tomato-based sauce that typically also contains olive oil, onion, and garlic. The preparation of sofrito modifies the bioactive compounds (carotenoids and polyphenols) in the ingredients to more bioavailable forms, promoting cis-lycopene formation and polyphenol bioaccessibility. To evaluate the health benefits of this cooking technique, the effect of consuming an acute dose of sofrito on the inflammatory status was studied. In a clinical trial, 22 healthy male subjects consumed a single dose of sofrito (240 g/70 kg) after three days without ingesting any tomato products and following a low-antioxidant diet the day before the intervention. Plasma carotenoids and total polyphenol excretion (TPE) were evaluated, as well as the inflammatory biomarkers C-reactive protein (CRP), interleukin-6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α). After the sofrito intake, a significant decrease in CRP (p = 0.010) and TNF-α (p = 0.011) was observed, but only TNF-α was inversely correlated with an increase in TPE and plasma β-carotene (not the major carotenoid, lycopene). The positive health effects of this tomato-based product may be attributed not only to lycopene, but to the bioactive compounds of all the ingredients.


Introduction
Sofrito is a sauce commonly used to prepare dishes in Mediterranean cuisine. It is based on tomato but also contains other ingredients, typically olive oil, onion, and garlic. The regular consumption of this sauce is included in a validated 14-item questionnaire to evaluate adherence to the Mediterranean diet [1,2].
Numerous studies have provided evidence for the protective role of tomato-based products and their bioactive compounds against the development of cardiovascular diseases and cancer [3,4], which is partly attributed to positive effects on inflammatory biomarkers [5][6][7].
The polyphenol and carotenoid profile of sofrito varies according to its composition [8], but as tomato is the principle ingredient, the major carotenoids are lycopene and β-carotene [8]. Carotenoids in food are mainly all-trans isomers, whereas cis-isomers predominate in the human organism [9]. Factors such as cooking practices and the food matrix promote the isomerization of carotenoids and their bioavailability [9][10][11], the latter being increased by the presence of lipids [12][13][14]. Our research group recently reported an enhanced formation of cis-lycopene in sofrito associated with the concentration of onion and the cooking time [15]. The total polyphenol content of tomatoes could be increased by processing [16], and the bioaccessibility of tomato polyphenols is enhanced by processing and oil addition [17][18][19]. Thus, due to its phytochemical content, sofrito seems to be a health-promoting component of the Mediterranean diet.
Although the impact of tomato products on health has been mainly associated with lycopene [4, 20,21], the other bioactive compounds present in sofrito could also be implicated [22,23]. Previous studies have reported inhibitory effects of dietary phytochemicals on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in macrophages, resulting in a decrease in pro-inflammatory cytokines and chemokines like interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF-α) [24,25]. As these molecules stimulate the production of C-reactive protein (CRP) in the liver, which activates NF-κB [26], their reduction could diminish the CRP level. In the current work, it was hypothesized that phytochemical compounds present in sofrito could improve the baseline inflammation level. Thus, the aim of this study was to evaluate the effect of a single dose of 240 g/70 kg of sofrito on the regulation of inflammatory biomarkers in healthy humans and to identify the biomarkers responsible for these changes.

Sofrito Samples
Sofrito samples were supplied by Gallina Blanca (GB Foods, Spain) and consisted of a mix of tomato (50%), onion (37%), extra virgin olive oil (12%), and salt. Every sample was packed in a glass jar that contained 350 g of the sofrito. The nutritional and phytochemical composition of sofrito are provided in Tables S1 and S2.

Participants
The study was carried out with twenty-two healthy male volunteers aged between 18 and 32. All participants provided written informed consent in advance. Only men were enrolled in the trial to avoid effects related to hormonal fluctuations during the menstrual cycle [27][28][29]. Exclusion criteria were chronic illness or homeostatic disorder, history of cardiovascular diseases, hypertension or dyslipidemia, toxic habits (such as use of tobacco, alcohol, and drugs), and tomato or onion allergy or intolerance.

Intervention
In this open, uncontrolled and acute nutritional study, all participants ingested a single portion of sofrito (240 g/70 kg body weight) in a state of fasting. Before the intervention, volunteers avoided eating tomatoes and their by-products for three days. One day before the intervention, they also followed a low antioxidant diet, which was continued until the last blood sample was drawn (24 hours after consumption of sofrito). The details of the diet are presented in Supplementary Material Table S3.
The study was run in the Department of Nutrition, Food Sciences and Gastronomy of the Food and Nutrition Torribera Campus, University of Barcelona (Spain), according to the principles of the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Clinical Investigation of the University of Barcelona (Barcelona, Spain). The clinical trial was registered and given the International Standard Randomized Controlled Trial Number (http://www.isrctn.com/) of ISRCTN17867378.

Dietary and Physical Activity Assessments
In order to complete the dietary register, subjects were asked to fill out a three-day food recall on the day of the intervention. These were analyzed using PCN Pro software (Programa de Càlcul Nutricional Professional, Santa Coloma de Gramenet, Barcelona). Physical activity was measured by the validated Spanish version of the Minnesota Leisure-Time Physical Activity Questionnaire [30].

Extraction of Biological Samples
Fasting blood and urine samples were drawn before sofrito consumption (baseline extraction) and up to 24 hours afterwards ( Figure 1). Blood samples were collected via venipuncture in the arm through EDTA tubes, and plasma was separated after centrifugation at 1902 g for 15 min at 4 • C. Plasma and urine were aliquoted and stored at −80 • C until the day of analysis.

Dietary and Physical Activity Assessments
In order to complete the dietary register, subjects were asked to fill out a three-day food recall on the day of the intervention. These were analyzed using PCN Pro software (Programa de Càlcul Nutricional Professional, Santa Coloma de Gramenet, Barcelona). Physical activity was measured by the validated Spanish version of the Minnesota Leisure-Time Physical Activity Questionnaire [30].

Extraction of Biological Samples
Fasting blood and urine samples were drawn before sofrito consumption (baseline extraction) and up to 24 hours afterwards ( Figure 1). Blood samples were collected via venipuncture in the arm through EDTA tubes, and plasma was separated after centrifugation at 1902 g for 15 min at 4 °C. Plasma and urine were aliquoted and stored at -80 °C until the day of analysis.

Clinical and Biochemical Evaluations
The diastolic and systolic blood pressure (DBP and SBP, respectively) and heart rate (HR) were measured in triplicate by a blood pressure monitor before and after the intake of a single dose of sofrito.
Biochemistry parameters in plasma were evaluated in an external laboratory (mdb.lab Durán Bellido, Barcelona). High density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol and triglycerides were analyzed by an enzymatic method. Urea and uric acid were analyzed by enzymatic and enzymatic/chromogen methods, respectively. Creatinine was determined by reaction kinetics of the Jaffe method (as modified by Larsen). Total proteins and albumin were measured by the endpoint biuret reaction and bromocresol green methods, respectively.

Analysis of Total Polyphenol Excretion in Urine
After a solid phase extraction (SPE) using a 96-well plate cartridge (Oasis MAX), total polyphenol excretion (TPE) analysis was performed in 2 mL of diluted (1:1) and acidified urine samples by the Folin-Ciocalteu method [31]. The spectrophotometry analysis was carried out using a Thermo Scientific Multiskan® Spectrum (Thermo Fisher Scientific, Vantaa, Finland, ref. 15019000) at a wavelength of 765 nm. The results are expressed as mg of gallic acid equivalent/L of urine (GAE/L). A cumulative urinary excretion curve for total polyphenols was calculated from 0 to 24 h.

Quantitative Analysis of Carotenoids in Plasma
Carotenoids were extracted by liquid-liquid extraction from plasma samples collected at 0 h and 24 h [32]. Chromatographic analysis of carotenoids was performed by HPLC-UV-DAD, using an HP Figure 1. Timeline of sample collection before and after intake of sofrito. On the left, baseline extraction of blood and urine. On the right, sample drawn after sofrito consumption: collection of blood at 24 h and cumulative urine at 0-3, 3-5, 5-12, and 12-24 h.

Clinical and Biochemical Evaluations
The diastolic and systolic blood pressure (DBP and SBP, respectively) and heart rate (HR) were measured in triplicate by a blood pressure monitor before and after the intake of a single dose of sofrito.
Biochemistry parameters in plasma were evaluated in an external laboratory (mdb.lab Durán Bellido, Barcelona). High density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol and triglycerides were analyzed by an enzymatic method. Urea and uric acid were analyzed by enzymatic and enzymatic/chromogen methods, respectively. Creatinine was determined by reaction kinetics of the Jaffe method (as modified by Larsen). Total proteins and albumin were measured by the endpoint biuret reaction and bromocresol green methods, respectively.

Analysis of Total Polyphenol Excretion in Urine
After a solid phase extraction (SPE) using a 96-well plate cartridge (Oasis MAX), total polyphenol excretion (TPE) analysis was performed in 2 mL of diluted (1:1) and acidified urine samples by the Folin-Ciocalteu method [31]. The spectrophotometry analysis was carried out using a Thermo Scientific Multiskan®Spectrum (Thermo Fisher Scientific, Vantaa, Finland, ref. 15019000) at a wavelength of 765 nm. The results are expressed as mg of gallic acid equivalent/L of urine (GAE/L). A cumulative urinary excretion curve for total polyphenols was calculated from 0 to 24 h.

Quantitative Analysis of Carotenoids in Plasma
Carotenoids were extracted by liquid-liquid extraction from plasma samples collected at 0 h and 24 h [32]. Chromatographic analysis of carotenoids was performed by HPLC-UV-DAD, using an HP 1100 HPLC system (Hewlett-105 Packard, Waldbronn, DE) containing a quaternary pump coupled to a DAD G1315B. The separation was carried out with Milli-Q water, methanol (MeOH) and methyl-tert-butyl ether (MTBE) (Panreac Quimica S.A., Barcelona, Spain), according to a procedure previously validated in our group [32]. A Waters RP column YMC Carotenoid S-5 µm (250 mm × 4.6 mm) and a precolumn YMC Guard Cartridge Carotenoid S-5 µm (20 mm × 4.0 mαm) were used.

Determination of Plasmatic Inflammatory Biomarkers
Plasmatic C-reactive protein (CRP) was measured by an immunoturbidimetric method from external services (mdb.lab Durán Bellido) at baseline and 24 h after consumption of sofrito.

Statistical Analysis
Normality of distribution was assessed by a Shapiro-Wilk test. In order to compare baseline and post-intervention values, a linear regression analysis was used for the normal variables (SDP and DBP, HR, HDL, uric acid, albumin and TNF-α). Non-normal variables were assessed by a non-parametric Wilcoxon signed-rank test. The log-transformed TPE variable was analyzed by a Bonferroni post hoc test to compare the excretion of total polyphenols at the different time points. Statistical analyses were performed with SPSS Version 23.0 for Windows (SPSS Inc., Chicago, IL, USA).
The correlation analysis was carried out using a correlation matrix that considers repeated measures [33] with software R, version 3.4.2. The Pearson coefficient (r) was calculated. Significant correlations (p < 0.05) are shown in the results. Table 1 provides anthropometric measurements of the participants, as well as the degree of adherence to the Mediterranean diet, level of physical activity and nutrient intake at the study baseline.

Clinical Measures
Clinical measures are shown in Table 2. Systolic blood pressure did not change after consumption of sofrito compared to baseline, whereas diastolic blood pressure decreased (p = 0.006). The total cholesterol was lower after the intervention, mainly due to a reduction of HDL (p = 0.005 and 0.015), although significant changes in LDL and triglycerides were not observed. Urea, total proteins, and albumin decreased significantly (p = 0.023, 0.011, and 0.015).

Phenolic Excretion in Urine
As a biomarker of polyphenol intake from the sofrito, the urinary excretion of total polyphenols from baseline until 24 hours after consumption was analyzed (Figure 2A). The TPE increased from baseline to 5 h after intake, and even more so from 12 to 24 h (p = 0.001), as illustrated by the cumulative curve in Figure 2B.

Quantification of Carotenoids in Plasma
Levels of carotenoids in plasma were evaluated before and after sofrito consumption (Table 3). All target carotenoids increased after the intervention, the predominant ones being 5-cis-lycopene, βcarotene and trans-lycopene. Total lycopene increased from 43% to 59% of total carotenoids. Xanthophylls, lutein, and cryptoxanthin increased significantly (p = 0.001 and 0.012) and zeaxanthin was detected in plasma after sofrito consumption but not at baseline. In all participants, a high concentration of carotenes was found at 24 h of sofrito intake. Notably, the changes in trans-β-carotene were highly significant (p <0.001), and the 9-cis-isoform and 13-cis-β-carotene were observed after the

Quantification of Carotenoids in Plasma
Levels of carotenoids in plasma were evaluated before and after sofrito consumption (Table 3). All target carotenoids increased after the intervention, the predominant ones being 5-cis-lycopene, β-carotene and trans-lycopene. Total lycopene increased from 43% to 59% of total carotenoids. Xanthophylls, lutein, and cryptoxanthin increased significantly (p = 0.001 and 0.012) and zeaxanthin was detected in plasma after sofrito consumption but not at baseline. In all participants, a high concentration of carotenes was found at 24 h of sofrito intake. Notably, the changes in trans-β-carotene were highly significant (p < 0.001), and the 9-cis-isoform and 13-cis-β-carotene were observed after the intervention but not at baseline. Other carotenes that increased were trans-lycopene (p < 0.001) and 13and 5-cis-lycopene (p = 0.005 and <0.001). The concentration of 9-cis-lycopene was detected after the consumption of sofrito but not at baseline. In addition, the total cis-lycopene was significantly higher (p < 0.001), as was the total lycopene (p < 0.001). In summary, the increase in carotenoid concentration was statistically significant after the consumption of sofrito, total carotenoids being three-fold higher compared to the baseline level (p < 0.001).

Inflammatory Biomarkers
After the consumption of sofrito, all the inflammatory biomarkers studied decreased compared to the baseline. However, the changes were only significant for CRP (p = 0.010) and TNF-α (p = 0.011) ( Figure 3A and 3C), but not IL-6 ( Figure 3B). In no case was IL-1β detected (data not shown).

Inflammatory Biomarkers
After the consumption of sofrito, all the inflammatory biomarkers studied decreased compared to the baseline. However, the changes were only significant for CRP (p = 0.010) and TNF-α (p = 0.011) ( Figure 3A and 3C), but not IL-6 ( Figure 3B). In no case was IL-1β detected (data not shown).

Correlations between Inflammatory Biomarkers and Bioactive Compounds
In order to evaluate the role of TPE in the decrease of inflammatory biomarkers, a correlation study was performed. The reduction of TNF-α, but not IL-6 or CRP, was inversely correlated with the excretion of total polyphenols. Figure 4 shows the negative correlation between TNF-α and urinary TPE (r = −0.42, p = 0.048).

Correlations between Inflammatory Biomarkers and Bioactive Compounds
In order to evaluate the role of TPE in the decrease of inflammatory biomarkers, a correlation study was performed. The reduction of TNF-α, but not IL-6 or CRP, was inversely correlated with the excretion of total polyphenols. Figure 4 shows the negative correlation between TNF-α and urinary TPE (r = −0.42, p = 0.048).  In addition, an inverse correlation was observed between the concentration of β-carotene in plasma and TNF-α, but not CRP and IL-6 (r = −0.41, p = 0.05) ( Figure 5). In addition, an inverse correlation was observed between the concentration of β-carotene in plasma and TNF-α, but not CRP and IL-6 (r = −0.41, p = 0.05) ( Figure 5).

Discussion
In this clinical trial, healthy individuals showed higher carotenoid levels in plasma 24 h after the consumption of sofrito. Arranz et al. reported higher plasma concentrations of cis-isomers of lycopene but not of other cis-carotenoids after the ingestion of tomato sauce without onion and garlic [12]. In the current study, both cis-and trans-isoforms of β-carotene and lycopene increased after sofrito

Discussion
In this clinical trial, healthy individuals showed higher carotenoid levels in plasma 24 h after the consumption of sofrito. Arranz et al. reported higher plasma concentrations of cis-isomers of lycopene but not of other cis-carotenoids after the ingestion of tomato sauce without onion and garlic [12]. In the current study, both cisand trans-isoforms of β-carotene and lycopene increased after sofrito intake. Not detected at baseline, 13 and 9-cis-isomer of β-carotene and 9-cis-lycopene were quantified after the intervention.
TPE levels had increased significantly at 12-24 h after sofrito intake compared to the baseline. In a previous study by our group, also in healthy volunteers, changes in polyphenol excretion were noted earlier, at 6-12 h after the consumption of a tomato-based product without onion [19]. Thus, the presence of polyphenols from onion in the sofrito could have a delaying effect on the urinary TPE [34].
It was initially hypothesized that phytochemicals in sofrito could affect the regulation of systemic pro-inflammatory markers through the inhibition of NF-κB, triggering a decrease in cytokines, chemokines and CRP [24][25][26]. After the sofrito intake, a significant reduction of CRP and TNF-α biomarkers (p = 0.010 and 0.011) in plasma was observed, but not of IL-6. The decrease in TNF-α was inversely correlated with TPE and the level of β-carotene.
In a previous study, both CRP and TNF-α decreased significantly in healthy volunteers after the consumption of tomato juice twice daily for 2 weeks [5]. In obese and overweight participants who consumed tomato juice for 20 days, the concentration of TNF-α decreased, and in obese subjects the level of IL-6 was significantly reduced, but no changes in CRP were observed in either case [35]. In contrast, in a parallel study, a significant decrease in CRP was observed in women (but not men) suffering from heart failure who consumed tomato juice for a month [36]. Gender-related differences in CRP have been previously described [37,38]. In a crossover trial, tomato paste attenuated the increase in IL-6 after a high-fat meal in healthy volunteers [6]. However, Valderas-Martínez et al. reported that healthy subjects showed a significant reduction in IL-6 only after consuming tomato sauce containing olive oil (a single dose), but not raw tomato or tomato sauce without olive oil [7]. A study in schoolchildren found that plasma β-carotene levels were inversely related to IL-6 and CRP, particularly the former, but not to TNF-α [39]. In patients with cardiovascular diseases, β-carotene was significantly correlated with IL-6 [40] and CRP [22]. In contrast, other authors did not find changes in these inflammatory biomarkers after interventions with tomato products or a high-tomato diet [41][42][43][44][45][46].
In summary, an acute effect on inflammatory biomarkers was observed at 24 h after the administration of a single dose of 240 g/70 kg of sofrito. Improvements in inflammatory biomarkers after the ingestion of tomato products have been attributed mainly to lycopene. However, the results reported here indicate that when the product contains other ingredients, such as onion and virgin olive oil, different bioactive compounds such as polyphenols and carotenoids such as β-carotene may be responsible for the anti-inflammatory effects.
A strong point of this work is that relatively few acute studies have been carried out on the impact of diet on inflammatory biomarkers in healthy humans. The significant reduction in TNF-α and CRP levels after the ingestion of a single dose of tomato-based sofrito, rich in bioactive compounds, suggests this Mediterranean sauce may contribute positively to the regulation of the inflammatory status, even in individuals with optimal health.
The main limitation of the study is the lack of controls. Nevertheless, the analyses were carried out 24 h after the intervention in the same conditions to avoid changes due to circadian rhythms, and the volunteers continued a low antioxidant diet after the consumption of sofrito.

Conclusions
The addition of sofrito to dishes and cuisine could contribute to the maintenance of health. An improvement in inflammatory biomarkers, particularly CRP and TNF-α, was observed after a single dose of sofrito, even though the subjects were healthy. Beneficial effects of sofrito intake could be partly attributed to the presence of bioactive compounds (polyphenols and carotenoids) from the ingredients. Although the health-protecting role of lycopene from tomato has been extensively studied, other carotenoids such as β-carotene (also found in large amounts in tomato by-products) seem to modulate the regulation of the inflammatory status.