Attenuation of Colonic Injury and Inﬂammation by Administration of a Phenolic Extract of Summer Savory ( Satureja hortensis L.) in Experimental Inﬂammatory Bowel Disease in Mice

Featured Application: Summer Savory extracts could be a promising pharmacological tool not only in the management of inﬂammation associated to Inﬂammatory Bowel Disease (IBD) but also in managing the impaired secondary inﬂammatory mechanisms related with colorectal cancer progression, with a potential clinical use as a supplement to standard of care therapy. Abstract: Summer Savory ( Satureja hortensis L.) is a plant traditionally used as a food spice in the Mediterranean region. Surprisingly, not much is known about the health beneﬁcial e ﬀ ects of its phenolic-rich extracts. The majority of publications have always focused on the properties of their essential oil. One of the main phenolic compounds of Summer Savory is rosmarinic acid, which has demonstrated anti-inﬂammatory outcomes in several animal models of inﬂammatory-mediated diseases. Inﬂammatory Bowel Disease (IBD) is a chronic inﬂammatory disease, in addition to Ulcerative Colitis and Crohn’s Disease, frequently related with increased morbidity and even mortality due to the complications associated, including colorectal cancer. Our work has shown, to our knowledge, for the ﬁrst time, that administration of a phenolic extract of Summer Savory in a mouse model of Ulcerative Colitis led to the reduction of several markers for intestinal injury, including reduction of inducible nitric oxide synthase (iNOS) and Cyclooxygenase-2 (COX-2 or prostaglandin-endoperoxide synthase) expression, two well-known mediators of tissue inﬂammation and progression to cancer and led also to a reduction of the mortality. Given the chemical constitution found in the extract and the preclinical evidence of a beneﬁcial e ﬀ ect of polyphenols in inﬂammatory processes, an opportunity arises for pharmacological modulation of pathways relevant for IBD and progression to cancer with phenolic-rich extracts.


Introduction
Summer Savory (Satureja hortensis L.) is a plant traditionally used as a food spice in Southern Europe and the Mediterranean region, yet, surprisingly, not much is known about the elucidation of The goal of this work was to characterize the chemical constitution and antioxidant effects of a Summer Savory aerial parts extract and to assess the anti-inflammatory effects (acute and chronic) that might be relevant for a pharmacological intervention in IBD, as well as in the prevention of CRC development.
We, therefore, screened the acute anti-inflammatory effect in a model of paw edema and further assessed the effect of the extract's administration in a mouse model of colitis induced by TNBS (2,4,6-Trinitrobenzenesulfonic acid).

Reagents and Chemicals
Ketamine (Imalgene ® 1000) and xilazine (Rompun ® 2%) were purchased through the supplier Bio2 Produtos Veterinários (Lisboa, Portugal). Other substances used throughout the experiments were purchased through Sigma-Aldrich, Sintra, Portugal (if not, that will be clearly stated throughout the publication).

Plant Related Material and Extract Preparation
Fresh samples of Summer Savory (Satureja hortensis L.) were acquired locally from a cultivar (Lisboa, Portugal). Water was used to wash plants (stems and leaves) that afterwards were sliced into thin slices. Consequently, sliced plant material (20 g) was extracted using 100 mL of ethanol (70%, v/v) for 24 h, in dark conditions, at room temperature and under stirring. The resulting extracts were filtered (Whatman, n • 1) and rotary evaporator at 40 • C (Heidolph LABOROTA 4001, Heidolph Instruments / Soquimica, Lisbon, Portugal) was used to eliminate ethanol. Finally, extracts were centrifuged (6000 g, 15 min, 4 • C) (Sigma 4K-15C), and the supernatants were split into 1 mL aliquots and were kept storage at −50 • C until future analyses.

Total Phenolic and Total Flavonoid Content
In order to determine total phenolic compounds the Kosar et al. method was used [37], assessing total phenolic concentration by the Folin-Ciocalteu method. When it comes to total flavonoid content, we followed Barros et al. method [38]. The outcome of total phenolic content was expressed as mg gallic acid equivalents (GAE) per mL extract and per g of dry plant, and total flavonoid content was expressed in µmol equivalents of catechin (CE) per mL of extract.

High-Performance Liquid Chromatography (HPLC)
HPLC of the extract was performed as previously described [15]. Briefly, the phenolic components of the aqueous extract were isolated using solid phase extraction C18 columns (500 mg/3 mL, Ref. 7020-03, J.T. Baker) in order to eliminate sugars and other non-phenolic components that could affect the chromatographic separation. An aliquot of the aqueous extract was added to the column and the adsorbed phenolic components were eluted with aqueous formic acid (0.1% v/v) and acetonitrile. The obtained extracts were combined concentrated to the initial volume and filtered through a 0.22 µm filter. The chromatographic separation was performed in a HPLC system (SpectraSystem, Thermo, Sigma-Aldrich, Sintra, Portugal), equipped with a diode array detector (DAD), and a Thermo C-18 column, and elution was performed with 0.1% formic acid (solvent A) and a mixture of 90% acetonitrile + 9.9% water + 0.1% formic acid (solvent B) at a flow rate of 0.8 mL/min. Spectra acquisition was made in the range of 190 nm to 700 nm with selective detection at 280, 320 and 360 nm. Identification of the main functional groups present in the extract was performed by comparison of their UV spectra with those of representative standards analyzed in the same conditions. Standard curves were determined for rosmarinic and ferulic acids by analyzing the corresponding standards in the concentration range of 0.05 to 1 mg/mL. CUPRAC assay followed the normal sample measurement procedure as previously stated [39]. Results were expressed as µmol ascorbic acid equivalents (AAE)/mL of extract.

Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was performed according to the procedure as previously stated [40]. A calibration curve of ferrous sulphate (0-1.25 mM) was utilized, and results were expressed as µmol Fe 2+ /mL of extract.

Superoxide Anion Radical-Scavenging Assay
The superoxide anion radical-scavenging assay was carried according to the procedure previously stated [42]. Results were expressed as µmol equivalents of ascorbic acid/mL of extract.

Animals
The paw edema protocol was performed using 46 Wistar rats (150-200 g, male) (Instituto de Higiene e Medicina Tropical, Lisbon, Portugal). Ad libitum food and water was guaranteed to all rats up to 12 h prior to the beginning of the protocol.

Edema Induction and Evaluation
Paw edema was induced by sub-plantar (intradermal) injection of 100 µL of a λ-carrageenan solution (1% in saline) into the rat left hind paw as previously described [43]. Paw volume measurements were performed as described: V 0 or basal volume is the volume of the hind paw measured immediately after carrageenan injection, and V 6 is the volume at 6 h post carrageenan administration. The increase in paw volume was measured as edema and expressed as relative percentage of the increase in the volume at 6 h compared to the initial volume, according to the following formula: % paw volume increase = [(V 6 − V 0 )/V 0 ] × 100.

Experimental Groups
All the animals were split into six groups randomly: (i) Control Group included animals in which edema protocol detailed previously was performed, except for the administration of 100 µL of apyrogenic saline instead of carrageenan. Animals were subjected to administration of water (1 mL/kg) by oral gavage (n = 6); (ii) Carrageenan Group included paw edema animals and administered with 1 mL/kg of water by gastric gavage (n = 8); (iii) Summer Savory group-paw edema animals and administered with Summer Savory extract (15 mg of phenolic acids/kg by gastric gavage) 30 min prior to injection of carrageenan (n = 8); (iv) indomethacin group included paw edema animals with and administered with indomethacin (10 mg/kg by gastric gavage) 30 min prior to injection of carrageenan (n = 8); (v) tempol group included paw edema animals and administered with tempol (30 mg/kg by gastric gavage) 30 min prior to injection of carrageenan (n = 8); and (vi) trolox group included paw edema animals and administered with trolox (10 mg/kg by gastric gavage) 30 min prior to injection of carrageenan (n = 8). The dose of the extract was selected from previous studies by our group related with the evaluation of the beneficial effects of different phenolic extracts in several models of Male CD-1 mice (Crl:CD1(ICR)), with weights ranging from 25-30 g (5-6 weeks of age) (Harlan, Spain), were kept according to the standard protocols. Thus, ad libitum to water and food, they were kept in an automatically controlled temperature/light room (20 • C with a 12 h light/dark cycle) at the Animal Facility of the Faculty of Pharmacy-University of Lisbon.

Induction of Colitis
Induction of colitis was performed by administration of TNBS as previously described (Rocha et al., 2019). Briefly, a 50% ethanolic (EtOH) solution of TNBS (2.5% m/v) was administered by intracolonic administration (4 cm above the anus). At day 4 post-induction, blood samples were collected by cardiac puncture under surgical anesthesia, followed by euthanasia by cervical dislocation and subsequent necropsy. The colon was removed and was observed for classification of diarrhea severity. Furthermore, the colon was washed with (phosphate buffered saline) PBS for a macroscopic observation of lesions and fixed in (paraformaldehyde) PFA for histological studies.

Experimental Groups
Mice were randomized into the experimental groups described below:

1.
Sham group (n = 6): the colitis induction protocol was performed as stated previously, except for the intracolonic administration that was performed with 100 µL of saline solution. Animals were administered daily with 10 mL/kg of water by gastric gavage until the end of the experiment.

2.
Ethanol group (n = 6): the colitis induction protocol was performed as stated previously except for the intracolonic administration that was performed with 100 µL of 50% (v/v) ethanol solution. Animals were administered daily with 10 mL/kg of water by gastric gavage until the end of the experiment. 3.
TNBS group (n = 10): the colitis induction protocol was performed as previously described, with the administration of 100 µL of a TNBS solution (2.5% TNBS in 50% ethanol). Animals were administered daily with 10 mL/kg of water by gastric gavage until the end of the experiment. 4.
TNBS + Summer Savory group (n = 10): the colitis induction protocol was performed as described previously. Animals were administered daily with Summer Savory extract (15 mg/kg of phenolic acids by oral gavage) until the end of the experiment.

Evaluation of Colitis Severity (Macroscopic Analysis)
Diarrhea severity was classified by an observer blinded to the experimental groups following the classification found in Table 1. A microscopic observation of the tissue was done, followed by colon measurement, as well as measurement of the extent of injury.  [44], as well as the immunohistochemistry studies, for measurement of COX-2 and iNOS expression. Colon histological damage was scored as follows: score 0-normal colon with no lesions, the mucosa is of uniform thickness, and the crypts are straight, normal crypt architecture, there is no cellular infiltration, edema, or exudate; score 1-colon with mild lesions, there are mucosal erosion and small superficial ulcers scattered along the length of the colon, with slight crypt loss and mononuclear cell infiltration; score 2-colon with moderate lesions, intestines have extensive erosion and ulceration, with moderate crypt loss and neutrophil infiltration; score 3-colon with very severe ulceration, much of the mucosa is thin with loss of crypts and markedly increased infiltration of neutrophils and acute inflammatory exudate. The intensity of the protein staining is relatable to the level of expression of iNOS and COX-2. The level of iNOS or COX-2 staining was quantitatively evaluated by determining the percentage of tissue area that was stained in brown, using the ImageJ (Fiji Is Just, 2020, NIH, Bethesda, Maryland, USA) software.

Statistical Analysis
The animal experiment results (in vivo) were represented as mean ± standard error of the mean (SEM) of n observations. In order to compare results among experimental groups, a one-way ANOVA test was performed, followed by a Bonferroni's test (post-hoc multiple comparisons) using GraphPad Prism version 8.2.1, GraphPad, San Diego, CA, USA). When p < 0.05 (CI of 95%), differences were considered statistically significant. Analysis of mortality onset was accomplished by Kaplan-Meier statistics, followed by a Mantel-Cox Test. When p < 0.05 (CI of 95%), differences were considered statistically significant.

Chemical Characterization
Initial chemical characterization of the Summer Savory extract was done using the Folin-Ciocalteu method. The resulting total phenolic and flavonoid compounds of the extract is displayed below in Table 2. The chromatographic profile resulted from the phenolic extract of Summer Savory is displayed in Figure 1 and shows that the predominant phenolics in Summer Savory ethanolic extract ( Figure 2) were hydroxycinnamic acids (53.5%), flavonol glycosides (23.3%), and flavones and flavanones (13.0%), considering the sum of the corresponding chromatographic areas.  Similar composition is reported for Summer Savory extracts obtained by Boroja et al. [18] using aqueous methanol or by Maškovićc et al. [19] using aqueous ethanol.
The main components found in the ethanolic extract of Summer Savory were rosmarinic acid (1 mg/mL) and isoferulic acid (0.75 mg/mL), corresponding to 19% and 15% of the chromatography area, respectively. These components were also predominant in other Satureja species hydroalcoholic extracts studied by other authors that also detected caffeic, isoferulic, chlorogenic acids, quercetin and apigenin glycosides, naringenin, and apigenin in relevant concentrations [17][18][19].
To determine the antioxidant properties of Summer Savory extract reducing capacity and free radical scavenging activity, several assays were performed ( Table 3). The antioxidant capacity of  Similar composition is reported for Summer Savory extracts obtained by Boroja et al. [18] using aqueous methanol or by Maškovićc et al. [19] using aqueous ethanol.
The main components found in the ethanolic extract of Summer Savory were rosmarinic acid (1 mg/mL) and isoferulic acid (0.75 mg/mL), corresponding to 19% and 15% of the chromatography area, respectively. These components were also predominant in other Satureja species hydroalcoholic extracts studied by other authors that also detected caffeic, isoferulic, chlorogenic acids, quercetin and apigenin glycosides, naringenin, and apigenin in relevant concentrations [17][18][19].
To determine the antioxidant properties of Summer Savory extract reducing capacity and free radical scavenging activity, several assays were performed ( Table 3). The antioxidant capacity of Similar composition is reported for Summer Savory extracts obtained by Boroja et al. [18] using aqueous methanol or by Maškovićc et al. [19] using aqueous ethanol.
The main components found in the ethanolic extract of Summer Savory were rosmarinic acid (1 mg/mL) and isoferulic acid (0.75 mg/mL), corresponding to 19% and 15% of the chromatography area, respectively. These components were also predominant in other Satureja species hydroalcoholic extracts studied by other authors that also detected caffeic, isoferulic, chlorogenic acids, quercetin and apigenin glycosides, naringenin, and apigenin in relevant concentrations [17][18][19].
To determine the antioxidant properties of Summer Savory extract reducing capacity and free radical scavenging activity, several assays were performed ( Table 3). The antioxidant capacity of Summer Savory extract was confirmed in the protocols tested. The outcomes match the ones demonstrated in previous studies, as well that ethanolic extracts of Summer Savory exhibit reducing capacity and are able to scavenge reactive oxygen species.

Paw Edema Evaluation
Intradermal injection of carrageenan unsurprisingly led to paw edema (evidenced by increased volume) at 6 h post-injection comparing to animals injected with only saline, therefore validating this local inflammation model. Administration of Summer Savory phenolic extract led to a statistically significant reduction of edema formation ( Figure 3). Comparison of these results with known anti-inflammatory and antioxidant compounds (indomethacin, trolox, and tempol) led to the conclusion that administration of a dose of 15 mg of phenolic acids/kg of this Summer Savory phenolic extract reduced the formation of paw edema at the same magnitude as the positive controls used.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 19 Summer Savory extract was confirmed in the protocols tested. The outcomes match the ones demonstrated in previous studies, as well that ethanolic extracts of Summer Savory exhibit reducing capacity and are able to scavenge reactive oxygen species.

Paw Edema Evaluation
Intradermal injection of carrageenan unsurprisingly led to paw edema (evidenced by increased volume) at 6 h post-injection comparing to animals injected with only saline, therefore validating this local inflammation model. Administration of Summer Savory phenolic extract led to a statistically significant reduction of edema formation ( Figure 3). Comparison of these results with known antiinflammatory and antioxidant compounds (indomethacin, trolox, and tempol) led to the conclusion that administration of a dose of 15 mg of phenolic acids/kg of this Summer Savory phenolic extract reduced the formation of paw edema at the same magnitude as the positive controls used.

Macroscopic and Functional Signs of Colitis Injury
Analysis of results from both the Sham and Ethanol Groups showed that animals did not exhibit macroscopic signs of colonic lesion and exhibited also 0% of mortality. Colitis induction in the TNBS/EtOH Group produced statistically significant differences in: the decrease of colon length, injury extent (ulcer) increase, and diarrhea severity increase, associated also with a 40% mortality rate. For groups treated with the Summer Savory extract, all these results of colonic injury were significantly attenuated when compared to untreated animals with colitis (Table 4, Figures 4-6).

Macroscopic and Functional Signs of Colitis Injury
Analysis of results from both the Sham and Ethanol Groups showed that animals did not exhibit macroscopic signs of colonic lesion and exhibited also 0% of mortality. Colitis induction in the TNBS/EtOH Group produced statistically significant differences in: the decrease of colon length, injury extent (ulcer) increase, and diarrhea severity increase, associated also with a 40% mortality rate. For groups treated with the Summer Savory extract, all these results of colonic injury were significantly attenuated when compared to untreated animals with colitis (Table 4, Figures 4-6).  ; EtOH Group (n = 6); TNBS + EtOH Group (n = 10); TNBS + Summer Savory Group (n = 10).     ; EtOH Group (n = 6); TNBS + EtOH Group (n = 10); TNBS + Summer Savory Group (n = 10).    In addition, mortality was significantly reduced in the group subjected to Summer Savory administration (40% vs. 20%, for TNBS and Summer Savory groups, respectively), as depicted in the Kaplan-Meier analysis in Figure 7.

Colon Injury: Histological Features and Inflammatory Markers
The histological analysis ( Figure 8) revealed that, even though control samples exhibit colon with no lesions (normal colon), a mucosa with uniform thickness, normal crypt architecture, and no signs of inflammation (score 0), the colitis sample exhibits a strong ulceration where crypts loss and thinner mucosa can easily be identified with a severe neutrophils' infiltration, consistent with a score of 3. The samples from animals treated with Summer Savory show erosion of the mucosa and small superficial ulcers disseminated across the length of the colon, with partial crypt destruction and only slight mononuclear cell infiltration, indicating a lower damage score of 1. In addition, mortality was significantly reduced in the group subjected to Summer Savory administration (40% vs. 20%, for TNBS and Summer Savory groups, respectively), as depicted in the Kaplan-Meier analysis in Figure 7. In addition, mortality was significantly reduced in the group subjected to Summer Savory administration (40% vs. 20%, for TNBS and Summer Savory groups, respectively), as depicted in the Kaplan-Meier analysis in Figure 7.

Colon Injury: Histological Features and Inflammatory Markers
The histological analysis ( Figure 8) revealed that, even though control samples exhibit colon with no lesions (normal colon), a mucosa with uniform thickness, normal crypt architecture, and no signs of inflammation (score 0), the colitis sample exhibits a strong ulceration where crypts loss and thinner mucosa can easily be identified with a severe neutrophils' infiltration, consistent with a score of 3. The samples from animals treated with Summer Savory show erosion of the mucosa and small superficial ulcers disseminated across the length of the colon, with partial crypt destruction and only slight mononuclear cell infiltration, indicating a lower damage score of 1.

Colon Injury: Histological Features and Inflammatory Markers
The histological analysis ( Figure 8) revealed that, even though control samples exhibit colon with no lesions (normal colon), a mucosa with uniform thickness, normal crypt architecture, and no signs of inflammation (score 0), the colitis sample exhibits a strong ulceration where crypts loss and thinner mucosa can easily be identified with a severe neutrophils' infiltration, consistent with a score of 3. The samples from animals treated with Summer Savory show erosion of the mucosa and small superficial ulcers disseminated across the length of the colon, with partial crypt destruction and only slight mononuclear cell infiltration, indicating a lower damage score of 1.

Discussion
Even though Summer Savory (Satureja hortensis) has been listed for many years as a medicinal herb and is a well-known herb in the food and culinary industry, scientific-based literature evaluating the potential beneficial effects of Summer Savory in health is surprisingly scarce, especially when compared to other aromatic herbs and even more obvious when particularizing for the study of the aerial parts as opposed to considerably more information regarding essential oil properties [1]. However, some studies, mainly from Mediterranean region countries in which Summer Savory is used commonly, have attributed antimicrobial, anti-inflammatory, antioxidant, antidiabetic, and antitumor effects to both seed (essential oil) and aerial parts extract of Summer Savory [47,48].
One of the few studies focusing on an alcoholic extract of the aerial parts of Summer Savory was a study from Boroja et al. in 2018 that evaluated the beneficial effects of administration of a methanolic extract of the aerial parts of Summer Savory in a cisplatin-induced model of oxidative kidney, liver, and testicular [18]. Injury was induced by injection of cisplatin (7.5 mg/kg, sigle dose i.p.) to wistar rats and Summer Savory extract was administered orally (doses of 50, 100, and 200 mg/kg/day for 10 days). Extract's administration ameliorated tissue morphology, impaired the rise in serum level of liver, renal and testes injury markers and reduced tissue oxidative stress with reduction of apoptosis evidenced by an increased Bcl-2/Bax ratio. In this study, and similarly to the situation described in our study, the principle constituent of the phenolic extract identified was rosmarinic acid.
In fact, several studies report high rosmarinic acid content in aromatic plants from the Lamiaceae family [49][50][51], and, although Satureja hortensis is not one of family members with higher concentration of rosmarinic acid, it normally has concentrations higher than plants, such as Rosmarinus officinalis and Lavandula angustifolia [52]. When compared to our study with spearmint extract, results from our present study shows that not only the concentration of rosmarinic acid is equivalent (1.0 mg/mL vs. 1.4 mg/mL, for Summer Savory and spearmint extracts, respectively) but also the antioxidant capacities, as well [15]. Given that antioxidant capacity is usually appointed as a main responsible for an alti-inflammatory activity, one of our previous work aimed to prove that anti-inflammatory activity is often independent of the anti-oxidant effect. In a study of the antiinflammatory effect of an extract of Rosmarinus officinalis and of rosmarinic acid, our team proved that the anti-inflammatory effect of both was equivalent for the same dose of rosmarinic acid, although the anti-oxidant activity was much higher for the extract [43]. Therefore, in the present work, characterization of the anti-oxidant effect of the extract was important to show anti-oxidant Figure 10. Evaluation of COX-2 and iNOS activation in the TNBS colitis model reveals that specimens from TNBS colitis group exhibits severe expression of COX-2 and iNOS (brown staining, arrow), while specimens from the animals treated with Summer Savory extract exhibited no expression of COX-2 and iNOS. Sham Group (n = 6); EtOH Group (n = 6); TNBS + EtOH Group (n = 10); TNBS + Summer Savory Group (n = 10). Original magnification ×100. Scale bar equals 50 µm.

Discussion
Even though Summer Savory (Satureja hortensis) has been listed for many years as a medicinal herb and is a well-known herb in the food and culinary industry, scientific-based literature evaluating the potential beneficial effects of Summer Savory in health is surprisingly scarce, especially when compared to other aromatic herbs and even more obvious when particularizing for the study of the aerial parts as opposed to considerably more information regarding essential oil properties [1]. However, some studies, mainly from Mediterranean region countries in which Summer Savory is used commonly, have attributed antimicrobial, anti-inflammatory, antioxidant, antidiabetic, and antitumor effects to both seed (essential oil) and aerial parts extract of Summer Savory [47,48].
One of the few studies focusing on an alcoholic extract of the aerial parts of Summer Savory was a study from Boroja et al. in 2018 that evaluated the beneficial effects of administration of a methanolic extract of the aerial parts of Summer Savory in a cisplatin-induced model of oxidative kidney, liver, and testicular [18]. Injury was induced by injection of cisplatin (7.5 mg/kg, sigle dose i.p.) to wistar rats and Summer Savory extract was administered orally (doses of 50, 100, and 200 mg/kg/day for 10 days). Extract's administration ameliorated tissue morphology, impaired the rise in serum level of liver, renal and testes injury markers and reduced tissue oxidative stress with reduction of apoptosis evidenced by an increased Bcl-2/Bax ratio. In this study, and similarly to the situation described in our study, the principle constituent of the phenolic extract identified was rosmarinic acid.
In fact, several studies report high rosmarinic acid content in aromatic plants from the Lamiaceae family [49][50][51], and, although Satureja hortensis is not one of family members with higher concentration of rosmarinic acid, it normally has concentrations higher than plants, such as Rosmarinus officinalis and Lavandula angustifolia [52]. When compared to our study with spearmint extract, results from our present study shows that not only the concentration of rosmarinic acid is equivalent (1.0 mg/mL vs. 1.4 mg/mL, for Summer Savory and spearmint extracts, respectively) but also the antioxidant capacities, as well [15]. Given that antioxidant capacity is usually appointed as a main responsible for an alti-inflammatory activity, one of our previous work aimed to prove that anti-inflammatory activity is often independent of the anti-oxidant effect. In a study of the anti-inflammatory effect of an extract of Rosmarinus officinalis and of rosmarinic acid, our team proved that the anti-inflammatory effect of both was equivalent for the same dose of rosmarinic acid, although the anti-oxidant activity was much higher for the extract [43]. Therefore, in the present work, characterization of the anti-oxidant effect of the extract was important to show anti-oxidant equivalence with other aromatic plants [14,15], but evaluation of its specific effect on inflammatory models was necessary to assess the anti-inflammatory activity.
Therefore, in the present work, in order to do a preliminary evaluation of the anti-inflammatory effect of the Summer Savory extract, we evaluated a single-dose effect of Summer Savory oral administration in an acute model of paw edema. Measurement of paw volumes after edema induction by carrageenan revealed that administration of Summer Savory extract impaired the increase in paw volume by 46.5% when compared to animals administered with vehicle. Additionally, the magnitude of that effect was not statistically different when compared to positive control groups administered with known antioxidant and anti-inflammatory properties (49% for indomethacin, 48% for Trolox, and 45.5% for tempol), indicating a potential benefit of this extract in inflammatory-mediated diseases.
The analgesic and anti-inflammatory effects of Summer Savory seed essential oil and hydroalcoholic and polyphenolic extracts was evaluated by Hajhashemi and colleagues [9]. Anti-nociceptive activity was evaluated in the mouse models of acetic acid and formalin-induced pain. Appraisement of anti-inflammatory activity was performed in the carrageenan-induced rat paw edema model was. Animals were pretreated with 50, 100, or 200 mg/kg, i.p. and 100 or 200 µL/kg, p.o, in the anti-nociceptive and anti-inflammatory models, respectively. Administration of hydroalcoholic and polyphenolic extracts or essential oil significantly decreased acetic acid-induced abdominal twitches, while hydroalcoholic extract additionally decreased pain responses in both early and late phases of the formalin test. In this test, the polyphenolic extract and essential oil only demonstrated beneficial effect in the late phase of the formalin test, while all three fractions reduced paw edema in the carrageenan model. However, even though one of the fractions tested was a polyphenolic extract, it was a seed extract which corresponds to a very different chemical constitution from a leave extract.
After the anti-inflammatory effect of Summer Savory was confirmed in an acute local model of inflammation, we aimed to access the positive effect of the extract in an experimental colitis mouse model. Administration of Summer Savory extract to animals with TNBS-induced colitis drove to a decrease of multiple markers of colon injury and inflammation: impairment of colon length decrease, reduction of injury extent, decrease of diarrhea severity, and reduction of mortality rate. Additionally, both macroscopical and histological signs of colon inflammation were decreased in animals administered with Summer Savory extract compared to colitis animals.
Surprisingly, even though no preclinical animal study was published until now that evaluated the beneficial effects of Summer Savory in experimental IBD, there is one publication reporting the results of a small clinical trial studying the administration of a commercial preparation of Summer Savory in Ulcerative Colitis patients [53]. This trial was a randomized, double-blind, placebo-controlled study that assessed the clinical benefit of commercial tablets of dried leaves of Summer Savory (500 mg daily) in Ulcerative Colitis. After 4 months, 85% (12/14) of patients that received supplementation with Summer Savory extract were in complete remission when compared to 46.2% (6/13) of patients on placebo. The flare up frequency was also statistically lower for patients treated with Summer Savory extract. This trial was very limited in terms of dimension, given that included only 27 patients total; nevertheless, it suggests a beneficial effect of Summer Savory in inflammatory bowel disease patients.
In our study, a relatively low dose phenolic extract of 15 mg/kg was used, which was derived from previous studies from our group that have been assessing the positive effects of different phenolic extracts in multiple models of inflammation with this dose of phenolic acids having generated matching results and is also within the dose-range of clinical translation [44][45][46]54].
Lately, we have been evaluating the protective effect of phenolic extracts originating from aromatic herbs and have demonstrated a beneficial effect of the administration of spearmint and pennyroyal phenolic extracts in this animal model of TNBS-induced Ulcerative Colitis [14,15]. Curiously, we demonstrated that rosmarinic acid was the principal compound in both of these experiments, like what we observed in the present study, further suggesting a relevant role of this phenolic acid in the beneficial effect. In addition, in the past, we have compared the beneficial role of an extract of rosemary and isolated rosmarinic acid in a local model of inflammation and established that rosmarinic acid was the principal responsible for the anti-inflammatory effect (Rocha et al., 2015). We have also identified in that study an important function of rosmarinic acid in the modulation of several critical pathways of inflammation in liver and lung inflammation models, in which administration of rosmarinic acid led to reduction of nuclear factor-kappa B (NF-kB) activation, as well as inhibition of the phosphatidylinositol 3-kinase (PI3k)/protein kinase B (Akt) and Glycogen synthase kinase 3beta (GSK-3beta) pathways.
In fact, Jin and colleagues have published their work in 2017 where they studied the effect of rosmarinic acid in dextran sulfate sodium (DSS)-induced experimental colitis and possible underlying mechanisms [55]. In this experiment, rosmarinic acid (30 or 60 mg/kg/day, p.o.) significantly reduced the severity of colitis, associated with decrease of inflammatory-related cytokines production (interleukins IL-6, IL-1β, and IL-22) and protein levels of COX-2 and iNOS. Furthermore, rosmarinic acid also inhibited NF-kB and signal transducer and activator of transcription 3 (STAT3) activation, with subsequent reduction of the activity of pro-survival genes.
Similarly, in our present study, we found that experimental colitis led to the induction of both iNOS and COX-2 expression and that administration of Summer Savory extract was able to impair that increase in expression, leading to statistically similar expression levels compared to control animal.
The effects of Summer Savory in COX-2 and iNOS expression were also characterized by Farzaneh and colleagues, who reported that Satureja hortensis extracts inhibited lipopolysaccharide (LPS)-induced inflammatory responses by J774.1 macrophages [56]. This study demonstrated that dichloromethane and hexane extracts od Summer Savory reduced nitric oxide (NO) production with more efficacy than other extracts, correlated with a decreased gene expression of iNOS, COX-2, IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α). Incubation of macrophages with the extracts also reduced IL-6 and IL-1β production by macrophages and reduced the expression of intercellular adhesion molecule 1 (ICAM-1) [56].
The beneficial effect of a Summer Savory aqueous extract (250 mg/kg) as anti-inflammatory was evaluated in a rabbit model of rhinosinusitis by measurement of nitric oxide (NO) metabolites and histological changes [57]. The results of this study demonstrated that both activity of NOS and concentration of NO metabolites were significantly reduced by topical administration of Summer Savory extract. Additionally, this study showed a reduction of edema and inflammation in the treated group when comparing to control [57].
Knowing that one of the main long-term consequences of IBD patients is the well-known increase of the risk for development of CRC [23][24][25] and its important link to chronic inflammation [24,26,27], it highlights even more the beneficial effects reported by our work. Research has actually highlighted the continuous effect of chronic inflammation and its association with the severity, extent and duration of IBD as the principal link between IBD and the elevated CRC risk [58].
Some studies have reported antiproliferative effects of S. hortensis extracts on leukemic cell lines [59]; however, we have previously evaluated the effect of Summer Savory extract in an in vitro based model with human colon adenocarcinoma HT-29 cells and concluded that this extract does not have any effect of HT-29 cell proliferation, although it partially inhibited cell invasion (unpublished data). Additionally, we further evaluated the effect on matrix metallopeptidases (MMP) activity, with no difference being observed when compared to controls (unpublished data), and, although a study has previously shown that MMP-2 and MMP-9 activity was inhibited by S. hortensis, those assays were only performed with the essential oil [60].
Rather than the more traditional mechanisms for cancer initiation and progression, inflammatory processes seem to be the main regulation of colitis-associated cancer, an imbalance in intestinal microbiota, and a crosstalk between various signaling pathways [61]; therefore, a pleiotropic approach seems to present itself as better pharmacological tool, not only targeting inflammation associated with colitis but also associated with the mechanism controlling cancer development and progression.
Several studies have already identified that extracts of plants known to possess high concentrations of rosmarinic acid exhibit anticancer effects in different in vitro and in vivo models of various types of cancer [62][63][64].
One study evaluated rosmarinic acid administration in a rat model of 1,2 dimethylhydrazine (DMH)-induced CRC [65]. Rosmarinic acid was administered (5 mg/kg) through the whole period of CRC induction and exhibited tumor reduction, reduction of stress oxidative markers, reduction mucosal bacterial enzymes activity, regulation of xenobiotic metabolizing enzymes, and up-regulation of apoptotic factors. In another study, rosmarinic acid antagonized activator protein-1-(AP-1)dependent activation of COX-2 expression in HT-29 colon cancer cells [66].
Given the well-known relevance of these two crucial inflammatory enzymes, the impairment of COX-2 and iNOS expression in our work confirmed the beneficial effect of Summer Savory not only on IBD pathogenesis but also on disease progression and the possible link to cancer proliferation. Their roles in angiogenesis, apoptosis, and metastatic processes and resistance to chemotherapy/radiation are well understood and of significant importance for IBD and CRC patients [67][68][69][70][71][72]. In fact, the role of COX-2 and iNOS in intestinal inflammation has pointed them out as great targets for chemoprevention of colon cancer [72].

Conclusions
For the first time, to our knowledge, our results show a beneficial effect of Summer Savory phenolic extract on the amelioration of experimental IBD, with reduction of its severity and mortality, as well as reduction of several injury markers and inflammatory mediators, including COX-2 and iNOS, known to have critical roles in intestinal inflammation and progression to CRC. Taking into consideration the role of inflammatory processes in the link between Ulcerative Colitis and progression to CRC, these results suggest that the beneficial effects evidenced by Summer Savory extract might provide an interesting pharmacological tool not only in the management of inflammation associated to IBD but also in the management of the impaired secondary inflammatory mechanisms related with CRC progression.

Conflicts of Interest:
The authors declare no conflict of interest.