**4. Discussion**

Gastritis is an inflammatory-based condition promoted by a variety of risk factors, which include stress, alcohol or drug abuse, and others; the most severe chronic form of gastritis is mainly caused by *Helicobacter pylori* infection. Unfortunately, the infection is diffused in several countries with unsustainable economic situation, and the conventional therapy against gastric inflammatory processes or for *H. pylori* eradication is not easily available.

This work investigates the anti-inflammatory and radical scavenger activities at the gastric level of eleven hydroalcoholic extracts obtained from plants widely used in Cameroon as spices and in the traditional medicine against a variety of diseases, including gastric disorders.

The study was carried out using two gastric cell models (AGS and GES-1 cells) stimulated with TNF<sup>α</sup>, which contributes to the inflammatory process in infected gastric epithelium [31]. AGS cells, a tumor gastric epithelial cell line, is a well-established in vitro model, while GES-1 cells are considered an in vitro model closer to the gastric epithelium of healthy subjects [32].

A prolonged oxidative stress may cause lipid peroxidation and DNA damages, leading to an increased risk to develop gastric cancer [33,34]; several extracts included in this study prevented H2O2-induced ROS generation in AGS and GES-1 cells, this e ffect could be explained with the presence of compounds able to counteract the excessive amount of ROS, derived from the unbalance between ROS production and endogenous antioxidant systems, acting as scavengers. The extracts displaying the highest antioxidant e ffect were those with the highest phenol content, as previously reported [25].

The investigation on the anti-inflammatory activity identified six extracts (XA, XP, DG, TT, AM, and AC) able to reduce the release and the gene expression of two NF-κB-dependent pro-inflammatory mediators, IL-8 and IL-6, which contribute to the amplification of the gastric inflammatory process [6,8,31]; the e ffect on the NF-κB pathway suggests that this transcription factor could be involved in the molecular mechanism responsible for the observed activity. In addition, the IC50s on IL-8 release in our experiments reflected in halved IL-8 mRNA level, suggesting the important contribution of the transcription, although not always statistically confirmed. The di fferent activity on the IL-8 promoter, especially evident in GES-1 cells, may be partly justified by the intervention of post-transcriptional regulatory mechanisms. The chemical analysis of the extracts has been previously reported by our group [24]; on the basis of this characterization, the hydroalcoholic extracts contain previously quantified secondary metabolites able to act as NF-κB inhibitors, such as pimaric acid in XP (8.73%), gingerol and shogaol in AM (0.7% and 2.05% respectively), chlorogenic acid and catechins, identified in some extracts [35–40]. Although further investigation is needed, these individual compounds could contribute, at least in part, to the anti-inflammatory activity displayed by the extracts. According to the literature, the anti-inflammatory e ffects could be linked to the antioxidant properties of the extracts, since it has been demonstrated that ROS are able to over-express IL-8 by activating oxidant-sensitive nuclear factors, such as NF-κB, in gastric epithelial cells [11].

Although the six extracts inhibited IL-8 release in both gastric cell models, only XP, TT, DG, and AC reduced IL-6 levels. Again, the mRNA analysis showed that all the extracts influenced the IL-6 at the transcriptional level, in particular DG, which obtained a statistically significant di fference with respect to the stimulated control. Our results allow to speculate that XA and AM do not inhibit other important transcription factors for IL-6, such as CREB, but further studies are needed to confirm this hypothesis.

NSAIDs are not used in the therapy of gastric inflammatory diseases, since they act on prostaglandin-endoperoxide synthase (cyclooxygenase) enzymes blocking also the PTGS1 (COX-1) isoform, which is involved in the protection of the gastric mucosa [14]. In this context, TT extract could be considered the most promising extract, for the ability to inhibit TNF α-induced *PTGS2* (*COX-2*) gene expression without a ffecting the *PTGS1* (*COX-1*) mRNA basal levels. Indeed, *Tetrapleura tetraptera* (TT) extract did not reduce the enzymatic activity of PTGS1 (COX-1) and PTGS2 (COX-2) isoforms in a cell-free system; however, these findings need further investigation.

In the literature, di fferent studies have demonstrated the beneficial e ffects of the fruits of this plant including antimalarial, anti-inflammatory, hypotensive, anti-insulin resistance, and antilipidemic properties [22,41,42].

This study demonstrates the antioxidant and anti-inflammatory activity, at the gastric level, of di fferent hydroalcoholic extracts from Cameroonian plants, selecting those with the most promising effects. XA, XP, TT, DG, AM, and AC are largely used and di ffused in some African countries as spices, thus making these plants interesting as functional foods, especially XP, TT, DG, and AC. Further studies are clearly needed to collect more evidence, especially for TT extract, although our findings provide a sound scientific support to the traditional use of these plants against gastric disorders. In particular, studies on the stability of the extracts in the gastric environment should reinforce the translational value of this work. Considering the order of the inhibitory concentrations (0.1–10 μg/mL) and the yields of extraction [23], it is plausible that the dietary consumption of 1–10 mg of Cameroonian spices

may achieve the bioactivity at gastric level. Thus, these products could be useful to alleviate gastric inflammation in countries where conventional therapy is not easily available.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/12/3787/s1, Figure S1: E ffect of the extracts on the NF-κB driven transcription in AGS cells. Data are expressed as percentage versus the stimulated control, which is arbitrarily assigned the value 100%., Figure S2: E ffect of the extracts on the NF-κB driven transcription in GES-1 cells. Data are expressed as percentage versus the stimulated control, which is arbitrarily assigned the value 100%, Figure S3: E ffect of the extracts on the IL-8 release in AGS cells. Data are expressed as percentage versus the stimulated control, which is arbitrarily assigned the value 100%, Figure S4: E ffect of the extracts on the IL-8 release in GES-1 cells. Data are expressed as percentage versus the stimulated control, which is arbitrarily assigned the value 100%, Figure S5: E ffect of the extracts on the IL-8 promoter activity in AGS cells. Data are expressed as percentage versus the stimulated control, which is arbitrarily assigned the value 100%, Figure S6: E ffect of the extracts on the IL-8 promoter activity in GES-1 cells. Data are expressed as percentage versus the stimulated control, which is arbitrarily assigned the value 100%, Figure S7: Effect of the extracts on the IL-6 release in GES-1 cells. Data are expressed as percentage versus the stimulated control, which is arbitrarily assigned the value 100%, Table S1: List of the primers used in the study.

**Author Contributions:** Conceptualization, A.P.A.N., M.D., J.-R.K., G.A.A. and E.S.; methodology, A.P.A.N., M.F., S.P., A.M., G.M. and G.B.; software, E.S.; validation, P.M., G.B. and M.D.; formal analysis, A.P.A.N.; investigation, A.P.A.N., A.D.T., M.F., A.M., G.M., S.P.; resources, A.P.A.N., M.F., A.M., G.M., S.P.; data curation, M.D., E.S.; writing—original draft preparation, A.P.A.N., M.F. and M.D.; writing—review and editing, M.D. and P.M.; supervision, M.D., J.-R.K. and G.A.A.; project administration, E.S.; funding acquisition, M.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by MIUR Progetto di Eccellenza.

**Acknowledgments:** The authors thank Dawit Kidane-Mulat, University of Texas, Austin, for providing GES-1 cells.

**Conflicts of Interest:** The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
