The potential of Thymus vulgaris aqueous extract to protect against delayed gastric emptying and colonic constipation in rats

Thyme is a rich source of bioactive phytochemicals and it is frequently used in folk-medicine to treat gastroenteritis irritations. The current study was performed to examine the potential of Thymus vulgaris aqueous extract (TV-AE) to protect against delayed gastric emptying (DGE) and colonic constipation in rats. Stomach ulcer was caused by a single oral dose administration of indomethacin (INDO) (30 mg kg−1 of body weight). Constipation was induced following intravenous intoxication of rats with vinblastine (VINB) (2 mg kg−1 of body weight) for one week. The effect of TV-AE at two graduated doses (100 and 200 mg kg−1) on DGE, gastrointestinal transit (GIT) and constipated rats and biochemical parameters was estimated using phenol red, charcoal meal test and colorimetric methods, respectively. The phytochemical-profile of TV-AE was explored using high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization-mass spectrometry (HPLC-PDA/ESI-MS). INDO and VINB caused a significant reduction in (P < 0.05) DGE and GIT and colonic motility dysfunction. TV-AE consumption remarkably (P < 0.05) attenuated the DGE (from 58.56% to 69.871%) and difficulty in evacuating stools (from 48.5 to 55.5 fecal pellets per rat) and balanced the GIT (65% to 71%). These GI-ameliorative effects were accompanied by reversed INDO/VINB-related oxidative changes, lipid-metabolism alteration and intracellular pathway moderation. HPLC-PDA/ESI-MS-analysis identified several chemical constituents including rosmarinic acid, quinic acid, luteolin-7-o-glucoside, protocatechuic acid and trans-cinnamic acid. Thus, TV-AE bioactive components may be used as medicinal substances to regulate/attenuate gastrointestinal–physiological activities and disturbances, which support its pharmacological use.


Introduction
Natural bioactive components are considered to be benecial elements designed to improve human health. 1,2 Plant products have the ability to transfer physiological benets, including reduction of cancer risks, inammatory states, cardiovascular diseases, diabetes and low-glycemic responses, neurodegenerative disorders, high blood cholesterol levels and reactive oxygen species and free radical overproduction. 3 Thymus vulgaris L., an aromatic herbaceous plant, is a medicinal herb belonging to the Lamiaceae family. It is commonly cultivated and used in many countries mainly as a food seasoning and a source of essential oil that are used in the food industry, in perfumery as a cosmetic and for medical purposes. 4 In traditional medicine, thyme is usually used in folk remedy in the prevention/treatment of a variety of disorders such as gastroenteric and broncho-pulmonary disruptions. 5 As reported by several researchers, thyme contains numerous phenolic compounds, especially thymol and carvacrol, which are found in its essential oil. Additionally, in wild-thyme, many other abundant phenolic compounds have been found such as caffeic and rosmarinic acid derivatives. 6 Thymus vulgaris extract has different pharmacological activities, which were conrmed via in vivo and in vitro experiments, including immunomodulatory activity, anti-inammatory effect, antibacterial ability, anthelmintic action, anti-oxidant activity, anti-thrombin capacity and potent antihypertensive ability. The therapeutic potential of Thymus vulgaris is based on its content of aliphatic phenols, avonoids, thymol, carvacrol, and eugenol, in addition to luteolin and saponins. [7][8][9] Functional gastrointestinal disorders (FGIDs) are dened by the perturbation of gastrointestinal tract sections such as gastroenteritis, ulcerative colitis, functional constipation and delayed gastric emptying (DGE). FGIDs, also known as disturbances of the gut-brain interaction, contain a number of separate idiopathic disruptions, which alter different regions of the GI-tract and involve visceral motility disorders related to an oxidative stress installation. 10,11 Excessive ROS generation may contribute to cell injuries by lipid-protein-DNA oxidation in various tissues. 12 In the GI tract, the cellular redox potential may even determine the fate of the differentiating cells. Wnt/b-catenin and notch signaling pathways are the major determinants of cell commitment for specialization. There may be a direct association between NADPH oxidase 1 (NOX-1)-dependent ROS fabrication, differential provocation of Wnt/b-catenin or notch signaling pathways, and intestinal proliferation. Modications in the redox capacities of GSH/GSSG and cysteine/cystine (Cys/ CySS) have been integrated with intestinal phenotypic transitions. The reducing abilities may favor cell proliferation, but oxidizing efficiencies can lead to cell growth reduction or inhibition. 13,14 There are no published reports in the literature about the effect of the extract of T. vulgaris on gastric emptying and functional constipation. Based on these considerations, this study was designed to characterize the various TV-AE phenolic components and to investigate the potentiality of TV-AE to protect the GI tract against gastric delayed emptying and colonic constipation in rats.

Chemicals
Clonidine, red phenol, NaCl, NaOH, charcoal meal, gum arabic, and haematoxylin/eosin were purchased from Sigma Chemical Co. (Sigma-Aldrich GmbH, Steinheim, Germany). Indomethacin and vinblastine were purchased from a local central pharmacy. All other reagents used were of analytical grade.

TV-AE preparation
T. vulgaris leaves were collected from the region of Jendouba (North-West of Tunisia) during the season of spring (2017). Voucher specimens were identied and authenticated by a taxonomist in the Higher Institute of Biotechnology of Beja Tunisia and deposited at the herbarium in the Faculty of Sciences, University of El Manar, Tunisia. The leaves were dried at 40 C in air, ground, and extracted with bi-distilled water. The obtained leaf extracts were dehydrated at the same temperature under vacuum and nally freeze dried.
2.3. TV-AE bioactive compound identication by liquid chromatography-high-resolution electrospray ionization mass spectrometry (LC-HRESIMS) assay 100 mg of TV-AE was dissolved in 100 mL of 10% methanol, ltered and then 1 mL was added to LC-MS vials. A reversephase column (Pursuit XRs ULTRA 2.8, C18, 100 Â 2 mm, Agilent Technologies, UK) was used to perform HPLC examinations. 20 mL of the sample was injected at a column temperature set at 30 C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). A gradient program was used for separation at a ow rate of 1 mL min À1 . The mobile phase consisted of an initial composition of 100% solvent A, with a gradient of 100% solvent B over 20 min, held at 100% solvent B for 5 min and 100% solvent A for 25 min. The drying gas ow rate was 1 mL min À1 at 320 C. MS was operated in the positive ion mode in the mass range of 100-2000 m/z. High-resolution mass spectral data was obtained on a Thermo Instruments ESI-MS system (LTQ XL/LTQ Orbitrap Discovery, UK) connected to a Thermo Instruments HPLC system (Accela PDA Detector, Accela PDA Autosampler and Accela Pump). 15

Animals
Male Wistar rats (180 and 220 g) were used at 13 weeks of age. The animals were obtained from the Society of Pharmaceutical Industries of Tunisia (SIPHAT, Ben-Arours, Tunisia), and isolated into different groups and acclimatized for two weeks with a standard pellet diet (standard pellet diet Badr-Utique-TN) and water ad libitum (22 AE 2 C; 12 h dark per light cycle). All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Tunis University and approved by the Animal Ethics Committee of National Institute of Health. 16 2.5. Experimental design 2.5.1. Acute DGE and CON inductions in rats. Acute UG was caused in the animals according to the procedure described by Oluwabunmi and Abiola. 17 Briey, the rats were administered with a single oral dose of indomethacin (30 mg kg À1 body weight). The animals were deprived of food for 24 h but had free access to water prior to ulcer induction. Various degrees of ulceration manifested 4 h aer indomethacin administration. Acute UG diagnosis was evaluated by different clinical signs, such as gastric juice volume, ulcer score and gastric pH. Colonic alterations were caused in vivo according to the method elucidated by Rtibi et al. 18 Briey, animals were intoxicated with intravenous injection of vinblastine (2 mg kg À1 of body weight in a saline solution) for 7 days. Diagnosis of VINB injuries was conrmed by several signs, such as fecal pellet, wet fecal weight, dry fecal weight and water content.
2.5.2. TV-AE and drug actions on GIT in healthy and treated rats. This manipulation was carried out according to our previous studies using the charcoal meal test. 19 Specically, aer 2 h of animal treatment with TV-AE at two doses (100 and 200 mg kg À1 ) or the standard molecule (1 mg kg À1 ), the colitis rats were provided the standard charcoal meal (10% charcoal in 5% gum arabic) by oral administration. Aer 30 min, the animals were anesthetized and their small bowel was excised. The distance travelled by the charcoal meal suspension was measured and expressed as a percentage of the total length. The peristaltic index was calculated using the following formula: Peristaltic index ¼ (distance travelled by charcoal meal/length of small-intestine) Â 100 % of inhibition ¼ mean distance travelled by the control À mean distance travelled by the test group/mean distance travelled by the control.
2.5.3. INDO and TV-AE effects on gastric emptying process. To assess the INDO/TV-AE actions on the gastric-emptying process, the red phenol method was used. In this context, the animals were divided into different groups of seven and administered 1 h before with the test meal (50 mg phenol red in 100 mL aqueous methyl cellulose) as follows: group 1 served as the control group (CONT) and received 1 mL of physiological solution (NaCl, 0.9%, p.o.). Groups 2 and 3 were treated only with the extract. Group 4 served as the INDO group without extract and drug. Groups 5 and 6 of INDO rats were pre-treated with two doses of TV-AE (100 and 200 mg kg À1 , b.w. p.o.).
Aer 1 h, the animals were anesthetized and euthanized. Their stomach and its contents were combined with 100 mL of 0.1 N NaOH. The supernatants were mixed with 4 mL of 0.5 N NaOH and the absorbance of the samples was read at 560 nm. Phenol red collected immediately from stomach organs aer test meal administration was used as the standard (0% of GE). 20 GE (%) was calculated according to the following formula: Gastric emptying rate (%) ¼ (1 À absorbance of treated/ absorbance of standard) Â 100.

VINB and TV-AE effects on colonic motor activity.
The animals were randomly divided into four groups. Healthy animals or the control group (CONT) received a physiological solution at a dose of 5 mL kg À1 (n ¼ 7). The VINB group was treated intravenously only with VINB (2 mg kg À1 b.w.) dissolved in a saline solution once per day for one week (n ¼ 7). Finally, the two other groups were treated by VINB in combination with TV-AE at two doses of 100 mg kg À1 and 200 mg kg À1 (n ¼ 7). On day 5, the faecal pellet weights (wet and dry weights) of the rats were measured for 24 h and the water levels were determined by the difference between the weights before and aer drying. 18 2.5.5. Oxidative and antioxidative marker assessment in rats. Gastric and colonic mucosal tissues were scraped and then placed in a phosphate buffered saline (PBS) solution, homogenized and centrifuged for 15 min at 9000 Â g. The obtained aliquots were stored at À80 C.
Lipid oxidation caused by ROS and free radicals in the UG/ UC rats led to MDA production, which was estimated with double heating technique adapted by Draper et al. 21 The expression of the results was reported as the mean of MDA nmol mg À1 of proteins. Thiol group (-SH) content was determined using Ellman's procedure 22 and the data was expressed as nmol of -SH per mg of proteins. Carbonyl residue generation aer protein oxidation was examined using the method by Levine et al. 23 The results were expressed as mmol carbonyl residue per mg protein.
Superoxide dismutase action was determined using the nicotinamide adenine dinucleotide (reduced) phenazine methosulphate nitroblue tetrazolium reaction system method. 24 The results were calculated and expressed in units of SOD per mg proteins. Catalase (CAT) activity was estimated using the procedure by Aebi 25 and the results were expressed as nmol min À1 mg À1 protein. GPx activity was analysed using the Flohé and Günzler design 26 and the data was expressed as nmol GSH min À1 mg À1 protein.
2.5.6. Lipid indicators and moderating intracellular pathways assessment in rats. Aer blood centrifugation at 3000 Â g for 10 min, the lipid marker (triglycerides, total cholesterol, and high-density cholesterol) contents were measured in the asobtained serum using commercially available kits purchased from Biomaghreb, Tunisia.
Gastric and colonic mucosal H 2 O 2 levels were determined according to the method by Dingeon et al. 27 and the data was expressed as mmol of H 2 O 2 per mg protein. Intracellular mediators (calcium and non-haem iron) levels were measured using commercial kits purchased from Biomaghreb, Tunisia. 28,29 The results were expressed as nmol of these parameters per mg protein.

Statistical analysis
A one-way analysis of variance test was used to determine the signicance between the different groups of all animals. Statistical analyses were calculated using StatView statistical soware. The data is representative of six to eight distinct observations. Differences were stated as mean AE SEM and designated signicant when the values of p were <0.05.

TV-AE consumption accelerates GIT in healthy and constipated rats
Chemotherapy-induced constipation (CIC) is a persistent problem in the treatment of different types of cancer, and is among the primary contributors to dose decreases, delays and inefficient treatment. 33 In the VINB rats, the overall gastrointestinal motor activity analysed by the progression of the test meal through the small-bowel was signicantly (P < 0.05) reduced (65.12 AE 1.66%) compared to those in the healthy animals (70.65 AE 3.63%). However, TV-AE administration at both doses resulted in a signicant (P < 0.05) improvement in gastrointestinal transit in both the healthy (77.92 AE 3.45 and 83.74 AE 2.88%, respectively) and VINB rats (68.90 AE 2.18 and 71.33 AE 3.46%, respectively) ( Table 2). Furthermore, the rats receiving VINB showed several signs, including severe difficulty in evacuating stools, which was observed in the CON model compared with the healthy group. This effect was conrmed by the faecal parameter disorders noticed in the VINB group at day 5, including faecal pellets and water content diminution (48.64 AE 3.21 fecal pellets and 27.66 AE 2.63% water content) in comparison with the control-group (56.23 AE 3.00 fecal pellets and   39.08 AE 3.10% water content) ( Table 3). These alterations were combined with histopathological-injuries, 34,35 which provoked loss of uidity/permeability. These eruptions were accompanied with equilibrium disruption of the absorption/secretion of water, electrolytes and nutrients, which led to diverse variations in the gastrointestinal physiological functions, such as GIT. TV-AE administration removed these disruptions, and thus allowed the construction of the mucosa, as shown by the histological study and the facilitation of intestinal and colonic contents advancement. 36,37 Actually, the exact mechanisms of the action of CIC remain unclear, but are believed to result from the association of intersecting processes, including GI dysmotility, secretory dysfunction, inammation and alterations in GI innervation. 33 TV-AE may contain many bioactive compounds that behave as purgatives/stimulate, which activates the gut musculature by acting on the nerve endings of the colon and causing movements that promote defecation. They also act on the walls of the intestine and increase mineral/liquid production, while lessening the absorption of sodium/chlorine. 38 In addition, interstitial cells of Cajal (ICCs) appear to play crucial roles in the physiological GI motility modulation and act as the pacemaker of gastrointestinal movement. In this respect, we think that TV-AE can depolarize the membrane potentials of ICCs. This excitation may lead to smooth muscle cells via a gap junction. These last cells may respond to this depolarization with the activation of voltagedependent Ca 2+ channels. 39 Therefore, this action of pacemaker potential depolarization may produce intensication of the intestinal motility rate. For this point, many recent research showed that diverse traditional herbal medicines such as Schisandra chinensis, 40 Ge-Gen-Tang, 41 Liriope platyphylla 42 and Citrus unshiu 43 demonstrated elicit excitatory/ inhibitory effects on the ICC pacemaker activity, which supports the notion that ICCs are critical in the control of smooth muscle motility in the GI tract.
Direct assessments of calcium currents in single smooth muscle cells by the whole cell voltage clamp methods in the rat and mouse colons showed a 50-70% decrease in Ca 2+ ion ux aer an inammatory response. Akbarali et al. 44 established that in addition to the down-regulation of Ca 2+ currents, the response to the ATP-sensitive K + channel agonist lemakalim improved in the inamed smooth muscle cells. Likewise, colonic irritation results in contracted contractions of colonic muscle strips in both human/animal models of colitis to depolarizing K + solutions. In particular, recent studies indicated that transient receptor potential vanilloid-4 (TRPV4) activation in the GI tract induces intracellular calcium accumulation, chemokine release enhancement, and colitis reinforcement. In contrast, anti-inammatory compounds signicantly diminished TRPV4expression in the upper-intestine. 45,46

TV-AE consumption facilitates the emptying process in healthy and INDO group
NSAIDs stimulate gastric ulcers via the suppression of prostaglandins, which are cytoprotective constituents to the gastric a Rats were intoxicated with VINB (2 mg kg À1 ) for one week. Then, the animals were treated with two increasing doses of TV-AE or reference molecule (clonidine, 1 mg kg À1 ). 2 h later, the rats were given the standard charcoal meal (10% charcoal in 5% gum arabic) or vehicle (NaCl, 0.9%). Data are expressed as mean AE standard error (n ¼ 7); *P < 0.05 versus control group, # P < 0.05 versus VINB-group. mucosal tissues, specically due to the COX pathway disturbance of arachidonic acid metabolism, resulting in the massive generation of leukotrienes and other factors such as that of the 5-lipoxygenase enzyme metabolic pathway. 47 INDO administration at a dose of 30 mg kg À1 produced acute ulcerative gastritis in the animals. This damage was manifested by an increase in the ulcer index and gastric juice volume and a signicant reduction in pH. These disruptive actions were accompanied with a delayed gastric emptying effect. 17 TV-AE at both concentrations induced a signicant (P < 0.05) acceleration of normal gastric emptying (81.52 AE 2.67% and 89.05 AE 3.44%, respectively) when the results are compared to that found in the healthy animals (70.25 AE 1.45%). In the ulcerative gastritis situation, the acquired data showed that the gastric emptying time analysed by the red phenol method strongly (P < 0.05) decreased in the gastric ulcer situation (58.56 AE 0.78%). In contrast, comparatively with the ulcerative gastritis group, TV-AE pre-treatment attenuated UG and remarkably accelerated (P < 0.05) the gastric emptying time (62.40 AE 0.99% and 69.87 AE 1.66%, respectively) (Fig. 2).
Natural small molecules produced from medicinal plants have been used for a long time to treat and prevent diverse ailments. Recently, the use of natural medicine in the treatment of various pathologies like peptic ulcer is an absolute requirement. Therefore, an alternative advance in recent days is the research of phytomedicaments serving as a tool in the prevention of peptic ulcers. 48 Several phytocomponents such as avonoids, tannins, terpenoids, and saponin have been reported in distinct anti-ulcer ndings as possible gastro-protective agents. Rosmarinic acid is known to be facilely absorbed from the gastrointestinal tract into the blood. It activates prostaglandin E2 generation, diminishes leukotriene B4 production in human leucocytes, and inhibits the complement system. It is also proven that caffeic acid and derivatives such as rosmarinic acid have therapeutic potential in the treatment/prevention of spasmogenic disruptions, peptic ulcers, and inammatory disorders. 49

TV-AE consumption relieves and ameliorates oxidative and antioxidative balance
In both INDO/VINB groups, the obtained results showed an oxidative stress installation, which is related with an enzymatic antioxidant disruption following a massive ROS/free radical generation. These injuries were conrmed by the increase in MDA production and carbonyl protein formation in the UG/UC conditions, and associated with the depletion of enzyme activities, such as SOD, CAT and GPx. However, TV-AE consumption at both different doses (100 and 200 mg kg À1 ) relieved oxidative damage and ameliorated/stimulated the antioxidant prole ( Fig. 3 and 4).
A recent study clearly exhibited the participation of oxidative stress/inammation in gastrointestinal problems such as ulcers and constipation. 50 This contribution was checked by the augmentation of intracellular ROS production, which induces damage to lipids, proteins and DNA and inammatory indicators. Iron catalyzes hydroxyl radical-mediated oxidative injury through its implication in the Fenton pathway, which leads to the reduction of this element in oxidative stress condition. Many authors showed that the phenolic compounds of thyme extract have greater antioxidant activity compared to a-tocophenol and butylated-hydroxylanisole. Rosmarinic acid (RA), the most important component of the thyme extract, has strong antioxidative activity since it acts as a free radical scavenger. 51 Further research suggested other possible mechanisms for the protection of RA against lipid peroxidation, including the scavenging effect of numerous radical species, interaction with peroxyl-radicals and partitioning into the LDL particle and terminating chain reactions of oxidative degradation of lipids by scavenging lipid radicals. 52 An alternative hypothesis is that antioxidant supplements modulate endogenous mechanisms that either decrease ROS production or increase the enzymes activities that decompose ROS. 53

TV-AE consumption improves lipid metabolisms/ intracellular mediators disruptions in intoxicated rats by drugs
To investigate the cell level damage and modications in lipid metabolism, we studied the effect of drugs on lipidemia indicators as well as intracellular mediators. Accordingly, we observed a signicant (P < 0.05) increase in H 2 O 2 production in both eruptions, which was accompanied by a depletion of calcium and free iron. TG, TC and HDL cholesterol were signicantly (P < 0.05) altered in the GU condition. However, the bioactive compounds and mineral constituents in TV-AE resulted in protection against all these disruptions (Table 4). Previous studies demonstrated that RA exhibited substantial H 2 O 2 scavenging action and inhibited H 2 O 2 -induced intracellular ROS generation. 54 The data also showed that the lipid components such as triglyceride, total cholesterol, and HDL cholesterol contents were remarkably different among the major groups (Table 5). A recent study reported that many bioactive components such as gallic acid, catechin, and epicatechin have cholesterol-lowering activities by reducing pancreatic cholesterol esterase activity, binding of bile acids, and inhibiting solubility of cholesterol in micelles, which may result in delayed cholesterol absorption. These avonoids may also reduce cholesterol absorption via interaction with cholesterol carriers and transporters across the brush border membrane in rats. 55 Abdulkarimi et al. 4 showed that thyme extract consumption decreased the plasma triglyceride, total cholesterol, LDL-c and VLDL-c, which in turn lowered the liver and abdominal lipids, and reduced the proportional liver and abdominal fat weights. These actions were exerted by the lower activity of the HMG-CoA reductase enzyme, diminished fat absorption from the gastrointestinal tract or the lipid catabolism for the gluconeogenesis process.

Conclusion
The current study demonstrated that TV-AE represents a source of bioactive functional components and may contribute an improvement in gastrointestinal physiological actions and their associated disorders in rats via various mechanisms. Consequently, this investigation supports the therapeutic potential and strong possibility of TV-AE as a functional food in phytomedicine, which should be further explored in clinical studies.

Financial disclosures
None declared.

Conflicts of interest
Only the authors are responsible for the content of this paper.

Acknowledgements
Financial support of Tunisian Ministry of Higher Education and Scientic Research is gratefully acknowledged. ). Results are expressed as mean AE SEM; n ¼ 7 in each group. Data was analyzed by Statview ANOVA. Data are expressed as mean AE standard error (n ¼ 7); *P < 0.05 versus control group, # P < 0.05 versus VINB/INDO-groups.