Gardenia jasminoides Ellis Fruit Extracts Attenuated Colitis in 2,4,6-Trinitrobenzenesulfonic Acid-Induced Rats

Ulcerative colitis (UC) is a relapsing inflammatory disease with an unknown precise etiology. The purpose of this study is to investigate the protective effects of Gardenia jasminoides Ellis fruit extracts (GFE) on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. GFE (50 mg/kg, 100 mg/kg) were administered orally for 7 days after induction. Meanwhile, the chemical components of GFE were performed by UPLC-QTOF-MS/MS. GFE significantly decreased DAI scores and ameliorated macroscopic and histologic damage. It also reduced the levels of MPO, NO, MDA, IL-1β, TNF-α, and IL-6, while increasing the level of SOD. Moreover, 56 components were identified in GFE using a UPLC-QTOF-MS/MS method, which can be categorized into six structural groups. Our results indicated that GFE has an ameliorative effect on TNBS-induced colitis in rats, which may further verify its anti-inflammatory and antioxidative properties. Therefore, GFE can be a promising protective agent of colitis that deserves further investigation.


Introduction
Ulcerative colitis (UC) is a relapsing inflammatory disease that affects the mucosal layer of the rectum and colon. e symptoms of UC include chronic diarrhea, stomach pain, blood in stool, and weight loss in the clinic [1,2]. It was found that UC occurs more and more commonly and worldwide in recent years, and the long-term abdominal discomfort already lowers the life quality of patients. Meanwhile, UC is widely accepted as a risk factor for colorectal cancer according to the clinical data [3]. Hence, World Health Organization lists UC as a miscellaneous problem.
Despite the precise cause of UC has not yet been completely elucidated, it is wildly considered that proinflammatory cytokines such as interleukin-1β (1L-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (1L-6) play an essential role in the process of the pathogenesis and progression of UC [4,5]. Current treatments of UC mainly rely on anti-inflammatory medicines such as aminosalicylates and immunosuppressive and corticosteroid agents. However, most of these drugs have lots of unacceptable adverse effects, such as poor tolerance and high recurrence rate, which are not quite suitable for long-term treatment [6]. erefore, it is still urgent to search new safe and effective agents for adjunct therapies.
Recently, more and more studies have focused on natural products as alternative or complimentary medicines for UC treatment. A series of natural products had been confirmed to be able to treat UC through anti-inflammatory actions, such as Forsythia suspensa Fructus, Eucommia ulmoides leaf, and brusatol, which can potentially enlarge the scope of drug candidates for UC treatment as well [7][8][9].
Gardenia jasminoides Ellis is a flowering plant belonging to Rubiaceae family. Its fruit has been used as food coloring in oriental countries [10]. As a natural plant, the fruit is used not only as a food but also as a medicine to treat hepatic disorders, headache, jaundice, inflammation, and hypertension [11][12][13]. Moreover, many bioactive ingredients from Gardenia jasminoides Ellis fruit extracts (GFE) such as iridoids, crocins, and organic acids have also shown anti-inflammatory effects. Specifically, geniposide and chlorogenic acid have shown good protective effects in rat colitis models [14,15]. Taken together, GFE has potential for the treatment of UC. erefore, the present study is to investigate whether GFE could have a protective effect in the rat colitis model induced by trinitrobenzene sulphonic acid (TNBS) and its mechanisms associated and to identify its bioactive ingredients by UPLC-QTOF-MS/MS. nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) assay kits were obtained from Beyotime Institute of Biotechnology, China. e interleukin-1β (IL-1β), necrosis factor-α (TNF-α), and interleukin-6 (IL-6) ELISA kits were obtained from MultiSciences (Lianke) Biotech, China. Acetonitrile, methanol, and formic acid were all of HPLC grade and were purchased from Merck, Germany. Ultrapure water was prepared by a Milli-Q system (Millipore, Bedford, USA).

Preparation of GFE.
e fruits (1500°g) were crushed, extracted with 70% aqueous ethanol for 2 hours under reflux, and extracted twice. e extract was concentrated by rotary evaporator and dried with lyophilizer. e yield of the extract was 21.39% (w/w). e dried extract was kept at −20°C.

Animals and Treatment.
Healthy male Sprague-Dawley (SD) rats (180-220 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China; Certificate of Conformity: SCXK2013-0004). Animals were housed in polypropylene cages at 22 ± 2°C, under 12 h day/night cycles. Rats were allowed free access to food and water. All procedures and experimental protocols were approved by Institutional Animals Ethics Committee of Jiangxi University of Traditional Chinese Medicine (certificate number: 201901112) and were carried out according to international standards and ethical guidelines on animal welfare.
Rat model of colitis was produced following the method described previously [16]. A catheter was inserted into the rat colon and then TNBS-ethanol solution (50%, v/v) was slowly administered at a dose of 0.5 mL/kg though the catheter. e control group of rats was treated with 50% ethanol solution. All rats were kept in a vertical position for 30 s to prevent from leaking out before returning to their cage. Rats were distributed into five groups of seven rats each: control group, TNBS group, SASP group (500 mg/kg), and GFE treated groups (50 mg/kg, 100 mg/kg). e TNBS and control groups were administered orally with 0.9% saline at the same volume. All drugs were administered orally for 7 consecutive days after induction. Rats were sacrificed under anesthesia to collect specimens on the 8th day.

Disease Activity Index Evaluation.
e rats' body weight, stool occult, and stool formation were recorded daily to calculate disease activity index (DAI) after modeling (Day 2). DAI was determined according to the previously scoring system [17].

Macroscopic Scoring of Colitis.
After sacrificing the rats, the colons were removed and then flushed with ice-cold normal saline for macroscopic observation. e macroscopic scores were determined according to the criteria described in previous studies with a total score of 4 [18] ( Table.1).

Histological Assessment of Colitis.
After the macroscopic assessment, about 0.5 × 0.5 cm 2 sections of colon tissues were fixed in 4% formalin, embedded in paraffin, and then stained with hematoxylin and eosin (H&E). e histopathological scores were assessed by the previous standard with a total score of 5 [19]. e other sections from the clean colon lesion were kept at −80°C for further analysis.

Measurement of MPO, SOD, MDA, NO, and Proinflammatory Cytokine in Colonic Tissues.
e activities of MPO, SOD, MDA, and NO were detected with the corresponding test kits for the supernatants of the colonic tissues. e levels of TNF-α, IL-1β, and IL-6 of the colonic tissues were measured using ELISA assay kits.

Statistical Analysis.
Results were presented as mean ± SD (standard deviation) using SPSS 20.0 software. Statistical differences were carried out by one-way ANOVA followed by Dunnett's test. P values < 0.05 were considered statistically significant.

UPLC-ESI-Q-TOF-MS/MS Profiles of GFE.
e components of GFE were analysed by UPLC-ESI-Q-TOF/MS. A total of 56 components were identified based on literatures and the detailed mass spectrometry data, while Table 2 shows the retention times, proposed formula, MS and fragments, and the identification results for the peaks labeled in the total ion flow chromatogram of GFE ( Figure 1) [20,21]. e components were mainly categorized into six structural groups, including iridoids, triterpenes, flavonoids, carotenoids, organic acids, and monoterpenoids.

Effects of GFE on DAI.
DAI score is a notable index to assess the severity of colitis. As shown in Figure 2, compared with the control group, the DAI score was significantly increased in TNBS group (p < 0.01) from Day 1, and treatment with GFE (50 mg/kg, 100 mg/kg) caused a significant decrease in the DAI score compared to the TNBS group at Day 3 and Day 4, respectively.

Macroscopic Changes.
Macroscopic changes are major indicators to evaluate the severity of colitis observed in rats administered with TNBS. In this study, macroscopic inspection of the colons displayed the presence of severe ulceration, edema, and tissue necrosis in TNBS group. In contrast, SASP and GFE (50 mg/kg, 100 mg/kg) treatment caused a remarkable loss in the macroscopic lesion in colonic tissue (Figure 3(a)). e corresponding macroscopic scores were also presented in Figure 3(b). e macroscopic score of TNBS group was dramatically increased compared with the control group (3.50 ± 0.55 vs. 0.29 ± 0.49, p < 0.01).
is indicated that the model was successfully established. Treatment with SASP or GFE (50 mg/kg, 100 mg/kg) significantly decreased macroscopic sores compared with TNBS group.

Histological Changes.
e histological study of colonic tissue showed inflammatory cell infiltration in the mucosa, loss of goblet cells, and epithelium and deformed or disappeared crypt in the TNBS group. e histologic sections of GFE (50 mg/kg, 100 mg/kg) or SASP groups showed moderate goblet cell loss, improvement of the crypt architecture, and mild inflammatory cell infiltration compared to those of the TNBS group (Figure 4(a)). e histological scores of GFE (50 mg/kg, 100 mg/kg) or SASP were also significantly decreased compared to the TNBS group (Figure 4(b)).

Histopathological scoring
Microscopic colon damage 0 Normal colonic tissue 1 Inflammation or focal ulceration limited to the mucosa 2 Focal or extensive ulceration and inflammation limited to the mucosa and the submucosa 3 Focal or extensive ulceration and inflammation limited with involvement of the muscularis propria 4 Focal or extensive ulceration and inflammation limited with involvement of the serosa 5 Extensive ulceration and transmural inflammation with involvement of the serosa  Figure 5(d)).

Discussion
UC is a chronic inflammatory bowel disease which affects a million people around the world. Current treatment of UC is generally anti-inflammatory or immunosuppressive agents. However, most of these agents have many side effects and are proved to be inadequate for treatment. us, more and more natural products, especially medicinal plants, are considered as alternative treatments. GFE has been considered to have anti-inflammatory effect in previous studies. erefore, we focused on the effect of GFE on TNBS-induced colitis in rats in the current study. TNBS-induced colitis model is one of the most appropriate models similar to human colitis in previous studies [1]. On this estimated model, DAI, macroscopic and histological changes were assessed in this study. GFE could reduce the DAI score and attenuate the macroscopic damage significantly induced by TNBS. e results of histological evaluation were consistent with macroscopic data, indicating that GFE treatment dramatically attenuated the destruction of the colonic tissue and its related inflammatory changes induced by TNBS. All these results above demonstrated that GFE had a noticeably protective effect against TNBS-induced colitis in rats. is efficacy was further proved by the investigation of biochemical and inflammatory biomarkers in colonic tissues.
It is known that reactive oxygen species (ROS) are involved in the initiation and progression of ulcerative colitis. Under normally physiological conditions, amounts of antioxidant systems control ROS. Nevertheless, excessive ROS released in inflammatory colitis lesions induce oxidative stress [22,23]. MDA is an important oxidative stress indicator which is produced by lipid peroxidation. It was documented that the tissue level of MDA was increased in TNBS-induced colitis, which can also be observed in our study [24]. Meanwhile, the intestinal cells possess more efficient antioxidant defense to reduce the harmful effects of oxidative stress. SOD is a crucial antioxidant enzyme which could reduce oxidative damage and scavenge free radicals. Several studies also showed that the colonic SOD level was decreased in the colitis model [25,26]. e change trends of colonic MDA and SOD levels in model rats were consistent with previous literatures, which indicated that the colitis model was established in our experiments successfully. GFE administrations (50 mg/kg, 100 mg/kg) significantly decreased the concentration of MDA and increased the activity of SOD. ese findings indicated that amelioration of colon damage with GFE at least partly was related to the reduction of oxidative stress.
NO is a free gaseous signaling molecule that is synthesized by inducible nitric oxide synthase. Previous studies have shown that overproduction of NO leads to colonic mucosal damage and implicates in the pathogenesis of colitis [27,28]. Hence, NO is an important indicator in inflammatory bowel disease. Meanwhile, the activity of MPO is also an important Evidence-Based Complementary and Alternative Medicine biomarker of neutrophils infiltration into the inflamed colonic tissue, which is commonly used to evaluate the severity degree of colonic inflammation in kinds of experimental studies and clinical trials [29,30]. In this study, the colonic MPO and NO levels were increased in TNBS group, revealing that consequent inflammation and recruitment occurred in rats induced by TNBS. Treatment with GFE showed remarkably decreased levels of MPO and NO in colonic tissue, thus indicating the effect of GFE treatment was associated with the anti-inflammatory property. Proinflammatory cytokines such as TNF-α, 1L-1β, and 1L-6 have essential roles in the pathological development of UC [31]. As previous studies demonstrated, the proinflammatory cytokine levels were increased in UC patients and TNBS-induced colitis rats model [32]. TNF-α could recruit leukocytes from the inflamed area and stimulate the expression of cytokines which is a key target molecule of taking part in intestinal inflammation [33,34], and 1L-1β and 1L-6 are also important indicators in the progression of UC. IL-1β could stimulate the recruitment of neutrophils to the inflamed colonic tissue [35]. IL-6 plays an important role in enhancing T-cell survival and apoptosis resistance in the lamina propria at the inflamed site [36,37]. erefore, targeting proinflammatory cytokines had become a therapeutic approach for UC patients. In the current study, treatment with GFE significantly decreased the colonic TNFα, 1L-1β, and 1L-6 levels. ese findings also indicated that GFE may influence the production of cytokines and inhibit immune inflammation.
Furthermore, this study explored the effective components in GFE by UPLC-ESI-Q-TOF/MS. 56 compounds of six main structural types in GFE, including iridoids, flavonoids,     Evidence-Based Complementary and Alternative Medicine triterpenes, monoterpenoids, carotenoids, and phenolic acids were identified based on UPLC-ESI-QTOF/MS analysis. According to previous studies, iridoids-type compound (e.g., geniposide and genipin), carotenoids-type compound (e.g., crocin), and phenolic acid-type compound (e.g., chlorogenic acid and gallic acid) have been demonstrated to exhibit good antioxidant activity and anti-inflammatory activity [38][39][40][41][42]. Geniposide and chlorogenic acid had even been proved to treat UC in animal models through inflammatory pathways. Hence, the effect of the type of iridoids, carotenoids, and phenolic acid  compounds from GFE on ulcerative colitis will be investigated in our further research.

Conclusion
Based on these findings of the current study, treatment with GFE could ameliorate TNBS-induced colitis in rats, and this protective effect of GFE may be mediated through its antiinflammatory and antioxidative properties. GFE could be a potential agent for colitis that deserves further investigation.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.