Rafeeq et al. BMC Complementary Medicine and Therapies (2021) 21:28 BMC Complementary https://doi.org/10.1186/s12906-021-03203-7 Medicine and Therapies RESEARCH ARTICLE Open Access Protective effect of 6-paradol in acetic acid- induced ulcerative colitis in rats Misbahuddin Rafeeq1* , Hussam Aly Sayed Murad1,2, Hossam Mohammed Abdallah3,4 and Ali M. El-Halawany3,4 Abstract Background: Ulcerative colitis is a gut inflammatory disorder due to altered immune response to gut microbiome, with interplay of environmental and genetic factors. TNF-α activates inflammatory response through a cascade of immune responses, augmenting pro-inflammatory mediators and proteases, activating chemotaxis, and infiltration of inflammatory cells, leading to ulceration and haemorrhage through cytotoxic reactive oxygen species. 6-Paradol, a dietary component in several plants belonging to the Zingiberaceae family, has shown anti-inflammatory and antioxidant activities. Current study evaluates the effect of 6-paradol in amelioration of ulcerative colitis in rats for the first time. Methods: 6-Paradol (95% purity) was obtained from seeds of Aframomum melegueta. Rats were divided randomly into six groups (n = 8). Group one was administered normal saline; group two was treated with the vehicle only; group three, sulfasalazine 500 mg/kg; and groups four, five, and six, were given 6-paradol (50, 100, 200, respectively) mg/kg orally through gastric gavage for 7 days. Colitis was induced on 4th day by intrarectal administration of 2 ml acetic acid (3%), approximately 3 cm from anal verge. On 8th day, rats were sacrificed, and distal one-third of the colon extending proximally up to 4 cm from anal orifice was taken for biochemical and gross examination. Two centimetres of injured mucosal portion was taken for histopathological investigations. SPSS (ver.26) was used for statistical analysis. Results: Colonic and serum glutathione (GSH) levels decreased, while colonic and serum malondialdehyde (MDA), colonic myeloperoxidase (MPO) activity, serum interleukin-6 (IL-6), serum tumour necrosis factor-α (TNF-α) levels, and colon weight to length ratio were increased significantly in the colitis untreated group compared to normal control. Treatment with 6-paradol considerably improved all these parameters, especially at a dose of 200 mg/kg (p < 0.001), revealing non-significant differences with sulfasalazine 500 mg/kg and normal control (p = 0.998). Sulfasalazine and 6-paradol in a dose dependent manner also markedly reversed mucosal oedema, atrophy and inflammation, cryptic damage, haemorrhage, and ulceration. There were non-significant differences between low and medium doses and between medium and high doses of 6-paradol for IL-6 and serum MDA levels. Conclusion: 6-Paradol demonstrated protection against acetic acid-induced ulcerative colitis, probably by anti- inflammatory and antioxidant actions. Keywords: 6-paradol, Spices, Ginger, Grain of paradise, Gut, IBD, Antioxidant * Correspondence: [email protected] 1Department of Pharmacology, Faculty of Medicine, King Abdulaziz University (KAU), Rabigh Campus, Jeddah 21589, Saudi Arabia Full list of author information is available at the end of the article © The Author(s). 2021, corrected publication 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Rafeeq et al. BMC Complementary Medicine and Therapies (2021) 21:28 Page 2 of 10 Background in the prevention and treatment of gut inflammatory Inflammatory bowel disease (IBD) is an idiopathic, disorders [13, 14]. Anti-inflammatory, antioxidant, chronic, and relapsing gut inflammatory disorder specu- gastroprotective, immunomodulatory, antinociceptive, lated to be caused by an altered immune response to the antimicrobial, antidiabetic, and anticancer activities gut microbiome. In addition, environmental and genetic have been reported from the extract of A. melegueta factors also play roles in its etiopathogenesis [1]. Two and its active components [15–17]. In addition, previ- major types of IBD include Crohn’s disease (CD) and ous reports also advocate anticancer, anti-diabetic, ulcerative colitis (UC). In UC, there is localized superfi- antioxidant, anti-inflammatory, and immunomodula- cial inflammation limited to the colon and rectum, while tory activities of 6-paradol, which is one of the major in CD, the whole intestine is involved with ulceration, constituents of grains of paradise [18–20]. Effects of haemorrhage and oedema. Moreover, the involvement is other individual active constituents from the Zingiber- transmural, meaning that all layers of the intestinal wall aceae family, such as such as gingerols, shogaols and are involved [2]. gingerdione, on ulcerative colitis and other inflamma- Tumour necrosis factor-α (TNF-α)alongwithIL-2and tory models have been investigated; however, no study INF-γ, are from T-helper1 (Th1) cells. A preponderance of has been conducted with paradol. Hence, the present these factors over mediators from T-helper2 (Th2) cells, study was designed to explore the protective effect such as IL-4, IL-5, and IL-6, results in the development and of 6-paradol in acetic acid-induced ulcerative colitis progression of allergic and autoimmune disease [3]. TNF-α in rats. plays a prudent role in the development of UC by augment- ing the inflammatory response through activation of a cas- Methods cade of immune responses [4]. TNF-α also activates the Plant material production of other chemical mediators, proteases, and A. melegueta seeds were procured from Harraz herbal pro-inflammatory markers, activating chemotaxis and infil- store, Cairo, Egypt, in 2018, and were identified by Dr. tration of inflammatory cells, leading to ulceration and Sherif El-Khanagry, Agriculture Museum, Dokki, Cairo. haemorrhage, by generation of cytotoxic reactive oxygen A specimen (voucher ID: AM-1039) was deposited in species [5, 6]. the herbarium of the Department of Pharmacognosy, The main symptoms include abdominal pain with Faculty of Pharmacy, King Abdulaziz University. cramping, diarrhoea, dysentery, constipation, fatigue and fever. The diseases are important causes of increased Extraction and isolation of 6-paradol from the seeds of healthcare expenditure and socioeconomic burden, as Aframomum melegueta the Quality Adjusted Life Years (QALYs) increase due to 6-Paradol was isolated as described briefly in our frequent relapses and exacerbations. The highest preva- previous report; pulverized seeds of A. melegueta lence of IBD is found in the US, Canada and Western (1.0 kg) were extracted using methanol by maceration Europe, with over 1 million in the US and 2.5 million in at room temperature until exhaustion. The combined Europe. Nevertheless, increasing numbers of patients are methanol extracts were evaporated under vacuum, also being diagnosed in the Middle East and Saudi yielding an oily brownish residue (TME, 50 g). TME Arabia [7, 8]. The therapy revolves around anti- was suspended in water (300 mL), followed by parti- inflammatory and immune-modulatory drugs, such as tioning with methylene chloride (500 mL X 3) to sulfasalazine, corticosteroids, methotrexate, and inflixi- yield, after evaporation, a CH2Cl2-soluble fraction mab (anti-TNF-α). Often, antibiotics and probiotics are (30 g). The remaining aqueous layer was freeze-dried also used for infections and abscess, healing of fistulas, and kept for further investigations. The CH2Cl2 frac- and resetting of the gut microbiome [9, 10]. tion was applied to a silica gel column (50 X 4 cm) Natural products have long been used for alleviating and gradient-eluted using n-hexane-EtOAc (5 until diseases and are considered as the major source of 80% v/v). One hundred millilitre fractions were col- most of the discovered medicines [11, 12]. Conse- lected and pooled based on TLC investigation into quently, searching for evidence for the use of herbal 10 sub-fractions (F1-F10). F1 (6 g) was chromato- medicines (especially those with historic use in the graphed on a silica gel column (40 X 4 cm) eluted treatment of GI disorders) in the treatment of IBD is with hexane-EtOAc (9.5:0.5 v/v) to obtain pure para- worthy and beneficial. 6-Paradol is a major phenolic dol (5 g). The identity of the isolated compound was dietary component that occurs in several plants confirmed by 1H and 13C NMR analysis recorded belonging to the Zingiberaceae family, such as on Bruker DRX-850 MHz Ultrashield spectrometers Zingiber officinale (ginger) and Aframomum melegueta (Bruker BioSpin, Billerica, MA, USA) using CDCl3 as (alligator pepper or