Background
Withania somnifera (Dunal), also known as Ashwagandha, is an important member of family Solanaceae, utilized as a medicine for more than 2500 years in Indian medicinal classic "Ayurveda" [
1]. Roots of this plant are considered most active for therapeutic purposes by virtue of significant accumulation of active constituents, withanolides [
2]. Several withanolides with cyclooxygenase inhibitory and lipid peroxidation inhibitory activity were isolated from leaves and fruits of
W. somnifera [
3,
4], besides anti-inflammatory [
5], anti-tumor [
6] and antioxidant [
7] activities. Immunomodulatory role of
W. somnifera roots and anti-inflammatory activity using adjuvant-induced arthritic rat models was also demonstrated [
8,
9]. Luvone et al [
10] had observed that methanolic extracts of
W. somnifera roots turns-on the synthesis of inducible nitric oxide synthase expression by acting at transcriptional level resulting in increased production of NO by macrophage, which was attributed to immunomodulatory and anti-inflammatory activity. Considering the various biological activities, roots of
W. somnifera can potentially be utilized for the effective treatment of various inflammatory conditions.
Inflammatory bowel disease (IBD) encompasses (i) various conditions that result in chronic inflammation of small and/or large intestine, alike in young, adults and old, (ii) Ulcerative Colitis (UC), characterized by chronic mucosal and sub-mucosal inflammation of large intestine and rectum and (iii) Crohn's Disease (CD), which is chronic transmural inflammation of all/any part of the gastro-intestinal tract involving mucosa, sub-mucosa, muscular and connective tissue [
11]. However, IBD, UC and CD are considered together because of similarities in their characteristics, pathology, complications, investigations, and treatment [
12].
Etiology of IBD largely remains unknown [
13]; however, development of tissue injury is attributed largely to distorted immune system and reactive oxygen species (ROS) [
14]. Sustained production of reactive oxygen and nitrogen species is due to dysfunction of immune system is believed to play an important role in the development of intestinal colon injury [
15]. It is therefore, logical to treat this disorder by controlling oxidative stress. Logically, antioxidant chemicals have been the main therapeutic strategy in IBD for last 50 years [
16]. The popular treatment of IBD involves use of (a) 5-aminosalicylate based compounds, which are potent ROS scavengers [
17], (b) broad spectrum antibiotics, (c) steroids and immuno-suppressants. These drugs are typically associated with side effects like nausea, anorexia, dizziness, headache, cytopenia, fever, myalgia, abnormal liver function, pleropericarditis, renal insufficiency and pulmonary toxicity. Plants are considered to be a goldmine to tackle complex inflammatory conditions with minimum of side effects. The effective herbs with antioxidant activity have been recently reviewed [
18]. Numbers of reports are available on anti-inflammatory activities of plant-derived crude extracts or compounds derived from different plants. For example, curcumin has been widely investigated for the treatment of experimental colitis to decrease inflammation in therapeutic dosages [
19‐
22]. Similarly, formulation containing a mixture of four herbal drugs (
Aegle marmeloes,
Coriandrum sativam,
Cyperus rotundus and
Vetiveria zinzaniods) showed significant inhibitory activity comparable with the standard drug prednisolone [
12] against IBD induced in experimental animal models. It has become, therefore, imperative to search for safer alternative strategies that modulate the entire inflammatory pathway.
Withania somnifera is a unique plant where a wide range of activities has been demonstrated including antagonism with several pro-inflammatory factors and immunomdulation. In Ayurveda, enema of
W. somnifera water extract is prescribed for intestinal ulcers, irritable bowel and rectal bleeding. All these attributes encouraged us to investigate its usefulness in the treatment of IBD [
23]. Ours is the first report where aqueous extract of
W. somnifera roots was assessed in Trinitro Benezyl Sulfonic Acid (TNBS) induced IBD in rat model, using formulation designed for rectal application. Mucosal adhesion of the formulation facilitated precise application of the extract on intestinal lesions.
Discussion
IBD is a common gastro-intestinal disorder marked with chronic inflammation of intestinal epithelium, damaging mucosal tissue and manifests into several intestinal and extra-intestinal symptoms, mainly related to oxidative stress, inflammation and autoimmune type.
TNBS-induced model is an experimental model of Th 1, like gut inflammation, mimics human Crohn's Disease and is widely used to investigate IBD [
28]. TNBS enema develops hapten-induced delayed type hypersensitivity and results in the development of chronic colitis involving granuloma with infiltration of inflammatory cells in all layers of the intestine. This is due to TNBS-induced over expression of IL-12, IFN-γ and IL-2, which supports the study that inducible colitis is due to Th-type 1 response [
31]. Ilan et al. [
32] studied the involvement of immune system in the pathogenesis of IBD after observing the similarities between human disease and TNBS-induced colitis. Their study revealed that IBD could be considered as an imbalance between pro-inflammatory and anti-inflammatory mediators. Monocytes/macrophages, polymorphonuclear leucocytes (PMNs) and endothelial cells are mainly involved in inflammatory response and their activation forces them to aggregate and infiltrate the tissue, where they undergo respiratory burst, which increases their oxygen use resulting into oxidative damage to the tissue and triggers the production of pro-inflammatory cytokines, ROS and other mediators of inflammation [
33].
Initiation and perpetuation of inflammatory cascade by ROS causes subsequent tissue damage through the activation of nuclear factor kappa B (NF-kB), which is a ubiquitous transcription factor involved in the regulation of several genes in immune and inflammatory responses [
34]. Oxidants are potent activators of NF-kB while the groups of structurally diverse anti-oxidants of herbal origin are capable of inhibiting NF-kB activation [
35]. Human body has its own anti-oxidant defense system, which involves enzymes such as superoxide dismutases, catalases and glutathione peroxidases. However, this enzymatic anti-oxidant defense system is often not sufficient, leading to increased free radicals and oxidative damage ultimately resulting in severe inflammation and cell death [
36].
The gastro-intestinal tract is a major site of generation of pro-oxidants, whose production is primarily due to the presence of plethora of microbes, food ingredients and interaction between immune cells. Not surprisingly, ROS has been implicated in the initiation and perpetuation of inflammatory disorders and pathogenesis of IBD. In one study, saliva of IBD patients were studied as an indicator of existence of oxidative and nitrosative stress. Excessive NO as well as higher levels of epidermal growth factor were found associated with the disease [
37]. Similarly increased ROS in colon mucosa of UC patients was demonstrated [
38,
39] and animal model exhibits increased oxidation and lipid peroxidation during initiation of colitis [
40].
W. somnifera extract seems to be a balanced combination of biologically active ingredients as demonstrated by several workers. The cyto-protective and anti-inflammatory activity has protected experimental animals against the induced diseases. Antibacterial activity demonstrated by
W. somnifera may also have significant role to play in IBD, especially for combating against the intestinal opportunistic pathogens known to play an important role in pathogenesis of IBD. Carminative and anti-diarrheal activity of
W. somnifera may also be useful in restoring the disturbed gastro-intestinal mobility. Anti-oxidant activity is thought to play a central role in preventing inflammation as well restoration of mucosal lining. In the present study, aqueous extracts of
W. somnifera roots have shown an excellent anti-oxidant activity, which is in total agreement with study carried out by Bhatnagar et al [
41].
W. somnifera exhibits inhibition of (a) cyclooxygenase (b) activation of NF-kB induced inflammatory markers (like tumor necrosis factor alpha (TNF-αalpha) and interleukins) [
42]. The withanolides constituting active ingredients of
W. somnifera roots shows promising antibacterial, anti tumor, immunomodulating and anti-inflammatory properties [
9].
Anti-ulcer activity of methanolic extract of
W. somnifera and its action against stress pyloric ligation induced gastric ulcer in rats has been reported [
41]. Treatment with
W. somnifera extract (100 mg/kg/day p.o.) for 15 days, significantly reduced volume of gastric secretion, free total acidity and ulcer index as compared to control group. Significant increase in total carbohydrates (TC) and its ratio to total protein (TP) was also observed, without significant change in total proteins. A significant increase in antioxidant enzymes (viz. catalase, superoxide dismutase (SOD)) and decrease in malondialdihyde (MDA) was observed. Increased MDA levels after TNBS enema decreased upon treatment with
W. somnifera formulation (Table
2).
W. somnifera extract was found effective antiulcerogenic agent, comparable of ranitidine hydrochloride [
41]. Treatment with
W. somnifera formulation has shown decrease in macroscopic scores for the IBD. Histopathology examination of
W. somnifera extract treated group revealed less damage compared to healthy animals (control group). Mesalamine treatment compared to
W. somnifera formulation has shown significant and comparable protection in the rats in our study as revealed by the decreased colon weight and better gain in body weight. In the light of above properties, WSREF can potentially cure local inflammation, modulate immune system and can be a logical choice in relieving IBD symptoms. Administration of Iranian folk herbal medicines
Ziziphora clinopoides and
Teucrium persicum were reported to boost body's antioxidant mechanism such as SOD and catalase, with concomitant decrease in pro-inflammatory factors like TNF-αalpha, IL-1β. These studies suggest potential dexterity of folk medicine in management of disease like IBD where free radicals and inflammation is the major pathophysiology [
43,
44].
Success of herbal extracts in repairing tissue damage in experimental colitis could be enhanced by changing the route of administration. Several plant extracts have been reported beneficial in IBD, however, to our knowledge there is no report on a rectally applicable formulation. The pluronic rectal gel formulation impregnated with WSRE is in liquid form that is easy to apply as enema. At body temperature, it turns into a gel, which covers the rectum surface and due to its mucoadhesive property it forms a layer that persist and slowly release WSRE at the lesions in the rectum. This feature enhanced the beneficial effects of WSRE and resulted in reduced inflammation, faster healing and mucorestoration as revealed by the microscopic observations. Not all the extracts with good antioxidant activity could be beneficial in IBD. Harputluoglu et al [
16] investigated oral application of
Gingko biloba extract in acetic acid-induced colitis. Despite of an excellent antioxidant activity and antagonistic activity against platelets activating factor, (known to play a key role in pathogenesis of IBD), the extract failed to show noticeable recovery in experimental colitis. Based on the present data, topical application of aqueous extract of
W. somnifera roots is strongly recommended in the treatment of distal colitis, especially for an early recovery in the damaged tissue. This data also substantiate the traditional way of treating colitis patients with enema of
Ashwagandha extract. Detailed molecular study to identify the precise nature of active molecules will further assist in understanding their mechanism of action.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PP, SG, SS and SJ carried out the study, designed experimental work, data collection and analysis. PP, SG and AH supervised the work and prepared the draft of manuscript. AP, KM and PK are involved evaluation of the data and corrected the manuscript. All the authors read and approved the final manuscript.