Extract of Laurus nobilis attenuates inflammation and epithelial ulcerations in an experimental model of inflammatory bowel disease

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are classified as chronic inflammatory disorders and typically require anti-inflammatory drug therapies, such as glucocorticoid regimens, non-steroidal anti-inflammatory drugs, and biologics, aimed at reducing inflammation in the bowel wall. However, each of these therapies is accompanied by a list of possible serious side effects. Because of this, there remains an urgent need to identify new pharmacologic options to reduce or prevent the pro-inflammatory events of IBD while minimizing adverse side effects and making available more cost-effective treatment modalities. We have previously identified several herbal extracts that demonstrate the potent bio-inhibitory activity of the innate immune response. In particular, Laurus nobilis (LN), more commonly called bay laurel, demonstrated significant anti-inflammatory function by inhibiting nuclear factor-κB activation. Based upon our original in vitro findings, we have now examined the effects of this herbal extract on a murine dextran sodium sulfate (DSS) model of IBD. Hema-toxylin and eosin-stained paraffin sections prepared from DSS-treated animals show clear epithelial damage, including ulcerations, extensive neutrophil infiltration into the mucosal layer, and granuloma formation. Tissue from DSS-treated animals that also received LN extract showed improved tissue morphology more closely resembling that from control animals. In addition, DSS-treated mice with co-administration of LN extract showed a significant reduction in CD4+ antibody staining within the mucosal layer in colonic sections indicating reduced lymphocyte infiltration. Based on these findings, we believe that the administration of LN extracts may be effective in reducing the intestinal epithelial damage seen in human IBD and warrants further investigation through clinical trials. Lay Summary: Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), manifest as chronic inflammation and ulceration of tissues lining the digestive tract. CD involves inflammation of the digestive tract's deeper layers, including the small and large intestines, and less commonly, the upper digestive tract. UC involves inflammation along the lining of the colon and rectum. Steroid or biological treatments for IBD are common, however, are limited due to significant side effects and/or prohibitive costs. In the present study, we provide evidence for use of the natural product, Laurus nobilis (bay leaf), as a safe and effective anti-inflammatory therapy for IBD.


Introduction
Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are idiopathic diseases that affect almost three million individuals in the United States. [1] This figure translates into 1.3% of the adult population and does not include children and adolescents under 18 years. Individuals afflicted with IBD are thought to possess a genetic predisposition towards susceptibility to environmental triggers, such as intestinal flora and specific antigens. [2] IBD is characterized by mucosal immune dysregulation and epithelial barrier dysfunction. Pathological changes include loss of crypts, erosions, ulcerations of the mucosal lining, and substantial leukocyte infiltration. As such, IBD is classified as a chronic inflammatory disorder in which the natural history of the disease involves periods of severe clinical presentation alternating with periods of clinical remission. Consequently, therapeutic strategies for treating IBD must consider both managing acute episodes and maintenance of long-term remission.
Current treatment regimens for IBD include anti-inflammatory drugs aimed at reducing inflammation in the bowel and restoring the normal intestinal lining. This could theoretically be accomplished by administering sufficient doses of anti-inflammatory therapeutics, such as non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids. However, many of these drugs can only be used for short-term treatments, as their long-term use causes gastrointestinal ulcerations and thrombotic events, [3] [4] and in the case of corticosteroids, adrenal insufficiency. [5] Because of this, there is an urgent need to identify alternative therapeutics for use as anti-inflammatory remedies, not only for improved outcomes but also to circumvent the serious side effects caused by current NSAID therapy.
Biologic therapies, [6] such as TNFα inhibiting monoclonal antibodies, [7] [8] are now becoming more widely used in various inflammatory diseases such as rheumatoid arthritis, [9] psoriasis, [10] and Crohn's disease, [11] and are designed to render substantial inflammatory inhibition. For example, infliximab has become a viable alternative for NSAID use, especially for steroid-refractory UC and CD. [12] [13] However, biologic therapies are often disadvantageous due to prohibitive expense because of costly manufacturing, challenging patient delivery methods, and generally pose serious, sometimes lifethreatening side effects. [8] [14] Natural products provide a logical starting base for identifying new compounds with targeted bioactivity because they have already undergone evolutionary selection to have propensity to interact with biological macromolecules, thus presenting as drug-like molecules. In fact, over 60% of current pharmaceuticals are of natural origin. [15] [16] There are numerous examples of immunomodulation by herbal medicines involving direct or indirect modification of the expression of cytokines. [17][18] [19] [20] In a previous study, we examined potential anti-inflammatory properties of 20 herbal preparations. [21] Using a well-studied cell culture model, we identified several herbal extracts that showed potent inhibition of an innate immune response, this being inhibition of nuclear factor-κB (NFκB) activation. Of the positive acting herbs we identified, aqueous extracts of both Rosa de Castillo and Laurus nobilis exhibited the smallest IC50 values toward NFκB inactivation. Moreover, of these two inhibitor extracts, the preparation of Laurus nobilis was the most stable as it remained fully active following repeated freeze-thaw cycles; Rosa de Castillo lost significant activity upon the first freezethaw exposure.
Laurus nobilis (LN), or more commonly called bay laurel, has been used for over 2000 years as an analgesic, antibacterial, and anti-fungal agent, as well as reputed to alleviate arthritis, rheumatism, and rashes, suggesting an anti-inflammatory function. [22] Anti-inflammatory activity was confirmed in essential oil from LN in a formalin-induced oedema rodent model. [23] LN was also shown to possess hypolipidemic properties by lowering plasma triglyceride and cholesterol levels in zebrafish. [24] Oral administration of LN in a 40-person clinical trial improved lipid profiles in individuals with T2DM by reducing LDL-cholesterol by 40% and triacylglycerides by 25%. [25] Khan, et al., [25] showed consumption of Laurel leaves improved both glucose and lipid levels in humans with T2DM. Importantly for future clinical trial consideration, one cohort ingested up to 3 g of powdered LN leaves per day for 30 consecutive days with no reported side effects.
Based upon our original in vitro findings indicating that extracts of LN provide significant anti-inflammatory activities, and because of the inherent stability of these aqueous preparations, we have now examined the effects of this extract on an in vivo model of IBD. [26] We hypothesize that LN, based on its documented anti-inflammatory properties, will attenuate the clinical and pathologic symptoms of IBD in an established animal model. We anticipate that these positive findings will provide us with proof-of-principle evidence to support future clinical trials and future studies to identify the bio-active compound(s) that mediate the anti-inflammatory activities.

Preparation of Laurus nobilis extracts
To maintain consistency between our preparations for direct quantitative comparisons in functional assays, we have designed an aqueous extraction protocol that is the basis of our standardization. Leaves of Laurel nobilis were sources from a local distributor (Rio Grande Herb Company, Albuquerque, New Mexico). Extracts were prepared by preparing powdered herbs using a mortar-pestle combination. Powdered herb (0.5 g) was mixed with ultra-pure water (10 ml, >15 megaΩ resistance) and heated at 85°C for 30 minutes. This aqueous fraction was then filter sterilized and stored at 4°C in the dark until use. Adherence to this standard protocol not only permits accurate quantitative comparisons between herbal extracts, but also affords us the opportunity to compare different harvests to account for seasonal variations. In addition, after freeze-drying and reconstituting the extract, consistent values were obtained in our bioactivity assays [21] , suggesting that the active ingredients in these extracts are stable and that this method is a reliable method for standardization.

Animal treatments
The animal model we used for this study is the dextran sodium sulfate (DSS) model of chemically induced IBD. [27] This model, first introduced in 1990, [28] is widely used to study both acute and chronic colitis. Noteworthy for our studies, this model has been used to evaluate natural products or extracts derived from medicinal herbs for their activities in preventing colitis [29] [30] and also to demonstrate synergy when natural products are combined. [31] Male C57BL/6J mice (8 weeks; Jackson Laboratories) were given drinking water, ad libitum, containing 3.5% dextran sodium sulfate (DSS, 40,000 Da, 3.5% (wt/vol), ICN Biochemicals) for a period of 5-6 days to induce an acute model of IBD. Freshly prepared Laurel extract was administered daily to the experimental group by gavage (200 µL). A volume of 200 μL was chosen because this amount of LN extract demonstrated maximal activity in our previous in vitro assays [21] and allows for an acceptable volume that does not cause unnecessary distress to mice when orally administered. Animals in control group (sham treatment) received drinking water without DSS and administered normal saline solution by gavage in parallel with experimental animals. After 5 days of treatment, animals were humanely sacrificed and colonic tissue removed and processed for histology and immunofluorescence staining. All studies involving the use of animals were reviewed and approved by the University of New Mexico Institutional Animal Care and Use Committee (Protocol no. 07UNM031).

Assessment of mouse weight and colon length
Mouse weight was measured daily from day 0 to 5 at approximately 10 a.m. every day and expressed as the relative change from day 0. Upon sacrifice on day 5, colons were isolated immediately after the last weight check by excision between the ileocecal junction and the distal rectum. The excised colon was placed on a non-absorbent surface and its length was measured with a ruler in such a manner that the organ was not stretched.

Histologic analysis
Tissues were excised and immersion-fixed in 10% buffered formalin, followed by paraffin embedding. Sections (5-10 μm) were prepared, deparaffinized, and stained with hematoxylin followed by counterstaining with eosin. Sections were examined by light microscopy.

Immunofluorescence
Intestinal tissue samples were excised, immersion-fixed in 10% buffered formalin and snap-frozen in liquid nitrogen. Cryosections (10-40 μm) were prepared and sections incubated with anti-CD4+ antibodies (R&D Systems, mouse CD4 monoclonal antibody, MAB554). Bound antibodies were detected using a FITCconjugated species-specific secondary antibody followed by examination and recording using a Zeiss Axioskop fluorescence microscope.

Results
In the present study, animal weights were monitored daily to assess the effects of DSS treatment and if LN extracts provided protection against the DSS-induced pathological course of events. As anticipated, administration of 3.5% DSS resulted in profound weight loss beginning at day 2 (~10%) with continued progression throughout the 5-day course of study (Fig. 1A). By the end of the study, all mice suffered ≥20% total body weight loss. Of the three mice treated with DSS, one was humanely sacrificed on day 4 due to substantial weight loss (~25%) and failure to thrive. By contrast, mice exposed to 3.5% DSS together with daily gavage of 200 μL LN extract demonstrated no or minimal weight loss (Fig. 1B), indicating that LN treatment protected mice from DSS-mediated intestinal damage allowing for proper nutrient absorption.  The colon lengths of all mice were measured at sacrifice day 5. The mean colon length of the DSS group at 47 mm was significantly (p = 0.003) shorter than that of control group with no DSS treatment at 58 mm (Fig. 2). The mean colon lengths of DSS-treated mice that received daily gavage with LN extract was recorded as 54 mm and was not statistically different from the control group (p = 0.285). However, the mean colon length from LN treated mice was statistically improved from mice treated with DSS alone (p = 0.04). These data indicate that LN extract is able to protect against DSSinduced colonic shortening which is a known pathologic feature of DSS-induced colitis. [32][33] The typical histological changes induced by acute DSSinduced colitis include mucin and goblet cell depletion, epithelial erosion and ulceration, and neutrophil infiltration into the lamina propria and submucosal space. [27] [26] To determine if LN extract administration protected against DSS-related histological changes, samples of colonic tissue were selected randomly from animal cohorts and prepared for histologic examination and immunodetection using the immune cell marker, CD4+. Examination of hematoxylin and eosin-stained paraffin sections prepared from control animals demonstrated normal epithelial architecture, including well-formed colonic folds, an abundance of goblet cells, and few inflammatory surveillance cells (Fig. 3A). By contrast, tissue from DSS treated animals show clear epithelial damage, including ulcerations, extensive neutrophil infiltration into the mucosal layer, and granuloma formation (Fig. 3B). Tissue from DSS treated animals that also received LN extract showed improved  Immunofluorescence analysis was next performed using an anti-CD4+ antibody to investigate if LN extract is able to quantitatively attenuate lymphocyte infiltration in DSS treated mice. To this end, cryosections were prepared from frozen tissues and incubated with anti-CD4+ antibodies. As expected, few CD4+ cells were identified in colonic tissue obtained from normal, control mice (Fig. 4A). However, DSS treated mice showed positive reactivity with anti-CD4+ largely within the mucosal layer indicating substantial lymphocyte infiltration resulting from DSS-induced colitis (Fig 4B). DSS treated animals with co-administration of LN extract demonstrated little anti-CD4+ staining indicating that Laurel extract treatment significantly reduced the DSSinduced pro-inflammatory response (Fig. 4C).

Discussion
Arguably, the most widely used mouse model of colitis uses daily administration of dextran sodium sulfate (DSS) to induce acute disease. [27] DSS is a water-soluble, negatively charged sulfated polysaccharide that is typically administered through the drinking water. The mechanism by which DSS induces intestinal inflammation is unclear but likely results from damage to epithelial luminal surfaces lining the large intestine thereby initiating a localized pro-inflammatory response. [34] The severity of colitis can vary based upon the duration of DSS administration, as well as the concentration of DSS used. [35] With DSS administration, pronounced weight loss is expected, along with altered stool consistency and hematochezia. [36] A weight loss greater than 20-30% is typically a significant physiological indicator of animal stress and calls for euthanasia administered according to institutional guidelines in accordance with IACUC recommendations. In this study we have identified that a simple aqueous extract of leaves from commercially available Laurus nobilis provides significant protection against inflammatory damage that occurs during the course of IBD. In the DSS-mouse model, oral treatment with LN extract prevented colonic shortening and epithelial damage, as a result of reducing infiltration of CD4+ immune cells. These data suggest that administration of LN extracts may be effective in reducing the intestinal epithelial damage seen in human IBD and warrants further investigation through clinical trials.
Future studies are aimed at identifying the active component or components in LN that are responsible for its anti-inflammatory activity. It is not yet clear if one component provides this activity or if multiple components are present which act in a synergistic manner for the full anti-inflammatory effect. Laurus nobilis, also known as bay laurel, true laurel, sweet bay, Grecian laurel or bay tree, is an aromatic evergreen tree or large shrub native to the Mediterranean region. Its fruit is a small black berry about 1 cm long containing a single seed. The fruit of Laurel contains up to 30% fatty oils and about 1% essential oils, including terpenes, sesquiterpenes, alcohols and ketones. The leaves contain about 1.3% essential oils, consisting of 45% eucalyptol, 12% terpenes, 3-4% sesquiterpenes, 3% methyleugenol and other α-and β-pinenes, α-phellandrene, linalool, geraniol and terpineol. Eucalyptol has been reported to reduce inflammation and pain in a mouse model of gouty arthritis, [37] as well as tobacco-induced lung inflammation [38] and amyloid beta-induced inflammation in a cell culture model of Alzheimer's disease. [39] Methyleugenol was found to reduce the extent of cerebral ischemic injury by providing an anti-inflammatory protection. [40] Most recently, the monoterpenes, terpinolene and α-phellandrene, were shown to attenuate both inflammation and oxidative stress in an in vitro model of wound healing, [41] suggesting the combined action of these two compounds may offer an important alternative clinical option for the treatment of wounds. Like eucalyptol, linalool [42] [43] and geraniol [44] are both able to reduce pulmonary inflammation. And importantly for our studies and future aims, geraniol was shown to reduce the histological and cytokine inflammatory profile of colitic mice. [45] Based on these collective findings, we believe that LN extracts provide anti-inflammatory activity through a

Additional information Author contributions
Both authors were involved in carrying out experimental procedures. RAO compiled the initial draft of the manuscript and NSC contributed to final editing.