WikiJournal Preprints/Extract of Laurus nobilis attenuates inflammation and epithelial ulcerations in an experimental model of inflammatory bowel disease
Natalie S. Correa; Robert A. Orlando, "Extract of Laurus nobilis attenuates inflammation and epithelial ulcerations in an experimental model of inflammatory bowel disease", WikiJournal Preprints, Wikidata Q102344224
Introduction[edit | edit source]
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. 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 (REF). IBD is characterized by mucosal immune dysregulation and epithelial barrier dysfunction. Pathological changes include loss of crypts, erosions and ulcerations of the mucosal lining, and substantial leukocyte infiltration. As such, IBD are classified as chronic inflammatory disorders in which the natural history of the diseases 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, and in the case of corticosteroids, adrenal insufficiency. 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, such as TNFα inhibiting monoclonal antibodies, are now becoming more widely used in various inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn's disease, 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. However, biologic therapies are often disadvantageous due to prohibitive expense because of costly manufacturing, challenging patient delivery methods, and generally pose serious, sometimes life-threatening side effects.
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. There are numerous examples of immunomodulation by herbal medicines involving direct or indirect modification of the expression of cytokines. In a previous study, we examined potential anti-inflammatory properties of 20 herbal preparations. 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 freeze-thaw exposure.
Laurus nobilis (LN), or more commonly called Bay Laurel, has been used for over 2000 years as an analgesic, anti-bacterial, and anti-fungal agent, as well as reputed to alleviate arthritis, rheumatism, and rashes, suggesting an anti-inflammatory function. Anti-inflammatory activity was confirmed in essential oil from LN in a formalin-induced edema rodent model. LN was also shown to possess hypolipidemic properties by lowering plasma triglyceride and cholesterol levels in zebrafish. 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%. Khan, et al., 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). 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.
Materials and Methods[edit | edit source]
Standardized preparation of herbal tea extracts[edit | edit source]
In order 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, suggesting that the active ingredients in these extracts are stable and that this method is a reliable method for standardization.
Animal treatments[edit | edit source]
The animal model we used for this study is the dextran sodium sulfate (DSS) model of chemically induced IBD. This model, first introduced in 1990, 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 and also to demonstrate synergy when natural products are combined. 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. Laurel extract was administered daily to the experimental group by gavage (200 µL). 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[edit | edit source]
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[edit | edit source]
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 and scored as described in the following table.
Immunofluorescence[edit | edit source]
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 FITC-conjugated species specific secondary antibody followed by examination and recording using a Zeiss Axioskop fluorescence microscope.
Results[edit | edit source]
Arguably, the most widely used mouse model of colitis uses daily administration of dextran sodium sulfate (DSS) to induce acute disease. 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. The severity of colitis can vary based upon the duration of DSS administration, as well as the concentration of DSS used. With DSS administration, pronounced weight loss is expected, along with altered stool consistency and hematochezia. 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 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 uL 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 DSS-induced colonic shortening which is a known pathologic feature of DSS-induced colitis.
The typical histological changes induced by acute DSS-induced colitis include mucin and goblet cell depletion, epithelial erosion and ulceration, and neutrophil infiltration into the lamina propria and submucosal space. 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 villi, 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 tissue morphology resembling that from control animals with much reduced neutrophil infiltration and an abundance of normal epithelial and goblet cells (Fig. 3C).
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, cyrosections 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 DSS-induced pro-inflammatory response (Fig. 4C).
Figure 2. Laurel extract treatment protects against colonic shortening in DSS-treated mice. Wild type mice (C57BL/6J) were given free access to distilled drinking water (no tx, n=3) or distilled water containing 3.5% dextran sodium sulfate (DSS, n=6) for 5 days. Once per day, either normal saline solution (DSS, n=3 and no tx, n=3) or an aqueous extract of Laurel tea (200 µL) (DSS + Laurel, n=3) was administered to animals by gavage. On day 5 animals were humanely sacrificed, colons resected from the ileocecal junction to the distal rectum and measured. Error bars represent standard deviations of mean values. p-values were calculated using a Student's t-Test. p-values ≤ 0.05 are considered statistically significant.
Figure 3. Laurel extract treatment attenuates leukocyte infiltration and morphological damage in colonic tissue from DSS-treated mice. Tissue samples (5 mm) were excised at the mid-point of the ascending colon from control (A, saline gavage only), DSS-treated (B) and DSS + LN-treated mice (C), fixed in Histochoice, embedded in paraffin and 5 µm sections prepared for routine H&E staining. Micrographs were taken at 100X and 200X magnification using bright field microscopy (Zeiss AxioSkop Microscope). Images were captured using SlideBook Image Acquisition software.
Figure 4. Laurel extract treatment reduces infiltration by CD4+ T-cells in DSS-treated mice. Tissue samples (5 mm) were excised at the mid-point of the transverse colon from sham treated, control (left panel, saline gavage only), DSS-treated (middle panel) and DSS + Laurel-treated mice (right panel). Cryosections were prepared and stained with murine anti-CD4 monoclonal antibodies; images were capture using a Zeiss AxioSkop Fluorescent microscopy and processed using SlideBook Image Acquisition software. Magnification, 100X.
Discussion[edit | edit source]
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, as well as tobacco-induced lung inflammation and amyloid beta-induced inflammation in a cell culture model of Alzheimer's disease. Methyleugenol was found to reduce the extent of cerebral ischemic injury by providing an anti-inflammatory protection. 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, suggesting the combined action of these two compounds may offer an important alternative clinical option for the treatment of wounds. Similar to eucalyptol, linalool and geraniol 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.
Based on these collective findings, we believe that LN extracts provide anti-inflammatory activity through a combined effect of multiple compounds working in synergy. By comparing the individual and combined effects of these compounds in an IBD model, new therapeutic modalities can be developed for the treatment of human IBD.
Additional information[edit | edit source]
Author contributions[edit | edit source]
Both authors were involved in carrying out experimental procedures. RAO compiled the initial draft of the manuscript and NSC contributed to final editing.
Acknowledgements[edit | edit source]
This work was supported by awards from the Science and Technology Corporation of the University of New Mexico (to R.A.O.) and the NIH MARC Program (to O.M.). N.S.C. is a recipient of the Ronald E. McNair Scholarship.
Competing interests[edit | edit source]
Authors have no competing interest.
Funding[edit | edit source]
This work was supported by an award from the Science and Technology Corporation of the University of New Mexico (to R.A.O.). N.S.C. is a recipient of the Ronald E. McNair Scholarship.
Ethics statement[edit | edit source]
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).
References[edit | edit source]
- Xu, Fang; Liu, Yong; Wheaton, Anne G.; Rabarison, Kristina M.; Croft, Janet B. (2019-02). "Trends and Factors Associated with Hospitalization Costs for Inflammatory Bowel Disease in the United States". Applied Health Economics and Health Policy 17 (1): 77–91. doi:10.1007/s40258-018-0432-4. ISSN 1175-5652. http://link.springer.com/10.1007/s40258-018-0432-4.
- Shin, Sung Jae; Noh, Choong-Kyun; Lim, Sun Gyo; Lee, Kee Myung; Lee, Kwang Jae (2017). "Non-steroidal anti-inflammatory drug-induced enteropathy". Intestinal Research 15 (4): 446. doi:10.5217/ir.2017.15.4.446. ISSN 1598-9100. PMID 29142512. PMC PMC5683975. http://irjournal.org/journal/view.php?doi=10.5217/ir.2017.15.4.446.
- White, William B. (2007-04). "Cardiovascular risk, hypertension, and NSAIDs". Current Rheumatology Reports 9 (1): 36–43. doi:10.1007/s11926-007-0020-3. ISSN 1523-3774. http://link.springer.com/10.1007/s11926-007-0020-3.
- Broersen, Leonie H. A.; Pereira, Alberto M.; Jørgensen, Jens Otto L.; Dekkers, Olaf M. (2015-06-01). "Adrenal Insufficiency in Corticosteroids Use: Systematic Review and Meta-Analysis". The Journal of Clinical Endocrinology & Metabolism 100 (6): 2171–2180. doi:10.1210/jc.2015-1218. ISSN 0021-972X. https://academic.oup.com/jcem/article/100/6/2171/2829580.
- Chan, Heyson Chi-hey; Ng, Siew Chien (2017-02). "Emerging biologics in inflammatory bowel disease". Journal of Gastroenterology 52 (2): 141–150. doi:10.1007/s00535-016-1283-0. ISSN 0944-1174. http://link.springer.com/10.1007/s00535-016-1283-0.
- Jacobsson, Lennart T. H.; Turesson, Carl; Gülfe, Anders; Kapetanovic, Meliha C.; Petersson, Ingemar F.; Saxne, Tore; Geborek, Pierre (2005-07). "Treatment with tumor necrosis factor blockers is associated with a lower incidence of first cardiovascular events in patients with rheumatoid arthritis". The Journal of Rheumatology 32 (7): 1213–1218. ISSN 0315-162X. PMID 15996054. https://pubmed.ncbi.nlm.nih.gov/15996054.
- Patel, Sheenal V.; Khan, David A. (2017-05-01). "Adverse Reactions to Biologic Therapy". Immunology and Allergy Clinics of North America. Biologic Therapies of Immunologic Diseases 37 (2): 397–412. doi:10.1016/j.iac.2017.01.012. ISSN 0889-8561. http://www.sciencedirect.com/science/article/pii/S0889856117300127.
- Gonzalez-Gay, Miguel A.; Gonzalez-Juanatey, Carlos; Vazquez-Rodriguez, Tomas R.; Miranda-Filloy, Jose A.; Llorca, Javier (2010-04). "Insulin resistance in rheumatoid arthritis: the impact of the anti-TNF-α therapy: TNF-α antagonist and insulin resistance in RA". Annals of the New York Academy of Sciences 1193 (1): 153–159. doi:10.1111/j.1749-6632.2009.05287.x. http://doi.wiley.com/10.1111/j.1749-6632.2009.05287.x.
- Channual, Jennifer; Wu, Jashin J.; Dann, Frank J. (2009). "Effects of tumor necrosis factor-α blockade on metabolic syndrome components in psoriasis and psoriatic arthritis and additional lessons learned from rheumatoid arthritis". Dermatologic Therapy 22 (1): 61–73. doi:10.1111/j.1529-8019.2008.01217.x. ISSN 1529-8019. https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1529-8019.2008.01217.x.
- Parmentier-Decrucq, Erika; Duhamel, Alain; Ernst, Olivier; Fermont, Catherine; Louvet, Alexandre; Vernier-Massouille, Gwenola; Cortot, Antoine; Colombel, Jean-Frédéric et al. (2009-10). "Effects of infliximab therapy on abdominal fat and metabolic profile in patients with Crohnʼs disease:". Inflammatory Bowel Diseases 15 (10): 1476–1484. doi:10.1002/ibd.20931. ISSN 1078-0998. https://academic.oup.com/ibdjournal/article/15/10/1476-1484/4643431.
- Doherty, Glen; Katsanos, Konstantinos H; Burisch, Johan; Allez, Matthieu; Papamichael, Konstantinos; Stallmach, Andreas; Mao, Ren; Berset, Ingrid Prytz et al. (2018-01-05). "European Crohn’s and Colitis Organisation Topical Review on Treatment Withdrawal ['Exit Strategies'] in Inflammatory Bowel Disease". Journal of Crohn's and Colitis 12 (1): 17–31. doi:10.1093/ecco-jcc/jjx101. ISSN 1873-9946. http://academic.oup.com/ecco-jcc/article/12/1/17/4060442.
- Eliadou, Elena; Day, Andrew S.; Thompson-Fawcett, Mark W.; Gearry, Richard B.; Rowbotham, David S.; Walmsley, Russel; Schultz, Michael; Inns, Stephen J. et al. (2015-10-16). "New Zealand Society of Gastroenterology Guidelines for the Management of Refractory Ulcerative Colitis". The New Zealand Medical Journal 128 (1423): 63–76. ISSN 1175-8716. PMID 26645757. https://pubmed.ncbi.nlm.nih.gov/26645757.
- McCamish, Mark; Yoon, William; McKay, James (2016-09). "Biosimilars: biologics that meet patients' needs and healthcare economics". The American Journal of Managed Care 22 (13 Suppl): S439–S442. ISSN 1936-2692. PMID 28719221. https://pubmed.ncbi.nlm.nih.gov/28719221.
- Bauer, Armin; Brönstrup, Mark (2014). "Industrial natural product chemistry for drug discovery and development". Natural Product Reports 31 (1): 35–60. doi:10.1039/C3NP70058E. ISSN 0265-0568. http://xlink.rsc.org/?DOI=C3NP70058E.
- Tibrewal, Nidhi; Tang, Yi (2014-06-07). "Biocatalysts for Natural Product Biosynthesis". Annual Review of Chemical and Biomolecular Engineering 5 (1): 347–366. doi:10.1146/annurev-chembioeng-060713-040008. ISSN 1947-5438. http://www.annualreviews.org/doi/10.1146/annurev-chembioeng-060713-040008.
- Spelman, Kevin; Burns, Jj; Nichols, Douglas; Winters, Nasha; Ottersberg, Steve; Tenborg, Mark (2006-06). "Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators". Alternative Medicine Review: A Journal of Clinical Therapeutic 11 (2): 128–150. ISSN 1089-5159. PMID 16813462. https://pubmed.ncbi.nlm.nih.gov/16813462.
- Tiwari, Ruchi; Latheef, Shyma K.; Ahmed, Ishtiaq; Iqbal, Hafiz M.N.; Bule, Mohammed Hussen; Dhama, Kuldeep; Samad, Hari Abdul; Karthik, Kumaragurubaran et al. (2018-05-08). "Herbal Immunomodulators - A Remedial Panacea for Designing and Developing Effective Drugs and Medicines: Current Scenario and Future Prospects". Current Drug Metabolism 19 (3): 264–301. doi:10.2174/1389200219666180129125436. http://www.eurekaselect.com/159454/article.
- Vanitha, Benny K. H. Tan and J. (2004-05-31). "Immunomodulatory and Antimicrobial Effects of Some Traditional Chinese Medicinal Herbs: A Review". Current Medicinal Chemistry. doi:10.2174/0929867043365161. Retrieved 2020-11-24.
- Aravindaram, Kandan; Yang, Ning-Sun (2010-08). "Anti-Inflammatory Plant Natural Products for Cancer Therapy". Planta Medica 76 (11): 1103–1117. doi:10.1055/s-0030-1249859. ISSN 0032-0943. http://www.thieme-connect.de/DOI/DOI?10.1055/s-0030-1249859.
- Orlando, Robert A.; Gonzales, Amanda M.; Hunsaker, Lucy A.; Franco, Carolina R.; Royer, Robert E.; Vander Jagt, David L.; Vander Jagt, Dorothy J. (2010-05-18). "Inhibition of Nuclear Factor κB Activation and Cyclooxygenase-2 Expression by Aqueous Extracts of Hispanic Medicinal Herbs". Journal of Medicinal Food 13 (4): 888–895. doi:10.1089/jmf.2009.1128. ISSN 1096-620X. PMID 20482259. PMC PMC3129691. https://www.liebertpub.com/doi/10.1089/jmf.2009.1128.
- Bower, Allyson; Marquez, Susan; Mejia, Elvira Gonzalez de (2016-12-09). "The Health Benefits of Selected Culinary Herbs and Spices Found in the Traditional Mediterranean Diet". Critical Reviews in Food Science and Nutrition 56 (16): 2728–2746. doi:10.1080/10408398.2013.805713. ISSN 1040-8398. PMID 25749238. https://doi.org/10.1080/10408398.2013.805713.
- Sayyah, M.; Saroukhani, G.; Peirovi, A.; Kamalinejad, M. (2003-08). "Analgesic and anti-inflammatory activity of the leaf essential oil ofLaurus nobilis Linn.". Phytotherapy Research 17 (7): 733–736. doi:10.1002/ptr.1197. ISSN 0951-418X. http://doi.wiley.com/10.1002/ptr.1197.
- Jin, Seori; Hong, Joo-Heon; Jung, Seung-Hyeon; Cho, Kyung-Hyun (2011-03). "Turmeric and Laurel Aqueous Extracts Exhibit In Vitro Anti-Atherosclerotic Activity and In Vivo Hypolipidemic Effects in a Zebrafish Model". Journal of Medicinal Food 14 (3): 247–256. doi:10.1089/jmf.2009.1389. ISSN 1096-620X. http://www.liebertpub.com/doi/10.1089/jmf.2009.1389.
- Khan, Alam; Zaman, Goher; Anderson, Richard A. (2009-01). "Bay leaves improve glucose and lipid profile of people with type 2 diabetes". Journal of Clinical Biochemistry and Nutrition 44 (1): 52–56. doi:10.3164/jcbn.08-188. ISSN 0912-0009. PMID 19177188. PMC 2613499. https://pubmed.ncbi.nlm.nih.gov/19177188.
- Eichele, Derrick D; Kharbanda, Kusum K (2017-09-07). "Dextran sodium sulfate colitis murine model: An indispensable tool for advancing our understanding of inflammatory bowel diseases pathogenesis". World Journal of Gastroenterology 23 (33): 6016–6029. doi:10.3748/wjg.v23.i33.6016. ISSN 1007-9327. PMID 28970718. PMC PMC5597494. http://www.wjgnet.com/1007-9327/full/v23/i33/6016.htm.
- Okayasu, Isao; Hatakeyama, Shigeru; Yamada, Masahiro; Ohkusa, Toshifumi; Inagaki, Yoshio; Nakaya, Rintaro (1990-03). "A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice". Gastroenterology 98 (3): 694–702. doi:10.1016/0016-5085(90)90290-H. https://linkinghub.elsevier.com/retrieve/pii/001650859090290H.
- Hausmann, M.; Obermeier, F.; Paper, D. H.; Balan, K.; Dunger, N.; Menzel, K.; Falk, W.; Schoelmerich, J. et al. (2007-05). "In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis". Clinical & Experimental Immunology 148 (2): 373–381. doi:10.1111/j.1365-2249.2007.03350.x. PMID 17437425. PMC PMC1868873. http://doi.wiley.com/10.1111/j.1365-2249.2007.03350.x.
- Chung, Ho-Lam; Yue, Grace-Gar-Lee; To, Ka-Fai; Su, Ya-Lun; Huang, Yu; Ko, Wing-Hung (2007-11-14). "Effect of Scutellariae Radix extract on experimental dextran-sulfate sodium-induced colitis in rats". World Journal of Gastroenterology 13 (42): 5605–5611. doi:10.3748/wjg.v13.i42.5605. ISSN 1007-9327. PMID 17948935. PMC 4172740. https://pubmed.ncbi.nlm.nih.gov/17948935.
- Camuesco, D.; Comalada, M.; Concha, A.; Nieto, A.; Sierra, S.; Xaus, J.; Zarzuelo, A.; Gálvez, J. (2006-06). "Intestinal anti-inflammatory activity of combined quercitrin and dietary olive oil supplemented with fish oil, rich in EPA and DHA (n-3) polyunsaturated fatty acids, in rats with DSS-induced colitis". Clinical Nutrition 25 (3): 466–476. doi:10.1016/j.clnu.2005.12.009. https://linkinghub.elsevier.com/retrieve/pii/S0261561406000033.
- Randhawa, Puneet Kaur; Singh, Kavinder; Singh, Nirmal; Jaggi, Amteshwar Singh (2014). "A Review on Chemical-Induced Inflammatory Bowel Disease Models in Rodents". The Korean Journal of Physiology & Pharmacology 18 (4): 279. doi:10.4196/kjpp.2014.18.4.279. ISSN 1226-4512. PMID 25177159. PMC PMC4146629. https://synapse.koreamed.org/DOIx.php?id=10.4196/kjpp.2014.18.4.279.
- Kim, Janice J.; Shajib, Md Sharif; Manocha, Marcus M.; Khan, Waliul I. (2012-02-01). "Investigating Intestinal Inflammation in DSS-induced Model of IBD". JoVE (Journal of Visualized Experiments) (60): e3678. doi:10.3791/3678. ISSN 1940-087X. PMID 22331082. PMC PMC3369627. https://www.jove.com/v/3678/investigating-intestinal-inflammation-in-dss-induced-model-of-ibd.
- Kiesler, Patricia; Fuss, Ivan J.; Strober, Warren (2015-03). "Experimental Models of Inflammatory Bowel Diseases". Cellular and Molecular Gastroenterology and Hepatology 1 (2): 154–170. doi:10.1016/j.jcmgh.2015.01.006. PMID 26000334. PMC PMC4435576. https://linkinghub.elsevier.com/retrieve/pii/S2352345X15000405.
- Vowinkel, Thorsten; Kalogeris, Theodore J.; Mori, Mikiji; Krieglstein, Christian F.; Granger, D. Neil (2004-04). "Impact of Dextran Sulfate Sodium Load on the Severity of Inflammation in Experimental Colitis". Digestive Diseases and Sciences 49 (4): 556–564. doi:10.1023/B:DDAS.0000026298.72088.f7. ISSN 0163-2116. http://link.springer.com/10.1023/B:DDAS.0000026298.72088.f7.
- Yan, Yutao; Kolachala, Vasantha; Dalmasso, Guillaume; Nguyen, Hang; Laroui, Hamed; Sitaraman, Shanthi V.; Merlin, Didier (2009-06-29). "Temporal and Spatial Analysis of Clinical and Molecular Parameters in Dextran Sodium Sulfate Induced Colitis". PLOS ONE 4 (6): e6073. doi:10.1371/journal.pone.0006073. ISSN 1932-6203. PMID 19562033. PMC PMC2698136. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006073.
- Chassaing, Benoit; Aitken, Jesse D.; Malleshappa, Madhu; Vijay‐Kumar, Matam (2014). "Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice". Current Protocols in Immunology 104 (1): 15.25.1–15.25.14. doi:10.1002/0471142735.im1525s104. ISSN 1934-368X. PMID 24510619. PMC PMC3980572. https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/0471142735.im1525s104.
- Yin, Chengyu; Liu, Boyu; Wang, Ping; Li, Xiaojie; Li, Yuanyuan; Zheng, Xiaoli; Tai, Yan; Wang, Chuan et al. (2020-05). "Eucalyptol alleviates inflammation and pain responses in a mouse model of gout arthritis". British Journal of Pharmacology 177 (9): 2042–2057. doi:10.1111/bph.14967. ISSN 0007-1188. PMID 31883118. PMC PMC7161556. https://onlinelibrary.wiley.com/doi/abs/10.1111/bph.14967.
- Kennedy-Feitosa, Emanuel; Okuro, Renata Tiemi; Pinho Ribeiro, Vanessa; Lanzetti, Manuella; Barroso, Marina Valente; Zin, Walter Araújo; Porto, Luís Cristóvão; Brito-Gitirana, Lycia et al. (12 2016). "Eucalyptol attenuates cigarette smoke-induced acute lung inflammation and oxidative stress in the mouse". Pulmonary Pharmacology & Therapeutics 41: 11–18. doi:10.1016/j.pupt.2016.09.004. ISSN 1522-9629. PMID 27599597. https://pubmed.ncbi.nlm.nih.gov/27599597.
- Khan, Andleeb; Vaibhav, Kumar; Javed, Hayate; Tabassum, Rizwana; Ahmed, Md. Ejaz; Khan, Mohd. Moshahid; Khan, M. Badruzzaman; Shrivastava, Pallavi et al. (2014-02). "1,8-Cineole (Eucalyptol) Mitigates Inflammation in Amyloid Beta Toxicated PC12 Cells: Relevance to Alzheimer’s Disease". Neurochemical Research 39 (2): 344–352. doi:10.1007/s11064-013-1231-9. ISSN 0364-3190. http://link.springer.com/10.1007/s11064-013-1231-9.
- Choi, Yoo Keum; Cho, Geum-Sil; Hwang, Sunyoung; Kim, Byung Woo; Lim, Ji H.; Lee, Jae-Chul; Kim, Hyoung Chun; Kim, Won-Ki et al. (2010-08-01). "Methyleugenol reduces cerebral ischemic injury by suppression of oxidative injury and inflammation". Free Radical Research 44 (8): 925–935. doi:10.3109/10715762.2010.490837. ISSN 1071-5762. https://doi.org/10.3109/10715762.2010.490837.
- de Christo Scherer, Marcella Malavazi; Marques, Franciane Martins; Figueira, Mariana Moreira; Peisino, Maria Carolina Oliveira; Schmitt, Elisângela Flávia Pimentel; Kondratyuk, Tamara P.; Endringer, Denise Coutinho; Scherer, Rodrigo et al. (2019-05-01). "Wound healing activity of terpinolene and α-phellandrene by attenuating inflammation and oxidative stress in vitro". Journal of Tissue Viability 28 (2): 94–99. doi:10.1016/j.jtv.2019.02.003. ISSN 0965-206X. http://www.sciencedirect.com/science/article/pii/S0965206X18301311.
- Ma, Jianqun; Xu, Hai; Wu, Jun; Qu, Changfa; Sun, Fenglin; Xu, Shidong (2015-12). "Linalool inhibits cigarette smoke-induced lung inflammation by inhibiting NF-κB activation". International Immunopharmacology 29 (2): 708–713. doi:10.1016/j.intimp.2015.09.005. https://linkinghub.elsevier.com/retrieve/pii/S1567576915301089.
- Kim, Min-Gu; Kim, Seong-Man; Min, Jae-Hong; Kwon, Ok-Kyoung; Park, Mi-Hyeong; Park, Ji-Won; Ahn, Hye In; Hwang, Jeong-Yeon et al. (2019-09). "Anti-inflammatory effects of linalool on ovalbumin-induced pulmonary inflammation". International Immunopharmacology 74: 105706. doi:10.1016/j.intimp.2019.105706. https://linkinghub.elsevier.com/retrieve/pii/S1567576919301286.
- Jiang, Kangfeng; Zhang, Tao; Yin, Nannan; Ma, Xiaofei; Zhao, Gan; Wu, Haichong; Qiu, Changwei; Deng, Ganzhen (2017-08-16). "Geraniol alleviates LPS-induced acute lung injury in mice via inhibiting inflammation and apoptosis". Oncotarget 8 (41): 71038–71053. doi:10.18632/oncotarget.20298. ISSN 1949-2553. PMID 29050341. PMC PMC5642616. https://www.oncotarget.com/article/20298/text/.
- De Fazio, Luigia; Spisni, Enzo; Cavazza, Elena; Strillacci, Antonio; Candela, Marco; Centanni, Manuela; Ricci, Chiara; Rizzello, Fernando et al. (2016-03-03). "Dietary Geraniol by Oral or Enema Administration Strongly Reduces Dysbiosis and Systemic Inflammation in Dextran Sulfate Sodium-Treated Mice". Frontiers in Pharmacology 7. doi:10.3389/fphar.2016.00038. ISSN 1663-9812. PMID 26973525. PMC PMC4776160. http://journal.frontiersin.org/Article/10.3389/fphar.2016.00038/abstract.