The Mechanism of Anxiolytic Effects of Moringa oleifera Leaf Extracts Associated with Significant Differential Expression of Crhb, Faah2a, Mao, and Pah Genes in Danio rerio
Mechanism of anxiolytic effects of Moringa oleifera leaf extracts
DOI:
https://doi.org/10.33736/bjrst.5669.2023Keywords:
Chronic behaviour study, Danio rerio, gene expression, Moringa oleifera, RT2 profiler PCR arrayAbstract
The search and development of new therapeutic agents from medicinal plants to alleviate anxiety is well justified due to the increasing cases of anxiety disorder and lack of effective treatment. Moringa oleifera has been used traditionally to treat anxiety. However, there is still lack of understanding on the mechanism for its anxiolytic effect. The purpose of this study was to investigate the anxiolytic effects and the mechanism of ethanolic extracts of the leaves of M. oleifera (MOLE) by observing behavioural changes of the Danio rerio and the differential gene expression analysis using custom RT2 Profiler PCR array. A 14-day chronic behaviour study was conducted using three concentrations of MOLE (500 mg/L, 1000 mg/L and 2000 mg/L) fluoxetine as the positive control. Stress-induced D. rerio treated with 1000 mg/L MOLE showed the lowest level of anxiety compared to other groups as evidenced by a decrease in freezing episodes and freezing time, increased entries into the light region. The fish also showed significant changes in the expression of crhb, faah2a, mao, and pah genes. MOLE with the presence of quercetin and kaempferol are believed to exert its anxiolytic effects through differential expression of gene (i) modulating the function of GABAA receptor (crhb), (ii) inhibiting the expression of nitric oxide synthase (NOS) and the production of nitric oxide, (iii) increasing the AEA levels in the brain (faah2a), (iv) increasing the level of dopamine levels in the brain (mao). These findings provide valuable insights into the potential use of MOLE as a treatment for anxiety-related disorders as well as the significance of the molecular pathways involved in its anxiolytic properties.
References
Ahmad, H., Rauf, K., Zada, W., McCarthy, M., Abbas, G., Anwar, F. & Shah, A.J. (2020). Kaempferol facilitated extinction learning in contextual fear conditioned rats via inhibition of fatty-acid amide hydrolase. Molecules, 25(20): 4683. DOI: 10.3390/molecules25204683
Alam, W., Khan, H., Shah, M., Cauli, O. & Saso, L. (2020). Kaempferol as a dietary anti-inflammatory agent: current therapeutic standing. Molecules, 25(18): 4073. DOI: 10.3390 /molecules25184073
Babaei, P., Kouhestani, S. & Jafari, A. (2018). Kaempferol attenuates cognitive deficit via regulating oxidative stress and neuroinflammation in an ovariectomized rat model of sporadic dementia. Neural Regeneration Research, 13(10): 1827. DOI: 10.4103/1673-5374.238714
Baldwin, D.S., Anderson, I.M., Nutt, D.J., Allgulander, C., Bandelow, B., den Boer, J.A., Christmas, D.M., Davies, S., Fineberg, N., Lidbetter, N., Malizia, A., McCrone, P., Nabarro, D., O’Neill, C., Scott, J., van der Wee, N. & Wittchen, H.U. (2014). Evidence-based pharmacological treatment of anxiety disorders, post-traumatic stress disorder and obsessive-compulsive disorder: A revision of the 2005 guidelines from the British Association for Psychopharmacology. Journal of Psychopharmacology, 28(5): 403-439. DOI: 10. 1177/0269881114525674
Bandelow, B., Zohar, J., Hollander, E., Kasper, S., Möller, H.J. & WFSBP Task Force On Treatment Guide (2008). World Federation of Societies of Biological Psychiatry (WFSBP) Guidelines for the pharmacological treatment of anxiety, obsessive-compulsive and post-traumatic stress disorders – first revision. The World Journal of Biological Psychiatry, 9(4): 248-312. DOI: 10.1080/15622970802465807
Benneh, C.K., Biney, R.P., Mante, P.K., Tandoh, A., Adongo, D.W., & Woode, E. (2017). Maerua angolensis stem bark extract reverses anxiety and related behaviours in zebrafish- involvement of GABAergic and 5-HT systems. Journal of Ethnopharmacology, 207: 129-145. DOI: 10.1016/j.jep.2017.06.012
Bhattacharya, A., Behera, R., Agrawal, D., Sahu, P.K., Kumar, S. & Mishra, S.S., (2014). Antipyretic effect of ethanolic extract of Moringa oleifera leaves on albino rats. Tanta Medical Journal, 42(2): 74. DOI: 10.4103/1110-1415 .137810
Bhutada, P., Mundhada, Y., Bansod, K., Ubgade, A., Quazi, M., Umathe, S. & Mundhada, D. (2010). Reversal by quercetin of corticotrophin releasing factor induced anxiety-and depression-like effect in mice. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 34(6): 955-960. DOI: 10.1016/j.pnpbp.2010.04.025
Bitencourt, R.M., Pamplona, F.A. & Takahashi, R.N. (2008). Facilitation of contextual fear memory extinction and anti-anxiogenic effects of AM404 and cannabidiol in conditioned rats. European Neuropsychopharmacology, 18(12): 849-859. DOI: 10.1016/j.euroneuro.2008.07.001
Blaser, R., Chadwick, L. & McGinnis, G. (2010). Behavioural measures of anxiety in zebrafish (Danio rerio). Behavioural Brain Research, 208(1): 56-62. DOI: 10.1016/j.bbr.2009.11.009
Burman, M.A., Szolusha, K., Bind, R., Kerney, K., Boger, D.L. & Bilsky, E.J. (2016). FAAH inhibitor OL-135 disrupts contextual, but not auditory, fear conditioning in rats. Behavioural Brain Research, 308: 1-5. DOI: 10.1016/j.b br.2016.04.014
Cachat, J.M., Canavello, P.R., Elegante, M.F., Bartels, B.K., Elkhayat, S.I., Hart, P.C. & Kalueff, A.V. (2010). Modeling stress and anxiety in zebrafish. Zebrafish Models in Neurobehavioral Research, 73-88.
Campanari, M.L., Bourefis, A.R., Buee-Scherrer, V. & Kabashi, E. (2020). Freezing activity brief data from a new FUS mutant zebrafish line. Data in Brief, 31: 105921. DOI: 10.1016/j. dib.2020.105921
Collymore, C., Tolwani, R.J. & Rasmussen, S. (2015). The behavioral effects of single housing and environmental enrichment on adult zebrafish (Danio rerio). Journal of the American Association for Laboratory Animal Science: JAALAS, 54(3): 280-285.
Coppin, J.P., Xu, Y., Chen, H., Pan, M.H., Ho, C.T., Juliani, R., Simon, J.E. & Wu, Q. (2013). Determination of flavonoids by LC/MS and anti-inflammatory activity in Moringa oleifera. Journal of Functional Foods, 5(4): 1892-1899. DOI: 10.1016/j.jff.2013.09.010
Davis, C.P. (2017). Anti-anxiety drugs (anxiolytics) side effects, list of names. Retrieved December 1, 2022 from https://www.medicinenet.com/anxi olytics_for_anxiety_drug_class/article.htm#what_are_anti-anxiety_anxiolytic_drugs.
Didycz, B. & Bik-Multanowski, M. (2018). Blood phenylalanine instability strongly correlates with anxiety in phenylketonuria. Molecular Genetics and Metabolism Reports, 14: 80-82. DOI: 10.1016/j.ymgmr.2017.12.003
Duarte, T., Fontana, B.D., Müller, T.E., Bertoncello, K.T., Canzian, J. & Rosemberg, D.B. (2019). Nicotine prevents anxiety-like behavioral responses in zebrafish. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 94: 109655. DOI: 10.1016/j.pnpbp.2019.109655
Egan, R.J., Bergner, C.L., Hart, P.C., Cachat, J.M., Canavello, P.R., Elegante, M.F., Salem, I.E., Brett, K.B., Anna, K.T., David, H.T., Sopan, M., Esther, B., Eric, G., Hakima, A., Zofia, Z. & Kalueff, A.V. (2009). Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behavioural Brain Research, 205(1): 38–44. DOI: 10.1016/j.bbr.2009.06.022
Filho, A.W., Filho, V.C., Olinger, L. & de Souza, M.M. (2008). Quercetin: further investigation of its antinociceptive properties and mechanisms of action. Archives of Pharmacal Research, 31: 713-721. DOI: 10.1007/s12272-001-1217-2
Falcon, E., Maier, K., Robinson, S.A., Hill-Smith, T.E. & Lucki, I. (2015). Effects of buprenorphine on behavioural tests for antidepressant and anxiolytic drugs in mice. Psychopharmacology, 232(5): 907-915. DOI: 10.1007/s00213-014-3723-y
Grundmann, O., Nakajima, J.I., Kamata, K., Seo, S. & Butterweck, V. (2009). Kaempferol from the leaves of Apocynum venetum possesses anxiolytic activities in the elevated plus maze test in mice. Phytomedicine, 16(4): 295-302. DOI: 10.1016/ j.phymed.2008.12.020
Gunduz-Cinar, O., MacPherson, K.P., Cinar, R., Gamble-George, J., Sugden, K., Williams, B., Godlewski, G., Ramikie, T.S., Gorka, A.X., Alapafuja, S.O., Nikas, S.P., Makriyannis, A., Poulton, R., Patel, S., Hariri, A.R., Caspi, A., Moffitt, T.E., Kunos. G. & Holmes, A. (2012). Convergent translational evidence of a role for anandamide in amygdala-mediated fear extinction, threat processing and stress-reactivity. Molecular Psychiatry, 18(7): 813-823. DOI: 10.1038/ mp.2012.72
Glover, V., Sandler, M., Owen, F. & Riley, G.J. (1977). Dopamine is a monoamine oxidase B substrate in man. Nature, 265(5589): 80-81. DOI: 10.1038/265080a0.
Haller, J., Goldberg, S.R., Pelczer, K.G., Aliczki, M. & Panlilio, L.V. (2013). The effects of anandamide signaling enhanced by the FAAH inhibitor URB597 on coping styles in rats. Psychopharmacology, 230(3): 353-362. DOI: 10.1007/s00213-013-3161-2
Haller, J., Barna, I., Barsvari, B., Gyimesi, P.K., Yasar, S., Panilio, L.V. & Goldberg, S. (2009). Interactions between environmental aversiveness and the anxiolytic effects of enhanced cannabinoid signalling by FAAH inhibition in rats. Psychopharmacology, 204(4): 607-616. DOI: 10.1007/s00213-009-1494-7
Howe, K., Clark, M.D., Torroja, C.F., Torrance, J. & Berthelot, C. (2013). The zebrafish reference genome sequence and its relationship to the human genome. Nature, 496(7446): 498-503. DOI: 10.1038/nature12111
Hsieh, C. H., Li, H.Y. & Chen, J.C. (2010). Nitric oxide and interleukin-1β mediate noradrenergic induced corticotrophin-releasing hormone release in organotypic cultures of rat paraventricular nucleus. Neuroscience, 165(4): 1191-1202. DOI: 10.1016/j.neuroscience.2009.12.003
Imran, M., Salehi, B., Sharifi-Rad, J., Aslam Gondal, T., Saeed, F., Imran, A., Shahbaz, M., Tsouh Fokou, P.V., Umair Arshad, M., Khan, H., Guerreiro, S.G., Martins, N. & Estevinho, L.M. (2019). Kaempferol: A key emphasis to its anticancer potential. Molecules, 24(12): 2277. DOI: 10.3390/molecules24122277
Jesse, C.R., Bortolatto, C.F., Savegnago, L., Rocha, J.B., & Nogueira, C.W. (2008). Involvement of l-arginine–nitric oxide–cyclic guanosine monophosphate pathway in the antidepressant-like effect of tramadol in the rat forced swimming test. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 32(8): 1838-1843.
Kalueff, A.V., Gebhardt, M., Stewart, A.M., Cachat, J.M., Brimmer, M., Chawla, J.S., Craddock, C., Kyzar, E.J., Roth, A., Landsman, S., Gaikwad, S., Robinson, K., Baatrup, E., Tierney, K., Shamchuk, A., Norton, W., Miller, N., Nicolson, T., Braubach, O., Gilman, C.P., Pittman, J., Rosemberg, D.B., Gerlai, R., Echevarria, D., Lamb, E., Neuhauss, S.C., Weng, W., Bally-Cuif, L. & Schneider, H. (2013). Towards a comprehensive catalog of zebrafish behaviour 1.0 and beyond. Zebrafish, 10(1): 70-86. DOI: 10.1089/zeb.2012.0861
Kao, T.K., Ou, Y.C., Raung, S.L., Lai, C.Y., Liao, S.L. & Chen, C.J. (2010). Inhibition of nitric oxide production by quercetin in endotoxin/cytokine-stimulated microglia. Life Sciences, 86(9-10): 315-321. DOI: 10.1016/ j.lfs.2009.12.014
Khawaja, T.M., Tahira, M. & Ikram, U.H. (2013). M. oleifera: A natural gift - a review. Journal of Pharmaceutical Sciences and Research, 2(11): 775-781.
Kim, H.Y., Kim, O.H. & Sung, M.K. (2003). Effects of phenol-depleted and phenol-rich diets on blood markers of oxidative stress, and urinary excretion of Quercetin and Kaempferol in healthy volunteers. Journal of the American College of Nutrition, 22(3): 217-223. DOI: 10.1080/ 07315724.2003.10719296
Lakshmi, B.V., Sudhakar, M. & Ramya, R.L. (2014). Anti-anxiety activity of M. oleifera assessed using different experimental anxiety models in mice. Journal of Pharmacy Research, 8(34): 3-8. DOI: 10.4103/0253-7613.84975
Lenman, A. & Fowler, C.J. (2007). Interaction of ligands for the peroxisome proliferator-activated receptor γ with the endocannabinoid system. British Journal of Pharmacology, 151(8): 1343-1351. DOI: 10.1038/sj.bjp.0707352
Maximino, C., Brito, T.M., Dias, C.A.G., de M., Gouveia, A. & Morato, S. (2010). Scototaxis as anxiety-like behavior in fish. Nature Protocols, 5(2): 209-216. DOI: 10.1038/nprot.2009.225
Müller, T.E., Nunes, M.E., Menezes, C.C., Marins, A.T., Leitemperger, J., Gressler, A.C.L., Loro, V.L. (2017). Sodium selenite prevents paraquat-induced neurotoxicity in zebrafish. Molecular Neurobiology, 55(3): 1928-1941. DOI: 10.1007 /s12035-017-0441-6
Mueller, T., Vernier, P. & Wullimann, M.F. (2004). The adult central nervous cholinergic system of a neurogenetic model animal, the zebrafish D. rerio. Brain Research, 1011(2): 156-69. DOI: 10.1016 /j.brainres.2004.02.073
National Institute for Health and Clinical Excellence (NICE) (2011). Anxiety: management of anxiety (panic disorder, with or without agoraphobia, and generalised anxiety disorder) in adults in primary, secondary and community care. London: The British Psychological Society and The Royal College of Psychiatrists.
OECD (2019), Test No. 203: Fish, Acute Toxicity Test, OECD Guidelines for the testing of chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264069961-en.
Prabsattroo, T., Wattanathorn, J., Iamsaard, S., Somsapt, P., Sritragool, O., Thukhummee, W. & Muchimapura, S. (2015). Moringa oleifera extract enhances sexual performance in stressed rats. Journal of Zhejiang University. Science. B, 16(3): 179. DOI: 10.1631/jzus.B1400197
Park, S.H., Sim, Y.B., Han, P.L., Lee, J.K. & Suh, H.W. (2010). Antidepressant-like effect of kaempferol and quercitirin, isolated from Opuntia ficus-indica var. saboten. Experimental Neurobiology, 19(1): 30-38. DOI: 10.5607/en. 2010.19.1.30
Piato, A.L., Capiotti, K.M., Tamborski, A.R., Oses, J.P., Barcellos, L.J., Bogo, M.R., Lara, D.R., Vianna, M.R. & Bonan, C.D. (2010). Unpredictable chronic stress model in zebrafish (D. rerio): behavioural and physiological responses. Progress in Neuropsychopharmacol and Biology Psychiatry 35(2): 561-567. DOI: 10.1016/j.pnpbp.2010.12.018
Pu, F., Mishima, K., Irie, K., Motohashi, K., Tanaka, Y., Kensuke, O., Takashi, E., Yoshhisa, E., Nobuaki, E., Katsuri, I. & Michihiro, F. (2007). Neuroprotective effects of quercetin and Rutin on spatial memory impairment in an 8-arm radial maze task and neuronal death induced by repeated cerebral ischemia in rats. Journal of Pharmacological Sciences, 104(4): 329-334. DOI: 10.1254/jphs.fp0070247
Qiagen (2014). RT2 Profiler PCR Array Handbook.
Qiagen (2018). RNeasy® Lipid Tissue Mini Handbook
Ren, J., Lu, Y., Qian, Y., Chen, B., Wu, T. & Ji, G. (2019). Recent progress regarding kaempferol for the treatment of various diseases. Experimental and therapeutic medicine, 18(4), 2759-2776. DOI: 10.3892/etm.2019.7886
Romero, M., Jiménez, R., Hurtado, B., Moreno, J. M., Rodríguez-Gómez, I., López-Sepúlveda, R. & Duarte, J. (2010). Lack of beneficial metabolic effects of quercetin in adult spontaneously hypertensive rats. European Journal of Pharmacology, 627(1-3): 242-250. DOI: 10.1016/j.ejphar.2009.11.006
Scriver, C.R. (2007). The PAH gene, phenylketonuria, and a paradigm shift. Human Mutation, 28(9): 831-845. DOI: 10.1002/humu.20526
Sevgi, S., Ozek, M. & Eroglu, L. (2006). L-NAME prevents anxiety-like and depression-like behavior in rats exposed to restraint stress. Methods and findings in experimental and clinical pharmacology, 28(2): 95-99. DOI: 10.1358/ mf.2006.28.2.977840
Spolidório, P.C.M., Echeverry, M.B., Iyomasa, M., Guimarães, F.S. & Del Bel, E.A. (2007). Anxiolytic effects induced by inhibition of the nitric oxide–cGMP pathway in the rat dorsal hippocampus. Psychopharmacology, 195: 183-192. DOI: 10.1007/s00213-007-0890-0
Sriraksa, N., Hawiset, T. & Khongrum, J. (2019). Effects of Moringa oleifera leaf extract on the acetylcholinesterase and monoamine oxidase activities in rat brains with streptozotocin-induced diabetes and sciatic nerve constriction. Naresuan Phayao Journal, 12(3): 13-22.
Stewart, A., Maximino, C., Marques De Brito, T., Herculano, A.M,, Gouveia, A. Morato, S., Cachat, J.M., Gaikwad, S., Elegante, M.F., Hart, P.C. & Kalueff, A.V. (2010). Neurophenotyping of adult zebrafish using the light/dark box paradigm. Zebrafish Neurobehavioural Protocols, 50: 157-167.
Sultana, B. & Anwar, F. (2008). Flavonols (kaempeferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants. Food Chemistry, 108(3): 879-884. DOI: 10.1016/j.foodchem.2007.11.053
Volke, V., Wegener, G., Bourin, M. & Vasar, E. (2003). Antidepressant-and anxiolytic-like effects of selective neuronal NOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole in mice. Behavioural Brain Research, 140(1-2): 141-147. DOI: 10.1016/s0166-4328(02)00312-1
Waters, P.J. (2003). How PAH gene mutations cause hyper-phenylalaninemia and why mechanism matters: Insights from in vitro expression. Human Mutation, 21(4): 357-369.
World Health Organization (2017). World Mental Health Day 2017: Mental health in the workplace. www.who.int/mental_health/world-mental-health-day/2017/en/. Accessed on 10 Jun 2022.
Downloads
Published
How to Cite
Issue
Section
License
Copyright Transfer Statement for Journal
1) In signing this statement, the author(s) grant UNIMAS Publisher an exclusive license to publish their original research papers. The author(s) also grant UNIMAS Publisher permission to reproduce, recreate, translate, extract or summarize, and to distribute and display in any forms, formats, and media. The author(s) can reuse their papers in their future printed work without first requiring permission from UNIMAS Publisher, provided that the author(s) acknowledge and reference publication in the Journal.
2) For open access articles, the author(s) agree that their articles published under UNIMAS Publisher are distributed under the terms of the CC-BY-NC-SA (Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License) which permits unrestricted use, distribution, and reproduction in any medium, for non-commercial purposes, provided the original work of the author(s) is properly cited.
3) For subscription articles, the author(s) agree that UNIMAS Publisher holds copyright, or an exclusive license to publish. Readers or users may view, download, print, and copy the content, for academic purposes, subject to the following conditions of use: (a) any reuse of materials is subject to permission from UNIMAS Publisher; (b) archived materials may only be used for academic research; (c) archived materials may not be used for commercial purposes, which include but not limited to monetary compensation by means of sale, resale, license, transfer of copyright, loan, etc.; and (d) archived materials may not be re-published in any part, either in print or online.
4) The author(s) is/are responsible to ensure his or her or their submitted work is original and does not infringe any existing copyright, trademark, patent, statutory right, or propriety right of others. Corresponding author(s) has (have) obtained permission from all co-authors prior to submission to the journal. Upon submission of the manuscript, the author(s) agree that no similar work has been or will be submitted or published elsewhere in any language. If submitted manuscript includes materials from others, the authors have obtained the permission from the copyright owners.
5) In signing this statement, the author(s) declare(s) that the researches in which they have conducted are in compliance with the current laws of the respective country and UNIMAS Journal Publication Ethics Policy. Any experimentation or research involving human or the use of animal samples must obtain approval from Human or Animal Ethics Committee in their respective institutions. The author(s) agree and understand that UNIMAS Publisher is not responsible for any compensational claims or failure caused by the author(s) in fulfilling the above-mentioned requirements. The author(s) must accept the responsibility for releasing their materials upon request by Chief Editor or UNIMAS Publisher.
6) The author(s) should have participated sufficiently in the work and ensured the appropriateness of the content of the article. The author(s) should also agree that he or she has no commercial attachments (e.g. patent or license arrangement, equity interest, consultancies, etc.) that might pose any conflict of interest with the submitted manuscript. The author(s) also agree to make any relevant materials and data available upon request by the editor or UNIMAS Publisher.
