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

  • MUHAMMAD FAIQ NAJMI Integrative Pharmacogenomics Institute, Universiti Teknologi MARA Selangor, Puncak Alam Campus, Bandar Puncak Alam, 42300 Selangor, Malaysia
  • MOHD SALLEH ROFIEE Integrative Pharmacogenomics Institute, Universiti Teknologi MARA Selangor, Puncak Alam Campus, Bandar Puncak Alam, 42300 Selangor, Malaysia
  • TEH LAY KEK Faculty of Pharmacy, Universiti Teknologi MARA Selangor, Puncak Alam Campus, Bandar Puncak Alam, 42300 Selangor, Malaysia
  • FARIDA ZURAINA MOHD YUSOF Faculty of Applied Science, Universiti Teknologi MARA, Shah Alam Campus, 40450 Shah Alam, Selangor, Malaysia
  • MOHD ZAKI SALLEH Integrative Pharmacogenomics Institute, Universiti Teknologi MARA Selangor, Puncak Alam Campus, Bandar Puncak Alam, 42300 Selangor, Malaysia
Keywords: Chronic behaviour study, Danio rerio, gene expression, Moringa oleifera, RT2 profiler PCR array


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.

Author Biography

MUHAMMAD FAIQ NAJMI, Integrative Pharmacogenomics Institute, Universiti Teknologi MARA Selangor, Puncak Alam Campus, Bandar Puncak Alam, 42300 Selangor, Malaysia




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 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,

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. Accessed on 10 Jun 2022.

How to Cite
MUHAMMAD FAIQ NAJMI, MOHD SALLEH ROFIEE, TEH LAY KEK, FARIDA ZURAINA MOHD YUSOF, & MOHD ZAKI SALLEH. (2023). 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. Borneo Journal of Resource Science and Technology, 13(2), 79-91.