Vol 68 No 2 (2023): Journal of the Chilean Chemical Society
Original Research Papers


Naureen Zahra
Institute of Molecular Biology and Biotechnology, The University of Lahore
Tahira Fareed
Institute of Molecular Biology and Biotechnology, The University of Lahore
Muhammad Hamza
Institute of Molecular Biology and Biotechnology, The University of Lahore
Basit Zeeshan
Faculty of Sustainable Agriculture, University Malaysia Sabah, Sandakan 90509, Sabah, Malaysia.
Abid Sarwar
Food and Biotechnology Research Center, PCSIR Labs Complex Lahore 54600, Punjab Pakistan
Tari Aziz
Department of Agriculture, University of Ioannina, 47100 Arta, Greece
Metab Alharbi
Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
Abdulrahman Alshammari
Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
Published August 22, 2023
  • TLR2, TRPV4, PTGS2, Citrus paradisi, Molecular docking (MD), Inflammation, Pain and Fever.
How to Cite
Zahra, N., Fareed, T., Hamza, M., Zeeshan, B., Sarwar, A., Aziz, T., Alharbi, M., & Alshammari, A. (2023). IN-VIVO AND IN-SILICO ANALYSIS OF ANTI- INFLAMMATORY, ANALGESIC, AND ANTI PYRETIC ACTIVITIES OF CITRUS PARADISI LEAF EXTRACT. Journal of the Chilean Chemical Society, 68(2), 5813-5821. Retrieved from


Medicinal plants recently gained attention due to the presence of many phytochemicals involved in various therapeutical activities. The main aim of this study was to determine the in-vivo and in-silico anti-inflammatory, analgesic, and anti-pyretic effects of Citrus paradisi leaf ethanolic extract using albino rats (n = 36). For inducing inflammation, pain, and fever in albino rat’s carrageenan, acetic acid dilution in distilled water and yeast dilution in saline were used. The four different concentrations (50, 100, 200, and 400mg/kg) of ethanolic extract of Citrus paradisi leaf were used to prevent inflammation, pain, and fever. Diclofenac and paracetamol were used as standard drugs in this study. The ethanolic extract of Citrus paradisi leaf showed efficient anti-pyretic and anti-inflammatory inhibition (90% and 80%, respectively) but less efficient analgesic inhibition (36%). Similarly, in-silico study was done using leaf bioactive compounds such as linalool, beta-pinene, geraniol, citral, and terpinene-4-ol as ligand molecules and proteins for anti-inflammatory, analgesic, and anti-pyretic activity were PTGS2, TRPV4, and TLR2, respectively. The process of docking was done using ligand and protein molecules. The results of in-silico study were the same as in-vivo study; the binding energy values of anti-inflammatory and anti-pyretic activity were more efficient than an analgesic. In summary, the ethanolic extract of Citrus paradisi leaf in in-silico and in-vivo studies  proved less efficient against pain while more efficient against inflammation and fever.



  1. Rather LJ, Mohammad FJSC, Pharmacy. Acacia nilotica (L.): A review of its traditional uses, phytochemistry, and pharmacology. 2015;2:12-30.
  2. Ishaq Muhammad, Syed Shams ul Hassan, Suet Cheung, Xiaoqing Li, Rui Wang, Wei-Dong Zhang, Shi-Kai Yan, Yan Zhang, Hui-Zi Jin. Phytochemical study of Ligularia subspicata and valuation of its anti-inflammatory activity. Fitoterapia, 2021, 148,104800.
  3. Naveed, M.; Batool, H.; Rehman, S.u.; Javed, A.; Makhdoom, S.I.; Aziz, T.; Mohamed, A.A.; Sameeh, M.Y.; Alruways, M.W.; Dablool, A.S.; et al. Characterization and Evaluation of the Antioxidant, Antidiabetic, Anti-Inflammatory, and Cytotoxic Activities of Silver Nanoparticles Synthesized Using Brachychiton populneus Leaf Extract. Processes 2022, 10, 1521.
  4. Saleem, A.; Afzal, M.; Naveed, M.; Makhdoom, S.I.; Mazhar, M.; Aziz, T.; Khan, A.A.; Kamal, Z.; Shahzad, M.; Alharbi, M. HPLC, FTIR and GC-MS Analyses of Thymus vulgaris Phytochemicals Executing in vitro and in vivo Biological Activities and Effects on COX-1, COX-2 and Gastric Cancer Genes Computationally. Molecules 2022, 27, 8512.
  5. Ishaq Muhammad , Yong Zhen Xiao , Syed Shams ul Hassan , Xue Xiao , Shi-Kai Yan , Yuan-Qiang Guo , Xian-peng Ma & Hui-Zi Jin (2020): Three new guaiane-type sesquiterpenoids and a monoterpenoid from Litsea lancilimba Merr, Natural Product Research.
  6. Kala CP, Dhyani PP, Sajwan BSJJoE, Ethnomedicine. Developing the medicinal plants sector in northern India: challenges and opportunities. 2006;2(1):32.
  7. World Health O. Quality control methods for medicinal plant materials. Geneva: World Health Organization; 1998.
  8. Shaheena S, Richa S. Comparative Pharmacognostic Evaluation of Leaves of Citrus sinensis Var. Jaffa and Citrus paradisi var. Redblush. Pharmacognosy Communications. 2018;8(1).
  9. Nunes CdR, Barreto Arantes M, Menezes de Faria Pereira S, Leandro da Cruz L, de Souza Passos M, Pereira de Moraes L, et al. Plants as Sources of Anti-Inflammatory Agents. 2020;25(16):3726.
  10. Hassan SSu, Abbas SQ, Muhammad I, Wu J-J, Yan S-K, Ali F, Majid M, Jin H-Z and Bungau S. Metals-triggered compound CDPDP exhibits antiarthritic behavior by downregulating the inflammatory cytokines and modulating the oxidative storm in mice models with extensive ADMET, docking and simulation studies. Front. Pharmacol. 2022, 13:1053744.
  11. Hassan, S.S.u.; Ali, F.; Ishaq, M.; Bano, I.; Hassan, M.; Jin, H.-Z.; Bungau, S. A Comprehensive In Silico Exploration of Pharmacological Properties, Bioactivities, Molecular docking, and Anticancer Potential of Vieloplain F from Xylopia vielana Targeting B-Raf Kinase. Molecules 2022, 27, 917.
  12. Ahsan, H., Ayub, M., Irfan, H.M. et al. Tumor necrosis factor-alpha, prostaglandin-E2 and interleukin-1β targeted anti-arthritic potential of fluvoxamine: drug repurposing. Environ Sci Pollut Res (2022).
  13. Naveed, M.; Bukhari, B.; Aziz, T.; Zaib, S.; Mansoor, M.A.; Khan, A.A.; Shahzad, M.; Dablool, A.S.; Alruways, M.W.; Almalki, A.A.; et al. Green Synthesis of Silver Nanoparticles Using the Plant Extract of Acer oblongifolium and Study of Its Antibacterial and Antiproliferative Activity via Mathematical Approaches. Molecules 2022, 27, 4226.
  14. Gupta V, Kohli K, Ghaiye P, Bansal P, Lather A. Pharmacological potentials of Citrus paradisi-an overview. International Journal of Phytotherapy Research. 2011;1:8-17.
  15. Adnan M, Umer A, Ahmad I, Khizar H, Shakeel S. In vitro Evaluation of Biological Activities of Citrus Leaf Extracts. Sains Malaysiana. 2014;43:185-94.
  16. Deng W, Liu K, Cao S, Sun J, Zhong B, Chun J. Chemical Composition, Antimicrobial, Antioxidant, and Antiproliferative Properties of Grapefruit Essential Oil Prepared by Molecular Distillation. Molecules (Basel, Switzerland). 2020;25(1).
  17. Paoli M, de Rocca Serra D, Tomi F, Luro F, Bighelli A. Chemical composition of the leaf essential oil of grapefruits (Citrus paradisi Macf.) in relation with the genetic origin. Journal of Essential Oil Research. 2016;28(4):265-71.
  18. Mushtaq H, Hanif S, Saeed U, Saeed S, Mushtaq A. A Study of Antimicrobial Effects of Citrus Paradisi 'Grape Fruit'. 2019:185-90.
  19. Ebhohimen IE, Edemhanria L, Awojide S, Onyijen OH, Anywar G. Chapter 3 - Advances in computer-aided drug discovery. In: Egbuna C, Kumar S, Ifemeje JC, Ezzat SM, Kaliyaperumal S, editors. Phytochemicals as Lead Compounds for New Drug Discovery: Elsevier; 2020. p. 25-37.
  20. Salehi B, Upadhyay S, Erdogan Orhan I, Kumar Jugran A, L D Jayaweera S, A Dias D, et al. Therapeutic Potential of α- and β-Pinene: A Miracle Gift of Nature. Biomolecules. 2019;9(11):738.
  21. Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31(5):986-1000.
  22. White JP, Cibelli M, Urban L, Nilius B, McGeown JG, Nagy I. TRPV4: Molecular Conductor of a Diverse Orchestra. Physiological reviews. 2016;96(3):911-73.
  23. Chen W-H, Tzen JTC, Hsieh CL, Chen YH, Lin T-J, Chen S-Y, et al. Attenuation of TRPV1 and TRPV4 Expression and Function in Mouse Inflammatory Pain Models Using Electroacupuncture. Evidence-Based Complementary and Alternative Medicine. 2012;2012:636848.
  24. Emílio-Silva MT, Mota CMD, Hiruma-Lima CA, Antunes-Rodrigues J, Cárnio EC, Branco LGS. Antipyretic Effects of Citral and Possible Mechanisms of Action. Inflammation. 2017;40(5):1735-41.
  25. Steiner AA, Chakravarty S, Robbins JR, Dragic AS, Pan J, Herkenham M, et al. Thermoregulatory responses of rats to conventional preparations of lipopolysaccharide are caused by lipopolysaccharide per se-- not by lipoprotein contaminants. American journal of physiology Regulatory, integrative and comparative physiology. 2005;289(2):R348-r52.
  26. Hassan M, Abbasi MA, Aziz ur R, Siddiqui SZ, Shahzadi S, Raza H, et al. Designing of promising medicinal scaffolds for Alzheimer’s disease through enzyme inhibition, lead optimization, molecular docking and dynamic simulation approaches. Bioorganic Chemistry. 2019;91:103138.
  27. Duke JA, Beckstrom-Sternberg SM, United S, Agricultural Research S. Dr. Duke's phytochemical and ethnobotanical databases. 1994.
  28. Dallakyan S, Olson AJ. Small-molecule library screening by docking with PyRx. Methods in molecular biology (Clifton, NJ). 2015;1263:243-50.
  29. Winnall WR, Ali U, O'Bryan MK, Hirst JJ, Whiley PAF, Muir JA, et al. Constitutive Expression of Prostaglandin-Endoperoxide Synthase 2 by Somatic and Spermatogenic Cells Is Responsible for Prostaglandin E2 Production in the Adult Rat Testis1. Biology of Reproduction. 2007;76(5):759-68.
  30. Lawhorn BG, Brnardic EJ, Behm DJ. Recent advances in TRPV4 agonists and antagonists. Bioorganic & Medicinal Chemistry Letters. 2020;30(8):127022.
  31. de Oliviera Nascimento L, Massari P, Wetzler L. The Role of TLR2 in Infection and Immunity. 2012;3(79).
  32. Qadir MI, Abbas K, Hamayun R, Ali M. Analgesic, anti-inflammatory and anti-pyretic activities of aqueous ethanolic extract of Tamarix aphylla L. (Saltcedar) in mice. Pakistan journal of pharmaceutical sciences. 2014;27(6):1985-8.
  33. Bose A, Mondal DS, Gupta J, Ghosh T, Dash G, Si S. Analgesic, anti-inflammatory and antipyretic activities of the ethanolic extract and its fractions of Cleome rutidosperma. Fitoterapia. 2008;78:515-20.
  34. Jucá MM, Cysne Filho FMS, de Almeida JC, Mesquita DdS, Barriga JRdM, Dias KCF, et al. Flavonoids: biological activities and therapeutic potential. 2020;34(5):692-705.
  35. Roghini R, Vijayalakshmi K. Free Radical Scavenging Activity of Ethanolic Extract of Citrus paradisi and Naringin -An In vitro Study. International Journal of Pharmacognosy and Phytochemical Research. 2018;10.
  36. Castro-Vazquez L, Alañón ME, Rodríguez-Robledo V, Pérez-Coello MS, Hermosín-Gutierrez I, Díaz-Maroto MC, et al. Bioactive Flavonoids, Antioxidant Behaviour, and Cytoprotective Effects of Dried Grapefruit Peels (Citrus paradisi Macf.). Oxidative Medicine and Cellular Longevity. 2016;2016:8915729.
  37. Khan R, Mallick N, Feroz Z. Anti-inflammatory effects of Citrus sinensis L., Citrus paradisi L. and their combinations. Pakistan journal of pharmaceutical sciences. 2016;29:843-52.
  38. Suntar I, Khan H, Patel S, Celano R, Rastrelli L. An Overview on Citrus aurantium L.: Its Functions as Food Ingredient and Therapeutic Agent. Oxid Med Cell Longev. 2018;2018:7864269.
  39. Kim SH, Hur HJ, Yang HJ, Kim HJ, Kim MJ, Park JH, et al. Citrus junos Tanaka Peel Extract Exerts Antidiabetic Effects via AMPK and PPAR-γ both In Vitro and In Vivo in Mice Fed a High-Fat Diet. Evidence-based complementary and alternative medicine : eCAM. 2013;2013:921012.
  40. Yu HY, Ahn JH, Park SW, Jung YS. Preventive effect of yuzu and hesperidin on left ventricular remodeling and dysfunction in rat permanent left anterior descending coronary artery occlusion model. PLoS One. 2015;10(1):e110596.
  41. Abe H, Ishioka M, Fujita Y, Umeno A, Yasunaga M, Sato A, et al. Yuzu (Citrus junos Tanaka) Peel Attenuates Dextran Sulfate Sodium-induced Murine Experimental Colitis. Journal of oleo science. 2018;67(3):335-44.
  42. Cirmi S, Navarra M, Woodside JV, Cantwell MM. Citrus fruits intake and oral cancer risk: A systematic review and meta-analysis. Pharmacological research. 2018;133:187-94.
  43. Manchope M, Casagrande R, Verri W. Naringenin: An analgesic and anti-inflammatory citrus flavanone. Oncotarget. 2016;8.
  44. Ferraz CR, Carvalho TT, Manchope MF, Artero NA, Rasquel-Oliveira FS, Fattori V, et al. Therapeutic potential of flavonoids in pain and inflammation: mechanisms of action, pre-clinical and clinical data, and pharmaceutical development. 2020;25(3):762.
  45. Lisa SR, Islam MK, Qais NJDUJoPS. Plants and Plant Constituents with Analgesic and Anti-inflammatory Activities: A Systematic Review. 2020;19(2):207-24.
  46. Al-Snafi A. Nutritional value and pharmacological importance of citrus species grown in Iraq. IOSR Journal of Pharmacy (IOSRPHR). 2016;06:76-108.
  47. Emílio-Silva MT, Mota CM, Hiruma-Lima CA, Antunes-Rodrigues J, Cárnio EC, Branco LGJI. Antipyretic effects of citral and possible mechanisms of action. 2017;40(5):1735-41.
  48. Sapkota B, Devkota HP, Poudel P. Citrus maxima (Brum.) Merr. (Rutaceae): Bioactive Chemical Constituents and Pharmacological Activities. Evidence-Based Complementary and Alternative Medicine. 2022;2022:8741669.
  49. Miya G, Nyalambisa M, Oyedeji O, Gondwe M, Oyedeji A. Chemical Profiling, Toxicity and Anti-Inflammatory Activities of Essential Oils from Three Grapefruit Cultivars from KwaZulu-Natal in South Africa. 2021;26(11):3387.
  50. Saini RK, Ranjit A, Sharma K, Prasad P, Shang X, Gowda KGM, et al. Bioactive Compounds of Citrus Fruits: A Review of Composition and Health Benefits of Carotenoids, Flavonoids, Limonoids, and Terpenes. 2022;11(2):239.

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