Exploring in vitro anti-inflammatory activity by COX inhibition of flavone isolated from the Calophyllum inophyllum Linn leaves in lipo-polysaccharide stimulated RAW264.7 macrophage cell line

 Corresponding author: Christy K. Jose. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Exploring in vitro anti-inflammatory activity by COX inhibition of flavone isolated from the Calophyllum inophyllum Linn leaves in lipo-polysaccharide stimulated RAW264.7 macrophage cell line Christy K. Jose 1, *, Francis Mathew 2 and Santhosh M. Mathews 3 1 Department of Pharmaceutical Chemistry, Pushpagiri College of Pharmacy, Perumthuruthy, Tiruvalla, Pathanamthitta Dist., Kerala, India. 2 Department of Botany, St. Thomas College, Ranni, Pathanamthitta Dist., Kerala, India. 3 Pushpagiri College of Pharmacy, Perumthuruthy, Tiruvalla, Pathanamthitta Dist., Kerala, India. World Journal of Advanced Research and Reviews, 2025, 26(01), 3328-3332 Publication history: Received on 04 February 2025; revised on 15 March 2025; accepted on 17 March 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.1.0837 Abstract Cell lines are being used in vaccine production, testing drug metabolism and cytotoxicity, antibody production, study of gene function, generation of artificial tissues and synthesis of biological compounds, thus, the field of scientific research is being revolutionized by cell lines. Inflammation is an inherent natural process of the immune system. When inflammation persists for a long period, various chronic diseases are triggered. Recent studies have reported the identification of novel natural compounds with anti-inflammatory properties and these compounds offer the potential to decrease excessive inflammation associated with various diseases. Activated macrophages produce different cytokines which, play an important role in the induction of acute and chronic inflammatory diseases. Lipopolysaccharides regulate inflammatory mediators. Thus, suppression of inflammatory mediator synthesis is one of the useful therapeutic strategies in the treatment of inflammatory diseases. COX-2 is not expressed under normal conditions in most cells, but inflammation mediators induce COX2 expression in a number of cellular systems. Pharmacological inhibition of COX by nonsteroidal anti-inflammatory drugs (NSAID) can provide relief from the symptoms of inflammation and pain. Shih-Chang Tsai et al explored the anti-inflammatory activity of acetone extract of Calophyllum inophyllum leaves and reported the plant can be used in prevention of inflammatory diseases, and its mechanism may be partially associated with blocking COX-2 and iNOS of RAW 264.7 cells. The present work aimed to demonstrate that the anti-inflammatory activity by COX inhibition in lipo-polysaccharide stimulated RAW264.7 macrophage cell line is due to the flavone isolated from the leaves of Calophyllum inophyllum. According to the current investigation, isolated flavones from Calophyllum inophyllum appear to have anti-inflammatory property. When compared to the IC50 value of the standard, diclofenac, the isolated flavone displayed a good IC50 value, which indicates that the ethanolic leaf extract of Calophyllum inophyllum has the potential to be developed as a non-steroidal antiinflammatory drug (NSAID). Keywords: Cell Lines; Inflammation; COX; RAW264.7; Calophyllum inophyllum; Flavone 1 Introduction Several advantages, which include cost effectiveness, easiness to use, unlimited supply of material and bypassing of ethical concerns associated with the use of animal and human tissue, offers immortal cell lines to be used in place of primary cells in research. Cell lines provide a consistent sample and reproducible results, since it provides a pure population of cells, which is valuable. The field of scientific research is being revolutionized by cell lines and they are being used in vaccine production, testing drug metabolism and cytotoxicity, antibody production, study of gene function, World Journal of Advanced Research and Reviews, 2025, 26(01), 3328-3332 3329 generation of artificial tissues (e.g., artificial skin) and synthesis of biological compounds e.g., therapeutic proteins [1]. Inflammation is an inherent natural process of the immune system. When inflammation persists for a long period, various chronic diseases such as autoimmune disorders, arthritis, cardiovascular diseases, diabetes, Parkinson’s, and cancer are triggered. Novel natural compounds with anti-inflammatory properties have been identified recently and studied, which offer the potential to decrease excessive inflammation associated with various diseases. Different cytokines such as interleukin-1β (IL-1ß), IL-6, reactive oxygen species (ROS), reactive nitrogen species (RNS), inducible nitric oxide synthase (iNOS), prostaglandin (PGE) and tumor necrosis factor-α (TNF-α), produced by activated macrophages plays an important role in the induction of acute and chronic inflammatory diseases [2]. The mRNA expression of inflammatory cytokines and mediators has been found to increase by exposure to bacterial lipopolysaccharides (LPS), a well-known component of the cell wall of gram-negative bacteria [3]. Lipopolysaccharides regulate inflammatory mediators such as TNF-α, IL-6, and NO [4]. Thus, suppression of inflammatory mediator synthesis is one of the useful therapeutic strategies in the treatment of inflammatory diseases. Cyclooxygenase (COX), also known as Prostaglandin G/H synthase, prostaglandin-endoperoxide synthase (PTGS, EC 1.14.99.1), is an enzyme that is responsible for oxidation of arachidonic acid to important biological mediators called prostanoids, including prostaglandins, prostacyclin and thromboxane. In the biosynthetic pathway to prostanoids from arachidonic acid, COX is the central enzyme. COX-1 (expressed form) and COX-2 (inducible form) are known to exist in isoenzymes. COX-1 is constitutively expressed in many tissues and is the predominant form in gastric mucosa and in kidney. COX-2 is not expressed under normal conditions in most cells, but inflammation mediators such as growth factors, cytokines and endotoxin induce COX2 expression in a number of cellular systems. Pharmacological inhibition of COX by nonsteroidal anti-inflammatory drugs (NSAID) can provide relief from the symptoms of inflammation and pain. Balsam, an oleoresin from the bark of Calophyllum inophyllum is used as cicatrisan [5]. It is reported that, traditionally the decoction of the leaves is used to relieve eye irritation and conjunctivitis [6, 7]. Kashman et al reported that the presence of calanolides in Calophyllum lanigerumis responsible for the anti-HIV activity [8]. From the leaves of Calophyllum lanigerum and Calophyllum inophyllum isoprenyl coumarins was isolated and are reported to be the active substances in inhibiting the HIV-1 reversed transcriptase activity. Shih-Chang Tsai et al explored the anti-inflammatory activity of acetone extract of Calophyllum inophyllum leaves and reported the plant can be used in prevention of inflammatory diseases, and its mechanism may be partially associated with blocking COX-2 and iNOS of RAW 264.7 cells [9]. The present work aimed to demonstrate the anti-inflammatory activity of flavone isolated from the leaves of Calophyllum inophyllum by COX inhibition in lipopoyssacharide stimulated RAW264.7 macrophage cell line. 2 Material and methods 2.1 In-vitro anti-inflammatory activity in cell lines 2.1.1 Principle RAW 264.7 cells were initially procured from National Centre for Cell Sciences (NCCS), Pune, India and were grown in Dulbecco’s modified Eagles medium, DMEM (Sigmaaldrich, USA). DMEM is a complete media with nutrients, proteins, amino acids, buffering system and vitamins, which provides the conditions and physicochemical properties of environment for the growth and maintenance of cell cultures. The cell line was cultured in 25 cm2 tissue culture flask with DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, sodium bicarbonate (Merck, Germany) and antibiotic solution containing: Penicillin (100U/mL), Streptomycin (100µg/mL) and Amphotericin B (2.5µg/mL). Cultured cell lines were kept at 37ºC in a humidified 5% CO2 incubator (NBS Eppendorf, Germany) until the cells were confluent. Serum is a complex mixture of proteins, source of minerals, lipids, hormones, growth and adhesion factors. Fetal bovine serum (FBS) and newborn calf serum (NCS) are most common. The amino acids like cysteine, L‐glutamine and tyrosine are essential for growth and cell proliferation. The major ions-Na+, K+, Mg2+, Ca2+, Cl-, PO43-, SO42-, HCO3--affect osmolarity of culture media. Antibiotic solutions like pencillin and streptomycin and antimycotic agents like kanamycin or amphotericin B are applied in cell cultures to reduce the frequency of contamination. The buffering system is essential to maintain proper pH. For establishing physiological pH for cells CO2 is dissolved in the culture medium. Carbon dioxide establishes equilibrium with bicarbonate ions. The bicarbonate buffers not only show low toxicity, but also help in glucose metabolism. Generally, most of cell lines are maintained at 37°C [10, 11]. World Journal of Advanced Research and Reviews, 2025, 26(01), 3328-3332 3330 2.2 Pro-inflammatory activation of RAW264.7 cells The RAW 264.7cells were grown to 60% confluency followed by activation with 1 µL lipopolysaccharide (LPS: 1µg/mL). Stimulation with lipopolysaccharide (LPS) is to trigger the inflammatory response. LPS stimulated RAW cells were exposed with different concentration (25, 50, 100 µg/mL) of sample solution. Diclofenac sodium, a standard antiinflammatory drug in varying concentration corresponding to the sample was added and incubated for 24 hours. After incubation the anti-inflammatory assay for quantification of COX concentration was performed using the cell lysate. 2.3 COX Inhibition assay The COX activity was assayed by the method of M. C Gierse Walker and J. K. Gierse 2010 [12] 1mL of reaction mixture containing Tris-HCl buffer (pH 8), glutathione 5 mM/L and hemoglobin 20µg/L was used for incubating 100µL cell lysate for 1 minute at 25°C. Arachidonic acid 200 mM/L was used for initiating the reaction. After 20 minutes of incubation at 37°C, 200µL of 10% trichloroacetic acid in 1 N hydrochloric acid was added for terminating the reaction. The supernatant taken after centrifugation was treated with 1% thiobarbiturate. COX activity was determined by reading absorbance at 632 nm. Diclofenac was used as standard and the reaction mixture without any tested compound served as control. COX activity inhibition was expressed in percentage using the following relation. % 𝐨𝐟 𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 = 𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝐜𝐨𝐧𝐭𝐫𝐨𝐥 − 𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝐭𝐞𝐬𝐭 𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝐜𝐨𝐧𝐭𝐫𝐨𝐥 ×𝟏𝟎𝟎 3 Result and Discussion Inflammation is a pathophysiological reaction that induces injury to living tissue to cause a localized build-up of plasmatic fluid and blood cells [13]. Inflammation is managed by a group of drugs known as NSAIDs, or non-steroidal anti-inflammatory medicines [14]. On continuous usage current anti-inflammatory medications have many side effects. Antioxidant-rich plants have been proposed as possible sources of chemicals with anti-inflammatory properties with the least side effects [15]. An important role in inflammatory processes is played by macrophages and they produce inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), which are generated by activated inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. When activated by appropriate stimuli, macrophages also produce different cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNFα). Thus, activated macrophages has a pivotal role in inflammatory diseases via, excess production of inflammatory mediators, such as NO and PGE2, as well as pro-inflammatory cytokines, to promote the inflammatory response [16, 17, 18]. The pro-inflammatory transcription factor NF-κB controls the up-regulation of iNOS and COX-2 during inflammation. Flavonoids that inhibit the induction of this factor are regarded as interesting tools for inflammation control [19, 20]. The standard used in this evaluation of the cyclooxygenase (COX) inhibitory activity was diclofenac, an antiinflammatory medication with very high COX inhibition. The percentage of inhibition and IC50 values of the standard and the isolated flavone is shown in Table 1 and its graphical representation is shown in Figures 1 and 2. Table 1 Percentage of inhibition and IC50 values of the standard and the isolated flavone Conc. of extracts (µg/mL) Percentage inhibition Diclofenac (Standard) Isolated flavone 25 43.35±0.0004 48.32±0.0006 50 65.21±0.0012 61.58±0.0006 100 84.91±0.0002 71.36±0.00011 IC50 31.07µg/mL 22.58 µg/mL World Journal of Advanced Research and Reviews, 2025, 26(01), 3328-3332 3331 Figure 1 Graphical representation of percentage inhibition of COX enzyme activity by Diclofenac standard Figure 2 Graphical representation of percentage inhibition of COX enzyme activity by the isolated flavones In the COX analysis, the Diclofenac exhibited 84.91 percentage of inhibition at a concentration of 100 μg/mL with an IC50 value of 31.07. Isolated flavone exhibited 71.36 percentage of inhibition at a concentration of 100 μg/mL and the IC50 value was obtained as 22.58μg/mL. According to the investigations, isolated flavone from Calophyllum inophyllum appears to have anti-inflammatory properties. When compared to the IC50 value of the standard, the isolated flavone displayed a good IC50 value, which indicates that the flavone from ethanolic leaf extract of Calophyllum inophyllum has the potential to be developed as a non-steroidal anti-inflammatory drug (NSAID). 4 Conclusion For the assessment of the anti-inflammatory potential the in vitro cyclooxygenase assay is commonly used. To estimate the predicted levels of COX inhibition of the compounds in vitro assay is used because it is a quick and simple method as compared to clinical research. In the current work, COX enzyme inhibition assay is performed for the isolated flavone from Calophyllum inophyllum leaves. It can be concluded that the isolated flavone appears to have anti-inflammatory properties, so it can serve as a lead and can further be modified to obtain various derivatives with more potent antiinflammatory activity with improved safety profiles. Compliance with ethical standards Disclosure of conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References [1] Kaur G, Dufour JM. Cell lines: Valuable tools or useless artifacts. Spermatogenesis. 2012; 2(1):1-5. World Journal of Advanced Research and Reviews, 2025, 26(01), 3328-3332 3332 [2] Guzik TJ, Korbut R, Adamek-Guzik T. Nitric oxide and superoxide in inflammation and immune regulation. J. Physiol Pharmacol. 2003; 54(4): 469-87. PMID: 14726604. [3] Kim KM, Kwon YG, Chung HT, Yun YG, Pae HO, Han JA, Ha KS, Kim TW, Kim YM. Methanol extract of Cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing NF-kappaB activation. Toxicol Appl Pharmacol. 2003; 190(1): 1-8. doi: 10.1016/s0041-008x(03)00152-2. PMID: 12831777. [4] Chang HY, Sheu MJ, Yang CH, Lu TC, Chang YS, Peng WH, Huang SS, Huang GJ. Analgesic effects and the mechanisms of anti-inflammation of hispolon in mice. Evid Based Complement Alternat Med. 2011; 2011: 478246. doi: 10.1093/ecam/nep027. Epub 2010 Oct 25. PMID: 19349477; PMCID: PMC3136186. [5] Munekazu I, Hideki T, Toshiyuki T, Shigetomo Y. Two xanthones from root bark of Calophyllum inophyllum. Phytochemistry. 1994; 35(2): 527-532. https://doi.org/10.1016/S0031-9422(00)94795-2. [6] Burkill IH, Birtwistle W, Foxworthy FW, Scrivenor JB. Watson JG. A dictionary of the economic products of the Malay peninsula. 2d ed. Published on behalf of the governments of Malaysia and Singapore by the Ministry of Agriculture and cooperatives. 1966. [7] Kartikar KR, Basu B Das. Indian medicinal plants Vol. 1. 2nd Edition. 1975. [8] Kashman Y, Gustafson KR, Fuller RW, Cardellina JH 2nd, McMahon JB, Currens MJ, Buckheit RW Jr, Hughes SH, Cragg GM, Boyd MR. The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum. J Med Chem. 1992; 35(15): 2735-43. doi: 10.1021/jm00093a004. [9] Tsai SC, Liang YH, Chiang JH, Liu FCL, Wen-Hsin C, Shu-Jen L, Wei W, Chieh-Hsi W, Jing-Ru. Anti-inflammatory effects of Calophyllum inophyllum L. in RAW264.7 cells. Oncology reports. 2012; 28: 1096-102. [10] Rodríguez-Hernandez C, García SE, Olvera-Sandoval C, Ramírez CFL, Muro AG, Francisco GB, Alma. Cell culture: History, Development and Prospects. International Journal of Current Research and Academic Review. 2014; 2: 188-200. [11] Swain PSP, Nanda PK, Nayak SK, Mishra SS, Basic techniques and limitations in establishing cell culture: a mini review. Advances in Animal and Veterinary Science. 2014; 2: 1–10. [12] Gierse Walker MC, Gierse JK, In-vitro assays for cyclooxygenase activity and inhibitor characterization. Cyclooxygenases: Methods in Molecular Biology (Methods and Protocols). 2010; 644: 131-144. [13] Bonheur YDD, Desire DDP, Ernestine N, Christophe M, Theophile D. Anti-inflammatory and antinociceptive effects of the stem bark aqueous extract of Rauwolfia vomitoria (Apocynaceae). World Journal of Pharmacy and Pharmaceutical Sciences. 2015; 4(7): 90-112. [14] Naz R, Ayub H, Nawaz S, Islam ZU, Yasmin T, Bano A, Wakeel A, Zia S, Roberts TH. Antimicrobial activity, toxicity and anti-inflammatory potential of methanolic extracts of four ethnomedicinal plant species from Punjab, Pakistan. BMC Complementary and Alternative Medicine. 2017; 17: 302. https://doi.org/10.1186/s12906-0171815-z). [15] Borquaye LS, Laryea MK, Gasu EN, Boateng MA, Baffour PK, Kyeremateng A, Doh G. Anti-inflammatory and antioxidant activities of extracts of Reissantia indica, Cissus cornifolia and Grosseria vignei. Cogent Biology. 2020; 6(1): 1785755. https://doi.org/10.1080/23312025.2020.1785755). [16] MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol. 1997; 15:323-50. doi: 10.1146/annurev.immunol.15.1.323. PMID: 9143691. [17] Korhonen R, Lahti A, Kankaanranta H, Moilanen E. Nitric Oxide Production and Signaling in Inflammation. Current Drug Targets - Inflammation & Allergy. 2005; 4(4): DOI: 10.2174/1568010054526359 [18] Ha YM, Chung SW, Kim JM, Kim DH, Kim JY, Lee EK, Lee J, Kim YJ, Yoo MA, Jeong KS, Chung HY. Molecular activation of NF-kappaB, pro-inflammatory mediators, and signal pathways in gamma-irradiated mice. Biotechnol Lett. 2010; 32(3): 373-378. doi: 10.1007/s10529-009-0165-4. PMID: 19915799. [19] Raso GM, Meli R, Di Carlo G, Pacilio M, Di Carlo R. Inhibition of inducible nitric oxide synthase and cyclooxygenase2 expression by flavonoids in macrophage J774A.1. Life Sci. 2001; 68(8): 921-931. doi: 10.1016/s00243205(00)00999-1. PMID: 11213362. [20] Kim JY, Park SJ, Yun KJ, Cho YW, Park HJ, Lee KT. Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-induced iNOS and COX-2 expression via the attenuation of NF-kappaB in RAW 264.7 macrophages. Eur J Pharmacol. 2008; 584(1): 175-184. doi: 10.1016/j.ejphar.2008.01.032. Epub 2008 Feb 5. PMID: 18295200.