Dual Anti-Inflammatory Activities of COX-2/5-LOX Driven by Kratom Alkaloid Extracts in Lipopolysaccharide-induced RAW 264.7 Cells

Dual Anti-Inflammatory Activities of COX-2/5-LOX Driven by Kratom Alkaloid Extracts in Lipopolysaccharide-induced RAW 264.7 Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dual Anti-Inflammatory Activities of COX-2/5-LOX Driven by Kratom Alkaloid Extracts in Lipopolysaccharide-induced RAW 264.7 Cells Siti Irma Rahmawati, Dwi Wahyu Indriani, Febby Nurdiya Ningsih, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4628929/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cyclooxygenase (COX) and lipoxygenase (LOX) enzymes play a crucial role in the production of pro-inflammatory eicosanoids, including prostaglandins and leukotrienes (LTs) via arachidonic acid (AA) pathways. Non-steroidal anti-inflammatory drugs (NSAIDs) typically work by inhibiting COX enzymes (COX-1, COX-2) to alleviate inflammatory responses in our bodies. However, the use of these selective COX inhibitors results in an upregulation of the AA pathway. This condition stimulates the LOX enzymes to increase LT production, exacerbating the severity of the disorders. In this study, the alkaloid extract derived from the leaf of Mitragyna speciosa (Kratom) demonstrated a dual inhibitory effect on COX-2/5-LOX enzymes in lipopolysaccharides (LPS)-induced RAW 264.7 macrophage cells. The alkaloid extract containing ~ 46% mitragynine inhibited COX-2 and 5-LOX activity at concentrations of less than 25 ppm with no toxicity to the cells. Above 25 ppm, the alkaloid extract exhibited toxicity to the cells ( e.g ,, ~ 46% viability at 50 ppm) and only inhibited COX-2 activity. In contrast, the Kratom crude extract containing ~ 5% mitragynine did not inhibit COX-2 or 5-LOX activity in LPS-induced RAW 264.7 macrophage cells at more than 25 ppm and did not exhibit toxicity to the cells even at 100 ppm. The alkaloid compounds in the Kratom leaf are likely responsible for this activity, as the alkaloid extract containing these biomolecules suppressed reactive oxygen species (ROS), nitric oxide (NO), inducible nitric oxide synthase (iNOS), and pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Molecular studies also suggested a strong binding affinity of Kratom alkaloids to the active sites of COX-2 and 5-LOX. The dual inhibitory activity of the Kratom alkaloids against COX-2 and 5-LOX provides insights into their potential as safer NSAIDs. Drug Discovery, Design, & Development Mitragyna speciosa cellular oxidative stress dual inhibition COX-2/5-LOX anti-inflammatory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Inflammation is a cellular defense response against toxic or foreign substances and involves the elimination of damaged cells to heal injured tissues or organs (Cock, 2024 ). It is characterized by activating signaling pathways that regulate the amounts of inflammatory mediators, including cytokines, chemokines, and lipid mediators in the local tissue cells (Giménez-Bastida et al., 2021 ). The process results in a pain response, in which people often use non-steroidal anti-inflammatory drugs (NSAIDs) to treat the pain (Wolfarth et al., 2022 ). These drugs commonly act to alleviate inflammatory symptoms by targeting cyclooxygenase enzymes (COX-1 and COX-2) in the arachidonic acid (AA) metabolic pathway (Charlier & Michaux, 2003a ). However, the long-term use of NSAIDs has side effects, including gastrointestinal issues (Tai & McAlindon, 2021), hepatotoxicity as well as injury (Sriuttha et al., 2018), widespread edema (Frishman, 2002), and an increased propensity to bleed (Schafer, 1995 ). For example, rofecoxib (Vioxx™) and valdecoxib (Bextra™) are two COX-2 inhibitors that were removed from the market due to their severe adverse effects, including cardiovascular disease, risk of stroke and cardiac arrest (Ahmadi et al., 2022 ). AA is the primary precursor for producing a cascade of pro-inflammatory metabolites in the inflammatory pathways. This process involves the enzymatic cleavage of membrane-bound AA (arachidonyl phospholipid) by phospholipase-A 2 , releasing free AA that is accessible to COX and Lipoxygenase (LOX) enzymes (Prasher et al., 2019 ). COX and LOX are widely recognized as pro-inflammatory enzymes that play a crucial role in producing pro-inflammatory eicosanoids, including prostaglandins (PGs) and leukotrienes (LTs). COX plays a vital role in inducing the formation of lipid mediators (Tsatsanis et al., 2006 ). Particularly, COX-2 catalyzes the oxidation of arachidonate into prostaglandins G2 (PGG2) during inflammation (Utar et al., 2011a ). The released arachidonate can also be further oxidized to produce a precursor for other prostaglandins, such as prostaglandin H2 (PGH2) and thromboxane. The overexpression of COX-2 is reported to implicate human cancers (Hyde & Missailidis, 2009 ). In addition, the AA pathway produces LTs via the LOX pathway, mediated by the 5-LOX enzyme. LTs are a distinct group of AA derivatives that also have a notable impact on the process of inflammation. They are synthesized by LOX enzymes via hydroperoxy eicosatetraenoic acids (HPETEs) (Mukhopadhyay et al., 2023 ). Among several LOX enzymes, 5-LOX plays a crucial role in the biosynthetic pathway, releasing 5-hydroperoxy eicosatetraenoic acid and leukotriene A4 (LTA4). Thereafter, 5-LOX enzyme converts LTA4 to lipoxins (LXs); i.e. , LXA4 and LXB4 (Wisastra & Dekker, 2014 ). LXs and other specialized pro-resolving mediators are synthesized and released to cease and overcome inflammation, resulting in tissue repair and regeneration (Kretzer et al., 2022 ). The 5-LOX route terminates upon the release of Leukotriene B4 (LTB4), which is a by-product that has a role in various inflammatory and allergic diseases, including atherosclerosis, cancer, and cardiovascular conditions (Mukhopadhyay et al., 2023 ). During the onset of inflammation, LTs and PGs are abundant (Kretzer et al., 2022 ; Wisastra & Dekker, 2014 ). The inhibition of COX enzymes (COX-1, COX-2), as typically performed by NSAIDs, results in an upregulation of the AA pathway. An inherent limitation of COX-2 inhibitors is their concurrent inhibition of COX-1, as selective COX-2 inhibitors typically also suppress COX-1 (Prasher et al., 2019 ). As a result, this condition increases the availability of AA, stimulating LOX enzymes to increase LT production (Fiorucci et al., 2001 ). In this respect, the decreased production of PGs by COX inhibitors shifts AA metabolism to the. alternative LOXs pathway. The AA-LOXs pathway produces LTs, and consequently, the use of NSAIDs is often linked to asthma and allergic reactions (Ahmadi et al., 2022 ). This condition is reportedly associated with undesirable side effects and the severity of its disorders (Gilroy et al., 1998 ). Furthermore, the long-term inhibition of COX-1 and COX-2 by non-selective NSAIDs leads to a decrease in the synthesis of PGs, which in turn impairs the function of the mucosa and causes damage to the gastrointestinal tract. Moreover, COX-2 is crucial for controlling renal function, as such, the use of COX-2 inhibitors can lead to significant adverse effects to individuals who are at risk of renal ischemia, liver cirrhosis, renal insufficiency, cardiovascular diseases, and congestive heart failure. Therefore, suppressing both COX-2 and 5-LOX to inhibit the production of PGs and LTs simultaneously may improve the efficacy of anti-inflammatory agents with fewer side effects. This approach has been considered an appealing choice for creating less risky NSAIDs (Charlier & Michaux, 2003b ). Currently, much effort is being invested in exploring anti-inflammatory compounds with dual inhibition activity targeting both COX-2 and 5-LOX enzymes, i.e. hybrid anti-inflammatory function. This strategy has been targeted to provide safer NSAIDS, wherein the active drugs exhibit good efficacy as anti-inflammatories with minimal side effects (Fiorucci et al., 2001 ). For instance, a novel anti-arthritic γ-sultam S-2474 was reported to display dual inhibition of COX-2/5-LOX without ulcerogenic effects in rats (Inagaki et al., 2000 ). Flavocoxid showed anti-inflammatory benefits by reducing the number of neuronal losses in Alzheimer's mice model due to its ability to inhibit COX-2 and 5-LOX enzymes (Bitto et al., 2017 ). Quercetin has recently been reported to demonstrate the ability to hinder the oxLDL-induced inflammatory process that leads to atherosclerosis by reducing the activity of the Toll-like Receptor or the nuclear factor kappa B (TLR/NF-κB) pathway, including COX-2 and 5-LOX (Gouda et al., 2023 ). Mitragyna speciosa Korth (family Rubiaceae); commonly known as 'Kratom', 'Ithang', or 'Thom' in Thailand, and 'Ketum' or 'biak-biak' in Malaysia; is a native tropical tree of Southeast Asia. It is found in the northern part of Peninsular Malaysia, as well as Central and South Thailand (Parthasarathy et al., 2013 ), and Indonesia. This plant has traditionally been used for its aqueous leaf extract to treat minor illnesses such as fever, diabetes, diarrhea, pain, wound healing, and opioid withdrawal symptoms (Garcia-Romeu et al., 2020 ; Hassan et al., 2013 ; Singh et al., 2016 ; Vicknasingam et al., 2020 ). Although no kratom-derived product has been approved by the US Food and Drug Administration (FDA) yet, kratom has attracted much attention in the United States of America and Europe due to its recreational effect and potential medical applications in relieving chronic pain, opioid use disorder symptoms, alcohol withdrawal, anxiety, and depression (Hassan et al., 2013 ; Veltri & Grundmann, 2019 ). These applications are due to the activity of alkaloids contained in the Kratom leaf, like mitragynine and its derivatives. An in vivo study reported that mitragynine exhibits an anti-inflammatory effect in the chronic and acute inflammation models (Mat et al., 2023 ; Wilson et al., 2021 ). This anti-inflammatory effect is believed to be achieved by suppressing COX-2 mRNA translation and lowering PGs synthesis (Utar et al., 2011a ). However, it remains unclear whether the activity of Kratom alkaloids or extracts obtained from the Kratom leaf affects 5-LOX enzyme activity during inflammation. In this study, the inhibition activity of Kratom extracts, i.e., methanolic-crude and alkaloid extract, against COX-2 and 5-LOX enzymes was evaluated in vitro using Lipopolysaccharides (LPS)-induced RAW 264.7 macrophage cells. Elucidating antioxidant and anti-inflammatory activities toward pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were also presented, aiming to understand the potential synergistic modulation of the Kratom extracts. Exploring the dual inhibition of COX-2/5-LOX is expected to provide insight into Kratom’s potential as a safer anti-inflammatory drug candidate. 2. Materials and Methods 2.1. Samples Preparation Kratom leaves were collected from the local plantation areas in Kapuas Hulu, West Borneo, Indonesia. The dried leaves were then pulverized and filtered through a 0.5 mm mesh filter. The powdered sample was placed in a sealed plastic bag and stored at 4°C for further analysis. Kratom extracts were prepared according to our previous protocols (Bayu et al., 2024 ), resulting in two types of extract, i.e. , methanolic-crude and alkaloid extract. Analysis of alkaloid compounds was performed using the chromatographic technique described in our previous report as well. The typical chromatograms are depicted in Fig. 1 . Mitragynine was confirmed using liquid chromatographic-high resolution mass spectrometry (LC-HRMS) and proton nuclear magnetic resonance ( 1 H NMR) spectroscopic analysis. LC-HRMS was conducted following the protocol described by Windarsih et al. ( 2022 ). The 1 H NMR spectrum was recorded on a Bruker AVANCE III 500 spectrometer (Billerica, MA, USA). The sample was dissolved in deuterated methanol and scanned for 62 scans. 2.2. Antioxidant Activities 2.2.1. Free Radical Scavenger Activity The ABTS (2,2’-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity was determined using a modified ABTS + radical cation decolorization assay (Tang et al., 2020 ). In brief, about 5 mL of 7 mM ABTS aqueous solution (Merck, USA) was reacted with 88 µL mM of 140 nM potassium persulfate solution (K 2 S 2 O 8 ) (Sigma-Aldrich, USA). The mixture was incubated in a dark place at room temperature for 12–16 hours to obtain ABTS + solution. The prepared ABTS + solution was then diluted with an analytical grade ethanol (Sigma-Aldrich, USA) to obtain an initial absorbance of 0.7 at 734 nm. Thereafter, an approximately 20 µL solution containing the extract or standard (Trolox, Sigma-Aldrich, USA) was added to 280 µL of the ABTS + -ethanolic solution. The mixture was transferred to a 96-well plate and incubated at 30°C for 5 minutes in the dark. Afterward, the absorbance was measured at 734 nm using a microplate reader (Tecan Spectrophotometer, Tecan Group Ltd, Switzerland). The percentage inhibition of free radical scavenging activity was calculated using the following equation: $$\% inℎibition= \frac{AB-AE}{AB} x 100$$ where AB = absorbance of the blank sample, and AE = absorbance of the extracts 2.2.2. Ferric Reducing Antioxidant Power (FRAP) Assay The FRAP method evaluates a sample’s capacity to convert Fe 3+ ions in Fe 3+ -TPTZ complexes (ferric-2,4,6-tripyridyl-s-triazine) into Fe 2+ ions (Tang et al., 2020 ). The FRAP method was carried out with a slight modification. In each well of a 96-well plate, approximately 20 µL of extract or ascorbic acid standard solution was mixed with 280 µL of FRAP dye solution, i.e. , a mixture of sodium acetate 300 mM, TPTZ solution and Fe solution in a ratio of 10:1:1. Subsequently, the solution was incubated at 37°C for 10 minutes. The absorbance was measured at 593 nm using a microplate reader (Tecan Spectrophotometer, Tecan Group Ltd, Switzerland). Ascorbic acid (Sigma-Aldrich, USA) was plotted at several concentrations ranging from of 0–50 µg/mL to make a standard curve. All data were expressed as ascorbic acid equivalents (AAE) per gram of dry weight (d.w.) kratom leaf (mg AAE/g d.w.) using standard curve equation. 2.2.3. Total phenolic content (TPC) TPC was determined using a modified Folin-Ciocalteu spectrophotometry method (Tang et al., 2020 ). In each well of a 96-well plate, 25 µL of extract was mixed with 25 µL of Folin-Ciocalteu reagent (Sigma-Aldrich, USA) solution, which was diluted with water at a ratio of 1:3, and 200 µL of water. The mixture was then incubated at room temperature for 5 minutes. Following incubation, the reaction mixture was basified by adding 25 µL of 10% sodium carbonate and incubated for another 60 minutes in the dark. The absorbance was then measured at 765 nm using a spectrophotometric plate reader (Tecan Spectrophotometer; Tecan Group Ltd, Switzerland). Gallic acid (Sigma-Aldrich, USA) was plotted at several concentrations ranging from 0 to 200 µg/mL to create a standard curve. The TPC of the extracts was expressed as gallic acid equivalents (GAE) per gram (dried weight; d.w.) kratom leaf (mg GAE/g d.w.) using a standard curve equation. 2.3. Anti-Inflammatory Activities 2.3.1. Cell Culture A RAW 264.7 macrophage cell line was purchased from the European Collection of Authenticated Cell Culture (ECACC; 91062702). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Thermo Fisher Scientific, NY, USA) with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution (Gibco, Thermo Fisher Scientific) at 37°C in 5% CO 2 incubator. The RAW 264.7 cells were sub cultured and plated until 70–80% confluency for assay. 2.3.2. Cell Viability Assay The cell viability test was carried out using a modified MTT assay(Kumar et al., 2018 ) involving the conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolilum bromide] (Sigma-Aldrich, USA). The RAW 264.7 macrophage cells were seeded, with 100 µL per well, onto 96-well microplates at 1 × 10 5 cells mL − 1 and cultured for 24 hours in a humidified 5% CO 2 incubator at 37°C to facilitate cell attachment (adherent cells). After that, the unattached cells (nonadherent cells) were removed carefully. The cells were subsequently treated with crude or alkaloid extracts in the final concentrations of 25, 50, 100, and 200 ppm (final media volume of 200 µL). The cells were then incubated in a humidified 5% CO 2 incubator at 37°C for 24 hours. Thereafter, about 20 µL of MTT reagent (5 mg mL − 1 ) was added to each well and incubated under 5% CO 2 at 37°C for 4 hours in a dark to form formazan crystals. The formazan crystals were dissolved in 150 µL dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA), and the mixture was incubated in the dark for another 15 minutes at 30 °C. Cell viability was evaluated by measuring the absorbance at 570 nm using a microplate reader and determined using the formula below: $$\text{c}\text{e}\text{l}\text{l} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}= \frac{{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}_{\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{c}\text{e}\text{l}\text{l}\text{s}}}{{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}_{\text{u}\text{n}\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{c}\text{e}\text{l}\text{l}\text{s}}} \text{x} 100\text{\%}$$ 2.3.3. Determination of LPS and H 2 O 2 -Induced Intracellular ROS and NO Production Reactive Oxygen Species (ROS) levels were measured by assessing the fluorescent signal generated from oxidized 2',7'- dichlorofluorescein diacetate (DCFH-DA), using a modified approach from a previous study (Kongkatitham et al., 2018 ). The RAW 264.7 macrophage cells were seeded into 96-well plates at a density of 5 × 10 4 cells per well (100 µL per well). The cells were pre-treated with different concentrations of the sample solutions; i.e. , crude (100, 50, and 25 ppm) and alkaloid extracts (25, 12.5, and 6.25 ppm); and the mixture was incubated for 1 hour. The mixture was then treated with 1 µg mL − 1 of LPS (Sigma Aldrich, USA) and directly incubated for another 24 hours. After the LPS induction, the treated cells were washed with Phosphate Buffer Saline (PBS; Sigma Aldrich, USA), and 100 µL of 10 uM DCFH-DA (Sigma Aldrich, USA) was added. The mixture was incubated in the dark at 37 °C for 45 min. Thereafter, the supernatant was gently removed and washed twice using PBS. Subsequently, the cells were incubated with 100 uM H 2 O 2 (Sigma Aldrich, USA) for 1 hour. The fluorescence was determined using Cytation 5 instrument (Biotek Instruments, Agilent, USA) at excitation and emission wavelengths of 485 nm and 528 nm, respectively. The medium and LPS-induced medium served as negative and positive control, respectively. The nitric oxide (NO) production assay was performed using a modified method from a previous study (Divate & Chung, 2017 ). As described above, the treated cells were incubated with LPS for 24 hours. Then, the medium from the LPS-induced treated cells was transferred to a centrifuge tube. The supernatant collected from LPS-treated cells was used to measure the levels of NO, TNF-α and IL-6. The supernatant was transferred 100 µL to another 96 well plate, followed by addition of 100 µL Gries reagent (Sigma Aldrich, USA). The mixture was incubated for 10 minutes and the NO production was measured at an absorbance of 540 nm. 2.3.4. Measurement of LPS-induced pro-inflammatory cytokines production (TNF-α and IL-6) The supernatant obtained from the LPS induction (section 2.3.3) was used to assess TNF-α and IL-6 levels in the samples using a mouse TNF-α and IL-6 ELISA kit (Elabscience, China). The protein content was first calculated using the Bradford reagent. All procedures were carried out in accordance with the manual instructions for the ELISA kit to determine the TNF-α and IL-6 levels in samples. 2.3.5. Measurement of COX-2 and 5-LOX activities The RAW 264.7 macrophage cells were cultured and treated with LPS (as described above in Determination of LPS and H 2 O 2 -induced Intracellular ROS and NO production). The cells were harvested, washed using PBS, and then lysed using RIPA Buffer (Sigma-aldrich, USA) as the lysis buffer. The protein content in the cell lysates was measured using Bradford reagent, while COX-2 and 5-LOX activities were quantified using an ELISA Kit (Elabscience, China). All reagents, including washing buffer, substrate solution, stop solution, and standards, were prepared according to the manufacturer's protocol. 2.4. Molecular docking simulation The three-dimensional (3D) protein structures of 5-LOX and COX-2 were retrieved from the Protein Data Bank (PDB) using their respective PDB IDs 3V99 and 5IKR. Selection criteria for the crystal structures included considerations such as, resolution, presence of the protein-ligand complex, active site residues, and source organism. Marvinsketch was utilized to model the 3D structures of the ligands, identify the most stable conformation, and save them in mol2 format. Molecular docking analyses were performed using Molegro Virtual Docker 6.0, taking into account the cavities in the protein structure that could serve as active sites for complex formation through bonding with native ligands. The protein-ligand complexes were evaluated based on their binding free energies, represented as docking scores. Post-docking analysis was conducted using BIOVIA Discovery Studio 2021. 2.5. Statistical analysis The statistical analysis was performed using OriginPro 2019 software (OriginLab, Japan), and all graphs were created using the same tool. The mitragynine content and antioxidant assay values were analyzed using two sample student t-tests, with p < 0.001 considered significant. Anti-inflammatory activity values were analyzed using a one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, with p < 0.01 and p < 0.001 considered significant. 3. Results 3.1. Antioxidant Activities of Kratom Extracts It is known that ROS could be produced as a by-product of AA metabolism catalyzed by 5-LOX and COX-2 enzymes (Liu et al., 2023 ). An oxidative defense system (antioxidant) in living organisms maintains the level of ROS, allowing ROS to perform their proper functions without negatively impacting the body (Neha et al., 2019 ; Snezhkina et al., 2019 ). As a result, antioxidant-containing components can ensure an adequate amount of ROS to carry out signaling and physiological tasks without causing inflammation (Jones et al., 2007 ; Neha et al., 2019 ; Snezhkina et al., 2019 ). In light of this, the antioxidant activity of the Kratom extracts was initially assessed to determine its efficiency in stabilizing ROS using several methods. Figure 2 A shows that crude or alkaloid extracts exhibited significant activity, with 94% free radical scavenging activity based on the ABTS assay at a concentration of 1,000 ppm. Interestingly, the FRAP assay revealed that the crude extract was 20 times more effective at scavenging free radical ferric iron (Fe 3+ ) than the alkaloid extract (Fig. 2 B). These two results indicate that both extracts contain active compounds with high antioxidant activity. TPC measurements supported this observation, revealing a high TPC content in the crude extract of Kratom leaf (7224 ± 46.4 mg GAE g − 1 ) (Fig. 2 C). 3.2. Cytotoxicity of Kratom Extracts on RAW 624.7 Macrophage Cells Before assessing the anti-inflammatory activities of Kratom extracts in RAW 264.7 macrophage cells, we evaluated their cytotoxicity using an MTT assay. This assay was conducted to determine suitable concentrations of the extracts that do not kill the cells. As shown in Fig. 3 , the crude extract was not cytotoxic on the RAW 264.7 cell line (91–100% cell viability) at concentrations of 25–200 ppm. In contrast, alkaloid extract exhibits dose-dependent cytotoxicity. No toxicity was observed at 25 ppm (~ 100% cell viability), but at 50–100 ppm, cell viability decreased by 10–46%. Furthermore, the alkaloid extract completely killed RAW 264.7 macrophage cells at 200 ppm. Alkaloids are widely recognized as highly active natural products, and their biological activity has already been shown to be advantageous as therapeutic agents when used in sufficient quantities (Nunes et al., 2020 ). As a result, the anti- inflammatory efficacy of crude and alkaloid extracts was evaluated at 25–100 ppm and 6.25-25 ppm, respectively. 3.3. Anti-inflammatory Activity of Kratom Extracts 3.3.1. The Effect on Intracellular Oxidative Stress In the previous discussion, Kratom extracts demonstrated significant antioxidant activity at a concentration of 1,000 ppm. Given that ROS are produced during metabolic reactions at the cellular level, we evaluated the activity of Kratom extracts in suppressing ROS production in RAW 264.7 macrophage cells. The crude and alkaloid extract concentrations were chosen at 25–100 ppm and 6.25-25 ppm, respectively, to minimize their detrimental influence on cells as previously stated. Oxidative stress was induced by adding H 2 O 2 into RAW 264.7 macrophage cells treated by LPS. The addition of H 2 O 2 exacerbates cellular oxidative stress as shown by an increase in ROS levels compared with control media (Fig. 4 A). Exposing the cells to Kratom extracts significantly reduced ROS production in a dose-dependent manner. When the cells were exposed to maximum concentrations of crude and alkaloid extract (100 ppm crude and 25 ppm alkaloid), ROS production was significantly reduced (50–64%). Fluorescence microscopic images revealed that the cells were destroyed after being exposed to the extracts (Fig. 4 C, D). Interestingly, the results indicate that the alkaloid extract is four times more effective at decreasing ROS generation than the crude extract. To further investigate this, the study was extended to evaluate the suppressive activity of Kratom extracts on the production of Reactive Nitrogen Species (RNS), which are also known to induce excessive inflammation at the cellular level. NO is an RNS that participates in various physiological functions, including the pathogenesis of inflammation. Therefore, observing NO production during inflammatory stimulation may provide more insight into the intracellular antioxidative activity of the extracts. The intracellular NO production in RAW 264.7 macrophage cells treated with LPS was approximately 1.7-fold higher than the untreated cells (Fig. 4 B). Similar to ROS, RNS decreased when LPS-treated cells were exposed to Kratom extracts. Previous studies have found that plant extracts high in phytochemical metabolites may compete with oxygen to capture nitrite free radicals, thereby limiting NO oxidation and displaying anti-inflammatory properties through NO inhibition (Gomathi et al., 2015 ; Ramya et al., 2015 ). Furthermore, the maximum NO attenuation was achieved at ~ 7–8 nM with 100 ppm and 25 ppm of crude and alkaloid extract, respectively. This result confirms that the alkaloid extract exhibits four times more activity than the crude extract in suppressing intracellular oxidative radicals. As a result, crude and alkaloid extracts derived from Kratom leaf have the ability to reduce cellular oxidative stress, hence lowering the risk of inflammation. This is supported by the observation that NO stimulates the expression of genes associated with inflammatory diseases, such as interleukins, 5-LOX, and COX 2 (Cumpstey & Feelisch, 2018 ; Laroux et al., 2001 ). 3.3.2. The effect on TNF-α and IL-6 Level Increased ROS/RNS production by inflammatory and host cells activates signal transduction cascades and alters the transcription factors such as NF-κB. These usually lead to the expression of inflammatory cytokines such as IL-6 and TNF-, chemokines, growth factors, and pro-inflammatory enzymes such as COX-2 and 5-LOX, which attract more inflammatory cells to the site of inflammation and even produce more reactive species (Cumpstey & Feelisch, 2018 ; Lei et al., 2015 ). TNF-α and IL-6 are the prominent pro-inflammatory cytokines released in RAW 264.7 macrophage cells after infection or exposure to LPS (Won et al., 2018 ; W. bin Zhang et al., 2019 ). These pro-inflammatory cytokines can exacerbate the inflammatory response and play an important role in the inflammatory process (Facchin et al., 2022 ). Furthermore, high levels of IL-6 and TNF-α have been linked to several inflammatory conditions. Therefore, measuring TNF-α and IL-6 can help determine the anti-inflammatory properties of bioactive components. Following the LPS-induced inflammation, treatment with the alkaloid extract inhibited TNF-α at concentrations greater than 12.5 ppm, whereas the crude extract inhibited TNF-α at concentrations greater than 50 ppm (Fig. 5 A). However, alkaloid extracts effectively reduced IL-6 level below 25 ppm, whereas crude extract inhibited IL-6 at all concentrations tested (Fig. 5 B). The alkaloid extract derived from Kratom leaf inhibited IL-6 levels in an inverse dose-dependent manner. Meanwhile, Kratom crude extract did not inhibit IL-6 levels in a dose-dependent manner. 3.3.3. The Effect on COX-2 and 5-LOX Activities To gain a better understanding of the crude and alkaloid extracts’ anti-inflammatory efficacy, their effect on COX-2 and 5-LOX activity was studied. Figure 6 A shows that LPS-induced RAW 264.7 cells treated with alkaloid extract had an average COX-2 inhibition of 33.46% across all concentrations. The alkaloid extract demonstrated inhibition of 5-LOX activity in LPS-induced RAW 264.7 macrophage cells at lower concentration (6.25 ppm) in an inverse dose-dependent manner (6.25 ppm = 5.5± 0.8 pmol/min/mg; 12.5 ppm = 14.2 ± 2.9 pmol/min/mg; p < 0.001 compared to media + LPS group) (Fig. 6 B). Conversely, the crude extract treatment exhibited only a 35.06% reduction in COX-2 activity at the lowest concentration (25 ppm) and did not show any activity on inhibition of 5-LOX at any concentration. 4. Discussion ROS are the intermediate products commonly produced during cellular metabolism. They are formed as by-products of cellular oxidative processes, including the metabolism of arachidonic acid by COX and LOX enzymes, and play a significant role as mediators in the modulation of inflammation (Cho et al., 2011 ; Liu et al., 2023 ). These species mainly include the hydroxyl radical (⋅OH), peroxyl (RO 2 ⋅), alkoxyl (RO⋅) superoxide anions (O 2 − ), oxy singlet oxygen ( 1 O 2 ), and hydrogen peroxide (H 2 O 2 ). At normal levels, ROS plays a critical role in cellular signaling pathways, such as cell metabolism, growth, differentiation, and death signaling (Zou et al., 2017 ). At moderate levels, they are defense molecules that destroy inflammation agents such as exogenous pathogens (Liu et al., 2023 ). However, ROS have a strong tendency to react with and damage cellular macromolecules such as proteins, lipids, and nucleic acids. This excessive oxidation can lead to increased inflammatory responses in the organs. Naturally, the level of ROS is maintained by components possessing antioxidant activity. The high antioxidant activity of crude and alkaloid extracts derived from Kratom leaf indicates that these extracts contain phytochemicals that are active in scavenging ROS. A significant amount of phenolics observed in the crude extract should contribute to the high antioxidant activity of Kratom’s crude extract (Fig. 1 C). Phenolic compounds are reported to be one of the effective nutrients in the prevention of oxidative stress (Huang et al., 2005 ; Parthasarathy et al., 2009 ). In this study, the TPC of the crude extract was greater compared to that of the alkaloid extract, which is in line with previous findings (P. Zhang et al., 2023 ). This is because the cascade process during alkaloid extraction involves acid-base reaction and the use of non-polar organic solvents, which separate non-polar alkaloids and polar compounds (non-alkaloids) such as phenolics. Phenolics are a group of polar compounds and, thus, are not extracted when non-polar organic solvents are used during the extraction process to obtain the alkaloid extract. Furthermore, the antioxidant activity of kratom's crude and alkaloid extracts can be attributed to the presence of alkaloid compounds such as mitragynine and its derivatives, including 7-hydroxy mitragynine, paynantheine, speciogynine, and speciociliatine (Fig. 1 ). These compounds have been reported to show antioxidant activity (Elahian et al., 2020 ). In this study, the alkaloid extract exhibited significant ABTS antioxidant activity and was found to contain a high content of mitragynine (45.9±0.9%). The presence of mitragynine was confirmed through LC-MS/MS analysis (Figure S1) and by the proton signals of mitragynine obtained in the 1 H NMR spectra (Figure S2). It should be noted that the lower FRAP value of the alkaloid extract compared to the crude extract does not necessarily indicate a lack of activity in the alkaloid extract. It might be a result of the fact that alkaloids, being weak bases, could potentially alter the redox reaction during the FRAP measurement. The FRAP assay requires an acidic condition (pH 3.6) to get the optimal redox reaction (Huang et al., 2005 ). In contrast, the ABTS assay is carried out under neutral pH conditions, wherein the chromophore reaction should not be significantly affected by the weak basicity of the alkaloid components. In general, kratom extracts exhibit high antioxidant activity (Fig. 2 ), which should be beneficial for scavenging ROS/RNS in LPS-H 2 O 2 -stimulated RAW 264.7 (Fig. 4 ) and for acting as anti-inflammatory agents by inhibiting inflammatory-related enzymes (Fig. 6 ). The evaluation of the anti-inflammatory activity of kratom extracts in vitro involved stimulating RAW 264.7 macrophage cells with LPS and assessing their ability to reduce intracellular free radicals (ROS and RNS), pro-inflammatory cytokines (TNF-α and IL-6) and inflammatory-related enzymes (COX-2 and 5-LOX). The ability of crude and alkaloid extracts to substantially decrease ROS in LPS-H 2 O 2 -stimulated RAW 264.7 cells without killing the cells should be associated with their antioxidant activity to scavenge free radicals (Fig. 2 A-B, 4 A). This is supported by the observation that these kratom extracts could also reduce NO levels in the LPS-treated RAW 264.7 macrophage cells (Fig. 4 B). It should be noted that LPS stimulation can activate NF-κB, which upregulates the inducible nitric oxide synthase (iNOS) enzyme to result in the enhancement of NO production in RAW 264.7 macrophage cells (Jones et al., 2007 ). This is corroborated by previous research that found plant extracts with a high concentration of phytochemical metabolites may compete with oxygen to capture nitrite free radicals, limiting nitric oxide oxidation and thus displaying anti-inflammatory properties through NO inhibition (Gomathi et al., 2015 ; Ramya et al., 2015 ). RAW 264.7 macrophages cells stimulated by LPS, will release various pro-inflammatory cytokines; e.g. , TNF-α, interleukin-1 beta (IL-1β), and interleukin 6 (IL-6) (Won et al., 2018 ; W. bin Zhang et al., 2019 ). TNF-α has the ability to induce apoptosis and trigger the release of other inflammatory cytokines such as IL-1, IL-6, and IL-10. Additionally, it stimulates T cells as well as other inflammatory cells. The alkaloid extract exhibited four times more potent (~ 50% reduction) four times more potently (~ 50% reduction) inhibition four times more potently (~ 50% reduction) than the crude extract did (Fig. 5 A). Furthermore, when the alkaloid extract concentration exceeded 6.25 ppm, TNF-α levels decreased. Nonetheless, the alkaloid extract exhibited significant suppression of IL-6 at the inverse concentration, whereas IL-6 levels were not reduced at 25 ppm of alkaloid extract exposure. In contrast, a crude extract exhibited IL-6 inhibition at concentrations ranging from 25–100 ppm but only exhibited TNF-α inhibition at 100 ppm. We hypothesized that each of the phytochemicals contained in the extracts possessed various activities on these pro-inflammatory cytokines. In particular, the alkaloid components manifest different mechanism of action, prompting further analysis to ascertain the individual magnitude of their effects. The AA pathway plays a key role in numerous inflammatory diseases, where its metabolism involves COXs and LOXs enzymes that lead to the production of a variety of bioactive mediators such as prostanoids and LTs (Wang et al., 2021 ). COX-2 catalyzes the conversion of arachidonate to inflammatory PGs, a process pivotal in inflammation. COX-2 inhibitors are among the most widely used medications due to their anti-inflammatory, antipyretic, and analgesic properties (Ahmadi et al., 2022 ). On the other hand, it has been reported that 5-LOX plays the most important role in the production of LTs, which also act as inflammatory mediators in respiratory, dermatological, and gastrointestinal disorders (Charlier & Michaux, 2003b ). The alkaloid extract demonstrated consistent inhibition of COX-2 across all tested concentrations (6.25-25 ppm), while crude extract concentrations higher than 25 ppm did not reduce COX-2 activity (Fig. 6 A). The alkaloid extract is mainly composed of mitragynine and its derivatives. Mitragynine has been found to inhibit the expression of COX-2 in a dose-dependent manner (Utar et al., 2011b ). Meanwhile, multiple components in the crude extract, besides alkaloids, might work synergistically or antagonistically to induce inflammation to some extent as the crude extract concentration increases. Similar to the interaction between artemisinin and casticin, which is antagonistic at a ratio of 1:3 (v/v), a synergistic interaction has been described for combination ratios ranging from 1:10 to 1:1000 (artemisinin to casticin, v/v) (Suberu et al., 2013 ). Interestingly, the alkaloid extract exhibited the 5-LOX enzyme inhibition in a dose-dependent manner, with 86% and 33% inhibition at 6.25 and 12.5 ppm, respectively (Fig. 6 B). Such activities were not observed when the crude extract was tested. Given that the alkaloid extract contained more mitragynine and its derivatives than the crude extract, these compounds could account for this activity. This hypothesis is supported by molecular docking results, which showed that mitragynine and some of its derivatives, like paynantheine and speciophylline, exhibited good binding affinity with COX-2 and 5-LOX proteins (Table S2). Moreover, mitragynine showed higher binding affinity compared to several opioids, including morphine and methadone. COX-2 possesses three regions of main active sites; i.e. , the hydrophobic pocket (Tyr385, Trp 387, Phe518, Ala201, Tyr 248, Leu352), the hydrophilic region (Arg120, Glu524, Tyr 355), the side pocket (His90, Arg513, Val523); while the binding pocket of 5-LOX is primarily Phe169, Phe610, Ala410, Ala672, Gln363, Gln413 and Ile406 (Bar et al., 2022 ). In this study, mitragynine demostrated multiple interactions with several main active sites of COX-2; i.e. , Arg120, Tyr355, Phe518, Leu 352, Trp 387, Val 523; and with 5-LOX; i.e. , Phe610, Ala410, Ile406, Gln413 (Figure S3A,B). In comparison, celecoxib, a selective COX-2 inhibitor (Goldenberg, 1999 ), formed multiple bonds with COX-2 active sites than with 5-LOX proteins (Figure S3C,D). Morphine showed more limited interactions with these two enzymes (Figure S3E,F), resulting in lower binding affinity compared to mitragynine, as indicated docking scores (Table S1). These findings suggest that an alkaloid extract derived from Kratom leaf effectively inhibits both COX-2 and 5-LOX enzymes, demonstrating dual anti-inflammatory activity. In contrast, the crude extract, similar to most NSAIDs, only inhibits COX-2 enzymes. However, it should be noted that our molecular docking study indicated variations in the binding affinity of each alkaloid compound, and thus, further investigations are warranted to determine the level of inhibitory activity of each component against COX-2 and 5-LOX enzymes. 5. Conclusion The alkaloid extract derived from kratom leaf, containing ~ 46% mitragynine, was found to exhibit significant antioxidant activity, effectively scavenging ROS (< 50%) and NO (34%) in vitro using RAW 264.7 macrophage cells induced with LPS. Its free radical scavenging activity correlated with the extract concentration, where a significant reduction of ROS (< 50%) and NO (34%) levels is observed at 25 ppm, without exhibiting toxicity to the cells. At a sufficient concentration (~ 12.5 ppm), the alkaloid extract also demonstrates activity in reducing the proinflammatory cytokines TNF-α and IL-6, with approximately 4-folds more activity than the crude extract. An interesting feature of the alkaloid extract is its dual anti-inflammatory activity, inhibiting both COX-2 and 5-LOX to some extent. This contrasts with the crude extract, which shows inhibition of COX-2 only. These results support previous reports that describe mitragynine, one of the alkaloids derived from kratom leaf, as being active in inhibiting COX-2. The additional information on the inhibition activity of the kratom alkaloid extract towards 5-LOX enzyme opens a new insight into the potential of the alkaloid compounds in Kratom to be developed as an alternative NSAID with fewer and safer side effects. Declarations Author Contributors All authors played significant roles in the research and manuscript preparation processes. S.I.R. contributed to the conceptualization, methodology, data analysis, investigation, writing- original draft, review, and editing. D.W.I. contributed to the conceptualization, methodology, data analysis, writing- original draft, review and editing, visualization. F.N.N. contributed to the investigation, writing- original draft, review, and editing. M.H. contributed to the investigation, and editing. P.A. contributed to the data analysis, review, and editing. A.R. contributed to the data analysis, review, and editing. F. contributed to in silico study. E.S. contributed to the data analysis and resources. N.L.P.I.D. contributed to the writing- review and editing, funding acquisition. A.B. contributed to the data analysis, investigation, review, and editing. M.Y.P. contributed to the supervision, writing- review and editing. Conflict of interest All authors declared that they have no conflict of interest. Acknowledgment This research was supported by the Research Organization for Health, National Research and Innovation Agency (BRIN), through the DIPA Research Organization for Health fiscal year 2024 (Project Number 6/III.9/HK/2024). The authors would like to acknowledge Jonathan Ardhianto Panggabean and Firmansyah Karim for their assistance with the extraction and chromatographic analysis. References Ahmadi, M., Bekeschus, S., Weltmann, K. D., von Woedtke, T., & Wende, K. (2022). Non-steroidal anti-inflammatory drugs: recent advances in the use of synthetic COX-2 inhibitors. In RSC Medicinal Chemistry . Royal Society of Chemistry. https://doi.org/10.1039/d1md00280e Bar, F. M. 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Evidence-Based Complementary and Alternative Medicine , 2018 . https://doi.org/10.1155/2018/7816923 Zhang, W. bin, Yang, F., Wang, Y., Jiao, F. zhou, Zhang, H. yue, Wang, L. wen, & Gong, Z. jiong. (2019). Inhibition of HDAC6 attenuates LPS-induced inflammation in macrophages by regulating oxidative stress and suppressing the TLR4-MAPK/NF-κB pathways. Biomedicine & Pharmacotherapy , 117 , 109166. https://doi.org/10.1016/J.BIOPHA.2019.109166 Zhang, P., Wei, W., Zhang, X., Wen, C., Ovatlarnporn, C., & Olatunji, O. J. (2023). Antidiabetic and antioxidant activities of Mitragyna speciosa (kratom) leaf extract in type 2 diabetic rats. Biomedicine & Pharmacotherapy , 162 , 114689. https://doi.org/10.1016/J.BIOPHA.2023.114689 Zou, Z., Chang, H., Li, H., & Wang, S. (2017). Induction of reactive oxygen species: an emerging approach for cancer therapy. In Apoptosis (Vol. 22, Issue 11, pp. 1321–1335). Springer New York LLC. https://doi.org/10.1007/s10495-017-1424-9 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryInformation26052024.docx Supplementary Materials - Dual Anti-Inflammatory Activities of COX-2/5-LOX Driven by Kratom Alkaloid Extracts in Lipopolysaccharide-induced RAW 264.7 Cells Highlight.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4628929","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":318201437,"identity":"05bd3167-244c-4bae-82ce-196f78d303be","order_by":0,"name":"Siti Irma Rahmawati","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie2RsQqDMBCGLwhxSdtVKDSvkNKhiw9jlji5dxQKuoidfQu31k251YcQCp3tVopDU5fSKboVmm887uP+nwOwWH4RB6CWg78RQOJxQCEwKQ7U91jtZij6DClilOVnYlB4umpwcVHheY1JB08flq5BEegALlo/qnKZCpIroKwzKLoLMqqisiWJRzIE6pmCHUcFQzFZAR2sKRIMRgUeExTdRdR9q7ZVphUZK2bswk/NtQ8OPt8z9+b1+qc8NQX7QibA5uy/GeYKFovF8g+8ANeZQYfiDOKnAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5376-7860","institution":"Research Center for Vaccine and Drugs, Research Organization for Health, National Research and Innovation Agency (BRIN), Jalan Raya Jakarta-Bogor Km. 46, 16911 West Java, Indonesia","correspondingAuthor":true,"prefix":"","firstName":"Siti","middleName":"Irma","lastName":"Rahmawati","suffix":""},{"id":318202653,"identity":"0bb130ec-abcd-465a-a613-044d2f6e9c1f","order_by":1,"name":"Dwi Wahyu Indriani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDCCA4wNDAkG/+UMGIAMEGBjSCBCy4cKZmNStDAwMM44w5y4ASFEQAvfjeQ2ad42tvTt0ofbHjD8smHgYyegRfJGIkgLT+7OvsR2A8a+NAY2ngf4tRjcTmy7zdsmkbvhDGObBGPPYQY2CQK2QLUYpBuQpOXmjDMJCWAtDD+I0CJ5/2H7jw8VBwx39gC1JDak8RD0C9+Z448NEgwOyJvzsD+T+PDHRk6+nYAtqCCxjYGHFPUg8IdUDaNgFIyCUTASAAC3tEbbXWn4XgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0470-5289","institution":"Research Center for Vaccine and Drugs, Research Organization for Health, National Research and Innovation Agency (BRIN), Jalan Raya Jakarta-Bogor Km. 46, 16911 West Java, Indonesia","correspondingAuthor":true,"prefix":"","firstName":"Dwi","middleName":"Wahyu","lastName":"Indriani","suffix":""},{"id":318202654,"identity":"d65a53fb-82bc-47d6-92c3-ffa92ecfd50e","order_by":2,"name":"Febby Nurdiya Ningsih","email":"","orcid":"https://orcid.org/0000-0003-1322-8445","institution":"Research Center for Vaccine and Drugs, Research Organization for Health, National Research and Innovation Agency (BRIN), Jalan Raya Jakarta-Bogor Km. 46, 16911 West Java, Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Febby","middleName":"Nurdiya","lastName":"Ningsih","suffix":""},{"id":318202655,"identity":"498bac2b-4837-4a5a-9c66-eb8db56abf55","order_by":3,"name":"Mutia Hardhiyuna","email":"","orcid":"https://orcid.org/0000-0003-2074-9409","institution":"Research Center for Vaccine and Drugs, Research Organization for Health, National Research and Innovation Agency (BRIN), Jalan Raya Jakarta-Bogor Km. 46, 16911 West Java, Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Mutia","middleName":"","lastName":"Hardhiyuna","suffix":""},{"id":318206420,"identity":"f5f1de46-5fd8-4cf0-9f24-5d79175d64c3","order_by":4,"name":"Firdayani","email":"","orcid":"https://orcid.org/0000-0001-9967-917X","institution":"Research Center for Vaccine and Drugs, Research Organization for 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Putra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYLCCBAMJOQMGxgYwh40hgaAGxoYPFRbGpGlpnHGmInEDkqX41eu2H3/+mLdNIn279OG2Bwy/bBj42AloMTuTY9gM1JK7sy+x3YCxL42BjecBAS0HchjBWjacYWyTYOw5zMAmQciW888fgrSkGxCv5UaCIdD7EglgLQw/iNLyxnDGhwoJw509QC2JDWk8hP1yPv3BhwSDOnlzHvZnEh/+2MjJtxOwBRUktjHwkKIeBP6QqmEUjIJRMApGAgAAKW5DoLhTxWkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9879-3293","institution":"Research Center for Vaccine and Drugs, Research Organization for Health, National Research and Innovation Agency (BRIN), Jalan Raya Jakarta-Bogor Km. 46, 16911 West Java, Indonesia","correspondingAuthor":true,"prefix":"","firstName":"Masteria","middleName":"Yunovilsa","lastName":"Putra","suffix":""}],"badges":[],"createdAt":"2024-06-24 09:10:03","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4628929/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4628929/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59028950,"identity":"f08e5492-11be-40a0-b75d-ea08c9e1cb33","added_by":"auto","created_at":"2024-06-25 13:52:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":216607,"visible":true,"origin":"","legend":"\u003cp\u003eTypical chromatogram of crude and alkaloid extracts derived from Kratom leaves\u003cstrong\u003e.\u003c/strong\u003e 7-OHMG, MG, PAY, SPG, and SPC are acronym for 7-hydroxymitragynine, mitragynine, paynantheine, speciogynine, and speciociliatine, respectively\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/0800b3e4145a141a316810d4.png"},{"id":59028951,"identity":"0d45160d-61f7-46c7-9064-5f4ca0f7e9e2","added_by":"auto","created_at":"2024-06-25 13:52:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":224160,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity of Kratom extracts at 1,000 ppm by using free radical scavenging ABTS (A) and FRAP assay (B) in comparison with their total phenolic content (C). The antioxidant value was processed statistically using One-way ANOVA with *** p \u0026lt; 0.001 indicating significant difference.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/43d3ac3c5d0d3c8472e9cb37.png"},{"id":59028955,"identity":"546cc56a-a9a1-4890-878d-c76ef29b5811","added_by":"auto","created_at":"2024-06-25 13:52:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":335566,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytotoxicity of Kratom extract in RAW 264.7 macrophage cells (A) and the cells images before (control media (B)) and after treated with the Kratom extracts (crude extract 50 ppm (C), alkaloid extract 50 ppm (D), alkaloid extract 25 ppm (E)). The percent viability data was processed statistically using One-way ANOVA; \u003cem\u003ei.e., \u003c/em\u003e*** p \u0026lt; 0.001 signifying significant difference compared to control media (without any treatment); \u003csup\u003e## \u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e### \u003c/sup\u003ep \u0026lt; 0.001 indicating significant difference from its group (either within crude extract or within alkaloid extract).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/ca436ecce00dea522c46a1be.png"},{"id":59031006,"identity":"0bf5531d-a2b1-42ac-a98f-55f0e3579d99","added_by":"auto","created_at":"2024-06-25 14:08:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":409284,"visible":true,"origin":"","legend":"\u003cp\u003eROS (A) and NO (B) production assessment in LPS-induced RAW 264.7 macrophage cells after treatment with the crude and alkaloid extracts derived from Kratom leaf. The fluorescence images of ROS intensity in the LPS-induced cells without (C) and with the treatment using alkaloid extracts at 25 ppm (D). The ROS and NO production data were processed statistically using One-way ANOVA (*** p \u0026lt; 0.001 indicating significant difference compared to control media (without any treatment); ## p \u0026lt; 0.01, ### p \u0026lt; 0.001 indicating significant difference from its group (either within crude extract or within alkaloid extract)).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/d0c3174159c1523d0e01ba5d.png"},{"id":59028952,"identity":"0a99c758-4716-4e6d-a5f2-e4fdca9549cc","added_by":"auto","created_at":"2024-06-25 13:52:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98101,"visible":true,"origin":"","legend":"\u003cp\u003eTNF-alevels (A) and IL-6 Levels (B) assessment in LPC-induced RAW 264.7 cells after being treated with the crude and alkaloid extracts derived from Kratom leaf. TNF-α levels and IL-6 Levels were processed statistically using One-way ANOVA with *** p \u0026lt; 0.001 indicating significant difference compared to control media (without any treatment); ## p \u0026lt; 0.01, ### p \u0026lt; 0.001 indicating significant difference from its group (either within crude extract or within alkaloid extract).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/78eb7e68c8548d7e282efd51.png"},{"id":59028956,"identity":"8f577d3c-a071-4068-94b5-4a3c4ede816e","added_by":"auto","created_at":"2024-06-25 13:52:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":120326,"visible":true,"origin":"","legend":"\u003cp\u003eThe inhibition profile of COX-2 (A) and 5-LOX activity (B) in RAW 264.7 macrophage cells after exposed with crude and alkaloid extract derived from Kratom leaf. The COX-2 and 5-LOX activity were processed statistically using One-way ANOVA (*** p \u0026lt; 0.001 indicating significant difference compared to control media (without any treatment); ## p \u0026lt; 0.01, ### p \u0026lt; 0.001 indicating significant difference from its group (either within crude extract or within alkaloid extract))\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/2646eea1579d4f4929c81be2.png"},{"id":59031887,"identity":"d495b09c-9be2-4aa0-8080-608fd97fd391","added_by":"auto","created_at":"2024-06-25 14:16:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2205052,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/4e410f10-f297-44ca-a658-2e1af632a1b2.pdf"},{"id":59029904,"identity":"4a0bbe21-19a6-446a-a94e-e7cec1fced3d","added_by":"auto","created_at":"2024-06-25 14:00:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1571522,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Materials - Dual Anti-Inflammatory Activities of COX-2/5-LOX Driven by Kratom Alkaloid Extracts in Lipopolysaccharide-induced RAW 264.7 Cells\u003c/p\u003e","description":"","filename":"SupplementaryInformation26052024.docx","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/7d2540f35baa8528e002006c.docx"},{"id":59028954,"identity":"c7caa5ee-5260-461c-934e-3efb16c8351b","added_by":"auto","created_at":"2024-06-25 13:52:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":298032,"visible":true,"origin":"","legend":"","description":"","filename":"Highlight.docx","url":"https://assets-eu.researchsquare.com/files/rs-4628929/v1/55c21a2a4152da68bd9dffa0.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eDual Anti-Inflammatory Activities of COX-2/5-LOX Driven by Kratom Alkaloid Extracts in Lipopolysaccharide-induced RAW 264.7 Cells\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInflammation is a cellular defense response against toxic or foreign substances and involves the elimination of damaged cells to heal injured tissues or organs (Cock, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It is characterized by activating signaling pathways that regulate the amounts of inflammatory mediators, including cytokines, chemokines, and lipid mediators in the local tissue cells (Gim\u0026eacute;nez-Bastida et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The process results in a pain response, in which people often use non-steroidal anti-inflammatory drugs (NSAIDs) to treat the pain (Wolfarth et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These drugs commonly act to alleviate inflammatory symptoms by targeting cyclooxygenase enzymes (COX-1 and COX-2) in the arachidonic acid (AA) metabolic pathway (Charlier \u0026amp; Michaux, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003a\u003c/span\u003e). However, the long-term use of NSAIDs has side effects, including gastrointestinal issues (Tai \u0026amp; McAlindon, 2021), hepatotoxicity as well as injury (Sriuttha et al., 2018), widespread edema (Frishman, 2002), and an increased propensity to bleed (Schafer, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). For example, rofecoxib (Vioxx\u0026trade;) and valdecoxib (Bextra\u0026trade;) are two COX-2 inhibitors that were removed from the market due to their severe adverse effects, including cardiovascular disease, risk of stroke and cardiac arrest (Ahmadi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAA is the primary precursor for producing a cascade of pro-inflammatory metabolites in the inflammatory pathways. This process involves the enzymatic cleavage of membrane-bound AA (arachidonyl phospholipid) by phospholipase-A\u003csub\u003e2\u003c/sub\u003e, releasing free AA that is accessible to COX and Lipoxygenase (LOX) enzymes (Prasher et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). COX and LOX are widely recognized as pro-inflammatory enzymes that play a crucial role in producing pro-inflammatory eicosanoids, including prostaglandins (PGs) and leukotrienes (LTs). COX plays a vital role in inducing the formation of lipid mediators (Tsatsanis et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Particularly, COX-2 catalyzes the oxidation of arachidonate into prostaglandins G2 (PGG2) during inflammation (Utar et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e). The released arachidonate can also be further oxidized to produce a precursor for other prostaglandins, such as prostaglandin H2 (PGH2) and thromboxane. The overexpression of COX-2 is reported to implicate human cancers (Hyde \u0026amp; Missailidis, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, the AA pathway produces LTs via the LOX pathway, mediated by the 5-LOX enzyme. LTs are a distinct group of AA derivatives that also have a notable impact on the process of inflammation. They are synthesized by LOX enzymes via hydroperoxy eicosatetraenoic acids (HPETEs) (Mukhopadhyay et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among several LOX enzymes, 5-LOX plays a crucial role in the biosynthetic pathway, releasing 5-hydroperoxy eicosatetraenoic acid and leukotriene A4 (LTA4). Thereafter, 5-LOX enzyme converts LTA4 to lipoxins (LXs); \u003cem\u003ei.e.\u003c/em\u003e, LXA4 and LXB4 (Wisastra \u0026amp; Dekker, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). LXs and other specialized pro-resolving mediators are synthesized and released to cease and overcome inflammation, resulting in tissue repair and regeneration (Kretzer et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The 5-LOX route terminates upon the release of Leukotriene B4 (LTB4), which is a by-product that has a role in various inflammatory and allergic diseases, including atherosclerosis, cancer, and cardiovascular conditions (Mukhopadhyay et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the onset of inflammation, LTs and PGs are abundant (Kretzer et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wisastra \u0026amp; Dekker, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The inhibition of COX enzymes (COX-1, COX-2), as typically performed by NSAIDs, results in an upregulation of the AA pathway. An inherent limitation of COX-2 inhibitors is their concurrent inhibition of COX-1, as selective COX-2 inhibitors typically also suppress COX-1 (Prasher et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As a result, this condition increases the availability of AA, stimulating LOX enzymes to increase LT production (Fiorucci et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In this respect, the decreased production of PGs by COX inhibitors shifts AA metabolism to the. alternative LOXs pathway. The AA-LOXs pathway produces LTs, and consequently, the use of NSAIDs is often linked to asthma and allergic reactions (Ahmadi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This condition is reportedly associated with undesirable side effects and the severity of its disorders (Gilroy et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Furthermore, the long-term inhibition of COX-1 and COX-2 by non-selective NSAIDs leads to a decrease in the synthesis of PGs, which in turn impairs the function of the mucosa and causes damage to the gastrointestinal tract. Moreover, COX-2 is crucial for controlling renal function, as such, the use of COX-2 inhibitors can lead to significant adverse effects to individuals who are at risk of renal ischemia, liver cirrhosis, renal insufficiency, cardiovascular diseases, and congestive heart failure. Therefore, suppressing both COX-2 and 5-LOX to inhibit the production of PGs and LTs simultaneously may improve the efficacy of anti-inflammatory agents with fewer side effects. This approach has been considered an appealing choice for creating less risky NSAIDs (Charlier \u0026amp; Michaux, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrently, much effort is being invested in exploring anti-inflammatory compounds with dual inhibition activity targeting both COX-2 and 5-LOX enzymes, \u003cem\u003ei.e.\u003c/em\u003e hybrid anti-inflammatory function. This strategy has been targeted to provide safer NSAIDS, wherein the active drugs exhibit good efficacy as anti-inflammatories with minimal side effects (Fiorucci et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). For instance, a novel anti-arthritic γ-sultam S-2474 was reported to display dual inhibition of COX-2/5-LOX without ulcerogenic effects in rats (Inagaki et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Flavocoxid showed anti-inflammatory benefits by reducing the number of neuronal losses in Alzheimer's mice model due to its ability to inhibit COX-2 and 5-LOX enzymes (Bitto et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Quercetin has recently been reported to demonstrate the ability to hinder the oxLDL-induced inflammatory process that leads to atherosclerosis by reducing the activity of the Toll-like Receptor or the nuclear factor kappa B (TLR/NF-κB) pathway, including COX-2 and 5-LOX (Gouda et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMitragyna speciosa\u003c/em\u003e Korth (family Rubiaceae); commonly known as 'Kratom', 'Ithang', or 'Thom' in Thailand, and 'Ketum' or 'biak-biak' in Malaysia; is a native tropical tree of Southeast Asia. It is found in the northern part of Peninsular Malaysia, as well as Central and South Thailand (Parthasarathy et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and Indonesia. This plant has traditionally been used for its aqueous leaf extract to treat minor illnesses such as fever, diabetes, diarrhea, pain, wound healing, and opioid withdrawal symptoms (Garcia-Romeu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hassan et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Vicknasingam et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although no kratom-derived product has been approved by the US Food and Drug Administration (FDA) yet, kratom has attracted much attention in the United States of America and Europe due to its recreational effect and potential medical applications in relieving chronic pain, opioid use disorder symptoms, alcohol withdrawal, anxiety, and depression (Hassan et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Veltri \u0026amp; Grundmann, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These applications are due to the activity of alkaloids contained in the Kratom leaf, like mitragynine and its derivatives.\u003c/p\u003e \u003cp\u003eAn \u003cem\u003ein vivo\u003c/em\u003e study reported that mitragynine exhibits an anti-inflammatory effect in the chronic and acute inflammation models (Mat et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wilson et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This anti-inflammatory effect is believed to be achieved by suppressing COX-2 mRNA translation and lowering PGs synthesis (Utar et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e). However, it remains unclear whether the activity of Kratom alkaloids or extracts obtained from the Kratom leaf affects 5-LOX enzyme activity during inflammation. In this study, the inhibition activity of Kratom extracts, i.e., methanolic-crude and alkaloid extract, against COX-2 and 5-LOX enzymes was evaluated \u003cem\u003ein vitro\u003c/em\u003e using Lipopolysaccharides (LPS)-induced RAW 264.7 macrophage cells. Elucidating antioxidant and anti-inflammatory activities toward pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were also presented, aiming to understand the potential synergistic modulation of the Kratom extracts. Exploring the dual inhibition of COX-2/5-LOX is expected to provide insight into Kratom\u0026rsquo;s potential as a safer anti-inflammatory drug candidate.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Samples Preparation\u003c/h2\u003e \u003cp\u003eKratom leaves were collected from the local plantation areas in Kapuas Hulu, West Borneo, Indonesia. The dried leaves were then pulverized and filtered through a 0.5 mm mesh filter. The powdered sample was placed in a sealed plastic bag and stored at 4\u0026deg;C for further analysis. Kratom extracts were prepared according to our previous protocols (Bayu et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), resulting in two types of extract, \u003cem\u003ei.e.\u003c/em\u003e, methanolic-crude and alkaloid extract. Analysis of alkaloid compounds was performed using the chromatographic technique described in our previous report as well. The typical chromatograms are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Mitragynine was confirmed using liquid chromatographic-high resolution mass spectrometry (LC-HRMS) and proton nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR) spectroscopic analysis. LC-HRMS was conducted following the protocol described by Windarsih et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum was recorded on a Bruker AVANCE III 500 spectrometer (Billerica, MA, USA). The sample was dissolved in deuterated methanol and scanned for 62 scans.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Antioxidant Activities\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Free Radical Scavenger Activity\u003c/h2\u003e \u003cp\u003eThe ABTS (2,2\u0026rsquo;-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity was determined using a modified ABTS\u003csup\u003e+\u003c/sup\u003e radical cation decolorization assay (Tang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In brief, about 5 mL of 7 mM ABTS aqueous solution (Merck, USA) was reacted with 88 \u0026micro;L mM of 140 nM potassium persulfate solution (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e) (Sigma-Aldrich, USA). The mixture was incubated in a dark place at room temperature for 12\u0026ndash;16 hours to obtain ABTS\u003csup\u003e+\u003c/sup\u003e solution. The prepared ABTS\u003csup\u003e+\u003c/sup\u003e solution was then diluted with an analytical grade ethanol (Sigma-Aldrich, USA) to obtain an initial absorbance of 0.7 at 734 nm. Thereafter, an approximately 20 \u0026micro;L solution containing the extract or standard (Trolox, Sigma-Aldrich, USA) was added to 280 \u0026micro;L of the ABTS\u003csup\u003e+\u003c/sup\u003e-ethanolic solution. The mixture was transferred to a 96-well plate and incubated at 30\u0026deg;C for 5 minutes in the dark. Afterward, the absorbance was measured at 734 nm using a microplate reader (Tecan Spectrophotometer, Tecan Group Ltd, Switzerland). The percentage inhibition of free radical scavenging activity was calculated using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\% inℎibition= \\frac{AB-AE}{AB} x 100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere AB\u0026thinsp;=\u0026thinsp;absorbance of the blank sample, and AE\u0026thinsp;=\u0026thinsp;absorbance of the extracts\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Ferric Reducing Antioxidant Power (FRAP) Assay\u003c/h2\u003e \u003cp\u003eThe FRAP method evaluates a sample\u0026rsquo;s capacity to convert Fe\u003csup\u003e3+\u003c/sup\u003e ions in Fe\u003csup\u003e3+\u003c/sup\u003e-TPTZ complexes (ferric-2,4,6-tripyridyl-s-triazine) into Fe\u003csup\u003e2+\u003c/sup\u003e ions (Tang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The FRAP method was carried out with a slight modification. In each well of a 96-well plate, approximately 20 \u0026micro;L of extract or ascorbic acid standard solution was mixed with 280 \u0026micro;L of FRAP dye solution, \u003cem\u003ei.e.\u003c/em\u003e, a mixture of sodium acetate 300 mM, TPTZ solution and Fe solution in a ratio of 10:1:1. Subsequently, the solution was incubated at 37\u0026deg;C for 10 minutes. The absorbance was measured at 593 nm using a microplate reader (Tecan Spectrophotometer, Tecan Group Ltd, Switzerland). Ascorbic acid (Sigma-Aldrich, USA) was plotted at several concentrations ranging from of 0\u0026ndash;50 \u0026micro;g/mL to make a standard curve. All data were expressed as ascorbic acid equivalents (AAE) per gram of dry weight (d.w.) kratom leaf (mg AAE/g d.w.) using standard curve equation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Total phenolic content (TPC)\u003c/h2\u003e \u003cp\u003eTPC was determined using a modified Folin-Ciocalteu spectrophotometry method (Tang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In each well of a 96-well plate, 25 \u0026micro;L of extract was mixed with 25 \u0026micro;L of Folin-Ciocalteu reagent (Sigma-Aldrich, USA) solution, which was diluted with water at a ratio of 1:3, and 200 \u0026micro;L of water. The mixture was then incubated at room temperature for 5 minutes. Following incubation, the reaction mixture was basified by adding 25 \u0026micro;L of 10% sodium carbonate and incubated for another 60 minutes in the dark. The absorbance was then measured at 765 nm using a spectrophotometric plate reader (Tecan Spectrophotometer; Tecan Group Ltd, Switzerland). Gallic acid (Sigma-Aldrich, USA) was plotted at several concentrations ranging from 0 to 200 \u0026micro;g/mL to create a standard curve. The TPC of the extracts was expressed as gallic acid equivalents (GAE) per gram (dried weight; d.w.) kratom leaf (mg GAE/g d.w.) using a standard curve equation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Anti-Inflammatory Activities\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Cell Culture\u003c/h2\u003e \u003cp\u003eA RAW 264.7 macrophage cell line was purchased from the European Collection of Authenticated Cell Culture (ECACC; 91062702). The cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; Gibco, Thermo Fisher Scientific, NY, USA) with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution (Gibco, Thermo Fisher Scientific) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The RAW 264.7 cells were sub cultured and plated until 70\u0026ndash;80% confluency for assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Cell Viability Assay\u003c/h2\u003e \u003cp\u003eThe cell viability test was carried out using a modified MTT assay(Kumar et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) involving the conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolilum bromide] (Sigma-Aldrich, USA). The RAW 264.7 macrophage cells were seeded, with 100 \u0026micro;L per well, onto 96-well microplates at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and cultured for 24 hours in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C to facilitate cell attachment (adherent cells). After that, the unattached cells (nonadherent cells) were removed carefully. The cells were subsequently treated with crude or alkaloid extracts in the final concentrations of 25, 50, 100, and 200 ppm (final media volume of 200 \u0026micro;L). The cells were then incubated in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C for 24 hours. Thereafter, about 20 \u0026micro;L of MTT reagent (5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added to each well and incubated under 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 4 hours in a dark to form formazan crystals. The formazan crystals were dissolved in 150 \u0026micro;L dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA), and the mixture was incubated in the dark for another 15 minutes at 30 \u0026deg;C. Cell viability was evaluated by measuring the absorbance at 570 nm using a microplate reader and determined using the formula below:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\text{c}\\text{e}\\text{l}\\text{l} \\text{v}\\text{i}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}= \\frac{{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}}_{\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d} \\text{c}\\text{e}\\text{l}\\text{l}\\text{s}}}{{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}}_{\\text{u}\\text{n}\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d} \\text{c}\\text{e}\\text{l}\\text{l}\\text{s}}} \\text{x} 100\\text{\\%}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Determination of LPS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-Induced Intracellular ROS and NO Production\u003c/h2\u003e \u003cp\u003eReactive Oxygen Species (ROS) levels were measured by assessing the fluorescent signal generated from oxidized 2',7'- dichlorofluorescein diacetate (DCFH-DA), using a modified approach from a previous study (Kongkatitham et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The RAW 264.7 macrophage cells were seeded into 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well (100 \u0026micro;L per well). The cells were pre-treated with different concentrations of the sample solutions; \u003cem\u003ei.e.\u003c/em\u003e, crude (100, 50, and 25 ppm) and alkaloid extracts (25, 12.5, and 6.25 ppm); and the mixture was incubated for 1 hour. The mixture was then treated with 1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of LPS (Sigma Aldrich, USA) and directly incubated for another 24 hours. After the LPS induction, the treated cells were washed with Phosphate Buffer Saline (PBS; Sigma Aldrich, USA), and 100 \u0026micro;L of 10 uM DCFH-DA (Sigma Aldrich, USA) was added. The mixture was incubated in the dark at 37 \u0026deg;C for 45 min. Thereafter, the supernatant was gently removed and washed twice using PBS. Subsequently, the cells were incubated with 100 uM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Sigma Aldrich, USA) for 1 hour. The fluorescence was determined using Cytation 5 instrument (Biotek Instruments, Agilent, USA) at excitation and emission wavelengths of 485 nm and 528 nm, respectively. The medium and LPS-induced medium served as negative and positive control, respectively.\u003c/p\u003e \u003cp\u003eThe nitric oxide (NO) production assay was performed using a modified method from a previous study (Divate \u0026amp; Chung, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As described above, the treated cells were incubated with LPS for 24 hours. Then, the medium from the LPS-induced treated cells was transferred to a centrifuge tube. The supernatant collected from LPS-treated cells was used to measure the levels of NO, TNF-α and IL-6. The supernatant was transferred 100 \u0026micro;L to another 96 well plate, followed by addition of 100 \u0026micro;L Gries reagent (Sigma Aldrich, USA). The mixture was incubated for 10 minutes and the NO production was measured at an absorbance of 540 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. Measurement of LPS-induced pro-inflammatory cytokines production (TNF-α and IL-6)\u003c/h2\u003e \u003cp\u003eThe supernatant obtained from the LPS induction (section 2.3.3) was used to assess TNF-α and IL-6 levels in the samples using a mouse TNF-α and IL-6 ELISA kit (Elabscience, China). The protein content was first calculated using the Bradford reagent. All procedures were carried out in accordance with the manual instructions for the ELISA kit to determine the TNF-α and IL-6 levels in samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5. Measurement of COX-2 and 5-LOX activities\u003c/h2\u003e \u003cp\u003eThe RAW 264.7 macrophage cells were cultured and treated with LPS (as described above in Determination of LPS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced Intracellular ROS and NO production). The cells were harvested, washed using PBS, and then lysed using RIPA Buffer (Sigma-aldrich, USA) as the lysis buffer. The protein content in the cell lysates was measured using Bradford reagent, while COX-2 and 5-LOX activities were quantified using an ELISA Kit (Elabscience, China). All reagents, including washing buffer, substrate solution, stop solution, and standards, were prepared according to the manufacturer's protocol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Molecular docking simulation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe three-dimensional (3D) protein structures of 5-LOX and COX-2 were retrieved from the Protein Data Bank (PDB) using their respective PDB IDs 3V99 and 5IKR. Selection criteria for the crystal structures included considerations such as, resolution, presence of the protein-ligand complex, active site residues, and source organism. Marvinsketch was utilized to model the 3D structures of the ligands, identify the most stable conformation, and save them in mol2 format. Molecular docking analyses were performed using Molegro Virtual Docker 6.0, taking into account the cavities in the protein structure that could serve as active sites for complex formation through bonding with native ligands. The protein-ligand complexes were evaluated based on their binding free energies, represented as docking scores. Post-docking analysis was conducted using BIOVIA Discovery Studio 2021.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed using OriginPro 2019 software (OriginLab, Japan), and all graphs were created using the same tool. The mitragynine content and antioxidant assay values were analyzed using two sample student t-tests, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 considered significant. Anti-inflammatory activity values were analyzed using a one-way analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s post hoc test, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Antioxidant Activities of Kratom Extracts\u003c/h2\u003e \u003cp\u003eIt is known that ROS could be produced as a by-product of AA metabolism catalyzed by 5-LOX and COX-2 enzymes (Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). An oxidative defense system (antioxidant) in living organisms maintains the level of ROS, allowing ROS to perform their proper functions without negatively impacting the body (Neha et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Snezhkina et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As a result, antioxidant-containing components can ensure an adequate amount of ROS to carry out signaling and physiological tasks without causing inflammation (Jones et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Neha et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Snezhkina et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In light of this, the antioxidant activity of the Kratom extracts was initially assessed to determine its efficiency in stabilizing ROS using several methods. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows that crude or alkaloid extracts exhibited significant activity, with 94% free radical scavenging activity based on the ABTS assay at a concentration of 1,000 ppm. Interestingly, the FRAP assay revealed that the crude extract was 20 times more effective at scavenging free radical ferric iron (Fe\u003csup\u003e3+\u003c/sup\u003e) than the alkaloid extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These two results indicate that both extracts contain active compounds with high antioxidant activity. TPC measurements supported this observation, revealing a high TPC content in the crude extract of Kratom leaf (7224\u0026thinsp;\u0026plusmn;\u0026thinsp;46.4 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Cytotoxicity of Kratom Extracts on RAW 624.7 Macrophage Cells\u003c/h2\u003e \u003cp\u003eBefore assessing the anti-inflammatory activities of Kratom extracts in RAW 264.7 macrophage cells, we evaluated their cytotoxicity using an MTT assay. This assay was conducted to determine suitable concentrations of the extracts that do not kill the cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the crude extract was not cytotoxic on the RAW 264.7 cell line (91\u0026ndash;100% cell viability) at concentrations of 25\u0026ndash;200 ppm. In contrast, alkaloid extract exhibits dose-dependent cytotoxicity. No toxicity was observed at 25 ppm (~\u0026thinsp;100% cell viability), but at 50\u0026ndash;100 ppm, cell viability decreased by 10\u0026ndash;46%. Furthermore, the alkaloid extract completely killed RAW 264.7 macrophage cells at 200 ppm. Alkaloids are widely recognized as highly active natural products, and their biological activity has already been shown to be advantageous as therapeutic agents when used in sufficient quantities (Nunes et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a result, the anti- inflammatory efficacy of crude and alkaloid extracts was evaluated at 25\u0026ndash;100 ppm and 6.25-25 ppm, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Anti-inflammatory Activity of Kratom Extracts\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. The Effect on Intracellular Oxidative Stress\u003c/h2\u003e \u003cp\u003eIn the previous discussion, Kratom extracts demonstrated significant antioxidant activity at a concentration of 1,000 ppm. Given that ROS are produced during metabolic reactions at the cellular level, we evaluated the activity of Kratom extracts in suppressing ROS production in RAW 264.7 macrophage cells. The crude and alkaloid extract concentrations were chosen at 25\u0026ndash;100 ppm and 6.25-25 ppm, respectively, to minimize their detrimental influence on cells as previously stated. Oxidative stress was induced by adding H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into RAW 264.7 macrophage cells treated by LPS.\u003c/p\u003e \u003cp\u003eThe addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exacerbates cellular oxidative stress as shown by an increase in ROS levels compared with control media (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Exposing the cells to Kratom extracts significantly reduced ROS production in a dose-dependent manner. When the cells were exposed to maximum concentrations of crude and alkaloid extract (100 ppm crude and 25 ppm alkaloid), ROS production was significantly reduced (50\u0026ndash;64%). Fluorescence microscopic images revealed that the cells were destroyed after being exposed to the extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Interestingly, the results indicate that the alkaloid extract is four times more effective at decreasing ROS generation than the crude extract.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate this, the study was extended to evaluate the suppressive activity of Kratom extracts on the production of Reactive Nitrogen Species (RNS), which are also known to induce excessive inflammation at the cellular level. NO is an RNS that participates in various physiological functions, including the pathogenesis of inflammation. Therefore, observing NO production during inflammatory stimulation may provide more insight into the intracellular antioxidative activity of the extracts. The intracellular NO production in RAW 264.7 macrophage cells treated with LPS was approximately 1.7-fold higher than the untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Similar to ROS, RNS decreased when LPS-treated cells were exposed to Kratom extracts. Previous studies have found that plant extracts high in phytochemical metabolites may compete with oxygen to capture nitrite free radicals, thereby limiting NO oxidation and displaying anti-inflammatory properties through NO inhibition (Gomathi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ramya et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, the maximum NO attenuation was achieved at ~\u0026thinsp;7\u0026ndash;8 nM with 100 ppm and 25 ppm of crude and alkaloid extract, respectively. This result confirms that the alkaloid extract exhibits four times more activity than the crude extract in suppressing intracellular oxidative radicals. As a result, crude and alkaloid extracts derived from Kratom leaf have the ability to reduce cellular oxidative stress, hence lowering the risk of inflammation. This is supported by the observation that NO stimulates the expression of genes associated with inflammatory diseases, such as interleukins, 5-LOX, and COX 2 (Cumpstey \u0026amp; Feelisch, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Laroux et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. The effect on TNF-α and IL-6 Level\u003c/h2\u003e \u003cp\u003eIncreased ROS/RNS production by inflammatory and host cells activates signal transduction cascades and alters the transcription factors such as NF-κB. These usually lead to the expression of inflammatory cytokines such as IL-6 and TNF-, chemokines, growth factors, and pro-inflammatory enzymes such as COX-2 and 5-LOX, which attract more inflammatory cells to the site of inflammation and even produce more reactive species (Cumpstey \u0026amp; Feelisch, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lei et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). TNF-α and IL-6 are the prominent pro-inflammatory cytokines released in RAW 264.7 macrophage cells after infection or exposure to LPS (Won et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; W. bin Zhang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These pro-inflammatory cytokines can exacerbate the inflammatory response and play an important role in the inflammatory process (Facchin et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, high levels of IL-6 and TNF-α have been linked to several inflammatory conditions. Therefore, measuring TNF-α and IL-6 can help determine the anti-inflammatory properties of bioactive components.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing the LPS-induced inflammation, treatment with the alkaloid extract inhibited TNF-α at concentrations greater than 12.5 ppm, whereas the crude extract inhibited TNF-α at concentrations greater than 50 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, alkaloid extracts effectively reduced IL-6 level below 25 ppm, whereas crude extract inhibited IL-6 at all concentrations tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The alkaloid extract derived from Kratom leaf inhibited IL-6 levels in an inverse dose-dependent manner. Meanwhile, Kratom crude extract did not inhibit IL-6 levels in a dose-dependent manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. The Effect on COX-2 and 5-LOX Activities\u003c/h2\u003e \u003cp\u003eTo gain a better understanding of the crude and alkaloid extracts\u0026rsquo; anti-inflammatory efficacy, their effect on COX-2 and 5-LOX activity was studied. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows that LPS-induced RAW 264.7 cells treated with alkaloid extract had an average COX-2 inhibition of 33.46% across all concentrations. The alkaloid extract demonstrated inhibition of 5-LOX activity in LPS-induced RAW 264.7 macrophage cells at lower concentration (6.25 ppm) in an inverse dose-dependent manner (6.25 ppm\u0026thinsp;=\u0026thinsp;5.5\u0026plusmn; 0.8 pmol/min/mg; 12.5 ppm\u0026thinsp;=\u0026thinsp;14.2 \u0026plusmn; 2.9 pmol/min/mg; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to media\u0026thinsp;+\u0026thinsp;LPS group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Conversely, the crude extract treatment exhibited only a 35.06% reduction in COX-2 activity at the lowest concentration (25 ppm) and did not show any activity on inhibition of 5-LOX at any concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eROS are the intermediate products commonly produced during cellular metabolism. They are formed as by-products of cellular oxidative processes, including the metabolism of arachidonic acid by COX and LOX enzymes, and play a significant role as mediators in the modulation of inflammation (Cho et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These species mainly include the hydroxyl radical (\u0026sdot;OH), peroxyl (RO\u003csub\u003e2\u003c/sub\u003e\u0026sdot;), alkoxyl (RO\u0026sdot;) superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), oxy singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). At normal levels, ROS plays a critical role in cellular signaling pathways, such as cell metabolism, growth, differentiation, and death signaling (Zou et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At moderate levels, they are defense molecules that destroy inflammation agents such as exogenous pathogens (Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, ROS have a strong tendency to react with and damage cellular macromolecules such as proteins, lipids, and nucleic acids. This excessive oxidation can lead to increased inflammatory responses in the organs.\u003c/p\u003e \u003cp\u003eNaturally, the level of ROS is maintained by components possessing antioxidant activity. The high antioxidant activity of crude and alkaloid extracts derived from Kratom leaf indicates that these extracts contain phytochemicals that are active in scavenging ROS. A significant amount of phenolics observed in the crude extract should contribute to the high antioxidant activity of Kratom\u0026rsquo;s crude extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Phenolic compounds are reported to be one of the effective nutrients in the prevention of oxidative stress (Huang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Parthasarathy et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this study, the TPC of the crude extract was greater compared to that of the alkaloid extract, which is in line with previous findings (P. Zhang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This is because the cascade process during alkaloid extraction involves acid-base reaction and the use of non-polar organic solvents, which separate non-polar alkaloids and polar compounds (non-alkaloids) such as phenolics. Phenolics are a group of polar compounds and, thus, are not extracted when non-polar organic solvents are used during the extraction process to obtain the alkaloid extract.\u003c/p\u003e \u003cp\u003eFurthermore, the antioxidant activity of kratom's crude and alkaloid extracts can be attributed to the presence of alkaloid compounds such as mitragynine and its derivatives, including 7-hydroxy mitragynine, paynantheine, speciogynine, and speciociliatine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These compounds have been reported to show antioxidant activity (Elahian et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the alkaloid extract exhibited significant ABTS antioxidant activity and was found to contain a high content of mitragynine (45.9\u0026plusmn;0.9%). The presence of mitragynine was confirmed through LC-MS/MS analysis (Figure S1) and by the proton signals of mitragynine obtained in the \u003csup\u003e1\u003c/sup\u003eH NMR spectra (Figure S2). It should be noted that the lower FRAP value of the alkaloid extract compared to the crude extract does not necessarily indicate a lack of activity in the alkaloid extract. It might be a result of the fact that alkaloids, being weak bases, could potentially alter the redox reaction during the FRAP measurement. The FRAP assay requires an acidic condition (pH 3.6) to get the optimal redox reaction (Huang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In contrast, the ABTS assay is carried out under neutral pH conditions, wherein the chromophore reaction should not be significantly affected by the weak basicity of the alkaloid components. In general, kratom extracts exhibit high antioxidant activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which should be beneficial for scavenging ROS/RNS in LPS-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated RAW 264.7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and for acting as anti-inflammatory agents by inhibiting inflammatory-related enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe evaluation of the anti-inflammatory activity of kratom extracts \u003cem\u003ein vitro\u003c/em\u003e involved stimulating RAW 264.7 macrophage cells with LPS and assessing their ability to reduce intracellular free radicals (ROS and RNS), pro-inflammatory cytokines (TNF-α and IL-6) and inflammatory-related enzymes (COX-2 and 5-LOX). The ability of crude and alkaloid extracts to substantially decrease ROS in LPS-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated RAW 264.7 cells without killing the cells should be associated with their antioxidant activity to scavenge free radicals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This is supported by the observation that these kratom extracts could also reduce NO levels in the LPS-treated RAW 264.7 macrophage cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). It should be noted that LPS stimulation can activate NF-κB, which upregulates the inducible nitric oxide synthase (iNOS) enzyme to result in the enhancement of NO production in RAW 264.7 macrophage cells (Jones et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This is corroborated by previous research that found plant extracts with a high concentration of phytochemical metabolites may compete with oxygen to capture nitrite free radicals, limiting nitric oxide oxidation and thus displaying anti-inflammatory properties through NO inhibition (Gomathi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ramya et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRAW 264.7 macrophages cells stimulated by LPS, will release various pro-inflammatory cytokines; \u003cem\u003ee.g.\u003c/em\u003e, TNF-α, interleukin-1 beta (IL-1β), and interleukin 6 (IL-6) (Won et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; W. bin Zhang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). TNF-α has the ability to induce apoptosis and trigger the release of other inflammatory cytokines such as IL-1, IL-6, and IL-10. Additionally, it stimulates T cells as well as other inflammatory cells. The alkaloid extract exhibited four times more potent (~\u0026thinsp;50% reduction) four times more potently (~\u0026thinsp;50% reduction) inhibition four times more potently (~\u0026thinsp;50% reduction) than the crude extract did (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, when the alkaloid extract concentration exceeded 6.25 ppm, TNF-α levels decreased. Nonetheless, the alkaloid extract exhibited significant suppression of IL-6 at the inverse concentration, whereas IL-6 levels were not reduced at 25 ppm of alkaloid extract exposure. In contrast, a crude extract exhibited IL-6 inhibition at concentrations ranging from 25\u0026ndash;100 ppm but only exhibited TNF-α inhibition at 100 ppm. We hypothesized that each of the phytochemicals contained in the extracts possessed various activities on these pro-inflammatory cytokines. In particular, the alkaloid components manifest different mechanism of action, prompting further analysis to ascertain the individual magnitude of their effects.\u003c/p\u003e \u003cp\u003eThe AA pathway plays a key role in numerous inflammatory diseases, where its metabolism involves COXs and LOXs enzymes that lead to the production of a variety of bioactive mediators such as prostanoids and LTs (Wang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). COX-2 catalyzes the conversion of arachidonate to inflammatory PGs, a process pivotal in inflammation. COX-2 inhibitors are among the most widely used medications due to their anti-inflammatory, antipyretic, and analgesic properties (Ahmadi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). On the other hand, it has been reported that 5-LOX plays the most important role in the production of LTs, which also act as inflammatory mediators in respiratory, dermatological, and gastrointestinal disorders (Charlier \u0026amp; Michaux, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe alkaloid extract demonstrated consistent inhibition of COX-2 across all tested concentrations (6.25-25 ppm), while crude extract concentrations higher than 25 ppm did not reduce COX-2 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The alkaloid extract is mainly composed of mitragynine and its derivatives. Mitragynine has been found to inhibit the expression of COX-2 in a dose-dependent manner (Utar et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e). Meanwhile, multiple components in the crude extract, besides alkaloids, might work synergistically or antagonistically to induce inflammation to some extent as the crude extract concentration increases. Similar to the interaction between artemisinin and casticin, which is antagonistic at a ratio of 1:3 (v/v), a synergistic interaction has been described for combination ratios ranging from 1:10 to 1:1000 (artemisinin to casticin, v/v) (Suberu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, the alkaloid extract exhibited the 5-LOX enzyme inhibition in a dose-dependent manner, with 86% and 33% inhibition at 6.25 and 12.5 ppm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Such activities were not observed when the crude extract was tested. Given that the alkaloid extract contained more mitragynine and its derivatives than the crude extract, these compounds could account for this activity. This hypothesis is supported by molecular docking results, which showed that mitragynine and some of its derivatives, like paynantheine and speciophylline, exhibited good binding affinity with COX-2 and 5-LOX proteins (Table S2). Moreover, mitragynine showed higher binding affinity compared to several opioids, including morphine and methadone.\u003c/p\u003e \u003cp\u003eCOX-2 possesses three regions of main active sites; \u003cem\u003ei.e.\u003c/em\u003e, the hydrophobic pocket (Tyr385, Trp 387, Phe518, Ala201, Tyr 248, Leu352), the hydrophilic region (Arg120, Glu524, Tyr 355), the side pocket (His90, Arg513, Val523); while the binding pocket of 5-LOX is primarily Phe169, Phe610, Ala410, Ala672, Gln363, Gln413 and Ile406 (Bar et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, mitragynine demostrated multiple interactions with several main active sites of COX-2; \u003cem\u003ei.e.\u003c/em\u003e, Arg120, Tyr355, Phe518, Leu 352, Trp 387, Val 523; and with 5-LOX; \u003cem\u003ei.e.\u003c/em\u003e, Phe610, Ala410, Ile406, Gln413 (Figure S3A,B). In comparison, celecoxib, a selective COX-2 inhibitor (Goldenberg, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), formed multiple bonds with COX-2 active sites than with 5-LOX proteins (Figure S3C,D). Morphine showed more limited interactions with these two enzymes (Figure S3E,F), resulting in lower binding affinity compared to mitragynine, as indicated docking scores (Table S1). These findings suggest that an alkaloid extract derived from Kratom leaf effectively inhibits both COX-2 and 5-LOX enzymes, demonstrating dual anti-inflammatory activity. In contrast, the crude extract, similar to most NSAIDs, only inhibits COX-2 enzymes. However, it should be noted that our molecular docking study indicated variations in the binding affinity of each alkaloid compound, and thus, further investigations are warranted to determine the level of inhibitory activity of each component against COX-2 and 5-LOX enzymes.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe alkaloid extract derived from kratom leaf, containing\u0026thinsp;~\u0026thinsp;46% mitragynine, was found to exhibit significant antioxidant activity, effectively scavenging ROS (\u0026lt;\u0026thinsp;50%) and NO (34%) \u003cem\u003ein vitro\u003c/em\u003e using RAW 264.7 macrophage cells induced with LPS. Its free radical scavenging activity correlated with the extract concentration, where a significant reduction of ROS (\u0026lt;\u0026thinsp;50%) and NO (34%) levels is observed at 25 ppm, without exhibiting toxicity to the cells. At a sufficient concentration (~\u0026thinsp;12.5 ppm), the alkaloid extract also demonstrates activity in reducing the proinflammatory cytokines TNF-α and IL-6, with approximately 4-folds more activity than the crude extract. An interesting feature of the alkaloid extract is its dual anti-inflammatory activity, inhibiting both COX-2 and 5-LOX to some extent. This contrasts with the crude extract, which shows inhibition of COX-2 only. These results support previous reports that describe mitragynine, one of the alkaloids derived from kratom leaf, as being active in inhibiting COX-2. The additional information on the inhibition activity of the kratom alkaloid extract towards 5-LOX enzyme opens a new insight into the potential of the alkaloid compounds in Kratom to be developed as an alternative NSAID with fewer and safer side effects.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors played significant roles in the research and manuscript preparation processes. S.I.R. contributed to the conceptualization, methodology, data analysis, investigation, writing- original draft, review, and editing. D.W.I. contributed to the conceptualization, methodology, data analysis, writing- original draft, review and editing, visualization. F.N.N. contributed to the investigation, writing- original draft, review, and editing. M.H. contributed to the investigation, and editing. P.A. contributed to the data analysis, review, and editing. A.R. contributed to the data analysis, review, and editing. F. contributed to in silico study. E.S. contributed to the data analysis and resources. N.L.P.I.D. contributed to the writing- review and editing, funding acquisition. A.B. contributed to the data analysis, investigation, review, and editing. M.Y.P. contributed to the supervision, writing- review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declared that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Research Organization for Health, National Research and Innovation Agency (BRIN), through the DIPA Research Organization for Health fiscal year 2024 (Project Number 6/III.9/HK/2024). The authors would like to acknowledge Jonathan Ardhianto Panggabean and Firmansyah Karim for their assistance with the extraction and chromatographic analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhmadi, M., Bekeschus, S., Weltmann, K. D., von Woedtke, T., \u0026amp; Wende, K. (2022). Non-steroidal anti-inflammatory drugs: recent advances in the use of synthetic COX-2 inhibitors. In \u003cem\u003eRSC Medicinal Chemistry\u003c/em\u003e. Royal Society of Chemistry. https://doi.org/10.1039/d1md00280e\u003c/li\u003e\n \u003cli\u003eBar, F. M. A., Sameti, M., Foudah, A. I., Haque, A., \u0026amp; Elsbaey, M. (2022). In vitro and in silico inhibition of COX-2 and 5-LOX by beta-carboline alkaloids from the seeds of Peganum harmala L. \u003cem\u003eSouth African Journal of Botany\u003c/em\u003e, \u003cem\u003e147\u003c/em\u003e, 926\u0026ndash;936. https://doi.org/10.1016/j.sajb.2022.03.044\u003c/li\u003e\n \u003cli\u003eBayu, A., Rahmawati, S. 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Springer New York LLC. https://doi.org/10.1007/s10495-017-1424-9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Research Center for Vaccine and Drugs, Research Organization of Healt, National Research and Innovation Agency ","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mitragyna speciosa, cellular oxidative stress, dual inhibition COX-2/5-LOX, anti-inflammatory","lastPublishedDoi":"10.21203/rs.3.rs-4628929/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4628929/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCyclooxygenase (COX) and lipoxygenase (LOX) enzymes play a crucial role in the production of pro-inflammatory eicosanoids, including prostaglandins and leukotrienes (LTs) via arachidonic acid (AA) pathways. Non-steroidal anti-inflammatory drugs (NSAIDs) typically work by inhibiting COX enzymes (COX-1, COX-2) to alleviate inflammatory responses in our bodies. However, the use of these selective COX inhibitors results in an upregulation of the AA pathway. This condition stimulates the LOX enzymes to increase LT production, exacerbating the severity of the disorders. In this study, the alkaloid extract derived from the leaf of \u003cem\u003eMitragyna speciosa\u003c/em\u003e (Kratom) demonstrated a dual inhibitory effect on COX-2/5-LOX enzymes in lipopolysaccharides (LPS)-induced RAW 264.7 macrophage cells. The alkaloid extract containing\u0026thinsp;~\u0026thinsp;46% mitragynine inhibited COX-2 and 5-LOX activity at concentrations of less than 25 ppm with no toxicity to the cells. Above 25 ppm, the alkaloid extract exhibited toxicity to the cells (\u003cem\u003ee.g\u003c/em\u003e,, ~\u0026thinsp;46% viability at 50 ppm) and only inhibited COX-2 activity. In contrast, the Kratom crude extract containing\u0026thinsp;~\u0026thinsp;5% mitragynine did not inhibit COX-2 or 5-LOX activity in LPS-induced RAW 264.7 macrophage cells at more than 25 ppm and did not exhibit toxicity to the cells even at 100 ppm. The alkaloid compounds in the Kratom leaf are likely responsible for this activity, as the alkaloid extract containing these biomolecules suppressed reactive oxygen species (ROS), nitric oxide (NO), inducible nitric oxide synthase (iNOS), and pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Molecular studies also suggested a strong binding affinity of Kratom alkaloids to the active sites of COX-2 and 5-LOX. The dual inhibitory activity of the Kratom alkaloids against COX-2 and 5-LOX provides insights into their potential as safer NSAIDs.\u003c/p\u003e","manuscriptTitle":"Dual Anti-Inflammatory Activities of COX-2/5-LOX Driven by Kratom Alkaloid Extracts in Lipopolysaccharide-induced RAW 264.7 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-25 13:52:16","doi":"10.21203/rs.3.rs-4628929/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f40e7046-f230-477a-97aa-556812c316cf","owner":[],"postedDate":"June 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33641857,"name":"Drug Discovery, Design, \u0026 Development"}],"tags":[],"updatedAt":"2024-09-23T07:36:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-25 13:52:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4628929","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4628929","identity":"rs-4628929","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}