Upregulation of HDAC5 gene expression for treatment of osteoarthritis: Chondroprotective properties of regular and Nano-formulated Boswellia serrata Roxb

Upregulation of HDAC5 gene expression for treatment of osteoarthritis: Chondroprotective properties of regular and Nano-formulated Boswellia serrata Roxb | 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 Article Upregulation of HDAC5 gene expression for treatment of osteoarthritis: Chondroprotective properties of regular and Nano-formulated Boswellia serrata Roxb Zobaida A. Mohamed, Marwa Saeed, Laura Acquaticci, Muhammed A. Saad, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7302080/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 Osteoarthritis is a serious disorder that destroys cartilage and joints and inhibits collagen formation. Natural products are widely recognized for their wide variety of therapeutic uses and great safety records. Using human chondrocyte cells (C20A4) and MTT assays, the potential benefits of regular and nano-formulated Boswellia serrata (Bs) methanol extract for treating osteoarthritis by influencing the expression of the HDAC5, IL1β, IL6, PGE2, NF-kB genes were evaluated. HDAC5 gene expression was significantly upregulated and survival rate of C20A4 was strongly elevated. IL1β, IL6, PGE2, NF-kB inflammation biomarkers gene expression was significantly suppressed by both conventional and Nano-formulations. In comparison to conventional extracts, Nano-formulations exhibited greater activity revealing that they might represent a more effective treatment strategy. HPLC-MS/MS technology of the Bs extract against 28 standard phenolic compounds e.g. p- coumaric acid, ferulic acid, rutin, myricetin and quercetin, proved the absence of these compounds in Bs gum resin. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Drug discovery Osteoarthritis (OA) Boswellia serrata HDAC5 IL1β IL6 PGE2 NF-kB human chondrocyte cells (C20A4) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Osteoarthritis (OA) is a chronic, progressive, degenerative condition that affects the entire joint, characterized by a reduction in joint space, the formation of osteophytes, subchondral sclerosis, and the presence of cysts 1 . The primary clinical manifestations of OA include joint pain and swelling, as well as a restricted range of motion 2 . OA collectively impair the quality of life for affected individuals 3 . This disease ranks among the most common diseases globally, and the disabilities it causes impose significant healthcare and socioeconomic challenges 4 . Due to its complex and multifactorial nature, halting the progression of OA is difficult; therefore, it is essential to adopt a strategy focuses on enhancing joint function, alleviating pain, and improving quality of life while minimizing adverse effects 5 , 6 . Currently, OA lacks a definitive cure, and earlier strategies primarily aimed at alleviating joint pain instead of preventing the disease's progression. As a result, various treatments, including oral and topical nonsteroidal anti-inflammatory drugs, opioids, and serotonin norepinephrine reuptake inhibitors, were utilized for symptom management and functional improvement, though their extended use poses significant health threats 7 . Recently, the emphasis has shifted towards slowing the progression of the disease by discovering disease-modifying drugs that interact with relevant biomarkers and therapeutic targets 8 . The inflammatory biomarkers Interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) serve as significant indicators of severe OA, with their concentrations closely associated with reductions in joint space and cartilage degradation 9 . Furthermore, numerous studies have established a connection between the onset and progression of OA and the altered functions of histone deacetylases (HDACs) 10 , 11 . Among these, HDAC5, a member of the class IIa HDAC family, has been shown to inhibit chondrocyte hypertrophy by functioning as a co-activator of HDAC4, which operates via parathyroid hormone-related protein 12 . Additionally, reduced levels of HDAC5 have been observed in the cartilage of mice undergoing medial meniscus surgery and in patients with advanced OA. The silencing of HDAC5 was linked to increased degeneration of articular cartilage and chondrosenescence, while the activation of HDAC5 in chondrocytes through metformin effectively reversed OA 10 . Recently, there has been a growing interest in plant-based natural remedies due to their safety and beneficial effects on bone health, which are linked to their chondroprotective and osteoprotective properties 13 , 14 , 15 . One such remedy is Boswellia serrata (Bs) gum resin, derived from the Bs tree native to the Indian highlands. This resin has a long history of use in treating arthritis and chronic inflammatory disorders 16 . It is known for its potent anti-inflammatory and antioxidant effects, attributed to the presence of six different boswellic acids reported in Fig. 1 . 17 , 18 , 19 , 20 Furthermore, an ethanol extract of Bs gum resin, rich in boswellic acids (FJH-UBS), has been shown to reduce inflammation and prevent the degradation of the extracellular matrix, demonstrating an anti-osteoarthritis effect in fibroblast-like chondrocyte cells derived from a 72-year-old white female with chondrosarcoma (SW1353) stimulated by interleukin (IL)-1β, as well as in animal models of osteoarthritis 21 . Additionally, the Bs extract applied to primary chondrocytes from osteoarthritis patients exhibited antioxidant properties through the activation of Nrf1 and Peroxisome Proliferator Activated Receptor alpha (PPARα) pathways, alongside anti-inflammatory effects by promoting growth/differentiation factor 15 (GDF15), decreasing cholesterol uptake, and regulating genes involved in fatty acid metabolism, while also reducing Toll-like receptor activation 22 . Moreover, the administration of Bs gum resin extract in a rat model of osteoarthritis induced by monosodium iodoacetate showed significant therapeutic potential by alleviating knee joint swelling, cartilage damage, and tissue deformation, as well as enhancing the expression of collagen type II alpha 1 and aggrecan, while downregulating nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, 5-lipoxygenase, prostaglandin E2 (PGE2), leukotriene B4, IL-6, TNF-α, and matrix metalloproteinases 3 and 13 8 . A revolutionary method for improving the bioavailability and efficacy of medications, particularly in the treatment of conditions like osteoarthritis, is the application of novel nanotechnology techniques 23 . By increasing the solubility and stability of bioactive compounds, nanoscale transporters facilitate improved absorption and retention at the target sites 24 . Nanocarriers provide a significant solution to multiple hurdles, predominantly by promoting a sustained drug release and intensified internalization, thereby augmenting the therapeutic potency 25 . In addition, recent nanotechnology-driven formulations have demonstrated promising outcomes in the reduction of key inflammatory factors, comprising IL-1β and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) thereby aiding in the mitigation of osteoarthritis symptoms and disease pathology 26 . These progressions underscore the vital role of nanotechnology in enhancing the efficacy of naturally derived therapeutic regimens for improved clinical outcomes 27 . So, the aim of the present study is to investigate the potential effects of Bs extract and nano-emulsion on the human chondrocyte cell line (C20A4) in an in-vitro model of OA by evaluating the gene expression of histone deacetylase 5 (HDAC5) and inflammation biomarkers IL1β, IL6, PGE2, NF-kB, and the survival rate of C20A4. Materials and Methods Materials Plant materials In April 2024, the gum resin of Boswellia serrata was purchased from ATOS Pharma, Egypt. The plant material was formally verified for authenticity by Prof. Abdulhalim Abdelmogeli Mohammed, specialist in flora and vegetation at the Agricultural Research and Extension Authority, Cairo. A voucher specimen representing the authenticated sample was archived at the Department of Pharmacognosy, Faculty of Pharmacy, Heliopolis University. The obtained resin was preserved in an airtight container under proper storage conditions until further experimental use. Chemicals and Reagents for cytotoxicity To perform the cytotoxicity assessment through the MTT method, RPMI-1640 growth medium, phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), and the C20A4 cell line were acquired from Sigma-Aldrich, Germany. The MTT dye solution was procured separately from Bio Basic Inc., Canada. Chemicals and Reagents for HPLC-ESI-MS/MS Delphinidin-3,5-diglucoside chloride and kaempferol-3-glucoside were obtained from PhytoLab, Vestenbergsgreuth, Germany, whereas the other analytical reference standards were sourced from Sigma-Aldrich, Milan, Italy. Stock solutions for each compound (1000 mg/L) were prepared by dissolving the respective pure standards in methanol of HPLC grade. All solvents employed in the study were of HPLC quality. Prior to chromatographic injection, the samples were passed through a Phenex™ RC syringeless filter (4 mm, 0.2 µm; Phenomenex, Castel Maggiore, BO, Italy). Chemicals and reagents for Nano emulsion (NanoEm) Sigma-Aldrich provided the chemicals required for NanoEm formulation, which included Tris, glycine, SDS, Tween 80, polyethylene glycol 600 (PEG 600), dimethyl sulfoxide (DMSO), phosphate-buffered saline tablets, and castor oil. All reagents were of analytical quality. Methods Extraction A portion of 10 g from the collected sample was subjected to ultrasonic-assisted extraction with 90% methanol (HPLC grade) until complete exhaustion. The resulting extract was filtered, and the solvent was completely removed under reduced pressure using a rotary evaporator maintained at 50°C. The obtained dry residue was weighed, and the required portion was forwarded for further analysis. Chondroprotective and osteoprotective activity - Cytotoxic activity (MTT assay) 28 . The MTT assay was employed to investigate the cytotoxic activity of Bs extracts (conventional and nano-formulated) in comparison with the standard Blank (Blk). Cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/mL (100 µL per well) and incubated at 37°C for 24 h, allowing a uniform monolayer to form. Upon reaching confluence, the growth medium was removed, and the plates were rinsed twice with washing medium. Test extracts were serially diluted in RPMI supplemented with 2% maintenance medium to prepare eight concentrations (10000, 5000, 2500, 1250, 625, 312.5, 156.25, and 78.12 µg/mL). A 0.1 mL portion of each dilution was dispensed into separate wells, while three wells containing only maintenance medium served as controls. Plates were incubated again at 37°C, and cells were monitored for toxic morphological changes, including monolayer disruption, rounding, shrinkage, and granule formation. Afterward, 20 µL of MTT solution (5 mg/mL in PBS) was pipetted into each well and gently agitated on a shaker at 150 rpm for 5 min. The plates were incubated for 4 h at 37°C in a 5% CO₂ atmosphere to permit the enzymatic reduction of MTT to formazan. Supernatants were then discarded, and crystals were solubilized with 200 µL DMSO under shaking (150 rpm, 5 min). Optical density values were determined at 560 nm with correction against a reference wavelength of 620 nm. Cell viability was expressed as percentage relative to untreated controls.The relative cell-viability percentage was evaluated by using the following relation: Relative cell-viability %= \(\:\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{c}\text{e}\text{l}\text{l}\text{s}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{w}\text{i}\text{t}\text{h}\:\text{t}\text{h}\text{e}\:\text{t}\text{e}\text{s}\text{t}\text{e}\text{d}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{a}\text{t}\:560\:\text{n}\text{m}}{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{u}\text{n}\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{c}\text{e}\text{l}\text{l}\text{s}\:\text{a}\text{t}\:560\:\text{n}\text{m}}\) × 100. Preparation and characterization of NanoEm 29 . The nanoemulsion (NanoEm) was prepared using castor oil, phosphate-buffered saline (PBS, 10 mM, pH 7.4), and Tween 80 in a ratio of 10:5:2.5, respectively. To begin, 50 mg of Bs powder was dissolved in 5 mL of PBS and subjected to ultrasonic treatment with a probe sonicator (cycle 1, amplitude 80%). Subsequently, 2.5 mL of Tween 80 was incorporated as the surfactant. The resulting mixture was then transferred to a cold-water bath, where castor oil (10 mL) was added dropwise under continuous homogenization (Daihan Homogenizer, ZS version, Korea) operated at 750 rpm for 11 minutes. The obtained nanoemulsion was employed for further characterization and subsequent experimental applications. Characterization of NanoEm included determination of droplet size, polydispersity index (PDI), and zeta potential using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) maintained at 25 ± 0.1°C. Samples were diluted with PBS (10 mM, pH 7.4) to achieve a particle count within the range of 50–300 KCPs. Morphological features were examined by transmission electron microscopy (TEM, model H-700, Hitachi Ltd., Japan). For TEM analysis, samples were diluted at a ratio of 1:50 with PBS (10 mM, pH 7.4), and a drop of the diluted suspension was placed on carbon-coated copper grids. After air drying for 5 minutes, the grids were negatively stained with 2% w/w phosphotungstic acid for 50 seconds, and excess stain was removed using filter paper. Additionally, the physicochemical properties of the NanoEm were evaluated. The pH was measured at room temperature using an Ohaus Starter 3100 bench pH meter (USA), which had been calibrated with standard buffer solutions of pH 4, 7, and 10. Viscosity was determined using a Lamy Rheology viscometer (B-One Plus, Germany) with spindle 6 at a rotation speed of 50 rpm for 3 minutes. Quantitative real-time polymerase chain reaction Total RNA from C20A4 cells was isolated using the RNeasy® Mini Kit (Catalog No. 74904, QIAGEN, Aarhus, Denmark) in accordance with the manufacturer’s protocol. At the conclusion of the extraction and purification steps, RNA quality and purity were verified by spectrophotometric measurement of the absorbance ratio at 260/280 nm using a Denovix spectrophotometer (AGBL, USA). The purified RNA (0.5–2 µg) was then subjected to reverse transcription into complementary DNA (cDNA) with the aid of the SuperScript IV One-Step RT-PCR kit (Catalog No. 204843, Qiagen QuantiTect RT kit), which is highly sensitive for such applications. For subsequent quantitative real-time PCR (qPCR), an optimized kit containing Taq polymerase, qPCR reaction buffer, primers, SYBR® Green I dye, and nucleotides was employed. The primer sequences utilized during the annealing stage are listed in Table 1 . Relative expression levels (RQ) of target genes, including HDAC5, NF-κB, prostaglandin EP2 receptor, IL-1β, and IL-6 were determined using the calculation algorithm provided in the Applied Biosystems software formula, ∆󠅄Ct = Ct gene test - Ct housekeeping control and ∆∆Ct = Ct sample1 - Ct calibrator, where (RQ) = \(\:{\text{2}}^{\text{-∆∆Ct}}\) . The outcomes were normalized to GADPH and presented as arbitrary units. Table 1 The primers sequence of studied genes in quantitative real-time polymerase chain reaction. Gene Primer Sequence HDAC5 Forward 5′- TCTTGTCGAAGTCAAAGGAGC − 3′ Reverse 5′- GAGGGGAACTCTGGTCCAAAG − 3′ NF-κB Forward 5′- GCAGCACTACTTCTTGACCACC-3′ Reverse 5′- TCTGCTCCTGAGCATTGACGTC-3′ EP2 Forward 5′-CCACCTCATTCTCCTGGCTA-3 Reverse 5′-CGACAACAGAGGACTGAACG-3 IL-1β Forward 5′- CCACAGACCTTCCAGGAGAATG-3′ Reverse 5′- GTGCAGTTCAGTGATCGTACAGG-3′ IL-6 Forward 5′- AGACAGCCACTCACCTCTTCAG-3′ Reverse 5′- TTCTGCCAGTGCCTCTTTGCTG-3′ HPLC-ESI-MS/MS 30 . High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was conducted using an Agilent 1290 Infinity system linked to a Triple Quadrupole 6420 (Agilent Technologies, Santa Clara, CA, USA). The system was fitted with an electrospray ionization (ESI) interface, which was operated alternately in positive and negative ionization modes. Polarity switching was performed within a single analytical run without complications. For each analyte, MS/MS parameters were tuned through flow injection analysis (FIA) by injecting 1 µL of a 10 mg L⁻¹ individual standard solution, using the Agilent Optimizer software. Chromatographic separation of the analytes was achieved on a Synergi Polar-RP C18 analytical column (250 mm × 4.6 mm, 4 µm; Phenomenex, Cheshire, UK), protected with a Polar RP guard cartridge (4 mm × 3 mm ID). The mobile phase consisted of two solvents: (A) water with 0.1% formic acid and (B) methanol with 0.1% formic acid. A gradient elution program at a flow rate of 0.8 mL/min was applied as follows: 0–1 min, isocratic at 20% B; 1–25 min, linear increase from 20–85% B; 25–26 min, isocratic at 85% B; and 26–32 min, return from 85–20% B. All solvents and prepared solutions were filtered prior to use through 0.2 µm polyamide membranes (Sartorius Stedim, Goettingen, Germany). The injection volume for each run was 2 µL. The column was maintained at 30°C, while the drying gas temperature in the ion source was set at 350°C. Additional source conditions included a drying gas flow of 12 L/min, nebulizer pressure of 55 psi, and capillary voltage of 4000 V. Data acquisition was carried out in dynamic multiple reaction monitoring (dynamic-MRM) mode. For quantitation, the most intense product ion was selected, while additional transitions were employed for confirmation. A retention time window of ± 2 min was assigned for each analyte. Detailed ion transitions along with optimized MS parameters for the studied compounds are provided in Table 2 and Fig. 2 . Statistical Analysis All results were presented as mean values accompanied by their corresponding standard deviations (SD). The Kolmogorov–Smirnov test was applied to determine whether the datasets followed a normal distribution. To assess differences among groups, a one-way analysis of variance (ANOVA) was conducted, and when significance was detected, Tukey’s post hoc test was employed for multiple pairwise comparisons. Statistical evaluations and preparation of graphical outputs were carried out using GraphPad Prism software, version 9 (GraphPad Software, Inc., San Diego, CA, USA). A probability value of less than 0.05 (p < 0.05) was considered the threshold for statistical significance. Outlier identification was performed using the Dixon test, and only values that failed this test were excluded from analysis. Table 2 HPLC–MS/MS acquisition parameters (dynamic-MRM mode) used for the analysis of the 28 marker compounds. No. Compounds Precursor ion, m/z Product ion, m/z Fragm-entor, V Collision energy, V Polarity Retention time (Rt, min) Delta retention time (ΔRt) 1 Gallic acid 169 125.2 * 97 12 Negative 6.96 2 2 Neochlorogenic acid 353 191.2 * , 179 82 12, 12 Negative 9.52 2 3 (+)-Catechin 289 245.2 * ,109.2 131 8, 20 Negative 11.44 2 4 Procyanidin B2 576.99 576.99 * , 321.2 160 0, 32 Negative 12.41 2 5 Chlorogenic acid 353 191.2 * , 127.5 82 12, 20 Negative 12.42 2 6 p -Hydroxybenzoic acid 137 93.2 * 92 16 Negative 12.86 2 7 (-)-Epicatechin 289 245.1 * , 109.1 126 8, 20 Negative 13.03 2 8 3-Hydroxybenzoic acid 137 93.2 * 88 8 Negative 13.59 2 9 Caffeic acid 179 135.2 * , 134.1 92 12, 24 Negative 13.65 2 10 Vanillic acid 167 152.4 * , 108.1 88 12, 20 Negative 14.32 2 11 Resveratrol 227 185 * 131 12 Negative 14.40 2 12 Syringic acid 196.9 182.2 * , 121.2 93 8, 12 Negative 15.28 2 13 Procyanidin A2 575 575 * , 285 170 0, 20 Negative 16.18 2 14 p -Coumaric acid 163 119.2 * , 93.2 83 12, 36 Negative 16.70 2 15 Ferulic acid 193 134.2 * , 131.6 83 12, 8 Negative 17.10 2 16 3,5-Dicaffeoylquinic acid 514.9 353.1 * , 191 117 8, 28 Negative 17.61 2 17 Rutin 609 300.2 * , 271.2 170 32, 50 Negative 17.73 2 18 Isoquercitrin 463 271.2 * , 300.2 155 44, 24 Negative 18.36 2 19 Delphindin-3,5-diglucoside 462.9 300.1 * 165 24 Negative 18.38 2 20 Phloridzin 435.39 273 * , 167 155 8, 28 Negative 18.83 2 21 Quercitrin 446.99 300.2 * , 301.2 160 24, 16 Negative 19.61 2 22 Myricetin 316.99 179.1 * , 182 150 16, 24 Negative 19.61 2 23 Naringin 578.99 271.3 * , 151.3 170 32, 44 Negative 19.62 2 24 Kaempferol-3-glucoside 447 284.2 * , 255.2 170 24, 40 Negative 19.77 2 25 Ellagic acid 301 301 * , 229 170 0, 24 Negative 21.41 2 26 Quercetin 300.99 151.2 * , 179.2 145 16, 12 Negative 21.87 2 27 Phloretin 272.99 167 * , 123 116 8, 20 Negative 22.30 2 28 Isorhamnetin 314.99 300.2 * , 196.1 145 16, 4 Negative 24.57 2 * These product ions were used for quantification. Results and discussion Chondroprotective and osteoprotective activity The data illustrated in Fig. 3 compare the survival rate in C20A4 of the Bs extract in both conventional and nano-formulation forms against the standard Blk., evaluated through the MTT assay on Human Chondrocyte Cell Line C20A4 cells. The extracts and the standard were tested at various concentrations (10000, 5000, 2500, 1250, 625, 3125, 156.25, 78.12 µ g/mL). Notably, the conventional Bs extract achieved an IC50 value of 674.5, which corresponds to 18.89% of the Blk. group's effect. In contrast, the nano-formulated version yielded an IC50 of 4274, equating to 119.69% of the standard, indicating enhanced cell viability with the Nano-formulation. Bs Extract Restores Epigenetic Balance and Suppresses Inflammatory Markers in C20A4 Cells: Enhanced Efficacy with Nano-formulation As shown in Fig. 4 , the C20A4 osteoarthritic chondrocyte model displayed a notable dysregulation in inflammatory and epigenetic markers. Specifically, HDAC5 (Histone Deacetylase 5), a key regulator of gene expression and anti-inflammatory response, was reduced by 10.07% relative to the normal control group. In contrast, there was a marked upregulation of NF-κB, a pro-inflammatory transcription factor, and the Prostaglandin EP2 receptor, which mediates inflammation and pain signaling. These were elevated to 111.75% and 109.29%, respectively, compared to the normal control. Upon treatment with Bs extract, both the conventional and Nano-formulated preparations produced significant modulatory effects. Notably, HDAC5 levels increased beyond the baseline of the normal control, reaching 124% and 131% in the conventional and Nano-formulated extract-treated groups, respectively. This suggests a potential epigenetic restoration or enhancement effect, particularly with the nanoformulation. Simultaneously, both treatments effectively reduced the elevated inflammatory markers. The conventional extract reduced NF-κB and EP2 receptor levels to 89% and 87%, respectively, while the nanoformulation demonstrated slightly greater efficacy, lowering them to 88% and 82% of normal control values. These findings indicate that Bs , especially in Nano-formulated form, has anti-inflammatory potential through suppression of key inflammatory pathways. When compared to the standard therapeutic agent Blk., the extract showed comparable effects. Blk. also elevated HDAC5 and reduced NF-κB and EP2 expression to similar levels. The only statistically notable difference was in EP2 receptor expression, where the Nano-formulated Boswellia achieved a further reduction to 82%, while Blk. reached only 89%. These findings highlight the potential of Bs , particularly in its Nano-formulated version, to counteract inflammation and restore epigenetic balance in osteoarthritic conditions. The extract's ability to elevate HDAC5 and suppress NF-κB and EP2 receptor expression supports its therapeutic value, possibly offering a comparable or even superior alternative to conventional treatments like Blk. for managing osteoarthritis-related inflammation. Nano-formulated Bs Exhibits Superior Suppression of Pro-inflammatory Cytokines IL-1β and IL-6 in Osteoarthritic Chondrocytes As depicted in Fig. 5 , the C20A4 osteoarthritic chondrocyte model demonstrated a pronounced pro-inflammatory response, with IL-1β and IL-6 levels increasing to 115% and 112%, respectively, relative to the normal control group. These cytokines are key mediators of inflammation, cartilage degradation, and joint damage in osteoarthritis. Treatment with the standard therapeutic agent, Blk., resulted in a modest reduction in IL-1β, lowering it to 92% of the level observed in C20A4 cells. However, it had no significant impact on IL-6, suggesting limited anti-inflammatory efficacy in this context. In contrast, treatment with Bs extract, both in conventional and Nano-formulated forms, led to notable reductions in both cytokines. The conventional extract decreased IL-1β to 70% and IL-6 to 90%, while the Nano-formulated extract produced an even more pronounced effect, reducing IL-1β to 64% and IL-6 to 82% compared to the C20A4 group. When benchmarked against the standard Blk., the conventional extract showed superior efficacy in reducing IL-1β, and the nanoformulation demonstrated superiority in suppressing both IL-1β and IL-6. Specifically, the nanoformulation reduced IL-1β and IL-6 to 70% and 85%, respectively, relative to the standard. These findings underscore the anti-inflammatory potential of Bs , particularly in its Nano-formulated form, in mitigating key inflammatory cytokines involved in osteoarthritis. The extract not only outperformed the standard treatment in reducing IL-1β but also showed added benefit in lowering IL-6 levels, highlighting its promise as a more effective therapeutic strategy. HPLC-MS/MS analysis of the dried extract Found to be strongly potent in gene expression downregulation of the inflammation biomarkers IL1β, IL6, PGE2, NF-kB, upregulation of HDAC5 gene expression and elevation of survival rate in C20A4, methanolic extract of Bs gum resin was analyzed by HPLC-ESI–MS/MS to examine the chemical profile. None of the standard 28 compounds was identified in Bs methanol extract, out of 28 bioactive polyphenol reference compounds Table 3 . The 28 compounds were quantified after the acquisition settings of the dynamic MRM mode were optimized. Table 3 lists the chosen ion transitions and mass spectrometer parameters for each molecule. Twenty-eight reference compounds belonging to different phenolic classes were used and the results are depicted in Table 2 . HPLC-MS/MS chromatogram of a standard mixture of 28 phenolic compounds plotted as overlapped multiple reaction monitoring (MRM) negative and positive transition of each analyte is shown in Fig. 2 . Table 3 Content (µg/g of dried extract) of phenolic compounds found in Bs methanol extract Phenolic compound Content µg/g Gallic acid n.d. Neochlorogenic acid n.d. Catechin n.d. procyanidin B2 n.d. Chlorogenic acid n.d. 4-Hydroxy benzoic acid n.d. Epicatechin n.d. 3-Hydroxy benzoic acid n.d. Caffeic acid n.d. Vanillic acid n.d. Syringic acid n.d. Procyanidin A2 n.d. P-Coumaric acid n.d. Ferulic acid n.d. Rutin n.d. Isoquercitrin n.d. Delphindin 3,5 diglucoside n.d. Phloridzin n.d. Naringin n.d. Quercitrin n.d. Myricetin n.d. Kaempferol-3-glucoside n.d. Ellagic acid n.d. Quercetin n.d. Phloretin n.d. Isorhamnetin n.d. TOT 0.00 Conclusions The present study demonstrates the promising therapeutic potential of Bs extract in modulating key molecular pathways involved in osteoarthritis, particularly through its ability to restore epigenetic balance and suppress pro-inflammatory markers. The C20A4 osteoarthritic chondrocyte model revealed significant disruptions in cellular homeostasis, marked by decreased expression of HDAC5, a crucial regulator of anti-inflammatory gene expression, and elevated levels of NF-κB and the Prostaglandin EP2 receptor, both of which play central roles in mediating inflammatory responses and cartilage degradation. Treatment with Bs extract, in both conventional and Nano-formulated forms, successfully reversed these alterations. Notably, HDAC5 expression was not only restored but enhanced, indicating potential epigenetic reprogramming in favor of anti-inflammatory activity. This was accompanied by a reduction in NF-κB and EP2 receptor expression, further supporting the extract’s role in attenuating inflammatory signaling. The Nano-formulated form of the extract consistently exhibited greater efficacy than its conventional counterpart, suggesting improved cellular uptake or bioavailability as a result of the nanotechnology-based delivery system. Parallel analysis of the pro-inflammatory cytokines IL-1β and IL-6 provided additional evidence of the extract’s anti-inflammatory properties. While the standard therapeutic agent modestly reduced IL-1β without affecting IL-6, Bs extract significantly suppressed both cytokines. The Nano-formulated preparation again showed superior performance, effectively reducing both IL-1β and IL-6 levels beyond the effects observed with the conventional extract or standard treatment. Taken together, these findings suggest that Bs exerts its therapeutic effects in osteoarthritic chondrocytes by targeting both epigenetic regulators and inflammatory mediators. The consistent superiority of the Nano-formulated version highlights the added value of advanced drug delivery systems in enhancing the bioactivity of phytotherapeutic agents. Overall, Bs , particularly in Nano-formulated form, represents a promising candidate for further development as a disease-modifying agent in osteoarthritis, with potential advantages over existing conventional therapies. In the C20A4 chondrocyte model, osteoarthritic downregulation of HDAC5 was not only restored but exceeded normal baseline following Bs treatment, with nano-formulations showing superior efficacy. This was accompanied by reduced NF-κB, EP2 receptor, IL-1β, and IL-6 expression, suggesting upstream transcriptional reprogramming rather than mere downstream cytokine blockade. The enhanced activity of the nano-formulated extract likely reflects improved bioavailability, positioning Bs as a promising disease-modifying therapy for osteoarthritis through epigenetic restoration and inflammation suppression. Declarations Acknowledgments The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through a small group Research Project under grant number RGP1/68/46 Author Contribution Zobaida A. Mohamed: Conceptualization; Methodology; Writing–original draft; Writing & editing. Marwa Saeed, Laura Acquaticci, Muhammed A. Saad, Yasmin Abo-zeid, Asmaa M. Elbakry: Conceptualization; Methodology; Supervision; Writing–review & editing. Mohamed Rabeh, Saad Alshehri: Funding Conceptualization; Methodology; Supervision; Writing–original draft; Reviewing & editing. Omar Sabry and Giovanni Caprioli: Conceptualization; Methodology; Supervision; Writing–original draft; Reviewing & editing. Funding This research was funded by King Khalid University, grant number RGP1/192/46 and The APC was funded by King Khalid University. Conflicts of interest The authors confirm that they have no conflicts of interest. Ethical approval Ethics approval was not required for this research. Data Availability Statement The authors confirm that the data supporting the findings of this study are available within the article and its supplementary material. References Kuyinu, E. L., Narayanan, G., Nair, L. S. & Laurencin, C. T. Animal models of osteoarthritis: classification, update, and measurement of outcomes. J. Orthop. Surg, Res. , 11, pp.1–27. (2016). Moon, S. M. et al. Aqueous extract of Codium fragile alleviates osteoarthritis through the MAPK/NF-κB pathways in IL-1β-induced rat primary chondrocytes and a rat osteoarthritis model no. 97pp.264–270 (Biomedicine & Pharmacotherapy, 2018). Loeser, R. F., Goldring, S. R., Scanzello, C. R. & Goldring, M. B. 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Oral Supplementation of Phosphatidylcholine Attenuates the Onset of a Diet-Induced Metabolic Dysfunction–Associated Steatohepatitis in Female C57BL/6J Mice. Cell. Mol. Gastroenterol. Hepatol. 17 (5), 785–800 (2024). Rabiei, M. et al. Nanotechnology application in drug delivery to osteoarthritis (OA), rheumatoid arthritis (RA), and osteoporosis (OSP). J. Drug Deliv. Sci. Technol. 61 , 102011 (2021). Bilia, A. R. et al. Nanocarriers: A successful tool to increase solubility, stability and optimise bioefficacy of natural constituents. Curr. Med. Chem. 26 (24), 4631–4656 (2019). Kim, M., Shin, M., Zhao, Y., Ghosh, M. & Son, Y. O. Transformative impact of nanocarrier-mediated drug delivery: overcoming biological barriers and expanding therapeutic horizons. Small Science, 4(11), p.2400280 (2024). Liu, Y. et al. Nanotechnology in inflammation: Cutting-edge advances in diagnostics, therapeutics and theranostics. Theranostics, 14(6), p.2490. (2024). Patel, J., Roy, H., Chintamaneni, P. K., Patel, R. & Bohara, R. Advanced strategies in enhancing the hepatoprotective efficacy of natural products: integrating nanotechnology, genomics, and mechanistic insights. ACS Biomaterials Sci. Eng. 11 (5), 2528–2549 (2025). Cheleschi, S. et al. Possible chondroprotective effect of canakinumab: an in vitro study on human osteoarthritic chondrocytes. Cytokine 71 (2), 165–172 (2015). Krisanti, E. A., Tobing, M. L. & Mulia, K. February. Preparation and characterization of nanoemulsion herbal drinks using natural deep eutectic solvent and virgin coconut oil. In AIP Conference Proceedings (Vol. 2710, No. 1, p. 040012). AIP Publishing LLC. (2024). Mustafa, A. M. et al. A new HPLC-MS/MS method for the simultaneous determination of 36 polyphenols in blueberry, strawberry and their commercial products and determination of antioxidant activity. Food Chem. no. 367 , 130743 (2022). Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":24504,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative examples of triterpene acids identified in \u003cem\u003eBoswellia serrata\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7302080/v1/1d2f949780c4a5b365c3aa46.png"},{"id":92413681,"identity":"7ece4064-09d5-494c-880e-8f297d5989ae","added_by":"auto","created_at":"2025-09-29 13:01:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61551,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC-MS/MS chromatogram of a standard mixture of 28 phenolic compounds plotted as overlapped multiple reaction monitoriative transition of each 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cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7302080/v1/a3428af042f3a48c44e15895.png"},{"id":92413683,"identity":"0016001b-c6c1-45f6-a577-c785b4513dae","added_by":"auto","created_at":"2025-09-29 13:01:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of the Effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e extract in regular and nano-formulations and the standard Blk on Histone deacetylase 5 (HDAC5) [F (20, 42) =15.33, p \u0026lt; 0.0001], nuclear factor kappa B (NF-kB) [F (20, 42) =8.575, p \u0026lt; 0.0001], and Prostaglandin EP2 receptor [F (20, 42) =16.28, p \u0026lt; 0.0001] using an in-vitro Human Chondrocyte Cell Line C20A4 cells. Each vertical-lined bar reflects the mean of the experiments ± S.D. Statistical analysis was performed by One-way ANOVA followed by Tukey's post-hoc test, with the criterion for statistical significance as follows: *p \u0026lt;0.05, **p \u0026lt;0.01, ***p \u0026lt;0.005, ****p \u0026lt;0.001, ns no significance.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7302080/v1/9868606bf84d610afa7b5274.png"},{"id":92413686,"identity":"cff314f8-ef74-42dc-baf5-2702ef92cb0f","added_by":"auto","created_at":"2025-09-29 13:01:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":57504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e extract in regular and nano-formulations and the standard Blk on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eInterleukin-1 beta (IL-1β)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e [F (20, 42) =67.25, p \u0026lt; 0.0001],\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e and Interleukin 6 (IL-6) \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e[F (20, 42) =7.849, p \u0026lt; 0.0001] activities using an in-vitro Human Chondrocyte Cell Line C20A4 cells. Each vertical-lined bar reflects the mean of the experiments ± S.D. Statistical analysis was performed by One-way ANOVA followed by Tukey's post-hoc test, with the criterion for statistical significance as follows: *p \u0026lt;0.05, **p \u0026lt;0.01, ***p \u0026lt;0.005, ****p \u0026lt;0.001, ns no significance.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7302080/v1/c3e29c992506cd3e5a4a837d.png"},{"id":104781648,"identity":"94824a1e-80f4-40f4-ab98-7eb618afc767","added_by":"auto","created_at":"2026-03-17 07:56:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1961412,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7302080/v1/4c425412-90db-4448-a654-7882a4489da5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eUpregulation of HDAC5 gene expression for treatment of osteoarthritis: Chondroprotective properties of regular and Nano-formulated Boswellia serrata Roxb\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a chronic, progressive, degenerative condition that affects the entire joint, characterized by a reduction in joint space, the formation of osteophytes, subchondral sclerosis, and the presence of cysts\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. The primary clinical manifestations of OA include joint pain and swelling, as well as a restricted range of motion\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. OA collectively impair the quality of life for affected individuals\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. This disease ranks among the most common diseases globally, and the disabilities it causes impose significant healthcare and socioeconomic challenges\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Due to its complex and multifactorial nature, halting the progression of OA is difficult; therefore, it is essential to adopt a strategy focuses on enhancing joint function, alleviating pain, and improving quality of life while minimizing adverse effects\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCurrently, OA lacks a definitive cure, and earlier strategies primarily aimed at alleviating joint pain instead of preventing the disease's progression. As a result, various treatments, including oral and topical nonsteroidal anti-inflammatory drugs, opioids, and serotonin norepinephrine reuptake inhibitors, were utilized for symptom management and functional improvement, though their extended use poses significant health threats\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Recently, the emphasis has shifted towards slowing the progression of the disease by discovering disease-modifying drugs that interact with relevant biomarkers and therapeutic targets\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe inflammatory biomarkers Interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) serve as significant indicators of severe OA, with their concentrations closely associated with reductions in joint space and cartilage degradation\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Furthermore, numerous studies have established a connection between the onset and progression of OA and the altered functions of histone deacetylases (HDACs) \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Among these, HDAC5, a member of the class IIa HDAC family, has been shown to inhibit chondrocyte hypertrophy by functioning as a co-activator of HDAC4, which operates via parathyroid hormone-related protein\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Additionally, reduced levels of HDAC5 have been observed in the cartilage of mice undergoing medial meniscus surgery and in patients with advanced OA. The silencing of HDAC5 was linked to increased degeneration of articular cartilage and chondrosenescence, while the activation of HDAC5 in chondrocytes through metformin effectively reversed OA\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecently, there has been a growing interest in plant-based natural remedies due to their safety and beneficial effects on bone health, which are linked to their chondroprotective and osteoprotective properties\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. One such remedy is \u003cem\u003eBoswellia serrata\u003c/em\u003e (Bs) gum resin, derived from the Bs tree native to the Indian highlands. This resin has a long history of use in treating arthritis and chronic inflammatory disorders\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. It is known for its potent anti-inflammatory and antioxidant effects, attributed to the presence of six different boswellic acids reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e Furthermore, an ethanol extract of Bs gum resin, rich in boswellic acids (FJH-UBS), has been shown to reduce inflammation and prevent the degradation of the extracellular matrix, demonstrating an anti-osteoarthritis effect in fibroblast-like chondrocyte cells derived from a 72-year-old white female with chondrosarcoma (SW1353) stimulated by interleukin (IL)-1β, as well as in animal models of osteoarthritis\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Additionally, the Bs extract applied to primary chondrocytes from osteoarthritis patients exhibited antioxidant properties through the activation of Nrf1 and Peroxisome Proliferator Activated Receptor alpha (PPARα) pathways, alongside anti-inflammatory effects by promoting growth/differentiation factor 15 (GDF15), decreasing cholesterol uptake, and regulating genes involved in fatty acid metabolism, while also reducing Toll-like receptor activation\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, the administration of Bs gum resin extract in a rat model of osteoarthritis induced by monosodium iodoacetate showed significant therapeutic potential by alleviating knee joint swelling, cartilage damage, and tissue deformation, as well as enhancing the expression of collagen type II alpha 1 and aggrecan, while downregulating nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, 5-lipoxygenase, prostaglandin E2 (PGE2), leukotriene B4, IL-6, TNF-α, and matrix metalloproteinases 3 and 13\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eA revolutionary method for improving the bioavailability and efficacy of medications, particularly in the treatment of conditions like osteoarthritis, is the application of novel nanotechnology techniques\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. By increasing the solubility and stability of bioactive compounds, nanoscale transporters facilitate improved absorption and retention at the target sites\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Nanocarriers provide a significant solution to multiple hurdles, predominantly by promoting a sustained drug release and intensified internalization, thereby augmenting the therapeutic potency\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. In addition, recent nanotechnology-driven formulations have demonstrated promising outcomes in the reduction of key inflammatory factors, comprising IL-1β and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) thereby aiding in the mitigation of osteoarthritis symptoms and disease pathology\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. These progressions underscore the vital role of nanotechnology in enhancing the efficacy of naturally derived therapeutic regimens for improved clinical outcomes\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSo, the aim of the present study is to investigate the potential effects of Bs extract and nano-emulsion on the human chondrocyte cell line (C20A4) in an \u003cem\u003ein-vitro\u003c/em\u003e model of OA by evaluating the gene expression of histone deacetylase 5 (HDAC5) and inflammation biomarkers IL1β, IL6, PGE2, NF-kB, and the survival rate of C20A4.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003ePlant materials\u003c/h2\u003e\u003cp\u003eIn April 2024, the gum resin of \u003cem\u003eBoswellia serrata\u003c/em\u003e was purchased from ATOS Pharma, Egypt. The plant material was formally verified for authenticity by Prof. Abdulhalim Abdelmogeli Mohammed, specialist in flora and vegetation at the Agricultural Research and Extension Authority, Cairo. A voucher specimen representing the authenticated sample was archived at the Department of Pharmacognosy, Faculty of Pharmacy, Heliopolis University. The obtained resin was preserved in an airtight container under proper storage conditions until further experimental use.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eChemicals and Reagents for cytotoxicity\u003c/h3\u003e\n\u003cp\u003eTo perform the cytotoxicity assessment through the MTT method, RPMI-1640 growth medium, phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), and the C20A4 cell line were acquired from Sigma-Aldrich, Germany. The MTT dye solution was procured separately from Bio Basic Inc., Canada.\u003c/p\u003e\n\u003ch3\u003eChemicals and Reagents for HPLC-ESI-MS/MS\u003c/h3\u003e\n\u003cp\u003eDelphinidin-3,5-diglucoside chloride and kaempferol-3-glucoside were obtained from PhytoLab, Vestenbergsgreuth, Germany, whereas the other analytical reference standards were sourced from Sigma-Aldrich, Milan, Italy. Stock solutions for each compound (1000 mg/L) were prepared by dissolving the respective pure standards in methanol of HPLC grade. All solvents employed in the study were of HPLC quality. Prior to chromatographic injection, the samples were passed through a Phenex\u0026trade; RC syringeless filter (4 mm, 0.2 \u0026micro;m; Phenomenex, Castel Maggiore, BO, Italy).\u003c/p\u003e\n\u003ch3\u003eChemicals and reagents for Nano emulsion (NanoEm)\u003c/h3\u003e\n\u003cp\u003eSigma-Aldrich provided the chemicals required for NanoEm formulation, which included Tris, glycine, SDS, Tween 80, polyethylene glycol 600 (PEG 600), dimethyl sulfoxide (DMSO), phosphate-buffered saline tablets, and castor oil. All reagents were of analytical quality.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eExtraction\u003c/h2\u003e\u003cp\u003eA portion of 10 g from the collected sample was subjected to ultrasonic-assisted extraction with 90% methanol (HPLC grade) until complete exhaustion. The resulting extract was filtered, and the solvent was completely removed under reduced pressure using a rotary evaporator maintained at 50\u0026deg;C. The obtained dry residue was weighed, and the required portion was forwarded for further analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChondroprotective and osteoprotective activity - Cytotoxic activity (MTT assay)\u003c/b\u003e\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe MTT assay was employed to investigate the cytotoxic activity of Bs extracts (conventional and nano-formulated) in comparison with the standard Blank (Blk). Cells were seeded in 96-well plates at a density of 1 \u0026times; 10⁵ cells/mL (100 \u0026micro;L per well) and incubated at 37\u0026deg;C for 24 h, allowing a uniform monolayer to form. Upon reaching confluence, the growth medium was removed, and the plates were rinsed twice with washing medium. Test extracts were serially diluted in RPMI supplemented with 2% maintenance medium to prepare eight concentrations (10000, 5000, 2500, 1250, 625, 312.5, 156.25, and 78.12 \u0026micro;g/mL). A 0.1 mL portion of each dilution was dispensed into separate wells, while three wells containing only maintenance medium served as controls. Plates were incubated again at 37\u0026deg;C, and cells were monitored for toxic morphological changes, including monolayer disruption, rounding, shrinkage, and granule formation. Afterward, 20 \u0026micro;L of MTT solution (5 mg/mL in PBS) was pipetted into each well and gently agitated on a shaker at 150 rpm for 5 min. The plates were incubated for 4 h at 37\u0026deg;C in a 5% CO₂ atmosphere to permit the enzymatic reduction of MTT to formazan. Supernatants were then discarded, and crystals were solubilized with 200 \u0026micro;L DMSO under shaking (150 rpm, 5 min). Optical density values were determined at 560 nm with correction against a reference wavelength of 620 nm. Cell viability was expressed as percentage relative to untreated controls.The relative cell-viability percentage was evaluated by using the following relation:\u003c/p\u003e\u003cp\u003eRelative cell-viability %= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{c}\\text{e}\\text{l}\\text{l}\\text{s}\\:\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d}\\:\\text{w}\\text{i}\\text{t}\\text{h}\\:\\text{t}\\text{h}\\text{e}\\:\\text{t}\\text{e}\\text{s}\\text{t}\\text{e}\\text{d}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\:\\text{a}\\text{t}\\:560\\:\\text{n}\\text{m}}{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\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{a}\\text{t}\\:560\\:\\text{n}\\text{m}}\\)\u003c/span\u003e\u003c/span\u003e \u0026times; 100.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation and characterization of\u003c/b\u003e \u003cb\u003eNanoEm\u003c/b\u003e\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe nanoemulsion (NanoEm) was prepared using castor oil, phosphate-buffered saline (PBS, 10 mM, pH 7.4), and Tween 80 in a ratio of 10:5:2.5, respectively. To begin, 50 mg of Bs powder was dissolved in 5 mL of PBS and subjected to ultrasonic treatment with a probe sonicator (cycle 1, amplitude 80%). Subsequently, 2.5 mL of Tween 80 was incorporated as the surfactant. The resulting mixture was then transferred to a cold-water bath, where castor oil (10 mL) was added dropwise under continuous homogenization (Daihan Homogenizer, ZS version, Korea) operated at 750 rpm for 11 minutes. The obtained nanoemulsion was employed for further characterization and subsequent experimental applications.\u003c/p\u003e\u003cp\u003eCharacterization of NanoEm included determination of droplet size, polydispersity index (PDI), and zeta potential using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C. Samples were diluted with PBS (10 mM, pH 7.4) to achieve a particle count within the range of 50\u0026ndash;300 KCPs. Morphological features were examined by transmission electron microscopy (TEM, model H-700, Hitachi Ltd., Japan). For TEM analysis, samples were diluted at a ratio of 1:50 with PBS (10 mM, pH 7.4), and a drop of the diluted suspension was placed on carbon-coated copper grids. After air drying for 5 minutes, the grids were negatively stained with 2% w/w phosphotungstic acid for 50 seconds, and excess stain was removed using filter paper.\u003c/p\u003e\u003cp\u003eAdditionally, the physicochemical properties of the NanoEm were evaluated. The pH was measured at room temperature using an Ohaus Starter 3100 bench pH meter (USA), which had been calibrated with standard buffer solutions of pH 4, 7, and 10. Viscosity was determined using a Lamy Rheology viscometer (B-One Plus, Germany) with spindle 6 at a rotation speed of 50 rpm for 3 minutes.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eQuantitative real-time polymerase chain reaction\u003c/h3\u003e\n\u003cp\u003eTotal RNA from C20A4 cells was isolated using the RNeasy\u0026reg; Mini Kit (Catalog No. 74904, QIAGEN, Aarhus, Denmark) in accordance with the manufacturer\u0026rsquo;s protocol. At the conclusion of the extraction and purification steps, RNA quality and purity were verified by spectrophotometric measurement of the absorbance ratio at 260/280 nm using a Denovix spectrophotometer (AGBL, USA). The purified RNA (0.5\u0026ndash;2 \u0026micro;g) was then subjected to reverse transcription into complementary DNA (cDNA) with the aid of the SuperScript IV One-Step RT-PCR kit (Catalog No. 204843, Qiagen QuantiTect RT kit), which is highly sensitive for such applications.\u003c/p\u003e\u003cp\u003eFor subsequent quantitative real-time PCR (qPCR), an optimized kit containing Taq polymerase, qPCR reaction buffer, primers, SYBR\u0026reg; Green I dye, and nucleotides was employed. The primer sequences utilized during the annealing stage are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Relative expression levels (RQ) of target genes, including HDAC5, NF-κB, prostaglandin EP2 receptor, IL-1β, and IL-6 were determined using the calculation algorithm provided in the Applied Biosystems software formula, ∆\u0026#917828;Ct\u0026thinsp;=\u0026thinsp;Ct gene test - Ct housekeeping control and ∆∆Ct\u0026thinsp;=\u0026thinsp;Ct sample1 - Ct calibrator, where (RQ) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{2}}^{\\text{-∆∆Ct}}\\)\u003c/span\u003e\u003c/span\u003e. The outcomes were normalized to GADPH and presented as arbitrary units.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe primers sequence of studied genes in quantitative real-time polymerase chain reaction.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer Sequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHDAC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward 5\u0026prime;- TCTTGTCGAAGTCAAAGGAGC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003cp\u003eReverse 5\u0026prime;- GAGGGGAACTCTGGTCCAAAG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNF-κB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward 5\u0026prime;- GCAGCACTACTTCTTGACCACC-3\u0026prime;\u003c/p\u003e\u003cp\u003eReverse 5\u0026prime;- TCTGCTCCTGAGCATTGACGTC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEP2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward 5\u0026prime;-CCACCTCATTCTCCTGGCTA-3\u003c/p\u003e\u003cp\u003eReverse 5\u0026prime;-CGACAACAGAGGACTGAACG-3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-1β\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward 5\u0026prime;- CCACAGACCTTCCAGGAGAATG-3\u0026prime;\u003c/p\u003e\u003cp\u003eReverse 5\u0026prime;- GTGCAGTTCAGTGATCGTACAGG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward 5\u0026prime;- AGACAGCCACTCACCTCTTCAG-3\u0026prime;\u003c/p\u003e\u003cp\u003eReverse 5\u0026prime;- TTCTGCCAGTGCCTCTTTGCTG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHPLC-ESI-MS/MS\u003c/b\u003e\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHigh-performance liquid chromatography coupled with tandem mass spectrometry (HPLC\u0026ndash;MS/MS) was conducted using an Agilent 1290 Infinity system linked to a Triple Quadrupole 6420 (Agilent Technologies, Santa Clara, CA, USA). The system was fitted with an electrospray ionization (ESI) interface, which was operated alternately in positive and negative ionization modes. Polarity switching was performed within a single analytical run without complications. For each analyte, MS/MS parameters were tuned through flow injection analysis (FIA) by injecting 1 \u0026micro;L of a 10 mg L⁻\u0026sup1; individual standard solution, using the Agilent Optimizer software.\u003c/p\u003e\u003cp\u003eChromatographic separation of the analytes was achieved on a Synergi Polar-RP C18 analytical column (250 mm \u0026times; 4.6 mm, 4 \u0026micro;m; Phenomenex, Cheshire, UK), protected with a Polar RP guard cartridge (4 mm \u0026times; 3 mm ID). The mobile phase consisted of two solvents: (A) water with 0.1% formic acid and (B) methanol with 0.1% formic acid. A gradient elution program at a flow rate of 0.8 mL/min was applied as follows: 0\u0026ndash;1 min, isocratic at 20% B; 1\u0026ndash;25 min, linear increase from 20\u0026ndash;85% B; 25\u0026ndash;26 min, isocratic at 85% B; and 26\u0026ndash;32 min, return from 85\u0026ndash;20% B. All solvents and prepared solutions were filtered prior to use through 0.2 \u0026micro;m polyamide membranes (Sartorius Stedim, Goettingen, Germany).\u003c/p\u003e\u003cp\u003eThe injection volume for each run was 2 \u0026micro;L. The column was maintained at 30\u0026deg;C, while the drying gas temperature in the ion source was set at 350\u0026deg;C. Additional source conditions included a drying gas flow of 12 L/min, nebulizer pressure of 55 psi, and capillary voltage of 4000 V. Data acquisition was carried out in dynamic multiple reaction monitoring (dynamic-MRM) mode. For quantitation, the most intense product ion was selected, while additional transitions were employed for confirmation. A retention time window of \u0026plusmn;\u0026thinsp;2 min was assigned for each analyte. Detailed ion transitions along with optimized MS parameters for the studied compounds are provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll results were presented as mean values accompanied by their corresponding standard deviations (SD). The Kolmogorov\u0026ndash;Smirnov test was applied to determine whether the datasets followed a normal distribution. To assess differences among groups, a one-way analysis of variance (ANOVA) was conducted, and when significance was detected, Tukey\u0026rsquo;s post hoc test was employed for multiple pairwise comparisons. Statistical evaluations and preparation of graphical outputs were carried out using GraphPad Prism software, version 9 (GraphPad Software, Inc., San Diego, CA, USA). A probability value of less than 0.05 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered the threshold for statistical significance. Outlier identification was performed using the Dixon test, and only values that failed this test were excluded from analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHPLC\u0026ndash;MS/MS acquisition parameters (dynamic-MRM mode) used for the analysis of the 28 marker compounds.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePrecursor ion, \u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProduct ion, \u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFragm-entor, V\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCollision energy, V\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePolarity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eRetention time (Rt, min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eDelta retention time (ΔRt)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGallic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e169\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e125.2\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNeochlorogenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e353\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e191.2\u003csup\u003e*\u003c/sup\u003e, 179\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12, 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e9.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(+)-Catechin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e289\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e245.2\u003csup\u003e*\u003c/sup\u003e,109.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8, 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e11.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProcyanidin B2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e576.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e576.99\u003csup\u003e*\u003c/sup\u003e, 321.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0, 32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e12.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChlorogenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e353\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e191.2\u003csup\u003e*\u003c/sup\u003e, 127.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12, 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e12.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-Hydroxybenzoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e137\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93.2\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e12.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(-)-Epicatechin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e289\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e245.1\u003csup\u003e*\u003c/sup\u003e, 109.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8, 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e13.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3-Hydroxybenzoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e137\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93.2\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e13.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCaffeic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e179\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e135.2\u003csup\u003e*\u003c/sup\u003e, 134.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12, 24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e13.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVanillic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e152.4\u003csup\u003e*\u003c/sup\u003e, 108.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12, 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e14.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResveratrol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e227\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e185\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e14.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSyringic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e196.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e182.2\u003csup\u003e*\u003c/sup\u003e, 121.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8, 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e15.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProcyanidin A2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e575\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e575\u003csup\u003e*\u003c/sup\u003e, 285\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0, 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e16.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-Coumaric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e163\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e119.2\u003csup\u003e*\u003c/sup\u003e, 93.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12, 36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e16.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFerulic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e193\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e134.2\u003csup\u003e*\u003c/sup\u003e, 131.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12, 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e17.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3,5-Dicaffeoylquinic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e514.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e353.1\u003csup\u003e*\u003c/sup\u003e, 191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8, 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e17.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRutin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e609\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e300.2\u003csup\u003e*\u003c/sup\u003e, 271.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e32, 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e17.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIsoquercitrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e463\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e271.2\u003csup\u003e*\u003c/sup\u003e, 300.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e44, 24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e18.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDelphindin-3,5-diglucoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e462.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e300.1\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e165\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e18.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhloridzin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e435.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e273\u003csup\u003e*\u003c/sup\u003e, 167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8, 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e18.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuercitrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e446.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e300.2\u003csup\u003e*\u003c/sup\u003e, 301.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24, 16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e19.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMyricetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e316.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e179.1\u003csup\u003e*\u003c/sup\u003e, 182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16, 24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e19.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNaringin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e578.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e271.3\u003csup\u003e*\u003c/sup\u003e, 151.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e32, 44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e19.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKaempferol-3-glucoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e447\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e284.2\u003csup\u003e*\u003c/sup\u003e, 255.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24, 40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e19.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEllagic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e301\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e301\u003csup\u003e*\u003c/sup\u003e, 229\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0, 24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e21.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuercetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e300.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e151.2\u003csup\u003e*\u003c/sup\u003e, 179.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16, 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e21.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhloretin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e272.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e167\u003csup\u003e*\u003c/sup\u003e, 123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8, 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e22.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIsorhamnetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e314.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e300.2\u003csup\u003e*\u003c/sup\u003e, 196.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16, 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e24.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e*\u003c/sup\u003e These product ions were used for quantification.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eChondroprotective and osteoprotective activity\u003c/h2\u003e\u003cp\u003eThe data illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e compare the survival rate in C20A4 of the \u003cem\u003eBs\u003c/em\u003e extract in both conventional and nano-formulation forms against the standard Blk., evaluated through the MTT assay on Human Chondrocyte Cell Line C20A4 cells. The extracts and the standard were tested at various concentrations (10000, 5000, 2500, 1250, 625, 3125, 156.25, 78.12 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL). Notably, the conventional \u003cem\u003eBs\u003c/em\u003e extract achieved an IC50 value of 674.5, which corresponds to 18.89% of the Blk. group's effect. In contrast, the nano-formulated version yielded an IC50 of 4274, equating to 119.69% of the standard, indicating enhanced cell viability with the Nano-formulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBs Extract Restores Epigenetic Balance and Suppresses Inflammatory Markers in C20A4 Cells: Enhanced Efficacy with Nano-formulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the C20A4 osteoarthritic chondrocyte model displayed a notable dysregulation in inflammatory and epigenetic markers. Specifically, HDAC5 (Histone Deacetylase 5), a key regulator of gene expression and anti-inflammatory response, was reduced by 10.07% relative to the normal control group. In contrast, there was a marked upregulation of NF-κB, a pro-inflammatory transcription factor, and the Prostaglandin EP2 receptor, which mediates inflammation and pain signaling. These were elevated to 111.75% and 109.29%, respectively, compared to the normal control.\u003c/p\u003e\u003cp\u003eUpon treatment with \u003cem\u003eBs\u003c/em\u003e extract, both the conventional and Nano-formulated preparations produced significant modulatory effects. Notably, HDAC5 levels increased beyond the baseline of the normal control, reaching 124% and 131% in the conventional and Nano-formulated extract-treated groups, respectively. This suggests a potential epigenetic restoration or enhancement effect, particularly with the nanoformulation.\u003c/p\u003e\u003cp\u003eSimultaneously, both treatments effectively reduced the elevated inflammatory markers. The conventional extract reduced NF-κB and EP2 receptor levels to 89% and 87%, respectively, while the nanoformulation demonstrated slightly greater efficacy, lowering them to 88% and 82% of normal control values. These findings indicate that \u003cem\u003eBs\u003c/em\u003e, especially in Nano-formulated form, has anti-inflammatory potential through suppression of key inflammatory pathways.\u003c/p\u003e\u003cp\u003eWhen compared to the standard therapeutic agent Blk., the extract showed comparable effects. Blk. also elevated HDAC5 and reduced NF-κB and EP2 expression to similar levels. The only statistically notable difference was in EP2 receptor expression, where the Nano-formulated \u003cem\u003eBoswellia\u003c/em\u003e achieved a further reduction to 82%, while Blk. reached only 89%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings highlight the potential of \u003cem\u003eBs\u003c/em\u003e, particularly in its Nano-formulated version, to counteract inflammation and restore epigenetic balance in osteoarthritic conditions. The extract's ability to elevate HDAC5 and suppress NF-κB and EP2 receptor expression supports its therapeutic value, possibly offering a comparable or even superior alternative to conventional treatments like Blk. for managing osteoarthritis-related inflammation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eNano-formulated Bs Exhibits Superior Suppression of Pro-inflammatory Cytokines IL-1β and IL-6 in Osteoarthritic Chondrocytes\u003c/h2\u003e\u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the C20A4 osteoarthritic chondrocyte model demonstrated a pronounced pro-inflammatory response, with IL-1β and IL-6 levels increasing to 115% and 112%, respectively, relative to the normal control group. These cytokines are key mediators of inflammation, cartilage degradation, and joint damage in osteoarthritis. Treatment with the standard therapeutic agent, Blk., resulted in a modest reduction in IL-1β, lowering it to 92% of the level observed in C20A4 cells. However, it had no significant impact on IL-6, suggesting limited anti-inflammatory efficacy in this context.\u003c/p\u003e\u003cp\u003eIn contrast, treatment with \u003cem\u003eBs\u003c/em\u003e extract, both in conventional and Nano-formulated forms, led to notable reductions in both cytokines. The conventional extract decreased IL-1β to 70% and IL-6 to 90%, while the Nano-formulated extract produced an even more pronounced effect, reducing IL-1β to 64% and IL-6 to 82% compared to the C20A4 group.\u003c/p\u003e\u003cp\u003eWhen benchmarked against the standard Blk., the conventional extract showed superior efficacy in reducing IL-1β, and the nanoformulation demonstrated superiority in suppressing both IL-1β and IL-6. Specifically, the nanoformulation reduced IL-1β and IL-6 to 70% and 85%, respectively, relative to the standard. These findings underscore the anti-inflammatory potential of \u003cem\u003eBs\u003c/em\u003e, particularly in its Nano-formulated form, in mitigating key inflammatory cytokines involved in osteoarthritis. The extract not only outperformed the standard treatment in reducing IL-1β but also showed added benefit in lowering IL-6 levels, highlighting its promise as a more effective therapeutic strategy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eHPLC-MS/MS analysis of the dried extract\u003c/h2\u003e\u003cp\u003eFound to be strongly potent in gene expression downregulation of the inflammation biomarkers IL1β, IL6, PGE2, NF-kB, upregulation of HDAC5 gene expression and elevation of survival rate in C20A4, methanolic extract of \u003cem\u003eBs\u003c/em\u003e gum resin was analyzed by HPLC-ESI\u0026ndash;MS/MS to examine the chemical profile. None of the standard 28 compounds was identified in \u003cem\u003eBs\u003c/em\u003e methanol extract, out of 28 bioactive polyphenol reference compounds Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The 28 compounds were quantified after the acquisition settings of the dynamic MRM mode were optimized. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e lists the chosen ion transitions and mass spectrometer parameters for each molecule. Twenty-eight reference compounds belonging to different phenolic classes were used and the results are depicted in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. HPLC-MS/MS chromatogram of a standard mixture of 28 phenolic compounds plotted as overlapped multiple reaction monitoring (MRM) negative and positive transition of each analyte is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eContent (\u0026micro;g/g of dried extract) of phenolic compounds found in \u003cem\u003eBs\u003c/em\u003e methanol extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenolic compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eContent \u0026micro;g/g\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGallic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNeochlorogenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCatechin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eprocyanidin B2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChlorogenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4-Hydroxy benzoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEpicatechin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-Hydroxy benzoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCaffeic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVanillic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSyringic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProcyanidin A2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-Coumaric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFerulic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRutin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsoquercitrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDelphindin 3,5 diglucoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhloridzin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaringin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQuercitrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMyricetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKaempferol-3-glucoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEllagic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQuercetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhloretin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsorhamnetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTOT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study demonstrates the promising therapeutic potential of \u003cem\u003eBs\u003c/em\u003e extract in modulating key molecular pathways involved in osteoarthritis, particularly through its ability to restore epigenetic balance and suppress pro-inflammatory markers. The C20A4 osteoarthritic chondrocyte model revealed significant disruptions in cellular homeostasis, marked by decreased expression of HDAC5, a crucial regulator of anti-inflammatory gene expression, and elevated levels of NF-κB and the Prostaglandin EP2 receptor, both of which play central roles in mediating inflammatory responses and cartilage degradation.\u003c/p\u003e\u003cp\u003eTreatment with \u003cem\u003eBs\u003c/em\u003e extract, in both conventional and Nano-formulated forms, successfully reversed these alterations. Notably, HDAC5 expression was not only restored but enhanced, indicating potential epigenetic reprogramming in favor of anti-inflammatory activity. This was accompanied by a reduction in NF-κB and EP2 receptor expression, further supporting the extract\u0026rsquo;s role in attenuating inflammatory signaling. The Nano-formulated form of the extract consistently exhibited greater efficacy than its conventional counterpart, suggesting improved cellular uptake or bioavailability as a result of the nanotechnology-based delivery system.\u003c/p\u003e\u003cp\u003eParallel analysis of the pro-inflammatory cytokines IL-1β and IL-6 provided additional evidence of the extract\u0026rsquo;s anti-inflammatory properties. While the standard therapeutic agent modestly reduced IL-1β without affecting IL-6, \u003cem\u003eBs\u003c/em\u003e extract significantly suppressed both cytokines. The Nano-formulated preparation again showed superior performance, effectively reducing both IL-1β and IL-6 levels beyond the effects observed with the conventional extract or standard treatment.\u003c/p\u003e\u003cp\u003eTaken together, these findings suggest that \u003cem\u003eBs\u003c/em\u003e exerts its therapeutic effects in osteoarthritic chondrocytes by targeting both epigenetic regulators and inflammatory mediators. The consistent superiority of the Nano-formulated version highlights the added value of advanced drug delivery systems in enhancing the bioactivity of phytotherapeutic agents. Overall, \u003cem\u003eBs\u003c/em\u003e, particularly in Nano-formulated form, represents a promising candidate for further development as a disease-modifying agent in osteoarthritis, with potential advantages over existing conventional therapies.\u003c/p\u003e\u003cp\u003eIn the C20A4 chondrocyte model, osteoarthritic downregulation of HDAC5 was not only restored but exceeded normal baseline following Bs treatment, with nano-formulations showing superior efficacy. This was accompanied by reduced NF-κB, EP2 receptor, IL-1β, and IL-6 expression, suggesting upstream transcriptional reprogramming rather than mere downstream cytokine blockade. The enhanced activity of the nano-formulated extract likely reflects improved bioavailability, positioning Bs as a promising disease-modifying therapy for osteoarthritis through epigenetic restoration and inflammation suppression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003eThe authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through a small group Research Project under grant number RGP1/68/46\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eZobaida A. Mohamed:\u003c/strong\u003e Conceptualization; Methodology; Writing\u0026ndash;original draft; Writing \u0026amp; editing. \u003cstrong\u003eMarwa Saeed, Laura Acquaticci, Muhammed A. Saad,\u003c/strong\u003e \u003cstrong\u003eYasmin Abo-zeid, Asmaa M. Elbakry:\u003c/strong\u003e Conceptualization; Methodology; Supervision; Writing\u0026ndash;review \u0026amp; editing. \u003cstrong\u003eMohamed Rabeh, Saad Alshehri:\u0026nbsp;\u003c/strong\u003eFunding Conceptualization; Methodology; Supervision; Writing\u0026ndash;original draft; Reviewing \u0026amp; editing.\u003cstrong\u003eOmar Sabry\u003c/strong\u003eand\u003cstrong\u003eGiovanni Caprioli:\u003c/strong\u003e Conceptualization; Methodology; Supervision; Writing\u0026ndash;original draft; Reviewing \u0026amp; editing.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003eThis research was funded by King Khalid University, grant number RGP1/192/46 and The APC was funded by King Khalid University.\u0026nbsp;\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003eThe authors confirm that they have no conflicts of interest.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003eEthics approval was not required for this research.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKuyinu, E. 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M. et al. A new HPLC-MS/MS method for the simultaneous determination of 36 polyphenols in blueberry, strawberry and their commercial products and determination of antioxidant activity. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003eno. 367\u003c/b\u003e, 130743 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"Osteoarthritis (OA), Boswellia serrata, HDAC5, IL1β, IL6, PGE2, NF-kB, human chondrocyte cells (C20A4)","lastPublishedDoi":"10.21203/rs.3.rs-7302080/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7302080/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoarthritis is a serious disorder that destroys cartilage and joints and inhibits collagen formation. Natural products are widely recognized for their wide variety of therapeutic uses and great safety records. Using human chondrocyte cells (C20A4) and MTT assays, the potential benefits of regular and nano-formulated \u003cem\u003eBoswellia serrata\u003c/em\u003e (Bs) methanol extract for treating osteoarthritis by influencing the expression of the HDAC5, IL1β, IL6, PGE2, NF-kB genes were evaluated. HDAC5 gene expression was significantly upregulated and survival rate of C20A4 was strongly elevated. IL1β, IL6, PGE2, NF-kB inflammation biomarkers gene expression was significantly suppressed by both conventional and Nano-formulations. In comparison to conventional extracts, Nano-formulations exhibited greater activity revealing that they might represent a more effective treatment strategy. HPLC-MS/MS technology of the Bs extract against 28 standard phenolic compounds e.g. \u003cem\u003ep-\u003c/em\u003ecoumaric acid, ferulic acid, rutin, myricetin and quercetin, proved the absence of these compounds in Bs gum resin.\u003c/p\u003e","manuscriptTitle":"Upregulation of HDAC5 gene expression for treatment of osteoarthritis: Chondroprotective properties of regular and Nano-formulated Boswellia serrata Roxb","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 13:01:48","doi":"10.21203/rs.3.rs-7302080/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":"8c6da411-780d-4ccf-89b9-2cab8e969e63","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55509244,"name":"Biological sciences/Biochemistry"},{"id":55509245,"name":"Biological sciences/Biotechnology"},{"id":55509246,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2026-03-12T12:41:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 13:01:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7302080","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7302080","identity":"rs-7302080","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}