**1. Introduction**

Osteoarthritis (OA) is a lingering joint illness accompanied by inflammation of the synovium and cartilage degeneration, causing physical disability in the elderly. Functional foods and medicines have been commonly utilized to care for OA, but pharmacological treatment of OA has limited effects [1,2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are usually utilized for reducing inflammation and pain in OA. However, a long-time consumption of NSAIDs adversely affects the gastrointestinal and cardiovascular systems [3,4]. To this day, the etiology of OA is not obvious, and no effectual therapeutic treatment has been developed for OA. Therefore, safer and more effective novel agents are needed for the treatment of OA [1].

The extracellular matrix (ECM) is mostly an organization of type I and II collagen, and aggrecan, which are the main constituents of ordinary cartilage that support the arthrodial cartilage to adapt to biomechanical forces during joint activity [5]. ECM is created and retained by the chondrocytes and is controlled by SOX9, which encodes the main transcription factor for ECM homeostasis [6,7]. Many studies have shown that inflammatory reactions generally play an enormous role in the OA and contributes to chondrocyte movement and phenotype and ECM degradation [7–9]. Overexpression of pro-inflammatory cytokines like IL-1β and IL-6 are implicated in the etiology of OA by upregulating the matrix metalloproteinases (MMPs) and triggering ECM collapse. In particular, IL-1β exerts inflammatory reactions by considerably upregulating the production of pro-inflammatory factors, and catabolic factors, for example, leukotriene B4 (LTB4), nitric oxide (NO), and MMPs to degrade the ECM [10–12].

MAPK mechanisms have been revealed to play an apparent part in terms of OA biology such as matrix composition and homeostasis of cartilage [13,14]. Additionally, NF-κB mechanisms are a core controller of pro-inflammatory and catabolic factor production. When the NF-κB mechanisms are activated, NF-κB p65 is phosphorylated in the cytoplasm and ultimately translocated to the nucleus [15,16]. Practically, transitions in these mechanisms have been identified to play a crucial role in articular chondrocyte function as well as form part of OA etiology and illness progression [17].

The fruit of *Terminalia chebula* Retz. (Fam. Combretaceae) has been widely utilized in Ayurvedic, Iranian medicine, and Unani as a treatment for diverse diseases such as asthma, bleeding piles, sore throat, vomiting, and gou<sup>t</sup> [18–20]. Moreover, *T. chebula* has been widely known to exhibit antioxidant e ffects by inhibiting ROS and NO production [21–24]. Clinical research has also proven that oxidative stress and inflammation contribute to OA, low back pain (LBP), and motor-related joint discomfort. The *T. chebula* fruit exhibits antioxidant e fficacy and downregulates inflammatory cytokines; however, its therapeutic e ffects warrant further investigation [25–29].

Meanwhile, recent preclinical and clinical studies have revealed that the standardized aqueous extract of *T. chebula* fruit (AyuFlex ®) could markedly suppress OA progression [25,30–35]. However, the underlying mechanism, the anti-arthritic e ffect of AyuFlex ®, remains obscure. Therefore, in our study, we devised experiments to clarify the e ffectiveness and applications of AyuFlex ®, and to evaluate the protective e ffectiveness of arthrodial cartilage in IL-1β-treated chondrocytes and MIA-incurred OA in a rat model.

### **2. Materials and Methods**

### *2.1. AyuFlex* ® *Preparation and Component Analyze*

AyuFlex ®, a water-soluble product derived from the edible fruits of *T. chebula* (Natreon Inc., New Brunswick, NJ, USA) [35], presents a phytochemical profile that includes ellagic acid as standardized using high-performance liquid chromatography (HPLC). Dimethyl sulfoxide (DMSO) was utilized to dissolve the AyuFlex ® and was then diluted in chondrocyte culture medium for in vitro studies.

### *2.2. Culture and Sample Processing of Primary Human Chondrocytes (HCHs)*

Primary human chondrocytes (HCHs) were provided by PromoCell Bioscience Alive GmbH (Heidelberg, Germany) and also retained in HCH culture medium complemented with fetal calf (FC) serum in CO2 incubator. When 80–90% confluence was reached, HCHs were subcultured, and cells of passage 1 were utilized for the experiment thereafter. HCHs was cultured in a 6-well plate at 1 × 10<sup>5</sup> cells per well. After 24 h, the HCH cells were exposed at each concentration of AyuFlex ® (5, 10, and 20 μg/mL) and in combination with IL-1β (10 ng/mL) in a humidified incubator for 24 h. HCHs exposed with growth media including only DMSO served as the vehicle control (final concentration of DMSO 0.1%).

### *2.3. Cell Viability Analysis*

Cell viability was conducted utilizing the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. HCHs were exposed with each concentration of AyuFlex ® (5, 10, and 20 μg/mL) during 24 h. The MTT reagen<sup>t</sup> (5 mg/mL) was dispensed into each well, and the cells were maintained in a humidified incubator for 3 h. The culture supernatants were suctioned from each well, and DMSO was utilized to melt the formazan crystals. The optical density (OD) was analyzed at a wavelength of 570 nm utilizing microplate reader equipment (Tecan, Mannedorf, Switzerland).

### *2.4. Western Blotting*

After lysing HCHs with CelLytic reagen<sup>t</sup> (Sigma-Aldrich, St. Louis, MO, USA), the lysates were kept at 4 ◦C and centrifuged at 10,000× *g* for 15 min. The content of the proteins was calculated by utilizing a Bradford reagen<sup>t</sup> (Bio-Rad Laboratories, Hercules, CA, USA). To separate, the proteins were applied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and then blotted to Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk in tris-bu ffered saline comprising 0.1% Tween-20 (TBS-T) for 1 h at 23 ◦C, and next with primary antibodies for β-actin, COL1A1, NF-κB p65, phospho-NF-κ<sup>B</sup> p65, ERK, phospho-ERK (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), 5-LOX, IL-6, MMP-2, -3, and -13, aggrecan (1:1000; Abcam, Cambridge, MA, USA), SOX9, COL2A1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), iNOS (1:1000; Invitrogen Life Technologies, Carlsbad, CA, USA), and leukotriene B4 (LTB4; 1:500; Enzo Life Sciences, Farmingdale, NY, USA) overnight at 4 ◦C. Following incubation with primary antibodies, the membranes were reacted with the goa<sup>t</sup> anti-rabbit or -mouse IgG(H+L)-horseradish peroxidase (HRP) secondary antibodies for 1 h at room temperature (RT). Protein bands were detected with the chemiluminescent (ECL) reagen<sup>t</sup> (GenDEPOT, Barker, TX, USA), and the intensity of bands was sensed utilizing a LuminoGraph chemiluminescent imaging instrument (Atto, Tokyo, Japan). As control for normalization, β-actin was utilized. Bands on the membranes were quantified utilizing the ImageJ program (developed at the NIH).
