Enteric-Coated Cologrit Tablet Exhibit Robust Anti-Inflammatory Response in Ulcerative Colitis-like In-Vitro Models by Attuning NFκB-Centric Signaling Axis

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the patients’ colorectal area culminating in an inflamed ‘leaky gut.’ The majority of UC treatments only provide temporary respite leading to its relapse. Therefore, this study investigated the efficacy of the enteric-coated ‘Cologrit’ (EC) tablet in alleviating UC-like inflammation. Cologrit is formulated using polyherbal extracts that have anti-inflammatory qualities according to ancient Ayurveda scriptures. Phytochemical profiling revealed the presence of gallic acid, rutin, ellagic acid, and imperatorin in Cologrit formulation. Cologrit treatment decreased inflammation in LPS-induced transformed THP-1 macrophages, and TNF-α-stimulated human colorectal (HT-29) cells through the modulation of NFκB activity, IL-6 production, and NFκB, IL-1β, IL-8, and CXCL5 mRNA expression levels. Cologrit also lessened human monocytic (U937) cell adhesion to HT29 cells. Methacrylic acid-ethylacrylate copolymer-coating of the enteric Cologrit tablets (EC) supported their dissolution, and the release of phytochemicals in the small intestine pH 7.0 environment in a simulated gastrointestinal digestion model. Small intestine EC digestae effectively abridged dextran sodium sulfate (2.5% w/v)-induced cell viability loss and oxidative stress in human colon epithelial Caco-2 cells. In conclusion, the enteric-coated Cologrit tablets demonstrated good small intestine-specific phytochemical delivery capability, and decreased UC-like inflammation, and oxidative stress through the regulation of TNF-α/NFκB/IL6 signaling axis.


Introduction
Ulcerative colitis (UC) is an idiopathic chronic inflammatory bowel disease of the colon and rectum. The disease's aetiology is mostly unknown, but it is frequently linked to genetic, dietary, lifestyle, microbial infection, and environmental factors, alone or in combination [1,2]. Adults between the ages of 30-40 are mostly affected by UC, which is characterized by relapsing and remitting mucosal inflammation [3]. Patients with inflammatory bowel syndrome-like UC show elevated levels of pro-inflammatory cytokines, chemokines, and adhesion molecules in their intestinal mucosa biopsies [4][5][6][7][8]. Individual patients' UC affects different regions of the colon, typically beginning in the rectum and progressing to the proximal segments. The disease can spread throughout the colon (known as pan-colitis), or be localized between the proximal and rectal regions of the colon (ulcerative proctitis) [9,10]. Topical, oral, and systemic administration of aminosalicylates, Table 1. Percentage of herbal extracts used in Cologrit formulation preparation.

CSIR-NISCAIR (Voucher No.)
Aegle The easiest oral route delivery of medicines is in the form of tablets. The term "tablet" refers to a pharmaceutical dose made up of active ingredients and excipients. Polymer coatings present on tablets improves their appearance, easier swallowing, and controls site-specific release of active ingredients. The enteric coating (EC) of the surface inhibits solubility of the tablet in the low pH conditions of the stomach, while supporting dissolution under neutral pH condition of the intestinal region [21]. Methacrylic acid-ethylacrylate copolymer (1:1), Type A (MMA) is an anionic polymer used as an EC on tablets. It is also referred to as "Methacrylic Acid Copolymer, Type C" in USP XXI/NF XVI [22]. MMA coating supports solubilization of the tablets at pH greater than 5.5 and preventing enzymatic digestion of the active ingredients [22].

In Vitro Release of Phytocontituents from EC Cologrit Tablet
EC Cologrit tablets were subjected to in vitro gastrointestinal digestion, whic cluded passing through the mouth (15 s), stomach (2 h), and intestine (2 h) [47]. Th tablets did not show any evidence for the EC solubilization, or cracking in mouth or s ach phases ( Figure 4A,B). In the intestinal phase, the MMA-coating ruptured with min of treatment exposing the tablet interior causing a complete dissolution within min ( Figure 4C).   UV spectroscopy analysis of the solubilized EC Cologrit tablets showed the presence of a specific absorption band between 307 and 371 nm, with a 'λ maxima' (λmax) at 339 nm with no interferences (Figure 5A,B). No Cologrit spectra was detected in the stomach phase 'chyme' between t = 0 and t = 120 min ( Figure 5A). However, a time-dependent absorbance spectrum for Cologrit tablet was observed in the intestinal phase 'digesta' indicating its dissolution ( Figure 5B). The EC Cologrit tablet disintegrated fastest between 40-60 min, after which the process slowed, indicating that the EC layer dissolved swiftly in the simulated digestive juice (pH = 6.8) ( Figure 5B). The findings were validated by HPLC analysis, which revealed the lack of Gallic acid, Rutin, Ellagic acid, and Imperatorin in stomach phase 'chyme' samples (t = 0, t = 60, and t = 120) (Table 4, Figure 5C). HPLC analysis of the intestinal phase revealed the presence of Gallic acid: t = 60 min (0.09 ± 0.06 µg/mg) and t = 120 min (0.12 ± 0.05 µg/mg); Rutin: t = 60 min (0.25 ± 0.02 µg/mg) and t = 120 min (0.29 ± 0.0 µg/mg); Ellagic acid: t = 60 min (0.21 ± 0.04 µg/mg) and t = 120 min (0.28 ± 0.07 µg/mg); and Imperatorin: t = 60 min (0.04 ± 0.02 µg/mg) and t = 120 min (0.07 ± 0.02 µg/mg) ( Table 4). When the HPLC profiles of EC Cologrit tablets disintegrated in the intestinal phase (t = 120 min) was compared to that of undigested Cologrit alone profile, the phytochemical release efficacy was determined to be 63.15% for Gallic acid, 85.29% for Rutin, 71.79% for Ellagic acid, and 4.57% for Imperatorin ( Figure 5; Table 4). The low detectable quantity of Imperatorin may be due to its conversion into xanthotoxol, which was not investigated in this work [48].
The efficacy of the GIT digested EC Cologrit tablet was analyzed in the human colonic Caco-2 cells. Digestae of the EC Cologrit tablet was found to be biocompatible in Caco-2 cells up to the tested concentration of 100 µg/mL and 24 h treatment time ( Figure 6A). The treatment of the Caco-2 cells with DSS caused a concentration-dependent loss of cell viability ( Figure 6B). DSS showing an inhibitory concentration 50% (IC 50 ) of 3.3% (w/v) ( Figure 6B). Hence for the study the treatment doe of 2.5% (w/v) was selected. Pre-and co-treatment of the Caco-2 cells with the undigested Cologrit showed a minor protection against the 2.5% (w/v) DSS-induced loss of cell viability (0 µg/mL: 64.27 ± 0.58%; p-value < 0.001).

Discussion
Ulcerative colitis is an inflammatory illness that causes inflammation and pain in the gastrointestinal tract. The condition produces severe discomfort and requires drastic lifestyle modifications. UC symptoms include 'Shula' (abdominal discomfort), 'Gudapaaka' (rectum burning), and 'Trishna' (extreme thirst), according to the ancient Ayurveda textbook 'Charak Samhita' [16]. Corticosteroids (CS) form a major part of UC medication, which attenuates the immune response giving relief in the form of anti-inflammatory drugs [49]. CS is administered to UC patients either orally or systemically and their longterm use can cause osteoporosis, depression, moon face, type 2 diabetes, and cataracts [1,49]. Because of its simplicity, and convenience, drug administration through the oral route is considered the best therapeutic option [50]. Oral drugs, on the other hand, are subjected to a harsh environment specifically during ongoing inflammatory bowel disease conditions. The orally delivered medicines may get degraded through hydrolysis, or oxidation in the presence of a variety of digestive enzymes in the stomach and small intestine regions. The medicines may also get denatured at varying pH conditions between acidic (pH 1-3) present in the stomach and neutral to slightly alkaline (pH 6-7.5) present in the duodenum, jejunum, and ileum [51]. As a result, pH-triggered release mechanisms are widely used in oral administration to improve the stability and controlled release of medications in the gastrointestinal region. In the present study, we focus on pH-responsive polymeric EC Cologrit tablets for oral delivery of anti-inflammatory and anti-oxidant phytochemicals to the UC specific regions of colon.
In our study, Cologrit was found to be biocompatible towards the intestinal epithelial cells. The medicine showed anti-inflammatory properties against bacterial lipopolysaccharide driven production of soluble TNF-α and IL-6 in the PMA-transformed THP1 macrophages. NFκB is a nuclear factor that plays a pivotal role between the stimulants and the pro-inflammatory cytokine release [60]. TNF-α signaling controls the expression of pro-inflammatory cytokines, chemokines, and adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through the NFκB pathway [42][43][44][45]. TNF-α interacts to the cell surface receptor TNFR1 and stimulates the recruitment of the adaptor proteins TRADD and Receptor-interacting serine/threonineprotein kinase 1 (RIP1) [61]. TRADD recruits TNFR-associated factor (TRAF) 2, as well as ubiquitin ligases, which promote RIP1 ubiquitination and the formation of a scaffold composed of ubiquitinated RIP1, NFκB essential modulator (NEMO), TRADD, and TNFR1 with linear polyubiquitin chains. This newly formed scaffold promotes downstream phosphorylation and degradation of the NFκB inhibitory IκB-α protein activating NFκB existing in the cytoplasmic pool [41,61]. Activated NFκB translocate to the cell nucleus and initiate the synthesis of pro-inflammatory cytokines.
In the present study, the anti-inflammatory activity of the Cologrit was found to be NFκB centric. TNF-α treatment controlled the release of NFκB from the THP-1-Blue NFκB reporter cells that was abolished by treatment with Cologrit. Similarly, TNF-α stimulated the mRNA expression of NFκB in the HT-29 cells that was reduced through treatment with Cologrit. Cologrit treatment also reduced the TNF-α prompted mRNA expression of pro-inflammatory cytokines and chemokines IL-1β, IL-8, and CXCL5. As a consequence, the reduction of IL-1, IL-8 in the HT-29 cells can be connected to downregulation of NFκB mRNA expression in the current study. Aegle marmelos (L.) Corrêa one of the main components of Cologrit has been found to reduction the mRNA expression of IL-6, TNF-α, TNF receptor-1 (TNFR1), TNFR1-associated death domain protein (TRADD) and NFκB [62][63][64]. Gallic acid, Imperatorin, Umbelliferon and Lupeol (triterpenoid) present in Aegle marmelos (L.) Corrêa and Cologrit are known to lowering TNF-α stimulated NFκB expression in various cell types by interfering with cell signaling pathway [64][65][66]. As a result, in the current investigation, suppression of IL-1, IL-8, as well as soluble TNF-α and IL-6 mRNA expression, can be linked to downregulation of NFκB mRNA expression. NFκB holds importance as a therapeutic target for controlling inflammation in UC [14]. Hence our results hold significance in the stherapeutic treatment of UC by controlling inflammation through TNF-α/NFκB signaling pathway modulation.
CXCL5, also known as epithelial cell-derived neutrophil activator 78 (ENA-78), is a pro-inflammatory chemokine that is produced by epithelial cells and has a role in neutrophil activation [67,68]. CXCL5, or ENA78, is significantly detected in colonic mucosa biopsy samples from UC patients [69,70]. TNF-α stimulates CXCL5 transcription via NFκB activation [71]. CXCL5 mRNA expression was found to be increased in the TNF-α stimulated HT-29 cells. This was reduced by treating the inflamed cells with Cologrit. TNF-α stimulates activation of NFκB through induction of IκB-α proteasomal degradation has been found to play a major role in the activation of adhesion molecules such as ICAM-1 [72]. The deactivation of the NFκB by blocking of TNF-α induced IκB-α proteasomal degradation has been found to reduce the production of adhesion molecules in the intestinal epithelial cells thereby reducing adhesion of immune cells [72]. In our study, Cologrit treatment inhibited the binding of TNF-α stimulated binding of U937 cells to the surface of HT-29 cells. This indicated a reduction in the production of adhesion molecules in the TNF-α induced HT-29 cells following treatment with Cologrit.
EC are designed to save the active ingredients of the tablets from dissolution and digestion within stomach under high acidic pH conditions [73]. Previous studies have shown that the Methacrylic acid-ethyl acrylate copolymer protects the dissolution of active ingredients under acidic conditions [74,75]. In the present study, we synthesized Cologrit EC tablets using MMA polymer showing integrity of coating at 6% (w/w) concentration. Using an in vitro GIT digestion model along with UC and HPLC analysis, we showcased the release of tablet ingredients at pH 7.0 and strong release phytochemicals. The controlled release of the phytochemicals Gallic acid, Ellagic acid and Rutin at the site of UC inflammation would significantly enhance their biological activity, as clearly seen in DSS stimulated Caco-2 cells showing cell protection against loss of cell viability and oxidative stress. Additionally, Gallic acid is known to holistically protect the probiotic gut microbiome population and eliminate pathogenic species like Firmicutes and Proteobacteria phyla through the amendment of carbohydrate, bile acid and amino acid metabolisms [76]. Additionally, Ellagic acid and Rutin are known to support the anti-inflammatory activity of the gut against the inflammation produced in UC regions [57,77]. Hence, their delivery at the site of disease without degradation in the stomach region by the EC tablet would enhance their biological efficacy in remediation of UC like inflammatory diseases.
Taken together, the study showed an efficacy of the polyherbal Cologrit and tablet in reducing inflammation, the loss of cell viability, and oxidative stress in the colonic epithelial cells. The anti-inflammatory activity of the Cologrit was identified as NFκB activation centric through which it controls the expression and release of pro-inflammatory cytokines. EC of the Cologrit tablets enhanced their site specific efficacy by protection against dissolution in high acidic pH of the stomach region. The EC tablets showed the same efficacy as the Cologrit, indicating no interference in the phytochemical release and biological efficacy from the EC polymer MMA. All of the plant chemicals present in the polyherbal Cologrit originating from the different plant components work tandemly in controlling gastrointestinal inflammation and oxidative stress. However, their individual role in the formulation, still need to be explored.

Conclusions
Ultimately, the study showcased the anti-inflammatory behavior of the enteric-coated Cologrit tablet in UC-like disease model. The mode of action for the Cologrit in the inflamed cells was found to be through the controlled NFκB activation by inducers such as TNF-α. Cologrit was found to function in the same way in both the macrophages and gut epithelial cells. The formation of EC Cologrit tablets enhanced their site specific activity through the inhibition of its dissolution in acidic pH conditions existing in stomach region. However, the tablets were found to be readily dissolved in the intestinal neutral pH, delivering its phytochemicals and showing efficacy in ameliorating UC-like cell damage and oxidative stress. Overall, Cologrit has been shown to be a unique herbal medicine for the control of ulcerative colitis, such as inflammatory bowel syndrome. In future studies, the plant components of the polyherbal Cologrit formulation could well be examined for their individual role in managing the various aspects of ulcerative colitis associated inflammation.

Preparation of Cologrit Formulation and Tablet
Cologrit powder (Batch no. PRFT/CHIN/0422/0196) was created by combining different quantities of the plant extracts listed in Table 1. The fruit of Aegle marmelos (L.) Corrêa and the bark of Holarrhena antidysenterica (L.) Wall. ex A. DC were ground separately, and then sieved through a #16 mesh sieve. Cuminum cyminum L. seeds, Trachyspermum ammi (L.) Sprague fruit, and Foeniculum vulgare Mill. fruit were mild-roasted individually for 30 min at 60 • C, then ground and sieved through a #16 mesh size sieve. In the formulation, a hydromethanolic [40% H 2 O and 60% CH 3 OH] extract of Cinnamomum camphora (L.) J. Presl leaf was also applied. All components were combined and blended for 15 min at 25 rpm in an octagonal blender into Cologrit formulation.
Cologrit tablets (Batch No. CHIH/COLA/0222/2320) were prepared by blending Cologrit powder with microcrystalline cellulose in an octagonal blender for 15 min at 25 rpm (Model: OB 25 L, Kevin process technologies Pvt. Ltd., Gujrat, India). The mixture was then mixed with excipients at the concentrations listed in Table 2. Granules were made in a fluidized bed dryer (Model: SC-12, Kevin process technologies Pvt. Ltd., Gujrat, India) and tablets were prepared on a 16-station rotary tableting machine with pre-compression (Model: FM-04, Falcon machineries, Gujarat, India). The tablet compression and storage were carried out in a conditioned room temperature of 25 ± 2 • C.

Enteric Coating of Cologrit Tablets
For 6% (w/w) EC, appropriate weight of MMA was dissolved in isopropyl alcohol (IPA) for 10 min while stirring at 200 rpm. Dichloromethane (DCM) was added to the prepared solution (Ratio of IPA: DCM set at 60:40), and stirred for another 30 min at 300 rpm [78]. The prepared solution was filtered and used to coat the Cologrit tablets.

Physical Evaluation of Tablet
A digital weighing balance (Model: CG 302, Aczet Pvt. Ltd., Mumbai, Maharashtra, India) was used to determine the average weight of 20 tablets. Average friability of 20 tablets at 25 rpm for 4 min was measured using test apparatus (Model: 903, Electronics India, Panchkula, Haryana, India). A hardness tester (Labotech, Ambala cantt, India) was used to determine the average hardness of ten tablets. A Vernier Calliper (Model: 532-119, Mitutoyo, Kawasaki, Japan) was used to measure the thickness and diameter of the tablets.
In the stomach phase, 20 mL of mouth phase bolus were mixed with 20 mL of simulated gastric fluid (chyme) containing NaCl (684 mM), HCl (72 mM) and pepsin (3.2 mg/mL), adjusted to pH 2.5, and incubated at 37 • C for 2 h on a shaker (100 rpm). 30 mL of stomach phase chyme with pH adjusted to 7.0 was mixed with small intestinal fluid (NaCl (150 mM) and CaCl 2 (10 mM), bile salt (5.0 mg), and lipase (60 mg/mL; digestae) for 2 h at 37 • C with constant stirring. The pH of the solution was kept at 7.0 by adding 10 mL of 0.1 N NaOH solution. The dissolved Cologrit concentration in the final digestae was 11.06 mg/mL. All the digestae were stored at −80 • C and used for biochemical analysis.

HPLC-Based Phytochemical Analysis
Individually, Gallic acid, Rutin, Ellagic acid, and Imperatorin standards were dissolved in methanol to make 1000 ppm standard stock solutions. Imperatorin was further diluted for preparing a 100 ppm working standard solution. All the other standards were diluted to a working standard solution of 25 ppm. 250 mg of Cologrit was sonicated for 30 min in 10 mL of water: methanol (20:80) mixture. The mixture was centrifuged at 10,000 rpm and filtered through a 0.45 µm nylon filter. HPLC analysis was performed on the standards' filtrate, and Cologrit tablet containing chyme and digestae samples collected under sub-Section 5.5.

UV Spectroscopy Analysis
During the digestion phase, chyme and digestae samples were collected every 20 min, and analyzed for Cologrit tablet dissolution using UV-1800 spectrometer (Shimadzu, Kyoto, Japan). A full spectrum UV scan (200-1000 nm) was performed using the samples and Cologrit specific absorbance spectra λmax were determined at 339 nm, with no optical interferences in the presence of biomolecules. Spectragryph software (Software-Entwicklung, Oberstdorf, Germany) was used to spectra and data processing.

Digestae Preparation for Cell-Based Assays
For the cell-based studies, fresh digestae containing 11.06 mg/mL of enzyme digested Cologrit tablet were prepared using the protocol described in sub-Section 5.5. Water only digestae was considered as untreated control. All of the digestae were adjusted to pH 7.0 and diluted in DMEM media (without supplements) in a 1:3 ratios for making stock solution. As required by the assay, additional dilutions were prepared in incomplete DMEM media.

Dose Response Analysis
Cologrit was suspended in 2% FBS containing DMEM at the concentrations of 1, 3, 10, 30, and 100 µg/mL. Following pre-incubation, HT-29 cells were plated at the concentration of 2 × 10 4 cells/well in 96 well plates and incubated overnight. Next day, the cells were treated with different concentrations of Cologrit for 24 h. Caco-2 cells were plated at the concentration of 1 × 10 4 cells/well in 96 well plates and pre-incubated overnight. Digestae w/wo Cologrit tablets were dissolved in serum-free DMEM medium at concentrations of 1, 3, 10, 30, and 100 µg/mL. In parallel, Caco-2 cells following pre-incubation were also treated for 24 h with 0.65 percent, 1.15 percent, 2.5 percent, and 5 percent (w/v) Dextran Sodium Sulfate (DSS) colitis grade (Mol. Wt. 36000-50000; MP Biomedicals, LLC, Illkirch, France) solution prepared in serum-free medium. At end of treatment time, media was removed and the cells were washed with sterile PBS. Cells were incubated for 3 h with a 10 µg/mL concentration of Alamar blue™, and fluorescence was measured at Ex. 560 nm/Em. 580 nm using Envision multimode plate reader (PerkinElmer, Waltham, MA, USA).

ELISA-Based Pro-Inflammatory Cytokines Analysis
For the IL-6 and TNF-α release estimation using ELISA assay, 1 × 10 5 THP-1 cells/mL were differentiated using 20 ng/mL PMA overnight. The next day, the PMA-containing media was replaced with fresh complete cell culture media, and the differentiated cells rested for 4 days with intermittent media change. Pre-treatment of Cologrit was given to the differentiated cell at the concentrations of 3, 10, 30 and 100 µg/mL for 24 h. Following day, the cells were co-treated with LPS (100 ng/mL) and varying concentrations of Cologrit for 6 h. Cell soup was collected and used for the quantification of cytokines according to manufacturer instructions.

Evaluation of NFκB Response
THP-1-Blue NFκB reporter cells were plated in 96-well plates at a seeding density of 5 × 10 5 /mL and pre-incubated overnight. Cells were subsequently co-treated for 24 h with 10 ng/mL TNF-α and Cologrit at 10, 30, and 100 µg/mL concentrations. The expression of NFκB was measured as Secreted Embryonic Alkaline Phosphatase (SEAP) and quantified using the QUANTI-Blue assay (InvivoGen, CA, USA) according to the manufacturer's instructions. The optical density at 630 nm readout was determined using an Envision multimode plate reader (PerkinElmer, Waltham, MA, USA).

Monocyte Adhesion Assay
HT-29 cells were plated at a density of 5 × 10 4 cells/well in 96-well plates. Pretreatment of the HT-29 cells was given with Cologrit at the concentrations of 3, 10, 30, and 100 µg/mL for 24 h. Next day, cells were co-treated with TNF-α (10 ng/mL) and varying concentrations of Cologrit for 24 h. U937 cells were pre-labelled with Calcein AM dye (0.5 µM) for 30 min at 37 • C. Subsequently, HT-29 cells were co-incubated with the pre-labelled U937 cells (1 × 10 5 cells/well) for 2 h. Non-adherent cells were washed with 1 × PBS, and fluorescence was measured with using Infinite 200Pro multimode plate reader (Tecan, Zurich, Switzerland) at Ex. 485 nm and Em. 530 nm.

Prophylactic Treatment with Cologrit Digestae
Caco-2 cells were pre-incubated with Cologrit digestae doses of 1, 3, 10, 30, and 100 µg/mL prepared in incomplete DMEM medium for 24 h. The cells were subsequently co-treated with 2.5% percent DSS and varying concentrations Cologrit digestae prepared in serum-free cell culture media. As negative and positive controls, water-based digestae w/wo 2.5 percent DSS were used. The treated cells were incubated for 24 h and then washed with PBS. The cells were treated with a 10 µg/mL of Alamar blue TM for 3 h. Fluorescence was measured at Ex. 560 nm and Em. 580 nm using Envision multimode plate reader (PerkinElmer, Waltham, MA, USA).

Oxidative Stress Analysis
Caco-2 cells were pre-treated with the Cologrit digestae concentrations of 1, 3, 10, 30, and 100 µg/mL prepared in serum-free DMEM media for 24 h. Next day, cells were co-treated with 2.5% DSS and varying concentrations of Cologrit digestae prepared in serum-free media for 24 h. Water based digestae w/wo 2.5% DSS were treated as negative and positive controls, respectively. At the end of incubation time, cells were washed with 10 µg/mL (100 µL) of 2 , 7 -dichlorofluorescein diacetate dye and incubated in dark for 45 min at 37 • C. Fluorescence was measured at Ex. 490 nm and Em. 520 nm using the Envision multimode plate reader (PerkinElmer, Waltham, MA, USA).

Statistical Analysis
GraphPad Prism 7 was used for statistical analysis (GraphPad Software, San Diego, CA, USA). The data were presented as mean ± standard deviation (SD). Significance between distinct treatment groups was determined using one-way ANOVA, followed by Dunnett's posthoc analysis. At a p-value of <0.05, the results were deemed statistically significant.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to institutional policies.