UP1306: A Composition Containing Standardized Extracts of Acacia catechu and Morus alba for Arthritis Management

Osteoarthritis (OA) is characterized by progressive articular cartilage degradation. Although there have been significant advances in OA management, to date, there are no effective treatment options to modify progression of the disease. We believe these unmet needs could be bridged by nutrients from natural products. Collagen induced arthritis in rats was developed and utilized to evaluate anti-inflammatory and cartilage protection activity of orally administered botanical composition, UP1306 (50 mg/kg) and Methotrexate (75 µg/kg) daily for three weeks. Objective arthritis severity markers, urine, synovial lavage, and serum were collected. At necropsy, the hock joint from each rat was collected for histopathology analysis. Urinary cartilage degradation marker (CTX-II), pro-inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6), and proteases (Matrix Metallopeptidase 3 (MMP3) and 13) were measured. Rats treated with UP1306 showed statistically significant improvements in arthritis severity markers, including uCTX-II (91.4% vs. collagen-induced arthritis (CIA)), serum IL-1β, TNF-α, and IL-6 levels as well as synovial MMP-13. The histopathology data were also well aligned with the severity score of arthritis for both UP1306 and Methotrexate. UP1306, a botanical composition that contains a standardized blend of extracts from the heartwood of Acacia catechu and the root bark of Morus alba, could potentially be considered as a dietary supplement product for the management of arthritis.


Introduction
Osteoarthritis (OA) is characterized by progressive articular cartilage degradation that will lead to joint pain, immobility, and other functional impairments [1]. Arthritis affects an estimated 54.4 million adults in the US, with projections of 78.4 million by the year 2040 [2]. Although there have been significant advances in OA management, to date, there are no effective treatment options to modify progression of the disease. The current approach mainly targets to relieve symptoms by reduction of pain and improvement of joint function using oral or topical over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs). This strategy is known to mask the primary cause leading to irreversible damage to the articular structure. Chronic uses of NSAIDs for symptomatic relief of OA are also limited due to their gastrointestinal, renal, and cardiovascular side effects [3]. Hence, these unmet needs could be bridged with natural products.
It has been a common practice to standardize the extract of a medicinal plant to be used alone or in combination with other functional bioactives or nutrients for multiple human ailments.

Composition
The Composition, UP1306, was prepared by mixing the standardized aqueous extract (that contains at least 65% catechins) from heartwood of A. catechu and M. alba root bark ethanol extract (that contains not less than 7% stilbenes and bioflavonoids) at a ratio of 1:2 by weight. The active contents in UP1306 are catechins (at least 15%), stilbenes, and bioflavonoids (at least 2%).

Model Induction and Treatment
Male Sprague Dawley rats (7-8 weeks old, n = 40) were purchased from Charles River Laboratories Inc. (Wilmington, MA, USA) and acclimated upon arrival for two weeks before being assigned randomly to their respective treatment groups: G1 = Normal control (−) (n = 10/group), G2 = collagen-induced arthritis (CIA) + Vehicle (0.5% Carboxy Methylcellulose) (n = 10/group), G3 = CIA + Methotrexate (+) (75 µg/kg) (n = 10/group), and G4 = CIA + UP1306 (+) (50 mg/kg) (n = 10/group). Treatment was initiated two weeks before model induction and lasted for an additional three weeks thereafter. Collagen type-II (Lot # 845) from bovine nasal septum and Incomplete Freund's adjuvant (IFA) (Lot # SLBR0642v) were purchased from Elastin Products Company (Owensville, MI, USA) and Sigma (St. Louise, MO, USA), respectively. All materials were kept at suitable temperature as recommended by the manufacturer. At the time of preparation, 60 mg of collagen was weighed and added to a pre-chilled 15 mL 0.1 M acetic acid in a 60 mL-sized flask with a magnetic stirrer to yield 4 mg/mL concentration [38,39]. The mixture was dissolved by gently stirring overnight at 4 • C. The next morning, the dissolved collagen was emulsified with equal volume of IFA (15 mL) to achieve a final concentration of 2 mg/mL Collagen. Rats sedated with isoflurane were then primed intradermally with 400 µL of the emulsified collagen at the base of their tail at two sites using a 1 mL syringe fitted with a 26 g needle. The dissolved mixture was kept in an ice bucket and stirred between groups at the time of injection to preserve uniform consistency. On the seventh day, rats were inoculated with a booster dose of 2 mg/mL type II collagen emulsified with equal volume of incomplete adjuvant at 100 µL/rat/site.

Clinical Observation
Clinical findings such as arthritis severity index, paw thickness, ankle diameter (using Digital Absolute, Model # PK-0505CPX, Mitutoyo Corporation, Kawasaki, Japan), and pain sensitivity (using Randall Selitto, IITC Life Science Inc., Woodland Hills, CA, USA) were monitored during the course of study. Urine was collected from overnight fasted rats using metabolic cages after three weeks of treatment post model induction. At Necropsy, serum from the cardiac and synovial lavage (100 µL of saline was injected into the articular cavity and aspirated back to the syringe) for biomarkers and ankle joint for histopathology were collected from each animal.

Histopathology
For histopathological examination, the ankle joints were kept in 10% formalin for 72 h. The fixed specimens were then decalcified with Calci-Clear Rapid for one and a half days and embedded in paraffin. Standardized 5 µm serial sections were obtained at the medial and lateral section in the sagittal plane of the joint and were stained with hematoxylin and eosin (HE) and Safranin O-fast green to enable evaluation of proteoglycan content. A modified Mankin system [40] was used to score structural and cellular alterations of joint tissues resulting from disease progression and/or treatment efficacy. The histological analysis was conducted at Nationwide Histology and slides were examined by a certified pathologist.

Urine CTX-II-
Rat urine samples were diluted 1:3 and the presence of CTX-II was measured using the Rat CTX-II ELISA kit from Mybiosource (product#: MBS2880519) as follows: diluted urine was added to a microplate coated with CTX-II antibody and allowed to bind for 2 h at 37 • C. A biotin-conjugated antibody against CTX-II was then added and allowed to bind to the CTX-II from the rat urine for 1 h at 37 • C. The microplate was washed thoroughly to remove unbound urine and antibody, before an enzyme-conjugated avidin antibody was added to bind to the biotin-conjugated antibody for specific detection. The avidin antibody was allowed to bind for 1 h at 37 • C. Washing was repeated, enzyme substrate was added, and the plate was developed for 30 min at 37 • C. After the addition of stop solution, the absorbance was read at 450 nm, multiplied by dilution factor, and the concentration of CTX-II calculated based on the absorbance readings of a CTX-II standard curve. CTX-II amount was normalized to the amount of Creatinine in the urine using the Creatinine Parameter Assay Kit from R and D Systems (product#: KGE005) as follows: urine was diluted 1:20, mixed with alkaline picrate (5 parts 0.13% picric acid: 1 part 1 N NaOH) in a microplate and incubated at room temp for 30 min. Absorbance was read at 492 nm and Creatinine amount in urine was calculated based on the absorbance readings of a Creatinine standard curve.
2.5.2. Serum IL-1β, TNF-α and IL-6 ELISA Rat serum was diluted 1:3 (IL-1β) or 1:2 (TNF-α and IL-6). Dilutions were selected based on the ELISA Kit manufactures suggestions for detection of protein level in the serum and the amount of serum available for each study subject. The presence of IL-1β, TNF-α, or IL-6 was measured using the Rat IL-1β, TNF-α, or IL-6 Quantikine ELISA kit from R and D Systems (product#: RLB00 for IL-1β, RTA00 for TNF-α, and R6000B for IL-6) as follows: diluted serum was added to a microplate coated with polyclonal IL-1β, TNF-α, or IL-6 antibody and allowed to bind for 2 h at room temperature. The microplate was washed thoroughly to remove unbound serum and then a polyclonal enzyme-conjugated IL-1β, TNF-α, or IL-6 antibody was added and allowed to bind for 2 h at room temperature. Washing was repeated, enzyme substrate was added, and the plate was developed for 30 min at room temperature. After the addition of stop solution, the absorbance was read at 450 nm, multiplied by dilution factor, and the concentration of IL-1β/TNF-α /IL-6 calculated based on the absorbance readings of an IL-1β/TNF-α /IL-6 standard curve. IL-1β/TNF-α /IL-6 amount was normalized to the amount of total protein in serum using the Pierce BCA Protein Assay kit from ThermoFisher Scientific (product#: 23225) as follows: serum was diluted 1:50, mixed with bicinchoninic acid (BCA) reagent in a microplate, and incubated at 37 • C for 30 min. Absorbance was read at 580 nm and protein concentration in serum was calculated based on the absorbance readings of a bovine serum albumin standard curve.

Synovial MMP-13 and Serum MMP-3 ELISA
Rat synovial fluid or serum was diluted either 1:2, 1:3, or 1:4. Dilutions were selected based on the ELISA Kit manufactures suggestions for detection of the level of protein in the serum and the amount of serum available for each study subject). The presence of MMP-13 or MMP-3 was measured using the Rat Matrix Metallo Proteinase 13 or 3 (MMP-13 or 3) ELISA kit from Mybiosource (product#: MBS702112 for MMP-13 and MBS729026 for MMP-3) as follows: diluted synovial fluid or serum was added to a microplate coated with MMP-13 or MMP-3 antibody and allowed to bind for 2 h at 37 • C. The samples were removed and then a biotin-conjugated MMP-13 or MMP-3 antibody was added and allowed to bind for 1 h at 37 • C. The microplate was thoroughly washed and an avidin-conjugated Horse Radish Peroxidase was added and allowed to bind for 1 h at 37 • C. Enzyme substrate was then added and the plate was developed for 30 min at 37 • C. After the addition of stop solution, the absorbance was read at 450 nm, multiplied by dilution factor, and the concentration of MMP-13 or MMP-3 calculated based on the absorbance readings of an MMP-13 or MMP-3 standard curve. MMP-13 or MMP-3 amount was normalized to the amount of total protein in synovial fluid using the Pierce BCA Protein Assay kit from ThermoFisher Scientific (product#: 23225) as follows: synovial fluid was diluted 1:20, mixed with bicinchoninic acid (BCA) reagent in a microplate, and incubated at 37 • C for 30 min. Absorbance was read at 580 nm and protein concentration in synovial fluid was calculated based on the absorbance readings of a bovine serum albumin standard curve.
All animal experiments were conducted according to institutional guidelines congruent with the guide for the care and use of laboratory animals.

Statistical Analysis
Data were analyzed using Sigmaplot (Version 11.0, San Jose, CA, USA). The results are represented as mean ± one SD. Statistical significance between groups was calculated by means of single factor analysis of variance followed by a paired t-test. p-values less than or equal to 0.05 (p ≤ 0.05) were considered as statistically significant. When normality test failed, for nonparametric analysis, data were subjected to Mann-Whitney sum ranks for t-test and Kruskal-Wallis one-way analysis of variance on ranks for ANOVA. Linear trapezoid rule was used to calculate AUC for days 12-18.

Arthritis Severity Index
Rats continued to show a slow progression of disease for the duration of the study. As seen in Figure 1, rats treated with Methotrexate and UP1306 showed statistically significant suppression in arthritis severity from day 13 and continued this significance for the duration of the study. At the end of the study, average severity scores of 3.15 ± 0.32, 1.85 ± 0.74, and 1.6 ± 0.70, were observed for CIA rats treated with Vehicle, UP1306, and Methotrexate, respectively. When the area under the arthritis severity curve was calculated, percent reductions of 58.9% (p = 0.01) and 51.1% (p = 0.02) with statistical significance were observed from Methotrexate and UP1306 treatment (Table 1).
Nutrients 2018, 10, x FOR PEER REVIEW 5 of 18 All animal experiments were conducted according to institutional guidelines congruent with the guide for the care and use of laboratory animals.

Statistical Analysis
Data were analyzed using Sigmaplot (Version 11.0, San Jose, CA, USA). The results are represented as mean ± one SD. Statistical significance between groups was calculated by means of single factor analysis of variance followed by a paired t-test. P-values less than or equal to 0.05 (p ≤ 0.05) were considered as statistically significant. When normality test failed, for nonparametric analysis, data were subjected to Mann-Whitney sum ranks for t-test and Kruskal-Wallis one-way analysis of variance on ranks for ANOVA. Linear trapezoid rule was used to calculate AUC for days 12-18.

Arthritis Severity Index
Rats continued to show a slow progression of disease for the duration of the study. As seen in Figure 1, rats treated with Methotrexate and UP1306 showed statistically significant suppression in arthritis severity from day 13 and continued this significance for the duration of the study. At the end of the study, average severity scores of 3.15 ± 0.32, 1.85 ± 0.74, and 1.6 ± 0.70, were observed for CIA rats treated with Vehicle, UP1306, and Methotrexate, respectively. When the area under the arthritis severity curve was calculated, percent reductions of 58.9% (p = 0.01) and 51.1% (p = 0.02) with statistical significance were observed from Methotrexate and UP1306 treatment (Table 1).

Figure 1.
Changes of Arthritis severity index: Rats (n = 10/group) were orally treated with UP1306 (50 mg/kg) and Methotrexate (75 μg/kg) daily for three weeks post model induction. Score 0: No sign of grossly visible arthritis (Normal); 1: Swelling and/or redness of one or two interphalangeal joints (or mild swelling and erythema of digits or ankles); 2: Involvements of three to four interphalangeal joints (or moderate swelling and erythema of digits or ankles); 3: Swelling of entire paw (or marked swelling of paws including digits); 4: Deformity and ankylosis (or sever swelling and erythema with limited motion in many joints). Data are mean ± SE. *p ≤ 0.05; **p ≤ 0.001; †p ≤ 0.0001 vs. CIA.

Ankle Diameter
A similar pattern in the reduction of ankle diameter was observed for rats treated with Methotrexate and UP1306 for the duration of the study ( Figure 2). These groups showed a statistically significant reduction in ankle width when the area under the curve was considered for days 12 to 18. Percent reductions of 71.6% and 59.7 % with statistical significance in paw edema were observed for rates treated with Methotrexate and UP1306, respectively (Table 1).

Ankle Diameter
A similar pattern in the reduction of ankle diameter was observed for rats treated with Methotrexate and UP1306 for the duration of the study (Figure 2). These groups showed a statistically significant reduction in ankle width when the area under the curve was considered for days 12 to 18. Percent reductions of 71.6% and 59.7 % with statistical significance in paw edema were observed for rates treated with Methotrexate and UP1306, respectively (Table 1).

Paw Thickness
In agreement with the severity score and ankle diameter, rats treated with Methotrexate and UP1306 showed a statistically significant reduction in paw swelling starting from day 13; this significance was maintained for the duration of the study (Figure 3). When the total area under the swelling curve (day 12-day 18) was considered, Methotrexate and UP1306 groups showed statistically significant reductions (69.6% and 61.1%) in paw edema compared to the vehicle treated CIA group, respectively (Table 1).

Paw Thickness
In agreement with the severity score and ankle diameter, rats treated with Methotrexate and UP1306 showed a statistically significant reduction in paw swelling starting from day 13; this significance was maintained for the duration of the study (Figure 3). When the total area under the swelling curve (day 12-day 18) was considered, Methotrexate and UP1306 groups showed statistically significant reductions (69.6% and 61.1%) in paw edema compared to the vehicle treated CIA group, respectively (Table 1).

Pain Sensitivity
Response to pressure as a measure of pain sensitivity was measured using the Randall-Selitto probe attached to an electronic monitor on priming day, boost day, and days 12, 14, 16, and 18. Both the left and right hind legs were monitored on those days and their average was used for data analysis. Changes from the vehicle-treated CIA rats have been reported as pain tolerance on those days. The highest pain tolerance was observed for rats in the Methotrexate group followed by the UP1306 group ( Figure 4). These reductions-such as 11.8%, 30.3%, 40.6%,and 41.6% for Methotrexate and 10.2%, 22.7%, 31.3%, and 31.3% for UP1306 on days 12, 14, 16, and 18, respectively-were statistically significant as of day 12 and remained significant for the rest of the duration of the study.

Pain Sensitivity
Response to pressure as a measure of pain sensitivity was measured using the Randall-Selitto probe attached to an electronic monitor on priming day, boost day, and days 12, 14, 16, and 18. Both the left and right hind legs were monitored on those days and their average was used for data analysis. Changes from the vehicle-treated CIA rats have been reported as pain tolerance on those days. The highest pain tolerance was observed for rats in the Methotrexate group followed by the UP1306 group ( Figure 4). These reductions-such as 11.8%, 30.3%, 40.6%,and 41.6% for Methotrexate and 10.2%, 22.7%, 31.3%, and 31.3% for UP1306 on days 12, 14, 16, and 18, respectively-were statistically significant as of day 12 and remained significant for the rest of the duration of the study.

Pain Sensitivity
Response to pressure as a measure of pain sensitivity was measured using the Randall-Selitto probe attached to an electronic monitor on priming day, boost day, and days 12, 14, 16, and 18. Both the left and right hind legs were monitored on those days and their average was used for data analysis. Changes from the vehicle-treated CIA rats have been reported as pain tolerance on those days. The highest pain tolerance was observed for rats in the Methotrexate group followed by the UP1306 group (Figure 4). These reductions-such as 11.8%, 30.3%, 40.6%,and 41.6% for Methotrexate and 10.2%, 22.7%, 31.3%, and 31.3% for UP1306 on days 12, 14, 16, and 18, respectively-were statistically significant as of day 12 and remained significant for the rest of the duration of the study.

Serum Pro-Inflammatory Cytokines
An increased production of cytokines is the integral part of collagen induced arthritis pathology. Rats treated with UP1306 showed statistically significant reduction in serum IL-1β level when compared to the vehicle treated CIA group ( Figure 5). There was a slight increase in serum IL-1β for the rats treated with Methotrexate. Similarly, marked changes in serum TNF-α and IL-6 levels were observed. As depicted in Figures 6 and 7, significant increases in serum TNF-α and IL-6 level were observed for the vehicle-treated CIA group compared to the normal control. UP1306-treated rats showed statistically a significant reduction in serum IL-6 (112.4% inhibitions, compared to diseased control) and TNF-α (266.6% inhibitions, compared to diseased control) levels when compared to vehicle-treated diseased rats (Figures 6 and 7). Methotrexate-treated rats showed modest but not statistically significant reductions in serum IL-6 (64.9% inhibitions, compared to a diseased control) and TNF-α (115.3% inhibitions, compared to diseased control) (Figures 6 and 7).

Serum Pro-Inflammatory Cytokines
An increased production of cytokines is the integral part of collagen induced arthritis pathology. Rats treated with UP1306 showed statistically significant reduction in serum IL-1β level when compared to the vehicle treated CIA group ( Figure 5). There was a slight increase in serum IL-1β for the rats treated with Methotrexate. Similarly, marked changes in serum TNF-α and IL-6 levels were observed. As depicted in Figures 6 and 7, significant increases in serum TNF-α and IL-6 level were observed for the vehicle-treated CIA group compared to the normal control. UP1306-treated rats showed statistically a significant reduction in serum IL-6 (112.4% inhibitions, compared to diseased control) and TNF-α (266.6% inhibitions, compared to diseased control) levels when compared to vehicle-treated diseased rats (Figures 6 and 7). Methotrexate-treated rats showed modest but not statistically significant reductions in serum IL-6 (64.9% inhibitions, compared to a diseased control) and TNF-α (115.3% inhibitions, compared to diseased control) (Figures 6 and 7).

Synovial MMP-13
A marked increase in the level of synovial MMP-13 level was observed for the vehicle-treated diseased rats, when compared to the normal control rats. As seen in Figure 8, rats treated with UP1306 showed a statistically significant reduction in MMP-13 level compared to vehicle-treated CIA rats. This inhibition for UP1306-treated rats was calculated as 146.7% vs. CIA treated rats. A moderate and non-significant reduction in synovial MMP-13 level was observed for the Methotrexate group.

Synovial MMP-13
A marked increase in the level of synovial MMP-13 level was observed for the vehicle-treated diseased rats, when compared to the normal control rats. As seen in Figure 8, rats treated with UP1306 showed a statistically significant reduction in MMP-13 level compared to vehicle-treated CIA rats. This inhibition for UP1306-treated rats was calculated as 146.7% vs. CIA treated rats. A moderate and non-significant reduction in synovial MMP-13 level was observed for the Methotrexate group.

Serum MMP-3
As seen in Figure 9, an increased level of MMP-3 was observed for vehicle-treated CIA rats, when compared to the normal control. On the other hand, percent inhibitions of 104.7% and 108.8% were observed for rats treated with Methotrexate and UP1306, respectively. Neither the increase in the CIA (compared to the normal control) rats nor the decreases observed in the treated rats (compared to the CIA rats) were statistically significant. There were bigger variations within the individual rats in the vehicle-treated CIA rats which could have an impact on the absence of statistical significance.

Serum MMP-3
As seen in Figure 9, an increased level of MMP-3 was observed for vehicle-treated CIA rats, when compared to the normal control. On the other hand, percent inhibitions of 104.7% and 108.8% were observed for rats treated with Methotrexate and UP1306, respectively. Neither the increase in the CIA (compared to the normal control) rats nor the decreases observed in the treated rats (compared to the CIA rats) were statistically significant. There were bigger variations within the individual rats in the vehicle-treated CIA rats which could have an impact on the absence of statistical significance.

Serum MMP-3
As seen in Figure 9, an increased level of MMP-3 was observed for vehicle-treated CIA rats, when compared to the normal control. On the other hand, percent inhibitions of 104.7% and 108.8% were observed for rats treated with Methotrexate and UP1306, respectively. Neither the increase in the CIA (compared to the normal control) rats nor the decreases observed in the treated rats (compared to the CIA rats) were statistically significant. There were bigger variations within the individual rats in the vehicle-treated CIA rats which could have an impact on the absence of statistical significance.

Urinary CTX-II
Significant urine CTX-II level changes both in disease inductions and treatment effects were observed in this study. As illustrated in Figure 10, a statistically significant increase in urine CTX-II level was observed for the vehicle-treated CIA group compared to the normal control confirming severity of disease. Treatment with UP1306 spared significant degradation of cartilage (up to 91.4%) compared to vehicle-treated diseased CIA rats. The positive control Methotrexate showed 70.4% inhibition in cartilage degradation compared to CIA with p = 0.06.

Urinary CTX-II
Significant urine CTX-II level changes both in disease inductions and treatment effects were observed in this study. As illustrated in Figure 10, a statistically significant increase in urine CTX-II level was observed for the vehicle-treated CIA group compared to the normal control confirming severity of disease. Treatment with UP1306 spared significant degradation of cartilage (up to 91.4%) compared to vehicle-treated diseased CIA rats. The positive control Methotrexate showed 70.4% inhibition in cartilage degradation compared to CIA with p = 0.06.

Histopathology Findings
The histopathology data was in alignment with the severity score of arthritis. When compared to the normal control rats, vehicle-treated rats showed severe synovitis, marked cartilage

Histopathology Findings
The histopathology data was in alignment with the severity score of arthritis. When compared to the normal control rats, vehicle-treated rats showed severe synovitis, marked cartilage degradation, synovial hyperplasia, pannus formation, and bone erosion (Figures 11-13). In contrast, rats treated with UP1306 had nearly normal morphology, with minimal alteration in matrix integrity, a smoother articulation cartilage surface, low levels of mononuclear cell infiltration, and synovial hyperplasia, as well as reduced articular bone damage. Except for inflammation, Methotrexate-treated animals also showed similar efficacy to that of the UP1306 group.     (B). CIA + Vehicle: 50X HE: Inflammation (i) and diffuse necrosis of the subchondral bone (SB) with effacement of the articular surfaces (*). There is complete loss of the bone architecture; (C). CIA + Methotrexate: 50×-HE-These images demonstrate an area of fibroplasia with monocytic inflammation (mi). SB-subchondral bone, C-cartilage with adhered inflammation (i), T-Tendon; (D). CIA + UP1306: 50× HE-Subchondral bone (SB) with the overlying cartilage (c) and adjacent bone being effaced (*) by inflammation; (i) T-tendon expanded by edema and infiltrated by inflammation.

Discussion
The CIA model in rats is the most commonly studied autoimmune model of RA with several pathological features resembling the immune-mediated polyarthritis in dogs and humans [41]. Its short duration between immunization and disease manifestations makes the model feasible for therapeutic efficacy evaluations. Following inoculation of heterogenic type II collagen (CII), rats mount both humoral and cellular responses to the antigen [38]. This sensitization subsequently leads to the host animal attacking its own type II collagen, which is predominantly present in the joint cartilage and hence results in erosive or non-erosive joint destruction. The pathophysiology of the disease is highly orchestrated and complex. Upon induction, rats will experience inflammatory pain and swelling, cartilage degradation, synovial hyperplasia, pannus formation, mononuclear cell infiltration, deformity, and immobility.
In the current study, rats started to show the pathognomonic signs of arthritis on day 12 post priming followed by a progressive increase in severity that approached near plateau on days 17 to 18. These symptoms were mitigated by oral treatment of an immune suppressant-Methotrexate and also a natural polyphenol composition-UP1306. Both treatment groups (Methotrexate and UP1306) showed measurable relief in arthritis severity, swelling, ankle width, and pain sensitivity when compared to the vehicle-treated diseased rats. When data for arthritis severity, paw thickness, and ankle diameter were pooled together for the duration of the study period from day 12-18 (where visible signs of arthritis were observed), CIA rats treated with Methotrexate and UP1306 showed statistically significant reduction in all of the cardinal signs of arthritis suggesting their application for symptomatic relief of arthritis.
TNF-α and IL-1β are the two primary cytokines involved in the initiation and progression of arthritis [42], mainly through (a) downregulation of the synthesis of major extracellular matrix components by inhibiting anabolic activities of chondrocytes [43,44]; (b) induction of additional cytokines (such as IL-6), chemokines, and extra cellular matrix degrading enzymes (MMPs and aggrecanases) [45,46]; (c) inhibition of anti-oxidant activity of the host [47]; and (d) induction of reactive oxygen species [48]. These processes facilitate maintenance of chronic inflammation and perpetual joint destruction in arthritic patients. For example, while injection of IL-lβ into the knee joints of rats caused joint inflammation and marked proteoglycan depletion [49,50], its blockade reversed the process [51][52][53]. Besides direct involvement in the inflammation process and cartilage degradation, dysregulation of IL-6 levels is also linked to the common clinical manifestations associated with rheumatoid arthritis pathology such as fever, fatigue, and weight loss [54]. Hence, modulating these pro-inflammatory cytokines at various stages of disease progression could alleviate the symptoms associated with arthritis and/or help to modify the disease. The anti-inflammation and cartilage protection activities of UP1306 observed in this study could be in part due to inhibition of these key pro-inflammatory cytokines.
Supplementation with UP1306 for three weeks resulted in significant reductions in the level of fundamental matrix proteolytic enzymes, such as MMP-13 and MMP-3. Along with aggrecan breakdown, degradation of collagen is a central feature of arthritis [55]. Pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 are known to play important roles in cartilage matrix degradation in articular cartilage through a cascade of events that lead to stimulation of aggrecanase and matrix metalloproteinase production [42]. When MMP enzymes are activated, MMP-3 is the first to be activated. It then triggers other MMPs, such as MMP1, MMP9, and MMP13, which leads to further degradation and amplification of extracellular matrix components. This leads to a simultaneously increased release and accumulation of degradants in the joints and causes joint inflammation. During the course of disease pathology, the major histocompatibility complex presents these fragments to T cells and promotes the activation and release of large amounts of inflammatory cytokines, such as IL-1β and IL-6, which in turn increases other MMP expression levels in the chondrocytes and synovial fibroblasts. Consequentially, all these processes result in augmented collagenase activity and worsening of joint inflammation. MMP-3 and MMP-13 have been found in increased levels at the sites of cartilage erosion in cases of rheumatoid arthritis and osteoarthritis [56]. Previous studies have shown that these MMP levels in OA patient's blood and synovial fluid were higher than in healthy people and the level was consistent with the extent of cartilage damage [57,58]. In fact, MMPs secreted into the synovial fluid can directly degrade the cartilage and bone composition leading to enhanced damage of surrounding articular structures [59]. In the current study, there was significant suppression of serum MMP-3 and MMP-13 levels by UP1306, which could provide a wholesome protection of cartilage degradation and improved pain relief. The reduction in MMPs observed in this study could partially be explained by (a) the effect of treatment materials in reducing the precipitating pro-inflammatory cytokines and/or (b) the activity of treatment materials by directly suppressing expression of these matrix degrading enzymes.
Urine C-terminal telopeptide of type II collagen (uCTX-II) has been by far the most studied and frequently referred biomarker of cartilage degradation that could be used for the purpose of diagnosis, determining the severity of disease or extent of disease progression, prognosis, and monitoring efficacy of treatment [60]. In clinical studies, high levels of CTX-II are a good predictor of increased risk of joint destruction [61]. Degradation and loss of articular cartilage are fundamental features of collagen-induced arthritis whereby increased CTX-II level directly correlated with the time course of paw swelling and arthritis severity. Our results were in accord with previous reports [35,62]. In the current study, substantiating the effects on paw swelling, paw thickness, arthritis severity, pro-inflammatory cytokines, and matrix degrading enzymes, rats treated with UP1306 showed significantly reduced levels of uCTX-II. These findings indicate that cartilage protection activity is one of the primary functions of UP1306 suggesting its usage in management of arthritic conditions. Together with symptoms and biomarkers, histopathological analyses of articular cartilage, synovial membrane, and subchondral bone have been used to evaluate arthritis disease progression or to measure outcome of therapeutic interventions [63]. In the current study, significant improvements in maintenance of the articular structural integrity of rats treated with UP1306 and Methotrexate were observed. These effects were demonstrated in the histopathology data as exhibited by limited loss, degeneration, or necrosis of chondrocytes, smoother articular cartilage surface, deeper and uniform stain of intracellular matrix, and close to normal contour of the subchondral bone. The changes in magnitude of histopathological severity scores for cartilage degradation, bone damage, inflammation, reactivity, and matrix integrity were computed and were found that UP1306 treatment resulted in 86.8%, 71.8%, 58.6%, 70.6%, and 87.1% inhibitions, respectively, when compared to vehicle-treated CIA rats.

Conclusions
Collectively, in the current study, UP1306 orally supplemented rats experienced (a) reduced inflammation as reflected by reduced arthritis index, paw thickness, paw edema, and reduced inflammatory cytokines (IL-1β, IL-6, and TNF-α), (b) decreased pain sensitivity, and (c) increased cartilage sparing activity and maintenance of articular structure as indicated by lower uCTX-II and cartilage degrading enzymes (MMP-13 and MMP-3). The aggregated effects that inhibit pro-inflammatory cytokines and cartilage matrix degrading enzymes indicate that the protective effect of UP1306 on arthritis could be partially mediated through the modulation of the immune system. These properties of UP1306 suggest its potential use as an alternative natural therapy for arthritis management.