Current Pharmaceutical Biotechnology
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Current Biotechnology Pharmaceutical
ISSN: 1389-2010 eISSN: 1873-4316
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Current Pharmaceutical Biotechnology, 2018, 19, 1156-1169
RESEARCH ARTICLE
Suppressive Effects of the Standardized Extract of Phyllanthus amarus on Type II Collagen-Induced Rheumatoid Arthritis in Sprague Dawley Rats
Javaid Alam
1, Ibrahim Jantan
2*, Endang Kumolosasi
1, Mohd A. Nafiah
3and Muhammed A. Mesaik
41Drug and Herbal Research Center, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia; 2School of Pharmacy, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia;
3Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjung Malim, Perak, Malaysia; 4Faculty of Medicine,University of Tabuk, Tabuk 71491, Saudi Arabia
A R T I C L E H I S T O R Y
Received: August 09, 2018 Revised: November 09, 2018 Accepted: December 04, 2018
DOI:
10.2174/1389201020666181211124954
Abstract: Background: Standardized extract of Phyllanthus amarus has been shown to possess inhibitory effects on cellular and humoral immune responses in Wistar-Kyoto rats and Balb/c mice.
Objective: In the present study, the standardized extract of P. amarus was investigated for its suppressive effects on type II collagen-induced rheumatoid arthritis (TCIA) in Sprague Dawley rats.
Method: The major components of the extracts, lignans and phenolic compounds were analysed by using a validated reversed phase HPLC and LC-MS/MS. A rheumatoid arthritis rat model was induced by administering a bovine type II collagen emulsion subcutaneously at the base of tail, on day 0 and 7 of the experiment. Effects of the extract on severity assessment, changes in the hind paw volume, bone mineral density, body weight and body temperature were measured. Concentrations of cytokines (TNF-α, IL-1β, IL-1α, IL-6) released, matrix metalloproteinases (MMP-1, MMP-3 MMP-9) and their inhibitor (TIMP-1), haematological and biochemical changes were also measured. ELISA was used to measure the cytokines and proteinases in the rat serum and synovial fluid according to manufacturer's instructions.
Results: The extract dose-dependently modulated the progression in physical parameters (i.e. decrease in body weight, increase in body temperature, reduced hind paw volume, reduced the severity of arthritis), bone mineral density, haematological and biochemical perturbations, serum cytokines production and levels of matrix metalloproteinases and their inhibitor in the synovial fluid. Histopathological examination of the knee joint also revealed that the extract effectively reduced synovitis, pannus formation, bone resorption and cartilage destruction.
Conclusion: The results suggest that the oral administration of a standardized extract of P. amarus was able to suppress the humoral and cellular immune responses to type II collagen, resulting in the reduction of the development of TCIA in the rats.
Keywords: Phyllanthus amarus, rheumatoid arthritis, pro-inflammatory cytokines, type II collagen-induced arthritis, immune response, metalloproteinases.
1. INTRODUCTION
Rheumatoid arthritis (RA) is a chronic and systemic autoimmune response to the multiple joints with unknown aetiology, progressive disability, systemic complications, early death and high socioeconomic costs. This symmetric polyarticular arthritis initially affects freely movable joints,
*Address correspondence to this author at the School of Pharmacy, Taylor’s University, 47500 Subang Jaya, Selangor, Malaysia; Tel: +6-016 2886445;
Fax: +6-0356295455;E-mail: [email protected]
i.e. diarthrodial like the shoulder, knee, hip and hand.
Inflamed synovium leads to pannus formation and the production of cytokines, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP) that destroy the local articular structures [1].
Cytokines, a diverse group of polypeptides, play a critical role in the pathogenesis of RA by acting on a phenomenon called cytokines redundancy. Among the pro-inflammatory cytokines, interleukin-1 alpha/beta (IL-1α/β) and tumour necrosis factor-alpha (TNF-α) play a significant role in
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triggering the intracellular molecular signalling pathway that leads to the activation of mesenchymal cell, recruitment of innate and adaptive immune system cells, activation of synoviocytes which in turn activate various mediators including TNF-α, IL-1, IL-6 and various matrix metalloproteinases (MMP-1, MMP-3, MMP-9), resulting in inflamed synovium, increase angiogenesis, bone erosion, cartilage damage and decrease lymphangiogenesis, which are characteristic features of RA [2, 3]. According to a recent report, 60-90% of RA patients used complementary and alternative medicine (CAM) like chiropractic and herbal therapies. Thus, there is a growing interest to discover new treatments from herbal source [4, 5]. In this regard, Phyllanthus amarus (family: Euphorbiaceae) is one of the potential natural immunomodulators, having broad-spectrum pharmacological properties for instance, the extract of P.
amarus, showed a significant inhibition on the cellular and humoral immune responses in Balb/c mice as well as suppressed Mac-1 expression, T- and B-cell proliferation, CD4+ and CD8+ T-cell expression in splenocytes and production of cytokines in Wistar-Kyoto rats [6, 7]. Similarly, its active constituents like hypophyllanthin and niranthin, significantly suppressed NF-kB and MAPKs signalling pathways as well as TLR4 and MyD88 expression in LPS- induced U-937 macrophages and human macrophages, respectively [8]. Phyllanthin, hypophyllanthin and corilagin rich extracts modulated the pain and inflammation by increasing the thermal and mechanical threshold in chronic musculoskeletal inflammatory pain model [9]. Gallic acid, on the other hand, ameliorated the fluoxetine-induced chronic liver damage in rats, by decreasing the serum and gene expression of TNF-α [10]. Likewise, geraniin proved to be antiosteoporotic in ovariectomy-induced bone loss model [11] and on migration inhibitory factor (MIF)-induced NF- κB nuclear translocation in peripheral blood mononuclear cells (PBMC), ellagic acid showed a profound inhibitory effect [12].
All these studies provide us the basis to investigate the suppressive effects of 80% ethanol extract of P. amarus in type II collagen-induced arthritis rat model. The type II collagen-induced arthritis model (TCIA) has been widely used as a model of human RA since they share both immunological and pathological features with RA.
2. MATERIALS AND METHOD 2.1. Chemicals and Reagents
Indomethacin, incomplete Freund adjuvant (IFA), Tween® 20 solution, Whatman® Grade1 filter paper and the chemical reagents used for tissue fixation and preparation, 10% neutral buffered formalin, xylene histological grade, absolute ethanol paraffin wax, were procured from Sigma Chemical Co. (St. Louis, MO, USA). Standard compounds phyllanthin, hypophyllanthin, gallic acid, ellagic acid, corilagin and geraniin of HPLC grade (>99% purity) were purchased from ChromaDex (CA, USA). Standard laboratory diet was obtained from Bell Laboratories, Inc. (Sudbury, UK). Bovine type II collagen was acquired from Chondrex, USA. Rat TNF- α, IL-1α, IL-1β, IL-6 duo set and rat MMP-9, TIMP-1 duo set ELISA kits were procured from R&D Systems (Minneapolis, MN, USA). Rat MMP-1and MMP-3 ELISA kits and
colorimetric activity assay kits were obtained from Cusabio, USA and Cayman Chemical (Michigan, USA), respectively.
2.2. Animals and Ethics
The male Sprague Dawley rats weighing 200-250 g, procured from the Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), were used and maintained under specific pathogen-free conditions at the experimental animal centre of Faculty of Pharmacy, UKM, Malaysia. The methods used in this study were approved by the UKM Animal Ethics Committee (UKM (AEC) PPI.1/138/1). The animals were kept in cages at 23 ± 2oC and fed with standard laboratory diet (Bell Laboratories, Inc., Sudbury, UK) and tap water throughout the experiments with 12 h dark/light cycle. One-week acclimatization was provided before the start of the experiment.
2.3. Plant Sample and Extract Preparation
The whole plants of P. amarus were obtained from Ma- rang, Kuala Terengganu, Malaysia. The plants were authen- ticated by Dr Abdul Latif Mohamad of the Faculty of Sci- ence and Technology, Universiti Kebangsaan Malaysia (UKM), and voucher specimens (P. amarus UKMB 30078) were deposited at the Herbarium of UKM, Bangi, Malaysia.
The whole plants of P.amarus (1 kg) were ground and ex- tracted with 80% EtOH (3 × 3 L) at room temperature for 72 h, then filtered through Whatman® grade1 filter paper (Sigma-Aldrich Corp). The filtrate was collected, and excess solvent was evaporated under reduced pressure using a ro- tary evaporator at temperature between 55 and 60°C. The yield of extract obtained was 108 g (10.8% w/w). The extract was resuspended in 2% Tween® 20 solution before being orally (PO) administered to rats. The possible endotoxin con- tamination of the extract was evaluated by Limulus Amebo- cyte Lysate (LAL) assay kit (Cambrex Bioscience, Walk- ersville, MD) following the manufacturer's recommended instructions. The extract was found to contain insignificant amounts of endotoxin which would potentially interfere with the investigations at the concentrations used. Endotoxin free reagents, sterile water, and buffers were used throughout the study as essential precautions to avoid any endotoxin con- tamination.
2.4. Subacute Oral Toxicity
The sub-acute oral toxicity of 80% ethanol extracts of Phyllanthus amarus was investigated in Sprague Dawley rats using the guide lines provided by Organization of Economic Cooperation and Development. Four groups (each containing six rats) of male Sprague Dawley rats were randomly di- vided. Three groups received a single dose of 80% ethanol extract of P. amarus (dose ranging from 100, 300, 500 mg/kg) while the fourth group did not receive any treatment.
The weight of the rats was measured weekly, and the rats were also observed for mortality and behaviour changes (ex- citability, convulsions, lethargy, sleep).
2.5. Induction of Arthritis
Solution of bovine type II collagen (Chondrex, USA) was prepared in 0.05 M acetic acid with gentle stirring overnight
at 4ºC. The solution was then added drop wise to equal vol- ume of incomplete freund adjuvant (IFA) (Sigma) using a homogenizer (Omni international/W/5 mm probe) for 2 to 3 min to form an emulsion. The emulsion was kept cool in an ice water bath and the mixing was repeated 3 times. Two hundred µL of this emulsion in 1mL hamilton syringe, was given subcutaneously on day 0 and 7 at the base of tail, in all experimental groups except the normal control.
2.6. Experimental Design
Arthritis was induced by administering the emulsion of bovine type II collagen (200 µL) subcutaneously on day 0 and 7. The extract in doses of 100, 200, 400 mg/kg and indomethacin (5 mg/kg) was given orally every morning starting from day 0 up to 28 days. The different doses of the extract were selected based on a previous study [7]. The extract and indomethacin were dispersed homogeneously in 2% tween-20. Normal and arthritic control group were given 2% of tween-20 solution orally. The animals were divided into the following groups (n = 6).
Group A: Normal control (normal rats with no immunization and treatment).
Group B: Arthritic control group (type II collagen emulsion (200 µL) on day 0 and day 7 with no treatment).
Group C: Positive control (type II collagen emulsion (200 µL) on day 0 and day 7 + Daily indomethacin (5 mg/kg) orally).
Group D: Extract 100 mg/kg (type II collagen emulsion (200 µL) on day 0 and day 7 + Daily 100 mg/kg of the extract).
Group E: Extract 200 mg/kg (type II collagen emulsion (200 µL) on day 0 and day 7 + Daily 200 mg/kg of the extract).
Group F: Extract 400 mg/kg (type II collagen emulsion (200 µL) on day 0 and day 7 + Daily 400 mg/kg of the extract).
2.7. Severity Assessment of Arthritis
Arthritis severity was evaluated by an arthritis score, arthritic index, percentage of arthritic limbs, time of arthritis first appeared by an independent observer without prior knowledge of the experimental groups which gives a semi- quantitative measure of inflammation [13]. Percentage of arthritis limbs = number of arthritic limbs in a group/ number of all limbs in a group x 100. Arthritis score: 0 = no sign of arthritis, 1 = swelling or redness of the paw or one digit, 2 = involvement of two joints, 3 = involvement of more than two digits, 4 = severe oedema of the entire paw. Arthritis Index: Total number of arthritic score in a group. Time of arthritis first appeared: It refers to the first day of onset of clinical symptoms of arthritis observed.
2.8. Measurement of Hind Paw Volume, Body Weight and Body Temperature
Plethysmometry (Ugo Basil, USA) was used to measure the hind paw volume on every alternative day starting from
day 0 that provided a quantitative measure of the hind paw volume. Similarly, body weight and body temperature were also recorded using Sartorius balance and Bio-TK8851 rodent thermometer on every alternate day.
2.9. Blood Analysis
Blood from all the groups was collected from retro-orbital plexus and analysed for complete blood count (RBC, WBC, platelet, differential count (%) (neutrophil, lymphocytes, monocytes, basophils, eosinophils)) using Sysmex high-end haematology analyser while total protein, creatinine, urea, uric acid, magnesium, calcium and phosphorous levels were also measured by Roche/Hitachi Cobas c 311/501 analysers using colorimetric assay
2.10. Bone Analysis
On day 28, animals were subjected to bone mineral density (BMD) of the hind paws by using dual-energy x-ray absorptiometry (DEXA), that used small dose of ionizing radiation to determine the bone mineral density. Briefly, the rats were anesthetized with I.P injection of ketamine (80 mg/kg)/xylazine (10 mg/kg) mix on day 28 and were imaged using the discovery W (S/N 88685) that measured the bone mineral density (BMD). Each scan was performed at a speed of 20 mm/sec. The BMD profile of hind paw was stored and analysed using window based software.
2.11. Serum Cytokine Analysis
On day 14 and 28, blood was collected from retro-orbital plexus and the whole blood was allowed to clot by leaving it undisturbed at room temperature for about 30 min. It was then centrifuged at 4000 rpm for 20 min in a refrigerated centrifuge. The resulting supernatant was collected and was subjected for analysis of TNF-α, IL-1α, IL-1β, IL-6, using commercially available ELISA kits. ELISA applied in this study were Rat TNF- α duo set (R&D), Rat IL-1α duo set (R&D), Rat IL-1β duo set (R&D), and Rat IL-6 duo set (R&D). The concentrations of the cytokines were calculated from the standard curves.
2.12. Synovial Fluid Analysis
Synovial fluid samples were collected from the knee joint on day 28 using Barton et al. perfusion method [14].
Samples were then analysed for matrix metalloproteinases (MPP-1, MMP-3, and MMP-9) and tissue inhibitor matrix metalloproteinase (TIMP-1), using commercially available ELISA kits. ELISA applied here were Rat MMP-9 duo set (R&D), Rat MMP-1 (Cusabio), Rat MMP-3 (Cusabio) and Rat TIMP-1 duo set (R&D).
2.13. Histopathology
Rats were killed by cervical dislocation. Knee joints were extracted and subjected to histopathological analysis.
Samples were washed with PBS and fixed in 10% neutral buffered formalin for 48 h and decalcified in 10% formic acid for 28 days, and embedded in paraffin. Section of 5 µm was prepared and stained with haematoxylin and eosin (H&E) [15].
2.14. HPLC Analysis and Standardization of Extract The lignans and phenolic compounds in the 80% ethanol extract of P. amarus including phyllanthin, hypophyllanthin, gallic acid, ellagic acid, corilagin and geraniin were identified and quantified by reversed phase HPLC method using two different chromatographic conditions, method 1 [16] and method 2 [17] as described previously. Briefly, the 80%
ethanol extract of P. amarus was standardized on a basis of standard compounds, phyllanthin, hypophyllanthin, corilagin and geraniin by using a reverse phase, Xbridge® C 18 column (5 μm, 250 mm × 4.6 mm i.d, Waters, Milford, USA) equipped with a 2998 photodiode array detector. The phenolic compounds were identified and quantified using method 1, with corilagin as an external standard, while identification and quantification of lignans were by using method 2. The detection was performed by comparing the retention times and ultraviolet-visible (UV-Vis) spectra of the peaks with those of the standard compounds. Limits of quantification (LOQ) and detection (LOD), precision and linearity were detected to validate HPLC for the standardization of extract.
2.15. LC-MS/MS Analysis of the Extract
Chromatographic separation was performed on Thermo Scientific C18 column (AcclaimTM Polar Advantage II, 3 x 150 mm, 3 µm particle size) on an UltiMate 3000 UHPLC system (Dionex). Gradient elution was performed at 0.4 mL/min and 40°C using H2O + 0.1% Formic Acid (A) and 100% ACN (B) with 22 min total run time. The injection volume of the sample was 1µL. The gradient started at 5% B (0-3min); 80% B (3-10min); 80% B (10-15min) and 5% B (15-22min). High-resolution mass spectrometry was carried out using a MicroTOF QIII Bruker Daltonic using an ESI positive/negative ionization with the following settings: - capillary voltage: 4500 V; nebulizer pressure: 1.2 bar; drying gas: 8 L/min at 200oC. The mass range was at 50-1000 m/z.
The accurate mass data of the molecular ions, provided by the TOF analyzer, were processed by Compass Data Analysis software (Bruker Daltonik GmbH).
2.16. Statistical Analysis
All the data were expressed as means ± SEM and were statistically analysed by using one-way and two-way analysis of variance (ANOVA) followed by Tukey/Dunnett and Bonferroni post hoc test. Results were considered statistically significant if P values were *< 0.05, **< 0.01, ***< 0.001.
3. RESULTS
3.1. Sub-Acute Toxicity of 80% Ethanol Extract of Phyllanthus amarus
No significant body weight changes were observed in the extract treated groups (100, 300, 500 mg/kg) relative to the control group. Similarly, no mortality and adverse clinical manifestations (e.g. diarrhoea, haematuria, restlessness, uncoordinated muscle movements) were perceived in the experimental animals during the four-week study period.
3.2. Effect of Phyllanthus amarus Extract on the Severity of Arthritis
The extract, at all three doses (100, 200, 400 mg/kg) significantly (p < 0.01, p < 0.001) decreased the arthritic index and percent arthritic limb in a dose-dependent manner compared with the arthritic group (Fig. 1A, B). In case of the time of arthritis first appeared, both 200 and 400 mg/kg doses showed a significant (p < 0.001) response and prolonged the time of arthritis first appeared compared with the arthritic group while the dose of 100 mg/kg showed a non-significant response (Fig. 1C).
3.3. Effects of Phyllanthus amarus Extract on Changes in Hind Paw Volume, Body Weight and Body Temperature Fig. 2 (A, B) shows the macroscopic and graphical presentations of the changes in hind paw volume that took place from day 0 to 28. The arthritic group showed a visible and considerable difference in the hind paw volume and physical appearance. A marked erthymena and swelling were observed in arthritic group with a significant increase in the hind paw volume as compared to the normal control. On contrary, the extract at 200 and 400 mg/kg doses showed a significant decrease as compared to the arthritic control starting from day 9 to day 27 (P < 0.001) (Fig. 2B).
However, at 100 mg/kg the extract showed a non-significant decrease in hind paw volume starting from day 7 to day 19 but showed a significant response on day 21 to day 27 as compared to the arthritic group (p < 0.01, p < 0.001 (Fig.
2B). The observation of the body weight on every alternate day revealed that there was a noticeable loss (25.6%) in the arthritic group. However, the percent loss (23.3%, 13.1% and 6.6%) in the treated groups (100, 200, 400 mg/kg, respectively) was significantly (p < 0.001) lowered than the arthritic group except at the dose of 100 mg/kg (Fig. 2C).
Similarly, measurement of body temperature showed that arthritic group exhibited a high percent rise in the body temperature (15.6%) with respect to the normal group. The rise in the body temperature (14.7%, 10.6%, 7.26%) in the treated group (100, 200, 400 mg/kg, respectively) followed a descending order, respectively. It could be concluded that 400 mg/kg dose showed a potentially strong response in reducing the hind paw volume, decreasing the percent weight loss and percent rise in temperature in type II collagen- induced arthritis rat model.
3.4. Effects of Phyllanthus amarus Extract on Haematological and Biochemical Perturbations
Haematological analysis revealed that the increase in white blood cell (WBC) count, platelet count, erythrocyte sedimentation rate (ESR) along with decrease in red blood cell (RBC) count and changes in differential count (i.e.
neutropenia and lymphocytosis) in arthritic control group were significantly altered in the treatment groups in a dose- dependent manner. The 200 and 400 mg/kg doses of the extract showed an equipotent effect in alerting all these haematological parameters (p < 0.001). However, at the dose of 100 mg/kg the extract showed a non-significant response as compared to the arthritic group (Table 1).
Fig. (1). Effects of standardized extract of Phyllantus amarus on severity of arthritis in type II collagen induced arthritis rats. (A) arthritic index; (B) % arthritis limbs; (C) time of arthritis first appeared. Statistical difference was determined using one-way ANOVA followed by a Dunnett post hoc test. Data are represented as mean ±SEM. (n=6). ≠≠≠ P<0.001, comparison with the normal. **P<0.01, ***P<0.001, compari- son with arthritic group.
Fig. (2). Effects of standardized extract of Phyllantus amarus on changes in body weight, body temperature and hind paw volume in type II collagen induced arthritis rats. (A) Macroscopic evidence of arthritis showing erythema and swelling markedly decrease in a dose dependant manner; (B) Hind paw volume on every alternative day; (C) Percent weight loss. (D) Percent rise in body temperature. For (C) and (D) statis- tical difference was determined using one-way ANOVA followed by a Dunnett post hoc test. For (B) statistical difference was determined using two-way ANOVA followed by a bonferoni post hoc test. Data are represented as mean ±SEM. (n=6). ≠≠≠ P<0.001, comparison with the normal. *P<0.05, **P<0.01, ***P<0.001, comparison with arthritic group.
Table 1. The haematological and biochemical parameters in normal, arthritic, treatment (100, 200,400 mg/kg) and positive control (indomethacin 5 mg/kg) groups.
Indomethacin Treatment Groups
Blood Analysis Normal Arthritic
5mg/kg 100 mg/kg 200 mg/kg 400 mg/kg RBC (x 10 12/l) 8.88 ± 0.14 5.18 ± 0.26 7.11 ± 0.24*** 5.65 ± 0.28 7.10 ± 0.33*** 7.28 ± 0.37***
WBC (x 10 9 /l) 8.61 ± 0.46 14.77 ± 0.41 8.4 ± 0.56*** 12.85 ± 0.71 10.53 ± 0.79*** 9.96 ± 0.76***
Platelets (x 10 9 /l) 390 ± 48.19 1083 ± 40.77 444.7 ± 28.45*** 861.2 ± 70.71** 597.8 ± 36.85*** 500.7 ± 29.07***
Differential Count (%)
Neutrophil 52 ± 1.23 15 ± 2.62 51.3 ± 2.23*** 19.5 ± 1.14* 40 ± 1.06*** 50 ± 1.60***
Lymphocytes 41 ± 1.22 72 ± 2.08 41.5 ± 2.52*** 69 ± 1.10 54 ± 1.88*** 42 ± 2.17***
Monocytes 4.3 ± 0.33 8.1 ± 0.83 4.3 ± 0.91 7.3 ± 0.55 6 ± 0.57 5.1 ± 0.47 Eosinophil 1.0 ± 0.00 1.3 ± 0.21 1.0 ± 0.00 1.0 ± 0.36 1 ± 0.25 1.1 ± 0.16 Basophil 1.5 ± 0.22 4.1 ± 1.01 1.8 ± 0.40 3.3 ± 0.33 2.0 ± 0.25 2.0 ± 0.36 ESR ( mm/hr) 4.1 ± 0.70 13.3 ± 0.42 6.6 ± 0.80 11.3 ± 0.42 9.3 ± 0.71*** 8.1 ± 0.60***
ALP (IU/L) 130.3 ± 3.69 196.2 ± 10.40 135.8 ± 4.72*** 170.8.0 ± 2.70* 162.7 ± 4.36** 140.5 ± 4.31***
ALT (IU/L) 32.0 ± 3.10 74.50 ± 1.97 43.33 ± 3.57*** 66.67 ± 3.33 60.50 ± 2.12** 49.50 ± 2.04***
AST (IU/L) 120.0 ± 2.23 200.8 ± 6.04 130.5 ± 5.09*** 187.3 ± 5.04 177.7 ± 3.31** 140.0 ± 4.07***
RF Factor (IU/ml) 4.50 ± 0.22 13.80 ± 0.50 6.66 ± 0.49*** 11.50 ± 0.56* 10.83 ± 0.70** 7.16 ± 0.70***
Total protein (g/dl) 5.78 ± 0.11 8.5 ± 0.18 6.18 ± 0.07*** 7.96 ± 0.18 7.75 ± 0.21** 6.53 ± 0.07***
C-Reactive protein (mg/dl) 2.66 ± 0.33 6.33 ± 0.42 2.8 ± 0.30*** 5.00 ± 0.36 4.5 ± 0.42* 3.16 ± 0.47***
Creatinine (mg/dl) 0.88 ± 0.06 0.20 ± 0.02 0.73 ± 0.04*** 0.30 ± 0.03 0.48 ± 0.03*** 0.63 ± 0.04***
Urea (mg/dl) 46.67 ± 1.11 26.50 ± 1.25 38.33 ± 1.94*** 27.83 ± 1.57 37.0 ± 2.78*** 40.17 ± 1.58***
Magnesium (mmol/l) 1.017 ± 0.06 0.45 ± 0.04 0.86 ± 0.04*** 0.50 ± 0.03 0.78 ± 0.05*** 0.80 ± 0.04***
Calcium (mmol/l) 2.26 ± 0.08 1.36 ± 0.14 2.00 ± 0.06*** 1.53 ± 0.14 1.81 ± 0.09* 1.91 ± 0.07**
Phosphorus (mmol/l) 2.58 ± 0.06 4.103 ± 0.09 2.65 ± 0.05*** 3.86 ± 0.07 3.6 ± 0.15** 2.73 ± 0.08***
Statistical difference was determined using one-way and two-way ANOVA followed Dunnett by a post hoc test. Data are represented as mean ± SEM. (n = 6). *P < 0.05, **P < 0.01,
***P < 0.001, comparison with arthritic group.
Likewise, the biochemical perturbations in the arthritic group such as increase in levels of alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate aminotrans- ferase (AST), total proteins, phosphorous and along with the decrease in creatinine, urea, magnesium and calcium were also favourably tempered in the extract-treated (100, 200, 400 mg/kg) groups. The 100 mg/kg dose of the extract showed a significant (p < 0.05) response in case of ALP level only and remained non-significant with the rest of the parameters while at higher doses (200 and 400 mg/kg) significantly (p < 0.01, p < 0.001) mitigated all these biochemical changes observed in the arthritic group, with 400 mg/kg dose being more potent among them (Table 1).
Similarly, serum C reactive protein (6.33 ± 0.42 mg/dl) and rheumatoid factor (RF) (13.80 ± 0.50 IU/ml) level were also significantly higher in the arthritic group as compared to the normal group. But the extract-treated group (100, 200, 400 mg/kg) showed a dose-dependent decrease in rheumatoid factor level with P values of < 0.05, < 0.01 and < 0.001, respectively. However, in case of C reactive protein, the 100 mg/kg dose showed non-significant response while the rest of the doses remained significant (p < 0.05, p < 0.001), respectively (Table 1). The extract of P. amarus, at the dose
of 400 mg/kg showed a dominant positive response in both haematological and biochemical perturbation observed in TCIA rat model.
3.5. Effect of Phyllanthus amarus Extract on Bone Mineral Density
The hind limbs of the arthritic group showed a significant decrease in the bone mineral density as compared to the vehicle control (Fig. 3). In the treatment groups, both 200 and 400 mg/kg dose of the extract showed significant (p<0.01, p<0.001) increase in the bone mineral density as compared to arthritic group. However, the manifestation of the 100 mg/kg dose was non-significant.
3.6. Effect of Phyllanthus amarus Extract on Serum Cytokines Levels
Circulating levels of serum cytokines (TNF-α, IL-6, IL-1β, IL-1α) were measured using ELISA. The levels of all these cytokines in serum of the arthritic group were higher significantly on day 14 and increased further on day 28. On the contrary, the treatment groups (100, 200, 400 mg/kg) showed significant reduction on day 14 and day 28 (Figs. 4A-D).
Fig. (3). Effects of the extract of Phyllantus amarus on bone mineral density of hind limbs of normal, arthritic, treatment (100, 200, 400 mg/kg) and positive control (Indomethacin 5 mg/kg) groups. Statistical difference was determined using one-way ANOVA followed by a dunnett post hoc test. Data are represented as Mean ± SEM. (n=6). **P<0.001, ***P<0.001, comparison with arthritic group.
Fig. (4). Effects of standardized extract of Phyllantus amarus on TNF-α, IL-6, IL-1β, and IL-1α level in the sera of normal, arthritic, treat- ment (100, 200, 400 mg/kg) and positive control (Indomethacin 5mg/kg) groups on day 14 and day 18. (A) TNF-α; (B) IL-6; (C) IL-1β; (D) IL- 1α. Statistical difference was determined using one-way ANOVA followed by a dunnett post hoc test. Data are represented as mean ±SEM.
(n=6). ≠≠≠ P<0.001, comparison with the normal. *P<0.05, **P<0.01, ***P<0.001, comparison with arthritic group on day 14 and day 18.
Fig. (5). Effects of standardized extract of Phyllantus amarus on MMP-3, MMP-1, MMP-9, and TIMP-1 level in the synovial fluid of normal, arthritic, treatment (100, 200, 400 mg/kg) and positive control (Indomethacin 5mg/kg) groups. (A) MMP-3; (B) MMP-1; (C) MMP-9; (D) TIMP-1. Statistical difference was determined using one-way ANOVA followed by a dunnett post hoc test. Data are represented as mean
±SEM. (n=6). ≠≠≠ P<0.001, comparison with the normal. *P<0.05, **P<0.01, ***P<0.001, comparison with arthritic group.
Table 2. Concentration ratio of MMPs toTIMP-1 arthritic, treatment (100, 200, 400 mg/kg) and positive control (Indomethacin 5 mg/kg) groups.
MMP-3/TIMP-1 MMP-1/TIMP-1 MMP-9/TIMP-1
Arthritic 4.12 ± 0.26 4.64 ± 0.15 3.11 ± 0.07
Treatment
100 mg/kg 3.77 ± 0.28 4.43 ± 0.06 3.07 ± 0.05
200 mg/kg 2.71 ± 0.15** 3.84 ± 0.13* 2.45 ± 0.11***
400 mg/kg 2.22 ± 0.37*** 2.84 ± 0.27*** 2.05 ± 0.14***
Positive Control (Indomethacin 5 mg/kg) 1.43 ± 0.14*** 2.75 ± 0.27*** 1.90 ± 0.09***
Statistical difference was determined using one-way ANOVA followed Dunnett by a post hoc test. Data are represented as mean ± SEM. (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, comparison with arthritic group.
On day 14, at doses of 200 and 400 m/kg, the extract showed lowered levels of TNF- α (p < 0.01, p < 0.001), IL-6 (p <
0.01, p < 0.001), IL-1β (p < 0.01, p < 0.001,) and IL-1α (p <
0.01, p < 0.001) as compared to the arthritic group, respectively. However, at the dose of 100 mg/kg the extract showed non-significant response (Figs. 4A-D). On day 28, all the doses (100, 200, 400 mg/kg) of the extract significantly reduced the levels of TNF-α (p < 0.01, p < 0.001, p < 0.001), IL-6 (p < 0.05, p < 0.001, p < 0.001), IL-1β (p < 0.01, p <
0.001, p < 0.001) and IL-1α (p < 0.01, p < 0.001, p < 0.001) as compared to the arthritic group, respectively (Fig. 4A-D).
3.7. Effect of Phyllanthus amarus Extract on Metalloproteinase and their Inhibitor in the Synovial Fluid
Synovial fluid analysis showed that the concentrations of proteinases (MMP-1, MMP-3, MMP-9) and their inhibitor (TIMP-1) were significantly high in the arthritic group as compared to the vehicle control (Fig. 5). In the treatment groups both 200 and 400 mg/kg doses of the extract showed significant (p < 0.001) reduction in the concentration of all of the proteinases as well as its inhibitors (TIMP-1) as compared to the arthritic group (Figs. 5A-D). However, the
manifestation of the 100 mg/kg dose was non-significant with respect to proteinases as well as its inhibitor. The arthritic group showed high ratio of MMP-3/TIMP-1, MMP- 1/TIMP-1 and MMP-9/TIMP-1 (4.12 ± 0.26, 4.64 ± 0.15, 3.11 ± 0.07) indicating increased activity of the proteinases, respectively (Table 2). On contrary, the 200 and 400 mg/kg doses of the extract showed significant (2.71 ± 0.15, 3.84 ± 0.13, 2.45 ± 0.11) decrease in the above-mentioned ratio of MMPs to TIMP-1, respectively (Table 2).
3.8. Histopathological Changes
Further evidence supporting the anti-inflammatory/
immunosuppressive effects of the standardized extract of P.
amarus on type II collagen-induced arthritis was obtained by histopathology analysis. At the end of the experiment, the knee joints of the all groups were subjected to histology study (H and E). The arthritis group exhibited noteworthy synovitis, marked cartilage damage, pannus formation and bone resorption (Fig. 6), while in the knee joint of the rats administered with the standardized extract of P. amarus, these pathological changes were markedly reduced. The group treated with 200 mg/kg showed moderate synovitis, mild cartilage damage only at the femur bone and no pannus formation and bone resorption. While the 400 mg/kg dose exhibited mild synovitis and with no cartilage damage, pannus formation and bone resorption as compared with arthritic group. However, the articular manifestation of 100 mg/kg dose of the extract was not significant.
3.9. HPLC and LCMS/MS Analyses
The lignans and phenolic compounds in the 80% ethanol extract of P. amarus namely phyllanthin, hypophyllanthin, gallic acid, ellagic acid, corilagin and geraniin were identified and quantified by reversed phase HPLC methods as previously reported. HPLC chromatograms of P. amarus for the identification of the lignans and phenolic compounds are shown in Fig. (7). The compounds identified by LC- MS/MS in 80% ethanol extract are given in Table 3.
4. DISCUSSION
The present work was aimed to investigate the anti- inflammatory and immunomodulatory effects of the stan- dardized extract of P. amarus in TCIA rat model. The TCIA model is a widely used model because it shares genetic, im- munological and pathological features with human RA [18].
In this study, the standardized extract of P. amarus effec- tively reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-1α) in serum of treated rats even at its low dose with the continuous treatment up to 28 days in the type II collagen-induced arthritis (Fig. A-D). In one of the recent in vitro study on the active constituents of P.
amarus, hypophyllanthin and niranthin, showed a decreased production of pro-inflammatory cytokines (TNF-α and IL- 1β) by suppressing NF-kB and MAPKs signaling pathways in U-937 macrophages. Similarly, TLR4 and MyD88 expres- sion were also markedly suppressed by hypophyllanthin and niranthin in LPS-activated human macrophages [8]. All these Fig. (6). Photomicrographs of the haematoxylin and eosin longitudinal section (HE 4X) from the knee joints of normal, arthritic, treatment (100, 200, 400 mg/kg), and indomethacin. In the normal photomicrograph there is no sign of synovitis (S), cartilage damage (C) and other manifestation of arthritis like pannus formation and bone resorption. Arthritic and extract 100 mg/kg photomicrographs shows severe synovi- tis (S), and marked cartilage damage (C), pannus formation and bone resorption (shown by arrows). The extract 200 mg/kg photomicro- graph shows moderate synovitis (S), mild cartilage damage only at the femur (F) bone and no pannus formation and bone resorption. The high dose (400mg/kg) extract and Indomethacin (5 mg/kg) photomicrographs shows a mild synovitis (S), and no cartilage damage, pannus formation and bone resorption. Arrows indicates Pannus formation and bone resorption, F= Femur, T= Tibia.
Fig. (7). Chromatogram (HPLC-UV, detection at 205-270 nm) of 80% ethanolic extract of Phyllanthus amarus. Identification of peaks: A:
phyllanthin (1); hypophyllanthin (2) with retention times 27.458 and 28.167 min, respectively. B: gallic acid (3); geraniin (4); corilagin (5);
ellagic acid (6) with retention times 7.543, 23.412, 26.376 and 33.112 min, respectively.
Table 3. Lignans and phenolic compounds identified by LC-MS/MS in 80% ethanol extract of Phyllanthus amarus.
Name of the Compound Compound Ionization Mode RT (minutes) OBS. M/Z MS/MS
Metaxalone 16 (+) 1.6 222.1122 116.0719
Pyridoxic acid 20 (+) 1.8 184.0631 116.0718
Gluconic acid 26 (-) 2 195.0517 175.1672,147.5931.
Naringenin 29 (-) 2.1 223.0463 195.0550,129.0271.
Quinic acid 30 (-) 2.2 191.0552 109.0293,104.1929,58.0881
D-(+)-Malic acid 39 (-) 2.6 133.0146 70.5902
Dinitramin 43 (-) 2.9 321.0829 253.0940, 183.9723, 133.0134
4-Oxoproline 47 (-) 3.1 128.0356 121.7174
Citrate 53 (-) 3.3 191.0206 117.0205
Stachydrine 17 (+) 3.6 166.0847 120.0810
6-Deoxy-L-galactose 56 (-) 4.1 199.0383 162.8396
Galloyl-glucopyranoside 59 (-) 4.2 331.0684 169.0142, 135.0455.
Gallic acid 79 (-) 6.5 168.0148 125.0248
Tri-o-methylellagic acid 14 (-) 7 343.0701 169.0152, 135.0464
Corilagin 96 (-) 7.9 633.0785 463.0587, 453.0706 301.0016.
Geraniin 112 (-) 8.5 951.0871 933.0763, 765.0671 463.0557, 301.0016.
Syringic acid 126 (-) 9.2 197.0470 125.0251
Quercetine 3-D-glucoside 132 (-) 9.2 463.0940 300.0308, 169.0156
N-acetyl-D-phenyalanine 137 (-) 9.5 206.0846 147.0461, 116.9290.
Phyllanthin 220 (+) 13.9 419.2689 142.1549, 149.0215
molecular signaling have a pivotal role in the production of pro-inflammatory cytokines during the innate and adaptive immune responses [19, 20].
In the severity assessment protocol, the arthritic group exhibited an ascension in arthritis index (total number of
arthritic score in a group), percent arthritic limbs and damp- ening the latency time (time of the arthritic first appeared) (Figs. 1A-C). On the contrary, the oral administration of the extract at doses of 100, 200 and 400 mg/kg reduced the se- verity of arthritis (arthritic index and percent arthritic limbs), more pronounced at high doses (Figs. 1A-B). The time of the
0.80 0.60
AU 0.40 0.20 0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 Minutes
1 2
5 6
3 4
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
Minutes 0.60
0.40 0.20 0.00
A
B
arthritic first appeared was approximately at day 9, 10, 12, 14 and 15 in arthritic, treatment (100, 200, 400 mg/kg) and positive control groups, respectively. Thus the extract dose- dependently increased the latency time for the induction of arthritis (Fig. 1C), indicating the strong immunosuppressive potential of P. amarus, which entailed inhibition of the immune system.
Similarly, changes in the body weight, body temperature and paw volume also revealed strong anti-inflammatory and immunosuppressant potential of the standardized extract of P. amarus. The macroscopic and graphical presentation showed marked erthyema and swelling in the arthritic group, with a decrease in the body weight and an increase in body temperature (Figs. 2A-D). On the contrary, at high doses, the extract significantly decreased the percent weight loss and percent rise in body temperature. It has been reported that a decrease in the body weights was restored after the admini- stration of anti-inflammatory drugs in the adjuvant-induced arthritis in rats [21]. In addition to this during inflammation, the pro-inflammatory cytokines (IL-6, TNF-α, IL-1), act on hypothalamus resulting in a rise in body temperature and weight loss [22]. In the present study, all these pro- inflammatory cytokines were high in the serum of the arthritic group but were significantly lowered dose-dependently in the treatment groups (Fig. 4A-D).
The blood analysis unveiled anaemia (reduced RBC), neutropenia (reduced neutrophil count), lymphocytosis (in- creased lymphocytes count), thrombocytosis (increased platelet count) and increased ESR in the arthritic group. In addition to this, an altered liver function test (increased ALP, ALT, AST, total proteins levels) and renal function test (in- creased creatinine, urea, calcium, phosphorous and magne- sium levels) was also observed in the arthritic group (Table 1).
These clearly evinced that RA, a multifactorial disease, not only disturb the balance in the immune system but affects various organs in the body [23]. The extract-treated groups significantly normalized the RBC, WBC, lymphocytes and platelet count. The increase in the leukocytes count is the consequence of systemic inflammatory reaction within the body and inflammatory cytokines may further induce the maturity and activation [24]. It has been reported that the rise in the WBC count in the conditions like arthritis is attributed to rise of colony-stimulating factors (CSF) mediated by IL- 1β [25]. Similarly, IL-6 also has a role in the hematopoiesis through JAK-dependent pathway [26]. All these cytokines were found in high concentrations in sera of the arthritic group and were ameliorated significantly in the treatment group (Fig. 4A-D). Increase ESR is attributed to an increase in the total protein and decrease in the RBC count in the ar- thritic group that were significantly altered in the treatment groups. Likewise, the biochemical perturbations in the ar- thritic group such as an increase in ALP, ALT, AST, total proteins, phosphorous level along with the decrease in creatinine, urea, magnesium and calcium were also favorably tempered in the extract-treated (100, 200, 400 mg/kg) groups. All these alterations in the arthritic group have been contributed to the chronic inflammation and oxidative stress observed in the RA patients [27, 28]. It is known that ROS play important role in the exposition of reduced antioxidant capacity and increased oxidative stress observed in RA [29, 30]. P. amarus in this regard have strong antioxidant poten-
tial, which might be responsible for attenuation of hemato- logical and biochemical changes [31, 32].
The standardized extract of P. amarus also dose- dependently decreased the levels of RF factor and C-creative protein. The rise in acute phase protein was most probably because of the increase level cytokines observed in arthritic group especially IL-6, which act on the liver to produce acute phase protein [33]. It has been reported that the expression of these cytokines (TNF-α, IL-1 and IL-6) were found high in collagen-induced arthritis mice [34]. Results in the present study also indicated that these cytokines (TNF-α, IL-1α/β) were significantly lowered in the treatment groups (100, 200, 400 mg/kg) on day 14 (except 100 mg/kg) and day 28 (Fig. 4A, C, D), exhibiting the anti-arthritic potential of the extract by reducing the severity and articular manifes- tation. The concentration of IL-6 was also significantly lowered on day 14 and day 28 at 200 and 400 mg/kg while on day 28 all the doses showed significant reduction as com- pared to arthritic control (Fig. 4B). Studies showed that RA patients resistant to DMARDs showed positive response, in terms of severity and erosive progression, to the use of monoclonal antibodies specific for IL-6 receptors [35]. Apart from this, IL-6 plays a significant role in the proliferation of fibroblast, chemokines production and leukocytes infiltration into the synovium membrane that leads to the formation of rheumatoid pannus, a characteristic feature of the arthritic knee joint [36].
Analysis of synovial fluids for the proteinases (MMP-3, MMP-1, MMP-9) and its inhibitor (TIMP-1) also revealed significant effect of the extract against type II collagen- induced arthritis. MMPs and TIMPs have a significant role in remodeling of the articular tissues in RA [37, 38]. Chon- drocytes, fibroblasts and macrophages are responsible for the production of these proteinase [39]. It has been reported that the degradation of bone and cartilage associated with RA is the result of the imbalance of these degrading enzymes and their secretions are regulated by cytokines like IL-1 and TNF-α [40-42]. High level of MMP-3 observed in RA not only results in the direct lysis of collagen but it also activates other proteinases like MMP-1, MMP-9, which further worsen normal articulation [43-45]. The biological activity of the proteinases is regulated by TIMPs and among them, TIMP-1 is the most widely distributed and suppresses the activity of all MMPs [46, 47]. Increase amount of TIMP-1 has been reported in synovial fluid of the RA patients and murine antigen induced arthritis [48, 49]. In the present study, the ratio MMPs to TIMP-1 in arthritic group were found high and were significantly reduced by the extract at the dose of 200 and 400 mg/kg (Table 2). The high concen- tration ratio of MMPs to TIMP-1in the arthritic group indi- cated that TIMP-1 was not sufficient to block the increased MMPs activity.
Remodeling of bone involves the synthesis of bone ma- trix by osteoblasts and bone resorption by osteoclast. Exces- sive osteoclast activity has been observed in rheumatoid ar- thritis [50, 51]. Increased bone loss has been correlated with inflammatory disease activity, increased levels of cytokines released during the inflammatory response in autoimmune disease, including TNF-α, and IL-6, which have been shown to stimulate bone degradation mainly through their action on
the osteoclasts [52, 53]. Increased levels of circulating TNF- α have also been implicated in inhibiting bone growth which itself mediates an increase in net bone loss manifested in rat model of postmenopausal osteoporosis and inflammatory autoimmune diseases [54, 55]. Several studies have shown that synovial tissues are source of a number of cytokines and inflammatory mediators that have the capacity to induce the recruitment, differentiation, and activation of osteoclasts.
These include interleukin (IL)-1α and IL-1β, tumor necrosis factor (TNF)-α, macrophage colony-stimulating factor, IL-6 and parathyroid hormone-related peptide [56, 57]. In the present study, we observed an increased level of cytokines (IL-6, IL-1α, IL-1β and TNF-α) in serum, proteinases and its inhibitors in synovial fluid and an increase activity (↑ arthritic index, ↑ % arthritic limbs, ↓ latency time, ↑ C-reactive protein,
↑ RF-factor, ↑ lymphocytosis) in arthritic group as compared to the treatment groups. It might be concluded that the pre- eminence of these pro-resorptive cytokines has subsidized the systemic bone loss by acting in an endocrine manner to affect bone mineral density of hind limbs (Fig. 3).
Furthermore, the histopathological changes in the arthri- tis group were remarkable and indicating severe synovitis, marked cartilage damage, pannus formation and bone resorp- tion which may be attributed to an increased level of cytoki- nes and the MMPs observed in the arthritic group (Fig. 7).
On the contrary, the extract at doses of 200 and 400 mg/kg significantly reduced the concentration of above-mentioned cytokines and MMPs. As a result, the histopathology of their knee joint revealed moderate to mild synovitis, mild to no cartilage damage and no pannus formation and bone resorp- tion (Fig. 6). Various in vitro and in vivo studies on the deg- radation of cartilage and bone in RA backed up the decisive role of cytokines in the remodeling of joints [58-60].
CONCLUSION
In summary, we found that the standardized extract of P.
amarus successfully alleviated the severity of arthritis in TCIA in Sprague Dawley rats. The severity of arthritis was measured based on the arthritic index, percent arthritic limb and the time of the symptoms of arthritis first appeared. The extract modulated physical changes (body weight, body temperature and paw volume), haematological and biochemical perturbations, bone mineral density, production of pro-inflammatory cytokines, proteinases and kept the knee joint histopathology to almost normal, most probably through its most consequential anti-inflammatory and immunosuppressive actions on the immune system extenuated by the amalgamated action of its active constituents that lead to the downregulation of the principle cytokines (TNF-α, IL- 1β, IL-1α, IL-6) involved in the pathogenesis of RA. Further studies on its active chemical compounds might contribute to the development of novel drugs for RA therapy in the future.
LIST OF ABBREVIATIONS
ALP = Alkaline Phosphatase ALT = Alanine Transaminase AST = Aspartate Transaminase
CAM = Complementary and Alternative Medicine
DMRD = Disease-modifying Antirheumatic Drug ESR = Erythrocyte Sedimentation Rate IL-1 = Interleukin-1
MMP = Matrix Metalloproteinase,
OBS = Observed
RA = Rheumatoid Arthritis RBC = Red Blood Cell
RF = Rheumatoid Factor
ROS = Reactive Oxygen Species
RT = Retention Time
TCIA = Type II Collagen Induced Arthritis TIMP = Tissue Inhibitors of Metalloproteinase TNF-α = Tumour Necrosis Factor-alpha WBC = White Blood Cell
ETHICS APPROVAL AND CONSENT TO PARTICI- PATE
Ethics approval has been obtained from Universiti Ke- bangsaan Malaysia Animal Ethics Committee (No.
PPI.1/138/1).
HUMAN AND ANIMAL RIGHTS
The male Sprague Dawley rats used in this study were procured from the Faculty of Science and Technology, Uni- versiti Kebangsaan Malaysia (UKM). All animal studies have been conducted in accordance to the guidelines of US for the Care and Use of Laboratory Animals https://grants.nih.gov/grants/olaw/Guide-for-the-Care-and- Use-of-Laboratory-Animals.pdf.
CONSENT FOR PUBLICATION Not applicable.
CONFLICT OF INTEREST
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
ACKNOWLEDGEMENTS
The authors greatly thank the Ministry of Agriculture and Agro-based Industries, Malaysia, for providing the grant under the NKEA Research Grant Scheme (NRGS) (no. NF- 2015-005).
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