Boswellia serrata for Rheumatoid Arthritis

Boswellia Serrata Extract in Arthritis

• Rheumatoid arthritis (RA) is a chronic inflammatory condition leading to joint destruction.
• Current treatments for RA provide symptomatic relief (NSAIDs) or modify the disease (DMARDs), but their effectiveness is limited by side effects.
• Boswellia serrata gum resin extract (BSE) was evaluated for its antioxidant and antiarthritic activity in collagen-induced arthritis.

Experiment and Methods

• Arthritis induced in male Wistar rats using the collagen-induced arthritis (CIA) method.
• BSE administered at 100 and 200 mg/kg body weight once daily for 21 days.
• Effects of treatment assessed via biochemical markers (articular elastase, MPO, LPO, GSH, catalase, SOD, NO).
• Inflammatory mediators (IL-1β, IL-6, TNF-α, IL-10, IFN-γ, PGE2) measured to determine the effect.
• Histological studies of joints were conducted to observe the effect.

Effects of BSE Treatment

• BSE caused significant changes in articular elastase, MPO, LPO, GSH, catalase, SOD, and NO levels.
• Oral administration of BSE significantly reduced inflammatory mediators IL-1β, IL-6, TNF-α, IFN-γ, PGE2) and increased IL-10.
• BSE's protective effects decreased arthritis scoring and improved bone histology.

Conclusion

• Protective effect might be mediated through modulation of the immune system.
• The extract inhibits proinflammatory cytokines and modulates antioxidant status

Introduction to Inflammation and Arthritis

• Inflammation is the initial immune-response to body infection or irritation when infected or irritated.
• Chronic inflammation can contribute to cancers, neurodegenerative disorders, and RA.
• RA destroys cartilage and bone within joints via inflammatory cells migrating to synovial and periarticular tissue.
• Imbalance between pro- and anti-inflammatory cytokines determines the degree of inflammation, resulting in cellular damage.
• Reactive oxygen species (ROS) and reactive nitrogen species are key modulators in RA.

Current RA Treatments

• Current RA treatments either relieve symptoms (NSAIDs) or modify the disease process.
• NSAID usage is limited due to side effects like gastrointestinal ulcers and opportunistic infections or cardiovascular complications.
• 100,000 hospitalizations and 16,500 deaths per year are linked to NSAID-induced ulcers and gastrointestinal bleeding in arthritis patients in US.
• Alternative anti-inflammatory remedies are re-emerging due to tolerability issues.
• Gum resin extracts of Boswellia serrata (BSE) have been an anti-inflammatory medicine in Ayurvedic medicine in India for centuries.
• BSE may treat inflammatory disorders like inflammatory bowel disease, rheumatoid arthritis and osteoarthritis based on recent studies.
• Acetone extract of Boswellia carterii gum resin decreased paw oedema, decreased arthritic scores, and suppressed local tissue TNF-a and IL-1β in Lewis rats.
• BSE is expected to have better tolerability than NSAIDs and lacks the adverse effects of corticosteroids.
• Study investigated the effect of Boswellia serrata gum resin extract (BSE) against collagen induced arthritis in Wistar rats.

Materials and Methods

• Freund's adjuvant complete (CFA) and other chemicals acquired for study.
• Boswellia serrata extract (BSE) was obtained from Herbosin CORPS, Meerut, U.P., India.
• Male Wistar rats (150-170g) randomly assigned to four groups of six animals each.
• Rats housed at 25±2°C and 45-55% humidity with 12 h light/dark cycles.
• Rats had free access to standard rodent pellet diet and water.
• Experimental study was conducted in accordance with the Institutional Animal Ethics Committee.

HPLC Analysis

• Ethanolic extract of Boswellic acid (BSE) isolated from gum resin separated on a C18 reverse phase column.
• Mobile phase consisted of Acetonitrile and 0.05% acetic acid in water (90:10 v/v gradient elution for 45 min).
• Analysis performed at 254 for KBBA, AKBBA and BBA, ABBA at 210 nm using 10mL of injection volume.
• BBA (beta bowswellic acid).
• ABBA (acetyle beta bowswellic acid).
• KBBA (keto beta bowswellic acid).
• AKBBA (acetyle keto beta bowswellic acid).

UPLC-MS/MS ESI-Q-TOF Conditions

• UHPLC was performed with a Waters ACQUITY UPLCTM system.
• UHPLC mobile phase consisted of methanol-water-glacial acetic acid (8:1:0.4, v/v/v), degassed.
• Q-TOF PremierTM was operated in V mode with resolution over 32,000 mass.
• Quantitation was performed using Synapt Mass Spectrometery (Synapt MS) with a scan time of 1.0 min.
• Accurate mass and composition for the precursor ions and for the fragment ions were calculated using the MassLynx V 4.1 software.

Drug Administration

• Ethanolic extract of Boswellic acid (BSE) isolated from gum resin of Boswellia serrata Roxb.
• Drugs prepared as a fine homogenized suspension in 2% gum acacia (w/v) for oral administration.

Induction of Collagen-Induced Arthritis (CIA)

• Collagen Type II was dissolved in 0.05 M acetic acid at a concentration of 2 mg/ml, emulsified with Freund's adjuvant complete (CFA).
• Rats immunized intradermally at about 1.5 cm distal from the base of tail.
• Group 1: Control (C).
• Group 2: Collagen-induced arthritis (CIA).
• Group 3: CIA + 100 mg/kg body weight Boswellia serrata extract (CIA + BSE100) daily.
• Group 4: CIA + 200 mg/kg body weight Boswellia serrata extract (CIA + BSE200) for 21 days starting from day 0 following by CIA.

Clinical Severity of Arthritis Measurement

• Thickness of hind paws measured with digital calliper (YAMAYO, Japan).
• Arthritis severity quantified daily using a clinical score from 0 to 4.
  • 0: no macroscopic signs of arthritis.
  • 1: swelling of one group of joints (wrist or ankle).
  • 2: two groups of swollen joints.
  • 3: three groups of swollen joints.
  • 4: swelling of the entire paw.
• At the end of experiment, animals were sacrificed by cervical dislocation.
• Arthritic and nonarthritic joints were removed and cut into small pieces.
• Homogenized in 5 vol. of 50 mM Tris-HCl buffer, pH 7.4 containing 0.1 M NaCl and 0.1% Triton X-100 and 1 vol. of fine glass powder.
• Crude extract was sonicated for 20 s and centrifuged to estimate TBARS and GSH.
• Resultant PMS was used to carry out elastase, MPO, catalase, SOD and NO assay.
• Resulting supernatant stored at -80°C until further analysis.

Articular Elastase (ELA) Protocol

• ELA levels in articular joints were evaluated as an index of polymorphonuclear leucocyte (PMNs) accumulation and activation.
• Tissue samples were first diluted and homogenized in a solution containing 20 mM potassium phosphate buffer pH 7.0 in a ratio of 1:10 (w/v).
• Samples were incubated for 24h at 37 °C with 0.1 M Tris-HCl buffer (pH 8.0), containing 0.5 M NaCl and 1 mM N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide.
• Amount of p-nitroanilide liberated was measured spectrophotometrically at 405 nm.

Myeloperoxidase (MPO) Assay

• Myeloperoxidase activity was analyzed as an index of neutrophils infiltration in the synovial tissue.
• Assay was carried out and expressed as U/g of protein.

Estimation of Thiobarbituric Acid Reactive Substances (TBARS) Protocol

• Tissue homogenate prepared in 0.15 M KCI (5%, w/v homogenate).
• Samples incubated for 0°C and 37°C for 1 h and incubated with subsequent reagents.
• Supernatant was taken and colour was developed by addition of TBA dissolved in NaOH.
• Absorbance read at 532 nm in a plate reader (Bio-Rad, USA).

Reduced Glutathione (GSH) Protocol

• Homogenized joint tissue (10%, w/v in phosphate buffer pH 7.4) was deproteinized.
• Supernatant was added to Tris buffer containing EDTA followed by the addition of DTNB.
• Measured in the groups following the method described earlier.
• Absorbance read in a microplate reader at 412 nm.

Total Superoxide Dismutases (SOD) Activity

• Measured in joints and adapted to microtiter plates.
• Reaction mixture consisted of phosphate buffer, xanthine and NBT.
• Incubation at room temperature for 15 min and then initiated by addition of xanthine oxidase mixture.
• SOD activity expressed in Units/mg protein.

Catalase Activity Protocol

• Catalase activity in the joint tissues was assayed using H2O2 as substrate.
• Reaction was adjusted to multiwell flat bottom plates by reducing the final volume.
• Reaction mixture included phosphate buffer, distilled water and 10% homogenate.
• Reaction started by adding H2O2, incubated at 37°C for 1 min, and stopped by addition of dichromate:acetic acid reagent.
• Tubes were kept in a boiling water bath, centrifuged, and read at 570 nm in a microplate reader.
• Enzyme activity was expressed as µmol H2O2 consumed/min/mg protein.

Nitric Oxide (NO) Measurement Protocol

• Joint tissues were washed with PBS (pH 7.4) and placed on ice.
• Sample added with Griess reagent and incubated at room temperature.
• Optical density measured at 540 nm in microplate reader.

Cytokine and PGE2 Level Protocol

• Cytokines: IL-1B, TNF-a, IFN-y, IL-6, IL-10.
• Tissues homogenized in a pH 7.6 buffer with Tris-HCl, KCI, NaCl, EDTA, EGTA, dithiothreitol and PMSF.
• Supernatants removed and assayed in duplicate using commercially available cytokine ELISA kits.
• Tissue cytokine concentrations expressed as pg/ml of protein.

Histological Examinations

• Rats were sacrificed, and knee joints were removed and fixed in formaldehyde.
• Samples were processed for paraffin embedding after decalcification in formic acid.
• Tissue sections were stained with haematoxylin-eosin for examination.

HPLC Analysis Results of Boswellia Serrata

• Ethanolic extract of Boswellia serrata showed KBBA, AKBBA at 254 nm and BBA, ABBA at 210 nm.
• Obtained using LiChroCART® C18 column with acetonitrile: 0.05% acetic acid in gradient elution mode.

UPLC/ESI-Q-TOF-MS/MS Results and Analysis

• MS identification via m/z values revealed boswellic acids in the extracts.
• Negative SIM mode identified acids with [M-H] ions.
• Results affirmed by Krüger et al. (2008) data.
• HPTLC validated extract composition data.

Clinical Severity of Disease Post Treatment

• Arthritis developed rapidly post collagen emulsification.
• Reduced by BSE treatment in collagen.

Effects on Elastase and Myeloperoxidase Activity

• Articular elastase and myeloperoxidase were assayed on day 21.
• Reduced infiltration through neutrophil activation
• Low levels in untreated group
• High levels in CIA group

Lowered TBARS Levels

• CIA TBARS levels significantly higher than control, indicates increased oxidative damage.
• BSE treatment decreased TBARS levels dose-dependently and lowers lipid peroxidation.

Recovery of GSH and SOD Levels

• BSE significantly restored GSH levels and SOD activity.
• The joints of CIA caused a marked decrease in the GSH and SOD levels.
• GSH and SOD are endogenous protection against hydrogen peroxide and SOD anions
• Indicates boosted antioxidant defence system

Effect of Boswellia serrata on Catalase Activity

• Significantly decreased in the joints in CIA group compared to control.
• Effectively treated with BSE, increased at both doses.

Impact on Nitric Oxide Levels

• Treatment with BSE declined the increase in the nitrite levels, more significant with higher doses.
• Significant increase observed in CIA group compared to control

Effect of BSE on Cytokine Levels

• Modulated by BSE through anti-inflammatory response.

Histological Results

• Massive cell infiltration observed in CIA group, decreased with BOSWELLIA SERRATA treatment.
• Shown through protective and mediatory effects and suppression of oxidization

Bone Observations

• Showed improved bone resorption
• Treatment was able to restore at a greater extent at higher doses.

Discussion on Anti-arthritic Activity

• BSE in collagen induced arthritis indicates reduction in free radical levels.
• Evaluation of myeloperoxidase activity to indicate proportional accumulation in tissue.
• Study suggests that BSE modulates inflammatory mediators.
• Boswellic acids in BSA reported as inhibitors of elastase

Boswellia Serrata Extract Benefits

• Shown through previous potential antioxidant
• Results indicate through these previous reports of experimental animals of significant increase in lipid peroxides and depletion of GSH and SOD levels.

This study investigates Boswellia serrata extract (BSE) as a potential anti-inflammatory treatment for rheumatoid arthritis (RA). It examines BSE's effects on reducing inflammation and oxidative stress, and neutrophil activity using a collagen-induced arthritis rat model. Various dosages were tested over a specific period to assess efficacy.