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In This Article
 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 » Discussion
 » Conclusion
 »  References
 »  Article Figures

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 Table of Contents    
RESEARCH ARTICLE
Year : 2016  |  Volume : 48  |  Issue : 2  |  Page : 155-161
 

Anti-inflammatory and anti-granuloma activity of Berberis aristata DC. in experimental models of inflammation


Department of Pharmacology, All Institute of Medical Sciences, New Delhi, India

Date of Submission24-Sep-2015
Date of Decision23-Dec-2015
Date of Acceptance17-Feb-2016
Date of Web Publication17-Mar-2016

Correspondence Address:
Surender Singh
Department of Pharmacology, All India Institute of Medical Sciences, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0253-7613.178831

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 » Abstract 

Objective: Berberis aristata (Berberidaceae) is an important medicinal plant used in traditional system of medicine for the treatment of rheumatoid arthritis and other inflammatory disorders. The aim of the present study is to scientifically validate the traditional use of BA in the treatment of inflammatory disorders.
Materials and Methods: Anti-inflammatory and anti-granuloma activity of BA hydroalcoholic extract (BAHE) were evaluated in experimental models, viz., carrageenan-induced paw edema, cotton pellet-induced granuloma formation, and complete Freund's adjuvant-induced stimulation of peritoneal macrophages in rats. Expression of inflammatory mediators, viz., tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, TNF-R1, and cyclooxygenase-2 (COX-2) was carried out in serum and peritoneal macrophages to derive the plausible mechanism of BAHE in activated peritoneal macrophages.
Results: Pretreatment with BAHE produced a dose-dependent reduction (P < 0.01) in carrageenan-induced paw edema and cotton pellet-induced granuloma model. BAHE treatment produced significant (P < 0.01) reduction in serum inflammatory cytokine levels as compared to control. Protein expression of pro-inflammatory markers, IL-1β, IL-6, TNF-R1, and COX-2, was found to be reduced in stimulated macrophages whereas anti-inflammatory cytokine, IL-10, was upregulated in peritoneal macrophages.
Conclusion: The result of the present study thus demonstrates the anti-inflammatory and anti-granuloma activity of BAHE which may be attributed to its inhibitory activity on macrophage-derived cytokine and mediators.


Keywords: Complete Freund's adjuvant, inflammation, macrophage, pro-inflammatory cytokines


How to cite this article:
Kumar R, Gupta YK, Singh S. Anti-inflammatory and anti-granuloma activity of Berberis aristata DC. in experimental models of inflammation. Indian J Pharmacol 2016;48:155-61

How to cite this URL:
Kumar R, Gupta YK, Singh S. Anti-inflammatory and anti-granuloma activity of Berberis aristata DC. in experimental models of inflammation. Indian J Pharmacol [serial online] 2016 [cited 2018 Apr 20];48:155-61. Available from: http://www.ijp-online.com/text.asp?2016/48/2/155/178831



 » Introduction Top


Inflammation is a highly regulated protective response which helps in eliminating the initial cause of cell injury and initiates the process of repair.[1] However, in chronic inflammatory disorders and granulomatous condition, there is an excessive inflammatory response characterized by the presence of activated and dysregulated macrophages which release multitude of mediators that play an important role in both maintenance and progression of the disease.[2] Rheumatoid arthritis (RA) is one of the chronic inflammatory disorders where macrophage activation and dysfunction are known to be central players in disease process, which is evident by the elevated levels of predominantly macrophage-derived pro-inflammatory cytokines, viz., tumor necrosis factor-α (TNF-α), interleukin-1 (IL-6), IL-1β, and cyclooxygenase-2 (COX-2), in inflamed synovial joint.[3] Current treatment regimens used in the treatment of RA start from symptomatic pain relief by nonsteroidal anti-inflammatory drugs to more potent biologics for inhibiting disease progression.[4] Most of these treatments have severe side effects such as gastrointestinal bleeding, ulcers, and opportunistic infections due to immunosuppression. Owing to chronic nature of disease and high side effect profile of current treatment agents, patients with musculoskeletal disorders are high users of complementary and alternative medicine.[5] Therefore, there is continuous research in exploration of natural agents which have high therapeutic value and less associated side effects.

Berberis aristata DC. (Berberidaceae) is an important medicinal plant used traditionally as an antimicrobial, antibacterial, antipyretic, immunostimulant, laxative, antihemorrhagic, and anti-inflammatory agent.[6] Topical instillation of aqueous extracts of BA resulted in significant reduction in inflammation and TNF-α level in endotoxin-induced uveitis and turpentine liniment-induced ocular inflammation in rabbits.[7],[8] Despite its widespread use in traditional medicine for treatment of various inflammatory disorders, only a few studies have evaluated the efficacy of this medicinal plant. Therefore, the present study was carried out to evaluate anti-inflammatory and anti-granuloma activity of this medicinal plant with special emphasis on ascertaining the immunomodulatory activity of BA hydroalcoholic extract (BAHE) on inflammatory markers in activated macrophages.


 » Materials and Methods Top


Animal

Adult male Wistar albino rats weighing 150–180 g from our institutional breeding stock were used in the study. They were housed at 25°C ± 2°C in clean polypropylene cages in batches of three with access to food and water ad libitum . The experimental protocol was approved by the Institutional Animal Ethics Committee, with animal ethics approval number 712/IAEC/13. All experiments were carried out in accordance with “Guidelines for care and use of animals in scientific research (Indian National Science Academy 1998, Revised 2000).”

Plant Material

BAHE of bark part has been provided by Natural remedies Pvt. Ltd., Bengaluru, Karnataka, India, bearing Batch No.: RD/2985. The total berberine content of BAHE was determined by high-performance liquid chromatography (HPLC). The total berberine content present in BAHE was found to be 6.7% w/w of dry extract [HPLC method and peak is presented in Supplementary Material and [Figure S1 [Additional file 1]].

Carrageenan Administration-induced Paw Edema in Rats

Overnight fasted animals were divided into five groups (n = 6). Dose of the test drug was selected on the basis of a pilot study carried out in our laboratory. Group I received vehicle (1% gum acacia per orally) and served as the control; Group II received indomethacin (3 mg/kg per orally); and Groups III, IV, and V received BAHE at a dose of 50, 100, and 200 mg/kg, respectively per orally. Sixty minutes after administration of the drug/vehicle, paw edema was induced by subplantar administration of 0.1 ml of 1% gamma-carrageenan (constituted in normal saline) into the left hind paw of the animal.[9] Paw edema was measured using a digital plethysmometer (Ugo Basile, Italy) at 1, 3, and 5 h postcarrageenan administration.

Cotton Pellet Implantation-induced Granuloma Formation

Five groups of male Wistar albino rats (n = 6) were used in the study. Grouping of animals and drug treatment was similar to that described under “carrageenan administration-induced paw edema.” Thirty minutes after administration of drug/vehicle, the animals were anesthetized with diethyl ether and a sterile cotton pellet (made of bleached cotton) weighing (30 ± 1 mg) saturated with normal saline was implanted subcutaneously bilaterally below the axilla. Animals were kept under aseptic conditions for the entire duration of the study. Drug/vehicle treatment was continued for the duration of six more days. On day 8, terminal blood collection was carried out and the cotton pellets were excised. Serum was separated by centrifugation at 3000 rpm and used for dot-blot study. The pellets were dried overnight at 60°C until a constant weight was recorded at two consecutive recordings. The difference between the initial and postimplantation weight was considered to be the dry weight of the granuloma tissue.[10]

Dot-blot Analysis for Estimation of Serum Tumor Necrosis Factor-alpha Level

Three microliters of serum from each sample was diluted with 7 μl of 10 mM phosphate buffer saline (pH = 7.2) and spotted onto nitrocellulose membrane and dried at room temperature. Nonspecific sites were blocked with blocking buffer (5% Blot-Quick blocking power [Genotech] in 10 mM phosphate buffered saline [PBS] containing 0.05% Tween-20) for 2 h at room temperature on a shaker. The membrane was washed thrice with washing buffer (10 mM PBS–0.1% Tween-20) for 10 min each. The membrane was then incubated with anti-TNF-α polyclonal primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, USA: Dilution of 1:500) overnight at 4°C. The wash was repeated and corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Bangalore Genei, Karnataka, India) was added at a dilution of 1:4000 and kept for 2 h at room temperature on a shaker. This was followed by three washes with washing buffer. The blots were then developed with nickel-enhanced diaminobenzidine substrate kit. Protein expression was evaluated by determining the intensity of darkness of dots by densitometer using Alpha-View imaging software (Alpha Imager EC Gel Doc System, CA, USA) as described by us earlier.[11]

Complete Freund's Adjuvant-induced Stimulation of Peritoneal Macrophages

Three groups (n = 6) of male Wistar albino rats were used in the study. After overnight fasting, Group I received vehicle (2% gum acacia) and served as the control, Group II received indomethacin (3 mg/kg), and Group III received BAHE (200 mg/kg) by gavage. Thirty minutes after administration of drug/vehicle, 0.1 mL of complete Freund's adjuvant (CFA) (0.05% w/v dead Mycobacterium butyricum in mineral oil) was injected into the peritoneal cavity using a 26-gauge needle.[12] Drug/vehicle treatment was continued for the duration of six more days.

Isolation and Culture of Rat Peritoneal Macrophages

On day 8, animals were sacrificed by an overdose of anesthetic ether and sterile RPMI 1640 media (10 mL) was injected into the peritoneal cavity by using a 21-gauge needle. After gently massaging the abdomen for 5 min, the injected media was aspirated under sterile conditions and transferred into a 15 mL centrifuge tube. The cellular content was pelleted out by centrifuging at 1000 g for 10 min at room temperature. The pellet was resuspended and cultured aseptically in 60 mm culture dishes at a cell number of approximately 106 cells/mL in RPMI 1640 medium with L-glutamine (GibcoBRL), containing NaHCO3 (2 g/L), 10% fetal calf serum (v/v), penicillin (100 U/mL), streptomycin (100 mcg/mL), and amphotericin B (250 ng/mL). The cells were maintained at 37°C in an atmosphere of 95% air and 5% CO2 for 2 h. The supernatant was then discarded and the adherent macrophages were washed twice with ice-cold 10 mM PBS, scraped off, and used for preparation of lysate for Western blot analysis.

Preparation of Cell Lysate

The macrophage pellets were lysed by strong vortexing in 100 μL of lysis buffer containing 50 mM Tris–HCl (pH = 7.4), 300 mM NaCl, 0.5% (v/v) Triton X-100, 5 mM ethylenediaminetetraacetic acid with 2 mM phenylmethylsulfonyl fluoride, 10 mcg/mL leupeptin, and 10 U/mL aprotinin. The lysed suspension was kept on ice for 30 min and then centrifuged at 10,000 rpm for 15 min at 4°C. The supernatant was collected and protein content was determined by using the Bradford method,[13] with BSA as the standard.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blotting

Seventy-five micrograms of protein was separated in 12% polyacrylamide gels (1.0 mm thick), overlaid with a 4% stacking gel in sodium dodecyl sulfate according to the standard procedure of Laemmli.[14] The proteins were transferred onto nitrocellulose membrane [15] under an electric potential. The membrane was incubated with blocking buffer (5% Blot-Quick blocking power [Genotech] in 10 mM PBS containing 0.05% Tween-20) for 2 h at room temperature on a shaker. The membrane was then washed thrice in 10 mM PBS–0.1% Tween-20 for 10 min each. Anti-TNF-R1, IL-1β, IL-6, IL-10, COX-2, or anti-β-actin polyclonal primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) was added at a dilution of 1:500 and the membrane was incubated overnight at 4°C. The wash was repeated and corresponding HRP-conjugated polyclonal secondary antibody (Merck Genei, Bangalore, India) was added at 1:3000 dilution and kept for 2 h at room temperature on a shaker. Following three washes with PBS–Tween buffer, the blots were developed with femtoLUCENT detection kit (Santa Cruz, CA, USA). Protein expression was evaluated by determining the intensity of darkness of protein bands by a densitometer (Alpha Imager EC Gel Doc System, CA, USA) using Alpha-View imaging software. Protein concentration of TNF-R1, IL-1β, IL-6, IL-10, and COX-2 was expressed as percentage integrated density value (%IDV) after normalization against β-actin.[11]

Statistical Analysis

All data were expressed as mean ± standard error. Difference between groups was analyzed by One-way ANOVA followed by Dunnett's multiple comparison(GraphPad in Stat; Version 3.05, (GraphPad Software, Inc., USA)). P < 0.05 was considered statistically significant.


 » Results Top


Effect of Berberis aristata Hydroalcoholic Extract on Paw Edema in Carrageenan-induced Paw Edema

Paw edema produced by subplantar administration of carrageenan was found to be persistent in all tested group throughout the observation period (1, 3, and 5 h) [Figure 1]. The standard drug indomethacin produced significant reduction in paw edema. Treatment with BAHE produced a significant reduction (P < 0.01) in paw volume throughout the observation period as compared to the control group. Maximum reduction in paw edema was observed in BAHE (200 mg/kg) group throughout the observation period.
Figure 1: Effect of Berberis aristata hydroalcoholic extract treatment in carrageenan induced paw edema in rats. Difference between initial paw volume and paw volume at observation points postcarrageenan administration was considered to be the increase in paw volume (depictive of paw edema). Each bar represents the mean ± standard error of the mean of six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *P < 0.05; **P < 0.01

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Effect of Berberis aristata Hydroalcoholic Extract on Granuloma Formation in Cotton Pellet Implantation-induced Granuloma

Subcutaneous implantation of sterile cotton pellet results in formation of granuloma tissue around the cotton pellet in all the experimental groups [Figure 2]. Treatment with BAHE produced a dose-dependent reduction in granuloma formation as seen by decrease in dry granuloma weight with increasing doses. Standard drug, indomethacin, treatment also significantly reduced granuloma formation. BAHE (200 mg/kg) demonstrated anti-granuloma activity similar to indomethacin treatment group.
Figure 2: Effect of Berberis aristata hydroalcoholic extract treatment on dry granuloma weight after subcutaneous cotton pellet implantation. Difference between initial weight (30 ± 1 mg) and weight after overnight drying was considered to be the dry weight of the granuloma tissue. Each bar represents the mean ± standard error of the mean of six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *P < 0.05; **P < 0.01

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Effect of Berberis aristata Hydroalcoholic Extract on Serum Cytokine Levels in Cotton Pellet Implantation-induced Granuloma

Immunoblot analysis was carried out to estimate serum levels of TNF-α, IL-1β, IL-6, and IL-10 [Figure 3]. Treatment with BAHE (200 mg/kg) demonstrated significant (P < 0.01) decrease in TNF-α, IL-1β, IL-6, and increase IL-10 level in serum as compared to the control. Indomethacin treatment significantly decreased IL-1β and IL-6 level as compared to control. However, Increased serum TNF-α level was observed in indomethacin treatment group as compared to control.
Figure 3: Effect of Berberis aristata hydroalcoholic extract treatment on serum tumor necrosis factor-alpha, interleukin-1β, interleukin-6, and interleukin-10 level after subcutaneous cotton pellet implantation. Each bar represents the mean ± standard error of the mean of six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *P < 0.05; **P < 0.01

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Effect of Berberis aristata Hydroalcoholic Extract on Inflammatory Mediators in Complete Freund's Adjuvant-induced Stimulation of Peritoneal Macrophages

Initially, the %IDV was calculated separately for IL-1β, IL-6, TNF-R1, COX-2, IL-10, and β-actin. Thereafter, normalization of individual protein expression was carried out by calculating the amount of protein expressed per unit of β-actin ([%IDV−protein/%IDV−β-actin] ×100). Comparison of normalized data demonstrated that treatment with BAHE (200 mg/kg) produced a significant reduction in macrophage expression of pro-inflammatory cytokine, IL-1β, IL-6, protein, and upregulated anti-inflammatory cytokine IL-10 as compared to the control [Figure 4]. BAHE (200 mg/kg) and indomethacin decreased protein expression of pro-inflammatory receptor, TNF-R1 as compared to control [Figure 5]. In addition, BAHE treatment also decreased expression of pro-inflammatory mediator COX-2 [Figure 6] as compared to control.
Figure 4: Effect of Berberis aristata hydroalcoholic extract treatment on interleukin-1β, interleukin-6, and interleukin-10 expression on complete Freund's adjuvant stimulated rat peritoneal macrophages. Each bar represents the mean ± standard error of the mean of six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *P < 0.05; **P < 0.01

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Figure 5: Effect of Berberis aristata hydroalcoholic extract treatment on tumor necrosis factor-R1 expression on complete Freund's adjuvant stimulated rat peritoneal macrophages. Each bar represents the mean ± standard error of the mean of six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *P < 0.05; **P < 0.01

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Figure 6: Effect of Berberis aristata hydroalcoholic extract treatment on cyclooxygenase-2 expression on complete Freund's adjuvant stimulated rat peritoneal macrophages. Each bar represents the mean ± standard error of the mean of six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *P < 0.05; **P < 0.01

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 » Discussion Top


Berberis aristata DC. is an erect, spinous, deciduous shrub widely distributed in the Himalayas and commonly known as “Indian Berberry” in English and “Daruhaldi” in Hindi. BA bark is used in various formulations intended for the treatment of osteoporosis, joint pain, fever, jaundice, hepatitis, and eye infections by traditional medicine practitioners.[6] In preclinical studies, aqueous extract of BA had demonstrated significant anti-inflammatory activity against endotoxin-induced uveitis in rabbits.[7] The principal chemical constituents present in the plant are alkaloids, i.e., berberine, oxyberberine, and oxycanthine.[6] Out of these phytoconstituents, only berberine is explored in various experimental models as therapeutic intervention. Recently, Wang et al . in 2014 demonstrated the antiarthritic and antiangiogenic activity of berberine in collagen-induced arthritis model and showed that the effect was due to inhibition of the phosphorylation of mitogen-activated protein kinase.[16] In the current study, we observed the anti-inflammatory and anti-granuloma potential in carrageenan-induced paw edema model, cotton pellet-induced granuloma model, and adjuvant-induced stimulation of peritoneal macrophages. The present study results showed that treatment with BA effectively inhibited the inflammation, granuloma formation, and serum TNF-α level and exhibited immunomodulatory activity on macrophage-derived mediators IL-1β, IL-6, IL-10, TNF-R1, and COX-2. Thus, our finding suggests that BA could be used as a potential agent in treatment of chronic inflammatory disorders.

Carrageenan-induced paw edema model is an experimental model, which have time-dependent, triphasic response and used in evaluating the activity of potential anti-inflammatory agent in autacoid system. The first phase (1–2 h after carrageenan administration) primarily attributed to histamine, the second phase (2–3 h after carrageenan administration) being attributed to serotonin and kinins, and the third phase (3 h onward) primarily attributed to prostaglandins.[17] In the present study, treatment with BAHE demonstrated anti-inflammatory activity at all observation points indicating inhibitory activity against multiple autacoid mediators. In cotton pellet-induced granuloma model, sterile cotton pellet was implanted at axial region of animal and increase in granuloma formation at the site of implantation was evaluated by measuring increase in dry weight of the implanted cotton pellet. Efficacy of drug in this model clearly highlights inhibitory activity on macrophage activation, infiltration, and aggregation and is considered a reliable in vivo model for studying chronic inflammatory condition present in RA patient synovium.[18],[19] In the current study, pretreatment with BAHE showed a dose-dependent reduction in granuloma formation, and efficacy was found to be comparable with standard drug indomethacin. BAHE treatment also inhibited the serum pro-inflammatory cytokine, TNF-α, IL-1β, IL-6 level and upregulated the anti-inflammatory cytokine, IL-10 level, as compared to control. Besides the anti-inflammatory activity, BAHE has shown immunomodulatory activity by inhibiting activated macrophages responsible for granuloma formation. To further validate the macrophage inhibitory activity of BAHE, expression of macrophage-derived cytokines, receptors, and mediators was studied directly on activated peritoneal macrophages in adjuvant-induced stimulation of peritoneal macrophages model.

Macrophages are the cells of the innate immunity system which possess broad pro-inflammatory, destructive, and remodeling capacities and considerably contribute in inducing inflammation and tissue destruction in chronic inflammatory disorders.[20] Various studies have already shown that there is a presence of abundant macrophages in the inflamed synovial membrane and at the cartilage-pannus junction in both experimental arthritis and RA patients, which secrete various classes of mediators (TNF-α, IL-1β, IL-6, IL-10, and COX-2), which play a pivotal role in disease progression and joint destruction.[21],[22] The importance of macrophage-derived proinflammatory cytokines, viz., TNF-α, IL-1β, and IL-6 in disease progression can be further evident by the success of biologics that targets these proinflammatory cytokines and helps in attenuating disease progression and joint destruction in RA.[23] IL-1β is a crucial mediator which promotes inflammation and joint destruction in the RA by influencing proteoglycan synthesis and degradation and inducing the production of matrix metalloproteinase-1 (MMP-1) and MMP-3, which enhances bone resorption. IL-6 plays an important role in various chronic inflammatory disorders and elevated levels of IL-6 were found in early acute inflammatory disease state in RA.[24] Macrophage also produces anti-inflammatory cytokine, IL-10, which regulates inflammatory state by reducing human leukocyte antigen-DR expression and antigen presentation, thus inhibits the production of pro-inflammatory cytokines, granulocyte-macrophage colony stimulating factor in dysregulated macrophages.[24],[25] In the present study, BAHE treatment attenuated the protein expression of both pro-inflammatory cytokine IL-1β, IL-6 and increased the expression of anti-inflammatory cytokine IL-10 as compared to control animals.

Pro-inflammatory cytokine, TNF-α plays a critical role in inflammatory disease cascade and inhibition of this cytokine plays an important role in attenuating disease progression. There are two different types of TNF receptors, i.e. TNF-R1 and TNF-R2. TNF-R1 has been implicated in most of the pathological actions of TNF-α. Therefore, inhibition of TNF-R1 can help in the attenuating disease progression.[22],[26] In the present study, treatment with BAHE and indomethacin decreased expression of TNF-R1 receptor in stimulated macrophages. Therefore, BAHE has dual mechanism on TNF-α by downregulating TNF-R1 receptor and inhibiting TNF-α protein.

RA is associated with high levels of prostaglandins which are synthesized by COX enzyme. In human, there are two subtypes of COX, constitutive COX-1 and inducible COX-2.[27] Previous studies had already shown that inducible COX-2 has been upregulated in vitro by various pro-inflammatory agents, such as lipopolysaccharide, IL-1, and TNF-α, and expression of COX-2 was detected in synovial tissue and infiltrating cells of human and rat arthritic joint.[28] Activated macrophages also express COX-2 on its surface that results in synthesis of increased levels of prostaglandins.[29] Therefore, inhibiting COX-2 can help in decreasing inflammation and attenuating joint destruction. In the present study, treatment with BAHE resulted in decreased expression of COX-2 in peritoneal macrophages, thus supporting the anti-inflammatory potential of BAHE. The results of present study thus demonstrate the anti-inflammatory and anti-granuloma activity of BAHE, which could have been primarily due to the presence of berberine and other berberine derivatives in the drug. However, due to the presence of multiple phytoconstituents in BAHE, efficacy cannot be solely attributed to berberine at this moment. Studies evaluating individual fractions of BAHE in similar experimental models are required to identify all active principles that could have contributed to this macrophage inhibitory activity.


 » Conclusion Top


Our findings suggest that BAHE has anti-inflammatory and anti-granuloma effects which are associated with inhibition of pro-inflammatory cytokines/receptor and mediators in activated macrophages. As chronic inflammation and activated macrophages are the central players in the pathogenesis of RA and other inflammatory disease, our findings support and validate the use of BAHE in treatment of chronic inflammatory disorders in traditional system of medicine.

Acknowledgments

We are thankful to Natural Remedies Pvt. Ltd., Bengaluru, Karnataka, India, for providing a generous gift of BA standardized extract and its HPLC standardization part.

Financial Support and Sponsorship

Nil.

Conflicts of Interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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