Combination of Zingiber officinale and Brassica oleraceae extracts shows better Anti-arthritic activity

Dr. Vaishali. R. Undale¹, Asawari S. Thakare², Pooja V. Katkar²

¹HOD, Pharmacology, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-411018.

²Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-411018.

*Corresponding Author E-mail: vaishali.undale@dypvp.edu.in, devya1317@gmail.com

ABSTRACT:

Rheumatoid arthritis (RA) is a chronic inﬂammatory joint disease characterised by synovial inflammation, auto-antibody production, cartilage and bone deformity, anti-citrullinated protein antibody and hyperplasia and can cause cartilage and bone damage as well as disability. worldwide about 1% of adult population is affected by rheumatoid arthritis. It is the one of the most leading cause of disability with no ultimate cure. Many of the currently available antiarthritic drugs serve to manage only the symptomatic relief and are associated with adverse effects. Number of herbs have proven for potential antiarthritic activity in preclinical and clinical studies conducted. Zingiber officinale and Braccica oleraceae are two drugs that act on different pathophysiological mechanisms of arthritis. Hence the aim of this study was to evaluate the synergistic potential of the combination in rheumatoid arthritis. Complete Freunds Adjuvant (CFA) 0.1ml was used as an inducer and both the drugs were evaluated individually and in 1:1 combination at (50,100, 200mg/kg) doses. Parameters like paw oedema, percentage inhibition of paw oedema and serum SGOT and SGPT were measured in order to evaluate the ability of drugs in improving situation of Rheumatoid arthritis. The results indicated dose dependent antiarthritic action of both the individual drugs as well as the combination. Also, the antiarthritic action of combinations was found to be significantly greater than that of individual drugs thereby justifying the synergistic effect of the two drugs.

KEYWORDS: Arthritis, Synovial inflammation, Anti-citrullinated protein antibody, Zingiber officinal, Brassica oleraceae, AST, ALT, Complete Freund’s Adjunvant (CFA).

INTRODUCTION:

Rheumatoid arthritis (RA) is an autoimmune systemic disease with chronic inflammation of the synovial joint and progressive destruction of cartilage and bone. Rheumatoid arthritis is a chronic systemic inflammatory disorder that may affect many tissue and organs- skin, blood vessels, heart, lungs but it attacks the joints, producing a nonsuppurative proliferative and inflammatory synovitis that often progresses to destruction of the articular cartilage and ankylosis of the joints^(I,II).

Rheumatoid arthritis is characterized by the infiltration of a variety of inflammatory cells in to the joint. As a symmetric disease, RA usually involves the same joints on both sides of the body^(III).Approximately 1% of worldwide adult population is affected by rheumatoid arthritis. The prevalence of RA is found to increase with age in both sexes with nearly 5% of female & 2% men ^(IV).

Zingiber officinale (Family: Zingiberaceae) is commonly known as Ginger. Zingiber officinale consists of Gingerol, Shogaol, Zingiberene, Zingerone. It is proven for astringent^(V) , analgesic^(VI) ,antibacterial^(VII), anti-inflammatory^(VIII), and antiarthritic^(IX), activities. It‘s use has been reported in Ayurveda for the treatment of diarrhea, dysentery, rheumatism^(X).

Brassica oleraceae (Family:Brassiciaceae) is commonly known as Broccoli. It consists of Sulforaphane, Glucoraphanin, Indole-3-carbinol, Glucobrassician. It is proven for anti-microbial^(XI), anti-cancer^(XIII), anti-bacterial^(XIII), and anti-oxidant^(XIV), activities. Though these herbs are proven for antiarthritic activity individually, the combination of two is not yet evaluated. In present study an effort is done to evaluate the anti-arthritic activity of alcoholic extract of Zingiber officinale (AEZO) and hydroalcoholic extract of Brassica oleraceae (HAEBO) in combination of 1:1 proportion in CFA induced arthritis in Wistar rats.

MATERIAL AND METHODS:

The authenticated extract of Braccica oleraceae were purchased from Rutvik Enterprises, Mumbai and extract of Zingiber officinale was gifted from ARI health care. The laboratory chemicals were purchased from local vendor while assay kits for SGOP/SGPT were purchased from Pathozyme Laboratories.

Wistar albino rats of either sex weighing between 180-200gm were purchased from Crystal Biological Solutions Pvt. Ltd. and acclimatized in institutional animal house for 15 days before the study. . They were housed in a clean animal cages maintained at a temperature of 22±1˚C and 60-70% relative humidity and 12 hours light and 12 hours dark cycle. After acclimatisation the rats were divided into 12 groups each containing 6 rats. The animals were provided with food and water ad libitum. The experimental protocol was approved by Institutional Animal Ethics Committee formed as per guidelines by CPCSEA (Protocal Approval Number: IAEC/Nov/19-19/p-22). Minimization of animal suffering and providing the utmost suitable environment to the animals was taken care of.

Experimental:

The in vitro and in vivo evaluation for antiarthritic activity was carried out as following

a) In-vitro Studies

Inhibition of protein denaturation^{XI, XIII}:

The In-Vitro antiarthritic activity was carried out by following the method described by Gautam RK et al. 2013.

The denaturation of tissue proteins is one of the well-documented causes of inflammatory and arthritic diseases. Production of auto antigens in certain rheumatic diseases may be due to in vivo denaturation of proteins. The mechanism of denaturation probably involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding. Agents that can prevent protein denaturation therefore, would be worthwhile for antiarthritic drug development. The increments in absorbance of test samples with respect to control indicates stabilization of protein i.e. inhibition of heat-induced protein (albumin) denaturation.

2ml of BSA solution, 28ml of phosphate buffer (PBS, pH 6.4) and 20ml distilled water were used as control solution (50ml). 2ml of BSA solution, 28ml of phosphate buffer and various concentrations of standard drug (10, 50, 100, 200, 400, 800, 1000 and 2000μg/ml) were served as Standard drug solution (50ml). 2ml of BSA solution, 28ml of phosphate buffer and various concentrations of plant extract (10, 50, 100, 200, 400, 800, 1000 and 2000μg/ml) were taken as Test solution (50ml). All of the above solutions were adjusted to pH, 6.4 using a small amount of 1N HCl. The samples were incubated at 37°C for 15 minutes and heated at 70°C for 5 minutes. After cooling, the absorbance of the above solutions was measured using UV-Visible spectrophotometer at 660nm.

b) In vivo Studies:

Evaluation of antiarthritic activity in CFA induces arthritis in Wistar rats:

Wistar rats of either sex weighing between 180-200gm were selected for the experiment. They were grouped in 12 groups and treated as follows-

Group 1: Normal (0.1ml normal saline)

Group 2: Disease control (Complete Freunds adjuvant)

Group 3: Diclofenac (50mg/kg)

Group 4: CFA+ AEZO (50mg/kg)

Group 5: CFA+ AEZO (100mg/kg)

Group 6: CFA+ AEZO (200mg/kg)

Group 7: CFA+ HAEBO (50mg/kg)

Group 8: CFA+ HAEBO (100mg/kg)

Group 9: CFA+ HAEBO (200mg/kg)

Group 10: CFA+ AEZO (50mg/kg) + HAEZO (50mg/kg)

Group 11: CFA+ AEZO (100mg/kg) + HAEZO (100 mg/kg)

Group 12: CFA+ AEZO (200mg/kg) + HAEZO (200mg/kg)

On the 0^th day, the basal paw volume of left hind paw of each animal was measured using Ugo Basil Plethysmometer 7141. On day one all the animals except normal group were injected with 0.1ml of Complete Freund’s Adjuvant in to the ankle joint of left hind paw. Dosing with standard drug Diclofenac (50mg/kg) and extracts (50, 100, 200mg/kg) was started on day one and continued for 28 days. Disease control and normal control group rats received normal saline throughout study while the rest experimental groups animals received respective treatment once daily by per oral route. Paw volume of injected paw was measured on 7^th, 21^st and 28^th day. On 29^th day the rats were anaesthetized with urethane and blood was withdrawn from the retro orbital plexus for evaluation of haematological parameters and liver parameters. The evaluation was done by using routine laboratory methods.

Statistical analysis:

The results are represented as Mean±SEM. The data was analyzed by One Way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test. p values <0.05 were considered as statistically significant.

Results:

From the previous acute toxicity studies conducted, it was found that all the extracts were safe up to 2000mg/kg body weight, so one tenth of this dose (i.e. 200mg/kg) was considered as the dose for evaluation of pharmacological studies. Observations such as paw volume, body weight and haematological parameters were recorded after the injection of CFA. The Freund’s adjuvant induced arthritic control group showed sign of arthritis development, that is increase in the paw volume. The Figures 1 and 2 shows inhibition of paw oedema on 21 days and 28 days after administration of Diclofenac and extracts. Paw volume of the injected footpad significantly increased and reached a peak at 28 days in disease control groups while both the extracts and standard drug showed significant decreases paw oedema on 28th day as compared to Disease control group. Standard drug Diclofenac at a dose of 50mg/kg significantly (p<0.01) decreased the paw volume from 1st day after the induction of CFA, whereas the extracts significantly decreased the paw volume after the 7th day. The RA factor is an immunoglobulin produced as an immune response to immune complex formation involved in pathophysiology of Rheumatic arthritis. The figure 3 showed decrease in RA factor in treatment groups as compared to disease control group. In percentage inhibition, as seen in figure No. 4 it was found that AEZO extract produced highest percentage inhibition 87.41% of paw volume as compared to HAEBO, which produced inhibition of 89.91% at the end of 28^th day. But combination of both extracts shows better % inhibition in paw volume, which is 76%. Standard drug Diclofenac showed the highest decreased the paw oedema respectively on 28th day. In the CFA induced arthritic animals the haematological perturbations such as an increase in the WBC count, a decreased RBC count were also favourably altered by Diclofenac and extracts treatment.

DISCUSSION:

CFA induced arthritis is the most widely used chronic test model in which the clinical and pathological changes are comparable with those seen in human rheumatoid arthritis. The Freund’s adjuvant model is chosen as, it develops chronic swelling in multiple joints with influence of inflammatory cells with erosion of joint cartilage and bone destruction. Chronic inflammation involves the release of number of mediators like cytokines (IL-1B and TNF-α). These mediators are responsible for pain, destruction of bone and cartilage that can lead to severe disability. Prostaglandins are mediator for acute inflammation but chronic inflammation are mediated by proinflammatory cytokine such as TNF-α. In the present study, the diclofenac, alcoholic and hydroalcoholic extract significantly suppressed the swelling of the paw in both acute and chronic phase which may be due to the suppression of inflammatory mediator released due to induction of Freund’s adjuvant. The significant reduction in the RA factor and WBC by the treatment with combination of extracts also signifies the antiarthritic activity of the combination than the individual treatment groups. Thus the study shows that combination of Zingiber officinale and Brassica oleraceae has more anti-arthritic activity as compared to individual drugs at the dose of 200mg/kg. activity.

ACKNOWLEDGEMENT:

The authors are thankful to ARI Healthcare for providing gist sample of Zingiber officinale, Dr. D.Y. Patil Unitech Society’s DYPIPSR for providing laboratory and animal house facility. These is no conflict of interest in this research work.

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Received on 13.07.2019 Modified on 10.09.2019

Research J. Pharm. and Tech 2020; 13(3):1175-1178.

DOI: 10.5958/0974-360X.2020.00216.4

Figure 1: Effect of treatment with combination of AEZO and HAEBO on paw volume in CFA induced arthritis in rats.	Figure 2: Effect of treatment with combination of AEZO and HAEBO on paw volume in CFA induced arthritis in rats.

Figure3: Effect of treatment with combination of AEZO and HAEBON on RA level in CFA induced arthritis in rats.	Figure 4: Percentage inhibition of paw edema due to treatment with AEZO and HAEBO combination in CFA induced arthritis in rats.

Figure 5: Effect of treatment with combination of AEZO and HAEBON on WBC count in CFA induced arthritis in rats.