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Indian J.L.Sci.2(2) : 17-22, 2013
ISSN : 2277-1743 (Print)
ISSN : 2278-7879 (Online)
ANTI-INFLAMMATORY EFFECT AND TOXICOLOGICAL EVALUATION OF
THYMOQUINONE (VOLATILE OIL OF BLACK SEED) ON ADJUVANT-INDUCED
ARTHRITIS IN WISTAR RAT
MEHTAB ALAMa1 AND VIKAS GALAVb
aKrish Biotech Research Pvt. Ltd., Kalyani, West Bengal, India bUniversity College of Medical Sciences & GTBH, University of Delhi, New Delhi, India ABSTRACT
Inflammatory disorder to be considered autoimmune disease which, affects the joints and is associated with swelling,
stiffness and pain. The study was subjected to evaluate therapeutic potential of thymoquinone (volatile oil of black seed) on
freund's complete adjuvant induced arthritis in rats. Arthritis was induced in rats by injecting 0.1ml of freund's complete
adjuvant into the left hind paw of the rat intradermally for 21 days. Thymoquinone orally administered to male and female wistar
rat at dose level of 2.5, 5.0, 10.0 mg/kg body weight for 35 days repeatedly, post induced did not produce any sign of toxicity,
mortality, pathological changes and significant blood parameters changes. The investigated result showed that the thymoquinone
(10 mg/kgb.wt) significantly (p<0.001) inhibited the FCA induced arthritis and showed significant anti-inflammatory activity.
Thymoquinone 10 mg/kgb.wt also tends to suppress the inflammation at p<0.05 level significance. Therefore, thymoquinone
treatment found to possess potent anti-inflammatory activity with no toxicity and the treatment significantly inhibited the
development phase of arthritis which, is further supported by its anti-inflammatory effect was comparable to that of standard
drug prednisolone (5 mg/kgb.wt).

KEYWORDS : Thymoquinone, Freund's adjuvant induced arthritis, Arthritis activity
Inflammation is a common clinical conditions and for medicinal purposes in many countries (Jansen, 1981).
rheumatoid arthritis (RA) is a chronic inflammatory This plant is one of the most extensively studied, both autoimmune disorder that affects about 2.1 million phytochemically and pharmacologically (Riaz et al., 1996; (Majithia & Geraci,2007., El-Dakhakhny, 1963) in Indians Siddiqui & Sharma, 1996). Most properties of whole seeds and Americans etc which, affects the joints and is or their extracts are mainly attributed to quinone associated with swelling, stiffness and pain. The drugs constituents, of which, thymoquinone is more abundant commonly in used for the treatment of inflammation and compound (F ilippo D ' A ntuono et al., 2002).
RA include glucocorticoids eg., cortisone, prednisolone Pharmacological action of Nigella sativa has been and NSAIDS drugs (eg. Ibuprofen), disease-modifying i n v e s t i g a t e d i n c l u d i n g i m m u n e s t i m u l a n t , anti-inflammatory and anti-rheumatic drugs (DMAIDs and antiinflammatory, anticancer, antimicrobial antiparasitic DMARDs; eg. Methotrexate (MTX) and leflunomide) and and antioxidant (Ibrahim Tekeoglu et al. 2007; Burits and biological response modifiers (eg. Tumor necrosis factor- alpha blocking agents). Such therapies are helpful controlling the symptoms of acute RA, but their effect on MATERIALS AND METHODS
chronic, prolong RA are unsatisfactory. Moreover, besides Male and female albino wistar rat (Rattus their high cost, the prolonged use of many of these norvegicus were maintained at 22 ± 3ºc, relative humidity is associated with severe adverse reactions and between 60 to 70 % and a light/dark cycle of 12 hr. The rat toxicity, including gastrointestinal disturbances and were provided with rat pellet feed (amrut brand, pranav agro Pune) and drinking water filtered through aquaguard water Thymoquinone (Nigella sativa seed) is a member filtration system ad libitum throughout the study period. All of the ranunculaceae family growing in countries bordering groups of rat were acclimatized 6 days prior to the start the mediterranean sea, India, Pakistan, and Iran. For many dosing. The thymoquinone sample was purchased from centuries, nigella sativa seeds (also called black seeds or sigma aldrich India and peanut oil from local market the black cumin) have been used as a food additive as well as dose volume maintained at 10 ml/kg body weight.
1Corresponding author
ALAM AND GALAV : ANTI-INFLAMMATORY EFFECT AND TOXICOLOGICAL EVALUATION OF THYMOQUINONE .
Complete Freund's Adjuvant Arthritis
Organ Body Weight Ratio
After randomization male and female rats (110 ± The vital organ such as liver, kidney, brain, heart, 20 g body weight) were divide in to six groups (I to VI) each lung, spleen, adrenal of rats and the male sex organ(testis, group consist 5 male and 5 female rats. On day one, all rats epididymis, prostrate and seminal vesicle) and female sex were injected into the sub plantar region of the left hind paw organs (ovary, uterus, cervix and vagina) were quickly with 0.1ml of freund's complete adjuvant. This consist of removed and weigh individually. The organ to body weight mycobacterium butyricum suspended in heavy paraffin oil by thorough grinding with motor and pestle to give a Biochemical Estimation
concentration of 6mg/ml. Dosing with the test and standard Different biochemical parameters like Alkaline substance was started on the first day and continued for 21 phosphatase (ALP) marker for bone destruction, Acid Phosphatase (ACP) the lysosomal enzyme activity, Serum Anti-inflammatory Studies
glutamate oxalo acetate transaminase (SGOT) and Serum Based on the subacute 28 days oral toxicity in rats.
glutamate pyruvate transaminase (SGPT) were estimated Group I served as arthritis control group and group II served by using ALP,ACP, SGOT and SGPT kit in Erba Mannhein as vehicle control group given a daily dose of normal saline EM 200 Clinical Chemistry Analyser. Bood samples were and peanut oil. The rats of group III, IV and V were given collected by sublingual rout, centrifused and supernatant thymoquinone mixed in peanut oil via gavage at dose level serum was collected. Different enzyme reagents were added 2.5, 5.0, 10 mg/kg body weight respectively for 35 days to the serum and estimated in an auto analyser.
post induced arthritis and group VI served as standard drug Haematological Study
(prednisolone) and given 5.0mg/kg body weight. Rats were Blood collected in EDTA tube was analyzed for observed for the paw swelling in the injected and contra red blood cells (RBC) and white blood cells (WBC) counts lateral hind paws of the rats were monitored daily using were determined according method of Winfrobe and liquid displacement plethysmometer (Ugo Basile, Italy).
Landsberg whereas, haemoglobbin and differential Increase in the extent of erythema and edema of the tissues leucocytes counts (DLC) were measured according to shows the severity of the inflammation. The difference in severity of arthritis between the experimental groups and Statistical Analysis
arthritis control group were statistically analyzed and Statistical significance were presented between toxicological effect and mortality throughout the study control and experimental values as mean ± SEM (n=5).
period. Body weight, food and water of individual rat were Statistical comparison of body weight changes was made recorded weekly for each group. After 56 day treatment as well as controls animals were sacrificed and blood collected directly from jugular vein in ethylene diamine tetra acetic acid (EDTA) solution and non-oxalate tubes for Organ Body Weight Ratio
the estimation of haematological and biochemical The absolute body weights of thymoquinone parameter respectively. All the animals were closely treated male and female rats no significant changes were observed for organs like ears, nose, tail, fore paws and hind observed while, comparable to controls rats. The relative organ weights (organ to body weight ratio) of animals Paw Edema
exposed to different dose of thymoquinone did not indicate Paw volumes of left hind limb were recorded and any significant changes and value are shown in (Table, 1).
Biochemical Study
using mercury column plethysmometer. On the day 21 , The results of serum biochemical parameters of rheumatoid arthritis becomes more evident and male rats are shown. There was no change in clinic- chemical parameters of male and female rats exposed to Indian J.L.Sci.2(2) : 17-22, 2013
ALAM AND GALAV : ANTI-INFLAMMATORY EFFECT AND TOXICOLOGICAL EVALUATION OF THYMOQUINONE .
Table 1: Relative Organ Body Weight of Male Rats Orally Administration Thymoquinone for
35 days post induced arthritis
Dose (mg/kg body weight)
Arthritis
Standard
Table 1 (continue): Relative Organ Body Weight of Female Rats Orally Administration Thymoquinone for
35 days post induced arthritis
Dose (mg/kg body weight)
(Arthritis
Standard
Control)
different dose of thymoquinone for 28 days and the values rats. While, the gross pathological examination observed were comparable to controls rats. A marked increase in the slightly uterus distention in two control and one treated activity of membrane marker enzymes ALP, AST and SGP female rat which, spontaneous and is physiological/cycle were observed in the serum of arthritis rats group V (Table, nature and did not effect on outcome of study (Table, 4).
Ant-inflammatory Effect
Haematology
The left hind paw injected with complete freund's The results of haematological parameters in male adjuvant become gradually swollen and reach edits peak at and female rats exposed to different doses thymoquinone 21st day. The results obtained for the different dose of are shown. There was no significance changes in Hb RBC, thymoquinone and the standard drug prednisolone 5.0 WBC and differential leukocyte count (DLT) (Table, 3).
mg/kg body weight in the complete freund's adjuvant- Histopathology
induced (FCA) paw edema test at specific time intervals. It Autopsy of treated animals after 56 days of was obvious that during 21st day treatment paw edema in exposure revealed no significance change in their vital disease control inflamed paw is increase in time dependent organs. Microscopic examination of liver, kidney, brain, manner and all administration groups significantly inhibited testes, and ovary of rats treated with the different doses of the development of joint swelling induced by complete thymoquinone for 28 days did not shown any significant Freund's adjuvant. Arthritic index and rheumatoid factor tissue damage and were comparable with those of controls were significantly (p<0.001) decreased start on 30 days in Indian J.L.Sci.2(2) : 17-22, 2013
ALAM AND GALAV : ANTI-INFLAMMATORY EFFECT AND TOXICOLOGICAL EVALUATION OF THYMOQUINONE .
Table 2: Serum Biochemical parameter in rats treated orally with thymoquinone for
35 days post induced arthritis
Dose mg/kg body weight
Arthritis
Standard
Parameter
2.5 (Low)
5.0 (Mid)
10.0(High)
Table 3: Haematological parameters in rats treated orally with thymoquinone for 35 days
post induced arthritis
Dose mg/kg body weight
Parameter
Arthritis
2.5 (Low)
5.0 (Mid)
10.0 (High)
Standard
Table 4: Histopathological Observation in tissue of rats treated orally with thymoquinone for 35 days
post induced arthritis
Number of Lesion
Dose mg/kg body weight
0 (Arthritis
0 (Vehicle
Standard
2.5(Low)
5.0(Mid)
10.0(High)
Control)
Control)
NAD= No Abnormality Detected, M= Male, F=Female treatment with thymoquinone 10.0 mg/kg, and inhibition of adjuvant-induced arthritis in rats is one of the prednisolone 5.0 mg/kg treated animal as compare to most suitable test procedures to screen anti-arthritic agents disease control treatment (Table, 5 & Figure 1 & 2).
since, it closely resembles human arthritis. Arthritis (RA), one of the chronic inflammatory diseases, systemic DISCUSSION
inflammatory disorder affecting the synovial joints and Most of the investigators have reported that typically producing symmetrical arthritis that leads to joint Indian J.L.Sci.2(2) : 17-22, 2013
ALAM AND GALAV : ANTI-INFLAMMATORY EFFECT AND TOXICOLOGICAL EVALUATION OF THYMOQUINONE .
Table 5: Effect Of Thymoquinone Mean Percentile In Paw Edema Volumes In Male Rats Induced By
Adjuvant Freund's Complete Adjuvent ( FCA)
Dose (mg/kg
body weight)
Value are expressed as a mean ± S.E.M (p< 0.001) as compared to control Table 5 (continue): Effect Of Thymoquinone Mean Percentile In Paw Edema Volumes In Female Rats
Induced By Adjuvant Freund's Complete Adjuvant ( FCA)
Dose (mg/kg
body weight)
Value are expressed as a mean ± S.E.M (p< 0.001) as compared to control prednisolone 5.0 mg/kg when comparable to the arthritis control and vehicle control group. Increased white blood cell count is a common feature of inflammatory reactions, especially those induced by microbial infection. So in arthritic group an increase in total leukocyte number was found. A significant reduction (p<0.001) in total leukocyte number was found in case of treated thymoquinone 10 and standard drug 5 mg/kg body weight. In present study it was found that the administration of thymoquinone and standard drug lead to inhibition leukocyte migration which, may have beneficial effect for joint preservation. Traditional medicine has maintained greater popularity all over developing world prompted by the increase awareness and interest in medicinal plant and the use is rapidly increase Figure 1 : Mean Percentile change In Paw Edema
generation by generation (Daswaniet al., 2006; Ogbonnia Volumes (Ml) In Male Rats Induced By FCA
et al., 2008). More often, this has led to indiscriminate use destruction. In this present study, the result demonstrated with out appropriate dose resulting in abuse. The incidence the effect of thymoquinone and prednisolone on FCA of adverse effects of these herbal remedies and sometimes induced arthritis model in rats, selected to evaluate their life-threatening conditions has been reported among efficacy against the proliferateive phase of inflammation, various ethnic groups (Elvin-Lewis, 2001; Chan, 2003).
the activity of membrane marker enzymes was The result presently conducted study revealed that daily significantly markedly increased in the adjuvant induced orally administration thymoquinone (volatile of Kalonji arthritic rats after treated with thymoquinone 10 mg/kg, and Seed) found to exhibit significant antiinflammatory and the Indian J.L.Sci.2(2) : 17-22, 2013
ALAM AND GALAV : ANTI-INFLAMMATORY EFFECT AND TOXICOLOGICAL EVALUATION OF THYMOQUINONE .
REFERENCES
. Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res., 2000. 14 (5): 323-
El-Dakhakhany M., 1963. Studies on the Egyptian Nigella sativa L: Somepharmacological properties of its seed's active principle incomparison to its dihydro- Figure 2 : Mean Percentile change In Paw Edema
compound and its polymer. Arzneim Forsch Drug Volumes (Ml) In Female Rats Induced By FCA
potent anti arthritic activity by significantly (p < 0.001) Elvin-Lewis M., 2001. Should we be concerned about altering the pathogenesis during arthritis without exerting 75 141-164.
any side effect and did not induce any treatment related Filippo D'Antuono L., Moretti A., Lovato A.F.S., 2002.
observable toxic effects, with regards to the haematological Seed yield, yield components, oil content and parameters, biochemical parameter and histopathological essential oil content and composition of Nigella when compared to its control group of animal treated with sativa L. and Nigella damascena L. Indust Crops corn oil (vehicle) only throughout the study.
Prod., 15: 59-69.
Ibrahim Tekeoglu1, Ali Dogan, Levent Ediz and Mustafa CONCLUSION
The result presently conducted study revealed that Effects of Thymoquinone (Volatile Oil of Black daily orally administration thymoquinone (volatile of Cumin) on Rheumatoid Arthritis in Rat Models.
K a l o n j i S e e d ) f o u n d t o e x h i b i t s i g n i f i c a n t Phytother. Res., 21, 895-897.
antiinflammatory and the potent anti arthritic activity by Ogbonnia, S., Adekunle, A. Bosa M. K and Enwuru V. N., significantly (p < 0.001) altering the pathogenesis during 2008. Evaluation of acute and subacute toxicity of arthritis without exerting any side effect during the Alstonia congensis Engler (Apocynaceae) bark repeated treatment and proved itself to be the traditionally and Xylopia aethiopica (Dunal) A. Rich used and recommended by the practitioner best for the (Annonaceae) fruits mixtures used in the treatment treatment for arthritis when compare to allopathic steroids of diabetes. African JBiotech., 7 (6): 701 -705.
drug. The study are also give advice to should not be used Riaz M., Syed M. and Chaudhary F.M., 1996. Chemistry of daily higher amount of kalonji seed in form of kalnoji oil themedicinal plants of the genus Nigella. Hamdard and kalonji majoon which, available in market. But give Medicus, 39: 404.
advise to use daily little amount which, is remedy for all Siddiqui A.A., Sharma P.K.R., 1996. Clinical importance of disease except death which, has been proved traditionally Nigella sativa L. A review. Hamdard Medicus, practionaer generation by generation.
ACKNOWLEDGEMENT
The author are grateful to the Director Lifescience Intelligentsia Foundation (LIFE) for the keen interest and constant encouragement and for the technical assistant Mr.
Shadab Alam and Mr. Saleem Ahmed Department Toxicology LIFE Hajipur District Vaishali, Bihar, India.
Indian J.L.Sci.2(2) : 17-22, 2013

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