Research Articles

2015  |  Vol: 1(2)  |  Issue: 2(Nov-Dec)

Ameliorative effect of Alpinia galanga rhizomes on Freund's complete adjuvant induced arthritis in mice


Sharad Bhandarkar1, Alok Pal Jain2*

1Institute of Pharmacy Abdul Majeed Central Education Society, Nagpur, Maharashtra, India

2RKDF College of Pharmecy, SRK University, Hoshangabad Road, Misrod, Bhopal - 462026 (MP) M.P., India

Corresponding author

Dr. Alok Pal Jain

RKDFCollege of Pharmecy

SRKUniversity, Hoshangabad Road,

Misrod, Bhopal - 462026 (MP)

Abstract

Arthritis is a inflammatory autoimmune disorder resulting in joint that is marked by swelling, pain, functional destruction and muscle wasting. The objective of present study was to evaluate rhizomes of Alpinia galanga for Freund’s complete adjuvant induced arthritis in mice model. Materials and methods: Ethanolic extract with the dose of 100 and 200mg/kg was tested for antiarthritic activity using Freund’s complete adjuvant model in mice. Activity was measured by paw edema measurement and observation of haematological parameters i.e. Leukocyte Count, Packed Cell Volume (PCV), and Rheumatoid Factor (RF), in the serum 28th days after CFA administration. Results: Antiarthritic potential of ethanolic extract found on dose dependent manner and 200mg/kg dose of extract was significantly observed. Results also supported by improved various inflammatory markers in plasma. Conclusion:  Study can be concluded that the effect of ethanolic extract may be due to presence of flavonoids in the extract.

Keywords: Alpinia galangal, Arthritis, Freund’s complete adjuvant 

Introduction

Arthritis is a chronic autoimmune disease related to inflammation resulting in joint that is marked by swelling, pain, functional destruction and muscle wasting. It is illustrated by localized and systemic inflammation with eminent plasma concentrations of pro-inflammatory cytokines. Freund’s complete adjuvant (CFA) is precise to enhance the humoral anti-type-II collage antibody response and to promote cellular immunity. However, it showed that, in mice, arthritis can be induced easily by a single injection of CFA. So, induction of this arthritis called as “adjuvant arthritis” (Morgan et al., 1997; Ratkay et al., 1993).

Alpinia galanga (Zingiberaceae) is commonly known as Kulanjan is distributed in throughout of India and Southeast Asia. It is well and richest source of essential oils like cineole, methyleugenol myrcene and methylcinnamate. It is also reported to presence of various flavonoids like galangin, alpinin, kampferide and 3-dioxy-4-methoxy flavones in rhizomes part (Cui, 2003). Traditionally it is used against rheumatism, bronchial catarrh, ulcers, throat infections, fever and dyspepsia. The rhizomes of the plant have been used in treatment of various ailments, such as stomach disorders, respiratory, cardiovascular and skin disorders (Chopra et al., 1956). Rhizomes have reported as antibacterial, antioxidant, immunostimulant, anti-protozoal, anti-fungal and expectorant action (Jain et al., 2012). In the present study we plan to evaluate ethanolic extract of A. galanga rhizomes for CFA induced arthritis in mice.  

Material and methods

Plant materials and extraction

The rhizomes of A. galanga were collected from local market and authenticated in the department of Botany, Dr. H. S. Gour University, Sagar (M.P.) India. A herbarium of the plant was deposited for authentication in the department. The dried crude plant materials were powdered and subjected to extraction with different organic solvents. Defating of the plant materials was done with petroleum ether. Ethanol extract was obtained by hot extraction in soxhlet extractor. Ethanol extract was then subjected for qualitative chemical screening to detect the presence of phytoconstituents.

Animals

Mice of either sex (150-200 gm) were selected for in vivo study. The food and water were supplied ad libitum. All the animals were kept under standard laboratory conditions in light and dark cycles and maintained under controlled temperature 27±20 for acclimatization. The experiment was conducted in accordance with Institutional Animal Ethical Committee (IAEC) CPCSEA, Government of India.

Acute toxicity study

Mice of either sex (150-200 gm) were used during investigation. The animals were fasted over night. According to the OECD guideline No-423 fixed dose method was adopted and the safest dose of the all extracts was 2000 mg/kg body weight calculated. On the basis of toxicity study, two different doses 100 and 200 mg/kg were selected for in vivo study.

Animals were divided in four groups each containing 6 animals per group. The treatment schedules of mice belonging to the different groups are shown below

·         Group 1: Control (Complete Freund’s adjuvant and normal saline solution)

·         Group 2: Indomethacin against FCA induced arthritis (10mg/kg p.o)

·         Group 3: Ethanol extract against FCA induced arthritis (100mg/kg p.o)

·         Group 4: Ethanol extract against FCA induced arthritis (200mg/kg p.o)

Freund’s complete adjuvant induced arthritis

The selected animals were anaesthetized and one ankle joint will be injected with 0.1 ml of Freund’s complete adjuvant (FCA) containing 0.1mg Mycobacterium tuberculosis; in another the contra lateral knee was injected 0.1 ml (0.9%) saline. The animals were allowed to free for recover. Animals were examined for visual observations of arthritis in peripheral joints and scores for arthritis (Ratkay et al., 1993). A confirmation of arthritic condition was considered when significant changes in redness and swelling were noticed in the paw. The clinical severity of the arthritis in each paw was quantified weakly by quantification of the paw volume change upto day 28th.  Animals paw volume was measured using mercury plethysmometer. Dosing with standard drug Indomethacin (10 mg/kg body weight) and ethanol extracts (100 and 200 mg/kg ) was started on the same day and continued up to 28th day.

Hematological parameters

Blood aamples collected from different groups were tested for measurement of Leukocyte Count, Packed Cell Volume (PCV), and Rheumatoid Factor (RF), in the serum 28th days after CFA administration.

Statistical analysis

Pharmacological data were represented as the mean ±S.E.M. for six rats and data were evaluated using the Tukey test. Values of P <0.05 were considered to be statistically significant.

Results

Phytochemical study

In the study of phytochemical screening of ethanol extract from A. galanga rhizomes revealed the presence of flavonoids, alkaloids, glycosides, proteins and carbohydrates. On the basis of acute toxicity study two consequence doses 100 and 200 mg/kg were selected for in vivo study.

FCA induced arthritis

The present study contains an in vivo experiment for evaluation of anti arthritis activity of A. galanga rhizomes. The mice received a single injection of CFA on initial day and were examined daily up to 28th day for symptoms of arthritis. A reference group was used for comparison. Redness and swelling in to the joints initiated to appear on 2 days earlier in mice injected with CFA alone. After the time duration of the experiment, mice injected with CFA alone developed arthritis, mice treated with ethanol extract and reference drug showed minor swelling, reduced oedema and redness. The control group showed gradually reduced oedema with time up to 28th days, but very slowly. The ethanol extract showed oedema inhibition in dose dependent manner. Ethanol extract with 200mh/kg showed most effective for oedema inhibition in mice.

Table 1. Effect of A. galanga ethanol extract on FCA induced mice paw edema

Treatment Groups

 

Paw Edema (ml) (Mean ±SEM)

0

week

1st

week

2nd

week

3rd

week

4th

week

Control with Saline solution

0.076±0.042

3.22±0.059

3.57±0.072

3.50±0.0428

3.07±0.068

Indomethacin against FCA induced arthritis (10mg/kg p.o)

0.062±0.072

1.43±0.073

1.14±0.027

0.85±0.037

0.27±0.061

Ethanol extract against FCA induced arthritis (100mg/kg p.o)

0.073±0.052

1.57±0.049

1.47±0.043

1.07±0.043

0.86±0.027

Ethanol extract against FCA induced arthritis (200mg/kg p.o)

0.048±0.061

 

1.23±0.072

1.14±0.072

0.84±0.018

0.46±0.061

Table 2. Effects of ethanol extract of A. galanga on hematological parameters

Animal groups

Rheumatoid Factor (IU/ml)

Leukocyte Count (Cu.mm/ml)

Packed Cell Volume (%)

Control with Saline solution

14.26±1.25

3145±352.4

29.24±2.65

Indomethacin against FCA induced arthritis (10mg/kg p.o)

42.18±3.26

4523±402.3

42.17±4.25

Ethanol extract against FCA induced arthritis (100mg/kg p.o)

35.24±2.56

5624±428.7

45.32±3.95

Ethanol extract against FCA induced arthritis (200mg/kg p.o)

41.27±3.14

5378±395.2

46.20±4.12

Both doses of the ethanol extract 100 and 200mg/kg produced a significant increase in the RF and Leukocyte count compared with untreated control group. There was no significant difference in the WBC counts. RF was negative in both treated and untreated rats.

Discussion and conclusion

The results of the paw volume and inflammatory markers from the site of CFA administration to the other paw can be correlated by a migratory phenomenon in CFA induced arthritis.

The possible mechanism by which the ethanol extract exhibited anti-arthritic activity could be to suppress the activation of nuclear factor (NF-kB). In inflammatory diseases, NF-kB is prominent and is responsible for improved expression of several pro-inflammatory mediators (Morgan et al., 2005; Riehemann et al., 1999).

In the present study, we select complete Freund’s adjuvant induced arthritis method in mice for induction of arthritis, because it is most suited and widely in use model for arthritis with clinical and laboratory utility due to accumulation of inflammatory cells, corrosion of joint cartilage and bone destruction comparable to human rheumatoid diseases (Singh and Majumdar, 1996).  

Oxygen derived free radicals are known to play an important role in the pathogenesis of different inflammatory disorders. The role of oxygen free radicals and associated activated oxygen free intermediates in the pathogenesis of arthritis has been identified with growing occurrence (Devi et al., 2007). Rhizomes of A. galanga already reported to have petent antioxidant activity (Jain et al., 2012). In conclusion, the anti arthritic effect of ethanol extract of A. galanga rhizomes showed in dose dependent manner. The plant contains various flavonoids constituents that may be responsible for the accelerated activity and mechanism behind may be its free radical scavenging action.

Acknowledgement

Authors are grateful to the head of the institution for providing necessary facilities for the research work.

Conflict of interest

Authors did not have any conflict of interest.

References

Chopra RN, Nayer SL, Chopra IC. 1956. Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi.

Cui Z. 2003. Determination of chemical constituents of the essential oil from Alpinia galanga (L.) by GCMS. Lixueban, 38: 104-107.

Devi PR, Kumari SK, Kokilavani C. 2007. Effect of Vitex negundo leaf extract on the free radicals scavengers in complete Freund's adjuvant induced arthritic rats. Indian Journal of clinical biochemistry, 22(1): 143-147.

Jain AP, Pawar RS, Lodhi S, Singhai AK. 2012. Immunomodulatory and anti-oxidant potential of Alpinia galanga Linn. rhizomes. Pharmacognosy Communications, 2(3): 30-37.

Morgan SL, Anderson AM, Hood SM, Matthews PA, Lee JY, Alarcon S. 1997. Nutrient intake patterns, body mass index, and vitamin levels in patients with rheumatoid arthritis. Arthritis Care & Research, 10: 9–17.

OECD: OECD guideline for the testing of Chemicals, Acute Oral Toxicity-Fixed dose procedure 423 adopted 17th December 2001.

Morgan ME, Flierman R, van Duivenvoorde LM, et al. 2005. Effective treatment of collagen-induced arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis & Rheumatology, 52: 2212–21.

Ratkay LG, Zhang L, Tonzetich J, Waterfield JD. 1993. Complete Freund’s adjuvant induces an earlier and more severe arthritis in MRL-lpr mice. The Journal of Immunology, 151: 5081–7.

Riehemann K, Behnke B, Osthoff K. 1999. Plant extract from stinging nettle (Urtica dioica), an antirheumatic remedy, inhibit the proinflammatory transcription factor NF-kB. FEBS Letters, 442: 89-94.

Singh S, Majumdar DK. 1996. Effect of fixed oil of Ocimum sanctum against experimentally induced arthritis and joint edema in laboratory animals. Pharmaceutical Biology, 34 (3): 218-222. 

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