Table of Contents 
ORIGINAL ARTICLE
Year : 2015  |  Volume : 22  |  Issue : 1  |  Page : 141-146  

Anti-arthritic activity of Dashanga Ghana (an ayurvedic compound formulation) against Freund's adjuvant induced arthritis in Charles Foster albino rats


1 Department of Rasa Shastra and Bhaishajya Kalpana, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
2 Drug Discovery Group, R&D Centre, The Himalaya Drug Company, Makali, Bangalore, India
3 SDM Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi, Karnataka, India

Date of Web Publication21-Dec-2015

Correspondence Address:
Galib Ruknuddin
Department of Rasa Shastra and Bhaishajya Kalpana, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat
India
Login to access the Email id


DOI: 10.4103/0971-6580.172279

PMID: 26862275

Rights and Permissions
   Abstract 


Introduction: Arthritis is the most common cause of disability, limiting the activities of adults throughout the world. Apart from the conventional treatment strategies using non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatic drugs, and glucocorticoids, newer and safer drugs are continuously being searched, as long-term usage of these drugs have resulted in adverse effects. Besides this, currently a number of medicinal plants are under scientific evaluation to develop a promising remedy in these cases. There is a need to investigate the complete therapeutic potential of these herbals for providing newer and safer treatment options with minimum side effects. Considering this, a polyherbal Ayurvedic compound formulation (Dashanga Ghana) has been studied in experimental animals to evaluate anti-arthritic activity. Materials and Methods: Dashanga Ghana has been prepared in the laboratory by following standard guidelines. Charles Foster albino rats were used to evaluate the activity through Freund's adjuvant induced arthritis model.
Results and Conclusions: Dashanga Ghana is found to possess significant anti-arthritic activity. Further studies are required to identify and characterize exact active phyto-constituents and to elucidate the exact mechanism of action, which is responsible for the observed pharmacological profile.

Keywords: Arthritis, Ayurveda, Dashanga Ghana, Freund's adjuvant, paw oedema


How to cite this article:
Ruknuddin G, Patgiri B J, Prajapati P K, Ashok B K, Ravishankar B. Anti-arthritic activity of Dashanga Ghana (an ayurvedic compound formulation) against Freund's adjuvant induced arthritis in Charles Foster albino rats . Toxicol Int 2015;22:141-6

How to cite this URL:
Ruknuddin G, Patgiri B J, Prajapati P K, Ashok B K, Ravishankar B. Anti-arthritic activity of Dashanga Ghana (an ayurvedic compound formulation) against Freund's adjuvant induced arthritis in Charles Foster albino rats . Toxicol Int [serial online] 2015 [cited 2017 Mar 30];22:141-6. Available from: http://www.toxicologyinternational.com/text.asp?2015/22/1/141/172279




   Introduction Top


Arthritis is the most common cause of disability, limiting the activities of adults throughout the world. The current treatment of arthritis is intended to minimize pain and inflammation with non-steroidal anti-inflammatory drugs, as well as to decelerate the progress of the disease by using disease modifying anti-rheumatic drugs. Because of the limitations and risks of conventional therapy, adverse reactions, chronic nature of the disease, and advanced age, people are exploring alternative measures to treat this condition. Alternative treatments, which are relatively safe and free of side effects, have been used both as adjunct and an alternative to conventional therapies in many countries.[1] Herbal therapies occupy a large section of alternative therapy. Ayurveda, the Indian traditional system of medicine, which is popular throughout the globe, has a large number of remedies that may be beneficial in cases of arthritis. Many such formulations are in common use even today.

Ayurvedic treasure house has a number of remedies, which can be beneficial in conditions of arthritis. The combination selected for this study (Dashanga Ghana) is based on theversion available inSharangadhara Samhita, whichis said to be significant in combating various types of inflammation.[2] Considering this, this study was planned to revalidate anti-arthritic activity of the compound in complete Freund's adjuvant induced arthritis in Charles Foster albino rats.


   Materials and Methods Top


Test formulation

The formulation composition (Dashanga Ghana) is a combination of 10 herbal ingredients of equal parts [Table 1]. Drugs such as Yashtimadhu, Tagara, Sukshmaila, Jatamansi, Haridra, Daruharidra, Shirisha, and Kushta were procured from pharmacy, Gujarat Ayurved University, Jamnagar, Gujarat, India. Raktachandana was procured from Moodbidri, Karnataka, India and Hribera from Palakkad, Kerala, India. Random samples of the collected drugs were subjected to pharmacognostical studies with an intention to check their identity and genuineness at Pharmacognosy Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda (IPGT and RA), Gujarat Ayurved University, Jamnagar, Gujarat, India. After establishing proper identity; dried individual ingredients were converted in to coarse powder (sieve 10). All the powders were mixed together thoroughly in specified proportions to prepare a homogenous blend, which was shifted to a stainless steel container, added with specified amounts (16 parts) of water and kept aside undisturbed throughout the night. On the following day, the contents were subjected to mild heat maintaining temperature in between 95 and 100°C. The contents were stirred constantly, in order to avoid the possibilities of settling down of the contents and their charring. When the volume was reduced to one quarter; the contents were filtered through a clean cotton cloth in to a stainless steel container to obtain decoction. The decoction wasfurther subjected to the process of reboiling with continuous stirring till the contents become semi solid. The semi-solid contents were shifted in to a tray and subjected to drying maintaining the temperature around 50°C. The dried contents thus received (Dashanga Ghana) were collected carefully with the help of scraper and used in the study.
Table 1: Formulation composition of Dashanga Ghana

Click here to view


Animals

Charles Foster albino rats weighing 200 ± 30 g of either sex were obtained from the animal house attached to the Pharmacology Laboratory, IPGT and RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. The animals were maintained on “Amrut” brand animal pellet feed of Pranav Agro Industries and tap water was given ad libitum. The temperature and humidity were kept at optimum and animals were exposed to natural day and night cycles. The experiments were carried out in conformity with the guidelines of the Institutional Animal Ethics Committee after obtaining its permission (IAEC03/08-11/PhD/02) and care was taken as per the Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines.

Acute toxicity study

Acute oral toxicity of Dashanga Ghana formulation was carried out in female Charles Foster albino rats as per the OECD 425 guidelines. The formulation was tested at the limit dose of 2000 mg/kg body weight. The result showed that Dashanga Ghana did not produce any changes in observed parameters and there was no mortality even at the limit dose. The animal dose was extrapolated from human therapeutic dose mentioned in classical literature.

Dose fixation

Dose of the test formulation was fixed by extrapolating the human dose to laboratory animals on body surface area ratio.[3] The adult human dose (500 mg) was converted to animal dose. On this basis the rat dose was found to be 45 mg/kg. The test drug was suspended in distilled water by making uniform suspension with 0.5% carboxy methyl cellulose with suitable concentration depending up on body weight of animals and administered orally with the help of gastric catheter sleeved to syringe. The drug was administered to overnight fasted animals.

Anti-arthritic activity

The selected animals were grouped into four groups of 6 rats each. To the first group (normal control) tap water was administered. Second group (arthritic control) was also administered with tap water orally and injected with Freund's adjuvant. Third group (test drug group) was administered with Dashanga Ghana in the dose of 45 mg/kg. The fourth group (reference standard [RS]) was administered with the standard drug dexomethasone (100 µg/kg). The test drug and RS were administered for 30 consecutive days. On day 1, the complete Freund's adjuvant was made into fine emulsion with the help of a syringe and 0.1 ml of it was injected beneath the plantar aponeurosis in the left hind paw and 0.05 ml subcutaneously into the root of the tail. The volumes of both the hind paws were measured with the help of plethysmometer just before the adjuvant injection (initial). The paw volume of left hind limb was measured at 2nd, 3rd, 5th, 10th, and 15th day, while of right hind limb on 15th, 20th, 25th, and 30th day. Paw volume of the 0 (initial) days were taken as the reference value for determining the increase in paw volume on the subsequent days. On 31st day, animals were weighed and sacrificed by over dose of ether anesthesia. Blood was collected from neck blood vessels and serum was separated for the estimation of biochemical parameters.[4] Parameters such as blood urea,[5] serum creatinine,[6] serum glutamate oxaloacetate transaminase,[7] serum glutamate pyruvate transaminase,[8] and serum alkaline phosphatase [9] were estimated by feeding requisite quantity of serum to the auto analyzer (Fully Automated Biochemical Random Access Analyzer, BS-200; Lilac Medicare Pvt. Ltd., Mumbai, Maharashtra, India) which was automatically drawn in to the instrument for estimating different parameters. References given in the kit literature mentioning the basis of the methods on which test procedures was carried out. Further both right and left synovial joints were dissected out and the histopathological slides were prepared by referring to standard procedure.[10] The slides were viewed under trinocular research Carl-Zeiss's microscope at various magnifications to note down the changes in the microscopic features.

Statistical analysis

The obtained data have been presented as a mean ± standard error of the mean, difference between the groups was statistically determined by Student's t-test for both paired and unpaired data for the treated group with the level of significance set at P < 0.05.


   Results Top


A normal progressive gain in body weight was observed in normal control group when the final values were compared with initial body weight. In arthritic control group significant decrease in body weight was seen in comparison to both initial values, as well as values of control group [Table 2]. Treatment with Dashanga Ghana showed gain in body weight similar to that of normal control group. In contrast an apparent and statistically significant decrease in body weight was found in dexomethasone treated group.
Table 2: Effect of the test drugs on body weight

Click here to view


An apparent suppression in paw volume was observed in Dashanga Ghana treated group and also in dexamethazone treated group at different time intervals. The observed suppression in paw oedema in Dashanga Ghana treated group at 2nd, 5th, and 10th day was statistically significant in comparison to arthritis control group. In dexomethasone (RS) administered group the suppression of oedema observed at all the time intervals is found to be statistically significant [Table 3].
Table 3: Effect of the test drugs on primary paw oedema (oedema of left hind paw)

Click here to view


Treatment with Dashanga Ghana apparently inhibited the secondary oedema at all the time intervals, however only the inhibition observed on 30th day was found to be statistically significant in comparison to arthritic control group. In dexomethasone treated group inhibition of secondary oedema at all-time intervals in comparison to arthritic control group was highly significant (P < 0.001) [Table 4].
Table 4: Effect of the test drugs on secondary paw oedema (oedema of right hind paw)

Click here to view


Statistically highly significant increase in blood urea was observed in arthritic control group in comparison to normal control group (P < 0.001) [Table 5]. Treatment with Dashanga Ghana significantly attenuated blood urea level. In dexamethsone treated group also significant attenuation of blood urea was observed. The other serum biochemical parameters are not altered to significant extent in arthritic control group and also in Dashanga Ghana treated group.
Table 5: Effect of the test drugs on serum biochemical parameters

Click here to view


Microscopic examination of both the joints [Table 6] showed normal intact joint cytoarchitecture in normal control rats [Figure 1]a. In Freund's adjuvant arthritis control rats, remarkable degenerative changes in the form of cartilage erosion, synovial membrane proliferation and hyperplasia, and pannus formation were observed in both the joints [Figure 1]b. These changes were found to be very much decreased in Dashanga Ghana treated group [Figure 1]c and dexamethsone treated group [Figure 1]d.
Table 6: Effect of the test drugs in synovial joints (histopathological observations)

Click here to view
Figure 1: (a) Photomicrographs of joint from control group (×100). (b) Photomicrographs of joint from Freund's adjuvant arthritis control group (×100). (c) Photomicrographs of joint from Dashanga Ghana treated group (×100). (d) Photomicrographs of joint from dexamethsone treated group (×100)

Click here to view



   Discussion Top


The Freund's adjuvant induced arthritis model in rats is the most common model used by several scientists to evaluate anti-arthritic activity of new drugs. This model closely resembles clinical arthritis and is the most widely used model of experimental arthritis which has been used for screening purposes in the disease produced in the rat by injection of complete Freund's adjuvant into certain dermal and tissue sites.[11] Therefore, this model is used with a relatively high degree of validity for evaluating agents with potential anti-arthritic activity. Researchers distinguished four phases of arthritis on the basis of biochemical markers of arthritis viz., days 1–4 with acute local inflammation and systemic effects, days 7–12 with remission of acute inflammation and periarthritis, days 12–28 with chronic inflammation, periarthritis, and osteogenic activity and day 35 onward with permanent articular deformity and minimal inflammation.[12] This model has been used as a model of subchronic or chronic inflammation in rats and is considerable relevance for the study of patho-physiology and pharmacological control of inflammatory processes.

Body weight was considered as an indirect index of health status and recovery from disease. A change in body weight of rats was also measured as one of the parameter to assess the course of the disease and the response to therapy of anti-inflammatory and anti-arthritic drugs. As the incidence and severity of arthritis is increased, a decrease in body weights of the rats also occurred during the course of the experimental period and this observation was supported by the findings of previous study on alterations in the metabolic activities of diseased rats.[13] It has been suggested that, the decrease in body weight during inflammation is due to deficient absorption of nutrients through the intestine and that treatment with anti-inflammatory drugs normalizes the process of absorption.[14] The evident restoration of the body weight of rats in the Dashanga Ghana treated group may involve improvement of intestinal absorption of the nutrients and a reduction in the distress caused by the severity of the arthritis.

The determination of paw swelling is apparently simple, sensitive, and quick procedure for evaluating the degree of inflammation and assessing of therapeutic effects of drugs. In this study, the rat was selected as an animal model since they develop a chronic swelling in multiple joints with an influence of inflammatory cells and followed by erosion of cartilage in joints and destruction of bones. Paw volumes of both hind limbs were recorded on the day of adjuvant injection and again measured at 2nd, 3rd, 5th, 10th, and 15th day, and 15th, 20th, 25th, and 30th day for primary and secondary lesions, respectively. The 15th day measurement is indicative of primary lesions and its onward measurement will aid in estimating secondary lesions. On 21st day, the secondary phase of rheumatoid arthritis becomes more evident and inflammatory changes spread systemically and become observable in the limb not injected with Freund's adjuvant. This is because of the manifestation of cell-mediated immunity.[15]

Dashanga Ghana produced significant effect on primary oedema and moderate effect on secondary edema. In contrast, dexomethasone administered group showed significant suppression of both primary and secondary oedema. This indicates presence of moderate to significant anti-arthritic activity in the test formulation and significant anti-arthritic activity in RS. This observation was further evidenced by histopathological study where joints from both test formulation and RS treated animals showed remarkable protection against Freund's adjuvant induced degenerative changes in the form of cartilage erosion, synovial membrane proliferation, hyperplasia, and pannus formation in both the joints.

Among the five serum biochemical parameters studied only one parameter was affected to significant extent by injection of Freund's adjuvant. Statistically significant increase in blood urea was observed in arthritic control group in comparison to normal control group. Increased blood urea level was reported in arthritic rats and it was hypothesized that substantial fraction of blood urea in arthritic rats comes from arginine synthesized in the kidney.[16] The test formulation significantly attenuated blood urea level. In dexamethsone treated group also significant attenuation of blood urea was observed.

Dashanga Ghana is a polyherbal compound formulation. Previously we have reported that this formulation have significant anti-inflammatory and analgesic activities.[17] Further, different fractions of the individual ingredients have been studied for analgesic, anti-inflammatory and anti-arthritic activities. Sirisha (Albizia lebbeck) has anti-inflammatory, analgesic activities.[18]Yastimadhu (Glycyrrhiza glabra) is anti-inflammatory, anti-arthritic and antioxidant.[19],[20],[21]Tagara (Valeriana wallichii) is analgesic.[22]Raktachandana (Pterocarpus santalinus) is anti-inflammatory, analgesic, and antioxidant.[23],[24]Jatamansi (Nardostachys grandiflora) is antioxidant.[25]Haridra (Curcuma longa) is anti-inflammatory, antirheumatic, analgesic, and antioxidant.[26],[27],[28],[29],[30]Daruharidra (Berberis aristata) is anti-inflammatory.[20]Kushta (Saussurea lappa) is anti-inflammatory, anti-arthritic, and antioxidant.[31],[32] These properties of individual drugs will contribute in expression of observed activity profile of the compound formulation.


   Conclusions Top


Dashanga Ghana possesses significant anti-arthritic activity and can be used in treating different forms of arthritis. Thus this study provides unequivocal evidence of traditional use of this formulation in the management of different inflammatory conditions including arthritis. However, further studies are required to identify and characterize exact active phytoconstituents and to elucidate the exact mechanism of action, which is responsible for the observed pharmacological profile with an intension to provide newer and safer treatment options with minimum side effects.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Soeken KL, Miller SA, Ernst E. Herbal medicines for the treatment of rheumatoid arthritis: A systematic review. Rheumatology (Oxford) 2003;42:652-9.  Back to cited text no. 1
    
2.
Sharangadhara, Sharangadhara Samhita, Uttara Khanda 11/4-6. 4th ed. Varanasi: Chaukhambha Orientalia; 2000.  Back to cited text no. 2
    
3.
Paget GE, Barnes JM. Evaluation of drug activities. In: Lawrence DR, Bacharach AL, editors. Pharmacometrics. Vol. 1. New York: Academic Press; 1964. p. 161.  Back to cited text no. 3
    
4.
Rosenthale ME. A comparative study of the Lewis and Sprague Dawley rat in adjuvant arthritis. Arch Int Pharmacodyn Ther 1970;188:14-22.  Back to cited text no. 4
    
5.
Tiffany, Talke HN, Schubert GE. Influence of non-enzymatic antioxidants on antioxidation status in acute haemorrhagic Necrotizing pancreatitis in rat. Bull Vet Inst Pulawy. 2005;49:133–9.  Back to cited text no. 5
    
6.
Slot C. Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest 1965;17:381-7.  Back to cited text no. 6
    
7.
Tietz NW. Clinical Guide to Laboratory Tests. 3rd ed. Philadelphia, PA: WB Saunders; 1995. p. 76.  Back to cited text no. 7
    
8.
Burtis CA, Ashwood ER. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, PA: WB Saunders; 1999. p. 652.  Back to cited text no. 8
    
9.
Wilkinson JH, Boutwell JH, Winsten S. Evaluation of a new system for the kinetic measurement of serum alkaline phosphatase. Clin Chem 1969;15:487-95.  Back to cited text no. 9
    
10.
Raghuramulu N, Nair KM, Kalyanasundaram S. A Manual of Laboratory Techniques. Hyderabad: National Institute of Nutrition; 1983. p. 246-53.  Back to cited text no. 10
    
11.
Newbould BB. Chemotherapy of arthritis induced in rats by mycobacterial adjuvant. Br J Pharmacol Chemother 1963;21:127-36.  Back to cited text no. 11
    
12.
Baumgartner WA, Beck FW, Lorber A, Pearson CM, Whitehouse MW. Adjuvant disease in rats: Biochemical criteria for distinguishing several phases of inflammation and arthritis. Proc Soc Exp Biol Med 1974;145:625-30.  Back to cited text no. 12
    
13.
Winder CV, Lembke LA, Stephens MD. Comparative bioassay of drugs in adjuvant-induced arthritis in rats: Flufenamic acid, mefenamic acid, and phenylbutazone. Arthritis Rheum 1969;12:472-82.  Back to cited text no. 13
    
14.
Patil KS, Suryavanshi J. Effect of Celastrus paniculatus Willd. Seed on adjuvant induced arthritis in rats. Pharmacogn Mag 2007;3:177-81.  Back to cited text no. 14
    
15.
Colpaert FC. Evidence that adjuvant arthritis in the rat is associated with chronic pain. Pain 1987;28:201-22.  Back to cited text no. 15
    
16.
Yassuda Filho P, Bracht A, Ishii-Iwamoto EL, Lousano SH, Bracht L, Kelmer-Bracht AM. The urea cycle in the liver of arthritic rats. Mol Cell Biochem 2003;243:97-106.  Back to cited text no. 16
    
17.
Galib R, Patgiri BJ, Prajapati PK, Ashok BK, Ravishankar B. Anti-inflammatory and analgesic activities of Dashanga Ghana: An Ayurvedic compound formulation. Int J Nutr Pharmacol Neurol Dis 2003;3:303-8.  Back to cited text no. 17
    
18.
Saha A, Ahmed M. The analgesic and anti-inflammatory activities of the extract of Albizia lebbeck in animal model. Pak J Pharm Sci 2009;22:74-7.  Back to cited text no. 18
    
19.
Akamatsu H, Komura J, Asada Y, Niwa Y. Mechanism of Anti-Inflammatory Action of Glycyrrhizin: Effect on Neutrophil Functions Including Reactive Oxygen Species Generation. Planta Med 1991; 57: 119-21.  Back to cited text no. 19
    
20.
Singh RH. A critical analysis of the studies done on indigenous anti-inflammatory and anti-arthritic drugs during post-independence era. Rheumatism 1978;13:99-108.  Back to cited text no. 20
    
21.
Rajesh MG, Latha MS. Protective activity of Glycyrrhiza glabra Linn. on carbon tetrachloride induced peroxidative damage. Indian J Pharmacol 2004;36:284-7.  Back to cited text no. 21
  Medknow Journal  
22.
Sah SP, Mathela CS, Chopra K. Elucidation of possible mechanism of analgesic action of Valeriana wallichii DC chemotype (patchouli alcohol) in experimental animal models. Indian J Exp Biol 2010;48:289-93.  Back to cited text no. 22
    
23.
Potnis VV. Anti-inflammatory activity of the creams containing turmeric and red sandal wood. Indian Drugs 1994;31:117-8.  Back to cited text no. 23
    
24.
Scartezzini P, Speroni E. Review on some plants of Indian traditional medicine with antioxidant activity. J Ethnopharmacol 2000;71:23-43.  Back to cited text no. 24
    
25.
Tripathi YB, Tripathi E, Upadhyay A. Antilipid peroxidative property of Nardostachys jatamanasi. Indian J Exp Biol 1996;34:1150-1.  Back to cited text no. 25
    
26.
Kohli K, Ali J, Ansari MJ, Raheman Z. Curcumin – A natural Anti Inflammatory Agent. Editorial Forum. Indian J Pharmacol 2005;37:141-7.  Back to cited text no. 26
  Medknow Journal  
27.
Deodhar SD, Sethi R, Srimal RC. Preliminary study on antirheumatic activity of curcumin (diferuloyl methane). Indian J Med Res 1980;71:632-4.  Back to cited text no. 27
    
28.
Chandra D, Gupta SS. Anti-inflammatory and anti-arthritic activity of volatile oil of Curcuma longa (Haldi). Indian J Med Res 1972;60:138-42.  Back to cited text no. 28
    
29.
Jacob A, Wu R, Zhou M, Wang P. Mechanism of the Anti-inflammatory Effect of Curcumin: PPAR-γ Activation. PPAR Research 2007;1-5.  Back to cited text no. 29
    
30.
Mullaicharam AR, Maheswaran A. Pharmacological effects of Curcumin. Int J Nutr Pharmacol Neurol Dis 2012;2:92-9.  Back to cited text no. 30
  Medknow Journal  
31.
Cho JY, Baik KU, Jung JH, Park MH.In vitro anti-inflammatory effects of cynaropicrin, a sesquiterpene lactone, from Saussurea lappa. Eur J Pharmacol 2000;398:399-407.  Back to cited text no. 31
    
32.
Gokhale AB, Damre AS, Kulkami KR, Saraf MN. Preliminary evaluation of anti-inflammatory and anti-arthritic activity of S. lappa, A. speciosa and A. aspera. Phytomedicine 2002;9:433-7.  Back to cited text no. 32
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


This article has been cited by
1 Protective effects of total flavonoids of Astragalus against adjuvant-induced arthritis in rats by regulating OPG/RANKL/NF-?B pathway
Xin-Yu Liu,Lu Xu,Ying Wang,Jin-Xia Li,Yu Zhang,Chong Zhang,Sha-Sha Wang,Xue-Mei Zhang
International Immunopharmacology. 2017; 44: 105
[Pubmed] | [DOI]



 

Top
  
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusions
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed1686    
    Printed16    
    Emailed0    
    PDF Downloaded45    
    Comments [Add]    
    Cited by others 1    

Recommend this journal