Users Online: 903

Home Print this page Email this page Small font sizeDefault font sizeIncrease font size

Home | About us | Editorial board | Search | Ahead of print | Current issue | Archives | Submit article | Instructions | Subscribe | Contacts | Login 

   Table of Contents      
Year : 2013  |  Volume : 3  |  Issue : 3  |  Page : 289-293

Diosgenin prevents hepatic oxidative stress, lipid peroxidation and molecular alterations in chronic renal failure rats

1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India
2 Department of Physics, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India

Date of Submission29-Jan-2013
Date of Acceptance13-Feb-2013
Date of Web Publication10-Jul-2013

Correspondence Address:
Elumalai Balamurugan
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2231-0738.114870

Rights and Permissions

Aim: Chronic renal failure (CRF) is one of the major contributors of cardiovascular pathological events and CRF associated uremic condition in rats elevates liver oxidative stress. The aim of the present study was to evaluate the preventive potential of diosgenin on antioxidant system and molecular protection potential in liver of CRF rats. Materials and Methods: CRF in rats was induced by feeding rats with diet containing 0.75% adenine for 5 weeks. Diosgenin was given orally at a dose of 40 mg/kg body weight (bw) of animal each and every day. The activities of antioxidant enzymes and lipid peroxidation level were determined by biochemical assays. Fourier transform infrared spectroscopy (FTIR) was employed to illustrate the molecular protection potential of diosgenin. Results: This study has shown adenine containing diet induced CRF in rats elevates liver oxidative stress by suppressing the activity of enzymatic antioxidant system, increased lipid peroxidation, and macromolecular structural alterations. In this study, treatment of diosgenin 40 mg/kg bw of rat significantly restores the enzymatic antioxidant system and reduces the lipid peroxidation level. Moreover, based on the FTIR study, we confirmed that diosgenin administration significantly protected the macromolecular changes including, the protein structural damage that occurred in liver. Conclusion: This study has proved the hepato protective action of diosgenin through antioxidant and molecular protection effect in CRF condition.

Keywords: Antioxidant, chronic renal failure, diosgenin, fourier transform infrared spectroscopy

How to cite this article:
Manivannan J, Arunagiri P, Sivasubramanian J, Balamurugan E. Diosgenin prevents hepatic oxidative stress, lipid peroxidation and molecular alterations in chronic renal failure rats. Int J Nutr Pharmacol Neurol Dis 2013;3:289-93

How to cite this URL:
Manivannan J, Arunagiri P, Sivasubramanian J, Balamurugan E. Diosgenin prevents hepatic oxidative stress, lipid peroxidation and molecular alterations in chronic renal failure rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2022 Aug 17];3:289-93. Available from:

   Introduction Top

Cardiovascular (CV) complications are the leading cause of death in patients with chronic kidney disease (CKD). [1] The elevated parathyroid hormone and disordered mineral metabolism (hyperphosphatemia) associated with secondary hyperparathyroidism complicate the clinical course of most patients with CKD and when advanced, are associated with markedly increased morbidity and mortality. [2]

Recent findings demonstrated that acute renal failure after kidney injury or bilateral nephrectomy triggers oxidative stress and causes damage to the liver and there was a gender difference with regard to the severity of hepatic oxidative stress and inflammatory response in acute uremia. [3] Chronic renal failure (CRF) patients have an increased plasma level of urea, which can be a source of cyanate, which can cause protein carbamoylation thereby changing biological activity of proteins. Therefore, in renal failure patients, cyanate can disturb metabolism and functioning of the liver. [4]

A recent evidence have shown that the most important frontline defense system composed of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) actively prevents the oxidative stress generated in liver during renal failure related pathological conditions with the help of an antioxidant molecule. [5] When reactive oxygen species (ROS) are produced at high-levels, mitochondrial derived ROS also causes deleterious effects by massive and irreversible oxidation of their principal targets (lipids, proteins, and DNA). [6]

Diosgenin is a naturally occurring steroidal saponin present in a variety of plants including, fenugreek (Trigonella foenum graecum) and roots of wild yam (Dioscorea villosa). [7] Molecular structure of diosgenin was shown in [Figure 1]. Diosgenin has been used in traditional medicine as an anti-hypercholesterolemia, anti-hypertriacylglycerolemia, anti-diabetes, and anti-hyperglycemia agent. [7],[8],[9],[10] Recent study on effect of diosgenin on vascular smooth muscle cells under Tumor necrosis factor (TNF-α) induced condition reports that, diosgenin abrogated TNF-α induced production of intracellular ROS and phosphorylation of mitogen activated protein kinases (MAPK). [11] Another finding suggested that diosgenin could have a beneficial role against aortic remodeling induced by oxidative stress in diabetic state, which was evidenced from the propensity of diosgenin to modulate the antioxidant defense and to decrease the lipid peroxidation in aorta. [12] Further, a study on membrane stabilizing effect of diosgenin, in experimentally induced myocardial infarction shown that anti lipid peroxidative potential of diosgenin may be due to its anti-oxidative activity. [13]
Figure 1: Structure of diosgenin

Click here to view

From this point of view, this present study intend to evaluate the preventive potential of diosgenin against CRF induced hepatic oxidative stress by means of fourier transform infrared spectroscopy (FTIR). A previous study reports that protective role of borneol, a natural terpene on liver metabolism in a nitric oxide deficient model of hypertension through interpretation of FTIR spectral information and results demonstrate that FTIR can successfully indicate the molecular changes that occur in liver during oxidative stress. [14] In this study, activity of enzymatic antioxidants, level of lipid peroxidation products, and macromolecular structure related alterations in liver of CRF rats treated with diosgenin were assessed.

   Materials and Methods Top

Animals and chemicals

Male albino Wistar rats, 7-10 weeks old (180-220 g) were procured from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University. The experimental study was approved by the Ethical Committee of Rajah Muthiah Medical College and Hospital, Annamalai Nagar, Tamil Nadu, India. Diosgenin was purchased from Sigma Aldrich (USA) and all other chemicals in this study used were of analytical grade.

Experimental groups and diosgenin treatment

CRF in Wistar rats was induced by fed the animals with diet containing 0.75% adenine for 5 weeks. Each of the following groups were consists of six animals. Diosgenin 40 mg/kg was administered orally every day by dissolved in oil as vehicle throughout the experiment.

Group I: Control (animals fed with rat chow).

Group II: Control + diosgenin 40 mg/kg body weight (bw) of animals.

Group III: Control animals fed with 0.75% adenine-CRF animals.

Group IV: CRF + diosgenin 40 mg/kg bw of animals.

Liver tissue homogenization

Liver tissues were sliced into pieces and homogenized in appropriate buffer in cold condition (pH 7.0) to give 20% homogenate (w/v). The homogenate was centrifuged at 560 × g for 10 min at 4°C in refrigerated centrifuge. The supernatant was separated and used for various biochemical estimations.

Antioxidant enzyme activity and lipid peroxidation products in liver

Liver antioxidant enzymes assay and lipid peroxidation assays were done for all the groups . SOD, CAT, GPx, were assayed with the methods of Kakkar et al., [15] Sinha, [16] Rotruck et al. [17] respectively. Total protein was assayed by the method of Lowry et al. [18] The level of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides were estimated by the methods of Niehaus and Samuelson, [19] Jiang et al. [20]

FTIR analysis

FTIR analysis was carried out as described previously. [21] A small and equal amount of liver samples were homogenized and freeze dried. Samples were stored under −80°C until used. For FTIR analysis, the samples were mixed with Potassium Bromide (KBr) at ratio of 1:100. The mixture was then subjected to a pressure of 1,100 kg/cm 2 to produce KBr pellets for use in FTIR spectrometer. Pellets of the same thickness were prepared by taking the same amount of sample and applying the same pressure. FTIR spectra of the region 4,000-400 cm−1 were recorded at the temperature of 25 ± 1°C on a Nicolet-Avatar-360 FTIR spectrometer.

Statistical analysis

Values are given as mean ± SD for six rats in each group. Data were analyzed by one-way analysis of variance followed by Duncan's multiple range test using SPSS version 11.5 (SPSS, Chicago, IL). The limit of statistical significance was set at P < 0.05.

   Results Top

Effect of diosgenin on enzymatic antioxidants and lipid peroxidation products

The activities of SOD, CAT, and GPx in liver of all the groups are presented in [Table 1]. The activities of these enzymatic antioxidants significantly decreased in CRF rats. Treatment with 40 mg/kg diosgenin significantly ( P < 0.05) restored the activity of these enzymatic antioxidants in liver.

[Table 1] also portrays the levels of TBARS, lipid hydro peroxides. Levels of lipid peroxidation products in liver were significantly elevated in CRF rats, whereas treatment of diosgenin significantly reduced the lipid peroxidation products.
Table 1: Effect of diosgenin on liver enzymatic antioxidants and lipid peroxidation

Click here to view

Molecular alterations

The representative FTIR spectra of the control liver have shown important macromolecular bonding frequency regions [Figure 2]. The spectral wave numbers were assigned from previous studies. [14],[22] Frequency shifts of macromolecules were illustrated in [Table 2]. The bands in 3,393 cm−1 region arise from N-H and O-H stretching modes of proteins, and intermolecular hydrogen bonding. In the experiment, Amide A appeared at 3,393 cm−1 in control and the band frequency was significantly shift in renal failure rats, whereas the diosgenin treatment protects the changes significantly and there was no significant change between control and diosgenin alone treated group. CH 2 symmetric stretching of lipids/fatty acids appears at 2853 cm−1 in control and in renal failure it was shifted increase in wave number significantly. However, the diosgenin treated group was significantly brought back the shifted peak to control level. The most important molecular bonds of proteins, amide linkages were significantly altered in renal failure group, in which the Amide I band was appeared in 1650 cm−1 in control. In the wave number of Amide II linkage, the control group appeared at 1542 cm − 1 and the significant change in frequency shift was observed in CRF rat liver. However, this change was significantly protected by diosgenin in Amide I but not in Amide II region.
Figure 2: Fourier transform infrared spectroscopy of control liver with important macromolecular bond frequency regions

Click here to view
Table 2: Effect of diosgenin on wave number alterations in liver tissue

Click here to view

   Discussion Top

The high-rate of CV complications observed in hemodialysis patients is only partly explained by traditional risk-factors, such as ageing, gender, hypertension, diabetes, smoking, dyslipidemia, and obesity. Indeed, non-traditional risk-factors including, malnutrition, inflammation, and oxidative stress have emerged. [23],[24] The oxidative stress generated during renal failure after kidney injury or bilateral nephrectomy activates oxidative stress and causes damage to the liver. [3] In this study, the enzymatic antioxidant system was focused in liver during the CRF condition.

Free radical scavenging enzymes such as SOD, CAT, and GPx are the first line of cellular defense against oxidative injury. SOD reduces superoxide anion to form H 2 O 2 and oxygen. CAT removes H 2 O 2 by breaking it down directly to oxygen. [5] The observed declined activities of SOD, CAT in renal failure rat liver may be due to the involvement of free radicals and oxidative stress, which is consistent with a previous study. [3] A decrease in activity of these enzymes leads to accumulation of superoxide anion and H 2 O 2 which in turn can form the toxic hydroxyl radical. Reduced activities of GPx might be due to reduced substrate because of increased utilization for protecting proteins from oxidative damage. Our results show that diosgenin prevented the decrease in the activity of all the above enzymic antioxidants in liver tissue of renal failure rats.

Lipid peroxidation, arising from the reaction of free radicals with lipids, has been linked with altered membrane structure and enzyme inactivation. Its end products measured as TBARS, lipid hydroperoxides were seen increased in liver tissue clearly indicating increased oxidative stress in renal failure. Diosgenin inhibits lipid peroxidation, which may be due to its radical scavenging and anti lipidperoxidative property. [13]

The FTIR spectroscopy monitors the vibration modes of functional groups present in proteins, lipids, polysaccharides, and nucleic acids in liver tissue. Shifts in peak positions indicate the molecular changes associated with macromolecules in a condition. In this analysis, the N-H stretching of proteins was changed significantly during renal failure, this indicates that the intra molecular hydrogen bonding in proteins of liver tissue may be disturbed. [14] This effect might be due to the oxidative stress generated during uremic condition as previously reported. [3] This effect was protected by diosgenin treated group indicates its antioxidant potential against oxidative stress generated during renal failure.

The wave number of fatty acid species changed significantly in renal failure liver, this indicates the acyl chain modification of membrane lipids. The frequency of the CH 2 bands of acyl chains depends on the degree of conformational disorder and level of flexibility. The position of these bands provides information about the lipid acyl chain flexibility (order/disorder state of lipids). [25] The shift of the peak position of these bands to higher values indicates that increase lipid order and acyl chain flexibility. A previous study indicates that this kind of changes may associate with oxidative stress generated in liver. [14] The above shift was significantly protected by diosgenin which indicates its protective effect on lipid species through its antioxidant and anti-lipid peroxidative potential in liver as already reported in aorta. [12] This effect further supports anti-lipid peroxidation potential of diosgenin on liver in uremia related oxidative stress and lipid peroxidation in an indirect way, which is consistent with lipid peroxidation parameter results.

Amide bands are the most important indicators of oxidative stress. The shift in Amide I and Amide II in renal failure group liver compared with control indicates the excess production of free radicals and production of free radical associated protein damage. This structural change might also occur due to elevated urea induced cyanate related toxicity as described previously. [4] However, diosgenin treatment significantly protects the Amide I and Amide II associated changes. According to findings of a previous study, all the constituent amino acid side-chains in proteins are susceptible to free radicals, but some are more vulnerable than others. Thus, exposure of proteins to free radical generating systems may induce secondary structural changes. Secondary structure is stabilized by hydrogen bonding of the peptide backbone and interference with the functional groups of the peptide bonds may cause structural modifications. Further the shift in Amide I and II regions corresponds to the alpha-helix protein conformational change. [26] The above effect may directly indicate the antioxidant potential of diosgenin, which actively scavenge free radicals and activate the defense system through which it protects the macromolecular damage and liver dysfunction.

Previous studies have shown that, natural products have promising strategy to protect tissue from oxidative stress and lipid peroxidation under various pathological conditions. [27],[28],[29] Consistent with the previous studies, [30],[31] this work also proven that natural molecules enhances the activity of enzymatic antioxidants thereby protects the tissue against oxidative stress.

   Conclusion Top

Over all study proves that the antioxidant potential of diosgenin prevents oxidative stress, lipid peroxidation mediated hepatic damage and macromolecular structural alterations in CRF rats. Thus, in future, diosgenin can be used as a hepato protective agent in renal failure patients.

   References Top

1.Foley RN, Parfrey PS, Sarnak MJ. Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis 1998;32:S112-9.  Back to cited text no. 1
2.Block GA, Klassen PS, Lazarus JM, Ofsthun N, Lowrie EG, Chertow GM. Mineral metabolism, mortality, and morbidity in maintenance hemodialysis. J Am Soc Nephrol 2004;15:2208-18.  Back to cited text no. 2
3.Golab F, Kadkhodaee M, Xu J, Soleimani M. Male susceptibility to hepatic damage in acute uremia in rats. Urology 2011;78:232.e1-e6.  Back to cited text no. 3
4.Soko³owska M, Niedzielska E, Iciek M, Bilska A, Lorenc-Koci E, W³odek L. The effect of the uremic toxin cyanate (CNO⁻) on anaerobic cysteine metabolism and oxidative processes in the rat liver: A protective effect of lipoate. Toxicol Mech Methods 2011;21:473-8.  Back to cited text no. 4
5.Silambarasan T, Raja B. Diosmin, a bioflavonoid reverses alterations in blood pressure, nitric oxide, lipid peroxides and antioxidant status in DOCA-salt induced hypertensive rats. Eur J Pharmacol 2012;679:81-9.  Back to cited text no. 5
6.Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology. Physiol Rev 2007;87:245-313.  Back to cited text no. 6
7.Raju J, Mehta R. Cancer chemopreventive and therapeutic effects of diosgenin, a food saponin. Nutr Cancer 2009;61:27-35.  Back to cited text no. 7
8.Juarez-Oropeza MA, Diaz-Zagoya JC, Rabinowitz JL. In vivo and in vitro studies of hypocholesterolemic effects of diosgenin in rats. Int J Biochem 1987;19:679-83.  Back to cited text no. 8
9.McAnuff MA, Harding WW, Omoruyi FO, Jacobs H, Morrison EY, Asemota HN. Hypoglycemic effects of steroidal sapogenins isolated from Jamaican bitter yam, Dioscorea polygonoides. Food Chem Toxicol 2005;43:1667-72.  Back to cited text no. 9
10.Son IS, Kim JH, Sohn HY, Son KH, Kim JS, Kwon CS. Antioxidative and hypolipidemic effects of diosgenin, a steroidal saponin of yam (Dioscorea spp.), on high-cholesterol fed rats. Biosci Biotechnol Biochem 2007;71:3063-71.  Back to cited text no. 10
11.Choi KW, Park HJ, Jung DH, Kim TW, Park YM, Kim BO, et al. Inhibition of TNF-α-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-κB signaling pathways. Vascul Pharmacol 2010;53:273-80.  Back to cited text no. 11
12.Pari L, Monisha P, Mohamed Jalaludeen A. Beneficial role of diosgenin on oxidative stress in aorta of streptozotocin induced diabetic rats. Eur J Pharmacol 2012;691:143-50.  Back to cited text no. 12
13.Jayachandran KS, Vasanthi HR, Rajamanickam GV. Antilipoperoxidative and membrane stabilizing effect of diosgenin, in experimentally induced myocardial infarction. Mol Cell Biochem 2009;327:203-10.  Back to cited text no. 13
14.Saravanakumar M, Manivannan J, Sivasubramanian J, Silambarasan T, Balamurugan E, Raja B. Molecular metabolic fingerprinting approach to investigate the effects of borneol on metabolic alterations in the liver of nitric oxide deficient hypertensive rats. Mol Cell Biochem 2012;362:203-9.  Back to cited text no. 14
15.Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984;21:130-2.  Back to cited text no. 15
16.Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972;47:389-94.  Back to cited text no. 16
17.Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973;179:588-90.  Back to cited text no. 17
18.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.  Back to cited text no. 18
19.Niehaus WG Jr, Samuelsson B. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968;6:126-30.  Back to cited text no. 19
20.Jiang ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem 1992;202:384-9.  Back to cited text no. 20
21.Sivakumar S, Sivasubramanian J, Raja B. Aluminium induced structural, metabolic alterations and protective effects of desferrioxamine in the brain tissue of mice: An FTIR study. Spectrochim Acta A Mol Biomol Spectrosc 2012;99:252-58.  Back to cited text no. 21
22.Cakmak G, Togan I, Severcan F. 17Beta-estradiol induced compositional, structural and functional changes in rainbow trout liver, revealed by FT-IR spectroscopy: A comparative study with nonylphenol. Aquat Toxicol 2006;77:53-63.  Back to cited text no. 22
23.Terrier N, Senécal L, Dupuy AM, Jaussent I, Delcourt C, Leray H, et al. Association between novel indices of malnutrition-inflammation complex syndrome and cardiovascular disease in hemodialysis patients. Hemodial Int 2005;9:159-68.  Back to cited text no. 23
24.Nanayakkara PW, Gaillard CA. Vascular disease and chronic renal failure: New insights. Neth J Med 2010;68:5-14.  Back to cited text no. 24
25.Severcan F. Vitamin E decreases the order of the phospholipid model membranes in the gel phase: An FTIR study. Biosci Rep 1997;17:231-5.  Back to cited text no. 25
26.Rice-Evans CA, Diplock AT, Symos MCR. Techniques in Free Radical Research. In: Burdon RH, van Knippenberg PH, eds. Laboratory techniques in biochemistry and molecular biology. New York: Elsevier; 1991. p. 207-21.  Back to cited text no. 26
27.Mullaicharam AR, Maheswaran A. Pharmacological effects of curcumin. Int J Nutr Pharmacol Neurol Dis 2012;2:92-9.  Back to cited text no. 27
  Medknow Journal  
28.Bhandari PR. Pomegranate (Punica granatum L). Ancient seeds for modern cure? Review of potential therapeutic applications. Int J Nutr Pharmacol Neurol Dis 2012;2:171-84.  Back to cited text no. 28
29.Johary A, Jain V, Misra S. Role of lycopene in the prevention of cancer. Int J Nutr Pharmacol Neurol Dis 2012;2:167-70.  Back to cited text no. 29
  Medknow Journal  
30.Begum N, Prasad NR, Thayalan K. Apigenin protects gamma-radiation induced oxidative stress, hematological changes and animal survival in whole body irradiated Swiss albino mice. Int J Nutr Pharmacol Neurol Dis 2012;2:45-52.  Back to cited text no. 30
  Medknow Journal  
31.Subash S, Subramanian P. Impact of morin (a bioflavonoid) on ammonium chloride-mediated oxidative damage in rat kidney. Int J Nutr Pharmacol Neurol Dis 2011;1:174-8.  Back to cited text no. 31
  Medknow Journal  


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]

This article has been cited by
1 Al2O3 nanoparticles trigger the embryonic hepatotoxic response and potentiate TNF-a-induced apoptosis—modulatory effect of p38 MAPK and JNK inhibitors
Gobichettipalayam Balasubramaniam Maadurshni, Ganeshmurthy Kanniamal Tharani, Inbamani Udayakumar, Manigandan Nagarajan, Jeganathan Manivannan
Environmental Science and Pollution Research. 2022;
[Pubmed] | [DOI]
2 Diosgenin Modulates Oxidative Stress and Inflammation in High-Fat Diet-Induced Obesity in Mice
Sahar Khateeb, Aishah Albalawi, Adel Alkhedaide
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 2022; Volume 15: 1589
[Pubmed] | [DOI]
3 Exposure to a “safe” dose of environmental pollutant bisphenol A elevates oxidative stress and modulates vasoactive system in hypertensive rats
Manigandan Nagarajan, Boobalan Raja, Jeganathan Manivannan
Human & Experimental Toxicology. 2021; : 0960327121
[Pubmed] | [DOI]
4 Antiglycation and antioxidant activities of the crude extract and saponin fraction of Tribulus terrestris before and after microcapsule release
Célia Cristina Malaguti Figueiredo, Amanda da Costa Gomes, Filipe Oliveira Granero, João Luiz Bronzel Junior, Luciana Pereira Silva, Valdecir Farias Ximenes, Regildo Márcio Gonçalves da Silva
Journal of Integrative Medicine. 2021;
[Pubmed] | [DOI]
5 Diosgenin: Recent Highlights on Pharmacology and Analytical Methodology
Mafalda Jesus,Ana P. J. Martins,Eugenia Gallardo,Samuel Silvestre
Journal of Analytical Methods in Chemistry. 2016; 2016: 1
[Pubmed] | [DOI]


    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
    Materials and Me...
    Article Figures
    Article Tables

 Article Access Statistics
    PDF Downloaded323    
    Comments [Add]    
    Cited by others 5    

Recommend this journal