Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 15  |  Issue : 3  |  Page : 150-157

Dissolution rate enhancement of irbesartan and development of fast-dissolving tablets


Department of Pharmaceutics, Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Chowdavaram, Guntur, Andhra Pradesh, India

Date of Submission25-Apr-2016
Date of Acceptance23-Jun-2016
Date of Web Publication5-Jan-2017

Correspondence Address:
Sasidhar R Lankapalli
Department of Pharmaceutics, Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Guntur 522019, Andhra Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-4315.197583

Rights and Permissions
  Abstract 

Objective
The objective of the present investigation was to enhance the solubility and dissolution rate of poorly soluble drug irbesartan by preparing it as solid dispersions and formulating it as fast-dissolving tablets (FDTs) using various excipients.
Background
Irbesartan is poorly soluble in water, and this low aqueous solubility in addition to its poor wettability leads to poor bioavailability of the drug.
Materials and methods
Solid dispersions were prepared using Soluplus, PEG 6000, and Kollidon as carriers. The dispersions were prepared using the solvent evaporation and kneading methods in a 1:1 ratio of drug and carrier. These formulations were characterized for solid state properties using X-ray powder diffraction and Fourier transform infrared spectroscopy spectral studies. Formulations were further evaluated for dissolution.
Results
The aqueous solubility of irbesartan in solid dispersions was improved by the presence of polymer Soluplus when compared with other carriers. Solid state characterization indicated that irbesartan was present as amorphous material in the formulation with carrier. This was due to efficient entrapment of the drug in polymer matrix. Thus, the solid dispersion prepared with Soluplus would be useful for delivering poorly soluble irbesartan with enhanced solubility and dissolution rate. Furthermore, the solid dispersions that were formulated as FDTs using superdisintegrants showed faster drug release with increased dissolution rate.
Conclusion
FDTs containing irbesartan solid dispersions prepared using the solvent evaporation method and croscarmellose sodium as superdisintegrant showed faster disintegration and increased dissolution rate.

Keywords: fast dissolving, irbesartan, solid dispersions, soluplus


How to cite this article:
Suryadevara V, Lankapalli SR, Sunkara S, Sakhamuri V, Danda H. Dissolution rate enhancement of irbesartan and development of fast-dissolving tablets. Egypt Pharmaceut J 2016;15:150-7

How to cite this URL:
Suryadevara V, Lankapalli SR, Sunkara S, Sakhamuri V, Danda H. Dissolution rate enhancement of irbesartan and development of fast-dissolving tablets. Egypt Pharmaceut J [serial online] 2016 [cited 2020 Apr 8];15:150-7. Available from: http://www.epj.eg.net/text.asp?2016/15/3/150/197583


  Introduction Top


Although various novel and advanced drug delivery systems have been introduced for therapeutic use, the popularity of oral dosage forms, particularly tablets, have not been eclipsed, because of its numerous advantages [1]. Two widely faced drawbacks in oral drug delivery are dysphagia and delivery of unpalatable drugs, which may be a problem for mainly geriatric, pediatric, and for nauseous patients [2]. Therefore, emphasis is laid on the development of viable dosage alternatives that has led to exploration of certain novel drug delivery systems such as fast-dissolving drug delivery. It is estimated that 50% of the population is affected by the said problem, which results in a high incidence of noncompliance and ineffective therapy. Traditional tablets and capsules administered with 250 ml of water may be inconvenient or impractical for such population. Hence, fast-dissolving/disintegrating tablets (FDDTs) are a perfect fit for them. FDDTs dissolve or more commonly disintegrate rapidly in the saliva without the aid of water [3],[4],[5]. The fast-dissolving/disintegrating dosage forms are well established in the management of pain, inflammation, vomiting, headache, and hypertension. Valuable research reports for formulation of rapidly disintegrating tablets are available. Moreover, various technologies for improving dissolution property of poorly water-soluble drugs have been documented to enhance bioavailability following oral absorption. Solubility of poorly soluble drug can be increased by formulating as solid dispersions using various excipients. Irbesartan is indicated for the treatment of hypertension. It may also delay progression of diabetic nephropathy and is also indicated for the reduction of renal disease progression in patients with type 2 diabetes,

hypertension, and microalbuminuria or proteinuria. Irbesartan shows low, pH-dependent solubility and is rapidly and completely bioactivated by ester hydrolysis to irbesartan during absorption from the gastrointestinal tract. Irbesartan has a terminal elimination half-life (t1/2) of ∼11‑15 h [6]. Soluplus is a novel polymer specially designed for solid solutions. Soluplus can increase the solubility and bioavailability of poorly soluble drugs. Soluplus is a polyvinyl caprolactum (57%)–polyvinyl acetate (30%)–poly ethylene glycol (PEG 6000–13%) graft copolymer. It is a polymeric solubilizer with an amphiphilic chemical structure, and because of its bifunctional characters it is able to act as a matrix polymer for solid solutions on one hand and on the other it is capable of solubilizing poorly soluble drugs in aqueous media [7],[8].

As Kollidon VA64 aides in stabilizing the active ingredient in amorphous form, solubility is improved. Kollidon VA64 is vinylpyrrolidone–vinyl acetate copolymer soluble in water and alcohol. The main applications of Kollidon VA64 are soluble binder for granulation and as a dry binder in direct compression technology, as a film-forming agent in sprays and as a pore former in coating and taste-masking applications, as well as solubilizer [9].

The aim of this work was to enhance the aqueous solubility and dissolution rate of irbesartan with carriers such as Kollidon VA64, Soluplus, and PEG 6000 using the solid dispersion technique and formulating the dispersions as FDDTs using superdisintegrants such as sodium starch glycolate (SSG), croscarmellose sodium (CCS) and evaluate the prepared FDDTs for their physiochemical parameters, in-vitro dissolution, and stability studies.


  Materials and methods Top


Materials

Irbesartan was obtained as a gift sample from Apotex Pharma Ltd (Bangalore, Karnataka, India). KollidonVA6 and Soluplus were obtained as a gift sample from Mylan Loboratories (Hyderabad, Telangana, India). SSG and CCS were commercially obtained from SD Fine-Chem Ltd (Mumbai, Maharashtra, India).

Saturated solubility studies

Saturated solubility studies of irbesartan were performed in different dissolution media. Irbesartan of 10 mg was weighed and transferred into different conical flasks containing 10 ml of different dissolution media (i.e. water pH 6.8, pH 7.2 phosphate buffer, and 0.1 N HCl) and were closed appropriately. All conical flasks were placed in a REMI incubator (ELLITE Scientific) shaker at 50 rpm and 37±1°C for 24 h. The conical flasks were removed from the incubator shaker and samples were filtered using Whatman filter paper. The clear solution obtained by means of filtration was suitably diluted with appropriate dissolution media and the absorbance values were noted at 244 nm using corresponding dissolution media as blank [10],[11].

Preparation of solid dispersion

The solid dispersions of irbesartan were prepared using the solvent evaporation and kneading methods using Soluplus, Kollidon, and PEG 6000 as carriers. Irbesartan, Soluplus, Kollidon, and PEG 6000 were weighed accurately in a ratio of 1:1 for drug and carrier. The obtained solid dispersions were subjected to X-ray powder diffraction (XRD) studies to confirm the conversion of crystalline drug into amorphous form and Fourier transform infrared spectroscopy for drug–carrier interaction studies. The drug content, solubility studies, dissolution, and stability studies were carried out for solid dispersions prepared [11],[12].

Characterization of irbesartan solid dispersions

X-ray powder diffraction

The powder crystallinity of irbesartan and its solid dispersions was determined using Bruker D8 Advance XRD with copper target instrument. The conditions were maintained at 40 kV voltages, with 40 mA current at room temperature. The scanning rate used was 0.1°/s over a range of 2θ values from 3° to 45°. The diffractograms are shown in [Figure 1].
Figure 1: PXRD data of Irbesartan Pure Drug, Kollidon VA64, solid dispersions prepared with Soluplus and Kollidon using the solvent evaporation method.

Click here to view


Differential scanning calorimetry

A differential scanning calorimeter (DSC 200F3; Shimadzu) was used to obtain the differential scanning calorimetry (DSC) curves of pure drug and formulations F4 and F8 representing the rates of heat flow. About 10 mg of sample was weighed in a standard open aluminum pan and were scanned from 40 to 400°C, at a heating rate of 10°C/min while being purged with dry nitrogen at a rate of 60 ml/min.

Drug content

Solid dispersions equivalent to 30 mg of drug were taken and dissolved in methanol and filtered using 0.45 μ membrane filters. Thereafter, the filtrate was suitably diluted with buffer, and drug content was analyzed against blank using a ultraviolet spectrophotometer at 244 nm. The concentration of drug present in solid dispersion is compared with that of standard solution containing 30 mg of pure drug. The percentage of drug present in the solid dispersions was calculated with respect to standard concentration obtained from calibration curve calculated for irbesartan.

In-vitro dissolution studies

Dissolution rate studies of pure irbesartan and irbesartan solid dispersions were performed in dissolution apparatus (LABINDIA DS8000) with rotating paddles at 50 rpm using 900 ml of pH 1.2 buffer and the temperature was maintained at 37±0.5°C throughout the experiment. A volume of 5 ml of the samples was drawn at various time intervals. The absorbance of the samples was measured at 244 nm for determining the amount of drug release at various intervals. Each time, the equal volume of buffer was replenished for maintaining the constant volume of dissolution medium. The dissolution studies were carried out in triplicate.

Preparation of irbesartan fast-dissolving tablets

Irbesartan fast-dissolving tablets (FDTs) were prepared using direct compression process, using optimized solid dispersion with superdisintegrant such as CCS and SSG along with microcrystalline cellulose as diluent. The compositions of various tablet formulations are given in [Table 2]. The prepared tablets were evaluated for various physical parameters and the results are given in [Table 3]. Physical parameters such as weight variation, hardness, friability, moisture uptake study, wetting time, water absorption ratio, and disintegration were evaluated for prepared tablets [13].
Table 1: Saturated solubility studies of irbesartan different dissolution media

Click here to view
Table 2: Composition of irbesartan fast-dissolving tablet formulations

Click here to view
Table 3: Physical<!--<query>Please provide footnote for ‘a’ given in Table [3].</query>--> parameters of irbesartan FDT formulations

Click here to view


Evaluation of fast-dissolving/disintegrating tablets

The moisture uptake study is carried out by keeping 10 tablets along with calcium chloride in a desiccator maintained at 37°C for 24 h to ensure complete drying of the tablets. The tablets are then weighed and exposed to 75% relative humidity (RH) at room temperature for 2 weeks. The required humidity can be achieved by keeping saturated sodium chloride solution in the desiccator for 24 h. The tablets are reweighed and the percentage increase in weight is recorded. If the moisture uptake tendency of a product is high, it requires special dehumidified area for manufacturing and packing [14].

Wetting time is determined by taking five circular tissue papers of 10 cm diameter and placing them in the petri dish with 10 cm diameter. A volume of 10 ml of water containing Amaranth water-soluble dye was added to the petri dish. One tablet was carefully placed on the surface of the tissue paper. The time required for water to reach the upper surface of the tablet was noted as the wetting time [15].

Water absorption ratio is determined by taking a piece of tissue paper folded twice and placed in a small petri dish (internal diameter=6.5 cm) containing 5 ml of buffer. A tablet was placed on the tissue paper. The wetted tablet was weighed. The test was carried out in triplicate. The water absorption ratio (R) was determined according to the following equation: water absorption ratio (R)=WaWb/Wa×100, where Wa is the weight of the tablet before the test and Wb is the weight of the tablet after water absorption [16].

Dispersion test of FDTs was carried out by taking a petri dish that was filled with 10 ml of buffer (pH 1.2) and the tablet was carefully placed in the center of the petri dish. and the time taken for the tablet to completely disperse into fine particles was noted [17].

In-vitro dissolution studies

Dissolution studies on each tablet formulation were performed in a calibrated 8 station dissolution test apparatus (LABINDIA DS8000) equipped with paddles (USP apparatus II method) using 900 ml of pH 1.2 buffer as a dissolution medium. The paddles were operated at 50 rpm and temperature was maintained at 37±1°C throughout the experiment. The samples (10 ml) were drawn at 5, 10, 15, 20, 30, and 45 min and replaced with an equal volume of the same dissolution medium to maintain the constant volume throughout the experiment. Samples drawn at various time intervals were suitably diluted with same dissolution medium and the amount of the drug dissolved was estimated using ELICO double beam UV spectrophotometer at 244 nm. The dissolution studies on each formulation were conducted in triplicate. From the dissolution profiles, various parameters such as T50, and DE30% were calculated. The dissolution parameter T50 was directly calculated from the dissolution profiles, whereas DE30% was estimated using trapezoidal rule to the dissolution profiles. The first order release rate constant was calculated by multiplying the slope value obtained from log percent drug undissolved versus time plot with 2.303.

Accelerated stability studies of irbesartan fast-dissolving tablets

The formulations that showed good in-vitro performance were subjected to accelerated stability studies. These studies were carried out by investigating the effect of temperature on the physical properties of tablets and chemical stability of tablets containing drugs. The tablet formulations such as F4 and F8 were subjected to accelerated stability studies. The above-mentioned formulations were kept in petri dishes after preparation and stored in thermostated oven at a temperature and RH of 25±2°C and 60±5% RH for 6 months and 40±2°C and 75±5% RH for 3 months, respectively. Thereafter, the tablets were evaluated for physical parameters and for drug content uniformity using a known spectrophotometric method as described earlier. These were further subjected to drug release studies.


  Results and discussion Top


Solubility studies

The effect of solubility of irbesartan in various media was assessed using saturation solubility studies. Saturated solubility studies revealed that irbesartan showed maximum solubility in 0.1 N HCl media compared with the other dissolution medium used. The drug concentration was measured at 244 nm using a ultraviolet spectrophotometer for all dissolution media. The results of solubility studies are given in [Table 1].

Preparation of solid dispersions

The solid dispersions of irbesartan were prepared with novel carriers such as Kollidon VA64, Soluplus, and PEG 6000 using the solvent evaporation and kneading methods. All dispersions were prepared under similar conditions to avoid batch-to-batch variations. The drug and carrier ratio was maintained as 1:1 constant for all formulations.

Characterization of solid dispersions

The XRD patterns of irbesartan and solid dispersions prepared with Soluplus and Kollidon as carrier using the solvent evaporation method are shown in [Figure 1]. The powder diffraction patterns of pure irbesartan showed characteristic high diffraction peaks. The diffraction patterns of solid dispersions prepared with Soluplus as carrier using the solvent evaporation method showed only few peaks with very weak intensities indicating the amorphous nature. DSC thermographic studies were carried out on irbesartan pure drug and solid dispersions prepared with Soluplus and Kollidon using the solvent evaporation method. These studies exhibited a sharp endothermic peak at 188.25°C for the pure drug irbesartan. For the solid dispersions, endothermic peaks were observed at 186.04 and 184.18°C. These studies revealed that there were no drug and excipient interactions, which were confirmed by obtaining similar thermographic peaks at respective temperature. Solid dispersions of irbesartan prepared with Soluplus and Kollidon VA64 also gave melting peaks but at slightly lower temperatures in the range of 184–186°C with a decrease in peak intensity as compared with pure irbesartan. This is consistent with a weak endothermic reaction between irbesartan and carriers. The decrease in intensity and broadening of peak indicates that the drug is dispersed with carrier. The DSC thermograms are shown in the [Figure 2],[Figure 3],[Figure 4].
Figure 2: DSC thermogram of irbesartan pure drug. DSC, differential scanning calorimetry.

Click here to view
Figure 3: DSC thermogram of irbesartan solid dispersions prepared with Soluplus. DSC, differential scanning calorimetry.

Click here to view
Figure 4: DSC thermogram of solid dispersions prepared with Kollidon. DSC, differential scanning calorimetry.

Click here to view


The dissolution studies for the prepared dispersions were carried out in 0.1 N HCl using the paddle method. The dissolution rate of the solid dispersions was more rapid compared with pure drug irbesartan. Among the prepared solid dispersions, the dispersions prepared with Soluplus as carrier using the solvent evaporation method released the drug at a faster rate compared with the other dispersions. The enhanced solubility and dissolution rate was due to intermolecular interactions and complexation originating from H bonding, ionic, and/or van der Waal’s interactions of hydrophilic carrier such as Soluplus with drug, which plays an important role in solubilization, stability, and maintaining supersaturation. Soluplus with multiple interaction sites increased the solubility of irbesartan in amorphous dispersions.

Preparation and evaluation of fast-dissolving tablets

The dispersions were prepared using the solvent evaporation method with Soluplus, Kollidon, and PEG 6000. Among the prepared solid dispersions, the dispersions prepared with Soluplus as carrier using the solvent evaporation method released the drug at a faster rate compared with the other dispersions. Hence, these dispersions were further formulated as FDTs using superdisintegrants such as CCS and SSG. The direct compression process was found to be suitable for compressing the tablet formulations as FDTs. The compositions of irbesartan tablet formulations are shown in the [Table 2]. All batches of tablets were compressed under identical conditions to minimize the processing variables. Thereafter, the compressed FDTs were further evaluated for physical parameters such as weight uniformity, hardness, friability, and drug content. These studies revealed that all tablet formulations were found to be stable and met I.P-specified limits for weight uniformity, friability, and drug content. The hardness of all tablet formulations was in the range of 2.5–3.0 kg/cm2. Weight uniformity of all tablet formulations was in the range of 198±3 mg/tablet. Friability losses of all tablet formulations were negligible and were in the range of 0.1–0.2%. Drug content estimated for all tablet formulations was highly uniform, with less than 2.5% variation. The results are given in [Table 3].

All tablet formulations were found to be stable within the I.P-specified limits for weight uniformity, friability, and drug content. Moisture uptake studies for FDTs were conducted to assess the stability of formulation. The results indicated that tablets containing a high concentration of superdisintegrant − that is, F4 and F8 (CCS 15%) − get softened and absorb more atmospheric moisture. The results are given in [Table 4].
Table 4: Evaluation parameters of irbesartan FDTs

Click here to view


The dissolution studies of FDTs were performed in 0.1 N HCl using the USP-II paddle method. All tablet formulations were found to release the drug at a faster rate compared with pure drug. The dissolution profiles of irbesartan FDDTs are shown in [Figure 5],[Figure 6],[Figure 7]. It was found that the tablet formulation F4 and F8 prepared with Soluplus and Kollidon with CCS 15% showed more rapid drug release when compared with pure drug and other formulations containing SSG. This can be attributed to improved wettability and dispersibility as well as increased amorphous fraction of drug. It was also found that, as the concentration of superdisintegrants increases, the tablets undergo rapid dissolution and drug release. This may be due to rapid intake of water by superdisintegrants, which leads to faster dissolution of the tablets and showed the improved dissolution profiles of poorly soluble irbesartan. The drug release of tablet formulations in the presence of various superdisintegrants was in the order of CCS>SSG. On the basis of the data obtained from the dissolution studies, various parameters such as T50, DE30%, and first order and zero order release rate constants were estimated. The dissolution rate of all formulations was found to be rapid when compared with pure irbesartan ([Figure 5],[Figure 6],[Figure 7]). The T50 and rate constants (K) values of the formulations indicated their rapid drug dissolution than that of pure drug. The kinetics of drug release from all formulations follow first order. The results of kinetic parameters evaluated are given in [Table 4].
Figure 5: Drug release profiles of irbesartan fast-dissolving tablet formulations.

Click here to view
Figure 6: Drug release profiles of irbesartan fast-dissolving tablet formulations.

Click here to view
Figure 7: Drug release profiles of irbesartan fast-dissolving tablet formulations.

Click here to view


Stability studies

The accelerated stability studies indicated that there were no visible and physical changes observed in the tablets after storage. It was also observed that there was no significant change in drug release from the tablets ([Table 5]). The drug release characteristics of the optimized tablets remained unaltered. Thus, the drug release characteristics of FDTs designed were found to be quite stable and are shown in [Figure 8].
Table 5: Dissolution parameters of irbesartan FDTs

Click here to view
Figure 8: Drug release profiles of optimized irbesartan fast-dissolving tablet formulations before and after stability studies.

Click here to view


Acknowledgements

The authors are thankful to Chebrolu Hanumaiah Institute of Pharmaceutical Sciences and Mylan Laboratories Ltd for providing the necessary research facilities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Ishikawa T, Watanabe Y, Utoguchi N, Matsumoto M. Preparation and evaluation of tablets rapidly disintegrating in saliva containing bitter-taste-masked granules by the compression method. Chem Pharm Bull (Tokyo) 1999; 47: 1451–1454.  Back to cited text no. 1
    
2.
Sammour OA, Hammad MA, Megrab NA, Zidan AS. Formulation and optimization of mouth dissolve tablets containing rofecoxib solid dispersion. AAPS PharmSciTech 2006; 7: E55.  Back to cited text no. 2
    
3.
Revathi V. Fast dissolving drug delivery system. Pharma Times 2007; 39: 22–23.  Back to cited text no. 3
    
4.
Bhyan B, Jangra S, Kaur M, Singh H. Orally fast dissolving films: innovations in formulation and technology. Int J Pharm Sci Rev Res 2011; 9: 50–56.  Back to cited text no. 4
    
5.
Chowdary YA, Soumya M, Madhu Babu M, Aparna K, Himabindu P. A review on fast dissolving drug delivery systems – a pioneering drug delivery technology. Bull Environ Pharmacol Life Sci 2012; 1: 8–20.  Back to cited text no. 5
    
6.
Pouleur HG. Clinical overview of irbesartan: a new angiotensin II receptor antagonist. Am J Hypertens 1997; 10: 318S–324S.  Back to cited text no. 6
    
7.
XXX. Soluplus®: A novel polymeric solubilizer for optimization of Carvedilol solid dispersions: formulation design and effect of method of preparation. Powder Technol 2013; 237: 406–414.  Back to cited text no. 7
    
8.
Djuric D. Soluplus®. Solubility enhancement with BASF pharma polymers. In: Reintjes T. (editor). Solubilizer compendium. BASF SE Pharma Ingredients & Services; 2011.  Back to cited text no. 8
    
9.
Ashis Kumar M, Vidyadhara S, Sasidhar RLC, Abhijit MD, Laxmana PS. Formulation and optimization of Ezetimibe containing solid dispersions using Kollidon VA64. Turk J Pharm Sci 2014; 11: 113–126.  Back to cited text no. 9
    
10.
Das A, Nayak AK, Mohanty B, Panda S. Solubility and dissolution enhancement of etoricoxib by solid dispersion technique using sugar carriers. ISRN Pharm 2011; 2011: 819765.  Back to cited text no. 10
    
11.
Arora SC, Sharma PK, Irchhaiya R, Khatkar A, Singh N, Gagoria J. Development, characterization and solubility study of solid dispersions of Cefuroxime Axetil by the solvent evaporation method. J Adv Pharm Technol Res 2010; 1: 326–329.  Back to cited text no. 11
    
12.
Ramu A, Vidyadhara S, Devanna N, Thirumala Naidu U, Lakshmi Kalyani P. Formulation and evaluation of irbesartan fast dissolving tablets. Asian J Pharm 2013; 7: 61–67.  Back to cited text no. 12
  Medknow Journal  
13.
Kaushik D, Dureja H, Saini TR. Formulation and evaluation of olanzapine mouth dissolving tablets by effervescent formulation approach. Indian Drugs 2004; 41: 10–12.  Back to cited text no. 13
    
14.
Banker GS, Anderson NR. In: Lachman L, Libermann HA, Kanig JL (editors). The theory and practice of industrial pharmacy. 3rd ed. New Delhi, India: Varghese Publishing House; 1987. pp. 293–299.  Back to cited text no. 14
    
15.
Controller of Publications. Indian Pharmacopoeia. New Delhi, India: Controller of Publications; 1996. p. 735.  Back to cited text no. 15
    
16.
Bi YX, Sunada H, Yonezawa Y, Danjo K. Evaluation of rapidly disintegrating tablets prepared by a direct compression method. Drug Dev Ind Pharm 1999; 25: 571–581.  Back to cited text no. 16
    
17.
Chaudhari PD, Chaudhari SP, Kolhe SR, Dave KV, More DM. Formulation and evaluation of fast dissolving tablets of famotidine. Indian Drugs 2005; 42: 641–649.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

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



 

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 and disc...
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed2046    
    Printed43    
    Emailed0    
    PDF Downloaded256    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]