J Interdiscip Dentistry
Home | About JID | Editors | Search | Ahead of print | Current Issue | Archives | Instructions |
Home Print this page Email this page Small font sizeDefault font sizeIncrease font size
Users Online: 270  | Login  | Contact us | Advertise | Subscribe  


 
Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 10  |  Issue : 2  |  Page : 61-66

Assessment of the antibacterial activity of Catharanthus roseus leaf extract on periodontal pathogens, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia: An In vitro study


Consultant Periodontist and Implantologist, Shivam Dental Clinic and Implant Center, Valsad, Gujarat, India

Date of Submission10-Jan-2020
Date of Acceptance30-May-2020
Date of Web Publication21-Aug-2020

Correspondence Address:
Dr. Khushboo Vijaysinh Rathod
Plot No. 7, Gurukrupa Society, Behind HR House, Tokarhada, Silvassa, U. T, Dadra and Nagar Haveli - 396 230
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jid.jid_1_20

Rights and Permissions
   Abstract 


Aim: The aim of this study was to evaluate the in vitro antimicrobial efficacy of Catharanthus roseus leaf extract against standard strains of periodontal pathogens, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia. Methodology: The C. roseus leaves were identified, shade dried, and ground, and ethanol extract was obtained. The antimicrobial efficacy of this extract was done using disc diffusion assay, minimum inhibitory concentration, and minimum bactericidal concentration. Results: C. roseus leaf extract showed antimicrobial activity against all the three test organisms. However, P. gingivalis was found to be more susceptible to C. roseus leaf extracts, since their growth was inhibited at relatively lower concentration as compared to A. actinomycetemcomitans and P. intermedia. Conclusion: Within the limitations of the study, the present results have provided preliminary scientific evidence for the development of antimicrobial products, and further studies and clinical trials need to be performed.

Keywords: Aggregatibacter actinomycetemcomitans, Catharanthus roseus, disc diffusion assay, minimum bactericidal concentration, minimum inhibitory concentration, Porphyromonas gingivalis, Prevotella intermedia


How to cite this article:
Rathod KV, Das S. Assessment of the antibacterial activity of Catharanthus roseus leaf extract on periodontal pathogens, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia: An In vitro study. J Interdiscip Dentistry 2020;10:61-6

How to cite this URL:
Rathod KV, Das S. Assessment of the antibacterial activity of Catharanthus roseus leaf extract on periodontal pathogens, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia: An In vitro study. J Interdiscip Dentistry [serial online] 2020 [cited 2020 Nov 30];10:61-6. Available from: https://www.jidonline.com/text.asp?2020/10/2/61/292914




   Clinical Relevance to Interdisciplinary Dentistry Top


Modern chemotherapeutic agents display a significant efficacy in improving periodontal health, but owing to undesirable side effects Hence, the development of natural form of therapies for the treatment of periodontal diseases is of great relevance.


   Introduction Top


Periodontitis being a chronic inflammatory disease is characterized by destruction of supporting structure of the teeth. Periodontal disease begins with gingivitis, the localized inflammation of the gingiva that is initiated by bacteria in the dental plaque, which is a microbial biofilm that forms on the teeth and gingiva. Bacterial plaque has been implicated as a primary etiologic factor in inflammatory periodontal disease.[1]

Although it is generally considered that periodontal disease has multifactorial etiology, it is known that some specific Gram-negative microorganisms in the subgingival plaque biofilm play a major role in the initiation and progression of periodontitis.[2] The incidence and progression of this disease are related to a substantial increase in Gram-negative anaerobic rods. Among them, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia are strongly implicated in the etiology of chronic periodontitis.[3] When these pathogens habitat the local periodontal tissues, an immune response is initiated by fibroblasts and macrophages by producing several cytokines as mediators of the inflammatory response and immune reaction.[4]

Routine practice of antimicrobial adjunct to conventional periodontal therapy was well-established owing to tissue penetrable nature of pathogenic bacteria.[5] Hence, the development of natural form of therapies for the treatment of periodontal diseases is of great importance, as the administration of systemic antimicrobials has been known to cause the development of antimicrobial resistance, interbacterial transfer of resistance determinants, and various side effects.[6] It has been observed that the appearance of formerly rare infections is on the rise, perhaps due to the incorrect or prevalent overuse of antimicrobials. Natural phytochemicals have demonstrated to be worthy substitutes to synthetic agents.[7]

It has been reported that in adults with periodontal disease, scaling and root planing along with the use of an adjunctive antimicrobial mediator increases patient outcomes over a period of time compared to scaling and root planing alone.[8] Modern chemotherapeutic agents display a significant efficacy in improving periodontal health, but owing to undesirable side effects such as tooth discoloration, taste alteration, and price of these substances, the usage of herbal products has increased lately and could be especially of high benefit to lower socioeconomic populations around the world.[9] Hence, the development of natural form of therapies for the treatment of periodontal diseases is of great relevance.

Catharanthus roseus (Madagascar periwinkle) is a species of Catharanthus native and endemic to Madagascar. Synonyms include Vinca rosea (basionym), Ammocallisrosea, and Lochnerarosea; other English names occasionally used include Cape periwinkle, rose periwinkle, rosy periwinkle, and “old maid.” In India, it is known as “nithyakalyani.”[10]

C. roseus is an important medicinal plant of the family Apocynaceae and is used to treat many fatal diseases. C. roseus also possesses good antioxidant potential. There are about two common cultivars of C. roseus which is named on the basis of their flower color that is the pink-flowered “rosea” and the white-flowered “alba.” C. roseus is extensively cultivated in northern India in order to meet their commercial need and due to the ever-increasing demand in the indigenous systems of the medicine and the pharmaceutical industry.[10]

C. roseus possesses known antibacterial, antifungal, antidiabetic, anticancer, and antiviral activities. The extracts have demonstrated significant anticancer activity against numerous cell types.[11]

The leaf juice of C. roseus produced a dose-dependent reduction in blood glucose of both normal and diabetic rabbits and comparable with that of the standard drug, glibenclamide. The plant shows the presence of various alkaloids, namely vincristine, which bind to tubulin dimers, inhibiting the assembly of microtubule structures. Disruption of the microtubules arrests mitosis in metaphase. The vinca alkaloids, therefore, affect all rapidly dividing cell types including cancer cells but also intestinal epithelium and bone marrow.[12]

Over centuries, cultures around the world have learned how to use plants to fight illness and maintain health. These readily available and culturally important traditional medicines form the basis of an accessible and affordable health care Regimen and are an important source of livelihood for indigenous and rural populations.[13]

Plant-based antimicrobial represents a vast untapped source of medicines and needs to be explored further. They have enormous therapeutic potential and are effective in the treatment of infectious disease.[14]

The literature on the antimicrobial effect of C. roseus on periodontal pathogens, especially P. gingivalis, A. actinomycetemcomitans, and P. intermedia, is not present. Hence, the present study was undertaken to evaluate thein vitro antimicrobial efficacy of C. roseus leaf extract against standard strains of P. gingivalis, A. actinomycetemcomitans, and P. intermedia.


   Methodology Top


Type of study: In vitro

Preparation of Catharanthus roseus leaf extract

The dried C. roseus leaves were procured from the Tropical Herbs and Spices, Coimbatore, Tamil Nadu, India. The specimen samples were examined and identified in ICMR, Belagavi, by a botanist. This study was conducted at the Department of Microbiology of Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre. The shade-dried leaves were used for the solvent extraction procedure. The dried leaf was powdered by hand crushing. About 10 g of this powder was soaked in 100 ml of ethanol for 48 h. The contents were then filtered through Whatman filter paper no. 1. The obtained filtrate was further processed in a Soxhlet apparatus, and the obtained dried extract was dissolved in DSMO for testing its antimicrobial potential. Extracts were stored at 4°C until further use [Figure 1].
Figure 1: Catharanthus roseus leaf extract

Click here to view


Microbial assessment

Anin vitro microbiological study was carried out to test C. roseus leaf extract on standard strains of P. gingivalis, A. actinomycetemcomitans, and P. intermedia in the laboratory.

Disc diffusion assay

Blood agar was used for the procedure. Agar plates were brought to room temperature before use. Using a loop or swab, colonies were transferred to the agar plates. Visually turbidity was adjusted with broth to equal that of a 0.5 McFarland turbidity standard that was vortexed. Within 15 min of adjusting the inoculum, a sterile cotton swab was dipped into the inoculum and rotated against the wall of the tube above the liquid to remove excess inoculum. Entire surface of agar plate was swabbed three times, rotating plates approximately 60° between streaking to ensure even distribution. Inoculated plate was allowed to stand for at least 3 min before making wells. Hollow tube of 5 mm diameter was taken and heated. It was pressed on above-inoculated agar plate and removed immediately by making a well in the plate. Likewise, five well on each plate was made. 75 μl, 50 μl, 25 μl, 10 μl, and 5 μl of extract were added into the respective wells on each plate. Plates were incubated within 15 min, then inverted and stacked, and incubated for 48–72 h in anaerobic jars. Plates were read-only if the lawn of growth was confluent or nearly confluent. Diameter of inhibition zones was measured to the nearest whole millimeter by holding the measuring device.

Minimum inhibitory concentration

In this study, serial tube dilution technique was followed. In the initial tube, 20 μl of extract was added into the 380 μl of thioglycollate broth. For dilutions, 200 μl of thioglycollate broth was added into the next 9 tubes separately. Then, from the initial tube, 200 μl was transferred to the first tube containing 200 μl of thioglycollate broth. This was considered as 10-1 dilution. From 10-1 diluted tube, 200 μl was transferred to the second tube to make 10-2 dilution. The serial dilution was repeated up to 10-9 dilution for each extract. From the maintained stock cultures of required organisms, 5 μl was taken and added into 2 ml of thioglycollate broth. In each serially diluted tube, 200 μl of the above culture suspension was added. The tubes were incubated in an anaerobic jar at 37°C for 24 h and observed for turbidity, which indicates the growth of the organisms. The turbidity in each tube was compared with a positive control, which contained only the pure bacterial culture. The minimum concentration of the extract in the tube, which does not show any turbidity, was considered as minimum inhibitory concentration (MIC) of the extract for that particular test organism.

Minimum bactericidal concentration

From the MIC dilution tubes, the first 3 or 5 tubes (which were sensitive in MIC) were spread across the center of a blood agar plate and allowed to dry for 20 min. After the plates had dried, a sterile spreading rod was used to evenly disperse the inoculum over the entire surface of the plate, which was then incubated at 37°C for 24 h, and colonies were counted on the next day. The minimum bactericidal concentration (MBC) was carried out to observe the bactericidal effect of C. roseus leaf extract against P. gingivalis, A. actinomycetemcomitans, and P. intermedia. If there was no growth of microorganisms, then the drug was known to have bactericidal effect. The MBC was recorded as the lowest concentration of the extract that prevents the growth of the bacteria and reduces the viability of the initial bacterial inoculum.


   Results Top


Disc diffusion assay

The onion extracts activity was seen at a higher concentration. With decreasing concentration of extract, the antimicrobial activity of onion was decreased. At 75 μL/mL, the zone of inhibition seen was 28, 26, and 18 mm for P. gingivalis, A. actinomycetemcomitans, and P. intermedia, respectively, which subsequently decreased, and at 5 μ/mL, no zone of inhibition was seen [Table 1] and [Figure 2].
Table 1: Disc diffusion assay values of Catharanthus leaf extract against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia

Click here to view
Figure 2: The inhibition zone is the area surrounded the hole, and there is no growth of inoculated microorganism (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia)

Click here to view


Minimum inhibitory concentration

In the present study, P. gingivalis, A. actinomycetemcomitans, and P. intermedia were sensitive to C. roseus leaf extract. MIC values of P. gingivalis and P. intermedia were sensitive until 25 μg/ml dilution and A. actinomycetemcomitans were sensitive until 50 μg/ml. P. gingivalis and P. intermedia were found to be more sensitive than A. actinomycetemcomitans. In MIC, as the compound gets diluted, the antimicrobial activity decreases. According to results, at a higher concentration of extract, its activity goes on increasing [Table 2] and [Figure 3].
Table 2: Minimum inhibitory concentration values of Catharanthus leaf extract against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia

Click here to view
Figure 3: Serial tube dilution technique followed to demonstrate the MIC procedure for the antibacterial activity of Catharanthus roseus leaf extract against Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans. If the test organism is sensitive then clear tube is seen and if it is resistant then turbid is seen

Click here to view


Minimum bactericidal concentration

MBC value of P. gingivalis showed a bactericidal effect until 12.5 μg/ml dilution. A. actinomycetemcomitans and P. intermedia showed a bactericidal effect until 50 μg/ml dilution. Catharanthus leaf extract showed greater bactericidal activity against P. gingivalis than A. actinomycetemcomitans and P. intermedia [Table 3] and [Figure 4].
Table 3: Minimum bactericidal concentration values of Catharanthus leaf extract against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia

Click here to view
Figure 4: Minimum bactericidal concentration plates for detection of antibacterial activity of Catharanthus roseus leaf extract against Porphyromonas gingivalis (i), Prevotella intermedia (ii), and Aggregatibacter actinomycetemcomitans(iii). As the compound is diluted, the number of colonies has increased

Click here to view



   Discussion Top


Antimicrobial resistance, considerable side effects, and the emergence of previously uncommon infections are the results of improper usage of synthetic antimicrobial agents.[15] Instead, plant chemicals are one of the most powerful and safe alternative chemotherapeutic agents to control many infections if they are supported by scientific-based evidence.[16] Recently, there is an increasing interest to investigate the effect of natural compounds, especially plant extracts, on the residence of the oral cavity. Many of the investigations have been focused on the ability of the compound to either promote the growth of beneficial organisms or inhibit the growth and metabolism of oral bacteria associated with certain diseases.

The present study reveals the antimicrobial activity of ethanol leaf extracts of C. roseus. The antimicrobial activity of C. roseus leaf extract was tested against two pathogenic bacteria, P. gingivalis, A. actinomycetemcomitans, and P. intermedia. The tested extract showed good antimicrobial activity against these pathogenic microorganisms. The antimicrobial activity was evaluated by disc diffusion assay, MIC, and MBC methods.

C. roseus shows the presence of various alkaloids. These alkaloids are responsible for many medically important properties of this plant. C. roseus possesses known antibacterial, antifungal, antidiabetic, anticancer, and antiviral activities. The extracts have demonstrated significant anticancer activity against numerous cell types.[17]

Shanmugaraju et al. in their study showed very strong antimicrobial activity of C. roseus against Staphylococcus spp. and Pseudomonas. They evaluated the antimicrobial activity by measuring the zone of inhibition. The strongest inhibition activity of the leaf extract was observed against Staphylococcus spp. (25 mm zone) at 100 mg/ml of leaf extract followed by Pseudomonas which showed a 20 mm inhibition zone at 100 mg/ml leaf extract. This study also illustrated that Gram-positive bacteria were more susceptible to this extract as compared to Gram-negative bacteria species.[10]

However, in this present study, we have tested the antibacterial activity of C. roseus extract against three Gram-negative bacteria. Further, investigations are required to test the antimicrobial activity of this extract against other periodontal pathogens, Gram-positive and Gram-negative bacteria.

The different parts of C. roseus (leaf, stem, flower, and root) and extracts have been subjected to antibacterial assay. According to Perez et al. study, the extracts of C. roseus did not exhibit antibacterial activity against Staphylococcus aureus. Moreover, leaf, stem, and flower extracts were also ineffective against Pseudomonas aeruginosa. The leaf extract did not exhibit activity against Corynebacterium diphtheriae; similarly, the crude extract of stem did not show activity against Shigella boydii. The most effective was the root extract, which exhibited broad-spectrum antibacterial activity against Salmonella typhimurium and S. boydii. The flower extract showed activity against C. diphtheria.[18]

Jaykumar et al. evaluated the antioxidant potential and antibacterial activity of C. roseus using anin vitro model. C. roseus leaves and root showed moderate sensitivity against Klebsiella pneumonia and least sensitive toward Escherichia coli, Salmonella typhi, Shigella sonnei, Bacillus cereus, Salmonella paratyphi, and S. aureus.[19]

Patil et al. conducted a study to investigate the antimicrobial properties of C. roseus plant against microorganisms such as P. aeruginosa, S. typhimurium, and S. aureus. They concluded that the extracts from the leaves of this plant showed good antibacterial properties against the test bacteria and can be used as a prophylactic agent in many of the diseases, which sometimes are of the magnitude of an epidemic.[17]

Wagay et al. investigated the antimicrobial activity and phytochemical analysis of acetone extract of C. roseus whole plant against the wound isolates. They concluded that maximum antibacterial activity was observed in crude ethanolic extract of C. roseus against P. aeruginosa.[20]

After extensive literature search and to the best our knowledge, this is the first study that determines thein vitro antibacterial activity of C. roseus leaf extract against three periodontopathogenic bacteria P. gingivalis, A. actinomycetemcomitans, and P. intermedia by disc diffusion assay, MIC, and MBC methods. These methods are used to identify the potency of an antibacterial drug and are considered as important issues in diagnostic laboratories to confirm the resistance of antimicrobial agent and also to monitor the activity of new antimicrobials.

The present in vitro antibacterial assessment study helps us to focus on an intervention approach to design and conduct a clinical trial to detect the beneficial effect of Catharanthus leaf extract on patients at risk for periodontitis. However,in vitro values of disc diffusion assay, MIC, and MBC may not hold good forin vivo studies due to their inherent limitations. The growth of microorganismsin vitro is exponential, whereas the growthin vivo can be very slow to none.[21] although these methods do not indicate the true activity of the drug at the locus of infection, these methodsin vitro serve as surrogate markers attempting to quantify the drug activity.[22]

Within the limitations of the present study, the lowest concentration of Catharanthus leaf extract was proven to be effective on three periodontogenic bacteria P. gingivalis, A. actinomycetemcomitans, and P. intermedia. However, as periodontitis is a polymicrobial disease, the susceptibility of various other periodontal pathogens to this extract must be evaluated. Further studies are required to (a) assess thein vivo efficacy of C. roseus leaf extract with other traditionally prescribed antimicrobials used for periodontal therapy and (b) evaluate thein vivo effect of C. roseus leaf extract in different formulations (gel, chips, strips, fibers, etc.) with variable concentrations.


   Conclusion Top


From the current study, it can be concluded that there is preliminary evidence for the antimicrobial activity of C. roseus leaf extracts against periodontal pathogens P. gingivalis, A. actinomycetemcomitans, and P. intermedia. Hence, C. roseus leaf extract can be used as an economical and suitable adjuvant to synthetic drugs and can be a potential therapeutic agent for periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Popova C, Panova VD, Panov V. Microbiology of periodontal diseases: A review. Biotechnol Biotechnol Equipment 2013;27:3754-9.  Back to cited text no. 1
    
2.
Newman MG, Takei HH, Carranza FA. Clinical Periodontology. Philadelphia: W.B. Saunders; 2002. p. 96-167.  Back to cited text no. 2
    
3.
Wolff L, Dahlén G, Aeppli D. Bacteria as risk markers for periodontitis. J Periodontol 1994;65:498-510.  Back to cited text no. 3
    
4.
Preshaw PM, Taylor JJ. How has research into cytokine interactions and their role in driving immune responses impacted our understanding of periodontitis? J Clin Periodontol 2011;38 Suppl 11:60-84.  Back to cited text no. 4
    
5.
Systemic antibiotics in periodontics. J Periodontol 1996;67:831-8.  Back to cited text no. 5
    
6.
Walker CB. The acquisition of antibiotic resistance in the periodontal microflora. Periodontol 2000 1996;10:79-88.  Back to cited text no. 6
    
7.
Abdollahzdeh SH, Mashouf R, Mortazavi H, Moghaddam M, Roozbahani N, Vahedi M. Antibacterial and antifungal activities of Punica granatum peel extracts against oral pathogens. J Dent 2011;8:1-6.  Back to cited text no. 7
    
8.
Bonito AJ, Lux L, Lohr KN. Impact of local adjuncts to scaling and root planing in periodontal disease therapy: A systematic review. J Periodontol 2005;76:1227-36.  Back to cited text no. 8
    
9.
de Oliveira SM, Torres TC, Pereira SL, Mota OM, Carlos MX. Effect of a dentifrice containing Aloe vera on plaque and gingivitis control. A double-blind clinical study in humans. J Appl Oral Sci 2008;16:293-6.  Back to cited text no. 9
    
10.
Shanmugaraju V, Bhakyaraj R. Antimicrobial potential activity of leaf extracts of Catharanthus roseus against human pathogens under laboratory conditions. Int J Curr Res Biol Med 2016;1:35-51.  Back to cited text no. 10
    
11.
El-Sayed A, Cordell GA. Catharanthamine, a new antitumor bisinod alkaloid from Catharanthus roseus. J Nat Prod 1981;144:289-93.  Back to cited text no. 11
    
12.
Graf WD, Chance PF, Lensch MW, Eng LJ, Lipe HP, Bird TD. Severe vincristine neuropathy in CharcotMarieTooth disease type 1A. Cancer 1996;77:1356-62.  Back to cited text no. 12
    
13.
Rajalakshmi NP, Banuraviganesh F. Antimicrobial activity and phytochemical screening of Catharanthus roseus. Int J Sci Res 2013;2:1-2.  Back to cited text no. 13
    
14.
Harborne JB, Baxter H, Moss GP. A handbook of bioactive compounds from plants. Phytochemical dictionary. 2nd ed. London:Taylor and Francis;1999.  Back to cited text no. 14
    
15.
Li M, Xu Z. Quercetin in a lotus leaves extract may be responsible for antibacterial activity. Arch Pharm Res 2008;31:640-4.  Back to cited text no. 15
    
16.
Mehta VV, Rajesh G, Rao A, Shenoy R, Mithun Pai BH. Antimicrobial efficacy of Punica granatum mesocarp, Nelumbo nucifera leaf, Psidium guajava leaf and Coffea canephora extract on common oral pathogens: An in vitro study. J Clin Diagn Res 2014;8:65.  Back to cited text no. 16
    
17.
Patil PJ, Ghosh JS. Antimicrobial activity of Catharanthus roseus – A detailed study. Br J Pharmacol Toxicol 2010;1:40-4.  Back to cited text no. 17
    
18.
Perez C, Pauli M, Bazerque P. An antibiotic assay by the agar-well diffusion method. Acta Biol Med 1990;2:708-12.  Back to cited text no. 18
    
19.
Jayakumar D, Mary SJ, Santhi RJ. Evaluation of antioxidant potential and antibacterial activity of Calotropisgigantea and Vincarosea usingin vitro model. Indian J Sci Technol 2010;3:720-3.  Back to cited text no. 19
    
20.
Wagay SA, Dwivedi SD, Sharma M, Tripathi J, Ahmad M. Antimicrobial activity of Catharanthus roseus. Chem Mater Res 2013;3:61-4.  Back to cited text no. 20
    
21.
Levison ME. Pharmacodynamics of antibacterial drugs. Infect Dis Clin North Am 2000;14:281-91.  Back to cited text no. 21
    
22.
Briethaupt H. The new antibiotics: Can novel antibacterial treatment combat the rising tide of drug resistant infections? Nat Biotechnol 1999;17:1165-9.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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
    Clinical Relevan...
   Introduction
   Methodology
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed570    
    Printed30    
    Emailed0    
    PDF Downloaded112    
    Comments [Add]    

Recommend this journal