• Users Online: 53
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 17  |  Issue : 2  |  Page : 53-58

Effects of Phyllanthus amarus on garlic-induced hepatotoxicity


1 Department of Biochemistry, Faculty of Science, University of Maiduguri, Maiduguri, Nigeria
2 Department of Biological Sciences, Faculty of Science, University of Abuja, Abuja, Nigeria
3 Department of Food Science and Technology, Faculty of Engineering, University of Maiduguri, Maiduguri, Nigeria

Date of Submission24-Sep-2018
Date of Decision01-Apr-2019
Date of Acceptance25-Apr-2019
Date of Web Publication29-Nov-2019

Correspondence Address:
Prof. L B Buratai
Department of Biochemistry, Faculty of Science, University of Maiduguri, Maiduguri
Nigeria
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njhs.njhs_11_18

Rights and Permissions
  Abstract 


Background: One of the most important organs that regulate various physiological processes in the body is the liver. The liver, if diseased or damaged by toxic agent(s) could contribute to the cause of morbidity and mortality globally. Phyllanthus amarus is highly valued in African traditional medicine for its hepatoprotective, anti-inflammatory and several related ailments.
Aim: The aim of the present study was to investigate the effects of the administration of P. amarus on garlic-induced hepatotoxicity in rats.
Settings and Design: Albino rats(n=30) weighing 170-200g were randomly divided into five Groups(I-V) of six. GroupI(control) received orally 1ml/kg body weight of distilled water while GroupsII, III, IV and V were administered orally same volume of garlic homogenate corresponding to 5g/kg body weight on a daily basis for 14days to induce liver toxicity. Similarly, GroupsIII, IV and V were orally administered with 100, 200 and 400mg/kg body weight of P. amarus aqueous leaf extract, respectively, for another 7days.
Materials and Methods: The levels of serum alkaline phosphatase(ALP), aspartate transaminase(AST), alanine transaminase(ALT) and total bilirubin(TB) were determined by standard methods. Histopathological analysis of the liver tissue was carried out as described by Druby and Wallington.
Statistical Analysis: Results were expressed as mean±standard errors of means. Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test was used to determine the difference between means at 95% level of significance.
Results: The results showed that all the doses of the extract of P. amarus significantly decreased(P<0.05) the levels of ALP, ALT and AST and TB relative to those administered 5g/kg body weight of garlic only(GroupII). The histopathological analysis of the liver samples also confirmed the hepatocurative potential of P. amarus against the hepatotoxicity caused by garlic. The ameriorative effect of P. amarus is suspected to be due to the presence of some antioxidants(phenols and flavonoids) as determined and quantified in the present work.
Conclusion: It was concluded that P. amarus showed hepatocurative effect having ameliorated the lobular necrosis and inflammation of the liver induced by garlic homogenate in albino rats.

Keywords: And antioxidants, garlic, hepatoprotection, hepatotoxicity, Phyllanthus amarus, Schum and Thonn


How to cite this article:
Hunaleyo H F, Buratai L B, Mofio B M, Lawan H K, Abdulrahman A A. Effects of Phyllanthus amarus on garlic-induced hepatotoxicity. Niger J Health Sci 2017;17:53-8

How to cite this URL:
Hunaleyo H F, Buratai L B, Mofio B M, Lawan H K, Abdulrahman A A. Effects of Phyllanthus amarus on garlic-induced hepatotoxicity. Niger J Health Sci [serial online] 2017 [cited 2019 Dec 10];17:53-8. Available from: http://www.chs-journal.com/text.asp?2017/17/2/53/272032




  Introduction Top


The liver although plays a central role in biotransformation and clearing chemicals, it is susceptible to the toxicity from these agents. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ.[1] Every chemical agent/drug is known to be associated with hepatotoxicity largely due to its ability to generate free radicals and to cause a disturbance in cell biochemically.[2] The fact that herbs are natural does not mean that they are harmless. In fact, there have been many reports of people suffering from serious health problems or even dying due to herbal remedies. Since substances consumed are metabolised through the liver, the liver is a prime target for the toxic effects of some herbs. People with normal liver function and have no history of prior liver disease have suffered adverse consequences to the liver as a result of taking certain herbs.[3]

Garlic(Allium sativum) is one of the wonder herbs from the lily family,[4] and it is the oldest known medicinal plant variety, or spice in existence and mankind recognised the curative qualities of this magic herb for over3000years. Meanwhile, it must not be assumed that since an herb has been used for thousands of years that the herb is possibly safe and truly effective for its claimed indication(s).[4]

Garlic has been an integral part of the diet for centuries, but it is taken for granted that garlic is safe in a wide range of doses. Garlic in high dose has the potential to induce liver damage while at low doses are considered safe.[5] Garlic is popularly consumed as a food condiment and has an important role in the treatment and management of several diseases. Nevertheless, unregulated and chronic consumption of garlic can cause damage to hepatocytes through the production of free radicals.[6]

Phyllanthus amarus(Family: Euphorbiaceae) is a widely distributed small, erect and tropical annual herb. This herb is commonly called stonebreaker, or is popularly called 'Chanka peidra' which translates to 'stone breaker'.[7],[8] P. amarus is, however, highly valued in African traditional medicine for its hepatoprotective, antidiabetic, anti-hypertensive, analgesic, anti-inflammatory and antimicrobial properties.[9]

However, in an extensive review of related literature, documented evidence to show that P. amarus can protect animal livers against garlic-induced hepatatoxicity is scarce. Against this background, the present study was designed to show the hepatocurative potentials of this plant extract against garlic-induced hepatotoxicity.


  Materials and Methods Top


Source and identification of plant materials

P. amarus herbs and dried cloves of A. sativum(Garlic) were obtained from the Botanical Garden of University of Maiduguri road and Maiduguri Monday Market, Nigeria, respectively. These plants were authenticated by a plant taxonomist in the Botanical Unit, Faculty of Science, University of Maiduguri, Borno State and the voucher number BCH#0080 of the herb deposited in the herbarium of Biochemistry Department of the University.

Preparation of garlic homogenate

Cold maceration(soaking) method was used to prepare the homogenate.[10] The garlic was peeled off, washed and weighed using a digital weighing balance. The weighed garlic(20g) was pounded in a clean mortar with pestle into a paste. The pounded garlic was soaked in distilled water for 48h(steering at intervals for proper mixing). After 48h, it was sieved into a conical flask using a clean sieve and a Whatman No. 1 filter paper. The filtrate was taken to the rotary evaporator to remove the excess amount of water, at a temperature of 60°C-80°C leaving behind the crude extract in the flask. The crude extract was further concentrated using the oven at a temperature of 30°C-40°C. Astock solution of 5g/ml was prepared from the concentrated paste-like garlic extract.[11] The stock solution was kept in the refrigerator at a temperature of 4°C until required.

Preparation of Phyllanthus amarus extract

The plant(P. amarus) leaves were gently sorted, air-dried at room temperature(28°C±2°C) for 2weeks until a constant weight was obtained. The dried leaves(40g) were pulverised into dry powder. The powder was extracted with distilled water using Soxhlet apparatus and concentrated by rotary evaporator at 65°C. It was then transferred into a suitable container and freeze-dried before further analysis.

Experimental animals

Matured Albino Wistar rats weighing(170-200g) of both sexes were obtained from Animal House of the Department of Biochemistry, Faculty of Science, University of Maiduguri, Borno State, Nigeria. Before the experiment, these animals were allowed to acclimatise for 14days in the animal house and maintained under standard conditions of temperature, light and relative humidity. Drinking water and standard pellet diet(Grand Cereal Nig. Ltd.,) were provided ad libitum. All experimental procedures were conducted in strict compliance with the guidelines for the care and use of laboratory animals.

Experimental procedures

Acute toxicity study(LD50)

The lethal dose in 50% of the total population(LD50) was interpolated using Lorke method.[12]



Where Do=highest dose that gave no mortality, D100=lowest dose that produced mortality.

Phytochemical analysis

Qualitative phytochemical tests were carried out on the aqueous leaf extract using standard procedures to identify the constituents as described by Sofowara,[13] Trease and Evans [14] and Harborne.[15] However, quantitative phytochemical analysis of total tannin content was determined by Van-Burden and Robinson [16] method, total saponin by the method of Obadoni and Ochuko,[17] total flavonoid content was determined by the Method of Bohm and Kocipai-Abyazan [18] and alkaloids using Harbone method.[19]

Hepatotoxicity induction and hepatocurative study

From the result of the LD50 test of the experimental animals, 30 albino rats of the Wistar strain weighing 170-200g were randomised into five groups of six rats such that the differences in the average weights between and within groups do not exceed±20% of the average weights of all the rats. GroupI, which served as the control was orally administered l. 0ml/kg/day of distilled water, while GroupsII-V were orally administered 5g/kg/day of fresh garlic homogenate for 14days to induce hepatotoxicity.

Following garlic hepatotoxicity induction by day 14, GroupII served as garlic control(negative control) while GroupsIII, IV and V were orally administered graded doses of 100, 200 and 400mg/kg/day, respectively, of P. amarus aqueous leaf extract for another 7days.

Chemical pathological assay of serum

At the end of 21days of treatment, the rats were sacrificed under diethyl ether anaesthesia(Sigma chemical Co; St Louis, USA) and the blood sample of each rat was collected separately into sterilised dried centrifuge tubes. The blood samples were allowed to clot and then spun. The supernatants(sera) were separated from the cell debris and decanted into new test tubes for some biochemical analysis, such as serum alkaline phosphatase(ALP), Alanine transaminase(ALT), Aspartate transaminase(AST) and total bilirubin(TB).

Histopathological study of the liver

The liver organ for each rat was cut to about 5mm thickness and fixed in 10% formalin. It was later processed and embedded in paraffin wax and then cut and stained with haematoxylin and eosin dye.[20] Photomicrographs were obtained using photographic microscope from the Department of Histology, University of Maiduguri Teaching Hospital, Maiduguri, Borno State, Nigeria.

Statistical analysis

Results were expressed as mean±standard errors of means. Statistical analysis was performed using one-way ANOVA, and Bonferroni post hoc test was used to determine the difference between means at 95% level of significance.


  Results Top


Acute toxicity study of P. amarus leaf extract showed no significant toxic effect in the albino rats[Table 1]. The phytochemical investigations of the leaf extract showed the presence of flavonoids, tannins, alkaloids, phenols, saponins and terpenoids[Table 2]. The animals were weak; eyes were red and showed decreased water and food intake after the administration of garlic. However, there was no vomiting and no death recorded. Garlic treatment(GroupII) as depicted in [Table 3] induced statistically significant(P<0.05) elevation in serum ALP, ALT, AST and TB when compared to the control group(GroupI). However, the increase in the levels of serum ALP, ALT, AST and TB in GroupII was significantly(P<0.05) decreased by the oral administration of P. amarus for 7days in a dose-related fashion. The tissue sections were examined under a light microscope, and the extent of necrosis was represented in photomicrographs of the histopathological examination in the liver tissue of GroupsI to V are presented as [Plate 1], [Plate 2], [Plate 3], [Plate 4], [Plate 5].
Table 1: Effect of aqueous leaf extract of Phyllanthus amarus on albino rats

Click here to view
Table 2: Qualitative and quantitative phytochemical components of aqueous leaf extract of Phyllanthus amarus

Click here to view
Table 3: Effects of aqueous leaf extract of Phyllanthus amarus aqueous on garlic induced hepatotoxicity in albino rats

Click here to view




  Discussion Top


Garlic(A. sativum) is popularly consumed in Nigeria and a major part of West Africa because of its health benefit in the treatment and management of several disease conditions.[21] However, excessive intake of garlic may cause liver damage [22] and haemolytic anaemia [23] as reported to have a significant increase in serum levels of ALT, AST and ALP and decrease in PCV, respectively. The release of these enzymes and their increased activity in the rats treated and observed in the present work was also reported earlierBuratai et al., (2000)[24] and Yahya et al., (2013).[37] The observed increase in the level of these enzymes was attributed to the excess garlic homogenate(5g/kg/day) administered which may have caused injury and alteration in their permeability[Table 3]. Garlic oil feeding(100mg/100g bw intragastrically) after 24h fasting was found lethal; and loss in body weight is highly due to increased toxicity which probably led to decreased food and water intake as reported by Joseph etal.[22] However, similar feeding of garlic oil was well tolerated by rats in the fed state.[22]

The administration of P. amarus aqueous leaf extract for 7days significantly alleviated or prevented the increased serum enzymes in garlic-induced liver injury. P. amarus plant has been reported to possess a membrane stabilising properties which consequently ameliorated the induced increases.[25] It, therefore, implies that the effect of any hepatocurative drug is dependent on its ability of reducing the harmful effect or restoring the normal hepatic physiology that has been disturbed by a known hepatotoxin.[25] Changes in the blood parameters such as TB can also be used to explain blood-relating functions of the plant extract or its products(2008).[26]

The present study has shown that aqueous extract of P. amarus is relatively safe in rats even at the concentration of 5000mg/kg body weight[Table 1]. However, phytochemical screening of P. amarus demonstrated the presence of flavonoids, saponins and tannins as well as the existence of phenolic compounds as indicated by high phenolic content value.[27] Garlic is a commonly used spice in medicine that can exert adverse effects when given at a high dose.[28] Unregulated and chronic consumption of garlic can cause damage to hepatocytes through the production of free radicals.[6] However, 5g/kg body weight of garlic homogenate which is a toxic dose [5] showed significant elevation of serum level of hepatic enzymes ALT, AST, ALP as well as serum TB as compared to the normal control group. Therefore, this study demonstrates that garlic homogenate administered at 5g/kg body weight to rats provoked a marked elevation in serum AST, ALT and ALP activities which indicates hepatocellular damage. The results of this corroborate with those of Rana etal.,[5] who reported elevated levels in serum levels of hepatic damage markers with marked histological damage in the rats liver administered 5g/kg/day of garlic homogenate. Meanwhile, the administration of 100mg, 200mg and 400mg of P. amarus aqueous leaf extract to the rats administered a high dosage of garlic successfully lowered the level of these enzymes as compared to Group II(negative control) as shown in [Table 2]. The hepatocurative potential of P. amarus can be explained based on the respective phytoconstituents detected in the extract. For example, flavonoids have been reported to exert antioxidant [29],[30] and anti-inflammatory [31] activities. The inhibitory activity of flavonoids on free radical release could be related to their hepatocurative effect since exogenous anti-oxidants may counteract the pathological effects of oxidative stress associated with chemical-induced hepatotoxicity, co-operating with natural systems such as glutathione, tocopherol or protective enzymes.[32] Moreover, saponins have been reported to exert hepatoprotective activity through the modulation of its antioxidant [33] and inflammatory activities [34] while tannins have been suggested to possess free radical scavenging, antioxidant, anti-inflammatory and hepatoprotective activities.[35]

Liver sections from excess garlic(5g/kg body weight) treated rats demonstrated the destruction of architectural pattern, moderate inflammation of the portal area and severe distortion of the liver parenchyma[Plate 2] when compared to the liver section of control rats(GroupI). This shows normal hepatocytes and normal architecture(normal portal tract, central vein and normal hepatocytes)[Plate 1]. The administration of P. amarus aqueous leaf extract in the dosage of 100, 200 and 400mg apparently restored the liver against garlic-induced damage[[Plate 3], [Plate 4], [Plate 5], respectively]. The hepatocurative or healing effect of the crude extract of P. amarus was thought to be due to the normalisation of impaired membrane function activity of the liver.[36] This healing or normalisation process as well as the improved structural integrity of some histopathological changes against the hepatotoxicity induced by garlic might also be associated with the presence of some antioxidative phytochemicals in P. amarus aqueous leaf extract.


  Conclusion Top


We thus concluded that the administration of aqueous extract of P. amarus to albino rats ameliorates garlic-induced liver toxicity. The data further lend credence to the prevention of garlic-induced increases in the hepatic biomarkers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
FriedmanSE, GrendellJH, McQuaidKK. Current Diagnosis and Treatment in Gastroenterology. NewYork: Lange Medical Books/McGraw-Hill; 2003. p.664-79.  Back to cited text no. 1
    
2.
Fernandez-ChecaJC, KaplowitzN. Hepatic mitochondrial glutathione: Transport and role in disease and toxicity. Toxicol Appl Pharmacol 2005;204:263-73.  Back to cited text no. 2
    
3.
MelissaP. Doctor Melissa Palmer's Guide to Hepatitis and Liver Disease. New York: Penguin Putnam Avery Press; 2004. p.335.  Back to cited text no. 3
    
4.
ConstanceCE. The Gift of Change: Embracing Challenges Today for a Promising Tomorrow. Florida: Balboa Press; 2013.  Back to cited text no. 4
    
5.
RanaSV, PalR, VaipheiK, SinghK. Garlic hepatotoxicity: Safe dose of garlic. Trop Gastroenterol 2006;27:26-30.  Back to cited text no. 5
    
6.
ObohG. Tropical green leafy vegetables prevent garlic-induced hepatotoxicity in the rat. JMed Food 2006;9:545-51.  Back to cited text no. 6
    
7.
BurkillHM. The Useful Plants of West Tropical Africa. 2nded., Vol.2. Kew, London, UK: Royal Botanic Gardens; 1994. p.119.  Back to cited text no. 7
    
8.
BharatiyaVB. Selected Medical Plants of India. India: Bombany Tafa Press; 1992. p.235-7.  Back to cited text no. 8
    
9.
AdeneyeAA, AmoleOO, AdeneyeAK. Hypoglycemic and hypocholesterolemic activities of the aqueous leaf and seed extract of Phyllanthus amarus in mice. Fitoterapia 2006;77:511-4.  Back to cited text no. 9
    
10.
TataraMR, SliwaE, DudekK, SiwickiAK, KowalikS, Luszczewska-SierakowskaI, etal. Influence of perinatal administration of aged garlic extract(AGE) and allicin to sows on some defence mechanisms in their piglets during postnatal life. Pol J Environ Stud 2005;14:378-81.  Back to cited text no. 10
    
11.
UviegharaKE. Procedures of Extraction of Garlic Extract. Abraka: Faculty of Pharmacy, Department of Pharmaceutical Chemistry Laboratory, Delta State University; 2014.  Back to cited text no. 11
    
12.
LorkeD. Anew approach to practical acute toxicity testing. Arch Toxicol 1983;54:275-87.  Back to cited text no. 12
    
13.
Sofowora AO. Medicinal Plants and Traditional Medicine in Africa. Ibadan; Nigeria: Spectrum Books Ltd.; 1993. p.320.  Back to cited text no. 13
    
14.
TreaseGE, EvansWC. ATextbook of Pharmacognosy. 13thed. London: Bacilluere Tinal Ltd.; 1989.  Back to cited text no. 14
    
15.
HarborneJB. Phytochemical Methods: AGuide to Modern Techniques of Plant Analysis. 3rded. London: Chapman A and Hall; 1998. p.1-301.  Back to cited text no. 15
    
16.
Van-BurdenJP, RobinsonWB. Formation of complexes between protein and tannic acid. JAgric Food Chem 1981;17:772-7.  Back to cited text no. 16
    
17.
ObadoniBO, OchukoPO. Phytochemical studies and comparative efficacy of the crude extract of some homeostatic plants in Edo and Delta states of Nigeria. Glob J Pure Appl Sci 2001;8:203-8.  Back to cited text no. 17
    
18.
BohmBA, Kocipai-AbyazanR. Flavonoid and condensed tannins from the leaves of Vaccinum raticulation and Vaccinum calcyimium. Pac Sci 1994;48:458-63.  Back to cited text no. 18
    
19.
HarboneJB. Phytochemistry. London: Academic Press; 1973. p.89-301.  Back to cited text no. 19
    
20.
AvwioroG. Histochemical uses of haematoxylin. Areview. JPharm Clin Sci 2011;1:24-34.  Back to cited text no. 20
    
21.
UmarIA, MadaiBI, BurataiLB, KarumiY. The effects of a combination of Garlic(Allium sativum) powder and wild honey on lipid metabolism in insulin-dependent diabetic rats. Niger J Expt Appl Biol 2000;1:37-42.  Back to cited text no. 21
    
22.
JosephPK, RaoKR, SundareshCS. Toxic effects of garlic extract and garlic oil in rats. Indian J Exp Biol 1989;27:977-9.  Back to cited text no. 22
    
23.
ObohG. Prevention of garlic-induced hemolytic anemia using some tropical green leafy vegetables. JMed Food 2004;7:498-501.  Back to cited text no. 23
    
24.
BurataiLB, IgbokweIO, AhmedAY, UmarIA. Effect of benzidine on the liver of the albino rat. Niger J Exp Appl Biol 2000;1:43-6.  Back to cited text no. 24
    
25.
RajkapoorB, VenugopalY, AnbuJ, HarikrishnanN, GobinathM, RavichandranV. Protective activity of Phyllanthus amarus seeds extracts in CCl4 treated rats; invitro & invivo. JMed Res 2008;12:38-49.  Back to cited text no. 25
    
26.
YakubuMT, AkanjiMA, OladijiAT. Effects of oral administration of aqueous extract of Fadogia agrestis(Schweinf. Ex hiern) stem on some testicular function indices of male rats. JEthnopharmacol 2008;115:288-92.  Back to cited text no. 26
    
27.
ZakariaZA, MohamedAM, JamilNS, RofieeMS, HussainMK, SulaimanMR.In vitro antiproliferative and antioxidant activities of the extracts of Muntingia calabura leaves. Am J Chin Med 2011;39:183-200.  Back to cited text no. 27
    
28.
Hamlaoui-GasmiS, MokniM, LimamN, N'guessan P, CarrierA, LimamF, etal. Grape seed and skin extract mitigates garlic-induced oxidative stress in rat liver. Can J Physiol Pharmacol 2012;90:547-56.  Back to cited text no. 28
    
29.
FerreiraJF, LuthriaDL, SasakiT, HeyerickA. Flavonoids from Artemisia annua L. as antioxidants and their potential synergism with artemisinin against malaria and cancer. Molecules 2010;15:3135-70.  Back to cited text no. 29
    
30.
TapasAR, SakarkarDM, KakdeRB. Flavonoids as nutraceuticals: Areview. Trop J Pharm Res 2008;7:1089-99.  Back to cited text no. 30
    
31.
SandharHK, KumarB, PrasherS, TiwariP, SalhanM, SharmaP. Areview of phytochemistry and pharmacology of flavonoids. Int J Pharm Sci 2011;1:24-41.  Back to cited text no. 31
    
32.
SanzMJ, FerrandizML, CejudoM, TerencioMC, GilB, BustosG, etal. Influence of a series of natural flavonoids on free radical generating systems and oxidative stress. Xenobiotica 1994;24:689-99.  Back to cited text no. 32
    
33.
ElekofehintiOO, AdanlawaIG, KomolafeK, EjelonuOC. Saponins from Solanum anguiri fruits exhibit antioxidant potential in wistar rats. Ann Biol Res 2012;3:3212-7.  Back to cited text no. 33
    
34.
AkkolEK, TatliII, AkdemirZS. Antinociceptive and anti-inflammatory effects of saponin and iridoid glycosides from Verbascum pterocalycinum var. mutense hub.-Mor. ZNaturforsch C 2007;62:813-20.  Back to cited text no. 34
    
35.
PithayanukulP, NithitanakoolS, BavovadaR. Hepatoprotective potential of extracts from seeds of areca catechu and nutgalls of Quercus infectoria. Molecules 2009;14:4987-5000.  Back to cited text no. 35
    
36.
GuptaAK, MisraN. Hepatoprotective activity of aqueous ethanolic extract of Chamomile capitula in paracetamol intoxicated albino rats. Am J Pharmacol Toxicol 2006;1:17-20.  Back to cited text no. 36
    
37.
YahyaF, MamatSS, KamarolzamanMF. Hepatoprotective activity of methanolic extract of Bauhinia purpurea leaves against paraceutamol-induced hepatic damage in rats. Evid Based Complement Alternat Med 2013;580:1-10.  Back to cited text no. 37
    



 
 
    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
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Tables

 Article Access Statistics
    Viewed146    
    Printed5    
    Emailed0    
    PDF Downloaded1    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]