copperleaf, Joseph’s coat, fire dragon, beef steak plant and match-me-if-you-can [10]. It is native to Fiji and nearby islands in the South Pacific, but has spread to most parts of the world most especially in the tropics of Africa, America and Asia. It is a popular outdoor plant that provides color throughout the year, although it is also grown indoors as a container plant. It has antimicrobial properties^9,11,12. Many cultivars are available with different leaf forms and colors: A. wilkesiana ‘Godseffiana’ has narrow, drooping, green leaves with creamy-white margins, 'Marginata' has coppery-green leaves with pink or crimson margins, 'Macrophylla' has larger leaves, variegated with bronze, cream, yellow and red, while 'Musaica' has green leaves that are mottled with orange and red^10,¹³. The leaf-poultice is used in the treatment of headache, swellings and colds. The seeds are essential components of a complex plant mixture used empirically by traditional healers in south-west Nigeria to treat breast tumours and inflammation¹⁴. The expressed juice or boiled decoction is used for the treatment of gastrointestinal disorders, fungal skin infections, malaria, hypertension and diabetes mellitus. However, the biochemical basis of the use of the leaves in the management of diabetes mellitus, as well as the biochemical impact of their administration on the diabetic is yet to be clearly understood. Thus, in the present study, the effect of aqueous extract of Acalypha wilkesiana ‘Godseffiana’ Muell Arg on plasma glucose level, chemistry and lipid profile, and atherogenic indices of alloxan treated Wistar rats were investigated.

MATERIAL AND METHODS:

Collection of Animals and Preparation of Plant Extract: Albino rats were collected from the animal house of the Department of Physiology, University of Nigeria, Enugu Campus, Enugu, Nigeria. Samples of the fresh Acalypha wilkesiana plants were collected from within the Choba and Abuja Campuses of University of Port Harcourt, Port Harcourt, Nigeria. After due identification at the University of Port Harcourt Herbarium, Port Harcourt, Nigeria, they were rid of dirt and the leaves were removed, oven dried at 55⁰C and ground into powder. The resultant powder was soaked in boiled distilled water for 12h, after which the resultant mixture was filtered and the filtrate, hereinafter referred to as the aqueous extract was stored for subsequent use. A known volume of this extract was evaporated to dryness, and the weight of the residue used to determine the concentration of the filtrate, which was in turn used to determine the dose of administration of the extract to the test animals.

Experimental Design: Studies were conducted in compliance with applicable laws and regulations. The rats were randomly sorted into five groups of five animals each, so that the average weight difference was ≤ 1.8g. The animals were housed in plastic cages. After a one-week acclimatization period on guinea growers mash (Port Harcourt Flour Mills, Port Harcourt, Nigeria), the animals were fasted overnight and diabetes was induced by injection of a freshly prepared solution of alloxan (140mg/kg body weight) in normal saline, via the tail vein of four groups, while the control rats were injected with normal saline alone. Seven days after administration of the alloxan, the animals were again fasted and blood collected via tail cutting¹⁵, for the determination of their fasting glucose levels. It was found that the rats had moderate diabetes, having hyperglycemia (that is, with blood glucose of over 180% of the control). Then the rats were kept for 3 days to stabilize the diabetic condition¹⁶ before commencing the treatment, which lasted for two days. The reference treatment group (reference) received daily by intra-gastric gavages, 50mg/kg body weight of Diabetmin^TM (metformin HCl), the first test group (Test 1) received daily by intra-gastric gavages 200mg/kg body weight of the Acalypha wilkesiana extract; the second group (Test 2) received 250mg/kg body weight of the Acalypha wilkesiana extract; while the test control and the control group received appropriate volumes of water by the same route. The dosage of administration of the extract was adapted from Ikewuchi and Ikewuchi¹⁷. The animals were allowed food and water ad libitum. The fasting glucose levels were taken each day, before and after administration. At the end of the treatment period, the rats were anaesthetized by exposure to chloroform. While under anesthesia, they were painlessly sacrificed and blood was collected from each rat into heparin sample bottles. Whole blood was immediately used to determine the triglyceride levels (using test strips), while the heparin anti-coagulated blood samples were centrifuged at 1000g for 10min, after which their plasma was collected and stored for subsequent analysis.

Determination of the Plasma Glucose Concentrations: The plasma glucose concentration was determined using the multiCarein^TM glucose strips and glucometer. The glucose contained in the sample reacts with the glucose oxidase enzyme in the glucose electrode strips to produce an electric current. The magnitude of the current produced by the electrodes is directly proportional to the glucose concentration.

Determination of the Plasma Lipid Profiles/Indices: Plasma triglyceride concentration (TG) was determined using multiCarein^TM triglyceride strips and glucometer (Biochemical Systems International, Arezzo, Italy). The test is based on lipase/glycerol kinase/glycerol phosphate oxidase/peroxidase/chromogen reaction. The intensity of the colour developed from the reaction is proportional to the concentration of triglycerides in the blood. Plasma total and high density lipoprotein cholesterol concentrations (TC and HDLC) were assayed enzymatically with Randox commercial test kits (Randox Laboratories, Crumlin, England). In the presence of magnesium ions, low density lipoproteins (LDL and VLDL) and chylomicrons fractions are precipitated quantitatively by the addition of phosphotungstic acid. After centrifugation, the cholesterol concentration of the high density lipoprotein (HDL) fraction, which remains in the supernatant, can be determined, as in total cholesterol. The cholesterol released by enzymatic hydrolysis is oxidized with the concomitant release of hydrogen peroxide, whose breakdown leads to the conversion of 4-aminoantipyridine to quinoneimine (the indicator) whose concentration can be determined spectrophotometrically at 500nm.

Plasma VLDL- and LDL-cholesterol (LDLC and VLDLC) concentrations was calculated using the Friedewald equation¹⁸ as follows:

[LDL cholesterol] (mg/dL) = [Total cholesterol] – [HDL cholesterol] –[ Triglycerides]/5

[VLDL cholesterol] (mg/dL) = [ Triglycerides]/5

While the plasma non-HDL cholesterol concentration was determined as reported by Brunzell et al.¹⁹:

[Non-HDL cholesterol] = [Total cholesterol] – [HDL cholesterol]

The atherogenic indices were calculated as earlier reported by Ikewuchi and Ikewuchi^20,21 using the following formulae:

[Total Cholesterol]

Cardiac Risk Ratio (CRR) = -----------------------------

[HDL Cholesterol]

[Total Cholesterol] -[HDL Cholesterol]

Atherogenic Coefficient (AC)= -------------------------------------------

[HDL Cholesterol]

[ Triglycerides]

Atherogenic Index of Plasma (AIP) =log ----------------------

[HDL Cholesterol]

Enzyme Assays: The plasma activities of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP), were determined using Randox Test kits (Randox Laboratories, Crumlin, England). ALT was measured by monitoring at 546nm, the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine. AST was measured by monitoring at 546nm, the concentration of oxaloacetate hydrazone formed with 2,4-dinitrophenylhydrazine. The activity of alkaline phosphatase (ALP) was determined by monitoring the degradation of p-Nitrophenylphosphate to p-nitrophenol, at 405nm.

Determination of Plasma Chemistry: Plasma total and conjugated bilirubin, urea and creatinine concentrations were determined using Randox test kits (Randox Laboratories, Crumlin, England). Direct (conjugated) bilirubin reacted with diazotized sulphanilic acid in alkaline medium to form a blue coloured complex, whose intensity was monitored at 546nm. Total bilirubin was determined in the presence of caffeine, which released albumin bound bilirubin, by the reaction with diazotized sulphanilic acid, with intensity of the resultant solution monitored at 578nm. Urease hydrolyzes urea to ammonia, which was quantified photometrically at 546nm, by Berthelot’s reaction. In the presence of a strong alkali, creatinine reacted with picric acid to form picramic acid which imparted a yellow-red color on the solution, whose intensity was monitored at 492nm. The amount of the complex formed was directly proportional to the creatinine concentration.

Plasma total protein was determined by the Biuret method, while plasma albumin was determined using bromcresol green (BCG) dye binding method²². Bromcresol green, a yellow dye, binds selectively to albumin at pH 4.2, to form an intense blue protein-dye complex with a maximum absorbance at 630nm. Alkaline copper solutions react with peptide bonds in protein to produce a violet color whose intensity at 560nm, is directly proportional to the amount of protein present.

Determination of Plasma Electrolytes: Plasma sodium and potassium concentration was determined by flame photometry. When elements or their compounds are heated at high temperatures, they gain energy and become excited, and so, when they fall back to their ground or original state, produces an emission spectrum which is characteristic of the element. The intensity of the emission is within certain limits, proportional to the concentration of the element in the solution. Plasma calcium concentration was determined by the cresol phthalein complexone method²³. Cresol phthalein complex develops the colour at pH 12; while complexing it with 8-hydroxyquinoline, after measuring the optic density at 575nm, eliminates magnesium interference. An excess of ethyleneglycol (diamine) tetra acetic acid (EDTA) was added for washing the calcium and the optical density was measured again. The difference was proportional to calcium level. The plasma albumin ‘corrected’ calcium levels were calculated²⁴ as follows:

Corrected calcium (mg/dL) = 4{measured calcium (g/L) + 0.02[40 – albumin (g/L)]}.

Plasma chloride concentration was determined by the titrimetric method²⁵. Mercuric nitrate was titrated against chloride to form mercuric chloride in the presence of an indicator diphenyl carbazone. Light violet colour is observed when the entire chloride ion in the sample has been used up and excess mercuric nitrate produces a violet colour. The end point of reaction is proportional to chloride concentration. Plasma bicarbonate concentration was determined by the titrimetric method²⁵. Hydrochloric acid (HCl) reacts with bicarbonate and liberates carbon (IV) oxide, leaving excess unreacted HCl in solution, which can be titrated with sodium hydroxide in the presence of phenol red indicator, to an orange coloured neutralization point. The amount of bicarbonate is inversely proportional to unreacted HCl.

Table1: Time course of the effect of aqueous extract of the leaves of Acalypha wilkesiana on plasma glucose levels of alloxan treated rats

Time

Magnitude

Control

Test control

Reference

Test 1

Test 2

Day 1

· B.A. (mmol/L)

· A.A. (mmol/L)

· % decrease

Day 2

· B.A. (mmol/L)

· A.A. (mmol/L)

· % decrease

3.498±0.188^a

2.568±0.137^a,‡

25.982±4.452^a

5.460±0.175^a,‡

5.916±0.351^a,‡

-8.250±4.635^a

5.723±1.391^b,c

3.705±0.077^b

21.385±11.750^a

10.875±2.310^b,‡

8.310±1.042^a

16.305±7.457^b

4.230±0.153^b

3.435±0.136^b,‡

18.133±4.895^a

5.955±0.595^a,‡

6.645±0.493^a,‡

-15.490±12.479^a,b

5.993±0.449^c,d

3.765±0.190^b,‡

34.600±7.108^a,b

6.750±0.378^c

6.630±0.195^a

0.726±4.185^a,b

9.090±2.164^d

4.005±0.273^b

46.516±9.225^b

9.765±2.306^b

7.965±1.352^a

13.326±4.642^b

Values are mean ± SEM, n=5, per group; B.A. = before administration; A.A. = after administration; Values in the same row with the different superscripts are significantly different at P<0.05: ^‡P<0.05 compared to B.A. value on Day 1; % reduction = percentage reduction from B.A. value for the day.

Table2: Effect of aqueous extract of the leaves of Acalypha wilkesiana on plasma lipid profile of alloxan treated rats

Lipid

Plasma concentration (mg/dL)

Control

Test control

Reference

Test 1

Test 2

Triglyceride

Total cholesterol

HDL cholesterol

VLDL cholesterol

LDL cholesterol

Non HDL cholesterol

113.561±19.411^a,b

430.009±24.980^a

196.953±17.064^a,c

22.382±3.843^a,b

210.673±34.588^a

233.056±31.591^a

68.516±2.748^a

453.075±29.938^a

148.714±4.842^a,b

13.497±0.538^a

290.872±26.402^a

304.367±26.518^b

81.900±7.357^b

444.444±20.292^a

193.502±19.869^c,d

16.141±1.460^b

253.962±33.704^a,b

270.106±32.321^a,b,c

73.710±6.559^a,b

225.469±39.892^b

158.745±9.416^a,b,c

14.519±1.307^a,b

52.213±33.589^c

66.725±34.204^d

76.936±2.748^b

318.489±33.243^c

156.877±9.569^b,d

15.165±0.515^b

146.393±39.507^b

161.558±39.392^c

Values are mean ± SEM, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

Statistical Analysis of Data: All values are quoted as the mean ± SEM. The values of the various parameters were analyzed for statistical significant differences between the groups, using the student’s t-test, with the help of SPSS Statistics 17.0 package. P<0.05 was assumed to be significant.

RESULTS:

The time course of the effect of aqueous extract of Acalypha wilkesiana on plasma glucose levels of alloxan treated rats is given in Table 1. Before administration on day 1, the plasma glucose levels of the test groups were significantly (P<0.05) higher than those of control and reference, but not different from test control. After administration on Day 1, the plasma glucose concentrations of the test groups were significantly (P<0.05) higher than that of control, but not different from the test control and reference. Before administration on day 2, the plasma glucose concentration of Test 2 was not significantly lower than test control, but was significantly (P<0.05) higher than control, reference and Test 1. After administration on day 2, there were no significant differences in the plasma glucose levels of all the animals. The percentage reduction in plasma glucose level of Test 2 after treatment on Day 1 was significantly higher than test control and reference, but not significantly higher than control and Test 1. On day 2, after administration, the percentage reduction in plasma glucose level of Test 2, was significantly (P<0.05) higher than control, but not different from test control, reference and Test 1. Compared to their corresponding values before treatment, the value after treatment on day 1, for control, reference and Test 1, were significantly (P<0.05) lower. Compared to corresponding values before treatment on day 1, the plasma glucose levels of the control (before and after treatment on day 2), test control (before treatment on day 2) and reference (before and after treatment on day 2), were significantly (P<0.05) higher.

Table 2 shows the effect of aqueous extract of Acalypha wilkesiana on the plasma lipid profiles of alloxan treated rats. The plasma triglyceride and VLDL levels of the test groups were not significantly different from those of control, test control and reference. The plasma total, LDL and non-HDL cholesterol concentrations of the test groups were significantly (P<0.05) lower than those of control, test control and reference, with Test 1 being significantly the least, while test control was the highest. The plasma HDL cholesterol levels of the test groups were higher though no significantly than the test control, and lower than the control and reference.

The effect of aqueous extract of Acalypha wilkesiana on the atherogenic indices of alloxan treated rats is shown in Table 3. The cardiac risk ratio and atherogenic coefficient of the test groups were significantly (P<0.05) lower than test control; only Test 1 was significantly lower than control and reference. There were no significant differences in the atherogenic index of plasma of all the animals.

The effect of aqueous extract of Acalypha wilkesiana on plasma marker enzymes is given in Table 4. There were no significant (P<0.05) differences in the plasma aspartate transaminase activities of all the animals. The plasma alanine transaminase activities of the test groups were significantly (P<0.05) higher than the control, but not different from the test control and reference groups.

Table3: Effect of aqueous extract of the leaves of Acalypha wilkesiana on atherogenic indices of alloxan treated rats

Index

Magnitude

Control

Test control

Reference

Test 1

Test 2

Cardiac risk ratio

Atherogenic coefficient

Atherogenic index of plasma

2.258±0.261^a

1.258±0.261^a

-0.616±0.059^a

3.040±0.142^b

2.040±0.142^b

-0.698±0.017^a

2.388±0.205^a,b

1.388±0.205^a,b

-0.736±0.041^a

1.390±0.189^c

0.390±0.189^c

-0.702±0.029^a

2.112±0.343^a

1.112±0.343^a

-0.670±0.037^a

Values are mean ± SEM, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

Table 4: Effects of aqueous extract of the leaves of Acalypha wilkesiana on plasma marker enzymes of alloxan treated rats

Enzyme

Activity (U/L)

Control

Test control

Reference

Test 1

Test 2

Aspartate transaminase

Alanine transaminase

Alkaline phosphatase

17.960±6.347^a

8.336±1.173^a

17.112±4.108^a,c

26.900±6.924^a

29.280±2.465^b

31.050±10.117^a,c

17.876±2.625^a

25.180±7.536^a,b

23.460±6.546^a

24.500±2.324^a

27.900±3.845^b

3.106±0.802^b

24.000±2.127^a

27.550±0.619^b

4.140±0.976^b,c

Values are mean ± SEM, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

Table 5: Effects of aqueous extract of the leaves of Acalypha wilkesiana on plasma chemistry of alloxan treated rats

Parameter

Magnitude

Control

Test control

Reference

Test 1

Test 2

Creatinine (µmol/L)

Urea (mmol/L)

Total bilirubin (µmol/L)

Direct bilirubin (µmol/L)

Unconjugated bilirubin (µmol/L)

Unconjugated/direct bilirubin ratio

Total protein (g/L)

Albumin (g/L)

Bicarbonate (meq/L)

Calcium (mg/dL)

Albumin corrected calcium (mg/dL)

Chloride (meq/L)

Potassium (mg/dL)

Sodium (mg/dL)

55.088±10.472^a

3.392±0.612^a

18.466±3.597^a

12.505±2.415^a

5.961±1.353^a

0.470±0.069^a,b

0.061±0.000^a

0.037±0.001^a

20.750±0.371^a

9.430±0.464^a,b

3.574±0.019^a,b

100.750±0.915^a,b

18.720±0.125^a

325.450±1.150^a

74.800±22.616^a,b

5.785±0.680^a,b

14.081±2.912^a,b

8.873±1.867^a,b

5.242±2.038^a,b

0.644±0.224^a,b

0.060±0.001^a

0.040±0.001^a,b

26.000±0.548^b,c

8.850±0.228^a

3.551±0.009^a

100.250±0.371^a,c

15.210±0.686^b

324.300±1.260^a,b

57.200±5.104^a

7.704±0.901^b

6.372±1.953^b

3.734±1.473^b

2.672±0.737^a,b

0.716±0.192^a

0.060±0.001^a

0.040±0.001^a,b

24.750±0.371^b

9.640±0.120^b

3.582±0.005^b

100.000±1.140^b,c

16.283±0.417^b

324.875±2.344^a,b

78.848±5.368^b

4.949±0.612^a

6.544±2.244^b

5.653±2.124^a,b

0.891±0.223^b

0.506±0.375^a,b

0.059±0.001^a

0.041±0.000^b

24.750±0.194^b

9.680±0.132^a,b

3.584±0.005^a,b

102.000±0.707^b

14.625±0.452^b

330.625±1.336^b

50.864±10.73^a,b

3.170±1.081^a,b

8.496±2.724^a,b

7.571±2.484^a,b

0.891±0.274^b

0.376±0.281^b

0.059±0.001^a

0.040±0.001^a

26.000±0.316^c

9.090±0.300^a,b

3.560±0.012^a,b

97.750±0.968^a

15.503±0.398^b

320.850±2.243^a

Values are mean ± SEM, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

The plasma alkaline phosphatase activities of the test groups were significantly (P<0.05) lower than the test control, control and reference groups.

The effect of aqueous extract of Acalypha wilkesiana on plasma chemistry of alloxan treated rats is shown in Table 5. The plasma creatinine level of Test 1 was significantly (P<0.05) higher than control and reference groups, but not different from test control and Test 2. The plasma urea level of Test 1 was significantly (P<0.05) lower than the reference, but not different from the control, test control and Test 2. The plasma total bilirubin concentration of Test 1 was significantly (P<0.05) lower than control, but not different from the test control, reference and Test 2. The plasma conjugated bilirubin concentrations of the test groups were not significantly different from the control, test control and the reference. The plasma unconjugated bilirubin and potassium concentrations of the test groups were significantly (P<0.05) lower than control, but not different from test control and reference. The unconjugated/conjugated bilirubin ratios of the test groups were not significantly different from control and test control; however, while that of Test 1 was not significantly different from the reference, that of Test 2 was significantly (P<0.05) lower. There were no significant differences in the plasma total protein levels of all the animals. The plasma albumin and sodium concentrations of Test 1 were significantly (P<0.05) higher than those of control and Test 2, but not different from test control and reference: Test 2 had the least sodium level of all the groups. The plasma bicarbonate levels of the test groups were significantly (P<0.05) higher than that of control: Test 1 was significantly (P<0.05) lower than Test 2, but not different from test control and reference, while Test 2 was significantly (P<0.05) higher than the reference, but not different from test control. The plasma calcium levels of Test 1 were significantly (P<0.05) higher than test control, but not different from control, reference and Test 2. The plasma chloride concentration of Test 1 was significantly (P<0.05) higher than that of test control and Test 2, but not different from control and reference.

DISCUSSION:

Alloxan induced diabetes mellitus is often characterized by decreased insulin level, hyperglycemia, elevated triglycerides and total cholesterol, and decreased high-density lipoprotein²⁶. The high percentage reduction in plasma glucose levels, produced by the extract in this study, supports the use of the plants leaves in the management of diabetes mellitus. The extract may exert its antihyperglycemic activity by stimulating insulin secretion from pancreatic β cells and insulin like activity, or by converting pro-insulin to insulin, or alternatively, by inhibiting hepatic gluconeogenesis. The hypoglycemic effect of the extract may have been produced by the saponins present in the leaves²⁷; saponins are a family of compounds with established hypoglycemic activity²⁸.

Elevated plasma total cholesterol level is a recognized and well-established risk factor for developing atherosclerosis and other cardiovascular diseases²⁹, and is found in diabetes mellitus. Therefore, a reduction in plasma total cholesterol level reduces the risk of cardiovascular diseases. Thus, the significantly lower plasma total cholesterol levels produced by the extract, connotes the ability of the extract to protect against cardiovascular complications. This hypocholesterolaemic effect of the leaf extract on alloxan treated rats, corroborates earlier report of the hypocholesterolaemic effect of the same extract on rats fed egg yolk supplemented diet¹⁷; and may be due to its content of saponins. Saponins have been reported to exhibit hypocholesterolaemic properties^28,30-32.

High plasma levels of LDL cholesterol are a risk factor for cardiovascular disease^29,33: while decreases in plasma LDL cholesterol have been considered to reduce risk of coronary heart disease^34,35. In this study, a significantly lower plasma LDL cholesterol levels was produced by the extract, indicating the likely cardio-protective effect of the extract, and further corroborating earlier report of a similar effect on rats fed egg yolk supplemented diets¹⁷.

Increases in plasma HDL cholesterol have been considered to reduce risk in coronary heart disease^34,36. High HDL exerts a protective effect by promoting reverse cholesterol transport through scavenging excess cholesterol from peripheral tissues, delivering it to the liver and steroidogenic organs for synthesis of bile acids and lipoproteins, and eventual elimination from the body^29,36,37; and inhibiting the oxidation of LDL as well as the atherogenic effects of oxidized LDL by virtue of its antioxidant^19,^29,36 and anti-inflammatory activity²⁹. So, the high plasma HDL cholesterol levels, recorded for the test groups, in the present study, are indicative of the cardioprotective effect of the extract. Many studies have shown that non-HDL cholesterolis a better predictor of cardiovascular disease risk than is LDL cholesterol^19,38,39. Therefore, the significantly lower plasma non HDL cholesterols observed in the test groups indicate the ability of the extract, to reduce cardiovascular risk and also corroborate an earlier report of a similar effect on rats fed egg yolk supplemented diet¹⁷.

Atherogenic indices are strong indicators of the risk of heart disease: the higher their value, the higher the risk of developing cardiovascular disease and vice versa^40-43. Low atherogenic indices are protective against coronary heart disease⁴³. In this study, the extract produced significantly lower cardiac risk ratio and atherogenic coefficient. This result corroborates an earlier report of a similar effect on rats fed egg yolk supplemented diet¹⁷.

The extract had no negative effect on the on the integrity and function of the liver and kidney of the diabetic rats. Rather, it (though not significantly) improved the plasma calcium, and lowered plasma urea and creatinine. This implies that the extract improves kidney function.

In conclusion, the treatment did not produce significant differences in the plasma marker enzyme activities and plasma chemistry compared to test control. All of these results indicate a dose dependent control of plasma glucose level by the extract, as well as a possible protective mechanism against the development of cardiovascular complications, via dyslipidemic conditions.

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Received on 18.10.2010

Accepted on 15.11.2010

Research J. Science and Tech. 2(6): Nov. -Dec. 2010: 153-159