Determination Of Antioxidant Properties

Published Date: 02 Nov 2017

CHAPTER 4

Most diseases are occurring due to oxidative stress. It is a good approach to cure the disease with something which is highly antioxidant. Various methods are regularly carried out in the determination of the antioxidant activity of organic compounds in plant extract. There is no single method can completely estimate the total antioxidant activity since different antioxidant compounds may possibly act in vivo through different mechanisms. The objective of this study was to assess the total antioxidant activity in Morinda citrifolia (M. citrifolia) fruit using three different assays; 1) 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay 2) β-carotene bleaching assay 3) Total phenols by Folin-Ciocalteu in order to provide data on antioxidant potential of the ability of the plant extracts.

4.1.1 DPPH free radical scavenging assay

The percentage of inhibition of M. citrifolia (Mengkudu) against the DPPH radicals is shown in Figure 4.1. Ascorbic acid which acts as the standard had the higher antioxidant activity with the percentage of inhibition of 88.99 ± 0.5%. The scavenging effect of M. citrifolia ethanolic extract was found to be 27.21 ± 1.24% at the concentration of 10.24mg/mL which showed significantly different when compared to ascorbic acid.

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Figure 4.1. DPPH free radical scavenging of Morinda citrifolia ethanolic extract. Data expressed as mean ± SD. Bars with different alphabets are significantly different (p<0.05).

4.1.2 Beta-carotene bleaching assay

The β-carotene bleaching method is one of the most frequently applied methods for determining the total antioxidant property of the extracts. In the β-carotene bleaching assay, linoleic acid produces hydroperoxides as free radicals during incubation at 50 °C and attacks the β-carotene molecules that cause reduction in the absorbance at 470 nm. The ability of the M. citrifolia extract was measured and compared with the control which contained no antioxidant component.

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Figure 4.2. Antioxidant activity of Morinda citrifolia ethanolic extract (Mengkudu) compared with BHT using β-carotene bleaching assay. Data expressed as mean ± SD. Bars with same alphabets are not significantly different (p>0.05).

As shown in Figure 4.2, the mean total antioxidant activity from M. citrifolia ethanolic extract was 78.96 ± 25.9% and for BHT was 92.56 ± 0.9%. M. citrifolia extract had lower antioxidant activity than had standard (BHT). The independent t-test analyses showed no significant difference (p>0.05) between means total antioxidant activity of M. citrifolia ethanolic extract and standard BHT.

4.1.3 Total phenolic content

Total phenol content (TPC) was determined in comparison with standard gallic acid. A linear calibration curve of Gallic acid with r2 value of 0.9827 was obtained as shown in Appendix A1. The means TPC of M. citrifolia extract was measured using Gallic acid equivalent (GAE) equation of y = 2.7593x + 0.00068 (R2=0.0.9827) whereby y = absorbance at 765nm and x = concentration of TPC in mg per ml of the extract. The results which expressed as milligrams gallic acid equivalent (GAE)/ 100 g dry weight (DW) sample showed the total phenolic content for M. citrifolia ethanolic extract evaluated using the Folin-Ciocalteu assay was 281.83 ± 14.78 mg GAE/100g DW.

4.2 Evaluation of anticoagulant properties in vitro

4.2.1 Effect of Morinda citrifolia extracts on prothrombin time (PT) in vitro

The mean baseline PT values of the respondents were varied between 9.4 and 14.2 seconds, with the mean of 11.38 ± 1.15 seconds. The mean PT values in the presence of 10mg/mL of both ethanolic and aqueous extracts were 14.24 ± 2.05 and 13.27 ± 2.11 respectively which statistically was not significant difference. Prolongation of the PT values of various concentration of both ethanol based (Ee) and aqueous based extract (Ae) were significant as compared to the control group with the p value of less than 0.05 (Table 4.1).

PT assays were markedly prolonged with the increment of the extracts concentration of 20, 30, 40 mg/mL. The mean PT of Ee was of 18.93 ± 2.64, 31.75 ± 8.07, and 58.27 ± 15.69 seconds respectively. Similar findings were observed with Ae where at concentration of 10, 20, 30 and 40 mg/mL the mean PT value were 13.27 ± 2.11, 19.83 ± 5.54, 32.64 ± 12.03, and 55.97 ± 14.54 seconds respectively. The blood coagulometer failed to measure any reading with both form of extracts concentration of 50 mg/mL. There were statistically significant differences of the mean PT value between the baseline (control) as compared to the M. citrifolia crude extracts concentration of 20, 30 and 40 mg/mL with all the p-value were less than 0.05.

Table 4.1. Prothrombin Time (PT) with various concentrations of both Morinda citrifolia ethanolic and aqueous based crude extracts.

Extract

Concentration (mg/mL)

Prothrombin time

(seconds)

Control

-

11.38 ± 1.15 a

Ee

10

14.24 ± 2.05 a

20

18.93 ± 2.64 b

30

31.75 ± 8.07 c

40

58.27 ± 15.69 d

50

> 60

Ae

10

13.27 ± 2.11 a

20

19.83 ± 5.54 b

30

32.64 ± 12.03 c

40

55.97 ± 14.54 d

50

> 60

Each value represents the mean ± S.D. The same alphabet are not significantly different (p>0.05) within groups using ANOVA test. The bold values indicate the clot was not observed in measured sample. No statistical tests were performed. Ee: Ethanol based extract; Ae: Aqueous extract

4.2.2 Effect of Morinda citrifolia extracts on activated partial thromboplastin time (APTT) in vitro

The effect of M. citrifolia crude extracts on APTT assays were as shown in Figure 4.3. The baseline mean APTT values of respondents were range between 24.1 to 34.3 seconds, with a mean of 30.06 ± 1.27 seconds. The mean APTT value in the presence of 10mg/mL of both ethanol and aqueous extracts were of 33.70 ± 3.65 and 32.08 ± 2.16 seconds respectively. Both Ee and Ae did not show significant differences of the APTT assays with the concentration extracts of 10mg/mL.

At the concentration of 20 mg/mL up to 40 mg/mL, significance differences from the baseline (control group) was observed with p<0.05. The mean APTT value of Ee at 20, 30, 40 mg/mL were 46.36 ± 7.58, 81.55 ± 16.97, and 118.03 ± 10.18 respectively. Similar results were observed with Ae where the mean APTT values were 41.91 ± 4.48, 71.29 ± 18.27, and 114.74 ± 11.53. At the concentration extracts of 50mg/mL, the blood coagulometer was failed to measure the APTT value of both forms of extracts.

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Figure 4.3. APTT with various concentrations of both ethanol and aqueous based Morinda citrifolia crude extracts. Each value represents the mean ± S.D. The same alphabet are not significantly different (p>0.05) within groups. The results were analyzed using ANOVA test. Ee: Ethanol based extract; Ae: Aqueous based extract

4.3 Effect of Morinda citrifolia extracts on the full blood count parameter and bleeding time in vivo

4.3.1 Full blood count

Table 4.2 indicates the blood count parameters such as red blood cells (RBC), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), and platelets. There was almost no significant difference (p>0.05) between all groups. However, RBC group the count was found to be significantly lower (p<0.05) at aspirin when compared to saline. In Hb group, the count was found to be significantly dropped at 750 mg/kg and aspirin when compared to saline.

Figure 4.4 illustrate the white blood cells and its components count including neutrophils, lymphocytes, monocytes, and eosinophils. There was no significant difference (p>0.05) between all groups. Although the counts were averagely decreased at 75 mg/kg, feeding M. citrifolia to the rats did not cause any significant difference.

Table 4.2. Full blood count parameter

RBC (x1012/L)

Hb (g/L)

MCV (fL)

MCHC (g/L)

Platelet (x109/L)

Saline

8.2 ± 0.98 a

152.8 ± 2.78 a

55.8 ± 1.3 a

333.8 ± 12.83 a

1020.8 ± 173.06 a

7.5 mg/kg

7.31 ± 0.5 a

144.67 ± 6.22 a

57.0 ± 3.63 a

348.83 ± 16.47 a

971.33 ± 201.44 a

75 mg/kg

7.75 ± 0.36 a

149.0 ± 6.45 a

56.0 ± 2.83 a

345.33 ± 13.82 a

1054.67 ± 104.16 a

750 mg/kg

7.83 ± 0.32 a

150.83 ± 6.23 b

56.67 ± 3.67 a

339.33 ± 10.37 a

1174.17 ± 94.85b

Aspirin

7.39 ± 0.68 b

141.17 ± 11.09 b

55.0 ± 4.15 a

350.17 ± 22.75 a

840.0 ± 205.46b

Each value represents the mean ± S.D. The same alphabet are not significantly different (p>0.05) within groups using ANOVA test. RBC = red blood cells, Hb = haemoglobin, MCV = mean corpuscular volume, MCHC = mean corpuscular haemoglobin concentration

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Figure 4.4. White blood cells and its components count. Each value represents the mean ± S.D. The same alphabet are not significantly different (p>0.05) within groups. The results were analyzed using ANOVA test.

4.3.2 Bleeding time

Bleeding time can also used to assess the platelet function by measuring the time it takes for bleeding to stop. Table 4.3 indicates the bleeding time of rats in all groups. There are significant different (p<0.05) between saline, 7.5, 75, and 750 mg/kg groups. The mean value of the bleeding time for saline, 7.5, 75, and 750 mg/kg were 310.0 ± 26.46, 430.0 ± 26.46, 513.3 ± 20.28, and 700.0 ± 10.0 respectively. The mean value of Aspirin group with 690.0 ± 30.0 was also significantly different while comparing with saline.

Table 4.3. Bleeding time (BT) with various concentrations of Morinda citrifolia extracts and controls.

Dosage

(mg/kg)

Bleeding time

(seconds)

Saline

-

310.0 ± 26.46 a

M. citrifolia

7.5

430.0 ± 26.46 b

75

513.3 ± 20.28 c

750

700.0 ± 10.0 d

Aspirin

30

690.0 ± 30.0 d

Each value represents the mean ± S.D. The same alphabet are not significantly different (p>0.05) within groups using ANOVA test.

4.4 Effect of Morinda citrifolia extracts on prothrombin time (PT) and activated partial thromboplastin time (APTT) in vivo

The effect of M. citrifolia crude extracts on PT and APTT in vivo were shown in Figure 4.5. There are no significant different between normal and three different groups of treatment for both PT assays. The mean value of PT for saline, 7.5, 75 and 750 mg/kg were 9.3 ± 0, 9.38 ± 0.33, 8.98 ± 0.71 and 9.88 ± 0.41 second respectively. Similar outcome also found in APTT assays which no significant different among the groups. The mean value of APTT for saline, 7.5, 75 and 750 mg/kg were 17.0 ± 1.97, 17.55 ± 1.31, 17.37 ± 1.13 and 18.12 ± 0.51.

Figure 4.5. Prothrombin time (PT) and Activated partial thromboplastin time (APTT) of experimental Sprague Dawley rats. Data expressed as mean ± SD.

4.5 Effect of M. citrifolia extract on platelet aggregation test in vivo

The effect of M. citrifolia extract on platetet aggregation test in experimental animal was conducted after several trials on human blood sample. As shown in Appendix the outcome founds that the M. citrifolia extract do have some anti-platelet activity by reducing the platelet aggregation of human blood samples.

Table 4.4 shows the mean and the percentage of inhibition of platelet aggregation in all experimental groups. There was no significant difference (p>0.05) of inhibition of platelet aggregation induced by ADP between saline, 7.5 mg/kg, 75 mg/kg, 750 mg/kg and aspirin groups. Administration of M. citrifolia to the rats caused the platelet aggregation induced by collagen to be reduced in all treatment groups. There were significant difference (p<0.05) between saline, 7.5 mg/kg, 75 mg/kg, 750 mg/kg and aspirin groups.

Table 4.4. Platelet aggregation with various concentrations of Morinda citrifolia extracts and controls.

ADP

Collagen

Impedance

% inhibition (Y)

Impedance

% inhibition (Y)

Saline

2.2 ± 2.68 a

0

7.5 ± 2.67 a

0

7.5 mg/kg

1.6 ± 0.89 a

27.27

6.0 ± 0.63 b

20

75 mg/kg

1.5 ± 0.55 a

31.82

5.17 ± 0.98 b

31.07

750 mg/kg

1.5 ± 0.84 a

31.82

4.67 ± 1.21 b

37.73

Aspirin

1.4 ± 1.14 a

36.36

4.2 ± 2.59 b

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Mean platelet aggregation of experimental rats and statistical analysis. Each value represents the mean ± S.D. The same alphabet are not significantly different (p>0.05) within groups using ANOVA test.