Chapter 3

Antioxidant activities

The potentially toxic and beneficial properties of pro-oxidants and antioxidants have made them the focus of many studies. Pro-oxidants may represent a threat to health, whereas antioxidants may counteracts these effects by scavenging pro-oxidants. Antioxidants are very important in industrial processes as well as in biological systems. They are known to possess anti-inflammatory, anti-cardiovascular disease,

antineurogenerative and anticancer properties. Imbalances between pro-oxidants and antioxidants in favor of the pro-oxidants may result in oxidative stress, which in turn result in oxidative damage of cellular components in the form of lipid peroxidation, protein denaturation or DNA conjugation. Oxidative stress has been associated with many diseases like cancer, post-ischemic and neural degradation, Parkinsons and Alzheimer disease, AIDS, and aging and cardiovascular diseases (Kool et al., 2007). Antioxidant assays were selected in two systems i.e. aqueous system and lipid system. Three major assays selected in aqueous system were DPPH scavenging assay, ABTS+ scavenging assay, DNA protection assay while total phenolic contents were determined by using Folin-Ciocalteu reagent. In lipid system TBARS (Thiobarbituric acid reactive substances) was used to evaluate antioxidant activity of crude extracts. Plant species were selected on the basis of previous screening (Inayatullah et al., 2007). Four plant extracts i.e. (L+F) S. nubicola, (S) S. nubicola, (L+S) H. nepalensis and (L+S) A. oblongifolium were selected on the basis of their antitumor and toxicity activities for further studies.

53

Chapter 3 Materials and methods 3.1: Determination of total phenolic contents

Antioxidant activities

Requirements: Folin-Ciocalteu reagent (2N, Sigma), Sodium carbonate (20%), Gallic acid (Sigma), Caffeic acid (Sigma), Rutin (Sigma), Trolox (S-6 methoxy-2, 5, 7, 8tetramethoxy chromane-2-carboxylic acid, Sigma) Procedure Total phenolic contents were determined by modifying the method of Singleton and Rossi (Singleton and Rossi, 1965). Six millilitre of instrument grade water was added to 10 ml volumetric flask followed by addition of 100 l of plant extract solution (initial concentrations 1000, 10,000 and 100, 000 ppm) in 100% methanol leading to final concentration of 10 ppm, 100 ppm and 1000 ppm. Folin-Ciocalteu reagent (Sigma, 500 l 1:10 dilution of 2N solution) was added immediately with vigorous shaking. After one minute freshly prepared aqueous solution of Sodium carbonate (1.5 ml, 20%) was added while shaking constantly. Volume was made up to 10 ml and flasks were kept at room temperature for one hour. Absorbance was measured in 1cm cuvette at 760 nm by using spectrophotometer (Carry WinUV, Varian). Gallic acid, caffeic acid, rutin and trolox were used as positive control and linear regression curves were drawn for each standard used. Data was expressed in terms of GAE/gms, mg of caffeic acid/gram of extract, mg of rutin/ gram of extract as well as mM Trolox/gram of extract. Data was analysed statistically by using ANOVA (SPSS version 11.1).

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Antioxidant activities

3.2. Antioxidant activity In order to investigate antioxidant capacity of the plant extracts, four antioxidant assays were selected, three in aqueous system ( DPPH assay, ABTS+ assay and DNA protection assay) and one in lipid system (TBARS). 3.2.1: DPPH assay (1,1- Diphenyl-2-picryl-hydrazyl radical) Requirements: DPPH (Sigma), Methanol (HPLC grade), Gallic acid, Caffeic acid, Trolox Procedure DPPH assay was performed according to the procedure described by Kulisic et al., (2004) modified by Obeid et al., (2005). DPPH solution was prepared by dissolving 32 mg in 1L 80% methanol. Three millilitre of DPPH solution was added to 1cm plastic cuvette followed by the addition of 200 l of test sample of concentrations (150 ppm, 1500 ppm and 15000 ppm) leading to the final concentration of 10 ppm, 100 ppm and 1000 ppm. Mixture was shaken well and kept in dark at room temperature for one hour. Absorbance was measured at 517 nm by using spectrophotometer (Carry WinUV, Varian). Absorbance of 80% methanol was considered as blank while negative control (DPPH solution) was also run simultaneously. Gallic acid, trolox and caffeic acid were used as positive controls. TEAC (trolox equivalent antioxidant capacity) values were calculated by using the standard regression curve. GAE/gram of extract as well as mg of caffeic acid per gram of extract was also calculated by using the standard regression curves. Percentage inhibition was measured according to the following formula

55

Chapter 3 %age inhibition = (Ac-As/Ac) x 100 where Ac = Absorbance of control As = Absorbance of sample 3.2.2: Calculation of EC50 value

Antioxidant activities

In order to calculate EC50 value, plant extract solution in methanol was further diluted and tested for DPPH assay to find out 50% inhibition. EC50 value was calculated by graph method. EC50 value for gallic acid, caffeic acid and trolox was also calculated. Data was analysed statistically by using ANOVA (SPSS version 11.1). 3.2.3: ABTS+ Assay Requirements: ABTS (Aldrich), Potassium persulfate, monosodium phosphate monohydrate, Disodium phosphate heptahydrate, Methanol (HPLC grade), Trolox Procedure ABTS+ assay was performed by modified method of Paixao et al., (2007). ABTS+ was dissolved in water (7mM) to get the stock solution. ABTS radical cation was produced by reacting the stock solution with 2.45 mM (final concentration) potassium persulfate solution. Solution was kept in dark at room temperature for 12 hours prior to use. Solution was diluted 50 fold with phosphate buffer (pH 8.04) and absorbance was set as 0.7 at 415 nm. Three millilitre of ABTS+ solution was added to 1 cm cuvette followed by the addition of 3l, 15l and 30 l of methanolic solution of plant extracts to get the final concentration as 1 ppm, 5 ppm and 10 ppm respectively. Trolox was used as positive control while ABTS+ solution served as negative control. Absorbance was measured at 415 nm. Data is expressed in terms of TEAC (Trolox equivalent antioxidant capacity). Percentage inhibition was measured according to following formula

56

Chapter 3 %age inhibition = (Ac-As/Ac) x 100 where Ac = Absorbance of control As = Absorbance of sample

Antioxidant activities

Data was analysed statistically by using ANOVA (SPSS version 11.1) 3.2.4: TBARS (Thiobarbituric acid reactive substances) assay Requirements: Thiobarbituric acid (TBA, Sigma), Copper chloride (Sigma), BHT (2-6di-ter-butyl-4 methyl phenol), Linoleic acid (Sigma), HCl (concentrated), Butanol (HPLC grade), Methanol (HPLC grade) Procedure TBARS for four plant extracts was performed by the method described by Kishida et al., (1993). Initial stock solution of methanol extracts were prepared as 10,000, 50, 000 and 100,000 ppm in 100% methanol. Three hundred microliter of CuCl2 solution (0.05 mM) was added to each test tube followed by the addition of 50 l of test solution and 100 l of linoleic acid. Mixture was vortexed for five seconds and incubated at 37C in shaking water bath for 20 hours. Reaction was stopped by the addition of BHT (20 l of 10mM in ethanol) to each test tube and freshly prepared solution of TBA (Thiobarbituric acid) (3ml, 0.67% in 0.1 M HCl) was added. TBA was dissolved in 0.1 M HCl by sonication and momentary heating. Mixture was vortexed for five seconds and tubes were kept in boiling water bath for 10 minutes. Tubes were allowed to cool and pink aqueous layer was transferred to another test tube containing 2.5 ml of 100 % butanol. Mixture was vortexed for five seconds and allowed to settle. Absorbance of pink layer was measured at 532 nm by using spectrophotometer (Carry WinUV, Varian). Butanol served as blank

57

Chapter 3

Antioxidant activities

while negative control (without any test substance) was run simultaneously. Trolox was used as positive control. Standard curve for trolox was used to calculate trolox equivalent values. Percentage inhibition was measured according to following formula %age inhibition = (Ac-As/Ac) x 100 where Ac = Absorbance of control As = Absorbance of sample Data was analysed statistically by using ANOVA (SPSS version 11.1) 3.3: DNA protection assay Requirements: Plasmid DNA (pBR322, Fermentas), Phosphate buffer, Methanol (HPLC grade), DNA ladder (Hind III digest, Fermantas), Ferrous sulphate, Hydrogen peroxide, Ethedium bromide, Bromophenol blue, Agarose (Sigma) Pro-oxidant and antioxidant potential of plant extracts was investigated by modified version of Tian and Hua (2002) method. Plasmid DNA (pBR322 Fermentas) was diluted two fold with phosphate buffer (pH 7.6) and treated with three different concentrations of the plant extracts (10 ppm, 100 ppm and 1000 ppm) in the final reaction volume of 15 l. Fenton reaction was induced by addition of 30% H2O2 (4 l) and 2mM FeSO4 (3 l). Four controls (untreated DNA, DNA treated with 2mM FeSO4, DNA treated with 30% H2O2, DNA treated with 2mM FeSO4 and 30% H2O2) were run simultaneously. Mixture was incubated at 37C in dark for one hour. Reaction was stopped by addition of 2 l of 6X bromophenol blue. Reaction mixture was run on 1% agarose gel and visualized by UV-transilluminator.

58

Chapter 3 Results 3.1: Determination of total phenolic contents

Antioxidant activities

Highly significant quantity of phenolic contents were found in crude extract of leaf and flower of S. nubicola in terms of equivalents of caffeic cid, gallic acid and rutin (Table 3.1). Phenolic contents were quite high in case of methanol extract of leaf and stem of A. oblongifolium too. While lowest phenolic contents were found in leaf and stem extract of H. nepalensis. Equivalents of standards were calculated on the basis of standard regression lines for caffeic acid (R2 = 0.987) (Fig 3.1), gallic acid (R2 = 0.987) (Fig 3.2) and rutin (R2 = 0.981) (Fig 3.3). Order of total phenolics in terms of mM of trolox also remained the same i.e. highest in case of S. nubicola and lowest in case of H. nepalensis (Table 3.1). Standard regression line of trolox was used to calculate TEAC (trolox equivalent antioxidant capacity) (R2 = 0.992) (Fig 3.4). 3.2: Statistical analysis Result of ANOVA presented highly significant difference in phenolic contents amongst the four extracts tested in terms of equivalent of standards.

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Chapter 3

Antioxidant activities

Regression line with Caffeic Acid

0.3 0.25

y = 0.001x - 0.0091 R = 0.9873

2

Absorbance

0.2 0.15 0.1 0.05 0 -0.05 0 50 100

150

200

250

300

Concentration

Fig 3.1. Regression line with caffeic acid with Folin-Ciocalteu reagent
Regression Line with Gallic Acid
0.4 y = 0.0013x - 0.005 R = 0.9874
2

Absorbance

0.3 0.2 0.1 0 -0.1 0 50

100

150

200

250

300

Concentration

Fig 3.2. Regression line with gallic acid with Folin-Ciocalteu reagent

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Chapter 3

Antioxidant activities

Regression line for Rutin

0.14 0.12

y = 0.0005x - 0.0006 R = 0.9813

2

Absorbance

0.1 0.08 0.06 0.04 0.02 0 -0.02 0 50 100

150

200

250

300

Concentration

Fig 3.3. Regression line with rutin with Folin-Ciocalteu reagent

Regression Line with Trolox

0.6 0.5 0.4 y = 5.5786x - 0.0008 R 2 = 0.9924

Absorbance

0.3 0.2 0.1 0 0 -0.1 0.02 0.04 0.06 0.08 0.1 0.12

Concentration (mM)

Fig 3.4. Regression line with trolox with Folin-Ciocalteu reagent

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Chapter 3

Antioxidant activities

Table 3.1: Total phenolic contents in terms of mg of caffeic acid, mg of gallic acid and mg of rutin per gram of crude extracts. Difference in superscript letters indicate significance level at p < .05

Extracts (L+F) S. nubicola (S) S. nubicola (L+S) H. nepalensis (L+S) A. oblongifolium

mg of crude extract per gm of dry weight 80 30 20 200

Total phenolic contents Caffeic acid 201.2 .32 120 .50c 69 .37d 151.5 .74b
a

Gallic acid 138.82 .22 82.53 .34c 47.692 .25d 104.35 .51b
a

Rutin 351.20 .58

a

Trolox 342.08 19.8a 187.50 1 2.80c 107.85 3.829d 233.03 13.05b

205.80 .90c 115.20 .66d 262.53 1.3b

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Antioxidant activities

3.2: DPPH scavenging activity Standard regression line for trolox, gallic acid and caffeic acid were used to calculate DPPH scavenging activity in terms of TEAC (trolox equivalent antioxidant capacity in terms of mM of trolox per gram of extract) GAE (gallic acid equivalent i.e. mg of gallic acid per gram of extract) and caffeic acid equivalent values (mg of caffeic acid per gram of extract) (Fig 3.5). R2 values remained as 0.994, 0.997 and 0.991 for trolox, gallic acid and caffeic acid respectively. Leaf and flower extract of S. nubicola presented highest TEAC, GAE and caffeic acid equivalent values (Table 3.2). Percentage inhibition remained highest in case of methanol extract of leaf and flower extract of S. nubicola (Fig 3.6). While lowest %age inhibition was observed in case of leaf and stem extract of H. nepalensis (Fig 3.10). EC50 value ranged from 5.29 0.04 to 25.1 0.17 (Fig 3.14)). Highly significant EC50 value was obtained in case of leaf and flower exract of S. nubicola. EC50 value remained as 16.59 and 14.31 in case of gallic acid and caffeic acid respectively. EC50 value was calculated by using regression lines for four crude extracts. Fig 3.7, 3.9, 3.11 and 3.13 present calculation of EC50 value for (L+F) S. nubicola, (S) S. nubicola, (L+S) H. nepalensis and (L+S) A. oblongifolium respectively. While Fig 3.8, 3.10 3.12 present pattern of DPPH scavenging in case of (S) S. nubicola, (L+S) H. nepalensis and (L+S) A. oblongifoilium respectively. Statistical analysis by using ANOVA revealed significant difference in EC50 value for crude extracts and three standards tested (Fig 3.14). 3.3: ABTS+ ASSAY Highly significant TEAC value was obtained in case of leaf and stem extract of A. oblongifolium (Table 3.2). Standard regression line for trolox was used to calculate

63

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Antioxidant activities

TEAC value (R2 = .996) (Fig 3.15). Percentage inhibition gradually increased with increase in concentration. 3.4: TBARS (Thiobarbituric acid reactive substances) assay Significant difference was observed in TEAC value for TBARS (Table 3.2) for four crude extracts tested. TEAC value remained in the range of 392.886 3.42 to 462.094 3.62. Standard curve for trolox was used to calculate TEAC value in case of TBARS (R2 = .911). Highest TEAC value in terms of mM of trolox per gram of crude extract was obtained in case of leaf and stem extract of A. oblongifolium i.e. 462.094. Overall result of the TBARS indicated potential of crude extracts to inhibit oxidation in lipid system. Pattern of percentage inhibition with trolox is presented in Figure 3.16.

64

Chapter 3 (a)
90

Antioxidant activities

Percentage scavenging

80 70 60 50 40 30 20 10 0 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035

Concentration

(b)
100 90 80 70 60 50 40 30 20 10 0 0 50 100 150 200 250 300

Percentage scavenging

Concentration

(c)
120

Percentage Scavengigng

100 80 60 40 20 0 0 50 100 150 200 250 300

Concentration

Fig 3.5: DPPH scavenging with three standards (a) trolox (b) gallic acid (c) caffeic acid 65

Chapter 3

Antioxidant activities

DPPH Assay with (L+F) S. nubicola

Percentage scavenging

100 80 60 40 20 0 0 2 4 6 8 10 12

Concentration (ppm)

Fig 3.6: Percentage inhibition with (L+F) S. nubicola in DPPH scavenging assay

EC50 value with (L+F) S. nubicola

60

Percentage inhibition

50 40 30 20 10 0 0

y = 9.33x + 0.72 R2 = 0.999

Concentration (ppm)

Fig 3.7: Regression line used to calculate EC50 in case of DPPH scavenging by (L+F) S. nubicola.

66

Chapter 3
DPPH assay with (S) S. nubicola

Antioxidant activities

Percentage scavenging

100 80 60 40 20 0 0 50 100 150

Concentration

Fig 3.8: Percentage inhibition with stem extract of S. nubicola in DPPH scavenging assay

EC50 with (S) S. nubicola Percentage scavenging

80 70 60 50 40 30 20 10 0 0 5 10 15 20 25

y = 3.4615x + 0.9117 R = 0.9979

2

Concentration (ppm)

Fig 3.9: Regression line with stem extract of S. nubicola used to calculate EC50 value in DPPH scavenging activity

67

Chapter 3

Antioxidant activities

DPPH assay with (L+S) H. nepalensis Percentage Scavenging

100 80 60 40 20 0 0 20 40 60 80 100 120

Concentration

Fig 3.10: Percentage inhibition with leaf and stem extract of H. nepalensis in DPPH scavenging assay

EC50 with (L+S) H. nepalensis

90 80 70 60 50 40 30 20 10 0 0

Percentage scavenging

y = 1.8907x + 3.728 R2 = 0.9823

10

15

20

25

30

35

40

45

Concentration (ppm)

Fig 3.11: Regression line with leaf and stem extract of H. nepalensis used to calculate EC50 value in DPPH scavenging activity

68

Chapter 3

Antioxidant activities

DPPH Assay with (L+S) A. oblongifolium

100 90 80 70 60 50 40 30 20 10 0 0 50 100 150

Percentage scavenging

Concentration

Fig 3.12: Percentage inhibition with leaf and stem extract of A. oblongifolium in DPPH scavenging assay

EC50 with (L+S) A. oblongifolium Percentage scavenging

60 50 40 30 20 10 0 0 2 4 6 8 10 12

y = 5.0807x + 4.1848 R = 0.9793

2

Concentration (ppm)

Fig 3.13: Regression line with leaf and stem extract of A. oblongifolium used to calculate EC50 value in DPPH scavenging activity

69

Chapter 3

Antioxidant activities

EC50 for standards and crude extracts in DPPH assay EC50 value (ppm)
30 25 20 15 10 5 0

a b d g e c f

al lic

Ca ac id

ffe ic a

Tr ol ci d

ox

(L +

S)

(L + A. ob lo ng

S)

(S ) H .n ep al en

S. sis

nu

(L + bi co l a

F)

S.

nu b

ifo liu

ic ol a

Standards and crude extracts

Fig 3.14: EC50 value for four methanol extracts and standards. Difference in letters present significance difference between EC50 value at p < .05.

70

Chapter 3

Antioxidant activities

Percentage scavenging with ABTS+ assay with trolox as standard

90 80 70 60 50 40 30 20 10 0 0

Percentage scavenging

y = 1.0339x + 2.486 R = 0.9836

2

10

20

30

40

50

60

70

80

90

Concentration (ppm)

Fig 3.15: Percentage inhibition with Trolox in ABTS+ scavenging activity

71

Chapter 3

Antioxidant activities

Fig 3.16: Percentage inhibition with trolox in case of TBARS (thiobarbituric acid reactive substances) assay.

72

Chapter 3

Antioxidant activities

Table 3.2: Result of DPPH assay, ABTS+ assay and TBARS in terms of TEAC (Trolox equivalent antioxidant capacity) in terms of mM of trolox per gram of extract. DPPH scavenging is also expressed in terms of GAE (Gallic acid equivalents i.e. mg of gallic acid per gram of extract) and equivalents of caffeic acid (mg of caffeic acid per gram of extracts). Difference in superscript letters indicate significance level at p < .05 ABTS+ assay Plant extracts (L+F) S. nubicola (S) S. nubicola (L+S) H. nepalensis DPPH scavenging activity Caffeic acid TEAC (mM) GAE (mg) (mg) 248.4 .49a 66.9 . 075c 26.7 0.18d 289.86 .51a 249.98 1.6a TEAC (mM) 149.23 3.5b 70.42 2.6c 54.74 1.6d 160.23 6.4a TBARS TEAC (mM) 400.314 6.94 c 420.062 8.90 b 392.886 3.42 d 462.094 3.62 a

123.75 .112c 106.64 .09c 94.5 .14d 176.76 .9b 81.24 .9d 149.85 .9b

(L+S) A. oblongifolium 140.4 1.2b

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Chapter 3

Antioxidant activities

3.5: DNA protection assay DNA protection activity was observed in case of leaf and flower extract of S. nubicola at 10 ppm and 100 ppm while at 1000 ppm pro-oxidant activity was obvious (Fig 3.17). Stem extract of S. nubicola showed DNA protection activity at 100 ppm and 1000 ppm while at 10 ppm pro-oxidant activity was observed. Leaf and stem extract of H. nepalensis showed pro-oxidant activity at 1000 ppm. While leaf and stem extract of A. oblongifolium could not show DNA protection activity at any concentration tested. Results of DNA protection assay are explained on the basis of Fenton reaction as described in chapter 1, page 21. Conclusion It is obvious that total phenolic contents by Folin-Ciocalteu reagent were higher in leaf and flower extract of S. nubicola and its DPPH scavenging activity also remained highest. (L+F) S. nubicola can further be selected for fractionation. Crude extract of A. oblongifolium showed highest potential for inhibition of lipid oxidation which can be considered in future to isolate active antioxidants in lipid system.

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Chapter 3

Antioxidant activities

10

11

12

13

14

15

16

17

Fig 3.17: DNA protection assay with different concentrations of crude extracts. Lane 1 is Hind III marker. Lane 2-5 presents controls (untreated DNA, DNA treated with 2mM FeSO4, DNA treated with 30 % H2O2, DNA treated with 2mM FeSO4 and 30 % H2O2). Lane 6-8 presents DNA treated with crude extract of leaf and flower of S. nubicola at 10, 100 and 1000 ppm. Lane 9-11 is treatment of DNA with different concentrations (10, 100 and 1000 ppm) of crude extract of (S) S. nubicola . Lane 12-14 presents treatment of DNA with three concentrations (10, 100 and 1000 ppm) of crude extract of (L+S) H. nepalensis. Lane 15-17 presents DNA treatment with three different concentrations (10, 100 and 1000 ppm) of crude extract of A. oblongifolium.

75