International Journal of Biological Macromolecules 67 (2014) 220–227
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Free radical scavenging activity and reducing power of Acacia nilotica wood lignin Keshaw Ram Aadil, Anand Barapatre, Sudha Sahu, Harit Jha ∗ , Bhupendra Nath Tiwary Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, (Central University) Bilaspur 495009, Chhattisgarh, India
a r t i c l e
i n f o
Article history: Received 14 November 2013 Received in revised form 6 March 2014 Accepted 22 March 2014 Available online 28 March 2014 Keywords: Acacia nilotica Lignin Antioxidant activity
a b s t r a c t Nine different fractions of lignin extracted by alkali, hot water and organosolv methods from Acacia wood powder were assessed for antioxidants activity. Results indicated that methanolic lignin fraction had highest polyphenol content of 393.30 ± 9.2 g/ml (GAE). The oraganosolv lignin with total phenols and phenolic hydroxyl group content exhibited significant antioxidant activity as compared to other lignin fractions. Antioxidant properties of acetone fractions revealed a high antiradical scavenging activity ( acetone > hot water > A1 > ethanol > Table 2 Total carbohydrates, phenolic hydroxyl and carboxyl groups content in all lignin fractions. Lignin fractions
Carbohydrates content (mg/g)
LS A1 A2 A3 A4 HWL AEL EEL MEL PEL
5.22 1.13 0.61 1.04 0.87 4.50 8.26 6.76 7.07 4.87
± ± ± ± ± ± ± ± ± ±
0.16bc 0.22a 0.24a 0.35a 0.08a 0.11b 0.07cd 0.51c 1.75cd 0.03b
Phenolic hydroxyl group (%) 0.52 0.74 2.73 0.14 0.53 1.79 2.41 3.05 2.79 3.29
± ± ± ± ± ± ± ± ± ±
0.02b 0.01c 0.04f 0.01a 0.03b 0.02d 0.03e 0.05g 0.07f 0.09h
Carboxyl group (% w/w) 1.53 1.31 2.73 2.62 1.70 1.61 1.59 2.10 1.76 1.43
± ± ± ± ± ± ± ± ± ±
0.04b 0.07a 0.10d 0.03d 0.08b 0.02b 0.09b 0.05c 0.04b 0.07ab
Values represent mean ± standard deviation (SD) of three independent experiments (n = 3). Values within in a column followed by different lower case letters are significantly different according to ANOVA (Tukey test).
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Table 3 Phenol contents, antioxidant activity (EC50 value) on DPPH radicals, ABTS radical, FRAP value, hydrogen peroxide scavenging and reducing power of Acacia lignin. Lignin Fractions LS A1 A2 A3 A4 HWL AEL EEL MEL PEL
TPC1 100.27 232.48 73.01 102.27 87.74 295.21 343.28 229.54 393.30 208.28
DPPH2 ± ± ± ± ± ± ± ± ± ±
f
5.6 6.2d 3.2g 3.5f 1.9g 7.3c 3.2b 9.07d 9.2a 4.1e
149.96 83.85 103.86 110.48 128.21 81.46 79.89 81.72 84.58 82.64
ABTS3 ± ± ± ± ± ± ± ± ± ±
d
4.4 0.4a 4.4b 1.2b 8.5c 0.9a 0.07a 00a 3.5a 0.6a
FRAP4
2.70 ± 0.1 2.84 ± 0.7a 3.25 ± 0.09c 3.15 ± 0.05b 3.95 ± 0.13d 2.78 ± 0.11a 2.95 ± 0.14a 2.77 ± 0.15a nd nd a
43.05 165.36 26.01 55.92 40.64 241.94 294.16 157.40 273.23 66.47
PMA5 ± ± ± ± ± ± ± ± ± ±
c
8.5 19.7b 4.8c 7.1c 8.5c 19.0 17.9a 55.5b 12.2a 10.5c
67.96 120.55 108.88 97.77 71.10 175.92 342.2 167.58 211.47 54.44
HPS6 ± ± ± ± ± ± ± ± ± ±
f
2.2 6.7d 6.7d 6.4e 2.2f 6.1c 12.9a 2.5c 1.9b 3.8f
178.61 139.2 107.67 175.08 162.20 161.23 114.7 121.4 110.7 101.8
RP7 ± ± ± ± ± ± ± ± ± ±
i
1.2 0.7f 1.4b 0.8h 0.5g 0.9g 1.6d 0.8e 1.2bc 0.7a
19.8 ± 1.3f 99.4 ± 1.2d 20.7 ± .0.4f 35.6 ± 1.7e 25.3 ± 0.6f 125.8 ± 2.2c 196 ± 0.9a 138.5 ± 7.4b 140.6 ± 1.7b 2.33 ± 0.3g
Values represent mean ± standard deviation (SD) of three independent experiments (n = 3). Values within in a column followed by different lower case letters are significantly different according to ANOVA (Tukey test). nd—not determined. 1 Total phenol content expressed as concentration of polyphenol (g) in term of gallic acid equivalent (GAE) per mg of extracted lignin fractions. 2 Concentration of test compound required to scavenge 50% inhibition of DPPH radical. 3 Concentration of test compound required to scavenge 50% inhibition of ABTS radical. 4 FRAP value was expressed as equivalent of gallic acid (mol). 5 Phosphomolybdate assay of each lignin fractions were expressed as equivalents of ascorbic acid (mmol/100 mg of sample). 6 Concentration of test compounds required to scavenge 50% of hydrogen peroxide (HPS-CUPRAC assay). 7 Reducing power of all extracted lignin fractions.
propanol > A3 > lignosulphonic acid > A4 > A2. A similar trend has been mentioned in available literature [3]. Further, the results revealed that phenolic content of Acacia wood lignin was higher in organosolv (208–393 g of GAE per mg of sample) and hot water (295.21 ± 7.33 g of GAE per mg) as compared to alkali lignin. Our observed value was significantly higher than the earlier reports in Miscanthus sinensis lignin (170–190 g of GAE per mg of lignin) [3]. The overall finding is suggestive of a higher extractability of phenolic contents in organosolv compared to hot water and alkali lignin [4].
3.3. DPPH radical scavenging activity The available report on lignin model compounds suggests that free phenolic hydroxyl groups are crucial for antioxidant activity while the aliphatic hydroxyl groups show a negative correlation [19]. The DPPH radical scavenging capacity of different lignin fractions were observed in the range of 49.91–93.85%, which is higher than that of ascorbic acid (28.25% at 200 M) but equivalent to tannic acid (83.42% at 50 M) used as standards. Among all the lignin fractions acetone fractions exhibited the highest inhibition percentage (93.85%). The DPPH scavenging activities of acetone, hot water and ethanol extracted lignin fractions were found to be more significant (p < 0.05) as compared to alkali lignins, whereas lignosulphonic acid showed the lowest DPPH scavenging activity. The lowest DPPH scavenging activity of commercial lignin could be due to its high molecular weight which enhances its hetrogenicity resulting into a decreased free radical scavenging activity [5,19,33]. The lowest EC50 value 79.89 ± 0.07 g/ml was observed in the acetone extracted lignin (Table 3). Lu et al. [2] have explained the observed higher EC50 (2070 ± 0.17 g/ml) of nanoscale lignin by assuming that organosolv pulping lignin contains a large number of active functional groups, which act as electron donors and neutralize the free radical. Our findings suggest that the purified Acacia lignin fractions, especially solvent fractions, have more free phenolic hydroxyl groups and low molecular weight as compared to commercial lignosulphonic acid. FTIR analyses of lignin fractions support our contention that the higher free radical scavenging activity of acetone (AEL) and ethanol (EEL) fractions might be due to C O of carboxyl or ketone groups that appears around at 1700 cm−1 in FTIR spectrum (Fig. 2). These might act as electron donating groups and provide stability to the compounds.
3.4. ABTS•+ radical scavenging activity The ABTS•+ free radical scavenging activity was quantified in terms of percent inhibition by each of the lignin fraction at a fixed concentration of 5 g/ml. A significant variation in the percent inhibition of different lignin fractions was observed (65.19–98.14%). The highest inhibition was observed in the lignosulphonic acid (98.14%) followed by the ethanol (95.35%) and alkali lignin (A1) (88.86%) at the same concentration. The EC50 value of different extracted lignin fractions ranged from 2.75 to 3.95 g/ml, while EEL exhibited the lowest EC50 value of 2.77 g/ml (Table 3). Though the reports on ABTS•+ radical scavenging activity of lignin is scanty, the available literature suggests that the organosolv lignins exhibited higher phenolic content and better antiradicals power than the alkali lignin [34]. A recent articles on lignin antioxidant activity provides evidence that the scavenging activity of lignin on ABTS•+ radical is mainly due to the electron or proton transfer mechanism, whereas for the DPPH radical test the combination of electron transfer with H atom transfer is considered [35]. The higher scavenging activity of oraganosolv lignin fractions (mainly EEL and AEL) for ABTS•+ radical was possibly due to higher phenolic hydroxyl and carboxyl groups as confirmed by analytical methods and FTIR analysis. 3.5. FRAP assay FRAP activity of nine lignin fractions varied considerably from 26.01 ± 4.89 to 294.16 ± 17.92 mol (Table 3). The highest FRAP value was exhibited by acetone fraction (294.16 ± 17.92 mol), followed by the methanolic (273.23 ± 12.25 mol) and hot water fraction (241.94 ± 19.03 mol) of lignin. The FRAP value of acetone fraction was significantly (p < 0.05) higher than alkali fractions. The order of FRAP value (mol) was as follows: AEL > MEL > HWL > A1 > EEL > PEL > A3 > LS > A4 > A2. The observed FRAP value of organosolv fractions of lignin especially in acetone fraction (AEL) might be due the higher phenolic or hydroxyl groups content as compared to alkali lignin fraction. As mentioned earlier [33], the hydroxyl groups and conjugated double bond of phenolic compounds like vitamin E and other phenolic acid are crucial for FRAP activity [36]. 3.6. Phosphomolybdate assay Antioxidant compound reduces the Mo(VI) to Mo(V) and produces green coloured phosphomolybdenum V complex, which
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Fig. 2. FTIR spectra of different lignin fractions extracted from Acacia wood. LS—lignosulphonic acid, A1—alkali lignin 0.1 N NaOH, A2—alkali lignin 0.2 N NaOH, A3—alkali lignin 0.3 N NaOH, A4—alkali lignin 0.4 N NaOH, HWL—hot water extracted lignin, AEL—acetone extracted lignin, EEL—ethanol extracted lignin, MEL—methanol extracted lignin, PEL—propanol extracted lignin.
shows a maximum absorbance at 695 nm. Different lignin fractions exhibited varying antioxidant activities as expressed in terms of ascorbic acid equivalent (mmol/100 mg). As shown in Table 3, acetone fraction of Acacia lignin exhibited the highest antioxidant power (342.22 mmol/100 mg, ascorbic acid equivalents), followed by the MEL (211.47 mmol/100 mg AAE) and HWL (175.92 mmol/100 mg AAE) fractions at a concentration of 150 g/ml, while lowest antioxidant effect was shown by PEL (54.44 mmol/100 mg AAE) fraction (p < 0.05). Based on the results, the order of overall antioxidant capacity of different lignin fractions was AEL > MEL > HWL > EEL > A1 > A2 > A3 > A4 > LS > PEL.
3.7. Determination of reducing power The reducing capacity of a compound serves as a significant indicator of its potential antioxidant activity. The reducing power assay is based on the mechanism of electron donating activity, which is main mechanism of phenolic antioxidant action [4]. Table 3 shows the reducing power of the extracts represented as EC50 value (g/ml) by interpolation from linear regression analysis of concentration vs. absorbance at 700 nm. Since the AEL extract showed a higher reducing power of 196 ± 0.93 at 250 g/ml as compared to other lignin fractions whereas the lowest reducing capacity was
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Table 4 Correlation between antioxidant activities/reducing power parameters and total phenolic content of Acacia wood lignin. Assays
TPC
EC50 a
EC50 b
FRAPc
EC50 d
EC50 e
RPf
TPC DPPH ABTS FRAP Phosphomolybdate HPS Reducing power
1 0.5498 0.2183 0.9147 0.5886 0.2007 0.7095
1 0.0674 0.4674 0.3123 0.4502 0.4054
1 0.176 0.0921 0.0132 0.1931
1 0.7695 0.0929 0.8861
1 0.1301 0.8573
1 0.0775
1
TPC—total phenol content expressed as concentration of polyphenol (g) in term of gallic acid equivalent per mg of extracted lignin fractions. a Concentration of test compound required to scavenge 50% inhibition of DPPH radical. b Concentration of test compound required to scavenge 50% inhibition of ABTS radical. c FRAP value was expressed as equivalent of gallic acid (mol). d Phosphomolybdate assay of each lignin fractions were expressed as equivalents of ascorbic acid (mmol/100 mg of sample). e Concentration of test compound required to scavenge 50% hydrogen peroxide. f Reducing power of lignin fractions.
shown by PEL of 2.33 ± 0.31 g/ml (Table 3). Significantly, the Acacia lignin fractions exhibited higher reducing capacity than the antioxidant compounds, such as gallic acid and tannic acid (25.77 and 44.64 g/ml, respectively) used as the standards. The observed FRAP value and reducing power capacity of oragnosolv lignin was significantly higher (p < 0.05) than other fractions, possibly due to the higher content of phenolic ( OH) groups in organosolv fractions of lignin, as confirmed by TPC assay and FTIR spectral analysis, suggesting their role as electron donors. 3.8. HPS–CUPRAC assay HPS–CUPRAC assay is a novel and recommended method for determination of scavenging activity of hydrogen peroxide by polyphenolic compounds [28]. Lignin fractions were found to show hydrogen peroxide scavenging (HPS) activity at a concentration of 200 g/ml. HPS scavenging percent of different lignin fractions ranged from 55.33 to 97.44%. Propanol, alkali (A2) and methanol fractions showed up to 90% HPS activity. The lowest EC50 value of 101.87 ± 0.7 was observed with propanol fraction. The EC50 value for hydrogen peroxide quenching of different lignin fractions were in the order of PEL < A2 < MEL < AEL < EEL< A1 < HWL< A4 < A3 < LS (Table 3). 3.9. Correlation with total phenol contents and EC50 values of antioxidant activities/reducing power In order to understand the interrelationship between Acacia lignin fractions and their antioxidant capacity, reducing power and phenolic contents correlation analysis was performed. As shown in Table 4, the TPC was moderately correlated with DPPH radical scavenging activity and antioxidant activity (R2 > 0.5498), whereas good correlation was observed with FRAP and reducing power (R2 = 0.9147 and 0.7095, respectively). The strong correlation of TPC with FRAP and reducing power assay suggested that phenolic contents affect the reducing power of Acacia lignin. However, a moderate correlation between TPC and antiradicals activity of Acacia lignin might not be attributed to the total phenol content alone but also to some other factors such as extraction methods and solubility as reported earlier [2,33]. 4. Conclusion The statistics of the present study put forward that Acacia lignin has the effective antioxidant activities. The results indicated the organosolv lignin fraction of Acacia to be more potent for antioxidant capacity. Some functional groups such as hydroxyl, methoxyl
and carboxyl groups are responsible for its antioxidant properties, based on which novel compounds may be extracted as the source of natural antioxidant to be used as possible food supplement and novel therapeutic products. Further studies on Acacia lignin as antioxidant in animal models are required to assess their potential benefits. Acknowledgements The funding of the present research by the University Grant Commission (UGC), New Delhi, India vide nos. F.41-543/2012 (SR) and F.40-183/2011, is gratefully acknowledged. Authors are also thankful to STIC-Cochin for FTIR analysis. References [1] M. Christiernin, Plant Physiol. Biochem. 44 (2006) 700–706. [2] Q. Lu, M. Zhu, Y. Zu, W. Liu, L. Yang, et al., Food Chem. 135 (2012) 63–67. [3] A. Garcia, A. Toledano, M.A. Andres, J. Labidi, Process Biochem. 45 (2010) 935–940. [4] T. Kalaivani, L. Mathew, Food Chem. Toxicol. 48 (2010) 298–305. [5] T. Dizhbite, G. Telysheve, V. Jurkjane, U. Viesturs, Bioresour. Technol. 95 (2004) 309–317. [6] W. Brand-Williams, M.E. Cuvelier, C. Berset, Lebensm. Wiss. Technol. 28 (1995) 25–30. [7] M.B. Arnao, A. Cano, M. Acosta, Food Chem. 73 (2001) 239–244. [8] I.F.F. Benzie, J.J. Strain, Anal. Biochem. 239 (1996) 70–76. [9] P.C. Wootton-Beard, A. Moran, L. Ryan, Food Res. Int. 44 (2011) 217–224. [10] K. Thaipong, U. Boonprakob, K. Crosby, L. Cisneros-Zevallos, D.H. Byrne, J. Food Compos. Anal. 19 (2006) 669–675. [11] A. Fardet, E. Rock, C. Remesy, J, Cereal Sci. 48 (2008) 258–276. [12] C.H. Chong, C.L. Law, A. Figiel, A. Wojdylo, M. Oziemblowski, Food Chem. 141 (2013) 3889–3896. [13] E.W.C. Chan, P.Y. Lye, Sy. Eng, Y.P. Tan, Free Radicals. Antioxid. 3 (2013) 2–6. [14] Z. Sroka, W. Cisowski, Food Chem. Toxicol. 41 (2003) 753–758. [15] J. Labaj, D. Slamenova, M. Lazarova, M. Kosikova, Nutr. Cancer—Int. J. 50 (2004) 198–205. [16] D. Slamenova, E. Horvathova, B. Kosikova, L. Ruzekova, J. Labaj, Nutr. Cancer 33 (1999) 88–94. [17] B. Koisikova, D. Slamenova, M. Mikulaisova, E. Horvathova, J. Labaj, Biomass Bioenergy 23 (2002) 153–159. [18] M.P. Vinardell, V. Ugartondo, M. Mitjans, Ind. Crops Prod. 27 (2008) 220–223. [19] V. Ugartondo, M. Mitjans, M.P. Vinardell, Bioresource Technol. 199 (2008) 6683–6687. [20] L.M. Bedoya, M. Beltran, R. Sancho, D.A. Olmedo, et al., Bioorg. Med. Chem. Lett. 15 (2005) 4447–4450. [21] C. Pouteau, P. Dole, B. Cathala, L. Averous, N. Boquillon, Polym. Degrad. Stab. 81 (2003) 9–18. [22] O. Goldschmidt, Anal. Chem. 26 (9) (1954) 1421–1423. [23] R.J.A. Gosselink, A. Abacherli, H. Semke, R. Malherbe, P. Kauper, A. Nadif, J.E.G. Van Dam, Ind. Crops Prod. 19 (2004) 271–281. [24] J.E. Hedge, B.T. Hofreiter, in: R.L. Whistler, J.N Be. Miller (Eds.), Carbohydrates Chemistry, 17, Academic Press, New York, NY, 1962. [25] V.L. Singleton, R. Orthofer, Methods Enzymol. 299 (1999) 152–178. [26] P. Prieto, M. Pineda, M. Aguilar, Anal. Biochem. 269 (1999) 337–341. [27] M. Oyaizu, Jpn. J. Nutr. 44 (1986) 307–315.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Free radical scavenging activity and reducing power of Acacia nilotica wood lignin Keshaw Ram Aadil, Anand Barapatre, Sudha Sahu, Harit Jha ∗ , Bhupendra Nath Tiwary Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, (Central University) Bilaspur 495009, Chhattisgarh, India
a r t i c l e
i n f o
Article history: Received 14 November 2013 Received in revised form 6 March 2014 Accepted 22 March 2014 Available online 28 March 2014 Keywords: Acacia nilotica Lignin Antioxidant activity
a b s t r a c t Nine different fractions of lignin extracted by alkali, hot water and organosolv methods from Acacia wood powder were assessed for antioxidants activity. Results indicated that methanolic lignin fraction had highest polyphenol content of 393.30 ± 9.2 g/ml (GAE). The oraganosolv lignin with total phenols and phenolic hydroxyl group content exhibited significant antioxidant activity as compared to other lignin fractions. Antioxidant properties of acetone fractions revealed a high antiradical scavenging activity ( acetone > hot water > A1 > ethanol > Table 2 Total carbohydrates, phenolic hydroxyl and carboxyl groups content in all lignin fractions. Lignin fractions
Carbohydrates content (mg/g)
LS A1 A2 A3 A4 HWL AEL EEL MEL PEL
5.22 1.13 0.61 1.04 0.87 4.50 8.26 6.76 7.07 4.87
± ± ± ± ± ± ± ± ± ±
0.16bc 0.22a 0.24a 0.35a 0.08a 0.11b 0.07cd 0.51c 1.75cd 0.03b
Phenolic hydroxyl group (%) 0.52 0.74 2.73 0.14 0.53 1.79 2.41 3.05 2.79 3.29
± ± ± ± ± ± ± ± ± ±
0.02b 0.01c 0.04f 0.01a 0.03b 0.02d 0.03e 0.05g 0.07f 0.09h
Carboxyl group (% w/w) 1.53 1.31 2.73 2.62 1.70 1.61 1.59 2.10 1.76 1.43
± ± ± ± ± ± ± ± ± ±
0.04b 0.07a 0.10d 0.03d 0.08b 0.02b 0.09b 0.05c 0.04b 0.07ab
Values represent mean ± standard deviation (SD) of three independent experiments (n = 3). Values within in a column followed by different lower case letters are significantly different according to ANOVA (Tukey test).
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Table 3 Phenol contents, antioxidant activity (EC50 value) on DPPH radicals, ABTS radical, FRAP value, hydrogen peroxide scavenging and reducing power of Acacia lignin. Lignin Fractions LS A1 A2 A3 A4 HWL AEL EEL MEL PEL
TPC1 100.27 232.48 73.01 102.27 87.74 295.21 343.28 229.54 393.30 208.28
DPPH2 ± ± ± ± ± ± ± ± ± ±
f
5.6 6.2d 3.2g 3.5f 1.9g 7.3c 3.2b 9.07d 9.2a 4.1e
149.96 83.85 103.86 110.48 128.21 81.46 79.89 81.72 84.58 82.64
ABTS3 ± ± ± ± ± ± ± ± ± ±
d
4.4 0.4a 4.4b 1.2b 8.5c 0.9a 0.07a 00a 3.5a 0.6a
FRAP4
2.70 ± 0.1 2.84 ± 0.7a 3.25 ± 0.09c 3.15 ± 0.05b 3.95 ± 0.13d 2.78 ± 0.11a 2.95 ± 0.14a 2.77 ± 0.15a nd nd a
43.05 165.36 26.01 55.92 40.64 241.94 294.16 157.40 273.23 66.47
PMA5 ± ± ± ± ± ± ± ± ± ±
c
8.5 19.7b 4.8c 7.1c 8.5c 19.0 17.9a 55.5b 12.2a 10.5c
67.96 120.55 108.88 97.77 71.10 175.92 342.2 167.58 211.47 54.44
HPS6 ± ± ± ± ± ± ± ± ± ±
f
2.2 6.7d 6.7d 6.4e 2.2f 6.1c 12.9a 2.5c 1.9b 3.8f
178.61 139.2 107.67 175.08 162.20 161.23 114.7 121.4 110.7 101.8
RP7 ± ± ± ± ± ± ± ± ± ±
i
1.2 0.7f 1.4b 0.8h 0.5g 0.9g 1.6d 0.8e 1.2bc 0.7a
19.8 ± 1.3f 99.4 ± 1.2d 20.7 ± .0.4f 35.6 ± 1.7e 25.3 ± 0.6f 125.8 ± 2.2c 196 ± 0.9a 138.5 ± 7.4b 140.6 ± 1.7b 2.33 ± 0.3g
Values represent mean ± standard deviation (SD) of three independent experiments (n = 3). Values within in a column followed by different lower case letters are significantly different according to ANOVA (Tukey test). nd—not determined. 1 Total phenol content expressed as concentration of polyphenol (g) in term of gallic acid equivalent (GAE) per mg of extracted lignin fractions. 2 Concentration of test compound required to scavenge 50% inhibition of DPPH radical. 3 Concentration of test compound required to scavenge 50% inhibition of ABTS radical. 4 FRAP value was expressed as equivalent of gallic acid (mol). 5 Phosphomolybdate assay of each lignin fractions were expressed as equivalents of ascorbic acid (mmol/100 mg of sample). 6 Concentration of test compounds required to scavenge 50% of hydrogen peroxide (HPS-CUPRAC assay). 7 Reducing power of all extracted lignin fractions.
propanol > A3 > lignosulphonic acid > A4 > A2. A similar trend has been mentioned in available literature [3]. Further, the results revealed that phenolic content of Acacia wood lignin was higher in organosolv (208–393 g of GAE per mg of sample) and hot water (295.21 ± 7.33 g of GAE per mg) as compared to alkali lignin. Our observed value was significantly higher than the earlier reports in Miscanthus sinensis lignin (170–190 g of GAE per mg of lignin) [3]. The overall finding is suggestive of a higher extractability of phenolic contents in organosolv compared to hot water and alkali lignin [4].
3.3. DPPH radical scavenging activity The available report on lignin model compounds suggests that free phenolic hydroxyl groups are crucial for antioxidant activity while the aliphatic hydroxyl groups show a negative correlation [19]. The DPPH radical scavenging capacity of different lignin fractions were observed in the range of 49.91–93.85%, which is higher than that of ascorbic acid (28.25% at 200 M) but equivalent to tannic acid (83.42% at 50 M) used as standards. Among all the lignin fractions acetone fractions exhibited the highest inhibition percentage (93.85%). The DPPH scavenging activities of acetone, hot water and ethanol extracted lignin fractions were found to be more significant (p < 0.05) as compared to alkali lignins, whereas lignosulphonic acid showed the lowest DPPH scavenging activity. The lowest DPPH scavenging activity of commercial lignin could be due to its high molecular weight which enhances its hetrogenicity resulting into a decreased free radical scavenging activity [5,19,33]. The lowest EC50 value 79.89 ± 0.07 g/ml was observed in the acetone extracted lignin (Table 3). Lu et al. [2] have explained the observed higher EC50 (2070 ± 0.17 g/ml) of nanoscale lignin by assuming that organosolv pulping lignin contains a large number of active functional groups, which act as electron donors and neutralize the free radical. Our findings suggest that the purified Acacia lignin fractions, especially solvent fractions, have more free phenolic hydroxyl groups and low molecular weight as compared to commercial lignosulphonic acid. FTIR analyses of lignin fractions support our contention that the higher free radical scavenging activity of acetone (AEL) and ethanol (EEL) fractions might be due to C O of carboxyl or ketone groups that appears around at 1700 cm−1 in FTIR spectrum (Fig. 2). These might act as electron donating groups and provide stability to the compounds.
3.4. ABTS•+ radical scavenging activity The ABTS•+ free radical scavenging activity was quantified in terms of percent inhibition by each of the lignin fraction at a fixed concentration of 5 g/ml. A significant variation in the percent inhibition of different lignin fractions was observed (65.19–98.14%). The highest inhibition was observed in the lignosulphonic acid (98.14%) followed by the ethanol (95.35%) and alkali lignin (A1) (88.86%) at the same concentration. The EC50 value of different extracted lignin fractions ranged from 2.75 to 3.95 g/ml, while EEL exhibited the lowest EC50 value of 2.77 g/ml (Table 3). Though the reports on ABTS•+ radical scavenging activity of lignin is scanty, the available literature suggests that the organosolv lignins exhibited higher phenolic content and better antiradicals power than the alkali lignin [34]. A recent articles on lignin antioxidant activity provides evidence that the scavenging activity of lignin on ABTS•+ radical is mainly due to the electron or proton transfer mechanism, whereas for the DPPH radical test the combination of electron transfer with H atom transfer is considered [35]. The higher scavenging activity of oraganosolv lignin fractions (mainly EEL and AEL) for ABTS•+ radical was possibly due to higher phenolic hydroxyl and carboxyl groups as confirmed by analytical methods and FTIR analysis. 3.5. FRAP assay FRAP activity of nine lignin fractions varied considerably from 26.01 ± 4.89 to 294.16 ± 17.92 mol (Table 3). The highest FRAP value was exhibited by acetone fraction (294.16 ± 17.92 mol), followed by the methanolic (273.23 ± 12.25 mol) and hot water fraction (241.94 ± 19.03 mol) of lignin. The FRAP value of acetone fraction was significantly (p < 0.05) higher than alkali fractions. The order of FRAP value (mol) was as follows: AEL > MEL > HWL > A1 > EEL > PEL > A3 > LS > A4 > A2. The observed FRAP value of organosolv fractions of lignin especially in acetone fraction (AEL) might be due the higher phenolic or hydroxyl groups content as compared to alkali lignin fraction. As mentioned earlier [33], the hydroxyl groups and conjugated double bond of phenolic compounds like vitamin E and other phenolic acid are crucial for FRAP activity [36]. 3.6. Phosphomolybdate assay Antioxidant compound reduces the Mo(VI) to Mo(V) and produces green coloured phosphomolybdenum V complex, which
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Fig. 2. FTIR spectra of different lignin fractions extracted from Acacia wood. LS—lignosulphonic acid, A1—alkali lignin 0.1 N NaOH, A2—alkali lignin 0.2 N NaOH, A3—alkali lignin 0.3 N NaOH, A4—alkali lignin 0.4 N NaOH, HWL—hot water extracted lignin, AEL—acetone extracted lignin, EEL—ethanol extracted lignin, MEL—methanol extracted lignin, PEL—propanol extracted lignin.
shows a maximum absorbance at 695 nm. Different lignin fractions exhibited varying antioxidant activities as expressed in terms of ascorbic acid equivalent (mmol/100 mg). As shown in Table 3, acetone fraction of Acacia lignin exhibited the highest antioxidant power (342.22 mmol/100 mg, ascorbic acid equivalents), followed by the MEL (211.47 mmol/100 mg AAE) and HWL (175.92 mmol/100 mg AAE) fractions at a concentration of 150 g/ml, while lowest antioxidant effect was shown by PEL (54.44 mmol/100 mg AAE) fraction (p < 0.05). Based on the results, the order of overall antioxidant capacity of different lignin fractions was AEL > MEL > HWL > EEL > A1 > A2 > A3 > A4 > LS > PEL.
3.7. Determination of reducing power The reducing capacity of a compound serves as a significant indicator of its potential antioxidant activity. The reducing power assay is based on the mechanism of electron donating activity, which is main mechanism of phenolic antioxidant action [4]. Table 3 shows the reducing power of the extracts represented as EC50 value (g/ml) by interpolation from linear regression analysis of concentration vs. absorbance at 700 nm. Since the AEL extract showed a higher reducing power of 196 ± 0.93 at 250 g/ml as compared to other lignin fractions whereas the lowest reducing capacity was
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Table 4 Correlation between antioxidant activities/reducing power parameters and total phenolic content of Acacia wood lignin. Assays
TPC
EC50 a
EC50 b
FRAPc
EC50 d
EC50 e
RPf
TPC DPPH ABTS FRAP Phosphomolybdate HPS Reducing power
1 0.5498 0.2183 0.9147 0.5886 0.2007 0.7095
1 0.0674 0.4674 0.3123 0.4502 0.4054
1 0.176 0.0921 0.0132 0.1931
1 0.7695 0.0929 0.8861
1 0.1301 0.8573
1 0.0775
1
TPC—total phenol content expressed as concentration of polyphenol (g) in term of gallic acid equivalent per mg of extracted lignin fractions. a Concentration of test compound required to scavenge 50% inhibition of DPPH radical. b Concentration of test compound required to scavenge 50% inhibition of ABTS radical. c FRAP value was expressed as equivalent of gallic acid (mol). d Phosphomolybdate assay of each lignin fractions were expressed as equivalents of ascorbic acid (mmol/100 mg of sample). e Concentration of test compound required to scavenge 50% hydrogen peroxide. f Reducing power of lignin fractions.
shown by PEL of 2.33 ± 0.31 g/ml (Table 3). Significantly, the Acacia lignin fractions exhibited higher reducing capacity than the antioxidant compounds, such as gallic acid and tannic acid (25.77 and 44.64 g/ml, respectively) used as the standards. The observed FRAP value and reducing power capacity of oragnosolv lignin was significantly higher (p < 0.05) than other fractions, possibly due to the higher content of phenolic ( OH) groups in organosolv fractions of lignin, as confirmed by TPC assay and FTIR spectral analysis, suggesting their role as electron donors. 3.8. HPS–CUPRAC assay HPS–CUPRAC assay is a novel and recommended method for determination of scavenging activity of hydrogen peroxide by polyphenolic compounds [28]. Lignin fractions were found to show hydrogen peroxide scavenging (HPS) activity at a concentration of 200 g/ml. HPS scavenging percent of different lignin fractions ranged from 55.33 to 97.44%. Propanol, alkali (A2) and methanol fractions showed up to 90% HPS activity. The lowest EC50 value of 101.87 ± 0.7 was observed with propanol fraction. The EC50 value for hydrogen peroxide quenching of different lignin fractions were in the order of PEL < A2 < MEL < AEL < EEL< A1 < HWL< A4 < A3 < LS (Table 3). 3.9. Correlation with total phenol contents and EC50 values of antioxidant activities/reducing power In order to understand the interrelationship between Acacia lignin fractions and their antioxidant capacity, reducing power and phenolic contents correlation analysis was performed. As shown in Table 4, the TPC was moderately correlated with DPPH radical scavenging activity and antioxidant activity (R2 > 0.5498), whereas good correlation was observed with FRAP and reducing power (R2 = 0.9147 and 0.7095, respectively). The strong correlation of TPC with FRAP and reducing power assay suggested that phenolic contents affect the reducing power of Acacia lignin. However, a moderate correlation between TPC and antiradicals activity of Acacia lignin might not be attributed to the total phenol content alone but also to some other factors such as extraction methods and solubility as reported earlier [2,33]. 4. Conclusion The statistics of the present study put forward that Acacia lignin has the effective antioxidant activities. The results indicated the organosolv lignin fraction of Acacia to be more potent for antioxidant capacity. Some functional groups such as hydroxyl, methoxyl
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