Accepted Manuscript Title: In vitro evaluation of antioxidant and cytotoxic activities of lignin fractions extracted from Acacia nilotica Author: Anand Barapatre Avtar Singh Meena Sowmya Mekala Amitava Das Harit Jha PII: DOI: Reference:
S0141-8130(16)30110-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.109 BIOMAC 5799
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-7-2015 21-12-2015 28-1-2016
Please cite this article as: Anand Barapatre, Avtar Singh Meena, Sowmya Mekala, Amitava Das, Harit Jha, In vitro evaluation of antioxidant and cytotoxic activities of lignin fractions extracted from Acacia nilotica, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.109 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro evaluation of antioxidant and cytotoxic activities of lignin fractions extracted from Acacia nilotica
Anand Barapatre1, Avtar Singh Meena2, Sowmya Mekala3, Amitava Das3, Harit Jha1*
1
Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central University),
Bilaspur, Chhattisgarh, INDIA. 495009. 2
Tumor Biology Laboratory, National Institute of Pathology, New Delhi, INDIA. 110029
3
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Uppal
Road, Hyderabad, TS – 500 007, INDIA
*
Corresponding author: Tel.: +91-9826630805; Fax: +91-7752-260148
Email ID: [email protected]
1
Highlights
nilotica is reported in traditional medicine for the treatment of various ailments.
Our study validate the efficiency of A. nilotica extracts as a traditional crude drug.
High radical deactivation index was seen in extracted A. nilotica wood fractions.
A. nilotica extracts show significant cytotoxicity against MCF-7 cancer cell line.
Abstract Lignin is one of the most important phytomacromolecule with diverse therapeutic properties such as anticancer, antimicrobial, anti-inflammatory and immune-stimulatory. The present study was carried out to evaluate the in vitro antioxidant, free radical scavenging and anti-proliferative/cytotoxic activities of eleven different lignin fractions, extracted from the wood of Acacia nilotica by Pressurized Solvent Extraction (PSE) and Successive Solvent Extraction (SSE) methods. Results indicate that the PSE fractions have high polyphenolic content and reducing power. However, the antioxidant efficiency examined by DPPH and ABTS radical scavenging assay was higher in SSE fractions. All lignin fractions revealed a significant ability to scavenge nitric oxide, hydroxyl and superoxide radicals. The extracted lignin fractions display high ferric ion reducing capacity and also possess excellent antioxidant potential in the hydrophobic (linoleic acid) system. Fractions extracted by polar solvent has the highest iron (Fe2+) chelating activity as compared to other factions, indicating their effect on the redox cycling of iron. Four lignin fractions depicted higher cytotoxic potential (IC50: 2-15 µg/mL) towards breast cancer cell line (MCF-7) but were ineffective (IC50: ≥100 µg/mL) against normal primary human hepatic stellate cells (HHSteCs). These findings suggest that the lignin extracts of A. nilotica wood has a remarkable potential to prevent disease caused by the overproduction of radicals and also seem to be a promising candidate as natural antioxidant and anti-cancer agents.
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Keywords: Acacia; Lignin; Fractionation; Free radical scavenging; Reactive oxygen species scavenging; Cytotoxicity.
1. Introduction Free radicals like reactive oxygen and nitrogen species (ROS/RNS) are the continuous product of anabolic and catabolic processes essential for energy supply, chemical signalling, detoxification, and immune function. The natural elimination of ROS and RNS is under the strict and controlled system [1]. However, overproduction, exposure to external oxidants or the failure of antioxidant defense mechanisms creates oxidative stress, which ultimately leads in damage to DNA, lipids and proteins [1-3]. Presently, the regulation of ‘redox’ status, along with the properties of dietary food and food components, is being explored extensively. Natural antioxidants present in the diet increase the resistance to oxidative damages and have a substantial impact on human health [2]. Worldwide, plant phenolics and polyphenols are frequently examined for their antioxidant properties, in terms of their ability to prevent damage from ROS/RNS (by radical scavenging) [4]. Lignin (polyphenolic in nature and 10-35% of the dry weight of lignocellulosic biomass) is the second highest available known biomolecule with high complexity due to its natural variability [5]. Lignin has large three-dimensional amorphous phenolic polymeric structures composed of three basic phenylpropanoid units, bound together through different types of inter-unit linkages [2]. The heterogeneity and diversity of lignin requires variation in the process of its isolation from lignocellulosic biomass. Chemical heterogeneity (i.e. composition, molecular weight, nature and the level of impurities) influences the chemical reactivity, thermal behavior and accessibility to solvents [6]. At an industrial scale, the production of kraft lignin is the highest. Kraft delignification leads to destruction of ether bonds in lignin and subsequent formation of stilbene, styrene, catechol and biphenyl like substructures. These compounds have high phenolic hydroxyl group moieties, ultimately resulting in modification of kraft lignin structure to enrich its antioxidant activity [5]. Solvent extraction method is frequently used for isolation and extraction of polyphenolic antioxidants. Extraction yield and antioxidant activity of the extracts are 3
strongly dependent on the solvents, due to the different antioxidant potential of compounds with different polarities [7]. Sequential successive solvent extraction (SSE) using different organic solvents, provides an effective process for reducing molecular and structural heterogeneity of antioxidants and increasing hydrogen-bonding capacity [6]. Recent studies suggest that Pressurized Solvent Extraction (PSE) is also an attractive and alternative technique for extracting organic compounds from solid, biological matrices and food applications, due to its high-throughput, automation and low solvent consumption than traditional methods [8]. Acacia nilotica, locally known as “Babul,” is a multipurpose deciduous tree of Mimosaseae family predominantly found in Central India [2, 9]. Different parts of A. nilotica have been well reported and used as a remedy against a variety of diseases in folk medicine. The traditional healers of different regions in India, particularly Chhattisgarh use Acacia species for the treatment of various cancers of mouth, bone and skin [9-11]. So far, several biologically active compounds, including condensed tannin and phlobatannin, protocatechuic acid, (+)-catechin, (-)-epigallocatechin-7-gallate, (-)-epigallocatechin, 5,7digallateumbelliferone, γ-Sitosterol, Gallic acid, niloticane, catechin, kaempferol, rutin, apigenin, understand and β-sitoterol have been isolated and reported from different parts of A. nilotica [12,13]. Among the Acacia species, A. nilotica, a plant with conventional medicinal properties was utilized for this study. Elevated temperature and high pressure facilitate the release of phenolic compounds and consequently potentiate the Acacia extracts as high levels of antioxidants and dietary supplements. Thus, based on this information and our previous studies, we investigated the free radicals, ROS/RNS scavenging and anticancerous properties of different A. nilotica lignin’s fractions isolated from wood and alkali extract of wood by ASE and SSE, respectively.
2. Materials and methods 2.1. Chemical and reagents Gallic acid, L-Ascorbic acid, DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and Methylthioazole tetrazolium 4
(MTT) were purchased from Sigma-Aldrich Inc. (Mumbai, India). Linoleic acid, ammonium thiocyanate, FeCl3, FeCl2, potassium ferricyanide, trichloroacetic acid (TCA), deoxyribose, ferrozine, NEDD (N-(1-naphthyl) ethylenediaminedihydrochloride), EDTA (ethylenediaminetetraacetic (nicotinamide
adenine
acid),
sodium
dinucleotide),
nitroprusside,
NBT
(nitro
sulphanilamide,
blue
tetrazolium),
NADH PMS
(phenazinemethosulfate) and all solvents were purchased from Merck and Hi-Media Pvt. Ltd. (Mumbai, India). All solutions were prepared with ultrapure Millipore water (Merck Millipore, Mumbai, India). Wood dust (18 mesh size) of A. nilotica wood was procured locally from saw mill of Bilaspur, Chhattisgarh, India.
2.2. Extraction of lignin fractions from A. nilotica A total of 11 distinct lignin fractions were extracted from A. nilotica wood, out of which six fractions were obtained by pressurized solvent extraction (PSE) system using the Dionex Accelerated Solvent Extractor (ASE 150, Thermo Scientific, India). Acetone (AC), chloroform (CH), ethanol (ET), ethyl acetate (EA1), methanol (ME) and water (AQ1) were used to extract different lignin fractions from A. nilotica wood through PSE. Extraction conditions comprise of temperature 60 °C, static time 7.5 min, rinse volume (60%), nitrogen purge time (300 s), static cycle 2, pressure 1700 psi and solvent volume 150 mL. Samples were immediately centrifuged for 10 min at 8000 rpm. The supernatant was recovered, vacuum dried and stored in tubes at -20 °C. Remaining five fractions were extracted by successive solvent extraction (SSE) method from alkali lignin extract. Alkali lignin extract of A. nilotica was prepared using 0.3 N NaOH, as described previously by Barapatre et al. [2]. Briefly, 250 mL of alkali lignin extract was extracted three times with n-hexane (25 mL), and the upper organic nhexane layer (HX) was separated and concentrated under vacuum. Then, the remaining aqueous layer was sequentially extracted three times with 25 mL of diethyl ether (DE). The DE layer was separated, and vacuum dried. The remaining aqueous layer was further extracted three times with ethyl acetate (EA2) and then with n-BuOH (BU).The remnant was treated as an aqueous fraction (AQ2).
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2.3. Total polyphenol content (TPC) Total polyphenol content (TPC) was determined by the method as described previously by Barapatre et al. [2]. The results were expressed as μg/mg Gallic acid equivalents (GAE) per mg of dry material.
2.4. Total reducing power assay Reducing power of all samples was determined as described previously by Barapatre et al. [2]. The EC50 values of extracts were calculated from the graph of A700 versus extract concentration. Ascorbic acid was used as a reference.
2.5. Free radical scavenging activity 2.5.1. DPPH The antiradical activity of lignin samples was measured based on their reaction with stable free radical DPPH* and subsequent reduction in λmax of DPPH* as described previously [2]. Ascorbic acid was used as a reference.
2.5.2. ABTS•+ The ABTS•+ radical scavenging assay was performed as described by Aadil et al. [9]. A sample of 25 µg was taken to determine the ABTS•+ scavenging the power. The % scavenging was presented as IC50 concentration (µg). Ascorbic acid was used as a reference.
2.6. Ferric reducing antioxidant power (FRAP) assay The FRAP assay of all lignin fractions was carried out as described by Aadil et al [9]. Concentration of the colored product (ferrous tripyridyltriazine complex) was measured at 593 nm. Gallic acid (GA) was taken as a standard and results were expressed in EC50 (µg).
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2.7. Iron (Fe2+) chelating activity The iron (Fe2+) chelating activity was determined according to the method of Singh et al. [14] with some modifications. The lignin fractions of 0.25 mL (final concentration 500 µg/mL) were mixed with 1.75 mL of methanol and 0.25 mL of 250 mM FeCl2 and incubated at 25 ºC for 10 min. This was followed by the addition of 0.25 mL of 2 mM ferrozine, which was allowed to react at room temperature for 10 min before determination of absorbance of the mixture at 562 nm. The solution devoid of sample solution and containing distilled water (0.25 mL) was used as a control. The blank solution contained distilled water (0.25 mL) instead of ferrozine solution, used for error correction because of the unequal colour of the sample solutions. The Fe2+ chelating ability was presented in % chelation and calculated as follows: Fe2+ chelating ability (%) = [A0 - (A1 - A2)] / A0 X 100 where, A0 stands for absorbance of the control, A1 for absorbance of the samples or standard, and A2 for absorbance of the blank. EDTA was used as a reference.
2.8. Hydroxyl radical (*OH) scavenging assay The hydroxyl radical scavenging activity was determined by the method of Mathew and Abraham [15] with a slight modification. One mL phosphate buffer (20 mM, pH 7.4) containing FeCl3 (1 mM), EDTA (1 mM), deoxyribose (2.8 mM), 0.1 mL ascorbic acid (1 mM) and 0.5 mL H2O2 (20 mM) was added to 1 mL of different lignin fractions (100 µg). The mixture was incubated at 37 ºC for 1 h and subsequently 1 mL of TBA (1%) and 1 mL of TCA (2.8%) were added. The mixture was incubated at 100 ºC for 20 min, cooled down to room temperature and measured at 532 nm. The control was made under the same method except replacing the test samples with absolute methanol. Hydroxyl radical scavenging activity was calculated as follows: HRSA (%) = [(A0 - A1) /A0] X 100 where, A0 is absorbance of the control, and A1 the absorbance of the samples. Ascorbic acid was used as reference at the same concentration as that of the sample. The result is shown as the inhibitory concentration (IC50) of the lignin fractions needed to get to HRSA 50%. 7
2.9. Superoxide radical scavenging The super oxide radical scavenging activity of different lignin fractions was measured by the reduction of NBT according to a previously reported method by Hazra et al. [16]. The non-enzymatic phenazinemethosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system generates superoxide radicals, which reduce nitro blue tetrazolium (NBT) to a purple formazan. The 1 mL reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 μM), NBT (50 μM), PMS (15 μM) and a sample solution of 100 μg/mL. The reaction mixture was incubated for 5 min at ambient temperature (25 ± 2 oC) and absorbance A562 nm was measured against an appropriate blank to determine the quantity of formazan generated. The results were presented in the form of inhibitory concentrations (IC50) needed to get 50% scavenging of SOD. Quercetin was used as positive control.
2.10. Nitric oxide radical (NO*) scavenging assay The nitric oxide radical scavenging assay was performed by the method of Kumari et al. [17] with slight modifications. The reaction mixtures (2.5 mL) containing sodium nitroprusside (25 mM, 0.5 mL) and the extract or standard solution (2 mL) in phosphate buffer saline (20 mM, pH 7.4) were incubated at ambient temperature (25 ± 2 o
C) for 2 h. After incubation, 1.5 mL of Griess reagent (containing 1% sulphanilamide,
0.1% NEDD and 5% H3PO3) was mixed and allowed to stand for 5 min at ambient temperature (25 ± 2 oC). A pink coloured chromophore was formed. The absorbance A570 was taken against the corresponding blank solutions and was represented as the IC50 concentrations of fractions. Ascorbic acid was used as a standard.
2.11. Determination of antioxidant scavenging activity in the linoleic acid system by the ferric thiocynate (FTC) method The method of Kikuzaki and Nakatani [18] was slightly modified to determine the antioxidant activity of the lignin fractions in the linoleic acid system. A mixture of 4 mL of an extracted lignin sample (100 μg/mL) in 99.5% ethanol, 4.1 mL of 2.51% linoleic acid in 99.5% ethanol, 8 mL of phosphate buffer (50 mM, pH 7.0) and 3.9 mL of water 8
was placed in a screw cap vial and then incubated at 40 °C in the dark for 1 h. A total of 9.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocyanate was added to 0.1 mL of this solution. Precisely 3 min after addition of 0.1 mL of FeCl2 (0.02 M in 3.5% HCl) to the reaction mixture, absorbance A500 was measured every 24 h for 360 h. The control was prepared with linoleic acid and without test sample. BHT was used as a positive control. Percent (%) inhibition of linoleic peroxidation = 100 - [(ΔA1 / ΔA0) X 100] where ΔA1 and ΔA2 are the change in absorbance of the sample and control respectively
2.12. Structure-antioxidant activity relationship of extracted sample based on FTIR data All extracted lignin fractions was characterized by FTIR for determination of its structure-antioxidant activity relationship. A small fraction of the sample was ground properly with KBr (1:100, w/w). The FTIR spectrum was obtained in the range of 400– 4000 cm−1 using FTIR spectrophotometer IRAffinity A1 (Shimadzu, Japan).
2.13. In vitro cytotoxicity assay on MCF-7 and primary human hepatic stellate cells (HHSteCs) control cell line The MTT assay, based on the conversion of the yellow tetrazolium salt-MTT to purple formazan crystals by metabolically active cells, provides a basis for quantitative determination of viable cells. Stock solutions of extracts were obtained by dissolving samples in DMSO followed by filtration through syringe filters (pore diameter 0.22 μm). Stock solutions were further diluted in culture media to obtain necessary concentrations for the experiments. DMSO serves as vehicle control and the cytotoxicity of DMSO in the concentrations present in dilutions of stock solutions was also evaluated. Briefly, MCF-7 or HHSteCs were seeded at a density of 104 cells per well in 96 well plates and allowed to adhere for 24 h at 37 ºC. The cells were then treated with increasing concentration of plant extracts (0.5-500 μg/mL). The cells were also tested with positive control Phenethylisothiocyanate (PEITC) and Tamoxifen (TAM) ranging from 0.2-200 µM for 72 h. Cytotoxicity was assessed by MTT assay as described by Meena et al. [19, 20]. Briefly, fifty microliter of MTT (1 mg/mL) was added to each well and 9
incubated at 37 °C for 4 h. Formazan crystals were solubilized in 50 μL of isopropanol by incubating in shaking condition at ambient temperature (25 ± 2 ºC) for 10 minutes. The absorbance was then determined by an ELISA reader (Bio-Rad, USA) at a wavelength of 570 nm using 630 nm as a reference filter. The results were presented in terms of % cell survival. Tests were performed five times, and the values were presented as mean ± SD. Cell survival (%) = (Acontrol – Asample) / AcontrolX 100
2.14. Statistical analysis Analysis of variance (ANOVA) test was carried out to test for the differences between the standard compound and extracted lignin fractions in the statistical program Graph pad prism 5.0. Significance of difference was defined at the 0.01 (p ME≈AC>CH and for solventsolvent fraction, it was BU>DE>EA>AQ2>HX. In the extraction of polyphenol through PSE, it was found that the extractable TPC was significantly higher when compared to conventional soxhlet extraction used in an earlier study by Aadil et al. [9]. The extractable TPC appeared to be higher in ET (61.16%), AC (4.26%) and AQ1 fractions (45.21%) while ME fraction had lower phenolic concentration. Our results are similar to Barros et al. [22], where they reported improved polyphenol extraction through PSE compared to the conventional soxhlet method. Barros et al. further suggested that under high pressure and temperature regime, the diffusion rates and disruption of some of the solute matrix interactions is enhanced thus increasing the quantity of polyphenols and antioxidants.
3.2. Total reducing power assay The reducing ability of the extracts was investigated by the reduction of Fe3+ ions to Fe2+. The reducing capacity of the compound served as an indicator of its potential antioxidant activity [9, 10]. As shown in Table 1, the reducing power of all fractions was presented as EC50 values (effective concentration) at the concentration of 100 µg/mL, where lower EC50 values represent the higher reducing power. The EC50 values of all lignin fraction range from 176-2412 µg/mL. Overall the results indicate that the ET, AQ1 and EA1 fractions from PSE, and DE from SSE had the lowest EC50 values among extracted lignin fractions, while CH and HX were the least potent fractions. The weakest 11
reducing potential were of CH and HX fractions might be due to the presence of hydrophobic compounds (i.e. with decreased water solubility) which may not be readily accessible in the system for the reduction of Fe3+. The lignin fractions extracted by PSE and SSE tested in this experiment showed significant reducing power in the range of 0.17-2.4 mg/mL, whereas Sultana et al. [23] reported the reducing power of ethanolic extract of A. nilotica bark at 10 mg/mL range. Kalaivani and Mathew [10] also reported that the different solvent extracts of A. nilotica leaves have a potent reducing ability and is concentration dependent.
3.3. Free radical scavenging activity (ABTS and DPPH) 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 [9]. The radical scavenging activity of phenolic compounds depends on the rate of abstracting the hydrogen atom from a phenol molecule by a free radical and, also on the stability of the radical formed. This abstracting ability was increased if some additional conjugation with substituents took place [2]. Table 1 shows the IC50 values of DPPH and ABTS*+ radical scavenging assays used to assess the antioxidant power of the extracted lignin fractions. As known, lower the IC50 value, the higher is the antioxidant capacity. In DPPH assay, IC50 of all fractions ranges from 59.15-1137.07 µg (Table 1). All fractions extracted by PSE depicted a potent free radical scavenging activity (IC50) at very low concentrations (≈60 µg/mL) except CH, whereas the DE fractions show highest among five extracts fractioned from alkali lignin fraction. In our previous study [9], it was reported that the lignin fraction extracted by organosolv method has significantly high antioxidant potential. It was also reported that the higher scavenging potential of organosolv lignin was due to the presence of a compound having high quantities of free phenolic hydroxyl groups and low molecular weight. Tung et al. [24] also found that the ethyl acetate (less polar solvent) fractions isolated from an ethanolic bark extract of A. confuse show higher DPPH scavenging potential as compared to polar fractions like butanol and water. 12
In ABTS*+ assay, the scavenging potentials (%) of different lignin fractions were observed in the range of 15.83-98.62%, and EC50 was estimated in the range of 12.68 to 78.95 µg/mL. The decreasing antioxidant scavenging activity order of lignin fractions in the assay can be ranked as DE>EA2≈BU>AQ1>ET>EA1>AQ2>ME>AC>HX>CH. On the other hand, it was found that the lowest IC50 value is obtained for DE (SSE fraction) and the highest for CH (PSE extract). Tsuda et al. [25] also found solvents such as ethyl acetate, provided slightly more active extracts than mixtures with ethanol or methanol, or methanol alone for tamarind seed coats. The average % scavenging for SSE fractions and PSE fractions were 78.42% and 58.04%, while the IC50 was 18.62 and 29.34 µg, respectively. In our previous study Aadil et al. [9] reported that the organosolv lignin fractions (mainly acetone and ethanolic fractions) exhibit highest ABTS*+ radical scavenging power at the 2.77 µg (IC50). As reported in literature, the nature of the solvent and its polarity alters its efficacy to extract a specific group of antioxidant compounds and influences the antioxidant properties of the extracts [6, 9].
3.4. Ferric reducing antioxidant power (FRAP) assay The ferric reducing antioxidant power (FRAP) assay measures the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) in the presence of antioxidants, which are reductants with half-reaction reduction potentials above Fe3+/Fe2+. This assay is also commonly used for the routine analysis of single antioxidants and total antioxidant activity of plant extracts [9]. In the present study, gallic acid was taken as a standard and out of eleven fractions, eight fractions exhibited potency in terms of ferric ion reduction. All fractions show the potential to reduce the Fe3+ ion in the ranges of 27.22 ± 0.8 µg to 110.92 ± 9.01 µg. The highest potential was exhibited by ET fraction while the lowest were shown by AQ2 and HX fractions. The order of IC50 values for FRAP was as follows: ET>DE>EA1≈EA2≈AQ1>BU>ME≈AC>CH>AQ2>HX. Pulido et al. [26] suggest that in the FRAP assay, the reducing ability of polyphenolics depends on the degree of hydroxylation and extent of conjugation of the phenolic compounds. Aadil et al. [9] also suggest that the high ferric reducing antioxidant power in organosolv lignin fractions of A. nilotica wood might be due to the presence of high phenolic and hydroxyl content. The 13
organosolv lignin fractions which were obtained by PSE and SSE were more potent than fractions which were used in our previous report [9]. This can be explained by the efficient extraction process applied in the present investigation.
3.5. Iron (Fe2+) chelating activity In vivo, metals, especially iron is a primary cause of ROS generation and plays a pivotal role in contributing to oxidative stress, DNA damage and cell death. Iron has been the target of many antioxidant therapies. Metal binding capacity was investigated by assessing the ability of the antioxidants to compete with the indicator ferrozine to complex with ferrous ions (Fe2+) in solution. The decreased absorbance of the reaction mixture indicates higher metal chelating capability [4]. The Fe2+ ion chelating activity (%) of all lignin fractions was tested at 500 µg/mL concentration (Table 1). PSE and SSE extracts show almost equal Fe2+ chelating capacity, and the average chelating by both extracts was ≈25%. Among all lignin fractions, BU (SSE extract) showed highest iron chelating activity while, CH fraction (PSE fraction) exhibited lowest chelating activity. Lignin fractions which were extracted by polar solvents showed high chelation ability. Among all, a total of five lignin fractions exhibited higher chelating ability than the standard, i.e. EDTA. Sulaiman et al. [27] reported the phenolic compound extracted from A. nilotica bark have excellent iron chelating power, they found the methanol extracted fraction (polar) shows highest chelating power than ethyl acetate and chloroform extracted fraction. Polyphenol specially, catecholate and gallate compounds binded with iron (II), and at pH 5-6, iron (Fe2+) is typically bound by two catecholate or three gallate ligands per metal ion. Mladěnka et al. [28] studied the chelating properties of different flavonoids and suggested that the iron chelation is strongly influenced by the unique phenolic structure, number and position of hydroxyl groups and the pH of the medium.
3.6. Hydroxyl radical (*OH) scavenging assay All fractions of A. nilotica wood depicted potent quenching potential against hydroxyl radical. Hydroxyl radical (*OH) is the most reactive (in vivo half-life 10-9 s) and 14
dangerous species among other ROS, which immediately attacks virtually any molecule in its neighborhood. The generation of *OH in the Fenton reaction is due to the presence of iron ions. Isolated extract inhibits the color formation in the deoxyribose assay, not by reacting with *OH but by chelating iron ions and preventing *OH formation [29]. The hydroxyl scavenging activity of the different lignin fractions were in the range of 11.0692.11%. The highest hydroxyl radical scavenging potential was exhibited by BU fraction having IC50 of 54.2 ± 0.18 μg while AQ1 has the lowest potential with IC50 452.15 ± 20.33 μg. Standard compound, i.e. ascorbic acid shows IC50 values at 34.52 ± 1.22 μg which was lower than the SSE fractions. Fractions extracted by SSE were more effective than PSE, and showed an average of 89.4% scavenging over average 51.5% exhibited by PSE fractions. Hydroxyl radical has the capacity to combine with nucleotides in DNA and cause strand breakage, which contributes to carcinogenesis, mutagenesis and cytotoxicity. The hydroxyl radical quenching ability of extracted lignin fractions indicate a possibility of inhibition of the propagation process of lipid peroxidation with a good scavenging ability of active oxygen species, thus reducing the rate of oxidative chain reactions.
3.7. Nitric oxide radical (NO*) scavenging assay The NO radical scavenging potential of different lignin fractions derived from the A. nilotica wood is shown in Table 2. Except for HX, all lignin fractions demonstrate substantial inhibition activity against NO* generation. The NO radical scavenging capacity of different lignin fractions was detected in the range of 6.61-65.49%. The IC50 values of three most potent fractions, AC, AQ1, ET, were 76.34, 86.62 and 86.88 µg/mL respectively, which is significantly lower than ascorbic acid (172.41 µg). It was found that the extracts which were obtained through PSE method have higher scavenging activity than SSE extracts and the average IC50 values were 110.87 and 225.05 µg respectively. Sulaiman et al. [27] also reported the ethyl acetate extracted fraction from A. nilotica bark have a significant NO* scavenging power.
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3.8. Superoxide radical scavenging The superoxide radical O2- is a highly toxic species that is generated by numerous biological and photochemical reactions, and via the Haber-Weiss’s reaction, it can generate the hydroxyl radical, which reacts with DNA bases, amino acids, proteins, and polyunsaturated fatty acids, and produces toxic effects. The toxicity of the superoxide radical is also due to the perhydroxyl intermediate (HO2) that reacts with polyunsaturated fatty acids. Superoxide radical also reacts with NO to generate peroxynitrite, which is known to be toxic to DNA, lipids and proteins [30]. In this assay O2- was generated by PMS-NADH coupling reaction system through the oxidation of NADH and evaluated by the reduction of NBT. The decrease in absorbance at 560 nm with test samples indicates the O2- quenching activity of polyphenols. The superoxide radical scavenging activity of different A. nilotica lignin extracts are presented in Table 2. AQ1 fraction (IC50 = 112.56 µg) was the most potent superoxide scavenger among all extracts which showed 44% decrease in NBT production at 100 µg/mL concentration. The CH and EA1 fractions exhibited lower superoxide scavenging activity among all. The IC50 values for both extracts were >500 µg/mL. Lignin fractions extracted from alkali lignin by SSE were more potent in superoxide scavenging than PSE fractions, in which all fractions have IC50 200 µM) respectively (Table 3). Out of eleven fractions, eight lignin fractions were cytotoxic (>50%) at concentrations ranging from 0.5 to 50 µg/mL. Moreover, at the same concentration AC, ME and BU ranged above 50%, which spotlighted non-cytotoxic effect of testing materials on MCF-7 cells. Interestingly, CH and DE lignin fractions were proved highly toxic to MCF-7 cells at a concentration of 50 µg/mL. The cell viability values (%) for both extracts were 7.99 ± 3.2 and 2.98 ± 0.57, respectively at 50 µg/mL. Although four extracts show potent inhibition activity at 50 µg/mL, at the 500 µg/mL concentration, all lignin fractions except BU and AQ2 showed a significant decrease in viable cell %. The differences in the profile of biological activities of extracts suggest variation in the constituents extracted with different solvents. Based on the results it was observed that the CH was the most potent cytotoxicity potential having 0.17 µg/mL IC50 value, whereas the aqueous (AQ1 and AQ2), ethyl acetate (EA1 and EA2) and DE extract showed great potential toward cytotoxic effect and had IC50
S0141-8130(16)30110-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.109 BIOMAC 5799
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-7-2015 21-12-2015 28-1-2016
Please cite this article as: Anand Barapatre, Avtar Singh Meena, Sowmya Mekala, Amitava Das, Harit Jha, In vitro evaluation of antioxidant and cytotoxic activities of lignin fractions extracted from Acacia nilotica, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.109 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro evaluation of antioxidant and cytotoxic activities of lignin fractions extracted from Acacia nilotica
Anand Barapatre1, Avtar Singh Meena2, Sowmya Mekala3, Amitava Das3, Harit Jha1*
1
Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central University),
Bilaspur, Chhattisgarh, INDIA. 495009. 2
Tumor Biology Laboratory, National Institute of Pathology, New Delhi, INDIA. 110029
3
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Uppal
Road, Hyderabad, TS – 500 007, INDIA
*
Corresponding author: Tel.: +91-9826630805; Fax: +91-7752-260148
Email ID: [email protected]
1
Highlights
nilotica is reported in traditional medicine for the treatment of various ailments.
Our study validate the efficiency of A. nilotica extracts as a traditional crude drug.
High radical deactivation index was seen in extracted A. nilotica wood fractions.
A. nilotica extracts show significant cytotoxicity against MCF-7 cancer cell line.
Abstract Lignin is one of the most important phytomacromolecule with diverse therapeutic properties such as anticancer, antimicrobial, anti-inflammatory and immune-stimulatory. The present study was carried out to evaluate the in vitro antioxidant, free radical scavenging and anti-proliferative/cytotoxic activities of eleven different lignin fractions, extracted from the wood of Acacia nilotica by Pressurized Solvent Extraction (PSE) and Successive Solvent Extraction (SSE) methods. Results indicate that the PSE fractions have high polyphenolic content and reducing power. However, the antioxidant efficiency examined by DPPH and ABTS radical scavenging assay was higher in SSE fractions. All lignin fractions revealed a significant ability to scavenge nitric oxide, hydroxyl and superoxide radicals. The extracted lignin fractions display high ferric ion reducing capacity and also possess excellent antioxidant potential in the hydrophobic (linoleic acid) system. Fractions extracted by polar solvent has the highest iron (Fe2+) chelating activity as compared to other factions, indicating their effect on the redox cycling of iron. Four lignin fractions depicted higher cytotoxic potential (IC50: 2-15 µg/mL) towards breast cancer cell line (MCF-7) but were ineffective (IC50: ≥100 µg/mL) against normal primary human hepatic stellate cells (HHSteCs). These findings suggest that the lignin extracts of A. nilotica wood has a remarkable potential to prevent disease caused by the overproduction of radicals and also seem to be a promising candidate as natural antioxidant and anti-cancer agents.
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Keywords: Acacia; Lignin; Fractionation; Free radical scavenging; Reactive oxygen species scavenging; Cytotoxicity.
1. Introduction Free radicals like reactive oxygen and nitrogen species (ROS/RNS) are the continuous product of anabolic and catabolic processes essential for energy supply, chemical signalling, detoxification, and immune function. The natural elimination of ROS and RNS is under the strict and controlled system [1]. However, overproduction, exposure to external oxidants or the failure of antioxidant defense mechanisms creates oxidative stress, which ultimately leads in damage to DNA, lipids and proteins [1-3]. Presently, the regulation of ‘redox’ status, along with the properties of dietary food and food components, is being explored extensively. Natural antioxidants present in the diet increase the resistance to oxidative damages and have a substantial impact on human health [2]. Worldwide, plant phenolics and polyphenols are frequently examined for their antioxidant properties, in terms of their ability to prevent damage from ROS/RNS (by radical scavenging) [4]. Lignin (polyphenolic in nature and 10-35% of the dry weight of lignocellulosic biomass) is the second highest available known biomolecule with high complexity due to its natural variability [5]. Lignin has large three-dimensional amorphous phenolic polymeric structures composed of three basic phenylpropanoid units, bound together through different types of inter-unit linkages [2]. The heterogeneity and diversity of lignin requires variation in the process of its isolation from lignocellulosic biomass. Chemical heterogeneity (i.e. composition, molecular weight, nature and the level of impurities) influences the chemical reactivity, thermal behavior and accessibility to solvents [6]. At an industrial scale, the production of kraft lignin is the highest. Kraft delignification leads to destruction of ether bonds in lignin and subsequent formation of stilbene, styrene, catechol and biphenyl like substructures. These compounds have high phenolic hydroxyl group moieties, ultimately resulting in modification of kraft lignin structure to enrich its antioxidant activity [5]. Solvent extraction method is frequently used for isolation and extraction of polyphenolic antioxidants. Extraction yield and antioxidant activity of the extracts are 3
strongly dependent on the solvents, due to the different antioxidant potential of compounds with different polarities [7]. Sequential successive solvent extraction (SSE) using different organic solvents, provides an effective process for reducing molecular and structural heterogeneity of antioxidants and increasing hydrogen-bonding capacity [6]. Recent studies suggest that Pressurized Solvent Extraction (PSE) is also an attractive and alternative technique for extracting organic compounds from solid, biological matrices and food applications, due to its high-throughput, automation and low solvent consumption than traditional methods [8]. Acacia nilotica, locally known as “Babul,” is a multipurpose deciduous tree of Mimosaseae family predominantly found in Central India [2, 9]. Different parts of A. nilotica have been well reported and used as a remedy against a variety of diseases in folk medicine. The traditional healers of different regions in India, particularly Chhattisgarh use Acacia species for the treatment of various cancers of mouth, bone and skin [9-11]. So far, several biologically active compounds, including condensed tannin and phlobatannin, protocatechuic acid, (+)-catechin, (-)-epigallocatechin-7-gallate, (-)-epigallocatechin, 5,7digallateumbelliferone, γ-Sitosterol, Gallic acid, niloticane, catechin, kaempferol, rutin, apigenin, understand and β-sitoterol have been isolated and reported from different parts of A. nilotica [12,13]. Among the Acacia species, A. nilotica, a plant with conventional medicinal properties was utilized for this study. Elevated temperature and high pressure facilitate the release of phenolic compounds and consequently potentiate the Acacia extracts as high levels of antioxidants and dietary supplements. Thus, based on this information and our previous studies, we investigated the free radicals, ROS/RNS scavenging and anticancerous properties of different A. nilotica lignin’s fractions isolated from wood and alkali extract of wood by ASE and SSE, respectively.
2. Materials and methods 2.1. Chemical and reagents Gallic acid, L-Ascorbic acid, DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and Methylthioazole tetrazolium 4
(MTT) were purchased from Sigma-Aldrich Inc. (Mumbai, India). Linoleic acid, ammonium thiocyanate, FeCl3, FeCl2, potassium ferricyanide, trichloroacetic acid (TCA), deoxyribose, ferrozine, NEDD (N-(1-naphthyl) ethylenediaminedihydrochloride), EDTA (ethylenediaminetetraacetic (nicotinamide
adenine
acid),
sodium
dinucleotide),
nitroprusside,
NBT
(nitro
sulphanilamide,
blue
tetrazolium),
NADH PMS
(phenazinemethosulfate) and all solvents were purchased from Merck and Hi-Media Pvt. Ltd. (Mumbai, India). All solutions were prepared with ultrapure Millipore water (Merck Millipore, Mumbai, India). Wood dust (18 mesh size) of A. nilotica wood was procured locally from saw mill of Bilaspur, Chhattisgarh, India.
2.2. Extraction of lignin fractions from A. nilotica A total of 11 distinct lignin fractions were extracted from A. nilotica wood, out of which six fractions were obtained by pressurized solvent extraction (PSE) system using the Dionex Accelerated Solvent Extractor (ASE 150, Thermo Scientific, India). Acetone (AC), chloroform (CH), ethanol (ET), ethyl acetate (EA1), methanol (ME) and water (AQ1) were used to extract different lignin fractions from A. nilotica wood through PSE. Extraction conditions comprise of temperature 60 °C, static time 7.5 min, rinse volume (60%), nitrogen purge time (300 s), static cycle 2, pressure 1700 psi and solvent volume 150 mL. Samples were immediately centrifuged for 10 min at 8000 rpm. The supernatant was recovered, vacuum dried and stored in tubes at -20 °C. Remaining five fractions were extracted by successive solvent extraction (SSE) method from alkali lignin extract. Alkali lignin extract of A. nilotica was prepared using 0.3 N NaOH, as described previously by Barapatre et al. [2]. Briefly, 250 mL of alkali lignin extract was extracted three times with n-hexane (25 mL), and the upper organic nhexane layer (HX) was separated and concentrated under vacuum. Then, the remaining aqueous layer was sequentially extracted three times with 25 mL of diethyl ether (DE). The DE layer was separated, and vacuum dried. The remaining aqueous layer was further extracted three times with ethyl acetate (EA2) and then with n-BuOH (BU).The remnant was treated as an aqueous fraction (AQ2).
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2.3. Total polyphenol content (TPC) Total polyphenol content (TPC) was determined by the method as described previously by Barapatre et al. [2]. The results were expressed as μg/mg Gallic acid equivalents (GAE) per mg of dry material.
2.4. Total reducing power assay Reducing power of all samples was determined as described previously by Barapatre et al. [2]. The EC50 values of extracts were calculated from the graph of A700 versus extract concentration. Ascorbic acid was used as a reference.
2.5. Free radical scavenging activity 2.5.1. DPPH The antiradical activity of lignin samples was measured based on their reaction with stable free radical DPPH* and subsequent reduction in λmax of DPPH* as described previously [2]. Ascorbic acid was used as a reference.
2.5.2. ABTS•+ The ABTS•+ radical scavenging assay was performed as described by Aadil et al. [9]. A sample of 25 µg was taken to determine the ABTS•+ scavenging the power. The % scavenging was presented as IC50 concentration (µg). Ascorbic acid was used as a reference.
2.6. Ferric reducing antioxidant power (FRAP) assay The FRAP assay of all lignin fractions was carried out as described by Aadil et al [9]. Concentration of the colored product (ferrous tripyridyltriazine complex) was measured at 593 nm. Gallic acid (GA) was taken as a standard and results were expressed in EC50 (µg).
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2.7. Iron (Fe2+) chelating activity The iron (Fe2+) chelating activity was determined according to the method of Singh et al. [14] with some modifications. The lignin fractions of 0.25 mL (final concentration 500 µg/mL) were mixed with 1.75 mL of methanol and 0.25 mL of 250 mM FeCl2 and incubated at 25 ºC for 10 min. This was followed by the addition of 0.25 mL of 2 mM ferrozine, which was allowed to react at room temperature for 10 min before determination of absorbance of the mixture at 562 nm. The solution devoid of sample solution and containing distilled water (0.25 mL) was used as a control. The blank solution contained distilled water (0.25 mL) instead of ferrozine solution, used for error correction because of the unequal colour of the sample solutions. The Fe2+ chelating ability was presented in % chelation and calculated as follows: Fe2+ chelating ability (%) = [A0 - (A1 - A2)] / A0 X 100 where, A0 stands for absorbance of the control, A1 for absorbance of the samples or standard, and A2 for absorbance of the blank. EDTA was used as a reference.
2.8. Hydroxyl radical (*OH) scavenging assay The hydroxyl radical scavenging activity was determined by the method of Mathew and Abraham [15] with a slight modification. One mL phosphate buffer (20 mM, pH 7.4) containing FeCl3 (1 mM), EDTA (1 mM), deoxyribose (2.8 mM), 0.1 mL ascorbic acid (1 mM) and 0.5 mL H2O2 (20 mM) was added to 1 mL of different lignin fractions (100 µg). The mixture was incubated at 37 ºC for 1 h and subsequently 1 mL of TBA (1%) and 1 mL of TCA (2.8%) were added. The mixture was incubated at 100 ºC for 20 min, cooled down to room temperature and measured at 532 nm. The control was made under the same method except replacing the test samples with absolute methanol. Hydroxyl radical scavenging activity was calculated as follows: HRSA (%) = [(A0 - A1) /A0] X 100 where, A0 is absorbance of the control, and A1 the absorbance of the samples. Ascorbic acid was used as reference at the same concentration as that of the sample. The result is shown as the inhibitory concentration (IC50) of the lignin fractions needed to get to HRSA 50%. 7
2.9. Superoxide radical scavenging The super oxide radical scavenging activity of different lignin fractions was measured by the reduction of NBT according to a previously reported method by Hazra et al. [16]. The non-enzymatic phenazinemethosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system generates superoxide radicals, which reduce nitro blue tetrazolium (NBT) to a purple formazan. The 1 mL reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 μM), NBT (50 μM), PMS (15 μM) and a sample solution of 100 μg/mL. The reaction mixture was incubated for 5 min at ambient temperature (25 ± 2 oC) and absorbance A562 nm was measured against an appropriate blank to determine the quantity of formazan generated. The results were presented in the form of inhibitory concentrations (IC50) needed to get 50% scavenging of SOD. Quercetin was used as positive control.
2.10. Nitric oxide radical (NO*) scavenging assay The nitric oxide radical scavenging assay was performed by the method of Kumari et al. [17] with slight modifications. The reaction mixtures (2.5 mL) containing sodium nitroprusside (25 mM, 0.5 mL) and the extract or standard solution (2 mL) in phosphate buffer saline (20 mM, pH 7.4) were incubated at ambient temperature (25 ± 2 o
C) for 2 h. After incubation, 1.5 mL of Griess reagent (containing 1% sulphanilamide,
0.1% NEDD and 5% H3PO3) was mixed and allowed to stand for 5 min at ambient temperature (25 ± 2 oC). A pink coloured chromophore was formed. The absorbance A570 was taken against the corresponding blank solutions and was represented as the IC50 concentrations of fractions. Ascorbic acid was used as a standard.
2.11. Determination of antioxidant scavenging activity in the linoleic acid system by the ferric thiocynate (FTC) method The method of Kikuzaki and Nakatani [18] was slightly modified to determine the antioxidant activity of the lignin fractions in the linoleic acid system. A mixture of 4 mL of an extracted lignin sample (100 μg/mL) in 99.5% ethanol, 4.1 mL of 2.51% linoleic acid in 99.5% ethanol, 8 mL of phosphate buffer (50 mM, pH 7.0) and 3.9 mL of water 8
was placed in a screw cap vial and then incubated at 40 °C in the dark for 1 h. A total of 9.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocyanate was added to 0.1 mL of this solution. Precisely 3 min after addition of 0.1 mL of FeCl2 (0.02 M in 3.5% HCl) to the reaction mixture, absorbance A500 was measured every 24 h for 360 h. The control was prepared with linoleic acid and without test sample. BHT was used as a positive control. Percent (%) inhibition of linoleic peroxidation = 100 - [(ΔA1 / ΔA0) X 100] where ΔA1 and ΔA2 are the change in absorbance of the sample and control respectively
2.12. Structure-antioxidant activity relationship of extracted sample based on FTIR data All extracted lignin fractions was characterized by FTIR for determination of its structure-antioxidant activity relationship. A small fraction of the sample was ground properly with KBr (1:100, w/w). The FTIR spectrum was obtained in the range of 400– 4000 cm−1 using FTIR spectrophotometer IRAffinity A1 (Shimadzu, Japan).
2.13. In vitro cytotoxicity assay on MCF-7 and primary human hepatic stellate cells (HHSteCs) control cell line The MTT assay, based on the conversion of the yellow tetrazolium salt-MTT to purple formazan crystals by metabolically active cells, provides a basis for quantitative determination of viable cells. Stock solutions of extracts were obtained by dissolving samples in DMSO followed by filtration through syringe filters (pore diameter 0.22 μm). Stock solutions were further diluted in culture media to obtain necessary concentrations for the experiments. DMSO serves as vehicle control and the cytotoxicity of DMSO in the concentrations present in dilutions of stock solutions was also evaluated. Briefly, MCF-7 or HHSteCs were seeded at a density of 104 cells per well in 96 well plates and allowed to adhere for 24 h at 37 ºC. The cells were then treated with increasing concentration of plant extracts (0.5-500 μg/mL). The cells were also tested with positive control Phenethylisothiocyanate (PEITC) and Tamoxifen (TAM) ranging from 0.2-200 µM for 72 h. Cytotoxicity was assessed by MTT assay as described by Meena et al. [19, 20]. Briefly, fifty microliter of MTT (1 mg/mL) was added to each well and 9
incubated at 37 °C for 4 h. Formazan crystals were solubilized in 50 μL of isopropanol by incubating in shaking condition at ambient temperature (25 ± 2 ºC) for 10 minutes. The absorbance was then determined by an ELISA reader (Bio-Rad, USA) at a wavelength of 570 nm using 630 nm as a reference filter. The results were presented in terms of % cell survival. Tests were performed five times, and the values were presented as mean ± SD. Cell survival (%) = (Acontrol – Asample) / AcontrolX 100
2.14. Statistical analysis Analysis of variance (ANOVA) test was carried out to test for the differences between the standard compound and extracted lignin fractions in the statistical program Graph pad prism 5.0. Significance of difference was defined at the 0.01 (p ME≈AC>CH and for solventsolvent fraction, it was BU>DE>EA>AQ2>HX. In the extraction of polyphenol through PSE, it was found that the extractable TPC was significantly higher when compared to conventional soxhlet extraction used in an earlier study by Aadil et al. [9]. The extractable TPC appeared to be higher in ET (61.16%), AC (4.26%) and AQ1 fractions (45.21%) while ME fraction had lower phenolic concentration. Our results are similar to Barros et al. [22], where they reported improved polyphenol extraction through PSE compared to the conventional soxhlet method. Barros et al. further suggested that under high pressure and temperature regime, the diffusion rates and disruption of some of the solute matrix interactions is enhanced thus increasing the quantity of polyphenols and antioxidants.
3.2. Total reducing power assay The reducing ability of the extracts was investigated by the reduction of Fe3+ ions to Fe2+. The reducing capacity of the compound served as an indicator of its potential antioxidant activity [9, 10]. As shown in Table 1, the reducing power of all fractions was presented as EC50 values (effective concentration) at the concentration of 100 µg/mL, where lower EC50 values represent the higher reducing power. The EC50 values of all lignin fraction range from 176-2412 µg/mL. Overall the results indicate that the ET, AQ1 and EA1 fractions from PSE, and DE from SSE had the lowest EC50 values among extracted lignin fractions, while CH and HX were the least potent fractions. The weakest 11
reducing potential were of CH and HX fractions might be due to the presence of hydrophobic compounds (i.e. with decreased water solubility) which may not be readily accessible in the system for the reduction of Fe3+. The lignin fractions extracted by PSE and SSE tested in this experiment showed significant reducing power in the range of 0.17-2.4 mg/mL, whereas Sultana et al. [23] reported the reducing power of ethanolic extract of A. nilotica bark at 10 mg/mL range. Kalaivani and Mathew [10] also reported that the different solvent extracts of A. nilotica leaves have a potent reducing ability and is concentration dependent.
3.3. Free radical scavenging activity (ABTS and DPPH) 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 [9]. The radical scavenging activity of phenolic compounds depends on the rate of abstracting the hydrogen atom from a phenol molecule by a free radical and, also on the stability of the radical formed. This abstracting ability was increased if some additional conjugation with substituents took place [2]. Table 1 shows the IC50 values of DPPH and ABTS*+ radical scavenging assays used to assess the antioxidant power of the extracted lignin fractions. As known, lower the IC50 value, the higher is the antioxidant capacity. In DPPH assay, IC50 of all fractions ranges from 59.15-1137.07 µg (Table 1). All fractions extracted by PSE depicted a potent free radical scavenging activity (IC50) at very low concentrations (≈60 µg/mL) except CH, whereas the DE fractions show highest among five extracts fractioned from alkali lignin fraction. In our previous study [9], it was reported that the lignin fraction extracted by organosolv method has significantly high antioxidant potential. It was also reported that the higher scavenging potential of organosolv lignin was due to the presence of a compound having high quantities of free phenolic hydroxyl groups and low molecular weight. Tung et al. [24] also found that the ethyl acetate (less polar solvent) fractions isolated from an ethanolic bark extract of A. confuse show higher DPPH scavenging potential as compared to polar fractions like butanol and water. 12
In ABTS*+ assay, the scavenging potentials (%) of different lignin fractions were observed in the range of 15.83-98.62%, and EC50 was estimated in the range of 12.68 to 78.95 µg/mL. The decreasing antioxidant scavenging activity order of lignin fractions in the assay can be ranked as DE>EA2≈BU>AQ1>ET>EA1>AQ2>ME>AC>HX>CH. On the other hand, it was found that the lowest IC50 value is obtained for DE (SSE fraction) and the highest for CH (PSE extract). Tsuda et al. [25] also found solvents such as ethyl acetate, provided slightly more active extracts than mixtures with ethanol or methanol, or methanol alone for tamarind seed coats. The average % scavenging for SSE fractions and PSE fractions were 78.42% and 58.04%, while the IC50 was 18.62 and 29.34 µg, respectively. In our previous study Aadil et al. [9] reported that the organosolv lignin fractions (mainly acetone and ethanolic fractions) exhibit highest ABTS*+ radical scavenging power at the 2.77 µg (IC50). As reported in literature, the nature of the solvent and its polarity alters its efficacy to extract a specific group of antioxidant compounds and influences the antioxidant properties of the extracts [6, 9].
3.4. Ferric reducing antioxidant power (FRAP) assay The ferric reducing antioxidant power (FRAP) assay measures the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) in the presence of antioxidants, which are reductants with half-reaction reduction potentials above Fe3+/Fe2+. This assay is also commonly used for the routine analysis of single antioxidants and total antioxidant activity of plant extracts [9]. In the present study, gallic acid was taken as a standard and out of eleven fractions, eight fractions exhibited potency in terms of ferric ion reduction. All fractions show the potential to reduce the Fe3+ ion in the ranges of 27.22 ± 0.8 µg to 110.92 ± 9.01 µg. The highest potential was exhibited by ET fraction while the lowest were shown by AQ2 and HX fractions. The order of IC50 values for FRAP was as follows: ET>DE>EA1≈EA2≈AQ1>BU>ME≈AC>CH>AQ2>HX. Pulido et al. [26] suggest that in the FRAP assay, the reducing ability of polyphenolics depends on the degree of hydroxylation and extent of conjugation of the phenolic compounds. Aadil et al. [9] also suggest that the high ferric reducing antioxidant power in organosolv lignin fractions of A. nilotica wood might be due to the presence of high phenolic and hydroxyl content. The 13
organosolv lignin fractions which were obtained by PSE and SSE were more potent than fractions which were used in our previous report [9]. This can be explained by the efficient extraction process applied in the present investigation.
3.5. Iron (Fe2+) chelating activity In vivo, metals, especially iron is a primary cause of ROS generation and plays a pivotal role in contributing to oxidative stress, DNA damage and cell death. Iron has been the target of many antioxidant therapies. Metal binding capacity was investigated by assessing the ability of the antioxidants to compete with the indicator ferrozine to complex with ferrous ions (Fe2+) in solution. The decreased absorbance of the reaction mixture indicates higher metal chelating capability [4]. The Fe2+ ion chelating activity (%) of all lignin fractions was tested at 500 µg/mL concentration (Table 1). PSE and SSE extracts show almost equal Fe2+ chelating capacity, and the average chelating by both extracts was ≈25%. Among all lignin fractions, BU (SSE extract) showed highest iron chelating activity while, CH fraction (PSE fraction) exhibited lowest chelating activity. Lignin fractions which were extracted by polar solvents showed high chelation ability. Among all, a total of five lignin fractions exhibited higher chelating ability than the standard, i.e. EDTA. Sulaiman et al. [27] reported the phenolic compound extracted from A. nilotica bark have excellent iron chelating power, they found the methanol extracted fraction (polar) shows highest chelating power than ethyl acetate and chloroform extracted fraction. Polyphenol specially, catecholate and gallate compounds binded with iron (II), and at pH 5-6, iron (Fe2+) is typically bound by two catecholate or three gallate ligands per metal ion. Mladěnka et al. [28] studied the chelating properties of different flavonoids and suggested that the iron chelation is strongly influenced by the unique phenolic structure, number and position of hydroxyl groups and the pH of the medium.
3.6. Hydroxyl radical (*OH) scavenging assay All fractions of A. nilotica wood depicted potent quenching potential against hydroxyl radical. Hydroxyl radical (*OH) is the most reactive (in vivo half-life 10-9 s) and 14
dangerous species among other ROS, which immediately attacks virtually any molecule in its neighborhood. The generation of *OH in the Fenton reaction is due to the presence of iron ions. Isolated extract inhibits the color formation in the deoxyribose assay, not by reacting with *OH but by chelating iron ions and preventing *OH formation [29]. The hydroxyl scavenging activity of the different lignin fractions were in the range of 11.0692.11%. The highest hydroxyl radical scavenging potential was exhibited by BU fraction having IC50 of 54.2 ± 0.18 μg while AQ1 has the lowest potential with IC50 452.15 ± 20.33 μg. Standard compound, i.e. ascorbic acid shows IC50 values at 34.52 ± 1.22 μg which was lower than the SSE fractions. Fractions extracted by SSE were more effective than PSE, and showed an average of 89.4% scavenging over average 51.5% exhibited by PSE fractions. Hydroxyl radical has the capacity to combine with nucleotides in DNA and cause strand breakage, which contributes to carcinogenesis, mutagenesis and cytotoxicity. The hydroxyl radical quenching ability of extracted lignin fractions indicate a possibility of inhibition of the propagation process of lipid peroxidation with a good scavenging ability of active oxygen species, thus reducing the rate of oxidative chain reactions.
3.7. Nitric oxide radical (NO*) scavenging assay The NO radical scavenging potential of different lignin fractions derived from the A. nilotica wood is shown in Table 2. Except for HX, all lignin fractions demonstrate substantial inhibition activity against NO* generation. The NO radical scavenging capacity of different lignin fractions was detected in the range of 6.61-65.49%. The IC50 values of three most potent fractions, AC, AQ1, ET, were 76.34, 86.62 and 86.88 µg/mL respectively, which is significantly lower than ascorbic acid (172.41 µg). It was found that the extracts which were obtained through PSE method have higher scavenging activity than SSE extracts and the average IC50 values were 110.87 and 225.05 µg respectively. Sulaiman et al. [27] also reported the ethyl acetate extracted fraction from A. nilotica bark have a significant NO* scavenging power.
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3.8. Superoxide radical scavenging The superoxide radical O2- is a highly toxic species that is generated by numerous biological and photochemical reactions, and via the Haber-Weiss’s reaction, it can generate the hydroxyl radical, which reacts with DNA bases, amino acids, proteins, and polyunsaturated fatty acids, and produces toxic effects. The toxicity of the superoxide radical is also due to the perhydroxyl intermediate (HO2) that reacts with polyunsaturated fatty acids. Superoxide radical also reacts with NO to generate peroxynitrite, which is known to be toxic to DNA, lipids and proteins [30]. In this assay O2- was generated by PMS-NADH coupling reaction system through the oxidation of NADH and evaluated by the reduction of NBT. The decrease in absorbance at 560 nm with test samples indicates the O2- quenching activity of polyphenols. The superoxide radical scavenging activity of different A. nilotica lignin extracts are presented in Table 2. AQ1 fraction (IC50 = 112.56 µg) was the most potent superoxide scavenger among all extracts which showed 44% decrease in NBT production at 100 µg/mL concentration. The CH and EA1 fractions exhibited lower superoxide scavenging activity among all. The IC50 values for both extracts were >500 µg/mL. Lignin fractions extracted from alkali lignin by SSE were more potent in superoxide scavenging than PSE fractions, in which all fractions have IC50 200 µM) respectively (Table 3). Out of eleven fractions, eight lignin fractions were cytotoxic (>50%) at concentrations ranging from 0.5 to 50 µg/mL. Moreover, at the same concentration AC, ME and BU ranged above 50%, which spotlighted non-cytotoxic effect of testing materials on MCF-7 cells. Interestingly, CH and DE lignin fractions were proved highly toxic to MCF-7 cells at a concentration of 50 µg/mL. The cell viability values (%) for both extracts were 7.99 ± 3.2 and 2.98 ± 0.57, respectively at 50 µg/mL. Although four extracts show potent inhibition activity at 50 µg/mL, at the 500 µg/mL concentration, all lignin fractions except BU and AQ2 showed a significant decrease in viable cell %. The differences in the profile of biological activities of extracts suggest variation in the constituents extracted with different solvents. Based on the results it was observed that the CH was the most potent cytotoxicity potential having 0.17 µg/mL IC50 value, whereas the aqueous (AQ1 and AQ2), ethyl acetate (EA1 and EA2) and DE extract showed great potential toward cytotoxic effect and had IC50
