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Antioxidant activity of the phenol rich fractions of leaves of Chukrasia tabularis A. Juss.
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Bioresource Technology 99 (2008) 7692–7698
Antioxidant activity of the phenol rich fractions of leaves of Chukrasia tabularis A. Juss. Rajbir Kaur a, Saroj Arora a,*, Bikram Singh b a
Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India b Natural Plant Product Division, Institute of Himalayan Bioresource Technology (IHBT), Palampur, Post Box No. 6-176061, Himachal Pradesh, India Received 24 October 2007; received in revised form 25 January 2008; accepted 30 January 2008 Available online 18 March 2008
Abstract The present study was designed to explore the antioxidant potential of chloroform, ethyl acetate, n-butanol and water fractions of 80% methanol extract of leaves Chukrasia tabularis by 2,20 -diphenyl-1-picrylhydrazyl (DPPH), deoxyribose degradation (non-site specific and site specific), reducing power and DNA nicking assays. The different fractions showed significant activities in all the free radical scavenging tests and these findings have also shown direct relationship between antioxidant activity and phenolic content. Among the fractions, ethyl acetate fraction exhibited highest inhibition of 93.14%, 89.99%, 87.04% in DPPH, non-site specific and site specific deoxyribose degradation assays, respectively and 91.20% reduction of ferricyanide to give Prussian blue coloured complex in reducing power assay at maximum concentration tested. This preliminary study indicates the antioxidant activity of the leaves of Chukrasia tabularis, and moreover the results showed correlation with the amount of phenolic content present in different fractions. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Chukrasia tabularis; Phenolic compounds; Free radical scavenging activities; DNA nicking assay; Total phenol content
1. Introduction The plants play a more comprehensive role in the human diet as they prevent diseases that are responsible for devastation of human population. Epidemiological studies have shown that a diet rich in fruits and vegetables is associated with a decreased risk of cardiovascular diseases and certain cancers (Bazzano et al., 2002; Block et al., 1992). These health effects have been attributed in part to the presence of phenolic compounds in dietry plants, which may exert their effects as a result of their antioxidant properties. The antioxidant activities of phenolics are mainly due to their redox properties that allow them to act as a reducing agents, hydrogen donors and singlet oxygen quenchers (Rice-Evans et al., 1996; Ramarathnam *
Corresponding author. Tel.: +91 0183 2451048, +91 0941 7285485; fax: +91 0183 2258819/2258820. E-mail address: [email protected] (S. Arora). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.01.070
et al., 1997; Khokhar and Apenten, 2003). In addition, they have a metal chelation potential (Amarowicz et al., 2004; Zhao et al., 2005; Othmana et al., 2007). Keeping this in view, coupled with the fact that Chukrasia tabularis has a high content of secondary metabolites including phenolic compounds, the present study was designed to estimate the antioxidant efficiency employing free radical scavenging tests viz. (2,20 -diphenyl-1-picrylhydrazyl (DPPH), non-site specific and site specific deoxyribose degradation, reducing power assays) together with inhibition of strand breaking of supercoiled deoxyribonucleic acid in DNA nicking assay. The total phenolic content was determined by Folin–Ciocalteu method. C. tabularis A. Juss. (Meliaceae) commonly known as Chittgong wood or lal devdari etc. is a medicinal plant that accumulates a variety of secondary metabolites including phenolic compounds, terpenes, limonoids, steroids, chuktabularins and tabularisins (Rastogi and Mehrotra, 1993; Nakatani et al., 2004; Fan et al., 2007; Zhang et al.,
R. Kaur et al. / Bioresource Technology 99 (2008) 7692–7698
2007). It has got reference in Ayurveda as astringent and antipyretic drug. Recent studies have also explored its antimalarial and antibacterial activities (Mackinnon et al., 1997; Nagalakshmi et al., 2001, 2003). 2. Methods
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Sigma Chemical Co. (St. Louis, MO, USA). Ellagic acid and 2-deoxyribose were obtained from Lancaster Synthesis Inc. USA. Supercoiled plasmid pBR 322 DNA and agarose was obtained from Genei, Bangalore. Bromophenol blue, EDTA, L-ascorbic acid, Tris (hydroxymethyl) aminomethane, Folin–Ciocalteu reagent, trichloroacetic acid (TCA) and gallic acid were of analytical grade.
2.1. Plant material 2.4. Determination of total phenol content Leaves were collected from the tree growing in the Guru Nanak Dev University campus, Amritsar. Botanical identification was made from Herbarium of Department of Botanical and Environmental Sciences, GNDU, Amritsar and voucher specimen was submitted in the herbarium. 2.2. Plant extract The leaves of plant were thoroughly washed with tap water, dried at room temperature and ground to fine powder. The powdered leaves were extracted with 80% methanol by employing maceration method. The 80% methanol extract was fractionated using different solvents viz. hexane, chloroform, ethyl acetate, n-butanol and water. The supernatant was filtered using Whatman No. 1 sheet, pooled and concentrated using vacuum rotary evaporator. The concentrated solutions were then lyophilized to get the dry form of respective fractions (Flow Chart 1). 2.3. Chemicals 2,20 -Diphenyl-1-picryl hydrazyl (DPPH), 2-thiobarbituric acid (TBA), ethidium bromide were obtained from Leaves Powder (750 g) Extracted with 80% methanol in water 80% Methanol Extract (151.3 g) Dissolved in Distilled Water Aqueous Methanol Extract Extracted with Hexane; thrice
Hexane Fraction (1.41 g)
Chloroform Fraction (2.36 g)
Remaining Extract Extracted with Chloroform; thrice
Remaining Extract Extracted with Ethyl Acetate; thrice
Ethyl Acetate Fraction (78.63 g)
Remaining Extract
Folin–Ciocalteu procedure given by Yu et al. (2002) was used to estimate the total phenol content in the different fractions of C. tabularis. Following this method, 0.1 ml aliquots of fractions were diluted to 1 ml with distilled water. To this solution 0.5 ml of Folin–Ciocalteu reagent (1:1) and 1.5 ml of 20% sodium carbonate solution was added. The mixture was incubated for 2 h at room temperature. The volume was raised to 10 ml with distilled water and the absorbance of blue coloured mixture was measured at 765 nm (Systronics 2202 UV–Vis Spectrophotometer). The amount of total phenol was calculated as mg/g (Gallic Acid Equivalents) from calibration curve of gallic acid standard solution. For the gallic acid, the curve absorbance versus concentration is described by the equation y = 0.0012x 0.0084 (R2 = 0.9908). Here, y = absorbance and x = concentration. 2.5. Free radical scavenging tests 2.5.1. DPPH radical scavenging activity DPPH, a stable nitrogen centered radical was used to assess the hydrogen donating ability of different fractions of C. tabularis as it offers a convenient and accurate method because of the relatively short time required for analysis. For assessing the DPPH radical scavenging activity, method described by Blois (1958) was used with slight modifications. In this assay, 0.2 ml of extract solution was added to 3 ml of 0.1 mM methanolic DPPH solution in a cuvette and absorbance was read at 517 nm. The decrease in absorption at ambient temperature was recorded for 10 min and correlated with the scavenging action of the test compound. The radical scavenging activity was expressed as the inhibition percentage and monitored as per the equation: % DPPH radical scavenging = (1 AS/AC) 100; AC = absorbance of control and AS = absorbance of sample solution. The DPPH solution without sample solution was used as control. IC50 value is the concentration of sample (lg/ ml) required to scavenge 50% DPPH free radical and was calculated from inhibition curve. Gallic acid being a phenolic compound was used as positive control.
Extracted with n-Butanol; thrice
n- Butanol Fraction (25.69 g)
Remaining Extract
Flow Chart 1. Extraction procedure of leaves of Chukrasia tabularis.
2.5.2. Reducing power assay The reducing power of different fractions was determined by the method of Oyaizu (1986). One milliliter of extract of different concentrations was mixed with 2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1%
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potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. A volume of 2.5 ml of 10% TCA was then added to the mixture and centrifuged at 3000 rpm for 10 min. 2.5 ml of supernatant was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl3 (0.1%) and the absorbance was measured spectrophotometrically at 700 nm. Increase in absorbance of the reaction mixture was interpreted as increase in reducing activity of the extract and the results were compared with gallic acid that was used as a positive control. The percentage reduction of the sample as compared to standard, i.e. gallic acid was calculated by using the formula [1 (1 AS/AC)] 100. Here, AC = absorbance of standard at maximum concentration tested and AS = absorbance of sample.
was analyzed on 1% agarose gel (prepared by dissolving 0.5 g of agarose in 50 ml of 1 TBE Buffer) followed by ethidium bromide staining. In this case, a phenolic compound, ellagic acid was used as a positive control.
2.5.3. Deoxyribose degradation assay Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and test compounds for hydroxyl radical generated by the Fe3+– ascorbate-EDTA–H2O2 system according to the method of Halliwell et al. (1987) and Arouma et al. (1987) with slight modifications. Depending upon the use of EDTA, this method comprised non-site and site specific scavenging of OH radicals. For non-site specific hydroxyl radical scavenging assay, the Haber–Weiss reaction mixture (1 ml) contained 2-deoxyribose (10 mM), Fe(III) chloride (10 mM), EDTA (1 mM), and H2O2 (10 mM) without or with the test extracts (10–100 lg/ml) in 50 mM potassium phosphate buffer, pH 7.4. In site-specific hydroxyl radical scavenging assay, EDTA was replaced with same amount of buffer. The reaction was triggered by adding ascorbic acid (1 mM) which served as a reducing agent by reducing Fe3+ to Fe2+ ions and subsequent incubation of the mixture for 1 h at 37 °C. Solutions of Fe(III) chloride, ascorbic acid and H2O2 were prepared in distilled water just prior to use. To 1 ml solution of above mixture, TBA in 25 mM NaOH (1 ml, 0.5%) and TCA (1 ml, 10% w/v aqueous solution) were added. The mixture was heated for 90 min on water bath at 80 °C and the amount of pink chromogen produced was spectrophotometrically measured at 532 nm. Here also, the gallic acid was used as a standard. The inhibitory effect on the hydroxyl radicals was calculated as: % hydroxyl radical scavenging capacity = (1 AS/AC) 100; AC = absorbance of control and AS = absorbance of sample solution.
The estimation of phenolic content among different fractions revealed that the ethyl acetate fraction exhibited higher phenol content of 983.5 mg/g GAE followed by n-butanol fraction (816.5 mg/g) > water fraction (608.8 mg/g) > chloroform fraction (400 mg/g GAE) whereas the crude extract had 816 mg/g GAE of phenol content. It was observed that the crude extract had low levels of phenolic content as compared to the ethyl acetate fraction.
2.5.4. DNA nicking assay The ability of different fractions to protect super coiled pBR 322 DNA from devastating effects of hydroxyl radicals generated by Fenton’s reagent was assessed by the DNA nicking assay described by Lee et al. (2002) with slight modifications. The reaction mixture contained 0.3 ll of plasmid DNA, 10 ll Fenton’s reagent (30 mM H2O2, 50 mM ascorbic acid, and 80 mM FeCl3) followed by the addition of extracts and the final volume of the mixture was brought up to 20 ll using distilled water. The mixture was then incubated for 30 min at 37 °C and the DNA
2.6. Statistical analysis Each experiment was performed at least three times and results are presented as the mean ± SE. IC50 was also calculated. 3. Results 3.1. Total phenol content
3.2. DPPH radical scavenging activity The DPPH scavenging activities of different fractions of 80% methanol extract of leaves of C. tabularis are shown in Fig. 1A. The ethyl acetate fraction showed highest DPPH radical scavenging activity of 93.14% at 100 lg/ml concentrations whereas chloroform, n-butanol and water fractions showed 36.99%, 41.78% and 15.37% inhibition, respectively, at the same concentration. The crude extract exhibited an inhibition of 92.91% at 180 lg/ml concentration (not shown in figure). Kinetic studies were carried out in order to determine the scavenging ability of fractions as a function of time. These studies indicated that at lower concentrations or at initial stages of reaction, the fractions showed a lesser tendency to reduce DPPH radicals but after 3 min of time interval or at higher concentrations the steady state was attained in less than 10 min. Furthermore, the different fractions showed radical scavenging ability in a dose dependent manner. The IC50 value of various fractions was compared with standard phenolic compound, i.e. gallic acid (Table 1). The lowest IC50 value was observed to be 37 lg/ml with ethyl acetate fraction followed by n-butanol (110 lg/ml), chloroform (132 lg/ml) and water (164 lg/ml) fraction. 3.3. Reducing power assay In this assay, reducing power of the fractions of C. tabularis steadily increased with increasing sample concentration (Fig. 1B). It was noted that among the different fractions, ethyl acetate fraction exhibited the maximum reducing power. The order of activity of various fractions
R. Kaur et al. / Bioresource Technology 99 (2008) 7692–7698
B 100 90 80 70 60 50 40 30 20 10 0
Reducing Power (%)
Inhibition (%)
A
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100 90 80 70 60 50 40 30 20 10 0
20 40 60 80 100 120 140 160 180 200
20 40 60 80 100 120 140 160 180 200
Concentration ( g/ml)
Concentration ( g/ml)
D
100 90 80 70 60 50 40 30 20 10 0
Inhibition (%)
Inhibition (%)
C
10 20 30 40 50 60 70 80 90 100
Concentration ( g/ml)
100 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 100
Concentration ( g/ml)
Fig. 1. Effect of different concentration of fractions of 80% methanol extract of Chukrasia tabularis in free radical scavenging tests: (A) DPPH assay, (B) reducing power assay, (C) non-site specific deoxyribose degradation assay and (D) site specific deoxyribose degradation assay. j = Gallic acid, N = nButanol fraction, x = Chloroform fraction, = Water fraction, d = Ethyl acetate fraction.
Table 1 IC50 value of different fractions of Chukrasia tabularis leaves in different free radical scavenging tests Assays
Extracts/ standard
IC50 (lg/ ml)
DPPH assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
21 132 37 110 164
Non-site specific deoxyribose degradation assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
<1 26.5 <1 128 45
Site specific deoxyribose degradation assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
55.5 94.5 8.0 60.5 117.5
Reducing power assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
56 336 101.5 200 472
3.4. Deoxyribose degradation assay Fig. 1C and 1D shows the hydroxyl radical scavenging ability of different fractions of methanol extract in non-site specific and site-specific hydroxyl radical scavenging assays. It has been found that in both, the fractions showed the scavenging ability in a dose dependent manner and out of different fractions; ethyl acetate was most active one. It showed maximum inhibition of 89.99% at 50 lg/ml followed by crude extract (82.52%) > chloroform fraction (62.42%) > water fraction (51.31%) > n-butanol fraction (36.31%). In site-specific assay, hydroxyl radical scavenging ability of different fractions followed the order as: ethyl acetate fraction (87.04%) > crude extract (79.69%) > gallic acid (50.55%) > n-butanol fraction (49.62%) > chloroform fraction (30.69%) > water fraction (24.34%) at concentration 60 lg/ml and was also found to be comparable to standard gallic acid. Table 1 shows the IC50 value of different fractions in site specific and non-site specific deoxyribose degradation assays. The lowest IC50 value was observed to be with ethyl acetate (<1 lg/ml in non-site specific and 8 lg/ml in site specific deoxyribose degradation assay). 3.5. DNA nicking assay
was as follows: ethyl acetate fraction > n-butanol fraction > chloroform fraction > water fraction at the highest concentration tested. The crude extract of leaves exhibited a reduction of 62.70% at 200 lg/ml (not shown in figure).
Fig. 2 shows the DNA damage protecting activity of various fractions of C. tabularis. The results are in confirmation with the hydroxyl radical scavenging ability of various fractions in deoxyribose degradation method. It has
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2
3
4
5
6
7
8
9 Form II Form III Form I
Fig. 2. Protective effect of different fractions of Chukrasia tabularis in DNA Nicking assay. Lane 1: Negative control (Distilled water + DNA), Lane 2: Control (DNA + Fenton’s reagent), Lane 3: Ellagic acid (250 mg/ ml) + FR as positive control, Lane 4: 80% methanol extract (250 mg/ml) + FR, Lane 5: chloroform extract (250 mg/ml) + FR, Lane 6: ethyl acetate extract (250 mg/ml) + FR, Lane 7: n-Butanol extract (250 mg/ml) + FR, Lane 8: water extract (250 mg/ml) + FR, Lane 9: control (DNA + Fenton’s reagent). Form II: ss nicked DNA; Form III: ds nicked and linear DNA and Form I: supercoiled DNA.
Densitometric Units
4500 4000 3500 3000 2500 2000 1500 1000 500 0 C
FR
EA
ME
CF
EAF
BF
WF
FR
Fractions Form II DNA
Form III DNA
Form I DNA
Fig. 3. Densitometric analysis of effect of different fractions of plant extract on pBR 322 DNA in the presence of hydroxyl radicals generated in Fenton’s reaction. Here, C = Control, FR = Fenton’s reagent, EA = Ellagic acid, ME = Methanol extract, CF = Chloroform fraction, EAF = Ethyl acetate fraction, BF = n-Butanol fraction and WF = Water fraction.
been found that when the plasmid DNA was dissolved in the Fenton’s reaction mixture, there was a time dependent increase of single stranded and double stranded nicked and linear forms of DNA (Forms II and III, respectively) due to hydroxyl radicals generated in reaction mixture. However, addition of various fractions to reaction mixture reduced the hydroxyl radical mediated strand breaking and conversion of supercoiled DNA to Forms II and III DNA. Densitometric analysis showed that ethyl acetate fraction and chloroform fraction showed significant protection of DNA from damage caused by hydroxyl radicals (Fig. 3).
4. Discussion During the last decade natural antioxidants, particularly phenolics, has been under very close scrutiny as potential therapeutic agents against a wide range of ailments including neurodegenerative diseases, cancer, diabetes, cardiovascular dysfunction, inflammatory diseases and also aging.
The medicinal actions of phenolics are mostly ascribed to their antioxidant capacity, free radical scavenging ability, chelation of redox active metal ions, modulation of gene expression and interaction with the cell signaling pathways. The free radical scavenging and antioxidant activities of phenolics are reported to depend upon the arrangement of functional groups about the nuclear structure. Both the number and configuration of H-donating hydroxyl groups are the main structural features influencing the antioxidant capacity of phenolics (Cao et al., 1997; Pannala et al., 2001). The present study is a step towards the exploration of natural antioxidants from various fractions of methanol extract of C. tabularis leaves employing free radical scavenging assays. The estimation of phenolic content of C. tabularis was done using Folin–Ciocalteu reagent that produced blue colour by reducing yellow hetero polyphosphomolybdatetungstate anions (Huang et al., 2005). It was observed that more was the number of hydrogen donating groups in the phenolic compounds; more was the intensity of blue coloured complex that indicated the higher total phenol content. DPPH assay is a preliminary test to investigate the antioxidant potential of extracts. As hexane extract did not show any activity so the results are not shown. Out of other fractions, the ethyl acetate fraction showed the maximum inhibition with IC50 value 37 lg/ml concentration in DPPH assay but the IC50 value of gallic acid which was used as standard was 21 lg/ml. Likewise in the reducing power assay, the ethyl acetate fraction exhibited maximum inhibition with low IC50 value (101.5 lg/ml). In deoxyribose degradation assay, deoxyribose was used as detector molecule (an important constituent of DNA) to detect damage by OH radicals in the presence or absence of EDTA (Gutteridge, 1987). In the present study, it was estimated that extracts showed their pronounced effects in the presence of EDTA, as they are capable to scavenge . OH present in the free solution and thus protect the degradation of deoxyribose (detector molecule) to thiobarbituric acid reactive material. It was further observed that the fractions were also efficient in chelating Fe(III) in the absence of EDTA and makes it unavailable to detector molecule and thus impaired the formation of OH radicals at a particular site. In both the assays, ethyl acetate fraction showed the strong inhibitory potential than the other fractions. The results of DNA nicking assay was also in confirmation with hydroxyl radical scavenging in deoxyribose degradation assay as the addition of extracts to the pBR322 DNA containing Fenton’s reaction mixture minimizes the formation of Form II (single stranded nicked DNA) and Form III (double stranded nicked and linear DNA) and thus maintain Form I (supercoiled) DNA by a hydrogen abstraction mechanism (Russo et al., 2000). It is well documented from the present study that the antioxidant potential of various fractions in different assays was linearly correlated to its phenolic content (Fig. 4A and
R. Kaur et al. / Bioresource Technology 99 (2008) 7692–7698
B 800 600 400 200 0 CF
EAF
BF
100 90 80 70 60 50 40 30 20 10 0
1200 1000 800 600 400 200 0 CF
WF
RP
EAF
BF
WF
Fractions
Fractions DPPH
TPC
1000
Inhibition (%)
1200
100 90 80 70 60 50 40 30 20 10 0
TPC
Inhibition (%)
A
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TPC
NSS
SS
TPC
Fig. 4. Correlation between total phenol content (TPC) and antioxidant activity: (A) correlation of TPC with DPPH assay and reducing power assay and (B) correlation of TPC with non-site specific and site-specific deoxyribose degradation assay.
B). The study of the literature revealed that the plant is rich in variety of polyphenolic compounds especially coumarins, limonoids, chuktabularins, tabularisins. So antioxidant activity might be due to these phenolic compounds (Rastogi and Mehrotra, 1993; Nakatani et al., 2004; Fan et al., 2007; Zhang et al., 2007). The observation is further substantiated by the similar reports in the literature (Yen and Duh, 1993; Siriwardhana et al., 2003; Karawita et al., 2005). It is concluded from the present investigation that the leaves of C. tabularis showed effective hydroxyl radical, DPPH radical and reduction potential ability by hydrogen or electron abstraction mechanism and thus may serve as a potent antioxidant. Further studies are in progress to evaluate the ability of fractions to scavenge other free radicals and to isolate and elucidate the structure of main components present in these fractions, which are conscientious for the activity of various fractions. Acknowledgements Financial assistance from University Grants Commission, New Delhi, India is duly acknowledged. Dr. P.S. Ahuja, Director, Institute of Himalyan Bioresource Technology (IHBT) Palampur (HP) is duly acknowledged for providing necessary lab facilities to pursue the work on fractionation of the active extracts. References Amarowicz, R., Pegg, R.B., Rahimi-Moghaddam, P., Barl, B., Weil, J.A., 2004. Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem. 84, 551–562. Arouma, O.I., Grootveld, M., Halliwell, B., 1987. The role of iron in ascorbate dependent deoxyribose degradation. J. Inorg. Biochem. 29, 289–299. Bazzano, L.A., He, J., Ogden, L.G., Loria, C.M., Vupputuri, S., Myers, L., Whelton, P.K., 2002. Fruit and vegetable intake and risk of cardiovascular disease in US adults: the first National Health and Nutrition Examination Survey Epidemiologic Follow-up Study. Am. J. Clin. Nutr. 76, 93–99.
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Bioresource Technology 99 (2008) 7692–7698
Antioxidant activity of the phenol rich fractions of leaves of Chukrasia tabularis A. Juss. Rajbir Kaur a, Saroj Arora a,*, Bikram Singh b a
Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India b Natural Plant Product Division, Institute of Himalayan Bioresource Technology (IHBT), Palampur, Post Box No. 6-176061, Himachal Pradesh, India Received 24 October 2007; received in revised form 25 January 2008; accepted 30 January 2008 Available online 18 March 2008
Abstract The present study was designed to explore the antioxidant potential of chloroform, ethyl acetate, n-butanol and water fractions of 80% methanol extract of leaves Chukrasia tabularis by 2,20 -diphenyl-1-picrylhydrazyl (DPPH), deoxyribose degradation (non-site specific and site specific), reducing power and DNA nicking assays. The different fractions showed significant activities in all the free radical scavenging tests and these findings have also shown direct relationship between antioxidant activity and phenolic content. Among the fractions, ethyl acetate fraction exhibited highest inhibition of 93.14%, 89.99%, 87.04% in DPPH, non-site specific and site specific deoxyribose degradation assays, respectively and 91.20% reduction of ferricyanide to give Prussian blue coloured complex in reducing power assay at maximum concentration tested. This preliminary study indicates the antioxidant activity of the leaves of Chukrasia tabularis, and moreover the results showed correlation with the amount of phenolic content present in different fractions. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Chukrasia tabularis; Phenolic compounds; Free radical scavenging activities; DNA nicking assay; Total phenol content
1. Introduction The plants play a more comprehensive role in the human diet as they prevent diseases that are responsible for devastation of human population. Epidemiological studies have shown that a diet rich in fruits and vegetables is associated with a decreased risk of cardiovascular diseases and certain cancers (Bazzano et al., 2002; Block et al., 1992). These health effects have been attributed in part to the presence of phenolic compounds in dietry plants, which may exert their effects as a result of their antioxidant properties. The antioxidant activities of phenolics are mainly due to their redox properties that allow them to act as a reducing agents, hydrogen donors and singlet oxygen quenchers (Rice-Evans et al., 1996; Ramarathnam *
Corresponding author. Tel.: +91 0183 2451048, +91 0941 7285485; fax: +91 0183 2258819/2258820. E-mail address: [email protected] (S. Arora). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.01.070
et al., 1997; Khokhar and Apenten, 2003). In addition, they have a metal chelation potential (Amarowicz et al., 2004; Zhao et al., 2005; Othmana et al., 2007). Keeping this in view, coupled with the fact that Chukrasia tabularis has a high content of secondary metabolites including phenolic compounds, the present study was designed to estimate the antioxidant efficiency employing free radical scavenging tests viz. (2,20 -diphenyl-1-picrylhydrazyl (DPPH), non-site specific and site specific deoxyribose degradation, reducing power assays) together with inhibition of strand breaking of supercoiled deoxyribonucleic acid in DNA nicking assay. The total phenolic content was determined by Folin–Ciocalteu method. C. tabularis A. Juss. (Meliaceae) commonly known as Chittgong wood or lal devdari etc. is a medicinal plant that accumulates a variety of secondary metabolites including phenolic compounds, terpenes, limonoids, steroids, chuktabularins and tabularisins (Rastogi and Mehrotra, 1993; Nakatani et al., 2004; Fan et al., 2007; Zhang et al.,
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2007). It has got reference in Ayurveda as astringent and antipyretic drug. Recent studies have also explored its antimalarial and antibacterial activities (Mackinnon et al., 1997; Nagalakshmi et al., 2001, 2003). 2. Methods
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Sigma Chemical Co. (St. Louis, MO, USA). Ellagic acid and 2-deoxyribose were obtained from Lancaster Synthesis Inc. USA. Supercoiled plasmid pBR 322 DNA and agarose was obtained from Genei, Bangalore. Bromophenol blue, EDTA, L-ascorbic acid, Tris (hydroxymethyl) aminomethane, Folin–Ciocalteu reagent, trichloroacetic acid (TCA) and gallic acid were of analytical grade.
2.1. Plant material 2.4. Determination of total phenol content Leaves were collected from the tree growing in the Guru Nanak Dev University campus, Amritsar. Botanical identification was made from Herbarium of Department of Botanical and Environmental Sciences, GNDU, Amritsar and voucher specimen was submitted in the herbarium. 2.2. Plant extract The leaves of plant were thoroughly washed with tap water, dried at room temperature and ground to fine powder. The powdered leaves were extracted with 80% methanol by employing maceration method. The 80% methanol extract was fractionated using different solvents viz. hexane, chloroform, ethyl acetate, n-butanol and water. The supernatant was filtered using Whatman No. 1 sheet, pooled and concentrated using vacuum rotary evaporator. The concentrated solutions were then lyophilized to get the dry form of respective fractions (Flow Chart 1). 2.3. Chemicals 2,20 -Diphenyl-1-picryl hydrazyl (DPPH), 2-thiobarbituric acid (TBA), ethidium bromide were obtained from Leaves Powder (750 g) Extracted with 80% methanol in water 80% Methanol Extract (151.3 g) Dissolved in Distilled Water Aqueous Methanol Extract Extracted with Hexane; thrice
Hexane Fraction (1.41 g)
Chloroform Fraction (2.36 g)
Remaining Extract Extracted with Chloroform; thrice
Remaining Extract Extracted with Ethyl Acetate; thrice
Ethyl Acetate Fraction (78.63 g)
Remaining Extract
Folin–Ciocalteu procedure given by Yu et al. (2002) was used to estimate the total phenol content in the different fractions of C. tabularis. Following this method, 0.1 ml aliquots of fractions were diluted to 1 ml with distilled water. To this solution 0.5 ml of Folin–Ciocalteu reagent (1:1) and 1.5 ml of 20% sodium carbonate solution was added. The mixture was incubated for 2 h at room temperature. The volume was raised to 10 ml with distilled water and the absorbance of blue coloured mixture was measured at 765 nm (Systronics 2202 UV–Vis Spectrophotometer). The amount of total phenol was calculated as mg/g (Gallic Acid Equivalents) from calibration curve of gallic acid standard solution. For the gallic acid, the curve absorbance versus concentration is described by the equation y = 0.0012x 0.0084 (R2 = 0.9908). Here, y = absorbance and x = concentration. 2.5. Free radical scavenging tests 2.5.1. DPPH radical scavenging activity DPPH, a stable nitrogen centered radical was used to assess the hydrogen donating ability of different fractions of C. tabularis as it offers a convenient and accurate method because of the relatively short time required for analysis. For assessing the DPPH radical scavenging activity, method described by Blois (1958) was used with slight modifications. In this assay, 0.2 ml of extract solution was added to 3 ml of 0.1 mM methanolic DPPH solution in a cuvette and absorbance was read at 517 nm. The decrease in absorption at ambient temperature was recorded for 10 min and correlated with the scavenging action of the test compound. The radical scavenging activity was expressed as the inhibition percentage and monitored as per the equation: % DPPH radical scavenging = (1 AS/AC) 100; AC = absorbance of control and AS = absorbance of sample solution. The DPPH solution without sample solution was used as control. IC50 value is the concentration of sample (lg/ ml) required to scavenge 50% DPPH free radical and was calculated from inhibition curve. Gallic acid being a phenolic compound was used as positive control.
Extracted with n-Butanol; thrice
n- Butanol Fraction (25.69 g)
Remaining Extract
Flow Chart 1. Extraction procedure of leaves of Chukrasia tabularis.
2.5.2. Reducing power assay The reducing power of different fractions was determined by the method of Oyaizu (1986). One milliliter of extract of different concentrations was mixed with 2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1%
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potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. A volume of 2.5 ml of 10% TCA was then added to the mixture and centrifuged at 3000 rpm for 10 min. 2.5 ml of supernatant was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl3 (0.1%) and the absorbance was measured spectrophotometrically at 700 nm. Increase in absorbance of the reaction mixture was interpreted as increase in reducing activity of the extract and the results were compared with gallic acid that was used as a positive control. The percentage reduction of the sample as compared to standard, i.e. gallic acid was calculated by using the formula [1 (1 AS/AC)] 100. Here, AC = absorbance of standard at maximum concentration tested and AS = absorbance of sample.
was analyzed on 1% agarose gel (prepared by dissolving 0.5 g of agarose in 50 ml of 1 TBE Buffer) followed by ethidium bromide staining. In this case, a phenolic compound, ellagic acid was used as a positive control.
2.5.3. Deoxyribose degradation assay Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and test compounds for hydroxyl radical generated by the Fe3+– ascorbate-EDTA–H2O2 system according to the method of Halliwell et al. (1987) and Arouma et al. (1987) with slight modifications. Depending upon the use of EDTA, this method comprised non-site and site specific scavenging of OH radicals. For non-site specific hydroxyl radical scavenging assay, the Haber–Weiss reaction mixture (1 ml) contained 2-deoxyribose (10 mM), Fe(III) chloride (10 mM), EDTA (1 mM), and H2O2 (10 mM) without or with the test extracts (10–100 lg/ml) in 50 mM potassium phosphate buffer, pH 7.4. In site-specific hydroxyl radical scavenging assay, EDTA was replaced with same amount of buffer. The reaction was triggered by adding ascorbic acid (1 mM) which served as a reducing agent by reducing Fe3+ to Fe2+ ions and subsequent incubation of the mixture for 1 h at 37 °C. Solutions of Fe(III) chloride, ascorbic acid and H2O2 were prepared in distilled water just prior to use. To 1 ml solution of above mixture, TBA in 25 mM NaOH (1 ml, 0.5%) and TCA (1 ml, 10% w/v aqueous solution) were added. The mixture was heated for 90 min on water bath at 80 °C and the amount of pink chromogen produced was spectrophotometrically measured at 532 nm. Here also, the gallic acid was used as a standard. The inhibitory effect on the hydroxyl radicals was calculated as: % hydroxyl radical scavenging capacity = (1 AS/AC) 100; AC = absorbance of control and AS = absorbance of sample solution.
The estimation of phenolic content among different fractions revealed that the ethyl acetate fraction exhibited higher phenol content of 983.5 mg/g GAE followed by n-butanol fraction (816.5 mg/g) > water fraction (608.8 mg/g) > chloroform fraction (400 mg/g GAE) whereas the crude extract had 816 mg/g GAE of phenol content. It was observed that the crude extract had low levels of phenolic content as compared to the ethyl acetate fraction.
2.5.4. DNA nicking assay The ability of different fractions to protect super coiled pBR 322 DNA from devastating effects of hydroxyl radicals generated by Fenton’s reagent was assessed by the DNA nicking assay described by Lee et al. (2002) with slight modifications. The reaction mixture contained 0.3 ll of plasmid DNA, 10 ll Fenton’s reagent (30 mM H2O2, 50 mM ascorbic acid, and 80 mM FeCl3) followed by the addition of extracts and the final volume of the mixture was brought up to 20 ll using distilled water. The mixture was then incubated for 30 min at 37 °C and the DNA
2.6. Statistical analysis Each experiment was performed at least three times and results are presented as the mean ± SE. IC50 was also calculated. 3. Results 3.1. Total phenol content
3.2. DPPH radical scavenging activity The DPPH scavenging activities of different fractions of 80% methanol extract of leaves of C. tabularis are shown in Fig. 1A. The ethyl acetate fraction showed highest DPPH radical scavenging activity of 93.14% at 100 lg/ml concentrations whereas chloroform, n-butanol and water fractions showed 36.99%, 41.78% and 15.37% inhibition, respectively, at the same concentration. The crude extract exhibited an inhibition of 92.91% at 180 lg/ml concentration (not shown in figure). Kinetic studies were carried out in order to determine the scavenging ability of fractions as a function of time. These studies indicated that at lower concentrations or at initial stages of reaction, the fractions showed a lesser tendency to reduce DPPH radicals but after 3 min of time interval or at higher concentrations the steady state was attained in less than 10 min. Furthermore, the different fractions showed radical scavenging ability in a dose dependent manner. The IC50 value of various fractions was compared with standard phenolic compound, i.e. gallic acid (Table 1). The lowest IC50 value was observed to be 37 lg/ml with ethyl acetate fraction followed by n-butanol (110 lg/ml), chloroform (132 lg/ml) and water (164 lg/ml) fraction. 3.3. Reducing power assay In this assay, reducing power of the fractions of C. tabularis steadily increased with increasing sample concentration (Fig. 1B). It was noted that among the different fractions, ethyl acetate fraction exhibited the maximum reducing power. The order of activity of various fractions
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B 100 90 80 70 60 50 40 30 20 10 0
Reducing Power (%)
Inhibition (%)
A
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100 90 80 70 60 50 40 30 20 10 0
20 40 60 80 100 120 140 160 180 200
20 40 60 80 100 120 140 160 180 200
Concentration ( g/ml)
Concentration ( g/ml)
D
100 90 80 70 60 50 40 30 20 10 0
Inhibition (%)
Inhibition (%)
C
10 20 30 40 50 60 70 80 90 100
Concentration ( g/ml)
100 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 100
Concentration ( g/ml)
Fig. 1. Effect of different concentration of fractions of 80% methanol extract of Chukrasia tabularis in free radical scavenging tests: (A) DPPH assay, (B) reducing power assay, (C) non-site specific deoxyribose degradation assay and (D) site specific deoxyribose degradation assay. j = Gallic acid, N = nButanol fraction, x = Chloroform fraction, = Water fraction, d = Ethyl acetate fraction.
Table 1 IC50 value of different fractions of Chukrasia tabularis leaves in different free radical scavenging tests Assays
Extracts/ standard
IC50 (lg/ ml)
DPPH assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
21 132 37 110 164
Non-site specific deoxyribose degradation assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
<1 26.5 <1 128 45
Site specific deoxyribose degradation assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
55.5 94.5 8.0 60.5 117.5
Reducing power assay
Gallic acid Chloroform Ethyl acetate n-Butanol Water
56 336 101.5 200 472
3.4. Deoxyribose degradation assay Fig. 1C and 1D shows the hydroxyl radical scavenging ability of different fractions of methanol extract in non-site specific and site-specific hydroxyl radical scavenging assays. It has been found that in both, the fractions showed the scavenging ability in a dose dependent manner and out of different fractions; ethyl acetate was most active one. It showed maximum inhibition of 89.99% at 50 lg/ml followed by crude extract (82.52%) > chloroform fraction (62.42%) > water fraction (51.31%) > n-butanol fraction (36.31%). In site-specific assay, hydroxyl radical scavenging ability of different fractions followed the order as: ethyl acetate fraction (87.04%) > crude extract (79.69%) > gallic acid (50.55%) > n-butanol fraction (49.62%) > chloroform fraction (30.69%) > water fraction (24.34%) at concentration 60 lg/ml and was also found to be comparable to standard gallic acid. Table 1 shows the IC50 value of different fractions in site specific and non-site specific deoxyribose degradation assays. The lowest IC50 value was observed to be with ethyl acetate (<1 lg/ml in non-site specific and 8 lg/ml in site specific deoxyribose degradation assay). 3.5. DNA nicking assay
was as follows: ethyl acetate fraction > n-butanol fraction > chloroform fraction > water fraction at the highest concentration tested. The crude extract of leaves exhibited a reduction of 62.70% at 200 lg/ml (not shown in figure).
Fig. 2 shows the DNA damage protecting activity of various fractions of C. tabularis. The results are in confirmation with the hydroxyl radical scavenging ability of various fractions in deoxyribose degradation method. It has
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2
3
4
5
6
7
8
9 Form II Form III Form I
Fig. 2. Protective effect of different fractions of Chukrasia tabularis in DNA Nicking assay. Lane 1: Negative control (Distilled water + DNA), Lane 2: Control (DNA + Fenton’s reagent), Lane 3: Ellagic acid (250 mg/ ml) + FR as positive control, Lane 4: 80% methanol extract (250 mg/ml) + FR, Lane 5: chloroform extract (250 mg/ml) + FR, Lane 6: ethyl acetate extract (250 mg/ml) + FR, Lane 7: n-Butanol extract (250 mg/ml) + FR, Lane 8: water extract (250 mg/ml) + FR, Lane 9: control (DNA + Fenton’s reagent). Form II: ss nicked DNA; Form III: ds nicked and linear DNA and Form I: supercoiled DNA.
Densitometric Units
4500 4000 3500 3000 2500 2000 1500 1000 500 0 C
FR
EA
ME
CF
EAF
BF
WF
FR
Fractions Form II DNA
Form III DNA
Form I DNA
Fig. 3. Densitometric analysis of effect of different fractions of plant extract on pBR 322 DNA in the presence of hydroxyl radicals generated in Fenton’s reaction. Here, C = Control, FR = Fenton’s reagent, EA = Ellagic acid, ME = Methanol extract, CF = Chloroform fraction, EAF = Ethyl acetate fraction, BF = n-Butanol fraction and WF = Water fraction.
been found that when the plasmid DNA was dissolved in the Fenton’s reaction mixture, there was a time dependent increase of single stranded and double stranded nicked and linear forms of DNA (Forms II and III, respectively) due to hydroxyl radicals generated in reaction mixture. However, addition of various fractions to reaction mixture reduced the hydroxyl radical mediated strand breaking and conversion of supercoiled DNA to Forms II and III DNA. Densitometric analysis showed that ethyl acetate fraction and chloroform fraction showed significant protection of DNA from damage caused by hydroxyl radicals (Fig. 3).
4. Discussion During the last decade natural antioxidants, particularly phenolics, has been under very close scrutiny as potential therapeutic agents against a wide range of ailments including neurodegenerative diseases, cancer, diabetes, cardiovascular dysfunction, inflammatory diseases and also aging.
The medicinal actions of phenolics are mostly ascribed to their antioxidant capacity, free radical scavenging ability, chelation of redox active metal ions, modulation of gene expression and interaction with the cell signaling pathways. The free radical scavenging and antioxidant activities of phenolics are reported to depend upon the arrangement of functional groups about the nuclear structure. Both the number and configuration of H-donating hydroxyl groups are the main structural features influencing the antioxidant capacity of phenolics (Cao et al., 1997; Pannala et al., 2001). The present study is a step towards the exploration of natural antioxidants from various fractions of methanol extract of C. tabularis leaves employing free radical scavenging assays. The estimation of phenolic content of C. tabularis was done using Folin–Ciocalteu reagent that produced blue colour by reducing yellow hetero polyphosphomolybdatetungstate anions (Huang et al., 2005). It was observed that more was the number of hydrogen donating groups in the phenolic compounds; more was the intensity of blue coloured complex that indicated the higher total phenol content. DPPH assay is a preliminary test to investigate the antioxidant potential of extracts. As hexane extract did not show any activity so the results are not shown. Out of other fractions, the ethyl acetate fraction showed the maximum inhibition with IC50 value 37 lg/ml concentration in DPPH assay but the IC50 value of gallic acid which was used as standard was 21 lg/ml. Likewise in the reducing power assay, the ethyl acetate fraction exhibited maximum inhibition with low IC50 value (101.5 lg/ml). In deoxyribose degradation assay, deoxyribose was used as detector molecule (an important constituent of DNA) to detect damage by OH radicals in the presence or absence of EDTA (Gutteridge, 1987). In the present study, it was estimated that extracts showed their pronounced effects in the presence of EDTA, as they are capable to scavenge . OH present in the free solution and thus protect the degradation of deoxyribose (detector molecule) to thiobarbituric acid reactive material. It was further observed that the fractions were also efficient in chelating Fe(III) in the absence of EDTA and makes it unavailable to detector molecule and thus impaired the formation of OH radicals at a particular site. In both the assays, ethyl acetate fraction showed the strong inhibitory potential than the other fractions. The results of DNA nicking assay was also in confirmation with hydroxyl radical scavenging in deoxyribose degradation assay as the addition of extracts to the pBR322 DNA containing Fenton’s reaction mixture minimizes the formation of Form II (single stranded nicked DNA) and Form III (double stranded nicked and linear DNA) and thus maintain Form I (supercoiled) DNA by a hydrogen abstraction mechanism (Russo et al., 2000). It is well documented from the present study that the antioxidant potential of various fractions in different assays was linearly correlated to its phenolic content (Fig. 4A and
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B 800 600 400 200 0 CF
EAF
BF
100 90 80 70 60 50 40 30 20 10 0
1200 1000 800 600 400 200 0 CF
WF
RP
EAF
BF
WF
Fractions
Fractions DPPH
TPC
1000
Inhibition (%)
1200
100 90 80 70 60 50 40 30 20 10 0
TPC
Inhibition (%)
A
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TPC
NSS
SS
TPC
Fig. 4. Correlation between total phenol content (TPC) and antioxidant activity: (A) correlation of TPC with DPPH assay and reducing power assay and (B) correlation of TPC with non-site specific and site-specific deoxyribose degradation assay.
B). The study of the literature revealed that the plant is rich in variety of polyphenolic compounds especially coumarins, limonoids, chuktabularins, tabularisins. So antioxidant activity might be due to these phenolic compounds (Rastogi and Mehrotra, 1993; Nakatani et al., 2004; Fan et al., 2007; Zhang et al., 2007). The observation is further substantiated by the similar reports in the literature (Yen and Duh, 1993; Siriwardhana et al., 2003; Karawita et al., 2005). It is concluded from the present investigation that the leaves of C. tabularis showed effective hydroxyl radical, DPPH radical and reduction potential ability by hydrogen or electron abstraction mechanism and thus may serve as a potent antioxidant. Further studies are in progress to evaluate the ability of fractions to scavenge other free radicals and to isolate and elucidate the structure of main components present in these fractions, which are conscientious for the activity of various fractions. Acknowledgements Financial assistance from University Grants Commission, New Delhi, India is duly acknowledged. Dr. P.S. Ahuja, Director, Institute of Himalyan Bioresource Technology (IHBT) Palampur (HP) is duly acknowledged for providing necessary lab facilities to pursue the work on fractionation of the active extracts. References Amarowicz, R., Pegg, R.B., Rahimi-Moghaddam, P., Barl, B., Weil, J.A., 2004. Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem. 84, 551–562. Arouma, O.I., Grootveld, M., Halliwell, B., 1987. The role of iron in ascorbate dependent deoxyribose degradation. J. Inorg. Biochem. 29, 289–299. Bazzano, L.A., He, J., Ogden, L.G., Loria, C.M., Vupputuri, S., Myers, L., Whelton, P.K., 2002. Fruit and vegetable intake and risk of cardiovascular disease in US adults: the first National Health and Nutrition Examination Survey Epidemiologic Follow-up Study. Am. J. Clin. Nutr. 76, 93–99.
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