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Antioxidant properties and stability of Aegle marmelos leaves extracts

Article  in  Journal of Food Science and Technology -Mysore- · February 2013

DOI: 10.1007/s13197-010-0221-z · Source: PubMed

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J Food Sci Technol (January–February 2013) 50(1):135–140
DOI 10.1007/s13197-010-0221-z

ORIGINAL ARTICLE

Antioxidant properties and stability of aegle marmelos leaves

extracts
Vanitha P. Reddy & Asna Urooj

Revised: 21 November 2010 / Accepted: 29 December 2010 / Published online: 26 January 2011
# Association of Food Scientists & Technologists (India) 2011

Abstract Aegle marmelos (AM) leaves were extracted being shifting towards finding alternatives for synthetic food
with methanol (ME), ethanol (EE), water (WE) and ingredients, natural substances having antioxidative properties
analyzed for antioxidant activities by DPPH radical need to be further explored (Sachindra et al 2010). The search
scavenging method, reducing power and in vitro inhibition for safe and effective naturally occurring antioxidants is now
by Fenton’s reagent—induced oxidation of lipid system. focused on edible plants especially spices and herbs
Stability of extracts to pH (4, 7 and 9) and temperature (Miliauskas et al 2004). Extracts from spices, herbs and hulls
(100 °C, 15 min.) was studied. The three extracts showed are reported to exhibit varying degrees of antioxidant activity
varying degree of efficacy in each assay in a dose (Koleva et al 2003, Miliauskas et al 2004, Dorman et al.
dependent manner. The inhibition of MDA formation in 2003a, b, Takeoaka and Dao 2003). Some of the common
Linseed oil by EE (47%) was significantly (P<0.05) higher spices have been evaluated and further screening of medicinal
than WE (28%) and ME (23%) but less than α- Tocopherol plants for their antiradical properties is of interest primarily to
(80%). WE showed maximum stability to high temperature. find out newer sources of natural antioxidants, functional
The antioxidant activity of EE at pH 4 was significantly foods and neutraceutical.
higher (P<0.05) compared with WE and ME. At pH 7, the Aegle Marmelos (Bilwa, AM), a medicinal plant is found
antioxidant activity of all the three extracts remained all over the sub Himalayan forests. Its history has been
unchanged. Data indicates that potential exists for the traced to the Vedic period, about 2000 BC. All parts of the
utilization of Aegle marmelos as a natural antioxidant. tree have medicinal qualities (Chanda et al 2008). It contains
volatile oils and pectin, the wood-ash is rich in minerals and
Keywords Aegle marmelos . Radical scavenging activity . is used in numerous products like candy, squash, coffee etc.
Reducing power assay . Lipid peroxidation . Stability Rana and Jain (1997) evaluated the anti fungal activity of
essential oil isolated from leaves of the Aegle marmelos
using spore germination assay and the oil established
Introduction variable efficacy against different fungal isolations. (Shoba
and Thomas 2001) reported the efficacy of methanol extract
The importance of natural antioxidants for use as food of AM in rodents against castor oil induced diarrhoea with
additives or nutritional supplements has already been reduction of both the induction time of diarrhoea and total
established (Bassiounny et al 1990, Mansour and Khalil weight of the feces. Aegle Marmelos is reported to have
2000, Reddy et al 2005, Roy et al 2010). Antioxidants or antidiarrheal (Shoba and Thomas 2001, Dhuley 2003),
ingredients having antioxdative properties are used exten- antiproliferative (Lampronti et al 2003), anti-inflammatory,
sively for improvement of food stability. With the focus is antipyretic (Arulet al. 1997), and hypoglycemic (Upadhyay
et al. 2004), (Kesari et al 2006, Narender et al. 2007) and
V. P. Reddy : A. Urooj (*) antioxidant (Sabu and Kuttan 2004).
Department of Studies in Food Science and Nutrition,
The fruit and bark of AM has been studied for antioxidant
University of Mysore,
Mysore 570006, India potency (Khangan and Raul 1996, Riyanto and Mustafa 2000,
e-mail: [email protected] Marzine and Gilbart. 2005), and the leaves are reported to be
136 J Food Sci Technol (January–February 2013) 50(1):135–140

antidiabetic and antilipidemic in rats (Upadhyay et al. 2004, Antioxidants extraction

Sabu and Kuttan 2004). Limited information is available in
the literature on the antioxidant aspects viz., components, A 15 g sample was extracted with 50 ml solvent, (methanol,
activity and their stability. Hence, the present study was ethanol and water separately) for 6 h in a mechanical shaker.
undertaken to analyze antioxidant components, activity and The extracts were filtered and filtrates were evaporated at 40º
stability of various solvent extracts of AM leaves. under reduced pressure to dryness in a rotary evaporator
(Superfit, India) .The residue of each extract was stored in air-
Materials and methods tight container at 4º until used. In case of water extract, the
sample was added to boiling water and extracted for 15 min
Plant material and filtered. The filtrate was freeze dried and stored at 4° C
until further use.
The selected plant material Aegle marmelos (AM) leaves
were collected locally during September and October Radical scavenging activity—(RSA)
period, washed and dried in a hot air oven at 55 °C. Dry
leaves were ground separately and passed through a 60 mesh The ability of extracts to scavenge DPPH radicals was
sieve and kept in air tight containers at 4 °C until further use. determined according to the method of Blois (1958).
Briefly, 1 ml of 0.1 mM DPPH solution was mixed with
Determination of antioxidant components 3 ml of extract (containing 100–1000 μg) in methanol. The
mixture was then vortex mixed vigorously and left for
In the dehydrated sample, different antioxidant components 30 min at room temperature in the dark. The absorbance
were estimated using standard methods. Ascorbic acid was was measured at 517 nm and activity expressed as %
determined according to the titrimetric method by the 2, 6- Radical Scavenging (RSA) relative to control using the
dichlorophenol-indophenol dye against KMnO4 (Ranganna following equation:
1999). α- Tocopherol was extracted by direct saponification
Absorbance of control Absorbance of sample
of dried sample and estimated based on formation of a red RSA% ¼ 100
Absorbance of control
complex from reaction of α,α′ –bipyridyl with ferrous ion
due to reduction of ferric ion by tocopherol (Freed 1966). β-
Carotene was quantified by open column chromatography,
followed by measuring the absorbance of elute at 450 nm Reducing power assay
against standard β-carotene (Ranganna 1999). Reduced
glutathione was determined based on the development of a The ability of extracts to reduce iron (III) to iron (II) was
yellow compound due to reaction of 5, 5-dithio (bis) nitro determined in this method. The dried extract (200 μg) in
benzoic acid with compounds containing sulphhydryll 1 ml of the corresponding solvent was mixed with 2.5 ml
groups (Beutler and Kelly 1963). Total phenols were of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of the
extracted from a weighed portion (50–500 mg) of dried potassium ferricyanide, (K3 Fe(CN)6: 10 g1−1) then the
sample with 5 ml of 1.2 M HCl in 50% aqueous methanol mixture was incubated at 50° for 30 min. Afterwards,
for 2 h and with 70% acetone shaken for 2 h and analyzed by 2.5 ml of trichloroacetic acid (100 g 1−1) was added to the
Folin-Ciocalteu micro method (Slinkard and Singleton mixture, which was then centrifuged at 1650 × g for
1967). Results were expressed as mg or g of Gallic acid 10 min. Finally supernatant solution (2.5 ml) was mixed
equivalent g−1 dry weight. The content of flavonoids was with distilled water (2.5 ml) and FeCl3 (0.5 ml) (1 g1−1)
determined by a pharmacopoeia method (Miliauskas et al and the absorbance was measured at 700 nm. Increased
2004) using Rutin as a reference compound. The amount of absorbance of the reaction mixture indicates increase in
Flavonoids in plant extracts in Rutin equivalents (RE) was reducing power (Yildirim et al. 2003).
calculated by the following formula,
Inhibition of lipid peroxidation in vitro
ðA:Mo :10Þ
X¼ Antioxidant activity
ðAo: MÞ

The protective action of the extracts against oxidation of

Where , X- flavonoids content , mg/g plant extract in lipid system was analyzed by the method of (Tamura and
RE; A- the absorption of plant extract solution ; Ao- the Yamagami 1994). An aqueous solution containing Linseed
absorption pf standard Rutin solution; M- the weight of oil (1.5 mg/ml) was poured into a 30 ml test tube and
plant extract; g; Mo – the weight of Rutin in the solution, g. diluted with 5 ml of Trizma–buffer solution (0.25 mM,
J Food Sci Technol (January–February 2013) 50(1):135–140 137

Table 1 Antioxidant components, total polyphenols and extract yield reacted solution (1 ml) mentioned above was mixed with
of different solvent extracts from Aegle leaves
0.2% (w/v) TBA (3 ml) and 0.05 M sulfuric acid (2.5 ml)
Antioxidant component Per 100 g and the mixture was heated for 30 min in 95° water bath.
After, the solution was then cooled in ice for 5 min; the
α- Tocopherol (mg)b 27 colored substances were extracted by 4.0 ml of 1-butanol.
β- carotene (μg) b 8600 The absorbance of 1-butanol layer was measured at
b
Glutathione (m. moles) 580 532 nm. Antioxidant activity (AOA) was expressed as
Ascorbic acid (mg) b 260 percentage inhibition of lipid peroxidation relative to the
Total Flavonoids (mg)a 2.4 control using the following equation:
Total polyphenols (g) b 2.4
Polyphenol contentc
ME 9.9 (27.8) Absorbance of control Absorbance of sample
AOA% 100
EE 11.5 (12.3) Absorbance of control
WE 8.9 (32.0)
a
mg/g extract, b per 100 g dry basis. c g of Gallic acid per 100 g of Heat and pH stability
extract. Values in parenthesis indicate yield in g of extract per 100 g of
dried leaves. ME methanol, EE ethanol, WE water extracts, n=4
The extracts were heated in a boiling water bath for
15 min and the residual antioxidant activity was
pH 7.4) containing 0.2% sodium dodecyl sulphate (w/v) determined by radical scavenging activity using DPPH
and 0.75 mM potassium chloride .Trizma buffer was as described previously. For pH stability, antioxidant
prepared by diluting 6.075 g of Tris (Trihydroxymethyl)- extracts were incubated for 24 h at pH 4, 7 & 9 and the
amino methane and 11.184 g of potassium chloride with residual antioxidant activity was determined during the
distilled water to 1 L after adjusting the pH of the solution to incubation period at different time intervals (0 min,
7.4 Lipid peroxidation was initiated by adding Fenton’s 30 min and 24 h) as radical scavenging activity against
reagent (FeCl3-1 μm and H2O2 0.5 μm). Incubation was DPPH (Arabshahi-Delouse et al. 2007).
continued for 16 h at 37° in a dark place. The reaction was
stopped by adding 50 μl of 1% BHT alcoholic solution. The Statistical analysis
solution obtained was used for antioxidant activity assay.
Mean values of 4 determinations (n=4) (p<0.05) was
Antioxidant activity assay subjected to one way ANOVA and Tukey’s multiple
comparison tests using SPSS software (version 11).
The dried plant extract (200 μg) in 100 μl of corresponding
solvent was mixed with the solution (9.9 ml) mentioned above
when necessary. BHT was used as a standard to evaluate the Results and discussion
antioxidative activity of samples. The reacted solution
obtained (1 ml) was used for TBA assay. Antioxidant component

TBA assay The Aegle leaves were found to be a good source of several
antioxidant components such as, β-carotene, glutathione,
The degree of oxidation of oil was measured by the 2- α-tocopherol, ascorbic acid and total polyphenols and
thiobarbituric acid (TBA) assay (Ohkawa et al 1994). The flavonoids (Tables 1 and 2).

Table 2 Comparison of antioxidant properties and polyphenols of Aegle extracts and standards

Solvent Radical scavenging activity DPPH, % Percent Inhibition of lipid oxidation (%) Reducing power (absorbance at 700 nm)

ME 81±2.064a 23±2.00a 0.232±0.003a

EE 87±1.00b 46±1.15b 0.246±0.004b
WE 93±0.57c 28 ±2.51a 0.349 ±0.003c
Standard 87±0.52ba 80±5.03cb 0.973 ±0.002dc
a
BHT, b
α- Tocopherol, c Ascorbic acid, -standard conc. used 150 μg and extracts 200 μg;
ME-methanol, EE- ethanol, WE-water extracts, n=4
138 J Food Sci Technol (January–February 2013) 50(1):135–140

100
pounds are reported to enhance OH. formation and inhibit
90 antioxidant enzymes (catalase and SOD) suggesting that the
80
naturally occurring substances can have pro-oxidant effects
under some reaction conditions (Sakihama et al 2002). At
70 200 μg concentration, WE showed maximum activity (92%)
60 followed by EE (88%) and ME (78%) Table 2. These
RSA %

percentages can be considered as a full absorption inhibition

50
of DPPH because after completing the reaction in the final
40 solution always possesses some yellowish color and there-
ME
30 fore its absorption inhibition compared to colorless methanol
EE
solution cannot reach 100% (Miliauskas et al., 2004).
20 WE
10 BHT Reducing power assay
0
0 100 200 300 400 500 600 700 800 900 1000 The reducing power (electron donating capacity) of
Concentration in ug bioactive compounds is associated with antioxidant activ-
ity (Siddharaju et al 2002, Yen et al 1993). In the present
Fig. 1 DPPH radical scavenging activities (RSA) of methanol,
ethanol and water extracts of Aegle leaves. BHT was used as positive study, the ability of extracts to reduce iron (III) to iron (II)
control. % RSA relative to control. ME methanol, EE ethanol, WE was determined and was compared with that of a standard
water extracts (ascorbic acid). All the three extracts showed some degree
of electron donating capacity (Table 3) in which EE extract
showed maximum reduction of iron (III) to iron (II)
Radical scavenging activity indicated by higher absorbance at 700 nm, but the
capacities were inferior to that of ascorbic acid. It was
Free radicals which are involved in the process of lipid interesting to note that despite containing the least amount
peroxidation are considered to play a major role in of phenolics, WE was the most potent reducing agent.
numerous chronic pathogens such as cancer and cardio-
vascular diseases among others (Dorman et al. 2003a, b). Antioxidant activity (AOA)
The DPPH radical scavenging has been widely used to
evaluate the free radical scavenging ability of various The reducing power assay and radical scavenging activity
natural products and has been accepted as a model assays are indicators of antioxidant activity. However,
compound for free radical originating in lipids (Porto et neither of these methods utilize a food or biologically
al 2000). relevant oxidizable substrate, hence no direct information
The radical scavenging activity (RSA) of the three solvent on the protective properties of the extracts can be
extracts (methanol, ethanol and water) as determined by determined (Dorman et al 2003a, b). In present study, the
DPPH assay at different concentrations is shown in Fig. 1. effect of various extracts on the % inhibition of lipid
The scavenging ability of three extracts against DPPH was peroxidation in Linseed oil (rich in PUFA) was determined
concentration dependent upto 300 μg., with increasing levels by TBA method. All the three extracts, were capable in
the RSA tended to decrease. This trend indicates that the preventing the formation of TBARS generated by Fenton’s
scavenging potency of the extracts might reach a saturation reagent (Table 3), however EE was the most effective in
point and at higher concentrations it may act as a pro- inhibiting lipid oxidation. A similar effect was not observed
oxidant. Similarly, at higher concentrations phenolic com- at higher concentrations of extracts (>500 μg) indicating a

Table 3 Effect of pH and Tem-

perature on the Antioxidant Solvent extract 0 min 30 min. 24 hrs Before heat treatment After heat treatment
Activity of Methanol,
Ethanol and Water Extracts 4ME 73±1.52al 71±1.73abl 70±1.00al 81±2.064 57±2.00
of Aegle Leaves 4EE 79±1.15bl 72±0.57am 75±.1.52bn 87±1.00 71±2.08
4WE 77±1.52al 68±1.52bm 72±2.00abm 93±0.57 89±1.2
7ME 67±1.52al 75±1.52bl 72±2.51al
Extracts conc. 200 μg; ME 7EE 73±2.64al 68±2.08al 71±2.64al
methanol, EE ethanol, WE water 7WE 80±1.00cl 72±2.08am 72±2.08am
extracts, n=4
J Food Sci Technol (January–February 2013) 50(1):135–140 139

pro oxidant role at higher concentrations. Sakihama (2002) by increasing the pH of media. The antioxidant activity of
reported that, the flavonoids and dihydroxycinnamic acids different extracts from cocoa by-products was found to be
can nick DNA via the production of radicals in the presence higher at alkaline pH (Azizah et al 1999). In an earlier
of Cu and O2. Phenoxyl radicals are reported to initiate study, it was observed that extracts from mint and carrot
lipid peroxidation while, Al, Zn, Ca, Mg and Cd have been leaves showed higher antioxidant activity at pH 9 than pH 4
found to stimulate phenoxyl radical-induced lipid perox- (Arabshahi-Delouse et al 2007). These differences might be
idation. due to different samples used, various compounds being
extracted in each case and the method used to evaluate
Heat stability antioxidant potency.

It is well known that many factors such as antioxidant

concentration, temperature and pH of the media, processing Conclusion
treatment influence the antioxidant activity (Gazzani et al
1998). Table 3 shows the effect of temperature (100° C, Results of the present study reveal that the AM is a
15 min.) on the stability of different solvent extracts. WE potential source of antioxidants which are responsible for
showed maximum stability as measured by RSA compared the antioxidant activity. The stability to heat and pH of the
to ME and EE after heat treatment and similar results were different extracts of AM leaves extracts with strong
found in case of Raphanus sativus where WE was stable antioxidant activity indicates its scope for utilization in
and radical scavenging activity increased after heat treat- food and biological systems. In addition to being consumed
ment (Reddy et al 2010). In a study conducted by Gazzani as healthy antioxidants, the compounds present in AM that
et al (1998), the antioxidant activity of a number of are responsible for antioxidant activity could be isolated
vegetable juices was stabilized by boiling, suggesting that and then used as food additives to delay the oxidative
the initial pro oxidant activity was due to pro oxidases like deterioration of foods and as nutraceutical in food for-
peroxidases which are inactivated at high temperatures. The mulations against degenerative diseases.
decline in antioxidant activity of extracts followed by
heating at 100 °C might be related to either loss of naturally Acknowledgment The authors thank the Department of Science and
occurring antioxidants present in the extracts or formation Technology, New Delhi, India. for the financial assistance. (Project no.
of novel compounds having prooxidant activity (Arabshahi- SR /SO/HS- 21/ 2004)
Delouse et al 2007). Plant extracts are known to exhibit
either a prooxidant or an antioxidant effect depending on
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