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J. Plant Biochem. Biotechnol.

(OctoberDecember 2013) 22(4):483487


DOI 10.1007/s13562-012-0178-2

SHORT COMMUNICATION

Isolation and characterization of potent bioactive fraction


with antioxidant and UV absorbing activity from Aloe
barbadensis Miller gel
Anirban Ray & S. Dutta Gupta & Sampad Ghosh

Received: 20 April 2012 / Accepted: 24 November 2012 / Published online: 8 December 2012
# Society for Plant Biochemistry and Biotechnology 2012

Abstract The methanolic extract of Aloe vera L. gel was


subjected to antioxidant guided fractionation with silica gel
column chromatography to screen the potent fraction. The
antioxidant capacities of different fractions were evaluated
using DPPH radical scavenging assay and correlated with
total phenol content. Total phenolic contents of different fractions were determined according to the Folin-Ciocalteu spectrophotometric method. The positive correlation was observed
between DPPH radical scavenging assay and total phenolic
contents indicating that phenolics in Aloe vera L. gel were
fundamental contributor of antioxidant activity. Third pooled
fraction was identified as potential fraction with highest antioxidant potential. This fraction indicated the presence of well
resolved fluorescent components. Characteristic UVvis
absorption and HPLC analysis indicates that aloin take
part in antioxidant potential attributed to aloe gel.
Keywords Aloe vera . Antioxidant-activity . UV
absorption . HPLC . Aloin
Abbreviations
FTIR
Fourier transform infrared spectroscopy
UV
Ultra violet
HPLC High performance liquid chromatography
Electronic supplementary material The online version of this article
(doi:10.1007/s13562-012-0178-2) contains supplementary material,
which is available to authorized users.
A. Ray (*) : S. Dutta Gupta
Agricultural and Food Engineering Department,
Indian Institute of Technology,
Kharagpur, West Bengal 721302, India
e-mail: anirbanrayiitkgp@gmail.com
S. Ghosh
Department of Chemistry, National Institute of Technology,
Jamshedpur, Jharkhand 831014, India

RP
LSD
DPPH

Reverse phase
Least significant difference
Diphenyl picrylhydrazyl

Aloe vera L. (Aloe barbadensis Miller) belongs to liliaceae


family (presently known as Asphodelaceae, APG III system
of 2009) that possesses therapeutic and antioxidant properties. Gels from Aloe vera leaves are widely used in skin care
products and as health supplement. The major components
of aloe may be divided in five groups (Choi and Chung
2003) e.g. Phenolics (anthraquinones, flavonoids), saccharides (mannose, glucomannan, acemannan etc.), vitamins
(B1, B2 etc.), enzymes (amylase, carboxypeptidase etc.)
and low molecular weight substances (cholesterol, salicylic
acid etc). The anthraquinones in Aloe vera mainly composed
of aloe emodin, aloin, aloetic acid, anthranol, barbaloin,
isobarbaloin, emodin, ester of cinnamic acid (Hamman
2008). Free radicals interact with cellular constituents such
as lipids and deoxyribonucleic acid, perpetuating cellular
damages (Rice-Evans et al. 1996). Free radicals can also
damage the genetic materials, create aging and wrinkles and
dismay the general resistance mechanism, increasing the
risk of melanoma (Kumar et al. 2009). Oxygen free radicals
are massively produced in diseases like cancer, cardiovascular diseases and diabetes mellitus (Zin et al. 2006). At
present antioxidant therapy (Rice-Evans et al. 1996) was
suggested a decade ago as an attractive new medical
strategy to lower down the oxygen free radical concentration in human body. Preliminary screening or fractionation is directed to isolate the bioactive fraction with potential
antioxidant activity, necessary for ayurvedic product (herbal
medicine) formulations. The information about the phenolic
antioxidants separation from aloe gel provides the valuable
information for ayurvedic product process optimization.

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J. Plant Biochem. Biotechnol. (OctoberDecember 2013) 22(4):483487

The objective of the present work is to evaluate antioxidative activities and UV absorbing potential of various
chromatographic fractions from leaf gel extract of Aloe vera
in relation to their total phenolic contents. Characterization
and preliminary identification was conducted to detect the
predominant compounds responsible for biological activity
(i.e. antioxidant and UV light absorbing potential) in aloe
gel.
The Aloe vera L. plants were identified by Prof. G. G.
Maity, Taxonomist, University of Kalyani and was grown in
the Agricultural and Food Engineering Departmental farm
of IIT Kharagpur, India. Plant material used in this study
was freeze dried parenchymatous leaf gel collected from
36 months old Aloe barbadensis Miller plants. The leaves
(1,801.6 gm) were harvested from 1st and 2nd whorl position of aloe plants. The removed gel (1,133.28 gm) was
stored at 20 C overnight and subjected to freeze drying
for three consecutive days. The freeze dried materials
(11.22 gm) were stored in deep-freezer at 20 C. To obtain
methanolic extracts, 11.22 g of freeze -dried material is
mechanically agitated in 300 ml of 90 % methanol for 4 h.
This step was repeated 3 times for complete extraction.
Then the mixture is centrifuged at 10,000 rpm for 10 min.
The supernatant was concentrated by subjecting to rotary
vacuum evaporation at 40 C to obtain the sticky brown
methanolic extracts (3.40 g).
Dried methanolic extract (0.65 g) was fractionated using
a silica gel-60 column (1.5 cm diameter and 18 cm height,
mesh size 120). 0.65 gm of extract was dissolved in 1 ml of
methanol and silica gel and introduced to the column ported
to Frac 920 G.E. healthcare fraction collector. Chloroformethanol (3:1, v/v) solution was used as mobile phase. Total
30 fractions were collected (each of them contained 2 ml) at
the time of chromatographic separation. The absorbance of
the fractions were measured using an UVVIS-NIR
Fig. 1 Antioxidant activity of
ten silica gel column
chromatographic pooled
fractions from methanolic
extracts of aloe gel. Plots and
bars show mean sd (n03)

Spectrophotometer (Perkin-Elmer, USA, Model Lamda900) for the wavelength range of 200400 nm. The fractions
having the same type of spectrum were pooled together.
There were total ten pooled fractions. The ten pooled fractions were concentrated to dryness at 40 C by rotary evaporation in vacuum. Dehydrated pooled fractions were
dissolved in 90 % methanol to get final concentration
of 1 mg/ml. This concentration of solutions was used for
biochemical estimations.
Total phenolic contents of each pooled fraction were
estimated following the method of (Zin et al. 2006). Absorbance of the reaction mixture was measured at 725 nm.
Freshly prepared methanolic solution (3.9 ml) of the DPPH
(6105 M) was incubated with 0.1 ml solution of pooled
fractions (1 mg/ml) for 45 min at room temperature (Ozsoy
et al. 2008). The change in absorbance was measured at
517 nm. Ascorbic acid was used as positive control. Radical
Scavenging Activity was calculated by the following formula: Percent inhibition (%)0(1At)/A0 100, Where, A0
absorption of blank sample at 517 nm, At absorption
of tested sample at 517 nm.
The absorbances of the pooled fractions in 95 % ethanol
were measured in UV-1800 UVVIS Shimadzu Spectrophotometer. Among the ten chromatographic fractions, only
first five fractions showed the considerable UV absorption,
are subjected to fluorescence spectral analysis using Perkin
Elmer LS-55 luminescence spectrometer. FTIR analyses of
the potent fraction was conducted by using Thermo Nicolet
Nexus 870 spectrometer, at a resolution of 3 cm1 .
Quantitative estimation was carried out for aloin as possible marker compound present in aloe gel. 3rd fraction was
analyzed with HPLC using following conditions: Column
was Phenomenex, Synergi Hydro-RP80A-C18 (4.6 mm
I.D.250 mm, 4 m). It was a temperature controlled column
(281 C). Mobile phase [methanol-water (60:40, v/v)] was

90.00
80.00
70.00

% Inhibition

60.00
50.00
40.00
30.00
20.00
10.00
0.00
Ascorbic
Acid

F1

F2

F3

F4

F5

Fractions

F6

F7

F8

F9

F10

J. Plant Biochem. Biotechnol. (OctoberDecember 2013) 22(4):483487

0.250

0.200

O.D. at 725 nm

Fig. 2 Elutes, following silica


gel column chromatography, of
leaf gel extract of Aloe vera:
samples were separated into ten
major fractions based on
absorbance of phenolics
following color development at
725 nm. Plots and bars show
mean sd (n03)

485

0.150

0.100

0.050

0.000
F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

Fractions

acidified with 0.1 % tri-fluoroacetic acid for better resolution


of peaks. Injection volume was 20 l at a time. Flow rate was
maintained at 1 ml/min. Detection was carried out at 220 and
254 nm but detection at 254 nm gives better resolution of
peaks. Aloin was used as standard marker anthraquinone for
qualitative and quantitative estimation.
Total phenol content assay and DPPH assay were
performed in three replicates, and values were expressed
as mean SD for triplicate determinations. Analysis of
variance and Fishers least significant difference (LSD)
tests at 95 % confidence interval were conducted to identify

significant differences among the means by ANOVA at


95 % confidence interval using MSTAT-C software (version
1.41, Michigan State University).
Pooled fractions are yellowish brown colored and highly
hydroscopic tacky material. Antioxidant potential of the ten
pooled fractions was estimated by DPPH free radical scavenging assay. Antioxidant assay values were shown in
Fig. 1. Total phenol content was estimated with ten pooled
fractions (Fig. 2). Among the fractions, 3rd pooled fraction
showed maximum phenolic absorption at 725 nm followed
by 4th, 5th, 6th and 2nd fractions. DPPH radical scavenging
assay and total phenolic content were well positively correlated. This observation indicated that the phenolics present
in aloe having the potential to scavenge DPPH free radicals
and phenolics are the major contributor of the antioxidant

25
rd

3 fraction

20

Intensity (a.u)

th

4 fraction

15
10
nd

2 fraction

th

5 fraction

5
st

1 fraction

0
400

450

500

550

Wave length ( )
Fig. 3 UV-Visible absorption profile (200700 nm) of 3rd pooled
fraction showing maximum absorptions in UV region (200400 nm)

Fig. 4 The fluorescence spectral profile of first five pooled fractions


(1st, 2nd, 3rd, 4th and 5th pooled fraction)

486

J. Plant Biochem. Biotechnol. (OctoberDecember 2013) 22(4):483487

25.5
24
22
20
18
16

2082.21

1013.91

14
12
%T
10

685.47

8
6
4
1637.72

2
0
3433.10

-2
-2.9
4000.0

3600

3200

2800

2400

2000

1800

cm-1

1600

1400

1200

1000

800

600

450.0

Fig. 5 FT-IR profile of the vacuum dried 3rd pooled chromatographic fraction

activity in aloe gel. This kind of positive correlation between


the phenolic contents and the antioxidant potential was also
observed by Zin et al. 2006 in other plant materials.
UV spectrum of the third pooled fraction revealed absorption peaks at 255 nm (UVC region), 270 nm (UVC region),
290 nm (UVB region) and 350 nm (UVA region) as shown in
Fig. 3. Absorption of UV radiation of the pooled fractions
affirms the presence of conjugated double bonds. Moreover, it
can be said that the absorption band between 320 and 380 nm
indicates the presence of phenolic compounds (Ozsoy et al.
2008). Prolong exposure to UVB (315280 nm) radiation can
cause various deleterious impacts on human skin and sometimes may cause skin cancer. In humans, over exposure to
solar UV radiation may produce deleterious impacts on the
skin, eye and immune system. UV radiation also increases the
chance of malignant melanoma by DNA damaging activity.
UV-mediated mutation was detected in 92 % of all melanoma
(Davies et al. 2002). This third pooled fraction contains the
phenolics having antioxidant and UVB protecting compounds
with higher concentration. So, this fraction may be used as an
Fig. 6 HPLC profile of
standard aloin (i) and third
pooled fraction (ii)

ingredient in the sun screen products to reduce sun light


induced skin damage, as an UVB protecting reagent and helps
in skin rejuvenation. UV protecting activity of aloe gel was
evaluated by Kumar et al. 2009. Present observation suggests
that presence of UV absorbing phenolic compounds attributes
the antioxidant potential and UV absorbing activity in
methanolic extract from aloe gel. Third pooled fractions shows
highest fluorescence emission and it gives characteristics
emission maxima at 448 nm (when excited at 365 nm)
(Fig. 4). After the forth pooled fraction, fluorescence behavior
was not detected among the samples. This suggests that
3rd pooled fraction contain highest concentration of fluorophore compounds presumably, flavonoids, flavonols and
anthraquinone moiety.
FTIR was conducted to identify predominant functional
groups present in the potent fraction. The assignments were
compared with the pre-existing reports for validation purpose.
FTIR profile (Fig. 5) indicates the presence of different polar
groups such as OH (corresponds to 3,433.10 cm1), 0CO
(corresponds to 1,637.72 cm1), -COC (corresponds to

i
AU

0.05
0.00
2.00

4.00

6.00

8.00

10.00

12.00

0.010

AU

ii
0.005
0.000
2.00

4.00

6.00
Minutes

8.00

10.00

J. Plant Biochem. Biotechnol. (OctoberDecember 2013) 22(4):483487

1,013.91 cm1) etc. and these functional groups are responsible


for the polar nature of the phenolic compounds in methanolic
extracts of aloe gel. The identification of the functional groups
implies the presence of specific compounds and functional
group detection also helps in physico-chemical characterization of any material of interest. Strong and broad intensity of
bands around 3,433 cm1 was assigned to phenolic OH
stretching frequency (Chun-hui et al. 2007). Phenolic OH
group stretching imply the presence of aloe specific phenolics
compounds e.g. flavonoids, anthraquinoes etc. Very broad with
high intensity band at phenolic region (at 3,433.10 cm1)
indicates the accumulation of phenolic compounds in high
degree. Since, total phenol contents were found high in third
fraction, the intensity of FTIR bands were also detected with
high intensity. Medium intensity and broad shaped bands were
observed due to 0CO stretching at 1,637.72 cm1 indicating
the presence of carbonyl compounds. Weak absorption peaks
in between 700 and 610 cm1 was might be due to C-H
bending indicating the presence of polymerized compounds
in the fraction.
RP-HPLC was conducted for biochemical investigation
of the fractions (Fig. 6). Since aloin is one important member of anthraquinones present in Aloe vera gel and the antioxidative activities of aloin were also supported by Tian and
Hua 2005, aloin was chosen as anthraquinone standard for
HPLC analysis. The retention time of aloin in present experiment was around 6.6 min. The retention time of aloin
was decreased than the previous report of Elamthuruthy et
al. 2005, due to shorter length of the column. The aloin
concentration present in potent pooled fraction was calculated from the standard graph. The highest concentration of
aloin was detected in 3rd fraction and the detected concentration was 672.1120.02 g/mg. Present finding suggests
that aloin take part in antioxidant activity in aloe gel and till
date there is little information available regarding aloin as a
contributor of antioxidative potential in Aloe vera L. gel.
Phenolic contents, UV radiation absorbing potential and
antioxidant potential of aloe gel are interlinked phenomena.
Qualitative and quantitative nature of the indigenous

487

phenolic compounds may attribute the UV absorbing and


antioxidant potential in Aloe gel. Present study and subsequent identification of the predominant compounds give a
rationale of UV absorption and antioxidant potential of
methanolic extracts of Aloe vera L. gel.
Acknowledgments This research was partly supported by Indian
Council of Agricultural Research National Innovation Agricultural
Project, India. Special thanks are given to Dr. A.P. Mitra, Department
of Agril & Food Engg., IIT-Kharagpur, India, for his kind help to carry
out HPLC. The authors would also like to thank Mr. Shahaank M.
Aswatha, Dept. of Computer Science & Engg., IIT-Kharagpur, India,
for the needful help provided in preparing the manuscript.

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