Food Chemistry 114 (2009) 1069–1072

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Activity guided isolation of antioxidants from the leaves of Ricinus communis L.

Pradeep Pratap Singh, Ambika, S.M.S. Chauhan *
Bio-Organic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India

a r t i c l e i n f o a b s t r a c t

Article history: An activity-guided isolation and purification process was used to identify the DPPH (l,l-diphenyl-2-pic-
Received 17 June 2008 rylhydrazyl) free radical scavenging components of the food plant (Ricinus communis L.) of Eri silkworm.
Received in revised form 15 September Dry leaves of R. communis L. were extracted with different solvents and tested for their antioxidant activ-
2008
ity against DPPH. The MeOH:water (8:2) extract showed strong DPPH radical-scavenging activity, and
Accepted 10 October 2008
was subjected to column chromatography over silica gel. Gallic acid, quercetin, gentisic acid, rutin, epi-
catechin and ellagic acid were isolated as active components and characterised by different spectroscopic
techniques.
Keywords:
Ricinus communis L.
Ó 2008 Elsevier Ltd. All rights reserved.
Antioxidant activity
DPPH
Eri silkworm

1. Introduction be used to understand the role of plant antioxidants in plant–

herbivore interactions and in human food (Arabshahi-Delouee &
Ricinus communis L. (Euphorbiaceae) is an annual, perennial Urooj, 2007; Srivastava, Kapoor, Thathola, & Srivastava, 2003). In
bush found nearly throughout India, which is widely cultivated the present communication, an activity-guided isolation and puri-
in the tropics and warm regions for castor oil (Scarpa & Guerci, fication process was used to isolate the DPPH radical-scavenging
1982). It possesses various biological activities such as hepatopro- secondary metabolites of R. communis L.
tective (Shukla, Visen, Patnaik, Kapoor, & Dhawan, 1992; Visen
et al., 1992), insecticidal (Upasani, Kotkar, Mendki, & Maheshwari,
2. Materials and methods
2003), contraceptive (Okwuasaba et al., 1997) and antifertility
activity (Sandhyakumary, Bobby, & Indira, 2003). Recently anti-
2.1. General procedures
inflammatory and free radical scavenging activities of the metha-
nolic extract of R. communis L roots in Wistar albino rats have been
All melting points were determined on a Thomas-Hoover
reported (Ilavarasan, Mallika, & Venkataraman, 2006).
Unimelt melting point apparatus and uncorrected. IR spectra were
The leaves of R. communis L. are the primary food for Eri silk-
recorded on a Shimadzu IR 435 spectrometer (Shimadzu, Kyoto,
worm (Philosamia ricini). Previously, we have reported the isolation
Japan) and the vmax is expressed in cm1; the electronic spectra
of secondary metabolites from the different primary food plants of
were obtained by Perkin–Elmer Lambda-35 UV–Vis spectropho-
polyphagus tasar silkworms such as Terminalia arjuna (Chauhan,
tometer (Perkin–Elmer, Waltham, MA), and the kmax is expressed
Mishra, Parkash, & Kaushik, 1998; Chauhan, Parkash, & Kaushik,
in nanometers. The 1H NMR spectra were recorded on a Bruker
1997), Shorea robusta (Chauhan, Singh, & Narayan, 2002) and Quer-
Avance 300 spectrometer (Bruker Biospin, Rheinstetten, Germany)
cus semicarpifolia (Chauhan, Singh, & Kumar, 2004). Herbivore in-
using TMS as internal standard (chemical shift in ppm). The sym-
sects sequester plant secondary metabolites for defence against
bols s, d, t, q and m stand for singlet, doublet, triplet, quartet and
predators or pathogens (Pasteels, Theuring, Witte, & Hartmann,
multiplet, respectively. The electrospray ionisation mass spectra
2003; Muller et al., 2001; Wheeler, Massey, & Southwell, 2002).
(ESI-MS) were recorded on a Waters Micromass LCT LC/MS (Waters
The sequestration of antioxidants from host plant is important
Corporation, Milford, MA).
for larval health, survival, cocoon formation and finally emergence
of the moth of silkworm (Bindu, Jaisankar, Hauer, Gutzeit, &
Kundu, 2006). Thus, activity-guided isolation of antioxidants can 2.2. Plant material

The leaves of R. communis L. were collected from a site near Uni-

* Corresponding author. Tel./fax: +91 011 27666845. versity of Delhi and were identified by the Department of Botany,
E-mail address: smschauhan@chemistry.du.ac.in (S.M.S. Chauhan). University of Delhi, Delhi, India. The leaves were naturally dried.

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.10.020
1070 P.P. Singh et al. / Food Chemistry 114 (2009) 1069–1072

2.3. Chemicals Table 1

Antioxidant activity of different solvent extracts and compounds isolated from the
dry leaves of R. communis L.
DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased from Sig-
ma–Aldrich (St. Louis, MO). Extract IC50 (lg/ml)a tIC50 (min1)a AE (103)
CH2Cl2 >40 ND ND
2.4. Isolation and extraction procedure CHCl3 >40 ND ND
MeOH 4.66 ± 0.19 20.1 ± 1.24 10.7
Acetone 6.04 ± 0.27 27.6 ± 1.16 5.99
The dry leaves (500 mg each) of R. communis L. were extracted MeOH:water (8:2) 2.70 ± 0.03 12.3 ± 0.11 30.2
with different solvents, viz, CH2Cl2, CHCl3, MeOH, acetone, MeOH: Water 5.85 ± 0.12 26.5 ± 0.82 6.44
water (8:2) and water. These extracts were tested for their antiox- Gallic acid (1) 3.15 ± 0.02 16.2 ± 0.98 19.5
idant activity against DPPH. The MeOH:water (8:2) extract of the Quercetin (2) 4.62 ± 0.13 22.5 ± 0.72 9.60
Gentisic acid (3) 2.92 ± 0.07 14.1 ± 0.15 24.4
dry leaves (1 kg) was concentrated under reduced pressure and the Rutin (4) 9.46 ± 0.42 42.8 ± 2.67 2.50
residue was column chromatographed over silica gel (60–120 Epicatechin (5) 5.82 ± 0.15 26.5 ± 1.82 6.47
mesh). The column was eluted with solvents of increasing polari- Ellagic acid (6) 2.52 ± 0.09 13.8 ± 0.12 28.7
ties, starting with petroleum ether, CHCl3, and variable concentra- Ascorbic acidb 11.1 ± 0.42 50.3 ± 2.47 1.78
tions of CHCl3 and MeOH. Forty fractions of 200 ml each were a
Values are the mean ± SD of triplicate determinations.
collected, verified by TLC and tested for antioxidant activity against b
Positive control IC50 = concentration of antioxidant needed to reduce the ori-
DPPH. Fractions 9–18, 23–26 and 34–40 were inactive or of low ginal amount of radical by 50%; tIC50 = time needed for the IC50 to reach 50% of the
original amount of radical scavenged; AE = antiradical efficiency (AE = 1/(IC50 tIC50)).
activity and were discarded. The different active fractions were
again separated by column chromatography to obtain pure
compounds.
Further elution of the column with CHCl3 yielded 12 mg of
2.5. Determination of DPPH radical scavenging capacity compound 2, which gave a positive FeCl3, as well as a positive van-
illin–HCl test for flavanoids. The presence of peaks at 3420 and
Antiradical activity of R. communis L. was determined using the 1725 cm1 in the IR spectrum, indicated the presence of hydroxyl
free radical DPPH, by minor modification of the reported method and carbonyl groups, respectively. The 1H NMR spectrum of com-
(Molyneux, 2004; Porto, Calligaris, Celotti, & Nicoli, 2000). Test pound 2 showed singlets at d 12.31, 10.36, 9.15, 8.93, 8.70 ppm
samples (10 ll) were added to 3 ml of a 6.1  105 M solution of for five hydroxyl groups. A double doublet at d 7.60 (J = 8.60 and
DPPH. Absorbance at 515 nm was monitored at 0 min, 1 min 1.80 Hz) ppm for H-60 , a doublet at 7.74 (J = 1.80 Hz) ppm for
and after every 1 min interval until a steady state was achieved. H-2, and three doublets at 6.91 (J = 8.34 Hz), 6.37 (J = 2.40 Hz)
Ascorbic acid was used as the positive control. IC50 was obtained and 6.19 (J = 1.80 Hz) ppm were due to H-5’, H-8’ and H-6’ protons,
by extrapolation from linear regression analysis. The percentage respectively. The M + 1 peak at 303.2120 in the ESI-MS spectrum
inhibition was calculated by the following equation: and comparison of mixed melting point with authentic sample of
quercetin confirmed that compound 2 is quercetin (2).
ðAcontrol 515nm  Asample 515nm Þ Fractions 19–22, rechromatographed over silica gel, on elution
%Inhibition ¼  100 with CHCl3:MeOH (94:6) gave 8 mg of compound 3. It gave posi-
Acontrol 515nm
tive FeCl3 and NaHCO3 tests for phenolic and carboxylic groups.
The presence of peaks at 3600 and 1680 cm1 in the IR spectrum
3. Results and discussion indicated the presence of hydroxyl and carbonyl groups. The 1H
NMR spectrum showed a singlet for one proton at d 15.10 ppm
3.1. Isolation and characterisation of antioxidants for a carboxylic group. The doublets at d 7.10 (J = 2.40 Hz) and
6.41 (J = 6.60 Hz) and the double doublet at 6.56 ppm were as-
Activity-guided isolation of the dry leaves of R. communis L. signed to H-3, H-4 and H-6 protons. The M + 1 peak was observed
indicated that MeOH:water (8:2) was the extract most active at 155.0142 in the ESI-MS spectrum and the above data indicated
against DPPH, with an IC50 of 2.70 lg/ml. Hence MeOH:water the compound as gentisic acid (3).
(8:2) was selected for the extraction and isolation of antioxidants. Further elution of the column with CHCl3:MeOH (90:10) affor-
The residue from the MeOH:water (8:2) extract was subjected to ded 40 mg of compound 4, which gave positive tests with vanil-
activity-guided fractionation on a silica gel column and eluted with lin–HCl and FeCl3 for flavonoids and phenolics, respectively. The
solvents of increasing polarities. Free radical-scavenging ability presence of peaks at 3406 and 1650 cm1 in the IR spectrum indi-
and IC50 values of the different solvent extracts and isolated com- cated the presence of hydroxyl and carbonyl groups, respectively.
pounds are shown in Table 1. The 1H NMR spectrum showed four singlets for one proton each
Active fractions 1–8 were again column chromatographed over at d 12.63, 10.82, 9.69 and 9.28 ppm, indicating the presence of
silica gel (60–120 mesh). On elution with CHCl3:petroleum ether phenolic hydroxyls. The six doublets at d 7.85 (J = 1.80 Hz), 7.49
(90:10) 11 mg of compound 1 was isolated as a white amorphous (J = 1.90 Hz), 6.41 (J = 2.20 Hz), 6.24 (J = 2.0 Hz), 5.25 (J = 7.0 Hz),
solid, which gave a positive test with FeCl3 and effervescence with and 1.12 (J = 7.0 Hz) were assigned to H-2’, H-5’, H-8, H-6, anomer-
NaHCO3 solution, indicating the presence of phenolic and carbox- ic-sugar proton and rhamnose methyl, respectively. A double dou-
ylic groups, respectively. The presence of peaks at 3225 and blet at d 7.52 ppm (J = 5.72 and 14.70 Hz) was assigned to the H-6’
1695 cm1 in the IR spectrum indicated the presence of hydroxyl proton and the multiplet between d 3.50–4.23 ppm was assigned
and carbonyl groups. The observation of a broad singlet at d 11.90 to the sugar protons. The M + 1 peak was observed at 611.5210
for one proton of carboxylic acid, a broad singlet at d 9.05 for three in the ESI-MS spectrum. The physical and spectroscopic data indi-
phenolic protons and a singlet at d 6.95 ppm for two aromatic pro- cated the compound 4 as rutin (4). The structure of compound 4
tons in the 1H NMR spectrum indicated that the compound may be was further confirmed by its hydrolysis. The products of hydrolysis
gallic acid (1). The observation of a molecular ion peak at 170.5100 were identified by different spectroscopic techniques and compar-
(M + 1) in the ESI-MS spectrum and comparison of mixed melting ison with the authentic sample. The aglycone thus obtained was
point and superimposable IR spectrum with authentic sample of characterized as quercetin (2). On the basis of the above data, the
gallic acid confirmed that compound 1 is gallic acid (1). compound was identified as rutin (4).
 P.P. Singh et al. / Food Chemistry 114 (2009) 1069–1072 1071

Fractions 27–33, were rechromatographed over silica gel and on
elution with CHCl3:MeOH (85:15) compound 5 was obtained. It
gave positive tests with vanillin–HCl and FeCl3. The presence
of peaks at 3538 cm1 in the IR spectrum indicated the presence
of a hydroxyl group. In the 1H NMR spectrum four doublets at d
6.85 (J = 1.58 Hz), 5.94 (J = 2.28 Hz), 5.87 (J = 2.19 Hz) and 4.57
ppm (J = 7.54 Hz) were assigned to the H-2’, H-8, H-6 and H-2 pro-
tons. The two double doublets at 2.90 (J = 5.42 and 14.70 Hz) and
2.55 (J = 8.10 and 15.20 Hz) were assigned to the CH2 protons.
The multiplets at d 6.73 for two protons and 4.02 ppm for one pro-
ton were assigned to the H-5’, H-6’ and H-3 protons. ESI-MS of the
compound showed a M + 1 peak at 291.7301. The above data indi-
cated that compound 5 was epicatechin (5).
Elution of the column with CHCl3:MeOH (80:20) yielded 20 mg
of compound 6. The IR spectrum of the compound indicated car-
bonyl absorptions at 1730 cm1 and hydroxyl absorption at
3570 cm1. The 1H NMR spectrum showed two aromatic protons
at d 7.51 and four hydroxyl signals at d 8.05 and 10.57 ppm, which
disappeared on deuterium exchange. ESI-MS of the purified com-
pound showed the M + 1 peak at 303.0774. The above spectral data
indicated that the compound 6 was ellagic acid (6) and its identity
was further confirmed by co-TLC with an authentic sample from
Sigma–Aldrich. The structures of compounds 1–6 are shown in
Fig. 1.
ity similar to the antioxidant activity reported from the roots of R.
communis L. (Ilavarasan et al., 2006). Ascorbic acid was used as po-
sitive control (Table 1).
The antioxidant activity of natural products, such as phenolic
acids and flavanoids, is due to the presence of free hydroxyls
(Cai, Sun, Xing, Luo, & Corke, 2006; Rice-Evans, Miller, & Paganga,
1996). The high antioxidant activity of phenolic acids, such as ella-
gic acid and gallic acid, can be attributed to the presence of the
o-dihydroxybenzene structure. The o-dihydroxybenzene structure
is important for enhanced antioxidant activity (Cai et al., 2006).
Chemical structure activity studies of the flavonols, by various
methods, have consistently shown that flavonols have superior
antioxidant activity to flavan-3-ols; this may be due to the pres-
ence of an o-dihydroxy group and 2,3-double bond in conjugation
with 4-oxo functions in the ring C. which increases the stabilisation
of the generated radical (Rice-Evans et al., 1996). Thus, quercetin
(2, IC50 = 4.62 lg/ml) possesses high antioxidant activity, in com-
parison to epicatechin (5, IC50 = 5.82 lg/ml), which has the same
number of hydroxyls but lacks the 2,3-double bond in conjugation
with 4-oxo function. The activity of rutin (4, IC50 = 9.46 lg/ml) was
found to be less in comparison to its aglycone (2), since glycosyla-
tion reduces the number of free hydroxyl groups and also the link-
age of sugar may hinder the access of free radical scavengers to the
radical centre of DPPH (Cai et al., 2006).

3.2. DPPH radical-scavenging activity

4. Conclusion

DPPH has been used to evaluate the antioxidative activity of
The DPPH-mediated in vitro study reveals that gallic acid, quer-
natural products in organic solvents. Hydrogen-donating ability
cetin, gentisic acid, rutin, epicatechin and ellagic acid are the major
of the antioxidant is responsible for its free radical-scavenging
phenolic compounds responsible for the antioxidant activity of the
activity. The DPPH method is based on the reduction of DPPH
dry leaves of R. communis L. The activity-guided isolation of antiox-
solution to DPPH-H in the presence of a hydrogen-donating antiox-
idants from the food plant of Eri silkworm may be used to under-
idant (Sentandreu, Navarro, & Sendra, 2008; Yamaguchi, Takamura,
stand the role of antioxidants in the host plant–insect herbivory
Matoba, & Terao, 1998). The MeOH:water (8:2) extract of R. com-
interactions.
munis L. leaves and the isolated compounds showed a concentra-
tion-dependent antiradical activity. The MeOH:water (8:2)
extract of the leaves was found to possess radical-scavenging activ- References

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