Antioxidant constituents of almond [Prunus dulcis (Mill.) D.A. Webb] hulls

Article abstract of DOI:10.1021/jf020660i

Almond hulls (Nonpareil variety) were extracted with methanol and analyzed by reversed phase HPLC with diode array detection. The extract contained 5-O-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid (cryptochlorogenic acid), and 3-O-caffe

Full text of DOI:10.1021/jf020660i

496 J. Agric. Food Chem. 2003, 51, 496−501
Antioxidant Constituents of Almond
[Prunus dulcis (Mill.) D.A. Webb] Hulls
GARY R. TAKEOKA* AND LAN T. DAO
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture,
800 Buchanan Street, Albany, California 94710
Almond hulls (Nonpareil variety) were extracted with methanol and analyzed by reversed phase HPLC
with diode array detection. The extract contained 5-O-caffeoylquinic acid (chlorogenic acid), 4-O-
caffeoylquinic acid (cryptochlorogenic acid), and 3-O-caffeoylquinic acid (neochlorogenic acid) in the
ratio 79.5:14.8:5.7. The chlorogenic acid concentration of almond hulls was 42.52 ( 4.50 mg/100 g
of fresh weight (n ) 4; moisture content ) 11.39%). Extracts were tested for their ability to inhibit the
oxidation of methyl linoleate at 40 °C. At an equivalent concentration (10 µg/1 g of methyl linoleate)
almond hull extracts had higher antioxidant activity than R-tocopherol. At higher concentrations (50
µg/1 g of methyl linoleate) almond hull extracts showed increased antioxidant activity that was similar
to chlorogenic acid and morin [2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one]
standards (at the same concentrations). These data indicate that almond hulls are a potential source
of these dietary antioxidants. The sterols (3â,22E)-stigmasta-5,22-dien-3-ol (stigmasterol) and (3â)-
stigmast-5-en-3-ol (â-sitosterol) (18.9 mg and 16.0 mg/100 g of almond hull, respectively) were
identified by GC-MS of the silylated almond hull extract.
KEYWORDS: Almond hulls; antioxidant; HPLC
INTRODUCTION
acid derivatives, coumarins, tocopherols, and polyfunctional
organic acids (17).
Autoxidation of polyunsaturated fatty acids not only lowers
the nutritional value of food (1) but is also associated with
membrane damage, aging, heart disease, and cancer in living
organisms (2). The addition of antioxidants has become popular
as a means of increasing the shelf life of food products and to
reduce wastage and nutritional losses by inhibiting and delaying
oxidation (3). Synthetic antioxidants such as 2+3-tert-butyl-4-
methoxyphenol (BHA) and 2,6-di-tert-butyl-4-methylphenol
(BHT) are widely used in the food industry. However, there
are serious concerns about carcinogenic potential of these
substances (4-7). Therefore, there is great interest in re-
placing them with natural antioxidants. Natural antioxidants are
primarily plant polyphenolic compounds that may be obtained
from plant parts such as oilseeds, grains, beans, vegetables,
fruits, leaf waxes, bark, and roots. Plant phenolics are multi-
functional and can act as reducing agents (free radical ter-
minators), metal chelators, and singlet oxygen quenchers.
Epidemiological studies have shown that consumption of
foods and beverages rich in phenolic content is correlated with
reduced incidences of heart disease (8). Other studies have
shown that phenolic compounds contained in fruits are potent
oxidation inhibitors of low-density lipoprotein (LDL) both in
vitro (9-14) and in vivo (15, 16). The most common plant
phenolic antioxidants include flavonoid compounds, cinnamic
The antioxidant activity of plant hulls from buckwheat (18),
navy bean (19), mung bean (20), peanut (21), rice (22, 23), and
sunflower (24) have been studied. The almond [Prunus dulcis
(Mill.) D.A. Webb] is one of the most versatile nuts in the world,
and California is the only place in North America where
almonds are grown commercially. In the past, almond hulls, a
byproduct of the almond industry, were removed from the
almonds after harvesting and used as supplemental livestock
feed. Almond hulls have been shown to be a rich source of
three triterpenoids (about 1% of the hulls), betulinic acid,
oleanolic acid, and ursolic acid, which have reported antiin-
flammatory, anti-HIV, and anticancer activities (25). Sang and
co-workers (26) identified 3-prenyl-4-O-â-glucopyranosyloxy-
4-hydroxylbenzoic acid, catechin, protocatechuic acid, and
ursolic acid in almond hulls. These constituents have beneficial
biological activities. Recently, there is interest in using almond
hulls as a natural source for sweetener concentrate and dietary
fiber. The aim of this study was to identify the phenolic
consituents in almond hulls and evaluate their potential anti-
oxidant activity.
EXPERIMENTAL PROCEDURE
Materials. The almond hulls (variety: Nonpareil) were supplied by
the Northern Merced Hulling Association (Ballico, CA). The dried hulls
were separated from the shells and ground in a Wiley mill to pass a
6.4 mm screen.
* To whom correspondence should be addressed. E-mail: grt@
pw.usda.gov.
10.1021/jf020660i This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society
Published on Web 12/07/2002

Antioxidant Constituents of Almond Hulls
J. Agric. Food Chem., Vol. 51, No. 2, 2003 497
Figure 3. HPLC chromatogram of Nonpareil almond hull extract
(methanol). Peaks: (1) unknown; (2) 3-O-caffeoylquinic acid; (3) unknown;
(4) 5-O-caffeoylquinic acid; (5) 4-O-caffeoylquinic acid.
Figure 1. Antioxidant activity of Nonpareil almond hull extract (methanol),
vitamin E, and morin.
Figure 2. HPLC chromatogram of chlorogenic acid and its isomers.
Peaks: (1) 3-O-caffeoylquinic acid; (2) 5-O-caffeoylquinic acid; (3) 4-O-
caffeoylquinic acid; (4) caffeic acid.
Figure 4. Antioxidant activity of almond hull methanol extract, chlorogenic
acid, and morin.
Chemicals. Methyl linoleate (MeLo) was purchased from Nu-Chek-
Prep, Inc. (Elysian, MN). Morin, stigmasterol, 5R-cholestane, and
â-sitosterol were supplied by Sigma Co. (St. Louis, MO). Chlorogenic
acid and R-tocopherol (vitamin E, 97%) were obtained from Aldrich
Chemical Co., Inc. (Milwaukee, WI). Solvents were of HPLC spec-
troquality grade unless otherwise stated.
in water and passed through a C18 solid-phase extraction cartridge
(Accubond, J&W Scientific, Inc., Folsom, CA), prewetted with
methanol and water. The cartridge was washed three times with 2 mL
aliquots of water and then eluted with 2 mL of methanol and stored in
the dark at 4 °C until used. The methanol eluent (0.5 mL) was diluted
with an equal volume of 0.07 M KH2PO4 solution (adjusted to pH )
2.5 with H3PO4) before HPLC injection. The remaining methanol eluent
was saved for other studies. The solvent system used was a linear
gradient of 2% to 40% methanol in 0.07 M KH2PO4 (pH ) 2.5) over
40 min with a flow rate of 1 mL/min. The sample loop volume was 20
µL, and the diode array detector was monitored at 325 nm.
Extraction. The ground almond hulls were sequentially extracted
with freshly distilled diethyl ether containing ca. 0.001% Ethyl
antioxidant 330 [(1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxy-
benzyl)benzene] followed by methanol in a Soxhlet extractor under
subdued light. The methanol extract was evaporated to dryness using
a rotary evaporator and stored in the dark at 4 °C until used.
Thin-Layer chromatography (TLC). Preparative thin-layer chro-
matography was performed on 0.25 mm thick, 20 × 20 cm silica gel
precoated plates (Alltech Associates, Deerfield, IL). A 50 µL volume
of methanol extract was applied as a single band along with chlorogenic
acid standard. The plate was developed with ethyl acetate-formic acid-
acetic acid-water (100:11:11:27, v/v) until the solvent front reached
13 cm. The plate was then air-dried and sprayed with natural product
reagent (1% methanolic diphenylboric acid ethylamino ester) followed
by 5% ethanolic poly(ethylene glycol) 4000 to visualize the spots under
UV light (27).
Derivatization. 5R-Cholestane (C27H48) was employed as the internal
standard. An aliquot (50 µL of a 1 mg/mL solution of 5R-cholestane
in pentane) was added to a 1.0 mL Wheaton V-vial equipped with an
open top black phenolic screw cap with Teflon-faced silicone liner
(Wheaton Science Products, Millville, NJ). The pentane was removed
under a stream of nitrogen. About 20 mg of almond hull extract was
added to the vial along with 200 µL of Sylon BFT (BSTFA + TMCS,
99:1; Supelco, Bellefonte, PA). Derivatized standards of chlorogenic
acid, stigmasterol, and â-sitosterol were prepared by adding a few
milligrams of each to V-vials along with 100 µL of Sylon BFT. The
samples were sonicated for 10 min in an ultrasonic bath and then heated
at 65 °C for 1 h.
High-Performance Liquid Chromatography (HPLC). The HPLC
system was used as previously described (28). The extract was dissolved

498 J. Agric. Food Chem., Vol. 51, No. 2, 2003
Takeoka and Dao
Figure 5. Mass spectra of the TMS derivatives of unknown from silylated almond hull extract (top) and chlorogenic acid standard (bottom).
Gas Chromatography (GC). Silylated almond hull extracts were
analyzed with a Hewlett-Packard 5890 Series II gas chromatograph
equipped with a flame ionization detector (FID). A 30 × 0.25 mm i.d.
DB-1 HT (df ) 0.1 µm) fused silica capillary column (J&W Scientific,
Folsom, CA) was employed. The oven temperature was held at 150
°C for 4 min and then programmed at 2 °C/min to 280 °C (final hold
20 min). The helium carrier gas linear velocity was 31.6 cm/s (150
°C). The injector and detector temperatures were 250 and 300 °C,
respectively. The split ratio of the injector was 1:20.
Gas Chromatography-Mass Spectrometry (GC-MS). A Hewlett-
Packard 5973 mass selective detector (MSD) was directly coupled to
a Hewlett-Packard 6890 gas chromatograph. The column and oven
temperature programming conditions were the same as described in
the previous section.
Determination of Chlorogenic Acid Content. Chlorogenic acid
content in almond hulls was determined by HPLC. Methanol extract
(0.5 g) was dissolved in 5 mL of water with stirring. The sample was
prepared in a similar manner as described in the HPLC section above.
The diluted extract (1 mL) was passed through a C18 SPE cartridge
prewetted with methanol and water. The cartridge was washed three
times with 2 mL aliquots of water, dried under a stream of nitrogen,
and eluted with 1 mL of methanol. The methanol eluent was treated
and analyzed as described in the HPLC section. Chlorogenic acid
content was quantified on the basis of an external standard curve of
chlorogenic acid standard.
Measurement of Antioxidant Activity. The antioxidant activity
experiments were performed according to a published procedure (29)
with some modification. Briefly, the methanolic extracts were added
to 1 g of methyl linoleate (MeLo), and methanol was evaporated under
a stream of nitrogen. Oxidation of MeLo was carried out in the dark at
40 °C. Sample aliquots (10 mg) were taken at regular intervals (24 h)
and dissolved in 5 mL of 2,2,4-trimethylpentane (isooctane) for
spectrophotometric measurements (Hewlett-Packard 8453 UV-vis
spectrophotometer) of conjugated diene absorption at 234 nm. Isooctane
was used as the blank. The antioxidant activity was determined by the
UV absorption maximum at 234 nm (conjugated dienes). All analyses
were carried out in duplicate.
Chlorogenic Acid Isomers. The chlorogenic acid isomers (1-, 3-,
and 4-O-caffeoylquinic acids) were synthesized from chlorogenic acid
(5-O-caffeoylquinic acid) according to the procedure reported by Nagels
et al. (30). Chlorogenic acid was dissolved in a saturated solution of
NaHCO3, and the temperature was raised to 90 °C. Heating was
stopped after 30 min, and the pH was adjusted to 3 with H2SO4. The
obtained mixture of isomeric caffeoylquinic acids was extracted with
ethyl acetate. The ethyl acetate phase was concentrated to dryness, and
the residue was dissolved in a mixture of methanol and 0.07 M KH2-
PO4 (50:50 v/v) with the solution filtered through a 0.45 mm disposable
filter membrane before HPLC analysis. The 3- and 4-isomers were
obtained in this way. The 1-isomer was present in only negligible
amounts (30).

Antioxidant Constituents of Almond Hulls
J. Agric. Food Chem., Vol. 51, No. 2, 2003 499
Figure 6. Mass spectra of the TMS derivatives of unknown from silylated almond hull extract (top) and stigmasterol standard (bottom).
RESULTS AND DISCUSSION
chlorogenic acid. High-performance liquid chromatography
(HPLC) of chlorogenic acid isomers and caffeic acid is
shown in Figure 2. Caffeic acid and the three chlorogenic acid
isomers gave four well-separated peaks. The 3-O-caffeoylquinic
acid eluted first at 22.7 min (isomer with the caffeoyl moiety
axial (30), followed by 5-O-caffeoylquinic acid (chlorogenic
acid) at 30 min (peak 2) and then 4-O-caffeoylquinic acid
(isomer with the caffeoyl moiety equatorial) with a retention
time of 31.2 min (peak 3), and the last eluted peak at 33.1
min (peak 4) is caffeic acid (hydrolysis product of chlorogenic
acid). The HPLC chromatogram of methanolic almond hull
extract showed five major compounds (Figure 3). Cochro-
matography of almond hull extract and chlorogenic acid isomers
also showed 5 separated peaks in which the UV absorbance
intensities of peaks 2, 4, and 5 were increased. Their on-line
UV-vis spectra and elution times were the same as those of
chlorogenic acid and its isomers. The presence of chlorogenic
acid was additionally confirmed by GC-MS of silylated almond
hull extract. Almond hulls contained 5-O-caffeoylquinic acid
(chlorogenic acid), 4-O-caffeoylquinic acid (cryptochlorogenic
acid), and 3-O-caffeoylquinic acid (neochlorogenic acid) in the
ratio 79.5:14.8:5.7. The total percentage of chlorogenic acid and
The methanol extract comprised 45.03 ( 0.80% (n ) 3) of
the fresh weight of the Nonpareil almond hulls (moisture content
) 11.39%). Antioxidant activity of the methanol extract is
shown in Figure 1. The methanol extract slowed the formation
of conjugated dienes (234 nm) during the oxidation of methyl
linoleate. Oxidation was strongly inhibited for the first 3 days
with 10 µg of almond hull extract compared with 10 µg of
Vitamin E. However, after 4 days, the antioxidant activity of
10 µg of almond hull extract was similar to that of vitamin E.
Antioxidant activity increased as the level increased to 30 µg
(Figure 1). This activity was even higher than 10 µg of morin.
Morin was reported as a flavonoid having greater antioxidant
activity than catechin, quercetin, rutin, luteolin, and kaempferol
(31). Additional study showed that vitamin E was a prooxidant
when its concentration was higher than 10 µg/1 g of MeLo (data
not shown).
Thin-layer chromatography of almond hull extract (methanol)
revealed a strong yellow fluorescent band under UV light with
an Rf ) 0.57 (same Rf value as chlorogenic acid standard) along
with several weak fluorescent bands. This result suggested that
the major phenolic compound present in almond hulls was

500 J. Agric. Food Chem., Vol. 51, No. 2, 2003
Takeoka and Dao
Figure 7. Mass spectra of the TMS derivatives of unknown from silylated almond hull extract (top) and â-sitosterol standard (bottom).
its isomers detected (at 325 nm) in almond hulls was 88%
compared to 80% in sweet potatoes (32). Peaks 1 and 3 which
comprised the remaining 12% have not yet been identified.
These percentage values should be considered as only ap-
proximate since the extinction coefficients of peaks 1 and 3
are unknown and may be very different from those of the
chlorogenic acid isomers. The chlorogenic acid content of
almond hulls was 42.52 ( 4.50 mg/100 g of fresh weight (n )
4). The concentrations of the other isomers were determined
using the chlorogenic acid standard curve. The approximate
levels of 4-O-caffeoylquinic acid and 3-O-caffeoylquinic acid
were 7.90 mg and 3.04 mg/100 g of fresh weight almond hulls,
respectively.
Since chlorogenic acid and its isomers are the major phenolic
compounds in almond hulls, we decided to compare the
antioxidant activity of almond hull extract with chlorogenic
acid standard. Chlorogenic acid was found to be the most
abundant phenolic acid in various plant extracts and also the
most active antioxidant constituent (13, 17, 32-36). It has been
shown that the antioxidant activities of 3-O-caffeoylquinic acid
and 4-O-caffeoylquinic acid are almost the same as chlorogenic
acid when assayed for scavenging activity on superoxide anion
radicals and inhibitory effect against oxidation of methyl
linoleate (37). The antioxidant activities of almond hull extract,
chlorogenic acid, and morin are shown in Figure 4. At 50 µg/1
g of MeLo, morin, chlorogenic acid, and almond hull extract
all showed similar strong antioxidant activity after 4 days of
MeLo oxidation. Again, this illustrated that the antioxidant
activity of phenolic compounds in almond hulls increased with
increasing concentration (in comparison to the 10 and 30 µg
levels shown in Figure 1).
Almond hull extract was silylated with BSTFA-TMCS (99:
1) to form trimethylsilyl (TMS) ether derivatives. The deriva-
tized extract was analyzed by GC and GC-MS. The majority
of the sample consisted of carbohydrates. Three peaks were
identified as chlorogenic acid (IDB-1 ) 3186), stigmasterol
(IDB-1 ) 3235), and â-sitosterol (IDB-1 ) 3295) by com-
paring retention indices and mass spectra (Figures 5-7) to those
of authentic standards. Semiquantitiative analysis was per-
formed using 5R-cholestane as the internal standard. The
concentrations of chlorogenic acid, stigmasterol, and â-sitosterol
were 27.8, 18.9, and 16.0 mg/100 g of almond hull (fresh
weight), respectively (n ) 2). These values should be con-
sidered approximate since response factors were not determined.
The chlorogenic acid concentration determined by HPLC is
probably more accurate. Stigmasterol and â-sitosterol, common

Antioxidant Constituents of Almond Hulls
J. Agric. Food Chem., Vol. 51, No. 2, 2003 501
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almond hulls. The latter compound was recently found in
almond nuts (39).
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