International Journal of Food Sciences and Nutrition,

September 2012; 63(6): 696702

Process development for the enrichment of curcuminoids in turmeric

spent oleoresin and its inhibitory potential against LDL oxidation
and angiotensin-converting enzyme

SURESH V. NAMPOOTHIRI, E.K. PRASEETHA, V.V. VENUGOPALAN, & A. NIRMALA MENON

Agroprocessing and Natural Products Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR,
Int J Food Sci Nutr Downloaded from informahealthcare.com by Florida State University on 02/22/13

Thiruvananthapuram, Kerala, India

Abstract
Turmeric (Curcuma longa) contains biologically active colouring constituents, curcuminoids, which are isolated from the
turmeric rhizome by solvent extraction. The mother liquor left after the separation of curcuminoids is known as turmeric spent
oleoresin (SOT). The present study developed a method for the enrichment of curcuminoids in SOT. By using this method,
curcuminoids in the SOT (8.4%) were doubled (17.5%). Presence of curcuminoids in enriched fraction was confirmed by high
performance liquid chromatography (HPLC) and liquid chromatography coupled with mass spectroscopy analysis. Further
studies on this fraction showed that it can effectively inhibit angiotensin-converting enzyme and low-density lipoprotein
oxidation with IC50 values of 19.45 mg/ml and 30.52 mg/ml, respectively. The results showed that curcuminoids enriched
For personal use only.

fraction (CEF) can reduce the risk of hypertension and cardiovascular diseases. In addition to this fraction, a turmerone-rich
hexane fraction was also separated from the spent oleoresin.

Keywords: turmeric spent oleoresin, curcuminoids, LDL oxidation, ACE, LC-MS, GC-MS

Introduction
Turmeric (Curcuma longa) is a rhizomatous plant,
which is widely used as a spice in food and
pharmaceutical industry because of its characteristic
colour, flavour and medicinal property. It belongs
to the family Zingiberaceae, native to tropical South
Asia. Turmeric has a characteristic deep orange-yellow
colour mainly due to the presence of curcumin (I),
demethoxycurcumin (II) and bis-demethoxycurcumin
(III) collectively known as curcuminoids. Based on
the variety, climatic and geographic conditions,
curcuminoids in turmeric vary from 2 to 9%.
Chemically, curcumin (diferuloyl methane) is a
polyphenol which has a distinctly earthy, slightly
bitter, hot peppery flavour and a mustardy smell.
Curcumin is well known due to its anti-carcinogenic
activity (Conney et al. 1991). It also shows anti-
inflammatory and immunomodulatory properties.
Moreover, curcumin is a good antioxidant and free
radical scavenger (Kapoor and Priyadarsini 2001).
Angiotensin-converting enzyme (ACE) is an enzyme
that converts angiotensin I to II, which is a potent
chemical that causes the muscles surrounding blood
vessels to contract, thereby narrowing the vessels. This
causes high blood pressure or hypertension. ACE
inhibitors are one of the most important groups of drugs
since they are used for controlling blood pressure,
treating heart failure and preventing strokes in people
with hypertension and diabetes (Lahogue et al. 2009).
Synthetic ACE inhibitors such as captopril and enapril
are currently using in the treatment of hypertension.
However, these inhibitors have side effects including
cough, taste disturbance and skin diseases. Therefore,
natural ACE inhibitors from medicinal plants receive
a great deal of attention in recent years.
In addition to this, individuals with hypertension have
a higher risk for atherosclerotic cardiovascular diseases
than other patients. Oxidation of low-density lipoprotein
(LDL) has been implicated as one of the main reasons

Correspondence: V.V. Venugopalan, Agroprocessing and Natural Products Division, National Institute for Interdisciplinary Science and
Technology (NIIST), CSIR, Thiruvananthapuram, Kerala, India. Tel: 919495111543. Fax: 91 0471 24917895. E-mail:
venugopalvv@yahoo.com

ISSN 0963-7486 print/ISSN 1465-3478 online q 2012 Informa UK, Ltd.

DOI: 10.3109/09637486.2011.652941

for human atherosclerosis (Wiztum and Steinberg
1991). It has been reported that dietary antioxidants
and free radical scavengers are able to prevent LDL
oxidation that can reduce the risk of atherogenesis
(Kinsella et al. 1993; Nakagawa et al. 2002).
Curcumin is isolated commercially from the
turmeric oleoresin (solvent extract of turmeric
rhizomes). The mother liquor left after the separation
of curcumin is known as turmeric spent oleoresin
(SOT), which has no economical importance.
Previous studies showed that still some more amount
of curcumin is present in the SOT, and it has some
biological and antioxidant activities (Jayaprakasa et al.
2004; Nagarajan et al. 2010). However, there were
no reports available regarding the ACE inhibition
and LDL oxidation prevention property of SOT. The
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objective of the present study was to develop an
improved method for the enrichment of curcuminoids
in SOT and its antioxidant and inhibitory potentials
against ACE and LDL oxidation.
Materials and methods
Chemicals and reagents
Gallic acid, quercetin, 2,2-diphenyl-1-pycrylhydrazyl
(DPPH), 2,2-azinobis-(3-ethylbenzothiazoline-6-
For personal use only.
sulphonic acid) (ABTS), Trolox (6-hydroxy-2,5,7,
8-tetramethychroman-2-carboxylic acid), LDL, thio-
barbituric acid (TBA), hippuryl-L -histidyl-L -leucine
(HHL) and ACE were purchased from Sigma-Aldrich
(St. Louis, MO, USA). high performance liquid
chromatography (HPLC) solvents, liquid chromato-
graphy coupled with mass spectroscopy (LC-MS)
solvents and aluminium chloride were purchased
from Merck (Germany). Folin Ciocalteu reagent was
purchased from Sisco Research Laboratories (India).
All other chemicals and solvents used were of standard
analytical grade. The SOT was procured from the
local spice processing industry.
Estimation of curcuminoids by spectrophotometric method
Curcuminoids content in SOT was estimated by
UV vis spectrophotometric method (ASTA 1997).
The curcuminoids have maximum absorbance
between 420 and 425 nm depending upon solvent.
About 0.1 g of SOT is accurately weighed, transferred
into a 100 ml standard flask and made up to the
mark using acetone. From this, 0.1 ml was pipetted
out and made up to 25 ml. The optical density (OD)
was then measured at 425 nm. The percentage of
curcuminoids was calculated using (Equation (1)) the
standard value of absorbance of 0.42 for a solution
containing 0.0025 g of curcumin per litre:
x 0:0025 100 25
% of curcumin 100; 1
0:42 1000 0:1 0:1
where x is the absorbance.
Enrichment of curcuminoids in turmeric spent oleoresin 697
Process development for the separation of curcumin-
enriched fraction from SOT
The SOT was homogenously mixed with cellulose
in the ratio 1:2 and made into a powder form. It
was extracted with hexane at room temperature and
filtered through Whatman No. 1 filter paper. The
residue obtained was separated into five portions and
extracted with hexane and ethyl acetate in the ratios
70:30, 60:40, 50:50, 40:60 and 30:70. After filtration,
each fraction was concentrated under vacuum and the
amount of curcuminoids was determined. Compared
to other fractions, hexane and ethyl acetate in the ratio
40:60 got maximum amount of curcuminoids, which
was named as curcuminoids enriched fraction (CEF).
HPLC instrumentation
The HPLC profiling of SOT and its enriched fraction
was done using Shimadzu (Japan) LC-8A System
equipped with a binary pump, a rheodyne injection
provided with 20 ml loop, a column temperature
controller, diode array detector (SPD-M10A) and a
C18 reversed-phase column (Phenomenex, Torrance,
CA, USA; ODS 2.5 mm, 50 4.6 mm), and the system
was controlled by CLASS-VP software. The standard
curcumin, spent oleoresin and the enriched fraction
were injected into the HPLC; the mobile phase was
optimized as gradient of 1% formic acid in water (A)
and methanol (B). The gradient elution was carried
out at a flow rate of 1 ml/min as follows: 0.01 min
75% A, 10 min 50% A, 20 min 0% A and 25 min 0%
A. The temperature of the column was maintained
at 358C. Chromatograms were monitored at 425 nm.
LC-MS instrumentation
The HPLC method is not satisfactory for complete
characterization of compounds with high sensitivity.
LC-MS has emerged as a more powerful tool for the
determination of many compounds (Xua et al. 2009).
LC-MS (Agilent Ion Trap 6310) was used to confirm
the presence of curcuminoids in the enriched fraction.
A C18 reversed-phase column (Phenomenex ODS
2.5 mm, 50 mm 4.6 mm) was used for the separ-
ation. Five microlitres of extracts (5 mg/ml in
methanol) and standards (1 mg/ml) were loaded and
injected by auto sampler and eluted through the
column with a gradient mobile phase system consist-
ing of 1% formic acid in water (A) and acetonitrile (B)
with the flow rate of 0.75 ml. The elution was
performed using the following gradient profile: 0 min
75% A, 0 10 min 50% A, 10 30 min 0% A and
30 35 min 0% A. UV detector was set at 425 nm.
MS (ESI APCI) was operated in an alternative
mode with a corona current of 1000 nA, nebulizer gas
pressure of 40 psi, dry gas flow of 5.0 l/min, dry gas
temperature of 3008C and a vaporizer temperature
of 200 C.
 698 S.V. Nampoothri et al.
GC-MS profiling of hexane fraction of spent oleoresin
Gas chromatography mass spectrometry (GC-MS)
analysis was carried out on a Shimadzu instrument
(GC-17A) equipped with QP-5050MS. The con-
ditions were as follows: fused silica capillary column
coated with methyl silicone (0.25 mm 50 m, film
thickness 0.25 mm), temperature programme created
between 80 and 2008C, held at 808C for 1 min and at
2008C for 20 min, rinsing rate of 58C/min, source
temperature of 1508C, injection port temperature at
2508C and flame ionization detector (FID) tempera-
ture at 3008C. For the carrier gas flow of 1 ml/min and
split ratio of 1:75 MS, conditions were electron impact
ionizing voltage 70 eV, electron impact multiplier at
2000 eV, scan speed of 690 amu/s and scan range of
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40 500 amu.
Estimation of total phenolic content
Total phenolic content (TPC) in the samples were
determined by using Folin Ciocalteu phenol reagent
(FCR) method (Singleton and Rossi 1965) with gallic
acid as standard. About 100 ml of different extracts
(three replicates), 500 ml FCR and 1 ml of sodium
carbonate (20%) were added and incubated at ambient
temperature (25 278C) for 90 min. The colour
For personal use only.
developed was measured at 760 nm using UV vis
spectrophotometer (UV-2450PC, Shimadzu, Japan).
Results were expressed as milligrams of gallic acid
equivalents (GAE) per gram of extract (mg GAE/g).
The active constituent in the FCR is a mixture of
phosphomolybdate and phosphotungstate, which
oxidizes phenols in the sample to produce blue colour.
The intensity of the colour is directly proportional
to the phenolic content:
observed concentration of sample
%TPC 100:
actual concentration of sample
Estimation of total flavonoid content
Total flavonoid content (TFC) was determined by a
colorimetric method (Jia et al. 1999). An aliquot of
4 ml of appropriate dilution of samples was added into
a volumetric flask containing 0.3 ml of 5% (w/v)
sodium nitrite and kept for 5 min, followed by reaction
with 0.3 ml of 10% (w/v) AlCl3 to form a flavonoid
aluminium complex. About 2 ml of 4.3% (w/v) NaOH
was added and the total volume was made up to 10 ml
with distilled water. The final solution was mixed well
again, and the absorbance was measured against a
blank at 510 nm with UV vis spectrophotometer. The
TFC of SOT and CEF was expressed as milligram of
quercetin equivalents per gram of sample:
observed concentration of sample
%TFC 100:
actual concentration of sample
Evaluation of antioxidant activity
DPPH free radical scavenging activity. The
antioxidant activity of SOT and CEF was measured
in terms of hydrogen donating or radical scavenging
ability using the stable DPPH method (Shimada
et al. 1992). Three millilitres of reaction mixture
contain 2.8 ml methanolic DPPH and 0.2 ml sample
at various concentrations. In control, methanol
was used in place of sample. The contents were
mixed well immediately and incubated for 30 min
at room temperature (25 298C). The degree of
reduction in absorbance was recorded in UVvis
spectrophotometer (UV-2450PC, Shimadzu, Japan)
at 517 nm:
A0 2 A1
% inhibition ;
A0 100
where A0 is the absorbance of the control and A1 is the
absorbance in the presence of sample.
ABTS radical scavenging activity. The assay was carried
out using improved ABTS decolouration method (Re
et al. 1999). The method is suitable for the study of both
water-soluble and lipid-soluble antioxidants, pure
compounds and food extracts. Different concentrations
of the standard (Trolox), ethanol and ABTS (900 ml)
were added in dark tubes to make the whole system to
1 ml. From this, 200 ml was transferred to the microplate
and absorbance was measured at 734 nm.
Biochemical evaluation
Inhibition of human LDL oxidation in vitro. Oxidation of
LDL leads to the production of malondialdehyde that
was measured by the reaction with TBA (Kotamballi
et al. 2002; Nampoothiri et al. 2011). LDL (50 mg/ml)
was incubated at different concentrations of extract,
and the oxidation of LDL was initiated by the addition
of 50 ml copper sulphate (2 mM) at 378C for 2 h. Final
volume of the reaction mixture was made up to 1.5 ml
with phosphate buffer (pH 7.4). After incubation,
500 ml of reaction mixture was mixed with 250 ml of
TBA (1% in 50 mM of NaOH) and TCA (0.28%).
Samples were again incubated at 958C for 45 min.
After cooling and centrifugation at 2000 rpm (10 min),
fluorescence was taken at 515 nm excitation and
553 nm emission. The result was expressed as
percentage of inhibition of LDL oxidation:
Oc 2 Os
% of inhibition 100;
Oc
where Oc is oxidation in the control and Os is oxidation
in the sample.
Inhibition of ACE. ACE inhibition activity was
assayed by the spectrophotometric method
 Enrichment of curcuminoids in turmeric spent oleoresin
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Figure 1. HPLC profile of standard curcumin at 425 nm.
(Hernandez-Ledesma et al. 2003; Pyo and Lee et al.
2007). About 50 ml of sample was treated with 100 ml of
For personal use only.
HHL (12.5 mM dissolved in a pH 8.3 buffer with 0.3 M
NaCl) and incubated for 5 min at 378C. A volume of
150 ml of ACE (20 mU prepared in deionized water)
was added to this and again incubated for 1 h at 378C.
The enzyme reaction was stopped by the addition of
250 ml of HCl (0.5 N). The released hippuric acid
(HA) was extracted with 1 ml ethyl acetate; after mixing
by centrifuge at 3000g for 10 min, 750 ml of organic
layer was taken and evaporated at 908C for 15 min.
The released HA was redissolved in 1 ml distilled
water and the absorbance was measured at 228 nm
using Microplate Reader (BioTek, Germany).
Statistical analysis
The experimental results were expressed as mean ^
standard deviation of triplicate measurements. The
data were subjected to one-way analysis of variance,
and the significance of differences between the means
was calculated by Duncans multiple range test using
SPSS for windows, standard version 7.5.1, SPSS and
the significance accepted at p , 0.05.
Results and discussion
Curcuminoids content in SOT
By UV vis spectrophotometric analysis, curcumi-
noids in SOT and CEF were found to be 8.4 ^ 0.2%
and 17.5 ^ 0.5%, respectively. Using the present
enrichment method, curcuminoids were almost
doubled. This CEF can used for colouring various
699
food stuffs and pharmaceutical formulations instead of
using synthetic dyes such as sunset yellow. Since most
of these synthetic dyes are carcinogenic and generate
other health problems.
HPLC and LC-MS profiling of CEF
The presence of curcumin in enriched fraction was
confirmed by HPLC profiling of standard curcumin
and CEF (Figures 1 and 2). It clearly shows the
presence of curcumin in CEF with a retention time of
22 min. This was further confirmed by LC-MS
analysis, by comparing with the M H ions and the
daughter ions in the mass spectrum of CEF and that of
standard curcuminoids (Figure 3).
Total phenolic content and total flavonoid content
TPCs of SOTand CEF were found to be 12.5 ^ 0.5%
and 19.1 ^ 0.9%, respectively, which indicate that
polyphenolic compounds were still present in the spent
oleoresin. Flavonoid content of samples was deter-
mined by aluminium chloride colorimetric method.
TFCs of SOT and CEF were 4.64 ^ 0.3% and
12.5 ^ 0.5%, respectively. These results showed that
on enriching curcuminoids, TPC and TFC also get
increased. Polyphenols such as curcumin possess
several biological activities, and the effects of these
compounds are mainly due to their capacity to transfer
electrons to free radicals and the chelate metal catalyst.
Evaluation of antioxidant activity
DPPH and ABTS radical scavenging activity. DPPH
and ABTS are powerful free radicals that are used to
evaluate the electron-donating capacity of antioxidants
(Duan et al. 2006). The antioxidants react with the
stable free radicals, i.e. a,a-diphenyl-b-picrylhydrazyl
(deep violet colour) and convert it to a,a-diphenyl-b-
picrylhydrazine with discolouration (Negi et al. 2010).
The degree of discolouration indicates the free radical
scavenging potentials of the sample per antioxidant.
Figure 2. HPLC profile of CEF at 425 nm.
 700 S.V. Nampoothri et al.
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For personal use only.
Figure 3. LC-MS profile of CEF. 1 denotes the MH ions of C1 (curcumin) and C2 (demethoxy curcumin). 2 denotes M H of C3
(bis-demethoxycurcumin). m/z 364.4, 301.4 and 286.4 are the daughter ions of curcuminoids.
Radical scavenging activity is numerically expressed in
IC50 values. It is the amount of antioxidants required
to scavenge 50% of free radical from a system at a
specified time. IC50 value is inversely related to
antioxidant activity. SOT and CEF significantly
scavenge the DPPH and ABTS radicals, among
them CEF was better. The activity of both of the
samples was lesser than that of standard antioxidant
gallic acid (Table I). The curcuminoids contain two
phenolic hydrogen in its structure, which can easily
give rise to the free radicals and reduce them into
neutral form. Hence, the radical scavenging activity of
SOT and CEF might be due to the hydrogen-donating
ability of the curcuminoids.
Inhibition of LDL oxidation. Oxidative modification of
LDL is known to play an important role in the
pathogenesis of atherosclerosis and coronary heart
diseases. The dietary antioxidants that protect LDL
from oxidation may therefore reduce atherogenesis
and coronary heart diseases. The SOTand its enriched
fraction can effectively prevent the LDL oxidation
(Table II), and thereby they can reduce the risk of
atherosclerosis. From these results, we found that
CEF was a potent inhibitor of LDL oxidation and its
IC50 value was almost close to standard ascorbic acid.
It was reported that polyphenols can prevent LDL
oxidation at in vitro conditions (Teissedre et al. 1996;
Seiichi et al. 2002). Since the curcuminoids are
polyphenolic compounds, the LDL oxidation
potential of CEF was due to the presence of
curcuminoids in it.
Inhibition of ACE. The importance of ACE in
hypertension is well established. By inhibiting this
enzyme, we can control the blood pressure. ACE
inhibitors are a group of drugs used primarily for the
treatment of hypertension and congestive heart failure.
Frequently prescribed ACE inhibitors include
captopril, enalapril and lisinopril. The common
adverse reactions of these drugs include hypotension,
cough, hyperkalaemia, headache, dizziness, fatigue,
nausea and renal impairment (Rossi 2006). Some
Table I. DPPH and ABTS free radical scavenging activities of
SOT and CEF.
Sample IC50 for DPPH (mg/ml) IC50 for ABTS (mg/ml)
SOT 62.62 ^ 0.5 62.96 ^ 0.7
CEF 41.00 ^ 0.2 41.92 ^ 0.1
Gallic acid 1.4 ^ 0.1
Trolox 7.80 ^ 0.3
 Enrichment of curcuminoids in turmeric spent oleoresin 701

Table II. Inhibition of LDL oxidation and ACE inhibition activity Table IV. Chemical composition of hexane fraction obtained
by SOT and CEF. by GC-MS analysis.

Sample IC50 for LDL (mg/ml) IC50 for ACE (mg/ml) Content
Serial no. Compound name Kovats indices (%)
SOT 41.97 ^ 0.3 21.60 ^ 0.2
CEF 30.52 ^ 0.4 19.45 ^ 0.1 1 a-Terpinene 1018 0.2
Ascorbic acid 24.5 ^ 0.1 2 p-Cymene 1026 0.2
Captopril 0.69 ^ 0.02 3 1,8-Cineole 1032 0.2
4 Carvatonacetone 1220 0.2
5 Carvacrol 1339 0.4
6 b-Elemene 1395 1.5
evidence also suggests that ACE inhibitors might 7 g-Elemene 1433 0.9
8 Cedrene 1435 0.8
increase inflammation-related pain (Fein 2009).
9 a-Bergamotene 1440 0.2
Rather than synthetic ACE inhibiting drug 10 a-Farnesene 1445 0.8*
molecules, natural plant extracts have some 11 b-Farnesene 1458 0.6
advantage, due to its less side effects, multifaceted 12 a-Aromadendrene 1461 1.1
activity and antioxidant properties. In the present 13 Ar-curcumene 1483 4.0
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14 Zingiberene 1495 1.0

study, SOT and CEF significantly inhibited the ACE
15 b-Sesquiphellandrene 1524 0.9
activity even though its activity was less than that of 16 Elemol 1540 2.1
standard captopril (Table II). 17 E-Nerolidol 1554 0.5
18 Ar-turmerone 1636 67
19 a-Turmerone 1643 3.8
GC-MS profiling of hexane fraction 20 b-Turmerone 1672 6.4
21 Curdione 1698 0.6
Hexane fraction was isolated from spent oleoresin 22 Farnesol* 1741 1.2
and it was analysed by GC-MS. Antioxidant property
of this fraction also studied (Table III). SOT contains * Correct isomer not identified.
volatile compounds and non-volatiles. Volatile con-
used. The good antioxidant property of CEF shows
For personal use only.

stituents in the SOT were extracted using hexane.

Twenty-two compounds corresponding to 94.6%
total hexane fraction were identified (Table IV). The
main compound was ar-turmerone (67%) followed
by beta-turmerone (6.4%), the monoterpene com-
pounds present only in traces and the sequiterpene
hydrocarbon contents were also low. The main
sequiterpene was ar-curcumene (4%) as in the case
of turmeric oil. Curdione and farnesol were also
present in the fraction. The antioxidant property of
this fraction was mainly due to the effect of major
compound (ar-turmerone), since the compound is
well known for its antioxidant and biological proper-
ties (Ji et al. 2004).
Conclusion
The curcuminoids in the SOT was estimated, and a
process has been developed for the enrichment of
curcuminoids. Its presence was confirmed by HPLC
and LC-MS analysis. Based on the above studies, the
CEF from SOT can be utilized as a natural inhibitory
agent against ACE and LDL oxidation. It was found to
be a promising alternative of synthetic drug being
Table III. Antioxidant activities of hexane fraction.
IC50 for IC50 for
DPPH ABTS
Sample TPC (%) TFC (%) (mg/ml) (mg/ml)
Hexane 6.98 ^ 0.7 1.15 ^ 0.4 153.55 ^ 0.8 154.64 ^ 0.9
fraction
that it can also be used as a natural antioxidant and a
colouring agent instead of using carcinogenic synthetic
antioxidants and dyes. However, there are still some
more biological studies needed regarding the use of
CEF in neutraceutical or pharmaceutical formulation.
Declaration of interest: The authors report no
conflict of interest. The authors alone are responsible
for the content and writing of the paper.
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