Correlation of In Vivo and In Vitro Assay Results

for Assessment of Free Radical Scavenging

Activity of Green Tea Nutraceuticals
Heba-Alla H. Abd-ElSalam, Medhat A. Al-Ghobashy, Muhammad Al-Shorbagy, Noha Nassar, Hala E. Zaazaa,

C: Food Chemistry
and Mohamed A. Ibrahim

Abstract: Green tea (GT)-derived catechins; epigallocatechin gallate (EGCG) in particular are commonly used nu-
traceuticals for their free-radical scavenging activity (FRSA). The influence of photodegradation on the protective power
of GT nutracenticals against oxidative stress was thoroughly explored. Photodegradation of GT extracts was carried out
and monitored using orthogonal stability-indicating testing protocol; in vitro and in vivo assays. Total polyphenol content
(TPC) and FRSA were determined spectrophotometrically while EGCG was selectively monitored using SPE-HPLC.
In vivo assessment of photodegraded samples was investigated via measuring a number of biomarkers for hepatic oxidative
stress and apoptosis (caspase-3, inducible nitric oxide synthase, nitric oxide, mitogen-activated protein kinase, glutathione,
thiobarbituric acid reactive substances, nuclear factor kappa beta, and nuclear factor erythroid 2-related factor) as well as
liver damage (alanine transaminase and aspartate transaminase) in serum of rats previously subjected to oxidative stress.
Results showed complete degradation of EGCG in photodegraded green tea samples with no correlation with either
TPC or FRSA. On the other hand, in vivo assay results revealed not only loss of activity but formation of harmful
pro-oxidants. Photostability was found crucial for the protective effect of GT extract against lead acetate insult. Results
confirmed that careful design of quality control protocols requires correlation of chemical assays to bioassays to verify
efficacy, stability, and most importantly safety of nutraceuticals.

Keywords: Folin-Ciocalteu, free radical scavenging activity, green tea nutraceuticals, photostability, SPE-HPLC

Practical Application: In this study, an orthogonal analysis protocol was designed to evaluate the photostability of a
model green tea nutraceutical formulation. Lack of agreement between chemical assay results required correlation to in
vivo activity in animal model. Results indicated that photodegraded green tea samples were not only ineffective but also
harmful to biological systems. Photodegradation products exerted prooxidant effect rather than the beneficial antioxidant
effect. Results of the study confirmed that inappropriate selection of analysis techniques and lack of correlation between
in vitro and in vivo assay results might lead to elusive conclusions about the quality of nutraceuticals.

Introduction is not regulated by a physician and is a lifelong habit for many

Green tea (GT) nutraceuticals are receiving increased attention
worldwide due to their free radical scavenging activity (FRSA) and
disease preventing properties (Singh and others 2011; Kapoor and
others 2013). They guard against the deleterious effects induced
by various compounds via modulating oxidative stress (Newsome
and others 2014) and their anti-inflammatory properties (Barnett
and others 2013). Recently, EGCG was found to prevent the pro-
gression of cell death cascades (Emoto and others 2014) and confer
protection against multiple organ damage (Priyadarshi and others
2003; Lin and others 2009). With all these beneficial effects, re-
quirements for registration of GT nutraceutical products are not as
demanding as those of pharmaceuticals (Bernal and others 2011)
that lead to the presence of substandard nutraceuticals in the mar-
ket. Taking in consideration that consumption of nutraceuticals
MS 20160492 Submitted 3/29/2016, Accepted 5/13/2016. Authors Abd-
ElSalam and Al-Ghobashy are with Bioanalysis Research Group, Faculty of Phar-
macy, Cairo Univ., Egypt. Authors Al-Ghobashy, Zaazaa, and Ibrahim are
with Analytical Chemistry Dept., Faculty of Pharmacy, Cairo Univ., Egypt. Au-
thors Al-Shorbagy and Nassar are with Pharmacology and Toxicology Dept., Faculty
of Pharmacy, Cairo Univ., Egypt. Direct inquiries to author Al-Ghobashy (E-mail:
medhat.alghobashy@cu.edu.eg).
of us and the complex nature of nutraceutical preparations, it has
been recommended that the quality and stability of such products
should be investigated carefully (Lockwood 2011; Abd-ElSalam
and others 2014).
Chemically, the main constituents of GT are polyphenolic cat-
echins and alkaloids. Catechins; epigallocatechin gallate (EGCG),
epicatechin, epigallocatechin, and epicatechin gallate represent up
to 30% of the dry weight of GT leaves (Hilal and Engelhardt 2007).
EGCG is the major antioxidant ingredient in GT forming 40%
of the total catechins content (Misaka and others 2013). EGCG
is commercially available in nutraceutical preparations in the form
of single ingredient formulation, EGCG-fortified preparations or
as part of crude GT extract. In literature, high-performance liquid
chromatography (HPLC) has been widely used for quantitative de-
termination of various GT components (Bernal and others 2011).
Evaluation of the total polyphenol content (TPC) and FRSA were
commonly employed to verify the quality of GT products (Wang
and others 2000; Kim and others 2011). The stability of catechins
has been investigated during preparation, fermentation, brewing
(Wang and Helliwell 2000; Kim and others 2011), storage of GT
leaves (Friedman and others 2009), and powder (Li and others
2011). Very few reports investigated the stability of catechins; in

C 2016 Institute of Food Technologists

 R

doi: 10.1111/1750-3841.13362 Vol. 81, Nr. 7, 2016 r Journal of Food Science C1707
Further reproduction without permission is prohibited
 Assessment of FRSA of GT nutraceuticals . . .

topical preparations (Scalia and others 2013) and tablet formula-
tion (Abd-ElSalam and others 2014). However, investigation of
the possible deleterious effects of degraded GT formulations on
biological systems has not been demonstrated. Recently, it has
been demonstrated that polyphenolic compounds such as gallic
acid can act as pro-oxidant and generate reactive oxygen species
(ROS) and hydrogen peroxide upon UV irradiation (Du and
C: Food Chemistry
Determination of TPC
TPC was determined spectro photometrically in terms of gallic
acid equivalent (GAE) as previously described (2008). Briefly, a set
of GA standard solutions (25.00 to 175.00 μg/mL) was prepared in
water and used for construction of the calibration curve. Aliquots
of equal volumes of each standard solution were mixed with
6.00 mL 0.2 N FC reagent then 5.00 mL of 0.7 M sodium car-

others 2014). This was employed by our group for catalytic degra- bonate. The absorbance was measured against a blank at 765.0 nm
dation of pharmaceuticals in industrial waste water using GA- after 40 min incubation at room temperature, away from light.
coated magnetic nanoparticles (Nadim and others 2015). Assay validation was carried out according to the pharmaceutical
In this work, the influence of photo-degradation on the protec- industry standards (ICH Guidelines 2005).
tive power of GT extract against oxidative stress was thoroughly
explored using a model GT nutraceutical formulation. Through- Determination of FRSA
out the study, TPC and FRSA were determined spectrophotomet- Spectrophotometric determination of the FRSA of GT sam-
rically while EGCG was selectively monitored using SPE-HPLC. ples was carried out using DPPH assay as previously de-
The kinetics of degradation was explored and correlation of the scribed (Brand-Williams and others 1995). GA was used
obtained results was investigated. The efficacy and safety of pho- as a positive control over a concentration range (1.00 to
todegraded samples were further assessed via measuring a set of 7.00 μg/mL) while the negative control sample was prepared
biomarkers for oxidative stress in plasma and tissues of rat animal similarly but using 1.00 mL methanol. Aliquots (1.00 mL)
model previously subjected to oxidative stress. Correlation of in of standard GA solutions were mixed with 3.00 mL of 4 mg%
vivo and in vitro assay results was carried out in order to recommend DPPH solution and incubated for 35 min at room temperature
a reliable approach for quality assessment of such nutraceutical away from light. The absorbance was measured at 517.0 nm against
preparations. methanol as a blank and the percentage inhibition was then cal-
culated as follows:
Materials and Methods
Instruments %Inhibition = (1−absorbance of test/absorbance of control) × 100
SPE experiments were carried out using a vacuum manifold
and manually packed cartridges (6.00 mL to 1.00 g) with HF The % Inhibition was plotted compared with GA concentration
Bondesil C18 (Agilent Technologies, Waldbronn, U.S.A.). Spec- and regression equation parameters were calculated. Assay perfor-
trophotometric analysis was carried out using UV-Visible spec- mance was validated according to ICH guidelines. Throughout
trophotometer controlled with UVProbe 2.21 software (Shimadzu this work, test samples were analyzed similarly and the percentage
Corporation, Kyoto, Japan). An HPLC system equipped with a inhibition was calculated. Results were expressed in terms of GA
UV-Vis detector and controlled with Class-VP Software (Shi- using the obtained regression equation.
madzu Corporation) was used in all determinations. Analysis was
carried out using HC-C18 column, 5 μm, 4.6 × 250 mm (Agi- Sample preparation for determination of EGCG
lent Technologies). Photo-stability studies were carried out using Optimization of sample preparation for EGCG determination
a Suntest CPS+ cabinet covering the wavelength range of visible was carried out as previously described by our group (Abd-ElSalam
light (Atlas, Germany). UV irradiation was carried out using a and others 2016). Briefly, aliquots of 0.25 g GT powder or tablets
calibrated UV lamp (Vilber Lourmat, France) housed in a home- were extracted in 50.00 mL water at 70 to 85 °C. The extract
made air-ventilated cabinet operated at 254 nm, 1012 μW/cm2 . was filtered using a double filter paper then a membrane filter
(0.45 μm). Caffeine was partially removed using dichloromethane
Chemicals, standards and samples (DCM), 2×50 mL and catechins were recovered using 4×25 mL
Caffeine, gallic acid (GA), Folin-Ciocalteu (FC), 1,1-diphenyl- ethyl acetate. The residue obtained after evaporation under vac-
2-picrylhydrazyl (DPPH), and lead acetate were purchased from uum, away from light was reconstituted in equal volume of phos-
Sigma (St. Louis, U.S.A.). GT powder and tablets labeled to con- phate buffer (0.25 M, pH 4.0). Aliquots of the reconstituted extract
tain 200 mg catechins per tablet (Technomad, Egypt) were ob- were then transferred to the SPE cartridges, pre-conditioned with
tained from the market. GT extract was prepared and standardized 2.00 mL methanol followed by 1.00 mL phosphate buffer. Elution
in-house and found to contain 6.75 g/100 g EGCG, and caffeine was carried out using 1.00 mL methanol after washing off the
2.90 g/100 g (Hilal and Engelhardt 2007; Abd-ElSalam and others residual caffeine using 2×1 mL DCM.
2014). All solvents and other chemicals were of HPLC grade and
were obtained from Sigma (St. Louis, U.S.A.). HPLC determination of EGCG
A previously reported set of analysis conditions (Jin and
Animals Row 2007) was optimized and validated. Briefly, separation
Adult male Wistar rats (180 ± 20 g) were purchased from was carried out using a C18 column and a mobile phase of
El-Nile Pharmaceutical Company (Cairo, Egypt). One-week ac- water: methanol: O-phosphoric acid (60:40:0.01 by volume,
climatization period was allowed at the animal facility of the Fac- pH 3.2 ± 0.05). Isocratic elution at 0.5 mL/min and detec-
ulty of Pharmacy, Cairo University (Cairo, Egypt). Animals were tion at 270 nm was carried out. System suitability parame-
then housed in groups at constant temperature (23 ± 2 °C), hu- ters were calculated in order to evaluate the efficiency of sep-
midity (60 ± 10%) and light/dark (12/12 h) cycles with light on aration (US Pharmacopoeia 2011). Determination of EGCG
at 5:00 am. All rats were allowed free access to water and food content was carried over a concentration range of 10.00 to
during the experimental period. 45.00 μg/mL. Regression equation was computed and used to

C1708 Journal of Food Science r Vol. 81, Nr. 7, 2016

Assessment of FRSA of GT nutraceuticals . . .

Table 1–Summary of regression equation and validation parameters for the chemical assays employed for analysis.

Parameter TPC FRSA EGCG content

Regression equation y= 2.152x2 +9.251x-0.048 y = 9.379x – 0.1443 y = 138296x+105619
Correlation coefficient (r) 0.999 0.999 0.999
Range 25.00–175.00 μg/mL 1.00–7.00 μg/mL 10.00–45.00 μg/mL
Accuracy 100.13 ± 1.46 99.86 ± 2.49 100.14 ± 0.94
Precision Repeatabilitya 99.41 ± 0.82 100.29 ± 1.07 98.88 ± 1.45

C: Food Chemistry
Intermediate precisionb 99.56 ± 1.60 101.47 ± 2.03 99.92 ± 1.47
LODc 10.00 μg/mL 0.88 μg/mL 0.64 μg/mL
LOQd 25.00 μg/mL 1.00 μg/mL 10.00 μg/mL
a
Intraday (n = 3), average of 3 determinations repeated 3 times within the day.
b
Interday (n = 3), average of 3 determinations repeated 3 times in 3 d.
c
Limit of detection = 3.3 × SD/slope.
d
Based on experimental determinations.

calculate EGCG content and assay validation was carried out (ICH iNOS, NO, as well as MAPK were determined in the hepatic tis-
Guidelines 2005). sue using the ELISA kits purchased from; Assay-Designs-Stressgen
(USA), Enzo Life Sciences (USA) and Life Sciences Advanced
Photostability study and kinetics of degradation Technologies (USA), respectively. The liver levels of glutathione
GT powder was extracted as previously described and the pho- (GSH) and TBARS were determined colorimetrically using Oxis-
tostability was investigated. Aliquot of 25 mL of the extract were elect cell biolabs kit purchased from Cell Biolabs, USA. Moreover,
placed in a set of petri dishes and were irradiated using either vis- NF-κB and Nrf-2 were determined in the hepatic tissues using
ible or UV light for 8 h under controlled temperature conditions. ELISA; ElAab, Wuhan, PRC, Wuhan ElAab Science, China and
Photodegraded samples were quantitatively transferred to a set of Immunoway, assay designs, Immunoway Biotechnology, China,
volumetric flasks and completed to original volume then analyzed respectively. All biomarkers were determined using the corre-
for TPC, FRSA, and EGCG content. In the case of UV irradi- sponding kits according to manufacturers’ instructions.
ation, 1 sample was withdrawn every hour for a total period of
8 h in order to investigate the kinetics of degradation. Results were Results
calculated relative to those obtained using an equivalent control Determination of TPC
sample that has not been suggested to the indicated stress. In vivo Colorimetric determination of TPC was carried out in terms of
investigation of the biological activity of UV-irradiated samples GA over a concentration range of 25.00 to 175.00 μg/mL using
was then carried out as will be described in detail. GA as a standard at 765 nm (Figure S1). Second order polynomial
regression was found superior to linear regression model with
Animal model respect to assay range and linearity. Various regression equation and
Experimental design. The experiment period spanned validation parameters were calculated to pharmaceutical industry
a total of 4 wk where animals were allocated into 5 groups standards as per ICH guidelines (Table 1).
(6 animals each): (i) control group; received saline by oral route
daily for up to 4 wk, (ii) Pb group; received intraperitoneal Determination of FRSA
lead acetate (100 mg/kg) every 24 h over the last 2 wk of the FRSA was measured using GA as a positive control over a con-
experiment (Rahman and Sultana 2006), (iii) Pb-GT group; centration range of 1.00 to 7.00 μg/mL, while methanol was used
received GT extract 300 mg/kg daily for 4 wk, 1 h before the as the negative control (Figure S2). The %Inhibition was calcu-
administration of lead acetate (100 mg/kg), (iv) Pb-PTGT group; lated as described and plotted compared with GA concentration.
received photodegraded GT extract (300 mg/kg, UV irradiation Regression equation and validation parameters were computed
for 8 h) daily for 4 wk, 1 h before the administration of lead acetate and summarized in Table 1 as per ICH guidelines.
(100 mg/kg) (Banji and others 2011; Haidari and others 2011),
and (v) PTGT group; received photodegraded GT extract Determination of EGCG content
(300 mg/kg, UV irradiation for 8 h) daily for 4 wk. In this experiment, SPE using C18 stationary phase was car-
Tissue processing and sampling. At the end of the exper- ried out in order to reduce the complexity of crude GT extracts.
imentation period, serum samples were drawn for analysis of ala- HPLC assay conditions were optimized to achieve good resolu-
nine transaminase (ALT) and aspartate transaminases (AST) levels. tion between peaks corresponding to GA, EGCG, and caffeine.
Rats were then euthanized and their livers were quickly dissected Previously reported developing systems of different compositions
and washed with ice-cold saline. Livers were homogenized in ice- and ratios were tried and optimum resolution was obtained us-
cold phosphate buffer saline (PBS, 0.02 M, pH 7.0 ± 0.05) and ing water: methanol: O-phosphoric acid (60:40:0.01 by volume,
used for determination of thiobarbituric acid reactive substances pH 3.2 ± 0.05) as the mobile phase (Figure 1). System suitability
(TBARS), nonprotein thiols (NPSH), inducible nitric oxide syn- parameters were calculated in order to evaluate the efficiency of
thase (iNOS), nuclear factor kappa beta (NF-κB), nuclear factor separation. Results showed good resolution, selectivity and sym-
like (Nrf)-2, nitric oxide (NO), mitogen-activated protein kinase metry of the obtained peaks. Excellent relative retention and ca-
(MAPK), and caspase-3 (Casp-3). pacity factor were also demonstrated as summarized in Table 2.
Determination of biomarkers. Serum ALT and AST lev- Calibration curve for the determination of EGCG was constructed
els were determined colorimetrically using a kit purchased from over a concentration range of 10.00 to 45.00 μg/mL. Regression
Teco Diagnostic (USA) while hepatic content of Casp-3 was de- equation and validation parameters were computed as per ICH
termined using ELISA (Uscn Life Science Inc., USA). In addition, guidelines and summarized in Table 1.

Vol. 81, Nr. 7, 2016 r Journal of Food Science C1709

 Assessment of FRSA of GT nutraceuticals . . .

Table 2–System suitability results of GA, EGCG, and caffeine by where kobs is the rate constant, Ct is concentration at a given
the proposed HPLC assay. time and Co is initial concentration. The Kobs and t0.5 were found
Gallic Reference to be –0.074 h−1 and 9.39 h in the case of TPC (r = 0.78),
Parameters acid EGCG Caffeine values while approximately a 10-fold increase in Kobs (0.781 h−1 ) and
Resolution (Rs ) 3.53 1.94 2.31 >2.00 10-fold decrease in t0.5 (0.89 h) values were noted in the case of
Peak symmetry (As ) 0.70 1.30 1.20 2.00 EGCG degradation (r = 0.95). The poor correlation coefficient
Retention factor (K´) 2.70 4.43 5.40 >2.00 obtained using TPC data was attributed to complex and possible
C: Food Chemistry

Separation factor (α) 1.31 1.62 1.29 >1.00 variation in chemical composition upon UV irradiation. This was
Number of theoretical plates (N) 5733 5295 5448 >2000
in agreement to our assumption that TPC is not a reliable marker
for monitoring quality of GT samples.

Determination of biomarkers
Photostability study and kinetics of degradation
Lead acetate-induced hepatic changes in TBARS,
In this experiment, GT samples were extracted as described in
NPSH, NO, and Nrf-2. Animals receiving lead acetate showed
order to reduce the complexity of the sample and remove possible
a 5-fold increase in hepatic TBARS and a 3-fold increase in both
interferences from other ingredients present in the extract. Initially,
NO and Nrf-2 as compared to their control counterparts. This was
TPC, FRSA, and EGCG content were determined for samples
paralleled with a 50% reduction in the hepatic NPSH content. Pre-
irradiated using either visible or UV light for 8 h. Figure 2 shows
treatment with GT extract suppressed lead acetate-induced hepatic
representative chromatograms of incubated samples and a control
damage shown as a decline in TBARS, NO, and Nrf-2 by 37%,
sample of equivalent concentration, that has not been subjected
to light. In the case of samples irradiated with visible light, the
percentage loss in TPC, FRSA, and EGCG content were 22.73 ±
0.54%, 16.48 ± 0.68%, and 60.88 ± 0.26%, respectively. On the
other hand, significantly higher values of 38.64 ± 0.36%, 22.50 ±
0.41%, and 99.07 ± 0.62% for TPC, FRSA, and EGCG content,
respectively were noted in the case UV irradiations (Table S1 and
Figure 3A).
Further investigations were carried out in the case of UV irra-
diation where samples were analyzed every hour for a total period
of 8 h. Results indicated almost complete degradation of ECGC
within 4 h of irradiation time. Surprisingly, this complete degra-
dation in EGCG content has not been reflected on the TPC and
FRSA. Abrupt increase in the percentage degradation of TPC
and FRSA was noted after 1 h of irradiation time followed by a
slight increase until the end of the incubation period. It should
be noted that there was no correlation between the percentage
loss in EGCG content and either TPC or FRSA (Table S1 and
Figure 3B). The decrease in TPC and EGCG content was found
to follow the first order reaction kinetics model that can be repre-
sented by the following equation:

Ct
ln = −kobs t
C0

Figure 2–Representative chromatograms showing SPE-HPLC analysis re-

sults of (A) control sample, (B) green tea extract after exposure to visible
Figure 1–Representative chromatogram of green tea extract prepared radiation for 8 h and (C) green tea extract after exposure to UV light for
using solid phase extraction and spiked with 15.00 μg/mL Gallic acid. 8 h.

C1710 Journal of Food Science r Vol. 81, Nr. 7, 2016

Assessment of FRSA of GT nutraceuticals . . .
37%, and 28%, respectively as compared to the Pb group. The
NPSH showed a 30% increase when GT extract was combined
with lead acetate. Pretreatment with PTGT further augmented
the lead induced deleterious effects clear as an increase in TBARS,
NO, and Nrf-2 by 50%, 60% and 40%, respectively as compared
to the lead acetate-treated rats, while the hepatic NPSH dropped
to 60% of the value observed with the lead acetate group. Sup-
plementation of PDGT alone in rats resulted in a significant spike
in hepatic levels of TBARS, NO, and Nrf-2 by 1.4 fold, 75%,
and 82%, respectively, while the NPSH level was reduced to 37%
of the basal value as compared to the control rats as shown in
Figure 4A to D).
Lead acetate-induced hepatic changes in NF-κB,
MAPK, iNOS, and Casp-3. Rats treated with lead acetate
showed nearly a 4-fold increase in hepatic levels of NF-κB,
MAPK, and Casp-3 and a 3-fold increase in iNOS as compared to
the control group. GT extract pretreatment amended lead acetate-
induced hepatic changes reflected as regression in the levels of the
above mentioned parameters by 40% in case of MAPK, iNOS,
and Casp-3 and by 50% in case of NF-κB as compared to the
Pb Group. Pretreatment with PTGT enhanced the Pb induced
elevations in NF-κB, MAPK, Casp-3, and iNOS by 30%, 40%,
40%, and 33%, respectively as compared to the Pb Group. Sole
C: Food Chemistry
administration of the PTGT to the rats caused an upsurge in hep-
atic levels of NF-κB, MAPK, Casp-3, and iNOS by 1.4, 1.32, 1.2
folds and 100% respectively as compared to the control group as
shown in Figure 5A to D.
Lead acetate-induced hepatic changes in serum ALT and
AST. Both ALT and AST serum activities showed an upsurge in
the lead acetate treated rats, which mounted to almost 2.5-folds of
the basal control values. Pretreatment with GT extract significantly
improved the lead acetate-induced enzymatic changes manifested
Figure 3–Bar graphs showing the results
of the percentage loss of free radical
scavenging activity, total polyphenol
content and EGCG content of the green
tea extract: (A) after 8 h exposure to
visible and UV irradiations in comparison
to control sample and (B) during 8 h
exposure to UV irradiations in
comparison to control sample.
Vol. 81, Nr. 7, 2016 r Journal of Food Science C1711
 Assessment of FRSA of GT nutraceuticals . . .
as a drop in the enzymatic activities of ALT and AST by 35%
and 54%, respectively as compared to the Pb Group. On the
other hand, pretreatment with the PTGT boosted the lead acetate
induced elevation in the enzymatic activities of ALT and AST by
67% and 60% as compared to the Pb Group. As expected, the sole
administration of the PTGT to the rats caused an elevation in ALT
and AST activities by 73% and 70%, respectively as compared to
C: Food Chemistry
the control group as summarized in Figure 6A and B.
Discussion
In literature, determination of the TPC and to lesser ex-
tent FRSA have been reported for evaluation of the quality of
nutraceuticals containing polyphenolic compounds (Bernal and
others 2011). However, in depth assessment of the stability of
nutraceuticals containing polyphenolic products has not been
thoroughly explored (Scalia and others 2013; Abd-ElSalam and
others 2014) and very few reports investigated the significance of
product quality on biological activity and safety (Lockwood 2011).
Determination of TPC, FRSA, and EGCG content
GT samples were prepared using a combination of LLE and SPE
as previously described. Prior to HPLC determinations, sample
clean-up using LLE was crucial to avoid permanent color change
of the C18 adsorbent caused by polymerized polyphenols that
might be present due to bad storage or processing of GT leaves.
Caffeine is the second major active component in GT extract and
has a higher relative retention on C18 column when compared
to EGCG, under the studied experimental conditions (Figure 1).
Thus, partial on-column decaffeination was carried out in order
to improve the dynamic capacity of the adsorbent toward EGCG.
The TPC and FRSA were determined in terms of GA follow-
ing previously reported protocols with slight modification (2011;
Figure 4–Effect of GT extract and PDGT on lead acetate-induced hepatic changes in (A) TBARS, (B) NPSH, (C) NO and (D) Nrf-2.
C1712 Journal of Food Science r Vol. 81, Nr. 7, 2016
Brand-Williams and others 1995). EGCG content was selectively
determined by HPLC after SPE. Monitoring the change in the
stability of EGCG in terms of an economic and available tech-
niques has been previously discussed in more detail by our group
(Abd-ElSalam and others 2014). Assay validation to pharmaceu-
tical industry standards was then carried out in order to ensure
reliability of the results (Table 1).
Photostability study and kinetics of degradation
Light induced degradation of EGCG has been previously re-
ported (Katiyar and others 2001) and attempts to improve its
photostability by inclusion of antioxidants into GT topical for-
mulations has also been demonstrated (Scalia and others 2013).
Results shown in Figure 3A and B and Table S1 clearly indicated
that the percentage loss in TPC and FRSA were not in agreement
to those observed with EGCG content. The 10-fold higher rate
of degradation in the case of EGCG confirmed such observation.
Lack of extensive decrease in TPC and FRSA could be attributed
to formation polyphenolic degradation products that might still
retain FRSA. This was in agreement to the previously reported
data which demonstrated that epimerization of catechins resulted
in almost no difference in FRSA (Xu and others 2004).
Light induced degradation of polyphenolic compounds was pre-
viously shown to occur through free radical-mediated mechanism
(Guo and others 1999). UV irradiation of polyphenolic com-
pounds has also been demonstrated to produce ROS and hydrogen
peroxide (Du and others 2014). Thus these compounds were con-
sidered as both antioxidants and pro-oxidants (A. and others 2015;
Nadim and others 2015). In this study, the decrease in caffeine
content in photodegraded samples could be attributed to the ac-
tion of ROS produced upon irradiation of EGCG in GT extracts
(Figure 3). Caffeine is a photo stable compound but liable for free
Assessment of FRSA of GT nutraceuticals . . .
radical-mediated degradation (Trovo and others 2013). Detailed
investigation of the mechanism of degradation of caffeine is not
under the scope of this study and is currently being explored by
our group. However, it raised a concern about not only losing
the efficacy of photodegraded preparations but also their safety.
This intriguing observation indicated also that in vivo assessment
of biological activity and safety of photodegraded samples should
be carried out.
Determination of biomarkers
Reported studies have previously shown that administration of
GT extract could guard against the deleterious effects of ROS
on multi-organ systems (Priyadarshi and others 2003; Lin and
others 2009). In the current investigation, we demonstrated that
photodegradation of GT extract is counter-protective in a lead
acetate model of oxidative stress in liver. This highlights the fol-
Figure 5–Effect of GT extract and PDGT on Pd acetate-induced hepatic changes in (A) NF-κB, (B) MAPK, (C) iNOS, and (D) Casp-3.
lowing findings (i) lead acetate induced an oxidant status in the
liver manifest by the increase in TBARS as well as NO/iNOS
in face of a decline in NPSH, events that were held in check
by GT extract co-administered with lead acetate and augmented
in presence of the photo-degraded product; (ii) in an attempt to
counteract the injurious oxidative effects induced by lead acetate,
an up-regulation of hepatic Nrf-2 was observed in this group that
C: Food Chemistry
was reduced by the photo-degraded product; (iii) both of NF-κB
as well as p42/44 MAPK and Casp-3 were induced by lead ac-
etate in the liver tissues, reduced by GT extract and boosted by
photodegraded product; and (iv) lead acetate increased AST and
ALT activities in rats sera which was further amplified by the pho-
todegraded product in contrast to GT extract protecting against
this enzymatic change.
In the current investigation, we provide evidence that photo
stability is a crucial factor in the protective effect elicited by GT
Figure 6–Effect of GT extract and PDGT on Pd
acetate-induced hepatic changes in: (A) serum
ALT and (B) serum AST.
Vol. 81, Nr. 7, 2016 r Journal of Food Science C1713
 Assessment of FRSA of GT nutraceuticals . . .
extract against lead acetate insult. Notably, reported studies indi-
cate that lead acetate administration results in tipping the prox-
idant/antioxidant status (Abdou and Hassan 2014; Omobowale
and others 2014). Indeed, in the current design, animals receiving
lead acetate showed an increase in TBARS. Noteworthy, ROS
play a crucial role in cellular injury via lipid peroxidation of cel-
lular membranes (Xu and others 2008; Abdou and Hassan 2014;
C: Food Chemistry
Omobowale and others 2014). By the same token, animals re-
ceiving lead acetate showed an increase in hepatic NO content,
which further augments tissue injury by formation of reactive
nitrogen species (RNS) (Poderoso and others 1999; Zhang and
Hogg 2004). In contrast, in the same group, nonprotein thiols
(NPSH) showed a decline in face of increasing oxidant burden in
an attempt of the system to counteract the deleterious effect of
TBARS. Furthermore, in a strenuous effort of the system to mend
the antioxidant / proxidant status, Nrf-2 showed an increment in
Pb group compared with the control counterpart. Noteworthy,
cells quickly augment their antioxidant capacity when confronted
with oxidative stressors to counteract increased ROS production
and maintain homeostasis. In this context, the transcription fac-
tor nuclear factor erythroid 2-related factor (Nrf-2) was elevated,
which under oxidative stress enhances the expression of a multi-
tude of antioxidant enzymes to restore redox homeostasis (Liu and
others 2014). Animals receiving GT extract, showed a decline in
TBARS, NO, Nrf-2 compared with an increment in GSH sug-
gesting the complete protection against hepatic ROS production
induced by concomitant lead acetate administration. Conversely,
animals receiving photo-degraded product in addition to lead ac-
etate showed worsening of oxidant/proxidant status, thus suggest-
ing the importance of shelf stability in the protective effects of
GT extract. A further support to the notion, the group receiv-
ing PDGT alone showed a significant tendency to disturb the
oxidant/proxidant milieu, albeit, to a less extent than those re-
ceiving PDGT on top of lead acetate.
Furthermore, ROS production triggers the translocation of
NF-κB to the nucleus which further generates more ROS in a
feed forward cycle and consecutively activates apoptotic pathway
(Lin and others 2009). Furthermore, evidence indicates that ROS
production activates pERK1/2 (Son and others 2011). Down-
stream from pERK1/2 activation, gene expression is regulated
via phosphorylation of transcription factors, which include the
pro-inflammatory transcription nuclear factor NF-κB (Zhao and
others 2012). Indeed, in the current investigation animals receiving
lead acetate showed an increase in NF-κB along with enhanced
p42/44 expression, the former further induces the expression of
iNOS (Whittle 2003), an event seen in the current investigation
following the receipt of lead acetate. Downstream from iNOS in-
duction, Casp-3 is triggered that mediates apoptosis (Daniel and
others 2013), which is further consolidated by findings from the
current investigation. Moreover, the buildup of iNOS might of-
fer an explanation for the increase in NO earlier observed in this
group in the present report that further contributes to the ox-
idative stress burden (Zhang and Hogg 2004). Various kinds of
antioxidants capable of decreasing NF-κB activity, reduce Casp-
3-mediated apoptosis and consequently lower tissue injury (Lin
and others 2009). The cotreatment of GT extract resulted in the
complete protection against hepatic apoptosis by reduction in the
production of ROS thus decreasing p42/44 NF-κB activity, and
hence suppressing caspase-3. In opposition, animals receiving the
photodegraded product concomitant to lead acetate showed in-
creased NF-κB, iNOS, as well as Casp-3, thus providing further
proof of concept to the shelf stability in the protective effects of
C1714 Journal of Food Science r Vol. 81, Nr. 7, 2016
GTE. This conception is further consolidated by the finding that
PDGT alone increased NF-κB, iNOS, as well as Casp-3, notwith-
standing those receiving PDGT atop of lead acetate.
AST and ALT are sensitive indicators of hepatic injury, which are
elevated in serum consequent to membrane damage and cellular
injury (Drotman and Lawhorn 1978) following ROS generation
and the subsequent cell necrosis (Gaskill and others 2005). Cer-
tainly, AST and ALT activities were elevated in sera of animals
receiving lead acetate which is in agreement with previous reports
(Ibrahim and others 2012; Mehana and others 2012). The latter
effect is further amplified by the photodegraded product signify-
ing a boosted damaging potential leading to further increase in
the leakage of liver enzymes, in contrast to GT extract protecting
against this enzymatic change possibly through the prevention of
intracellular leakage of the enzymes as a result of cell membrane
stability and cellular regeneration (Thabrew and others 1987).
Conclusions
The purpose of stability studies is to provide evidence on the
variation of quality of a drug product with time under the influ-
ence of a variety of environmental factors. Forced degradation help
identify possible routes of degradation and enable the development
of stability-indicating assay protocols. However, inappropriate se-
lection of analysis techniques and lack of correlation between in
vitro and in vivo assay results might lead to elusive conclusions about
quality of nutraceuticals. In this study, an orthogonal analysis pro-
tocol was designed to evaluate the stability of EGCG in a model
GT formulation. Lack of agreement between assay results required
correlation to in vivo activity in animal model. Results indicated
that photodegraded GT samples were not only ineffective but also
harmful to biological systems. Photodegradation products exerted
prooxidant effect rather than the beneficial antioxidant effect.
Author Contributions
MAA HEZ MYA, and NNN designed the study. HHA MAA
MYA and NNN performed the laboratory work. All authors con-
tributed to data analysis and manuscript editing. MAA had pri-
mary responsibility for the final manuscript. All authors read and
approved the final manuscript.
Conflict of interest
Authors declare no commercial conflict of interest.
Ethics statement
This study was carried out in strict accordance with the rec-
ommendations in the Guide for the Care and Use of Laboratory
Animals (Ilar 1996) and was performed in accordance to the ethical
procedures and policies approved by Research Ethical Committee
(REC) of Faculty of Pharmacy, Cairo Univ. Cairo, Egypt. The
REC approved the current protocol and the approval number is
AC 1199. Euthanasia was done under sodium pentobarbital anes-
thesia, and all efforts were made to minimize suffering.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publisher’s website:
Figure S1. Absorption spectra of Folin-Ciocalteu reagent against
water as a blank (––) and of a standard solution of gallic acid after
reaction with Folin-Ciocalteu, measured at 765.0 nm ( ).
Figure S2. Absorption spectra of DPPH reagent against methanol
as a blank ( ) and of GA after reaction with DPPH reagent (––)
Table S1. The percentage loss of TPC, percentage loss of an-
tioxidant activity and percentage loss of EGCG for extracts after
visible light and UV irradiations.
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