Inhibitory property of the Piper betel phenolics against photosensitization-induced biological damages

Article abstract of DOI:10.1016/j.bmc.2007.12.052

The Piper betel phenolics, allylpyrocatechol (APC) and chavibetol (CHV), were found to protect photosensitization-mediated lipid peroxidation (LPO) of rat liver mitochondria effectively, APC being significantly more potent. The better activity of APC vis-a-vis CHV could be attributed to its higher reactivity with ¹O₂, as revealed from the rate constant values of ¹O₂ quenching by the respective phenolics. APC also prevented the detrimental effects of the Type II photosensitization-induced toxicity to mouse fibroblast L929 cells. The results suggest that APC may play an important role in protecting biological systems against damage, by eliminating ¹O₂ generated from certain endogenous photosensitizers.

Full text of DOI:10.1016/j.bmc.2007.12.052

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2932–2938
Inhibitory property of the Piper betel phenolics
against photosensitization-induced biological damages
Soumyaditya Mula,a Debashish Banerjee,b Birija S. Patro,a Sayanti Bhattacharya,b
Atanu Barik,c Sandip K. Bandyopadhyayb and Subrata Chattopadhyaya,*
aBio-Organic Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
bDepartment of Biochemistry, Dr. B.C. Roy Post Graduate Institute of Basic Medical Sciences & IPGME&R, 244B,
Acharya Jagadish Chandra Bose Road, Kolkata 700 020, India
cRadiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
Received 21 September 2007; revised 24 December 2007; accepted 24 December 2007
Available online 31 December 2007
Abstract—The Piper betel phenolics, allylpyrocatechol (APC) and chavibetol (CHV), were found to protect photosensitization-med-
iated lipid peroxidation (LPO) of rat liver mitochondria effectively, APC being significantly more potent. The better activity of APC
1
1
`
vis-a-vis CHV could be attributed to its higher reactivity with O2, as revealed from the rate constant values of O2 quenching by the
respective phenolics. APC also prevented the detrimental effects of the Type II photosensitization-induced toxicity to mouse fibro-
blast L929 cells. The results suggest that APC may play an important role in protecting biological systems against damage, by elim-
1
inating O2 generated from certain endogenous photosensitizers.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
might be useful from the point of view of low cost and
less toxicity.
Photosensitization is a widely occurring phenomenon in
biological systems due to the ubiquitous nature of visible
light and a number of endogenous and exogenous com-
pounds which can act as photosensitizers.1 The poten-
tially damaging event is primarily triggered due to the
generation of various radical and non-radical reactive
oxygen systems (ROS). Generation of free radicals in
skin by solar ultraviolet light accelerates skin cancer,
photoaging, and other light-related skin pathologies.2
Cellular exposure to UV light also leads to iron release
resulting in excessive production of ROS and ultimately
to pathogenesis.3 Photosensitization of the skin during
drug therapy is a very well-known and frequently re-
ported effect.4 Several classes of commonly used chemi-
cals are able to react with the UVA/UVB photons
yielding stable toxic products or producing free radi-
cals.5 Therefore, there is a need to develop suitable for-
mulations that can prevent photo-induced biological
damages. To this end, exploration of edible herbs/plants
that are also credited with various medicinal attributes
The Piper betel plant is widely growing in the tropical
humid climate of South East Asia, and its leaves, with
a strong pungent and aromatic flavor, are widely con-
sumed as a mouth freshener. The leaves are credited
with wound healing, digestive, and pancreatic lipase
stimulant activities in the traditional medicine.6 During
our exploration of non-toxic and affordable herbal
medicinal formulations, the P. betel leaf extract and its
constituent phenolics were found to show impressive
gastroprotective,7 and anti-inflammatory as well as
immunomodulatory properties.8
Recently, we have also found that the P. betel leaf ex-
tract can prevent the photosensitization-induced dam-
ages to lipids and proteins.9 Hence, the present work
was undertaken with the primary aim to: (i) investigate
whether its major antioxidant10 phenolics, chavibetol
(CHV), allylpyrocatechol (APC), are responsible for
the activity, (ii) rationalize the differential potency of
CHV and APC mechanistically, and (iii) evaluate the
protecting efficacy against photosensitization-induced
cellular damage. The chemical structures of CHV and
APC are shown in Figure 1.
Keywords: Cells; Lipids; Photosensitization; Protection.
*
Corresponding author. Tel.: +91 22 25593703; fax: +91 22
25505151; e-mail: schatt@barc.gov.in
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.052

S. Mula et al. / Bioorg. Med. Chem. 16 (2008) 2932–2938
2933
OH
OMe
APC
CHV
a
OH
OH
a
13
12
11
10
9
b
c,e
Chavibetol (CHV)
Allylpyrocatechol (APC)
c
8
c,e
Figure 1. Chemical structures of chavibetol (CHV) and allylpyrocate-
chol (APC).
7
6
5
c
2. Results
4
3
d,e
2.1. Prevention of photosensitization-induced lipid perox-
idation by CHV and APC
2
1
0
The individual protective capacity of CHV and APC
against photo-induced peroxidation of rat liver mito-
chondria was studied by assaying the TBARS and
LOOH formed during the lipid peroxidation (LPO).
Our studies showed that exposure of mitochondria to
methylene blue (MB) plus light led to enhanced forma-
tion of TBARS as a function of illumination time (data
not shown). Exposure of mitochondria to photo-irradi-
ation for 0.5 h led to a significant (p < 0.001) increase in
the TBARS concentration (12.81 0.22 nmol/mg pro-
tein) as against the control value (2.29 0.05 nmol/mg
protein) without photo-exposure.
1
2
3
4
5
Treatments
b
a
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
b
b
When the mitochondria were irradiated for 0.5 h in the
presence of various concentrations (30, 60, and
180 lM) of APC or CHV, significant protection was ob-
served. Even at a very low concentration (30 mM), APC
showed measurable (ꢀ32%, p < 0.01) prevention in
TBARS formation, which increased in a concentra-
tion-dependent manner. The protections offered by 60
and 180 lM of APC were ꢀ48% (p < 0.01) and 86%
(p < 0.001), respectively. The protective capacity of
CHV also showed a similar trend, although it was less
effective than APC at all the test concentrations. For
example, CHV (30, 60, and 180 lM) could prevent
ꢀ22% (p < 0.05), 40% (p < 0.01), and 72% (p < 0.001)
of photo-induced LPO. Under similar conditions, the
protections offered by the positive controls, sodium
azide (10 mM) and vanillin (2 mM), were 73% and
34%, respectively. The results with CHV and APC are
summarized in Figure 2a. The protections offered (for
a 30 min photo-exposure) by some other known antiox-
idants under similar conditions are shown in Table 1.
c
1
2
3
4
5
Treatments
Figure 2. (a) Photosensitization-induced formation of TBARS in rat
liver mitochondria and prevention by CHV and APC. A mixture of
mitochondrial protein (0.5 mg/mL), methylene blue (50 lg/mL) with-
out and with different concentrations of CHV or APC was incubated
for 30 min and the amounts of TBARS were estimated. The values are
means SEM (n = 5). 1, control mitochondria; 2, mitochon-
dria + MB + hm; and 3–5, mitochondria + MB + hm in the presence
of CHV or APC (30, 60, and 180 lM, respectively). ap < 0.001
compared to normal; bp < 0.05, cp < 0.01, dp < 0.001 compared to
photo-irradiated experimental control; ep < 0.05 compared to CHV
treatment. (b) Photosensitization-induced formation of LOOH in rat
liver mitochondria, as revealed from the absorbance at 560 nm and its
prevention by APC, CHV (each 180 lM) and sodium azide (10 mM).
A mixture of mitochondrial protein (0.5 mg/mL), methylene blue
(50 lg/mL) without and with the test samples was incubated for 30 min
and the amounts of LOOH were estimated. The values are mean-
Photo-irradiation of the rat liver mitochondria expect-
edly led to the increased formation of LOOH, which,
however, was prevented by CHV and APC. Following
photo-irradiation for 0.5 h, the LOOH concentration in-
creased more than 2-fold (p < 0.001). This was revealed
from the increase in the absorbance at 560 nm from
0.032 0.003 (unirradiated control) to 0.078 0.009
(after photo-irradiation). Addition of APC, CHV (each
180 lM) or sodium azide (10 mM) to the mitochondria
prior to photo-irradiation reduced the absorbance at
s
SEM (n = 5). 1, control mitochondria; 2, mitochondria + MB + hm;
and 3–5, mitochondria + MB + hm in the presence of APC, CHV or
NaN3, respectively. ap < 0.001 compared to normal; bp < 0.01,
cp < 0.001 compared to photo-irradiated experimental control.
reduction in LOOH formation by 87% (p < 0.001),
61.5% (p < 0.01), and 50% (p < 0.01), respectively, com-
pared to the photo-irradiated sample. The formation of
LPO by photosensitization of rat liver mitochondria,
and its prevention by APC, CHV, and sodium azide
are shown in Figure 2b.
560 nm
0.039 0.006, respectively. These corresponded to
to
0.010 0.003,
0.030 0.005,
and

2934
S. Mula et al. / Bioorg. Med. Chem. 16 (2008) 2932–2938
Table 1. Protective activitiesa of some known antioxidants against
photosenstitization-induced oxidation of rat liver mitochondria
2.3. Steady-state photolysis of APC
The steady-state photolysis of APC in the presence of
visible light + MB, but not with visible light alone, led
to its degradation as revealed from the HPTLC analy-
ses. The extent of degradation increased in a time-
dependent manner. As shown in a typical HPTLC pro-
file (Fig. 4) the photolysis of APC led to the formation
of three major products together with several minor
ones. Two of these products were identified as 1 and 2
and their structures are shown in Figure 5. The photo-
sensitized degradation was not noticed under an aerobic
condition and was markedly suppressed by sodium azide
(data not shown).
Antioxidant (concn)
% Protectionb against lipid peroxidation
Mannitol (100 mM)
Glutathione (10 mM)
20.1 2.12
35.6 4.44
Ascorbic acid (10 mM) 25.7 3.26
SOD (1000 U/assay) 8.4 0.60
a The values are for photo-exposure for 30 min.
b The values are means SEM (n = 5).
2.2. Singlet oxygen scavenging activity of APC and CHV
The luminescence of singlet oxygen at 1270 nm de-
creased progressively in the presence of increasing con-
centrations of CHV and APC. A plot of the inverse of
the lifetime of singlet oxygen against the concentrations
of the test compounds showed excellent linearity
(Fig. 3a and b). The rate constant for total (physical
and chemical) 1O2 quenching (kQ) by a quencher Q
(CHV, APC or NaN3) was determined by the Stern–
Volmer equation: kobs = kd + kQ [Q], where kobs and kd
2.4. Cytotoxicity of APC against L929 cells
The cytotoxicity of APC (5, 10, and 20 lM) against the
L929 cells following incubation of the cells for 20 h was
evaluated by the MTT method.11 The preincubation
time (20 h) of the L929 cells was chosen based on a
time-dependent trial study (data not shown). APC did
not show any cellular toxicity at 5 and 10 lM concentra-
tions. However, at a concentration of 20 lM, it showed
ꢀ25% cell killing. For carrying out the photocytotoxic-
ity experiments, the cytotoxicity of MB (9 and 18 lM)
against the L929 cells was also determined. It was found
that MB (9 lM) was non-toxic to the cells, while its tox-
icity even at a higher concentration (18 lM) was also
1
are the decay rate constants of O2 phosphorescence,
measured in the presence and absence of CHV and
APC, respectively. Under similar conditions, the rate
constants for CHV, APC, and the positive control,
NaN3 were 5.36 · 106, 8.42 · 106, and 2.41 ·
108 MÀ1 sÀ1, respectively.
a
80000
60000
40000
20000
0.000
0.002
0.004
0.006
0.008
concentration (M)
60000
Figure 4. A typical HPTLC chromatogram of the products, obtained
after photolysis of APC for 4.5 h in the presence of MB. The HPTLC
analysis was carried out using silica gel plates using 20% EtOAc/
hexane as the solvent. The products were detected at 310 nm.
b
50000
40000
30000
20000
10000
O
OH
O
HO
O
OH
O
OH
CHO
0.000
0.002
0.004
0.006
0.008
2
1
concentration (M)
Figure 3. Stern–Volmer plot for the reaction 1O2 with (a) APC and (b)
CHV in acetonitrile.
Figure 5. Chemical structures of two of the products formed by the
reaction of 1O2 with APC.

S. Mula et al. / Bioorg. Med. Chem. 16 (2008) 2932–2938
2935
Table 2. Evaluation of cytotoxicity of different concentrations of APC
and MB
Control
APC 5 μM
APC 10μM
Treatment
% Survivala
110
100
90
80
70
60
50
40
30
20
10
0
Control L929 cells
100.2 3.2
96.2 2.8
96.5 5.4
75.7 3.5b
102.2 2.6
95.3 4.3
c
c
c
c
APC (5 lM)-treated L929 cells
APC (10 lM)-treated L929 cells
APC (20 lM)-treated L929 cells
MB (9 lM)-treated L929 cells
MB (18 lM)-treated L929 cells
d
d
a
a
a The L929 cells were treated with different concentrations of APC or
MB and the % survival was determined by the MTT assay. The
values are means SEM (n = 5).
b
b p < 0.05.
marginal (5%). Hence these were chosen for the further
experiments. The results are summarized in Table 2.
2.5. Prevention of photocytotoxicity of L929 cells by APC
0
5
10
15
Irradiation time (min)
For this, the effect of the light plus MB combination on
the proliferation of L929 cells in the presence of APC
was investigated by the MTT assay. The experiments
were carried out at three photo-exposure periods (5,
10, and 15 min) using two different concentrations of
APC (5 and 10 lM) and MB (9 and 18 lM). It was
found that with 9 lM MB, the damage was less (14–
21%) even for a photo-exposure period of 15 min. The
protection offered by APC under this condition was
measurable (6–14%), but not very significant. However,
survival of the L929 cells reduced progressively on
increasing the photo-exposure time in the presence of
MB (18 lM). For example, the percentages of cell sur-
vival were ꢀ67.6, 64.4 (p < 0.05), and 48.9 (p < 0.01),
respectively, for the photo-exposure of 5, 10, and 15.
Preincubation of the cells with APC (5 and 10 lM) im-
proved the cell survival over the entire photo-exposure
period significantly, better protection being observed
with 10 lM of APC. The survival of the L929 cells incu-
bated with APC (10 lM) ꢀ94.2%, 92.2%, and 82.9%
during photo-exposure of 5, 10, and 15 min. The corre-
sponding figures for the APC (5 lM)-pretreatment were
ꢀ89.1%, 88.3%, and 85.4%, respectively. The results
(Fig. 6) revealed that compared to the photo-irradiated,
untreated cells, those treated with APC had significantly
better viability for photo-exposure up to 10 (p < 0.05)
and 15 min (p < 0.001). However, when the cells were
pretreated with a higher concentration APC (20 lM),
the percentages of cellular survival were less (data not
shown). This may be attributed to the cytotoxicity of
APC at the higher concentration. Under the same condi-
tion, vanillin (50 lM) increased the cell viability by only
12.5% for a photo-exposure up to 15 min.
Figure 6. Time-dependent photosensitization-induced killing of L929
cells and its prevention by APC. The L929 cells (2 · 104 cells per well)
in serum free RPMI solution containing APC (0–10 lM) were
irradiated for 0–15 min with visible light in the presence of MB
(18 lM). Following incubation in FCS for 20 h, the cell viability was
determined. The values are means SEM (n = 5). ap < 0.05, bp < 0.01
c
with respect to unirradiated cells; p < 0.05, dp < 0.001 with respect to
the respective irradiated, but non-treated, cells.
conditions,15 causes maximum damage to membrane
lipids, DNA, etc.,16 due to its relatively long half-life
in the cellular milieu. The cellular membrane can play
an important role in the photosensitization. In particu-
lar the peroxidation of polyunsaturated fatty acids,
which are extremely sensitive to ROS, has been sug-
gested as the primary event that, through chain reac-
tions, triggers metabolic pathways leading to
inflammatory response and to cell death.14,17,18 Products
of lipid oxidation can activate membrane phospholipa-
ses19,20 and can induce alterations of the membrane lipid
packing, favoring the penetration of water molecules in-
side the bilayer.21 The induced alterations in membrane
permeability, transport systems, loss of membrane
bound enzymes, etc., can eventually lead to cell lysis
and death under specific conditions.22 Adverse effect of
photosensitization to specific carriers for succinate, cit-
rate, and oxalacetate has been reported.23
Very recently, we have identified10 CHV and APC as the
major antioxidants of the P. betel leaf extract, which
also showed protecting activity against photosensitiza-
tion.9 However, this does not guarantee protective effi-
cacy of CHV and APC against photosensitization,
since different ROS are involved in the latter process.
Consequently, in the present study, the capacity of
CHV and APC in preventing photosensitization-in-
duced LPO in rat liver mitochondria and scavenging
1O2 was assessed. Subsequently, the study was extended
to cellular system using the most active compound,
APC. Using methylene blue as the sensitizer, the extent
of oxidation of lipids by photosensitization was investi-
gated by measuring the amounts of TBARS formed in
3. Discussion
The photosensitization generates several reactive oxygen
species (ROS) via type I (superoxide radicals) and pre-
1
dominantly type II (singlet oxygen, O2)12 processes
leading to damages to various sub-cellular structures
and molecules.13,14 Among these, singlet oxygen, pro-
duced under various normal and pathophysiological

2936
S. Mula et al. / Bioorg. Med. Chem. 16 (2008) 2932–2938
the absence and presence of CHV and APC. Methylene
blue (MB) is an ideal photosensitizer for the investiga-
tion as it does not interfere with spectrophotometric
measurement of microsomal lipid peroxidation. The
combination of MB, light, and oxygen mainly generates
1O2 and can induce oxidative damage in membrane.22 It
was found that while both CHV and APC inhibited the
TBARS formation in a concentration-dependent man-
ner, the efficacy of APC was significantly better
(p < 0.05). Given that the TBA assay is fairly non-spe-
cific, the lipid-protecting capacities offered by a fixed
concentration (180 lM) of the test compounds were also
assayed by measuring LOOH, the relatively unstable
product of LPO. Addition of the compounds to the
mitochondria prior to photo-irradiation could inhibit
the formation of LOOH very effectively. Lipid peroxida-
tion is a complex multistep process wherein the initially
formed lipid radicals get converted to TBARS via the
intermediacy of LOOH and other products. CHV and
APC could efficiently prevent the photosensitization-in-
duced LPO at its various stages, as is revealed from our
results.
to L929 cells was assessed. As such APC was non-toxic
to the mouse fibroblasts L929 cells up to a concentration
of 10 lM, although it showed toxicity at 20 lM concen-
tration. The photosensitization-mediated cell killing
could be prevented very efficiently by APC at very low
concentrations, while another naturally occurring phe-
nolic, vanillin, was much less effective. Interestingly,
the protective effect of APC was more pronounced at
a higher concentration of MB. UV screening by APC
has apparently no connection with its protective effect
against photosensitization since the investigations were
conducted using light devoid of UVB (APC absorbs
only in the UVB region, 204 and 280 nm). There can
1
be little doubt that APC quenches O2 with a high reac-
tivity and thus removes the ROS efficiently from biolog-
ical systems, protecting them under photosensitization
conditions.
It is worth mentioning that the fungicidal and nemato-
cidal activity of P. betel and allergic reaction of eugenol
has been reported earlier.24 Also, reactive oxygen species
(ROS) are crucial for hydroxychavicol toxicity toward
jB epithelial cells.25 Possibly, the toxicity of APC de-
pends on the cell types explaining our results. Earlier,
we have found APC to be strong scavenger of various
ROS in cell free systems.10 Our results are consistent
with previous reports where catechols were found to
produce less ROS compared to other benzene diols.26
In our previous studies, we did not observe any side ef-
fect of the whole P. betel extract or its constituents (APC
and CHV) to rats up to a dose of 0.5 g/kg body
weight.7,10,27
1
Given that sodium azide a specific O2 scavenger could
prevent the photosensitization-induced LPO, the
1
involvement of O2 in the process was apparent. Our
studies on the 1O2 scavenging activity of CHV and
APC also confirmed this. Kinetic measurements re-
vealed that APC was ꢀ1.5-fold more efficient than
CHV in scavenging 1O2. Earlier, APC was found to
scavenge the superoxide radicals more effectively than
CHV.10 These factors, taken together, might account
for their relative efficacy in preventing the photosensiti-
zation-induced LPO.
Overall, the present studies revealed its high potency as
an in vitro antioxidant against photosensitization-in-
duced biological damages. All these results suggested
its possible use as a natural, non-toxic protector against
photosensitization-induced oxidative biological dam-
ages. Currently, there is a growing interest of the
plant-based or herbal medicines all over the World.
However, lack of proper quality control of these often
leads to spurious drugs. Variation in the profile/concen-
trations of the active principles in the plant-based or
herbal preparations primarily contributes to this. It is
well established that factors such as habitation, time of
collection, maturity of the plants, etc., affect the concen-
trations of their bioactive chemical constituents. Hence,
for a proper quality control of the plant-based drugs, it
is essential to identify the bioactive constituents. From
this perspective, the identification of APC and CHV as
the active principles for the preventive property of P. be-
tel leaf extract against photosensitization is extremely
important. The results suggested that to promote P. be-
tel as an effective antioxidant, it is essential to assay the
content of the CHV and especially APC in the samples.
The steady-state photolysis experiments showed that
irradiation of APC with visible light in the presence of
MB led to its time-dependent depletion. The process re-
quired an aerobic condition and was markedly sup-
pressed by sodium azide (data not shown). All these
data confirmed that the photosensitized degradation of
APC is mediated by 1O2. Formation of a number of deg-
radation products in the reaction (as revealed by the
HPTLC analysis) suggested that the degradation pro-
ceeds through a complex process. One of the degrada-
tion products, 1, is produced by an expected oxidative
cleavage of the olefinic function of APC, while 2 is
formed by dimerization of two APC molecules at the
allylic position, followed by oxygenation of the dimeric
product at the ortho-position of the phenolic moieties.
1
The chemical reaction with O2 appears to contribute a
large share to the process of 1O2 quenching by APC. For
the present, however, there is no evidence against a pos-
1
sible involvement of physical quenching of O2 in the
APC–1O2 interaction. Thus, the measured rate constant
may actually be for the total reaction, which includes the
physical quenching, if any, in addition to the chemical
quenching.
4. Experimental
4.1. Materials
Subsequently, we extended the work to cellular system
wherein the protective activity of the best candidate,
APC, against the photosensitization-induced damage
Following our reported procedure APC and CHV were
isolated from the methanol extract of P. betel leaves and

S. Mula et al. / Bioorg. Med. Chem. 16 (2008) 2932–2938
2937
fully characterized.10 Ascorbic acid and 2-thiobarbituric
acid (TBA) (both from Himedia Lab. Pvt. Ltd, India),
ethylenediamine tetraacetic acid (EDTA) (E. Merck, In-
dia), trichloroacetic acid (TCA) (Thomas Baker, India),
and potassium phosphate, sodium azide, KOH, and
HCl (all from SRL, India) were used. Tetraethoxypro-
pane, guanidine hydrochloride, glutathione, a-tocoph-
erol, Superoxide dismutase (SOD) (type I, from bovine
liver, sp. Act. 26,000 U/mg protein), methylene blue
(MB), hematoporphyrin, RPMI 1640, thizolyl blue tet-
razolium (MTT), fetal calf serum (FCS), and penicillin
were obtained from Sigma Chemicals, USA. All other
reagents were of analytical grade. Mouse fibroblasts
L929 cells were obtained from National Centre for Cell
Science (NCCS), Pune, India.
positive controls. The lipid hydroperoxide (LOOH)
was assayed as described earlier.27
4.4. Assay of singlet oxygen scavenging by CHV and APC
Singlet oxygen was produced by photosensitization of
hematoporphyrin (120 lM, OD at 532 nm 0.521) in ace-
tonitrile, and its luminescence at 1270 nm in the absence
and presence of various concentrations of CHV and
APC was detected with a transient luminescence spec-
trometer model TL 900 (Edinburgh Instr., UK). Hema-
toporphyrin was excited using the pump beam from an
Nd-YAG-pumped optical parametric oscillator laser
(5 ns full-width half-maximum pulse), and the signals
were passed through a cutoff filter.
Stock solutions of ascorbic acid and EDTA were pre-
pared in deaerated water just prior to use. The test sample
was used as an aqueous solution containing 0.1% ethanol.
All solutions were made with triply distilled water.
4.5. Assay of photosensitized degradation of APC
A solution of APC (0.13 M) and MB (100 mM) in water
(containing 0.1% methanol) was exposed to visible light
as above. Aliquots were taken from the samples at dif-
ferent intervals of irradiation and subjected to high-per-
formance thin layer chromatography (HPTLC) on a
silica gel plate C18 analytical column (YMC,
25034.6 mm id) using 20% EtOAc/hexane as the solvent.
The extent of APC degradation was measured by a de-
crease in the area of APC peak measured at 280 nm.
4.2. Animals and preparation of mitochondria
The rats were bred in the BARC Laboratory Animal
House Facility and procured after obtaining clearance
from the BARC Animal Ethics Committee. All the exper-
iments were conducted with strict adherence to the ethical
guidelines laid down by the Committee for the Purpose of
Control and Supervision of Experiments on Animals
(CPCSEA) constituted by the Animal Welfare Division
of Government of India on the use of animals in scientific
research. Mitochondria were isolated from male Wistar
rats weighing 250 20 g as described earlier.27
The preparative scale photolysis was carried out for 24 h
in 4 sets each containing APC (10 mg), the other condi-
tions remaining same. The reaction mixture was ex-
tracted with CHCl3 (3· 5 mL), the combined organic
extracts were dried and concentrated in vacuo. The res-
idue was subjected to preparative thin layer chromatog-
raphy (silica gel, 10% EtOAc/hexane) to isolate 1 and 2.
4.3. Photosensitization-induced lipid peroxidation assay
The system for exposing mitochondria to photosensitiza-
tion was simple, physiologically relevant, and similar to
that described earlier.9 In brief, the reaction mixture (final
volume 1.0 mL) containing mitochondrial protein (final
concentration 0.5 mg/mL), MB (50 lg/mL), and APC or
CHV (0–180 lM, final concentration) in KH2PO4–
KOH buffer (pH 7.4) was kept in a trap maintained at
37 ꢁC and irradiated for 30 min with a 100 W tungsten
lamp. The distance from the light source and the trap
was 15 cm. The light intensity of the system, as measured
4.5.1. 2-(3,4-Dihydroxyphenyl)ethanal (1). IR: 2720,
1
1720 cmÀ1; H NMR: d 4.20 (d, J = 2.0 Hz, 2H), 5.35
(br s, 2H), 7.26–7.36 (m, 1H), 7.51–7.56 (m, 1H), 7.69–
7.78 (m, 1H), 9.98 (t, J = 2.0 Hz, 1H); MS (m/z, rel.
int.): 152 (M+, 48), 125 (100).
4.5.2. 3,4-Bis[4-hydroxy-5-oxo-6-peroxocyclohexa-1,3-
dienyl]hexa-1,5-diene (2). IR: 3443, 1698 cmÀ1
;
1H
NMR: d 3.23–3.32 (m, 2H), 3.77 and 3.82 (two s, 2H),
4.96–5.11 (4H), 5.81–5.92 (m, 2H), 6.43–6.62 (m, 2H),
6.77–6.85 (m, 2H); MS (m/z, rel. int.): 327 (M+1+, 16),
295 (18), 162 (100).
by a Lux meter, was calculated to be 32.45 W mÀ2
.
The light-induced lipid peroxidation of the rat liver
mitochondria was assayed by the TBA method de-
scribed elsewhere.28 For estimating the thiobarbituric
acid reactive substrates (TBARS), the reactants were
heated for 15 min on a boiling water bath with the
TBA reagent (0.5% TBA/10% TCA/6 mM EDTA/
0.63 M HCl). After cooling, the precipitate formed was
removed by centrifugation at 1000g for 10 min. The
absorbance of the sample was determined at 532 nm
against a blank that contained all the reagents except
the mitochondria. Malonaldehyde standard was pre-
pared by the acidic hydrolysis of tetraethoxypropane.
For correction of endogenous TBARS, fresh samples
were boiled without photo-irradiation, and the values
were subtracted. Various antioxidants were used as the
4.6. Cell line maintenance and evaluation of cytotoxicity
of APC
The L929 cells were routinely seeded at a density of
0.1–3 · 106 cells/mL and grown in RPMI 1640 medium
supplemented with 10% heat-inactivated FCS, 2 mM
glutamine, 100 IU/mL penicillin, and 100 lg/mL strep-
tomycin in a humidified 5% CO2 atmosphere at 37 ꢁC.
Cells were passaged every 3–4 d so as to maintain the
cell density below 0.4 · 106 cells/mL. The cell density
and viability were determined by the Trypan blue dye
exclusion test. Subcultures were obtained by trypsiniza-
tion (0.25% trypsin in PBS). The cells were discarded
after 3 weeks of subculture.

2938
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MTT dye reduction assay.11 The L929 cells (2 · 104 cells
per well) were grown in 96-well microtiter plates for 2 h
in serum free RPMI solution (180 lL) containing APC
(0, 5, 10, and 20 lM). Fetal calf serum (20 lL) was
added to the cells and incubated for another 20 h. Sub-
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washed once with PBS. The cell viability was deter-
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18 lM) in PBS (200 lL) and incubated for 2 h. After
removal of MB solution, the cells were washed once
with PBS (200 lL), and 100 lL PBS was added. The
microplate was covered with a glass plate and irradi-
ated with a 100 W tungsten lamp at room temperature
for different times (0, 5, 10, and 15 min). Subsequently,
PBS solution was removed and the cells were incubated
with freshly added RPMI solution containing 10% FCS
(200 lL) for 72 h. To determine cell toxicity, the med-
ium was removed by aspiration and 100 lL of PBS
containing MTT (0.5 mg/mL) was added to each well.
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5. Statistical analysis
The data were analyzed using a paired t-test for the
paired data or one-way analysis of variance (ANOVA)
followed by a Dunnett’s multiple comparisons post test.
In addition, Bonferroni correction was also carried out
for knowing the simultaneous statistical inference
among the groups under investigations. The IC50 values
of the test samples were estimated using the Probit anal-
ysis and the significance level of the analyses was also
investigated by the chi-square test. A probability value
of p < 0.05 was considered significant.
References and notes
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