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ISSN: 2320-5407 Int. J. Adv. Res. 5(10), 1257-1281
1257
Journal Homepage: - www.journalijar.com
Article DOI: 10.21474/IJAR01/5645
DOI URL: http://dx.doi.org/10.21474/IJAR01/5645
RESEARCH ARTICLE
EFFECT OF DIFFERENT HIBISCUS SABDARIFFA EXTRACTS ON GLUTATHIONE AND ITS
RELATED ENZYMES IN MAMMALIAN LUNG TISSUE AND CULTURE CELLS UNDER INDUCED
OXIDATIVE STRESS.
Hamed R R1, Hussein F E2, *Guneidy R A1, Awad H3 and Ali E A A1.
1. Department of Molecular Biology, National Research Centre, Cairo, Egypt.
2. Department of Cytology and Histology, Faculty of Science, Cairo University.
3. Department of Tanning Materials and Leather Technology, National Research Centre, Cairo, Egypt.
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Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History
Received: 17 August 2017
Final Accepted: 19 September 2017
Published: October 2017
Key words:-
Oxidative stress, polyphenols,
antioxidant and prooxidant abilities,
antioxidant enzymes, glutathione, lung
tissue, fibroblast.
Oxidative stress refers to the excessive ROS production and the
antioxidant system cannot be able to neutralize them. HPLC analysis of
aqueous, 30% and 70% ethanol extracts of the polyphenol rich H.
sabdariffa calyx (the highest phenolic, flavonoid, anthocyanin contents
and antioxidant capacities) showed that cyanidin 3-O-glucoside
chloride, chlorogenic acid and delphinidin derivatives were the major
constituents. Production of H2O2 (prooxidant capacity) increased by
increasing the ethanol extracts concentration and incubation time at pH
7.4. Gallic acid was the greatest in the production of H2O2.
Pretreatment of rats with H. sabdariffa ethanol extract followed by tert-
butyl hydroperoxide (t-BHP) injection partially blocked the effect of
the t-BHP, as indicated by the oxidative stress markers. In rat lungs
homogenates, glutathione, malondialdehyde levels and lactate
dehydrogenase activity returned back almost to the normal level. The
antioxidant enzymes catalase and glutathione S-transferase (GST) were
increased, while glutathione peroxidase (GPx) and glutathione
reductase (GR) were not affected. Histological studies indicated the
presence of sever lung injury with t-BHP and H. sabdariffa extracts. In
human lung fibroblast cells, H. sabdariffa extracts (150 µg and 300 µg)
have almost no effect on GST activity while, resolved the inhibitory
effect of H2O2 on GR and GPx activity.
Copy Right, IJAR, 2017,. All rights reserved.
……………………………………………………………………………………………………....
Abbreviations:-
CAT:
Catalase
CDNB:
1-chloro-2, 4-dinitrobenzene
DMSO:
Dimethylsulfoxide
DPPH:
1,1 Diphenyl -2-picryl-hydrazyl
GPx:
Glutathione peroxidase
GR:
Glutathione reductase
GSH:
Reduced glutathione
GST:
Glutathione S- transferase
Corresponding Author:- Hamed R R.
Address:- Department of Molecular Biology, National Research Centre, Cairo, Egypt.
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IC50:
Inhibitor concentration causing 50% inhibition
LDH:
Lactate dehydrogenase
MDA:
Malonyldialdehyde
MTT:
3, (4, 5-dimethylthiazol-2-yl) 2, 5 diphenyltetrazoliumbromide
ROS:
Reactive oxygen species
SE:
Standard error
TAC:
Total anthocyanin content
TBA:
2-thiobarbituric acid
t-BHP:
Tert-butyl hydroperoxide
TFC:
Total flavonoid content
TPC:
Total phenolic content
Introduction:-
In the biological system, reactive oxygen species (ROS) are highly reactive molecules resulted from normal cellular
metabolism and several environmental factors. Several mechanisms counteract oxidative stress by producing
antioxidants. A shift in the balance between oxidants and antioxidants in favor of oxidants is termed as “oxidative
stress”. Oxidative stress refers to the excessive ROS production and the antioxidant system cannot be able to
neutralize them. This imbalanced protective mechanism can lead to the damage of cellular molecules such as DNA,
proteins, and lipids. Reactive oxygen species such as hydroxyl radicals, superoxide radicals and the non-radical
hydrogen peroxide play important roles in the pathophysiology of a large number of diseases and oxidative damage
(Pham-Huy et al., 2008).
In the process of cell respiration, mitochondrial oxidative metabolism produces ROS species and organic peroxides
(Hussain et al., 2016). Lung is a site of major ROS production having a large surface that is constantly in contact
with air oxygen and air pollutants that influenced the structure and function of the lung surfactant system (Juvin et
al., 2002 and Anseth et al., 2005). Lung cells are recruited when air pollutants induce lung inflammation and
released ROS, enhancing inflammation associated with tissue damage and other pathological effects (Macchionea
and Garcia, 2011).
Reactive oxygen species has led to the evolution of an antioxidant defense system to protect lung tissue from
substantial damage (Tkaczyk & Vìzek, 2007). Antioxidants are naturally occurring substances that combat
oxidative damage in biological entities. Antioxidants (reducing agent) are separated into two classes based on their
mode of action as enzymatic and non-enzymatic antioxidants (Abd El-Aal, 2012).
Glutathione (GSH) is an important protective antioxidant against free radicals and other oxidants and has been
implicated in inflammatory responses, modulation of redox-regulated signal transduction, regulation of cell
proliferation, remodeling of the extracellular matrix (ECM), apoptosis and mitochondrial respiration. The GSH
redox system is crucial in maintaining intracellular GSH/GSSG homeostasis, which is important to the normal
cellular physiological processes, and represents one of the most important antioxidant defense systems in lung cells.
This system uses GSH as a substrate in the detoxification of peroxides such as H2O2 and lipid peroxides, a reaction
which involves glutathione peroxidase (GPx). This reaction generates GSSG which is then reduced to GSH by
glutathione reductase (GR) in a reaction requiring NADPH (Rahman and MacNee, 2000). The most important
antioxidant enzymes in lungs are superoxide dismutase (SOD), catalase (CAT) and GPx (Patra et al., 2008). Other
antioxidants, such as glutathione-S-transferase (GST) and the redox proteins have crucial roles in the antioxidant
defense system (Birben et al., 2012).
Phytochemicals such as polyphenols have been reported to be able to modulate ROS production, oxidative stress and
inflammatory processes. Antioxidants such as polyphenols are natural compounds present in plants with numerous
biological activities (Hussain et al., 2016). On the other side, antioxidants can exert a prooxidant activity in the
presence of transition metal ions, generated phenoxyl radicals and ROS; induce cellular lipid peroxidation, DNA
damage and apoptosis. Recently, the prooxidant property of the antioxidant compounds is not necessarily harmful
for the biological systems and can be used therapeutically to treat oxidative stress condition (e.g. cancer treatment)
(Eghbaliferiz and Iranshahi, 2016).
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Species of the genus Hibiscus (family: Malvaceae) includes more than 300 species of annual herbs, shrubs or trees
(Da-Costa-Rocha et al., 2014). Pharmacological investigations of the genus Hibiscus indicated their activities as
anti-hypertensive, anti-inflammatory, hepatoprotective, anti-tumor, anti-diabetic, anti-convulsivant,
immunomodulators, antioxidant and anti-mutagenic agents (Rosa et al., 2007). The calyx of Hibiscus sabdariffa is a
tropical plant, local name karkade (Arabic), roselle or red sorrel (English), rich in phenolic compounds with
antioxidant activity and used in folk medicines against many complaints (Hirunpanich et al., 2006; Wang et al.,
2011; Hassan et al., 2014).
This study was concerned with the investigation of the effect of different H. sabdariffa extracts on glutathione and
its related antioxidant enzymes in rat lung tissue and human lung fibroblast (MRC-5) subjected to oxidative stress
inducer. It was also concerned with the investigation of their possible roles in the antioxidant and prooxidant
abilities involved in their biological potential in protection from oxidative stress condition.
Materials and Methods:-
Materials:-
Chemicals:-
Folin-Ciocateu's (FC) reagent, Bovine serum albumin fraction ΙV (BSA), reduced glutathione (GSH), oxidized
glutathione (GSSG), and 1-chloro-2, 4-dinitrobenzene (CDNB) were purchased from Merck Company. Delphinidin
HCl, cyanidin HCl, α-tocopherol, ascorbic acid, 1, 1 diphenyl-2-picryl-hydrazyl (DPPH), 3, 4, 5-trihydroxy benzoic
acid (gallic acid), nicotinamide adenine dinucleotide phosphate reduced form (NADPH) were products of Sigma-
Aldrich Company. Doxorubicin, [3, (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazoliumbromide] (MTT) and
xylenol orange were purchased from Sigma Chemical Company. Dulbecco’s Modified Eagle's/ F12 Medium
(DMEM-F12), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serums (FBS) were purchased
from Biowest. Butylated hydroxytoluene and ammonium ferrous sulphate were purchased from El-Gomhouria
Company.
Plant material:-
The dried calyces of H. sabdariffa (family Malvaceae) were collected from different localities and markets at
various periods from 2013 to 2016. The plants were botanically identified by the Botany Department, National
Research Center, Egypt.
Methods:-
Phytochemical analyses:-
The dried calyces of H. sabdariffa were grounded, powdered and homogenized at a ratio 1:50 (w/v) with aqueous,
30% and 70% ethanol. The mixture was allowed to stands at 95°C for 15 min. The plant homogenates were
centrifuged at 1000 g for 10 minutes, filtered through Whatman No. 1 filter paper and saved at -20°C for further
analyses. Total phenolic content (TPC) was determined using the standard colorimetric method described by
Singleton and Rossi, 1965. The total phenolic content of plant extract was represented as mg of gallic acid
equivalent (GAE)/g calyx using the standard gallic acid calibration curve. The total flavonoid content (TFC) was
measured according to the colorimetric method of Dewanto et al., 2002. Total flavonoid content was expressed as
mg rutin equivalent (mg rutin/g calyx). Total anthocyanins (TAC) were measured according to the method described
by Fuleki and Francis, 1968. The free radical scavenging activity of H. sabdariffa extract was determined using
DPPH (1, 1 diphenyl -2-picryl-hydrazyl) according to the method described by Brand-Williams et al., 1995, with
some modification (Leong and Shui, 2002). IC50 concentrations were calculated after constructing the percent
inhibition versus log extract concentrations curve.
Determination of H. sabdariffa phenolic and anthocyanin compounds using high performance liquid
chromatography analysis:-
H. sabdariffa extracts and hydrolysable calyces were subjected to high performance liquid chromatography (HPLC)
analysis in the National Research Center, central lab, Cairo, using an Agilent Technologies 1100 series liquid
chromatograph equipped with an auto sampler and a diode-array detector (Lee et al., 2005 & Kim et al., 2006).
Determination of hydrogen peroxide production (prooxidant activity)
The ability to produce hydrogen peroxide (H2O2) by different H. sabdariffa extracts (30, 70% ethanol and aqueous)
and some phenolic compounds were investigated in sodium phosphate buffer pH 7.4, deionized water and RPMI
1640 media using the ferrous ion oxidation – xylenol orange (FOX) assay (Long et al., 1999 and Banerjee et al.,
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2003). The FOX assay was calibrated using a standard H2O2 solution to cover the range of 2-20µM. A known
volume of different H. sabdariffa extracts (500µl) were added to 500µl of deionized water and 100mM sodium
phosphate buffer, pH 7.4. The reaction mixtures were incubated at 37°C for 12, 36 and 48 h with shaking in the
dark. Different H. sabdariffa extracts (50, 150, 300 µg/ml) diluted in RPMI 1640 media incubated at 37°C for the
indicated period up to 48h with shaking in the dark. Different phenolic compounds (250 µM) such as gallic acid,
protocatechuic acid, caffeic acid, catechin. quercetin, delphinidin, α-tocopherol and the powerful antioxidant
standard compound (citric acid) were incubated in 50 mM sodium phosphate buffer, pH 7.4 at 37°C for 12, 36 and
48h with shaking in the dark.
Biological materials:-
Experimental animals:-
Fifty adult male albino rats weighing between 100-120 g were obtained from the animal house of the National
Research Center, Giza, Egypt. The animals were kept under a 12 h light-dark cycle at room temperature. All animals
were allowed to adapt to the environment for 2 weeks before the beginning of the experiment for normalization of
laboratory condition.
Experimental Design:-
The rats were divided into 6 groups and each group includes 8 animals. Group 1: control group, healthy rats. Group
2: was given distilled water only for 5 consecutive days, on day 5, (0.2 mmol/kg body weight) of tert-
butylhydroperoxide (t-BHP) was injected intra-peritoneal. Group 3, 4: were orally administered daily with aqueous
extract of H. sabdariffa (250 mg/kg body weight) for 5 consecutive days, on day 5, (0.2 mmol/kg body weight) of t-
BHP was injected intraperitoneal to group 4. Group 5, 6: were orally administered daily with 30 % ethanol extract
of H. sabdariffa (250 mg/kg body weight) for 5 consecutive days, on day 5, (0.2 mmol/kg body weight) of t-BHP
was injected intraperitoneal to group 6. The rats were sacrificed after 18 h of t-BHP intra-peritoneal injection. The
animals were killed. The lungs were immediately removed and weighed and washed using ice-cold saline. Tissues
of the lungs were homogenized with ratio 1:2 (w/v) in 25mM Tris- HCl buffer, pH 8, containing 1mM EDTA and
2mM β-mercaptoethanol. The lung homogenates were centrifuged at 10,000 xg for 20 min at 4°C and and stored at
−20°C for further analyses.
Cell culture:-
Human lung fibroblast cells (MRC-5) were purchased from Vacsera, Dokki , Egypt and cultured in DMEM-F12
medium which was supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100U/ml streptomycin.
Growth curves for MRC-5 was determined under baseline conditions prior to investigation of cytotoxicity. The
cytotoxicity effect of 30% ethanol extracts of H. sabdariffa against MRC-5 was investigated using MTT [3, (4, 5-
dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide] (MTT assay) assay, which yields a blue formazan product
in living cells, but not in dead cells (Kang et al., 2005).
Experimental Design:-
For experiments, cells were cultured in 75 cm2 flasks supplemented with 10% heat-inactivated FBS, 100U/ml
penicillin and 100U/ml streptomycin and incubated at 37°C, 5% CO2 until confluent (80-90٪). The flasks were
subjected to eight different treatments. 30% ethanolic extracts of H. sabdariffa were dissolved in DMSO (0.5%, v/v,
final concentration) and added to culture medium to achieve the differently designed concentrations. The cells pre-
treated with fresh media without serum containing the following different concentrations: 30% ethanol extracts of H.
sabdariffa (150, 300µg/ml), hydrogen peroxide (0.05, 1mM/ml), 30% ethanol extracts of H. sabdariffa (150µg/ml)
containing 1mM/ml of hydrogen peroxide, 30% ethanol extract of H. sabdariffa (300µg/ml) containing 0.05mM/ml
hydrogen peroxide, different possible controls as DMSO only and control (the media alone without serum). The
cells were incubated for 24 h at 37°C, 5% CO2 .Media was removed and the cells were harvested (Awad et al.,
2014). After incubation, the medium was removed. Cells were scraped. The cells washed twice with cold phosphate
buffer saline. Cells were lysed in 0.1 M potassium phosphate buffer, pH 8 containing 5mM EDTA and 5 mM β-
mercaptoehanol.The lysate was sonicated for 30 sec three times, centrifuged at 2000 rpm and preserved at -20°C for
further analyses.
Biochemical analyses:-
Protein concentration was determined by the method of Bradford, 1976 using bovine serum albumin as a standard.
The total GSH was measured calorimetrically using the method of Saville, 1958. Level of lipid peroxidation was
determined by measuring the formation malondialdehyde (MDA) using the method of Ohkawa et al., 1979.
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Enzyme Assays:-
The Glutathione S- transferase (GST) activity was determined according to the method described by Habig et al.,
1974. Glutathione peroxidase (GPx) was measured according to the method described by Weinhold et al., 1990.
The Glutathione reductase (GR) activity was determined following the decrease in absorbance at 340 nm according
to the method described by Zanetti, 1979.
Catalase (CAT) activity was assayed according to the method described by Aebi, 1984. Total Lactate dehydrogenase
(LDH) activity was measured as described by Fountain et al. (1970).
Histological analysis:-
The lung tissue of the experimental rats were immediately removed and fixed in 10% neutral-buffered formal saline
for 72 h at least. Serial sections of 6 µm thick were cut and stained with Haematoxylin and eosin (Carleton, 1980).
Statistical analysis:-
Data were analyzed using means ± standard deviation. Differences between two groups were determined by t-test. P
< 0.05 significant, P < 0.01 highly significant and P > 0.05 insignificant.
Resulte:-
Phytochemical analyses:-
The results in Table 1 showed that the 70% ethanol extract of H. sabdariffa at 95°C for15 min have the highest
phenolic content (39.19 ± 6.63 mg gallic acid equivalent /g dry calyx), flavonoid contents (1.33 ± 0.09 mg rutin
equivalent/g dry calyx) and anthocyanin content (9 ± 3.8 mg anthocyanin/g dry calyx). The antioxidant capacity
using DPPH scavenging activity showed that 30% ethanol and aqueous H. sabdariffa extracts at 95°C have a
powerful antioxidant capacity with IC50 values (5.68 ± 0.9 mg dry calyx /ml and 6.44 ± 1.163 mg dry calyx/ml,
respectively) compared to 70% ethanol H. sabdariffa extract (IC50=11.04 ± 3.3 mg dry calyx /ml).
Analysis of H. sabdariffa phenolic and anthocyanin compounds using high performance liquid
chromatography analysis:-
HPLC analysis of H. sabdariffa calyx phenolic compounds extracted with 30% ethanol, 70% ethanol, H2O and
hydrolyzed calyx were monitored at 280 nm, 320 nm and 360 nm. The results showed that chlorogenic acid was the
highest concentration in the aqueous, 70% ethanol and 30% ethanol extracts representing 3.304, 1.543 and 1.105
mg/g calyx, respectively. Considerable amounts of caffeic acid were also observed in the three examined extracts.
Alcoholic extraction of H. sabdariffa calyx indicated the presence of protocatechuic acid, gallic acid, quercetin and
kaempferol in a concentration ranged from 0.0312 to 0.355 mg/g calyx. Protocatechuic acid and kaempferol were
not found in aqueous extract. Cinnamic acid (0.0118 mg/g calyx) and sinapic acid (0.0113 mg/g calyx) were found
only in aqueous extract. Hydrolysis of the calyx caused a decrease in the concentration of catechin to 0.2874 mg / g
calyx. However, caffeic acid (2.088 mg/g calyx) and quercetin (0.696 mg/g calyx) increased in concentration by
hydrolysis of H. sabdariffa calyx. Chlorogenic acid, rutin and kaempferol could not be detected after hydrolysis. H.
sabdariffa pigment has been reported to be rich in delphinidin-3-sambubioside (70% of the anthocyanins) and
cyanidin-3-sambubioside as the major pigments, with delphinidin-3-monoglucoside and cyanidin-3-monoglucoside.
The content of cyanidin 3-O-glucoside chloride in the 30% ethanol , 70% ethanol and aqueous extracts of H.
sabdariffa was found to be 13.8, 14.29, 17.7 mg/g calyx, respectively. The content of delphinidin derivative
determined in 30% ethanol, 70% ethanol and aqueous extract of H. sabdariffa was found to be 2.08, 1.653 and1.69
mg/g calyx, respectively (Table 2).
Hydrogen peroxide production (prooxidant activity):-
For the three H. sabdariffa examined extracts, no H2O2 was produced by incubation in deionized water at 37°C up to
48 h. 30% and 70% ethanol extracts produced H2O2 in 100 mM phosphate buffer, pH 7.4 after 24 h of incubation
(0.58 ± 0.12 µg/ml and 5.9 ± 0.28 µg/ml, respectively) and its production increased by increasing the incubation
time to 48 h (2.3 ± 0.33 µg/ml and 7.9 ± 0.05 µg/ml, respectively). Production of H2O2 of the three H. sabdariffa
extracts was observed in RPMI 1640 media only at a concentration more than 50 µg/ml. After 48 h of incubation,
H2O2 concentration was found at the concentration of 150 and 300 µg/ml of the aqueous extract (0.21 µg/ml, 0.45
µg/ml, respectively) (Fig.2 b). H2O2 concentration was 0.54 µg/ml and 0.45 µg/ml with 300 µg/ml of both H.
sabdariffa alcoholic extracts after 24 h incubation (Fig.2 a). After 48h of incubation H2O2 generation was increased
with 30% and 70% ethanol extracts at concentrations of 150 µg/ml (0.26 µg/ml and 0.53 µg/ml, respectively) and
300 µg/ml (0.93 µg/ml and 0.92 µg/ml, respectively) (Fig.2 b). The ability of some phenolic compounds (250µM)
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which are abundant in H. sabdariffa extracts as gallic acid, protocatechuic acid, caffeic acid, catechin, quercetin,
delphinidin, α-tocopherol and the standard antioxidant compound citric acid was incubated in pH 7.4 at 37°C. As
shown in Fig.3, all polyphenols and the standard antioxidant compound citric acid produced amount of H2O2 in
phosphate buffer after 24h. The production of H2O2 by gallic acid was the greatest. The yield of H2O2 was in the
following decreasing order gallic acid > caffeic acid > quercetin > delphinidin > protocatechuic acid > catechin >
citric acid > α-tocopherol. The production of H2O2 of all the examined phenolics was increased by increasing the
period of incubation time to 48h, except quercetin and protocatechuic acid. They reached almost the same level at
the equivalent concentration after 36h of incubation (Fig.4).
Animal study:-
Effect of H. sabdariffa extracts on some oxidative stress, lipid peroxidation and tissue damaged biomarkers:-
The lung non-enzymatic antioxidant defense was evaluated by total glutathione (GSH) level (Table 3). Rat group
injected with t-BHP showed significant decrease )0.05< p < 0.1 ( in the level of GSH in lung homogenate (85.35 ±
9.4 nmol /g tissue) compared with the control group (111.56 ± 9.06 nmol /g tissue). Oral administration with H.
sabdariffa ethanol extract showed highly significant decrease (p < 0.001) in the concentration of GSH (62.55 ± 6.63
nmol /g tissue) compared to the control group. However oral administration with H. sabdariffa aqueous extract
significantly (p < 0.05) increased the concentration of GSH (126.92 ± 20.74 nmol /g tissue) compared to the control
group. Also the t-BHP treated rats after oral administration with aqueous and ethanol extracts showed significant
increase in the concentration of GSH (135.9 ± 9.3 nmol/ g tissue and 112.45 ± 8.43 nmol /g tissue, respectively )
compared to the t- BHP treated group.
The effect of H. sabdariffa extracts on t- BHP induced oxidative stress in rats using malondialdehyde (MDA) as a
marker of lipid peroxidation are shown in Table 3. The MDA levels insignificantly decreased (p > 0.1) from 1647.8
± 133 to 1329.6 ± 115.8 nmol/ g tissue in lung homogenate of rats treated with t-BHP. Pretreatment with aqueous
extract of H. sabdariffa insignificantly decreased the MDA level (p > 0.1) in the lungs injected with either t-BHP or
not (1451.7 ± 133.8 nmol/ g tissue, 1453.8 ± 144.7 nmol/ g tissue, respectively) compared to the control group. The
reverse is true for the rats treated with H. sabdariffa ethanol extract where insignificant increase (p > 0.1) in the
MDA level was detected (2124.9 ± 225 nmol/ g tissue) compared to control group. However, pretreatment with the
ethanol extract of H. sabdariffa before injection of t-BHP significantly decreased )0.05< p < 0.1) the MDA level
(1227.6 ± 161.9 nmol/g tissue) compared to t-BHP treated rats.
Lactate dehydrogenase (LDH) activity in all the experimental groups was increased (Table 3). Significant increase
in LDH activity was observed in t-BHP treated rats (p < 0.01) and rats administrated with aqueous (p < 0.05) H.
sabdariffa extract (99.14 ± 11.99 unit/ g tissue, 80.14 ± 9.66 unit/ g tissue, respectively) compared to the control
group (57.56 ± 3.79 unit/ g tissue). Treatment with ethanol H. sabdariffa extract showed insignificant increase in the
LDH activity (63.58 ± 5.7 unit/ g tissue, p > 0.1) compared to the control group. In rats injected with t-BHP ,
pretreatment with aqueous and ethanol H. sabdariffa extracts, LDH activity insignificantly decreased ( 84.98 ±
7.56 unit/ g tissue, 75.89 ± 6.16 unit/ g tissue, respectively ) compared to the t-BHP treated rats.
Significant increase in protein concentration (p < 0.01) was observed in the t-BHP treated rats and rats administrated
with either aqueous or ethanol H. sabdariffa extract (55.5 ± 7.2 mg/ g tissue, 63.26 ± 8.2 mg/ g tissue, 58.6 ± 5.6
mg/ g tissue, respectively) compared to the control group (31.5 ± 3.4 mg/ g tissue). Pretreatment with ethanol or
aqueous extracts of H. sabdariffa before injection of t-BHP insignificantly increased the protein concentration (66.6
± 7.2 mg/g tissue, 64.7 ± 7.8 mg/g tissue, respectively) compared to the t-BHP treated rats (Table 3).
Effect of H. sabdariffa extracts on the activity of the antioxidant enzymes:-
In the lung tissue homogenates of in the t-BHP treated rats insignificant decrease in glutathione S-transferase (GST)
activity was observed (1.199 ± 0.063 unit/ g tissue) compared to the control group (1.388± 0.21 unit/ g tissue, p >
0.1). Pretreatment with aqueous extract of H. sabdariffa before injection with t-BHP insignificantly increased the
GST activity (1.229 ± 0.149 unit/ g tissue, p > 0.1) compared to the t-BHP treated rats (1.199 ± 0.063 unit/ g tissue).
Treatment with H. sabdariffa aqueous extract have almost no effect on GST activity (1.308 ± 0.139 unit/ g tissue)
when compared to control group. Oral administration of H. sabdariffa ethanol extract insignificantly increased the
GST activity in lung tissue of rats either injected with t-BHP or not (1.313 ± 0.116 unit/ g tissue, 1.488 ± 0.143 unit/
g tissue, respectively, p > 0.1), (Table 4).
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Decrease in the activities of glutathione peroxidase (GPx) was observed in lung tissue of all the examined groups
(Table 4). GPx activity significantly decreased in the t- BHP treated rats (3.22 ± 0.324 unit/ g tissue, p < 0.01) and
that administered aqueous extract of H. sabdariffa (3.11 ± 0.291 unit/ g tissue, p < 0.05) compared to the control
group (5.616 ± 0.917 unit/ g tissue). Insignificant decrease in the GPx activity was observed in rats administered 30
% H.sabdariffa ethanol extract (3.68 ± 0.705 unit/ g tissue) compared to the control group. Insignificant increase
was observed in the GPx activity of both aqueous and ethanol H. sabdariffa extracts before injection with t-BHP
(3.84 ± 0.31 unit/ g tissue, 3.46 ± 0.433 unit/ g tissue, respectively) compared to the t- BHP treated rats group.
A highly significant decrease in glutathione reductase (GR) activity in lung tissue of t- BHP treated rats (1.97 ±
0.264 unit/ g tissue, p < 0.01) was observed compared to the control group (3.79 ± 0.459 unit/ g tissue). Oral
administration with aqueous and ethanol extracts of H. sabdariffa caused insignificant decrease (p > 0.1) in the GR
activity (2.77 ± 0.53 unit/ g tissue, 3.23± 0.399 unit/ g tissue, respectively) compared to the control group. The GR
activity was significantly increased in rats orally administered H. sabdariffa aqueous extract before t-BHP injection
(4.14 ± 0.389 unit/ g tissue, p < 0.001), but insignificantly increased in rats orally administered ethanol extract of
H. sabdariffa before t-BHP injection (2.84 ± 0.509 unit/ g tissue, p > 0.1) when compared to the t- BHP treated rats.
While when compared to the control group, GR activity increased in lung tissue of orally administered H. sabdariffa
aqueous extract but decreased with ethanol extract administration (Table 4).
Data of catalase (CAT) activity in the lung of rats of all the experimental groups under investigation were increased
(Table 4). The CAT activity was significantly increased in t- BHP treated rats (3.21 ± 0.376 unit/ g tissue, p < 0.05)
and rats orally administered H. sabdariffa aqueous extract (3.47 ± 0.23 unit/ g tissue, p < 0.01) but insignificantly
increased in rats orally administered H. sabdariffa ethanol extract, (3.5 ± 0.625 unit/ g tissue, p > 0.1) when
compared to the control group (2.1 ± 0.374 unit/ g tissue). Aqueous H. sabdariffa extract administration have
almost no effect on t-BHP injected rats, while ethanol H.sabdariffa extract administration significantly increased
CAT activity after t-BHP injection (4.38± 0.477 unit/ g tissue, 0.05< p < 0.1) as compared to control and t- BHP
treated rats (Table 4).
Histological examination:-
Injection of rats with t-BHP caused severe thickening of the alveolar walls and blood capillaries walls and
infiltration of inflammatory cells in the alveolar wall. Treatment with aqueous extract of H. sabdariffa showed mild
cellular infiltration close to the bronchiole and blood vessel walls thickening with severe dilatation and congestion.
Treatment with ethanol extract of H. sabdariffa showed focal aggregation of inflammatory cells that invades the
wall of the bronchiole under the epithelium and increase in alveolar septae thickening. Pretreatment of the rats with
H. sabdariffa aqueous extract followed by t-BHP injection, showed focal aggregation of inflammatory cells and
thickening of blood vessels walls but the alveolar septae look normal. Pretreatment of the rats with H. sabdariffa
ethanol extract followed by t-BHP injection showed marked thickening with hypertrophy of muscle fibers in the
bronchiole wall, the blood vessels show dilatation with congestion and fibrous tissue surrounded the bronchiole
walls and the blood vessels (Fig. 4, 5).
Effect of H. sabdariffa ethanol extracts on human lung fibroblast (MRC-5) cells:-
Cytotoxicity of different concentrations of 30% ethanol H. sabdariffa extract ( 37.5µg, 75.5 µg , 150 µg , 300 µg
,and 600 µg /ml) against normal human lung fibroblast (MRC-5 cells) showed less than 10% reduction of cell
viability, i.e. H. sabdariffa ethanol extract had almost no toxicity up to 600 µg. Glutathione level and the catalytic
activities of GST, GPx, GR and protein concentration were also examined in human lung fibroblast cell (MRC-5)
homogenates of six preparations 1) control 2) control DMSO 3) cells subjected to two concentrations of H2O2 (0.05
and 1mM) 4) cells treated with two concentrations of 30 % H. sabdariffa ethanol extract (150 and 300 µg dry
weight) 5) cells treated with 300 µg H. sabdariffa extracts in the presence of 0.05mM H2O2 6) cells treated with150
µg H. sabdariffa extracts in the presence of 1mM H2O2 (Fig. 6). At low concentration of H2O2 (0.05 mM), the level
of GST did not increase in the cells, however, increasing H2O2 concentration to 1mM increased the GST activity by
35%. Increasing H. sabdariffa extract concentrations from 150 µg to 300 µg increased the percent inhibition of GST
activity from 41% to 65.2%. The two H. sabdariffa extracts (150 µg and 300 µg) have almost no effect on the
activity of GST in the cells subjected to 0.05 and 1mM H2O2. H2O2 at concentration of 0.05mM or 1mM decreased
GR and GPx activities. Treatment of MRC-5 cells with 300 µg of H. sabdariffa extracts increased the catalytic
activity of GPx, while using 150 µg of H. sabdariffa extracts have no effect on GPx activity. Increasing H.
sabdariffa extract concentrations from 150µg to 300 µg caused an increase in the GR and GPx activities. Both
concentrations of H. sabdariffa extract resolved the inhibitory effect of H2O2 on GR and GPx activity. H2O2 did not
affect the GSH level at its high concentration 1mM while the protein concentration increased. Treatment of cells
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with 150µg of H. sabdariffa extracts decreased GSH level and increased the protein concentration. However
treatment of cells with 300 µg of H. sabdariffa extracts decreased the protein concentration. Addition of 1mM H2O2
to 150 µg H. sabdariffa extracts increased GSH level. Addition of 1mM H2O2 to 150 µg H. sabdariffa extracts
decreased the concentration of protein and addition of 0.05mM H2O2 to 300 µg H. sabdariffa extracts decreased the
concentration of protein.
Discussion:-
Phytochemicals have been observed to be protective and defensive against many diseases. Most of phytochemicals
possess antioxidant activity, scavenge reactive oxygen species (ROS), chelate metal ions, inhibit oxidases and
activate antioxidant enzymes. They include phenols, phenolic acids, flavonoids and many sterols (Pacôme et al.,
2014). H. sabdariffa is an important source of vitamins, minerals, and bioactive compounds, such as organic acids,
phytosterols and polyphenols, some of them have antioxidant properties (Sayago-Ayerdi et al., 2007). Phenolic
acids can occur in the free form (aglycones), but usually they are found in conjugated forms, as glycosides and
esters. Also, they can be linked by ester bonds to the cell wall polysaccharides or they are associated with dietary
fiber, namely lignin (Bravo, 1998 and Garcia-Salas et al., 2010).
In our study, chlorogenic acid had the highest concentration in the aqueous, 70% ethanol and 30% ethanol extracts
representing 3.304, 1.543 and 1.105 mg/g calyx, respectively. Considerable amounts of caffeic acid were also
observed in the three examined extracts. Owoade et al. (2016) reported the presence of chlorogenic acid, ferulic
acid, naringenin, rutin and quercetin in H. sabdariffa extract. Pacôme et al. (2014) identified 18 compounds in H.
sabdariffa petals: phenolic acids (chlorogenic acid and protocatechuic acid) and flavonoids (gossypetrin, sabdaretin,
gossypetin, luteolin, gossytrin, hibiscetin, rutin, hibiscetrin, myricetin, eugenol, nicotiflorine, quercitrin, quercetin,
kaempferol, astragalin and cyranoside). Phenolic acids also exist as insoluble bound complexes, which are either
high molecular weight polyphenols or single phenolic compounds strongly bound to cell wall proteins or
polysaccharides, and are not extractable by organic solvents (Arranz et al., 2010). Bound phenolic acids are
typically liberated using base hydrolysis, acid hydrolysis or both (Mattila & Kumpulainen, 2002). Polyphenols are
largely metabolized following ingestion in the stomach, small and large intestine, and liver with incomplete
understanding of the absorption and metabolism of all polyphenols. Caffeic acid (2.088 mg/g calyx) and quercetin
(0.696 mg/g calyx) increased in concentration by alkaline hydrolysis of H. sabdariffa calyx, while chlorogenic acid,
rutin and kaempferol could not be detected after hydrolysis. Chlorogenic acid isomers can be hydrolyzed to quinic
and caffeic acids (Sánchez Maldonado et al., 2014). This may be the reason for increasing caffeic acid from 0.862
to 2.088 mg/g calyx and complete degradation of chlorogenic acid after alkaline hydrolysis. The amount of caffeic
acid released upon hydrolysis is lower than the amount expected from hydrolysis of chlorogenic acid. This may be
due to hydrolysis with NaOH without protection using ascorbic acid and ethylenediaminetetraacetic acid (EDTA)
that resulted in complete degradation of caffeic acid (Ross et al., 2009). HPLC showed the complete disappearance
of rutin and the increase of quercetin from 0.1 to 0.69 mg/g calyx indicating the hydrolysis of rutin to quercetin
(Table 2). This result is in accordance with the previous study of Wang et al. (2011) where acidic and enzymatic
hydrolysis of rutin could produce isoquercitrin and quercetin. The observed decrease of catechin from 1.25 to 0.287
mg/g calyx, could be due to the degradation by alkaline hydrolysis or introducing of two double bonds in the C ring
due to the presence of transition metals. One should note that H. sabdariffa contain iron, phosphorus, calcium,
manganese (Sultan et al., 2014). The main constituents of Hibiscus anthocyanins were identified as delphinidin,
delphinidin-3-glucoxyloside (Lin et al., 2007; Maganha et al., 2010 and Kuo et al., 2012). H. sabdariffa pigment
has been reported to be rich in delphinidin-3-sambubioside and cyanidin-3-sambubioside as the major pigments,
with delphinidin-3-monoglucoside and cyanidin-3-monoglucoside (Wong et al., 2002 and Frank et al., 2005). Our
results indicated the presence of delphinidin derivatives and cyanidin 3-O-glucoside chloride in 30% ethanol, 70%
ethanol and aqueous extracts. Cyanidin 3-O-glucoside chloride concentration represented 74.3%, 72.7% and 69%,
respectively of total phenolic compounds. The content of delphinidin derivatives represented 9.1%, 8.4%, 6.6%,
respectively of total phenolic compounds (Table 2).
The most important feature discussed for polyphenols is their antioxidant activity (Spanou et al., 2012 and Stagos
et al., 2012). Actually, polyphenols can act as free radical scavengers and can interact with metal ions to form a
chelate through their aromatic hydroxyl group. Emerging evidence indicates that these polyphenols may also behave
as prooxidants initiating a reactive oxygen species (ROS) production, cellular DNA damage and cell death
(Akagawa et al., 2003 and Mileo and Miccadei, 2016). Polyphenols such as caffeic acid, ferulic acid and apigenin,
may have a prooxidant effect by activating ROS production and oxidative stress conditions (León-González et al.,
2015).
Caffeic acid
Caffeic acid
Caffeic acid
Quinic acid
Quinic acid
Caffeic acid
Chlorogenic acid
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The prooxidants activity of dietary flavonoids is linked to the total number of hydroxyl groups in the molecule, 2, 3-
double bond in ring C and 4-oxo arrangement of flavons (Procházková et al., 2011). In H. sabdariffa, delphinidin
(3 OH in the B ring) and quercetin (2, 3 double bond in the ring C beside 4-oxo arrangement) could be considered a
strong prooxidant. In the present study, the prooxidant activity resulted from H2O2 production was monitored with
the H. sabdariffa extracts. It showed that gallic acid was the greatest in the production of H2O2 due to the presence of
three adjacent OH group. Production of H2O2 by the phenolic compounds tested indicated that, the production of
H2O2 is related to their structures and only ortho and para phenolic compounds may undergo autoxidation. Akagawa
et al. (2003) investigated the production of hydrogen peroxide by polyphenols, epigallocatechin, epigallocatechin
gallate, epicatechin, epicatechin gallate and catechin which are abundant in green tea and black tea and chlorogenic
acid and caffeic acid which are abundant in coffee. Flavonoids generally occur in foods as O-glycosides with sugars
bound at the C3 position. Glycoside modification of the OH substitutions leads to inactivation of transition metal-
initiated prooxidant activity of a flavonoid (Rahal et al., 2014). Transition metals could also mediate the prooxidant
activity of polyphenols, through the reduction of metal ions involved in redox-cycling and promoting the generation
of hydroxyl radicals through Fenton-reaction (León-González et al., 2015). In our results, ethanol extracts of H.
sabdariffa increased the production of H2O2. It had to mentioned that most buffers and H. sabdariffa extracts
contained trace metal ions. Iron, copper, zinc and lead were detected in H. sabdariffa calyx as reported by Bakare-
Odunola and Mustapha, 2014. Also, high manganese levels (453.71 ± 14.07 mg/kg) followed by iron
(219.8mg/kg) in H. sabdariffa flower was pointed out in the research of Mihaljev et al. (2014).
Lungs are vulnerable target for oxidative stress (OS), because of their location, anatomy and function (Bargagli et
al., 2009). The lung is a highly vascularized organ, vulnerable to pathogens, pollutants, oxidant, gases and toxicants,
thus making it susceptible to OS (Piao et al., 2008). Lung under hypoxic condition increased inflammatory cell-
derived prooxidants, lipid peroxidation products and change antioxidant enzymes activities (Hoshikawa et al.,
2001; Minko et al., 2002 and Araneda et al., 2005). Hypoxia induced cell membrane structure and function
alterations, caused acute lung injury and damage to components of the extracellular matrix (Nagato et al., 2012).
Tert- butyl hydroperoxide (t-BHP) is a short-chain analog of lipid hydroperoxide and used as a model substance to
investigate the mechanism of cell injury resulting from acute oxidative stress in cells and tissues. There are two
pathways by which t-BHP is metabolized to free radical intermediates, first, provided by cytochrome P450 or by
hemoglobin in erythrocytes, leads to production of peroxyl and alkoxyl radicals (Davies, 1989). These radicals
subsequently initiate lipoperoxidation of membrane phospholipids and membrane permeability alterations. The other
pathway employs glutathione peroxidase; t-BHP is converted to tert-butanol and glutathione disulphide (GSSG)
(Crane et al., 1983 and Kucera et al., 2014). Kucera et al. (2014) reported that t-BHP induced hepatocytes injury,
changes of cell viability and oxidative stress. Hydrogen peroxide is present in exhaled air of humans and rats. Also,
it is one of the major ROS that induce OS (Halliwell et al., 2000). It easily react with intracellular ions such as iron
and copper to generate the highly reactive hydroxyl radicals and attack cellular components to cause various
oxidative damages and inflammation leading to chronic diseases such as aging, atherosclerosis, rheumatoid arthritis
and cancer (Piao et al., 2008).
Inflammation is an important protective response (is a natural defense mechanism) to cellular/ tissue injury
(Hussain et al., 2016). The purpose of this process is to destroy and remove the injurious agent and injured tissues,
thereby, promoting tissue repair. When this crucial and normally beneficial response occurs in an uncontrolled
manner, the result is excessive cellular/tissue damage that results in chronic inflammation and destruction of normal
tissue. Reactive oxygen species liberated by phagocytes recruited to sites of inflammation, are proposed to be a
major cause of the cell and tissue damage, including apoptosis, associated with many chronic inflammatory diseases.
Lung cells, alveolar epithelial type II cells, are susceptible to the injurious effects of oxidants (Rahman and
Macnee, 2000).
Glutathione (GSH), ubiquitously present in all cell types, is the major non-enzymatic antioxidant and regulator of
intracellular redox homeostasis (Liu et al., 2002). The antioxidant GSH has been shown to be critical to the lungs'
antioxidant defenses, protecting airspace epithelium (membrane integrity) from oxidative/free radical-mediated
injury and inflammation. Alterations in the levels of GSH in the lung lining fluid have been shown in various
inflammatory conditions (Rahman and Macnee, 2000).
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The present results showed that t-BHP reduced the level of GSH an index of OS, in rat lung. Pretreatment with H.
sabdariffa aqueous extract and 30% ethanol extract before injection with t-BHP effectively blocked these
phenomena, as indicated by slight increase in GSH level. The group treated with 30% ethanol H. sabdariffa extract
showed a significant decrease in GSH level. Liu et al. (2002) reported a slight decrease in GSH level in rat liver
treated with protocatechuic acid alone. H. sabdariffa 30% ethanol and aqueous extracts contain gallic acid, catechin,
chlorogenic acid, caffeic acid, rutin and quercetin. However, protocatechuic acid and kaempferol could not be
detected in aqueous extract. H. sabdariffa ethanol extract contain protocatechuic acid, higher amount of quercetin
and caffeic acid beside the possible deglycosylation of rutin due to rutin hydrolysis in the rat stomach resulting in
production of ROS. One should note that gallic acid, caffeic acid, quercetin and delphinidin are strong producers of
H2O2 in buffer (pH 7.4). H2O2 is one of the factors that induce acute lung injury and it can easily cross membranes.
GSH homoeostasis may play a central role in the maintenance of the integrity of the lung airspace epithelial barrier
(Rahman et al., 2001 and Nagato et al., 2012). In the present investigation, the increase in protein concentration
and LDH activity were observed in rat lungs treated with ethanol extract, aqueous extract or t-BHP alone or with
both. A slight decrease in LDH was observed in lungs of rats pre-treated with H. sabdariffa aqueous or ethanol
extracts followed by t–BHP injection (84.98 unit/ g tissue, 75.89 unit/ g tissue, respectively) compared to the t-BHP
treated rats (99.14 unit/ g tissue) . This may indicate slight protection by aqueous extract and ethanol extract to lung
tissue. These results are in agreement with other studies that showed that the total protein concentration and LDH
activity were increased in animals submitted to lung injury (Dal-Pizzol et al., 2006 and da Cunha et al., 2011). The
increased protein concentration may indicate an increase in permeability of the alveolar capillary membrane (da
Cunha et al., 2013). The increase in MDA, a marker for Lipid peroxidation was only observed in rat lungs treated
with H. sabdariffa ethanol extract alone. This behaviour was also observed in GSH concentration. However,
insignificant decrease in MDA level was observed in rat lungs treated with either aqueous extract or t-BHP alone.
A reduction in the activities of GST, GPx and GR in t-BHP treated rat lungs was observed in this study. This
reduction may be due to an overwhelming oxidative modification of the enzymatic proteins by excessive generation
of highly reactive free radicals, leading to loss of integrity and function of cell membranes (Kalava &Mayilsamy,
2014). Our result is in accordance with the previous study of Yen et al. (2004) that reported the reduction in the
activity of GR and GPx in rat livers on t-BHP induction. Treatment with H. sabdariffa aqueous extract has almost no
effect on GST activity compared to control. Oral administration of H. sabdariffa ethanol extract insignificantly
increased the GST activity in lung tissue of rats either injected with t-BHP or not. In Majid et al. (1991) study, oral
feeding of ellagic acid at varying dose levels for eight weeks resulted in a dose-dependent significant increase in the
hepatic GST activity as well as in the hepatic and pulmonary GSH levels. However, the pulmonary GST activity
was not affected, indicating that ellagic acid has a differential effect on GST activity in different organs. After
ellagic acid administration, its elimination from the body through the bile and urine in the form of glutathione
conjugates. Majid et al. (1991) deduced that the in vitro inhibition of GST by ellagic acid is due to the fact that GST
has to conjugate ellagic acid with GSH in addition to conjugating CDNB with GSH. In vivo, ellagic acid enhances
the hepatic GST activity. This increase may be due to the greater need of GSH and GST for the elimination of
ellagic acid itself from the body. Since the lung is constantly exposed to the environment, there are significant
differences in the kinetic, structural, and immunological properties among the tissue specific GSTs. Furthermore,
different forms of GSTs differ among themselves in their ability to conjugate various xenobiotics to GSH.
GSTs function is to catalyze the conjugation of the sulfur atom of GSH to an electrophilic center of endogenous and
exogenous toxic compounds, increasing their solubility and excretion (Habig and Jakoby, 1981). GSTPi (GSTP1)
is responsible for more than 90% of the GST activity within the adult human lung epithelial cell population. Among
the human GST classes, GSTP1 was expressed more abundantly in the respiratory tissue (Yan et al., 2010). Most of
GSTs composed of two identical subunits. A complete active site of each subunit composed of one site of binding
for GSH (G site) and one site which binds a number of hydrophobic substrates (H site) adjacent to the G site (Wilce
and Parker, 1994). Glutathionylation of proteins is considered as a primary line of defense. This reversible
modification reaction can be performed non-enzymatically, but it has proposed that GSTP1 may enhance this
modification of thiol proteins during oxidative stress and/or nitrosative stress (NS). Because of their GSH high
affinity and specificity, GSTs are structurally well equipped to accommodate diverse GSH-bound substrates such as
glutathionylated proteins. GSTP1 can potentially serve as a cellular NO donor and/or carrier (Vasieva, 2011),
regulating the activities of other thiol redox-modifying enzymes (e.g. GR) (Mazzetti et al., 2015). Decrease in GPx
and GR activity of lungs in rat treated with either ethanol extract or aqueous extract may be due to H. sabdariffa
calyx had been analyzed to contain phytic acid, tannin and glycosides such as delphinidin-3-monoglucoside and
delphinidin which are toxic to animal and human tissues at high doses (Morton, 1987; Nnamonu et al., 2013).
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Catalase is not essential for some cell types under normal conditions; it plays an important role in cell adaptation to
OS (Mates et al., 1999). In the present study, CAT activity was significantly higher in rat lung treated with aqueous
extract (p < 0.01) or t-BHP alone (p < 0.05 ) or with rat lungs pretreated with ethanol extract followed by t-BHP
injection (0.05< p < 0.1). Insignificant increase in catalase activity of rat lungs treated with ethanol extract or
pretreated with aqueous extract followed by t-BHP injection (p > 0.1) was observed. The activation of CAT activity
could contribute to the detoxification of ROS (Ilieva et al., 2014). Pretreatment with ethanol extract followed by t-
BHP injection partially blocked the effect of the t-BHP. The OS markers, GSH, MDA, LDH levels returned back
almost to the normal level. However, OS enzyme markers including GR and GPx did not return back to the normal
level. CAT, the enzyme detoxifying the effect of high concentration of H2O2 increased significantly (0.05 < p < 0.1).
Pretreatment with aqueous extract followed by t-BHP injection increased the GSH level almost to the normal level
(abolish the effect of t-BHP). MDA was not affected; however, LDH level and protein concentration (indicating the
increase in permeability of the alveolar capillary membrane) could not block the effect of t-BHP. GR activity
returned back to almost the normal level, however, GPx and CAT activity could not abolish the effect of t-BHP. The
changes in the examined OS markers and antioxidant enzymes activities suggested lung injury in rat lungs injected
with t-BHP or pretreated with both H. sabdariffa aqueous extract and ethanol extract. However, pretreatment with
H. sabdariffa extracts followed by t-BHP injection could abolish the effect of t-BHP in some respects.
the histopathological analyses were done to verify the presence of inflammatory infiltrates and so validate the lung
injury model. The injection of rats with t-BHP caused severe thickening of the alveolar walls and blood capillaries
walls and infiltration of inflammatory cells in the alveolar wall. Loss of alveolar epithelial integrity results in the
accumulation of protein-rich and highly cellular edema fluid in the alveoli (Proudfoot et al., 2011). This is
confirmed with the increase in the total protein concentration and LDH activity in lung homogenate, decrease in the
GSH level, decrease in GPx and GR activity and increase in CAT activity. Treatment with aqueous extract of H.
sabdariffa showed mild cellular infiltration close to the bronchiole and blood vessel walls thickening with severe
dilatation and congestion and treatment with ethanolic extract of H. sabdariffa showed focal aggregation of
inflammatory cells that invades the wall of the bronchiole under the epithelium and increase in alveolar septae
thickening. This may be due to the phenolics and flavonoids present in the extracts which are strong producers of
H2O2 (a major ROS) resulting in production of ROS that induce acute lung injury. This behavior agreed with the
increase in protein concentration and LDH activity in rat lungs treated with either ethanol extract or aqueous extract
and the decrease in GSH level in rat treated with ethanol extract only. The antioxidant enzymes, GPx and GR
activity decreased while an increase in CAT activity in rat lungs pretreated with either ethanol extract or aqueous
extract was observed. Pretreatment of the rats with H. sabdariffa aqueous extract followed by t-BHP injection,
showed focal aggregation of inflammatory cells and thickening of blood vessels walls but the alveolar septae look
normal. The accumulation of excess fibrous connective tissue (the process called fibrosis) resulted in the end stage
of organ failure. The pathogenesis of fibrosis in many diseases is thought to involve aberrant or over-exuberant
wound-healing processes initiated to protect from injurious stimuli (Sakai &Tager, 2013). Fibroblasts play a key
role during wound healing that produce large quantities of extracellular matrix components, cytokines and repair
growth factors (Quesnel et al., 2010). One of the key steps in the tissue response is the deposition of collagen fibers
at the sites of injury where lung fibroblasts release procollagen peptides into the extracellular space in order to create
a scar. So collagen deposition must not be viewed as a late response to abnormal healing, but as an early
phenomenon (Gonzalez-Lopez &Albaiceta, 2012). Initiation of fibrous tissue surrounded the bronchiole walls and
the blood vessels was observed in histopathological examination of the rat lungs pretreated with ethanol extract
followed by t-BHP injection which could indicate the start of lung tissue repair including fibroblast. Pathological
accumulations of fibroblasts are the main source of the extracellular matrix proteins accumulation in the fibrotic
area. Lung injury resulted in fibroblast proliferation, differentiation and migration. Therefore, fibroblast cells were
chosen for the present cell line study.
Toxicity, cytotoxicity and genotoxicity were reported as a result of addition of phenolic compounds from plant
sources to the cell culture media. This was attributed mainly to the H2O2 production by the oxidation of the phenolic
compounds in some plants such as green tea, grape seed, apple extract (Halliwell, 2014). Polyphenols can be
oxidized in cell culture media. Cell culture media are frequently deficient in antioxidants especially tocopherols and
ascorbate and also deficient in selenium (Halliwell, 2008). Cells require transition metal ions, especially iron and
copper, in order to grow. The components of the cell culture medium, including the various inorganic salts,
vitamins, and amino acids, contribute to, but are not completely essential for the generation of H2O2 by a polyphenol
(Babich et al., 2011). The widely used Dulbecco’s modified Eagle’s medium (DMEM) contains added iron (III)
nitrate, Fe (NO3) 3. Free iron is also present in Ham’s FIO medium, often used to study the oxidation of low density
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lipoprotein (LDL) in vitro (Firth &Gieseg, 2007). In other media, iron is mostly supplied in transferrin-bound form,
usually in the widely used calf serum. Whereas transferrin-bound iron will not normally catalyze free radical
reactions, contaminating (or added) free iron ions can be prooxidant, as can be copper and many other transition
metal ions (Aruoma &Halliwell, 1987 and Halliwell, 2014).
In the present results, H. sabdariffa extracts (aqueous, 30% ethanol and 70% ethanol) in RPMI 1640 media produce
substantial amounts of H2O2. H2O2 increased by increasing the concentration to 300 µg/ml of H. sabdariffa extract.
Insignificant changes in H2O2 production was observed by increasing the extract concentration to 300µg either in 30
or 70% ethanol. Halliwell et al. (2000) reported that ascorbate-induced apoptosis in HL-60 cells was significantly
greater in DMEM medium than in RPMI medium, correlating with the increased rates of H2O2 generation on
addition of ascorbate to DMEM. This result is in accordance with the previously reported effects relative to
epigallocatechin and epigallocatechin gallate, catechin and quercetin, showing that phenolic compounds are
oxidized in cell culture media and lead to H2O2 generation (Long et al., 2000). Caffeic, chlorogenic and ferulic
acids, were effective inducing DNA cleavage in human promyelocytic leukemia HL-60 cells in the presence of Cu
(II) ions. Other phenolic compounds, such as piceatannol, a stilbene naturally present in grapes and rhubarbrhizome,
among others, can interact with copper ions in their oxidized forms, Cu (II), promoting the Haber-Weiss and Fenton
reactions that generate ROS, inducing DNA cleavage (León-González et al., 2015). One should note that the values
of H2O2 produced was due to the phenolic compounds in the extract and any H2O2 produced in the control was
decomposed to O2 and water by the addition of CAT. H2O2 (strong ROS) at the level of 0.05mM or 1mM caused a
decrease in GR (44%, 59.7%) and GPx (65.7%, 49%) activity, respectively, however it did not affect the GSH level
at its high level (1mM) and increased the protein concentration. This may indicate that the antioxidant capacity of
the cells could not compensate the prooxidant effect of the phenolic compounds. Treatment of MRC-5 cells with
low concentration of H2O2 (0.05 mM) and high concentration of H. sabdariffa extract (300 µg) abolished the
inhibitory effect of H2O2 to either GR or GPx activity. The activity of GR and GPx increased almost to the normal
level. Increasing the concentration of H2O2 to 1mM and decreasing the H. sabdariffa extract to 150 µg resolved the
inhibitory effect of H2O2 to both GR and GPx activity; however they did not go back to the normal level. Addition
of H. sabdariffa extract (300µg/ml) to the cell culture is associated with a high decrease in protein concentration.
This could be due to toxicity of polyphenol, anthocyanin, flavonoid and other compounds producing ROS in the
media in presence of metal ions. However, decreasing the H. sabdariffa extract concentration to 150µg/ml and
increasing the H2O2 concentration to 1mM increased the protein concentration. This may confirm that this effect is
mainly due to H. sabdariffa and not to H2O2. GST activity decreased by 41%, 65.2% by increasing the H. sabdariffa
extract concentration from 150 µg to 300 µg, respectively. One has to note that in the DMSO control, an increase in
the GST activity was observed. This could be due to the detoxification by GST needed to cell survival. DMSO is a
toxic material at high concentration but its use at low level did not affect the growth. At low concentration of H2O2,
the level of GST did not increase, however at high concentration (1mM) it caused increase in GST required for
detoxification and cell survival. Pretreatment of MRC-5 cells with high or low concentration of H. sabdariffa extract
almost did not affect the GST activity. GSH level decreased in cells treated with 150µg H. sabdariffa extract by
60.8%. However, addition of 1mM H2O2 to 150 µg H. sabdariffa extracts increased GSH level and goes back to
almost the normal level. This was also detected in the rat lungs pretreated with ethanol extract followed by t-BHP
injection.
It must be realized that cells in culture are different from those in vivo in many ways. The normal cell matrix (which
has important influences on cell morphology and function) is absent, as are other cell types that normally surround
the cells in question and communicate with them. Cells that survive and grow in culture are often not representative
of cells in vivo in terms of gene expression, enzyme levels and metabolism. Most cells in vivo are exposed to low O2
concentrations except for the cells lining the respiratory tract, skin epidermis and cornea of the eye, however cell
culture is commonly performed under 95% air 5% CO2. Levels of O2 to which cells in culture are exposed can affect
many of their properties. The production of ROS rates by cellular enzyme systems and by leakage of electrons from
electron transport chains are limited at normal cellular levels, elevated O2 will thus increase ROS production in cells
in culture. Increased ROS production might cause increased cell proliferation or lead to senescence, cell death, or
adaptation. For human fibroblasts, a value of 40-50 doublings was originally suggested. Cell culture media are
frequently deficient in the antioxidants that are normally obtained in the human diet and delivered to cells by the
bloodstream (Halliwell, 2014).
Phytochemicals such as polyphenols have been reported to be able to modulate the inflammatory processes
(Hussain et al., 2016). Much attention is currently focused on the role of natural polyphenols on modulating
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intracellular ROS levels. Several polyphenols were demonstrated to interfere with enzymes driving the epigenetic
alterations which modulate inflammation process, as such; it will be a challenge for future anti-inflammatory
therapies (Mileo and Miccadei, 2016). Anti-inflammatory effects of polyphenols can be realized via binding to
various receptors. Flavonoid (e.g.resveratrol, epigallocatechin gallate and curcumin) induced receptor stimulation
can modulate active state of different kinases thus influence differentiation and apoptosis, cell survival (inhibition of
apoptosis) and inflammatory response. In a study of Trebatická and Durackova (2015), conducted on human
brain, polyphenols can be inhibiting the inflammation mediated by macrophages through the reduction of
proinflammatory cytokines formation and inhibition of platelet aggregation.
Declarations:-
Acknowledgements:-
The authors thank all subjects who participated in this study.
Funding:-
This work was supported by the National Research Centre of Egypt, Project of Polyphenols as a potential strategy of
multidrug resistance prevention: adjuvant therapy (No. ).
Availability of data and materials:-
Data and materials related to this work are available upon request.
Competing interests:-
The authors declare that they have no competing interests.
Consent for publication:-
All authors approve the manuscript for publication.
Ethics approval and consent to participate:-
All procedures performed in this study were in accordance with the ethical standards of the institutional and/or
national research committee. All experiments were carried out in accordance with the Egyptian laws and National
Research Centre guide lines, and the informed consent was obtained from all of the patients.
Table 1:- Phytochemical analyses of H. sabdriffa extracts
Extraction
solvent
TPC
mg GAE /g dry
plant
TFC
mg RE /g dry
plant
TAC
mg anthocyanin E/g
dry plant
Antioxidant capacity
using DPPH IC50*
30% ethanol
20.96 ± 3.69
(14.8, 27.6)
0.39 ± 0.07
(0.28, 0.52)
4.24 ± 0.33
(3.67, 4.8)
5.68 ± 0.9
(4.7, 7.5)
70% ethanol
39.19 ± 6.63
(26, 47)
1.33 ± 0.09
(1.15, 1.58)
9 ± 3.8
(2.7,15.9)
1.04 ± 3.3
(6.4, 17.6)
H2O
19.08 ± 2.67
(13.7, 21.9)
0.43 ± 0.03
(0.38, 0.495)
3.2 ± 0.23
(2.8, 3.6)
6.44 ± 1.16
(4.1, 7.8)
Values are represented as mean ± SE of 3 to 4 experiments
Values between parentheses are the highest and the lowest value
IC50 (amount of extract which cause 50% inhibition of DPPH free radical)
Antioxidant capacity is represented by IC50 (mg dry plant/ml)
Table 2:- Characterization of phenolic and anthocyanin compounds extracted from H. sabdariffa calyx using high
performance liquid chromatography analysis.
Extraction solvent
Area
Rt
Compound
Hydrolyzed
Calyx
H2O
70%
ethanol
30%
ethanol
0.1388
0.1469
0.144
0.125
3208.7
5.40
Gallic acid
0.1232
0
0.355
0.212
1618.7
9.30
Protocatechuic acid
0.2874
1.257
0.4562
0.821
608.1
17.50
Catechin
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0
0
0
0
1529.4
21.80
Syringic acid
0
0
0
0
2294.6
23.50
Vanillic acid
0
0
0
0
2779.3
35.80
Coumarin
0
0.0118
0
0
2192.4
42.00
Cinnamic acid
0
0
0
0
2761
50.90
Chrysin
0
0
0
0
1296.9
16.50
Gentisic acid
0
3.304
1.543
1.105
1054.7
19.60
Chlorogenic acid
2.088
0.862
0.826
0.665
2808.5
20.20
Caffeic acid
0.0373
0
0
0
3798.6
30.90
Ferulic acid
0.0186
0.0113
0
0
2897.5
32.30
Sinapic acid
0.0434
0.0499
0.0926
0
2590.5
39.60
Rosmarinic acid
0
0.1898
0.1486
0.051
1668.9
35.30
Rutin
0.696
0.0689
0.1066
0.0762
3582.2
42.70
Quercetin
0
0
0.0312
0.0388
3848
45.60
Kaempferol
-
17.7
14.29
13.8
1307.8
9.88
Cyanidin 3-O–glucoside
chloride
-
2.08
1.653
1.69
6193.7
7.11
Delphinidin derivatives
Values are represented as mg/g calyx
Rt: Retention time
Table 3:- Effect of different extracts of H. Sabdariffa on GSH, protein, malondialdehyde (MDA) levels and
lactate dehydrogenase activity (LDH) of lung tissue treated with tert-butyl hydroperoxide (t-BHP).
Treatment
GSH
(nmol/g tissue)
Protein
(mg/g tissue)
MDA
(nmol/g tissue)
LDH
(unit/g
tissue)
Control
111.56 ± 9.06
31.5 ± 3.4
1647.8 ± 133.1
57.56 ± 3.79
t-BHP (0.2 mmol/kg)
85.35 ± 9.4
55.5 ± 7.2
1329.6 ± 115.8
99.14 ±
11.99
t-BHP vs. control
p < 0.1 < 0.05
p < 0.01
p > 0.1
p < 0.01
Aqueous H. sabdariffa (250 mg/Kg)
126.92 ± 20.74
63.3 ± 8.2
1453.8 ± 144.7
80.14 ± 9.66
Aqueous H. sabdariffa vs. control
p < 0.05
p < 0.01
p > 0.1
p < 0.05
Aqueous H. sabdariffa + t-BHP
135.86 ± 9.3
64.7 ± 7.8
1451.7 ± 133.8
84.98 ± 7.56
Aqueous H. sabdariffa + t-BHP vs. control
p < 0.1 < 0.05
p < 0.01
p > 0.1
p < 0.01
Aqueous H. sabdariffa + t-BHP vs. t-BHP
p < 0.01
p > 0.1
p > 0.1
p > 0.1
30% ethanol H. sabdariffa (250 mg/Kg)
62.55 ± 6.63
58.6 ± 5.6
2124.9 ± 225
0.58 ± 5.736
30% ethanol H. sabdariffa vs. control
p < 0.001
p < 0.01
p > 0.1
p > 0.1
30% ethanol H. sabdariffa + t-BHP
112.45 ± 8.43
66.6 ± 7.2
1227.6 ± 161.9
75.89 ± 6.16
30% ethanol H. sabdariffa + t-BHP vs.
control
p > 0.1
p < 0.01
p < 0.1 < 0.05
p < 0.05
30% ethanol H. sabdariffa + t-BHP vs. t-
BHP
p < 0.1 < 0.05
p > 0.1
p < 0.1 < 0.05
p > 0.1
Values are represented as mean ± SE of n=7
P < 0.01 highly significant, P < 0.05 significant and P > 0.1 insignificant
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Table 4:- Effect of different H. sabdariffa extracts on the activity of some antioxidant enzymes in lung tissue of rats
treated with tert-butylhydroperoxide (t-BHP)
Values are represented as mean ± SE of n=7
P < 0.01 highly significant, P < 0.05 significant and P > 0.1 insignificany
(a)
Treatment
GST
unit/g tissue)
GPx
(unit/g tissue)
GR
(unit/g
tissue)
CAT
(unit/g
tissue)
Control
1.388 ± 0.21
5.616 ± 0.917
3.79 ± 0.459
2.1 ± 0.374
t-BHP (0.2 mmol/kg)
1.199 ± 0.063
3.22 ± 0.324
1.97 ± 0.264
3.21 ± 0.376
t-BHP vs. control
p > 0.1
p < 0.01
p < 0.01
p < 0.05
Aqueous H. sabdariffa (250 mg/Kg)
1.308 ±
0.1397
3.11 ± 0.291
2.77 ± 0.53
3.47 ± 0.23
Aqueous H. sabdariffa vs. control
p > 0.1
p < 0.05
p > 0.1
p < 0.01
Aqueous H. sabdariffa + t-BHP
1.229 ± 0.149
3.84 ± 0.307
4.14 ± 0.389
3.1 ± 0.377
Aqueous H. sabdariffa + t-BHP vs. control
p > 0.1
p > 0.1
p > 0.1
p < 0.1 < 0.05
Aqueous H. sabdariffa + t-BHP vs. t-BHP
p > 0.1
p > 0.1
p < 0.001
p > 0.1
30% ethanol H. sabdariffa (250 mg/Kg)
1.488 ± 0.143
3.68 ± 0.705
3.23 ± 0.399
3.5 ± 0.625
30% ethanol H. sabdariffa vs. control
p > 0.1
p > 0.1
p > 0.1
p > 0.1
30% ethanol H. sabdariffa + t-BHP
1.313 ± 0.116
3.46 ± 0.433
2.84 ± 0.509
4.38 ± 0.477
30% ethanol H. sabdariffa + t-BHP vs.
control
p > 0.1
p > 0.1
p > 0.1
p < 0.01
30% ethanol H. sabdariffa + t-BHP vs. t-
BHP
p > 0.1
p > 0.1
p > 0.1
p < 0.1 < 0.05
0.005 0.005 0.005 0.005 0.005
0.54
0.005
0.43 0.45
0
0.1
0.2
0.3
0.4
0.5
0.6
50 150 300
H2O2 concentration ( µM)
Extract concentration (µg/ml)
water 30% ethanol 70% ethanol
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(b)
Fig. 1: H2O2 concentration in RPMI cell culture medium containing 50,150 and 300 µg/ml of H. sabdariffa different
extracts: a) after 24h of incubation and b) after 48h of incubation.
Fig 2:- Production of H2O2 by some phenolic compounds and the standard antioxidant compound citric acid (250
µM) after 24 h of incubation at pH 7.4.
0.005
0.21
0.45
0.005
0.26
0.93
0.005
0.53
0.92
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
50 150 300
H2O2 concentration ( µM)
Extract concentration (µg/ml)
water 30% ethanol 70% ethanol
6.2
0.98
3.84
0.946
3.77
2.6
0.499 0.548
0
1
2
3
4
5
6
7
H2O2 concentration (µM)
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Fig. 3:- Effect of incubation time on H2O2production by different phenolic compounds and the standard antioxidant
compound citric acid.
0
1
2
3
4
5
6
7
8
9
10
012 24 36 48
H2O2 concentration (µM)
Incubation time (h)
α-tocopherol Citric acid
Delphindin Catechin
Gallic acid Protocateuchic acid
Caffeic acid Quercetin
(a)
(b)
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Fig. 4:- Histological findings for (A) control ( thin walls of the alveoli (arrow), (B) rat lung treated with H.
sabdariffa ethanol extract (focal aggregation of inflammatory cells (arrowhead) that invades the wall of the
bronchiole under the epithelium (arrow), (C) rat lung injected with t-BHP (severe thickening of the alveolar walls
and blood capillaries walls (arrow)) and (D) rat lung pretreated with ethanol extract followed by t-BHP (marked
thickening with hypertrophy of muscle fibers in the bronchiole wall (arrowhead). The blood vessels show dilatation
with congestion (arrow), (H&E section).
(c)
(d)
(b)
(a)
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Fig. 5:- Histological findings for (A) control ( thin walls of the alveoli (arrow), (B) rat lung treated with H.
sabdariffa ethanol extract (focal aggregation of inflammatory cells (arrowhead) that invades the wall of the
bronchiole under the epithelium (arrow), (C) rat lung injected with t-BHP (severe thickening of the alveolar walls
and blood capillaries walls (arrow)) and (D) rat lung pretreated with ethanol extract followed by t-BHP (marked
thickening with hypertrophy of muscle fibers in the bronchiole wall (arrowhead). The blood vessels show dilatation
with congestion (arrow), (H&E section).
(c)
(d)
1 mM
H2O2
150 µg extract
1mM H2O2+
150µgextract
-80
-60
-40
-20
0
20
40
60
% change of GSH level
0.05mM
H2O2
300µg
extract
0.05 mM
H2O2+300µ
g extract
1 mM
H2O2
150 ug
extract
1mM H2O2
+ 150 µg
extract
-100
-50
0
50
100
150
200
% change of protein
0.05mM
H2O2
1 mM
H2O2
150 ug
extract
300µg
extract
0.05 mM
H2O2+300µ
g extract
1mM H2O2
+ 150 µg
extract
-200
-150
-100
-50
0
50
100
150
200
250
% change of GPx activity
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Fig. 6: Effect of H. sabdariffa extracts (150 and 300 µg) on glutathione, protein concentration and some
antioxidant enzymes catalytic activities of MRC-5 cells subjected to H2O2 (0.05 and 1 mM).
0.05mM
H2O2
1 mM H2O2
150 µg
extract
300µg
extract
0.05 mM
H2O2+300
µg extract
1mM H2O2
+ 150 µg
extract
-80
-60
-40
-20
0
20
40
60
80
% change of GST activity
0.05mM
H2O2 1 mM
H2O2
150 ug
extract
300µg
extract 0.05 mM
H2O2+300µg
extract 1mM H2O2
+ 150 µg
extract
-200
-150
-100
-50
0
50
100
150
200
% change of GR activity
Gallic acid
Protocatechuic acid
Rosmarinic acid
Caffeic acid
Chlorogenic acid
Ferulic acid
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Fig. 7:- Structure of the major H. sabdariffa phenolic compounds as indicated from the present HPLC analysis.
Conclusion:-
Phenolic compounds can act as antioxidants or prooxidants depending on their chemical structure, concentration,
environmental conditions such as the cell type and redox stress. Lungs are vulnerable target for OS, because of their
location, anatomy and function. Evaluation of the OS condition is a hot topic in biology and medicine. Oxidative
stress is viewed as an imbalance between the production of ROS and their elimination by protective mechanisms,
which can lead to chronic inflammation. H2O2 production by all the examined H. sabdariffa extracts indicated the
involvement of flavonoids and their related structures (results of HPLC analysis) in their prooxidant activities. The
changes in the examined OS markers and antioxidant enzymes activities suggested lung injury in rat lungs injected
with t-BHP or pretreated with both H. sabdariffa aqueous extract and ethanol extracts. However, pretreatment with
H. sabdariffa extracts followed by t-BHP injection could abolish the effect of t-BHP regarding GSH, protein levels,
antioxidant enzyme activities and some histopathological observations. Further research studies should not only
focus on understanding the mechanism of phenolics to exert their effects, ROS production but also their effects on
other organs.
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