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 R 1 , Hussein F E 2 , * Guneidy R A 1 , Awad H 3 and Ali E A A 1 . 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. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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 ( 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.

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)  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, H 2 O 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 H 2 O 2 was produced by incubation in deionized water at 37°C up to 48 h. 30% and 70% ethanol extracts produced H 2 O 2 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 H 2 O 2 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, H 2 O 2 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). H 2 O 2 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 H 2 O 2 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) 1262 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 H 2 O 2 in phosphate buffer after 24h. The production of H 2 O 2 by gallic acid was the greatest. The yield of H 2 O 2 was in the following decreasing order gallic acid > caffeic acid > quercetin > delphinidin > protocatechuic acid > catechin > citric acid > α-tocopherol. The production of H 2 O 2 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).  (Fig. 6). At low concentration of H 2 O 2 (0.05 mM), the level of GST did not increase in the cells, however, increasing H 2 O 2 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   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.

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The prooxidants activity of dietary flavonoids is linked to the total number of hydroxyl groups in the molecule, 2, 3double 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 H 2 O 2 production was monitored with the H. sabdariffa extracts. It showed that gallic acid was the greatest in the production of H 2 O 2 due to the presence of three adjacent OH group. Production of H 2 O 2 by the phenolic compounds tested indicated that, the production of H 2 O 2 is related to their structures and only ortho and para phenolic compounds may undergo autoxidation. 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) 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 H 2 O 2 in buffer (pH 7.4). H 2 O 2 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 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 H 2 O 2 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 H 2 O 2 (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 H 2 O 2 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 H 2 O 2 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 1268 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 H 2 O 2 . H 2 O 2 increased by increasing the concentration to 300 µg/ml of H. sabdariffa extract. Insignificant changes in H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 produced was due to the phenolic compounds in the extract and any H 2 O 2 produced in the control was decomposed to O 2 and water by the addition of CAT. H 2 O 2 (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 H 2 O 2 (0.05 mM) and high concentration of H. sabdariffa extract (300 µg) abolished the inhibitory effect of H 2 O 2 to either GR or GPx activity. The activity of GR and GPx increased almost to the normal level. Increasing the concentration of H 2 O 2 to 1mM and decreasing the H. sabdariffa extract to 150 µg resolved the inhibitory effect of H 2 O 2 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 H 2 O 2 concentration to 1mM increased the protein concentration. This may confirm that this effect is mainly due to H. sabdariffa and not to H 2 O 2 . 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 H 2 O 2 , 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 H 2 O 2 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 O 2 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% CO 2 . Levels of O 2 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 O 2 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 1269 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.

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. H 2 O 2 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.