Anthocyanin-rich extract from Hibiscus sabdariffa calyx counteracts UVC-caused impairments in rats

Abstract

Context: Ultraviolet radiation (UV) was reported to cause oxidative stress. Hibiscus sabdariffa L. (Malvaceae) calyx is commonly used in traditional Asian and African medicines and possesses strong antioxidant capacity due to its anthocyanin (ANTH) content.

Objective: This study researched the possible protective role of Hibiscus sabdariffa calyx extract (HSCE) in UVC exposure of rats.

Material and methods: Levels of serum enzymes, renal function tests, and some oxidant/antioxidant biomarkers of skin, lens, and retina tissues were monitored. Rats were exposed to UVC 4 h daily for 40 d and simultaneously received HSCE containing 2.5, 5, and 10 mg doses of ANTH in drinking water.

Results: Significant (p < 0.05) increases in the levels of serum aminotransferases, lactate dehydrogenase, urea, creatinine, and uric acid were noted after UVC exposure. In skin, lens, and retina tissues, total oxidant status, oxidative stress index, lipid peroxidation, and protein oxidation escalated markedly (p < 0.05) whereas total antioxidant status, reduced glutathione, and superoxide dismutase decreased dramatically (p < 0.05) related to UVC. Co-administration of HSCE with each ANTH dose significantly (p < 0.05) reversed aforementioned parameters (except total oxidant status) almost in all tissues. The LD₅₀ of HSCE in rats was determined to be above 5000 mg/kg.

Discussion and conclusion: Our data revealed that HSCE has a remarkable potential to counteract UVC-caused impairments, probably through its antioxidant and free radical-defusing effects. Therefore, HSCE could be useful against some cutaneous and ocular diseases in which UV and oxidative stress have a role in the etiopathogenesis.

• Anthocyanins
• lens
• oxidative stress
• photoaging
• photocarcinogenesis
• retina
• skin
• ultraviolet radiation

Introduction

Ultraviolet radiation (UV) leads to different health problems including erythema, edema, pigmentation, premature skin aging (photoaging), and melanogenesis (Biesalski & Obermueller-Jevic, 2001; Ichihashi et al., 2003; Mishra et al., 2012; Yan et al., 2011). The UV spectrum range consists of UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280 nm). Among them, the most powerful and dangerous one is UVC which has sterilizing, non-ionising, and biocidal effects. Although cellular damage of UVA and UVB are well documented, UVC damage is rarely reported owing to its almost complete absorption via the ozone layer under normal conditions (Basti et al., 2009). However, during the past few decades, depletion of the ozone layer gave rise to an elevation in the level of UVC arriving to the biosphere (Osman et al., 2010). Therefore, detrimental effects of UVC on humans have appeared increasingly. For example, escalations of skin cancers have been reported related to UVC (Rorsman & Tegner, 1988).

The skin is the first and main organ suffering from sun-originated UV and artificial sources (Inal & Kahraman, 2000). Eyes also suffer from UV directly. UV causes increase of reactive oxygen species (ROS) and initiates photo-oxidation in skin and eyes (Aly & Ali, 2014; Barg et al., 2014). When ROS increase excessively, antioxidant deficiency occurs, and then cellular components are damaged (Nishi et al., 1991). Aforementioned deficiency may increase through insufficient intake of both oral and topical antioxidants (Zondervan et al., 1996). In recent years, the use of natural compounds which possess especially antioxidant and anti-inflammatory properties has created considerable interest as protective agents for reducing UV-induced skin damage (Aly & Ali, 2014; Bai et al., 2014; Sierra et al., 2013; Svobodová et al., 2003). One of the materials being used as a source of natural compounds are plant extracts (Tülüce et al., 2012).

Hibiscus sabdariffa L. (Malvaceae) (HS) is traditionally used as an important medicinal plant in some parts of the world, especially Asia and Africa (Essa & Subramanian, 2007). HS extracts were demonstrated to have a broad range of therapeutic effects (Ali et al., 2005). These effects have been attributed to various constituents of HS including anthocyanins (ANTH), flavonoids, polysaccharides, organic acids, and some minerals. The importance of the HS calyx that we investigated in the current study is mainly based on its ANTH content (Wong et al., 2002). ANTH were reported to possess antioxidant (Ajiboye et al., 2011), antihypertensive (Wahabi et al., 2010), hypocholesterolemic, hepatoprotective, and many other beneficial activities (Hirunpanich et al., 2006). To the best of our knowledge, there are no available data regarding the protective effect of HS or ANTH against UV.

The present study was conducted to determine protective effect of HS calyx extract (HSCE) on UVC-induced toxicity through monitoring some serum enzymes and renal function markers as well as oxidant/antioxidant status of skin, lens, and retina tissues for the first time in the literature.

Materials and methods

Plant material and extraction procedure

In the autumn, dried whole samples of HS were obtained from an herbal store in Sanliurfa, Turkey. The plant sample was authenticated by a taxonomist Assoc. Prof. Esat Cetin, PhD. Calyces of HS (400 g) were powdered in an electrical mill. Powder (200 g) was added to an aqueous solution consisted of distilled water (750 ml), ethanol (500 ml), and methanol (500 ml) for 24 h at 40 °C. Then, the blend was filtered and stored in a deep freeze for 2 h. The plant extract was concentrated under reduced pressure and lyophilized (Cryodos; TelStar Industrial, Terrassa, Spain) to obtain the dry residue. ANTH content of this residue was calculated through spectrophotometric analysis at 520 and 700 nm according to the method of Giusti and Wrolstad (2001). The LD₅₀ of HSCE was determined to be above 5000 mg/kg in rats (Onyenekwe et al., 1999), indicating that the extract is essentially non-toxic.

Animals

Experiments were performed on 35 Sprague–Dawley male rats (296 ± 5 g) of approximately the same age. Animals were housed in polypropylene cages (seven per cage) and kept under standard conditions (12/12 light/dark cycle at 21 ± 2 °C and 50–70% humidity). They were acclimated to laboratory conditions for 20 d before treatments. Standard pellet feed and tap water were provided ad libitum. Dorsal areas of rats were shaved with a safety razor and this operation was repeated before the UV periods. Animals received humane care according to the criteria outlined in “The Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science and published by the National Institutes of Health. The ethical regulations were followed in accordance with national and institutional guidelines for the protection of animal welfare during experiments. This study was approved by the Ethic Committee of the Harran University.

Experimental design

Rats were randomly assigned to five equal groups (n = 7): control, UVC, UVC + ANTH_2.5, UVC + ANTH₅, and UVC + ANTH₁₀. All animals except those of controls were exposed to UVC irradiation (1.25 mW/cm²) 4 h daily for 40 d. In addition, rats of UVC + ANTH_2.5, UVC + ANTH₅, and UVC + ANTH₁₀ groups received drinking water supplemented with HSCE containing 2.5, 5, and 10 mg ANTH, respectively.

Exposure to UVC

In this experiment, UVC was produced by a lamp (Mazda TG model, Toshiba, Tokyo, Japan) with 30 W power and 90 cm length. Rats were exposed to UVC in a 182 × 68 × 50 cm³ box which was painted flat dark as described by Brainard et al. (1986) and Tülüce et al. (2012). The lamp was mounted on the lid of the box in a distance of 44 cm to the animals. The animals were subjected to UVC light radiated from the lamp for 40 d. The intensity of the UVC coming from the lamp was measured by a spectrophotometer to have a wavelength with a peak value of 254 nm. In this experiment, energy of the UVC light emitted from the lamp per cm² for 1 s was found as 0.0014 J/cm². This amount was adjusted according to Tülüce et al. (2012).

Preparation of skin, lens, retina, and serum samples

At the end of the treatments, rats were anesthetized with ketamine (100 mg/kg ip). Blood samples were taken from the animals into tubes with K₃EDTA by injectors from the heart under light anesthesia then rats were euthanized with inhalation of CO₂. The serum samples were obtained by centrifuging blood samples at 3000 rpm for 15 min at 4 °C. Skin tissue was dissected and put in Petri dishes for washing with physiological saline (0.9% NaCl). After the eye enucleation, lens and retina were carefully removed using a posterior approach and rinsed with 6.3-mM EDTA phosphate buffer. All tissue samples were kept at −80 °C until analysis. Tissues were homogenized in ice-cold phosphate buffer solution for 5 min using both an ultrasonic (Bandelin, UW 2070, Sigma, St. Louis, MO) and mechanic (Heidolph, silent crusher M, VWR Corporate, Radnor, PA) homogenizers and then centrifuged at 7000g for 15 min. All processes were carried out at 4 °C. Skin, lens, and retina supernatants were used to determine protein concentration and oxidant/antioxidant parameters. In addition, some serum biochemical constituents were measured in serum.

Biochemical analysis

In the current study, oxidant/antioxidant status was monitored by measuring total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), malondialdehyde (MDA), protein carbonyl (PC), and reduced glutathione (GSH) levels as well as superoxide dismutase (SOD) activity in skin, lens, and retina tissues. Activities of some serum marker enzymes including aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), and lactate dehydrogenase (LDH) were measured to determine the protective role of HSCE on the damage of especially skeletal muscle and liver tissues. Creatinine, urea, and uric acid levels were assessed to determine renal functions.

Determination of serum biochemical constituents

Activities of AST, ALT, GGT, and LDH as well as levels of urea, creatinine, and uric acid were determined in serum by an autoanalyzer (Roche, Indianapolis, IN; Cobas Integra 400 plus) using the kits.

Determination of TAS level

TAS levels were assessed spectrophotometrically (Shimadzu, Kyoto, Japan) at 660 nm using kits (Rel Assay Diagnostics kit, Mega Tıp, Gaziantep, Turkey) developed by Erel (2004). This method was based on the bleaching of the distinct color of 2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] radical cation via the action of antioxidants. The results were expressed as mmol Trolox Eq/l.

Determination of TOS level

TOS levels were determined spectrophotometrically (Shimadzu, Kyoto, Japan) at 530 nm using kits (Rel Assay Diagnostics Kit; Mega Tıp, Gaziantep, Turkey) developed by Erel (2005). In this new colorimetric method, oxidants presented in the sample oxidize the ferrous ion-o-dianisidine complex, yielding ferric ion. The oxidation reaction was enhanced by the presence of excess glycerol in the reaction medium. The ferric ion and xylenol orange generated a colored complex. The results were given as micromolar hydrogen peroxide equivalents per liter (mmol H₂O₂ Eq/l).

Calculation of OSI value

OSI was defined as the ratio of the TOS level to the TAS level (Bilgili et al., 2013). Specifically, OSI (arbitrary unit) = TOS (mmol H₂O₂ Eq/l)/TAC (mmol Trolox Eq/l).

Determination of SOD activity

SOD activity was assessed by the Cayman Chemical SOD Assay kit (Cat. no 706002) measuring the dismutation of superoxide radicals generated by xanthine oxidase and hypoxanthine.

Determination of GSH level

GSH concentration was assayed by reacting with O-phthaldialdehyde (OPT, 10 mg/10 ml methanol) according to the modified method of Lee and Chung (1999). Pure reduced GSH was used as a standard for calibration. GSH samples were measured by using spectrofluorimetry (Jasco 6000*, USA), with an excitation at 345 nm and an emission at 425 nm.

Determination of MDA level

MDA (a major oxidation product of peroxidized polyunsaturated fatty acids) was estimated using the modified thiobarbituric acid-reactive substance method by Hegde et al. (2003). MDA was measured by spectrofluorimetry, with excitation at 520 nm and emission at 555 nm. Calculations were performed using a linear regression from tetraethoxypropane for the MDA standard curve.

Determination of PC level

PC (a useful marker of protein oxidation) was measured by Cayman Chemical Protein Carbonyl Assay Kit (Cat. no. 10005020) as the carbonyl content in the samples. This kit is based on the principle that utilizes the 2,4-dinitrophenylhydrazine reaction to measure the protein carbonyl content in homogenate (Levine et al., 1990). The amount of protein–hydrozone produced is quantified spectrophotometrically at an absorbance of 360 nm by a 96-well plate reader (Spectra Max M5). The carbonyl content was standardized to protein concentration.

Determination of protein

The protein content in the samples was measured by the method of Lowry et al. (1951) with bovine serum albumin as the standard.

Statistical analysis

The statistical analyses were realized using the Minitab 13 for windows (Minitab, State College, PA). All data were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) statistical test and Tukey's posttest were used to determine the differences between means of the experimental groups accepting the significance level at p < 0.05.

Results

Effect on levels of serum marker enzymes

Results for levels of serum marker enzymes are demonstrated in Table 1. UVC application increased the activities of AST, ALT, LDH significantly (p < 0.05) and GGT slightly (p < 0.05) compared with the control. Combined treatment of HSCE with three different ANTH doses to UVC-treated rats significantly (p < 0.05) ameliorated levels of these enzymes. In UVC + ANTH_2.5 and UVC + ANTH₅ groups, GGT was decreased remarkably even when compared with those of controls.

Table 1. The photoprotective effect of anthocyanin-rich Hibiscus sabdariffa L. calyx extract on serum marker enzymes of rats exposed to UVC (n = 7).

Parameters	Control	UVC	UVC + ANTH2.5	UVC + ANTH5	UVC + ANTH10
AST (U/l)	98.97 ± 5.51	156.35 ± 28.91^a	96.04 ± 7.53^b	135.75 ± 10.13^b	103.59 ± 5.45^b
ALT (U/l)	47.29 ± 6.19	74.14 ± 9.50^a	56.29 ± 5.82^ab	58.31 ± 11.91^b	54.60 ± 5.04^b
GGT (U/l)	1.96 ± 0.23	2.03 ± 0.45	1.23 ± 0.15^ab	1.44 ± 0.42^ab	1.58 ± 0.48
LDH (U/l)	809.06 ± 255.37	1317.67 ± 359.02^a	644.59 ± 206.00^b	704.99 ± 211.35^b	741.52 ± 103.41^b

Each value represents the mean ± SD. ^ap < 0.05 compared with the control group. ^bp < 0.05 compared with the UVC group.

Effect on serum renal function markers

Serum renal function markers (urea, creatinine, and uric acid) are presented in Table 2. While the levels of urea, creatinine, and uric acid increased significantly (p < 0.05) in the UVC group compared with the control, HSCE supplementation with almost each ANTH dose resulted in marked (p < 0.05) improvements of aforementioned parameters.

Table 2. The protective effect of anthocyanin-rich Hibiscus sabdariffa L. calyx extract on serum renal function markers of rats exposed to UVC (n = 7).

Parameters	Control	UVC	UVC + ANTH2.5	UVC + ANTH5	UVC + ANTH10
Urea (mg/dl)	22.92 ± 5.96	37.78 ± 4.09^a	26.78 ± 2.94^b	29.47 ± 3.08^b	28.68 ± 1.58^b
Creatinine (mg/dl)	0.25 ± 0.05	0.41 ± 0.06^a	0.29 ± 0.03^b	0.32 ± 0.03^ab	0.30 ± 0.05^b
Uric acid (mg/dl)	1.80 ± 0.21	3.40 ± 0.53^a	2.52 ± 0.17^ab	4.19 ± 0.77^a	3.77 ± 1.04^a

Each value represents the mean ± SD. ^ap < 0.05 compared with the control group, ^bp < 0.05 compared with the UVC group.

Effect on oxidant/antioxidant status

As shown in Table 3, TAS and GSH levels of skin, lens, and retina tissues were significantly (p < 0.05) lower, TOS, OSI, MDA, and PC values of these tissues were dramatically (p < 0.05) higher in the UVC group compared with control. Co-administration of HSCE with all ANTH doses remarkably (p < 0.05) amended levels of foregoing parameters except TOS, especially in skin and retina tissues in UVC-treated rats. With regard to SOD, its activity diminished markedly (p < 0.05) in the UVC group compared with control in all tissues, and rose again notably (p < 0.05) with each ANTH dose of HSCE in lens tissue.

Table 3. The photoprotective effect of anthocyanin-rich Hibiscus sabdariffa L. calyx extract on UVC-induced oxidative stress in rats (n = 7).

Tissues	Parameters	Control	UVC	UVC + ANTH2.5	UVC + ANTH5	UVC + ANTH10
Skin	TAS (mmol Trolax Eq/l)	1.25 ± 0.36	0.67 ± 0.07^a	0.91 ± 0.09^ab	0.84 ± 0.26	0.94 ± 0.22^b
	TOS (mmol H2O2 Eq/l)	6.95 ± 1.56	10.90 ± 3.30^a	8.99 ± 1.66	8.70 ± 2.74	8.40 ± 1.58
	OSI (arbitrary unit)	5.83 ± 1.84	16.06 ± 4.15^a	9.94 ± 1.69^ab	10.45 ± 2.51^ab	9.13 ± 1.85^ab
	MDA (nmol/g)	4.99 ± 1.61	9.42 ± 1.39^a	5.71 ± 1.27^b	7.81 ± 0.71^ab	6.25 ± 1.97^b
	PC nmol/mg prot.	0.43 ± 0.09	1.03 ± 0.14^a	0.52 ± 0.14^b	0.60 ± 0.18^b	0.56 ± 0.14^b
	GSH nmol/g	1.22 ± 0.12	0.55 ± 0.18^a	0.94 ± 0.28^b	0.89 ± 0.26^ab	1.09 ± 0.21^b
	SOD U/g	7.12 ± 0.34	3.57 ± 0.87^a	4.68 ± 1.10^a	4.94 ± 1.54^a	5.34 ± 1.54^a
Lens	TAS (mmol Trolax Eq/l)	1.16 ± 0.14	0.67 ± 0.15^a	0.88 ± 0.11^ab	0.76 ± 0.18^a	0.79 ± 0.11^a
	TOS (mmol H2O2 Eq/l)	5.86 ± 1.40	8.69 ± 1.68^a	7.35 ± 0.90	6.98 ± 2.10	7.54 ± 1.06
	OSI (arbitrary unit)	5.14 ± 1.33	13.34 ± 3.67^a	8.51 ± 1.92^ab	9.30 ± 2.75^a	9.71 ± 2.13^a
	MDA (nmol/g)	7.25 ± 1.24	13.16 ± 4.36^a	8.99 ± 1.47	7.38 ± 2.82^b	8.92 ± 1.68
	PC nmol/mg prot.	0.44 ± 0.04	1.39 ± 0.31^a	0.73 ± 0.18^ab	0.98 ± 0.32^a	0.98 ± 0.31^a
	GSH nmol/g	13.92 ± 4.37	6.30 ± 1.81^a	11.03 ± 2.99^b	8.38 ± 2.41^a	8.03 ± 1.39^a
	SOD U/g	2.45 ± 0.17	1.52 ± 0.19^a	2.36 ± 0.70^b	2.20 ± 0.43^b	2.37 ± 0.10^b
Retina	TAS (mmol Trolax Eq/l)	2.12 ± 0.18	1.09 ± 0.06^a	1.45 ± 0.10^ab	1.68 ± 0.20^ab	1.94 ± 0.30^b
	TOS (mmol H2O2 Eq/l)	4.81 ± 1.11	7.44 ± 2.24^a	5.76 ± 1.58	5.92 ± 1.99	5.52 ± 1.17
	OSI (arbitrary unit)	2.25 ± 0.40	6.79 ± 1.80^a	4.01 ± 1.26^ab	3.53 ± 1.10^ab	2.92 ± 0.90^b
	MDA (nmol/g)	30.16 ± 9.74	51.67 ± 6.29^a	34.75 ± 8.51^b	40.79 ± 3.36^ab	39.97 ± 2.18^b
	PC nmol/mg prot.	0.25 ± 0.07	0.76 ± 0.17^a	0.44 ± 0.09^ab	0.38 ± 0.12^b	0.51 ± 0.14^ab
	GSH nmol/g	4.90 ± 0.75	2.81 ± 0.29^a	3.39 ± 0.23^ab	3.67 ± 0.12^ab	3.59 ± 0.20^ab
	SOD U/g	21.86 ± 5.72	10.94 ± 1.78^a	17.46 ± 3.09^b	13.93 ± 3.10^a	13.51 ± 4.04^a

Each value represents the mean ± SD. ^ap < 0.05 compared with the control group. ^bp < 0.05 compared with the UVC group.

Discussion

Today, as UV triggers many skin diseases including skin cancers, it has become one of the main problems of our century. Thus efforts are needed to provide effective protection against its detrimental influences. Some experimental trials have investigated the medical effects of several plants in this regard. The current study was conducted to investigate whether anthocyanin-rich HSCE could prevent some alterations related to UVC exposure in rats for the first time. In daily life, as skin and eyes are the organs to be directly in contact with UVC due to thinning of ozone layer and other artificial sources such as sterilization lamps, we decided to monitor alterations in oxidant/antioxidant status of these organs. We also measured some serum enzymes and renal function markers. Although many in vivo studies were performed to research the influences of UVA and UVB on mammals, there were only three such trials investigated the in vivo effects of UVC until current study (Altinas et al., 2007; Özkol et al., 2011; Tülüce et al., 2012). Therefore, we compared the data of this study with the results of foregoing three studies and some other UVC applied in vitro trials as well as UVA and UVB administrated in vivo investigations.

Enzymes as macromolecules leak from the injured tissues into the body fluids. Thus, measurement of these molecules in serum has been found to be important in the evaluation of related tissue damage. In the present study, significant increases of AST, ALT, LDH, and slight elevation in GGT activity were determined in the UVC group compared with the control group. Escalated activities of these enzymes might be particularly attributed to the damaged cell structures of skeletal muscle and liver tissues (Aldrich, 2003). UVC may have given rise to their more leakage into the serum due to autolytic breakdown or cellular necrosis resulted from increased oxidative stress (Recknagel et al., 1989). In some earlier studies increased serum enzymes were shown to be associated with escalated ROS (Tuluce et al., 2011). While significant escalations in serum AST, ALT, and LDH activities of the present trial were also reported in the study of Altinas et al. (2007), slight increases of these enzymes were observed in another previous study (Özkol et al., 2011) in which UVC was administrated for 1 h twice a day for 4 weeks. The reason for this discrepancy may be due to the length of the UVC application period. Co-administration of HSCE with each ANTH dose significantly decreased AST, ALT, GGT, and LDH activities. GGT diminished remarkably in UVC + ANTH_2.5 and UVC + ANTH₅ groups even when compared with those of controls. Since HSCE possesses tissue protective (Carvajal-Zarraba et al., 2012) and antioxidant (Sarkar et al., 2014) features mainly due to its ANTH content, it might have decreased activities of foregoing enzymes through preventing their leakage from cells to interstitial fluid. HSCE may have performed such an action by rejuvenating the cell membranes that directly or indirectly suffered from UVC during this study.

The dramatic elevation in renal function biomarkers such as urea, creatinine, and uric acid has been known to be an indicator of impaired renal function. Thus, in the current study, drastic elevations of these markers in UVC administrated rats indicate UVC-caused nephrotoxicity. Our results of increased urea and creatinine levels are in accord with Altınas et al. (2007) in which the effect of UVC was determined on urea and creatinine levels on the 7th, 15th, 30th, 45th, 60th, and 75th days of the experiment in mice. However, in a previous study (Özkol et al., 2011), unchanged levels of serum urea and creatinine were observed probably due to different lengths of the UVC application period. Marked decrements of urea, creatinine, and uric acid levels in the HSCE-treated groups reveal nephroprotective properties of HSCE. Results of the present study are in agreement with some earlier reports where HS calyx caused a decrease in creatinine levels and improved kidney functions (Ademiluyi et al., 2013; Mohagheghi & Maghsoud, 2011). These improvements might be attributed to an activity of some bioactive phytochemicals especially ANTH present in the HS calyx.

Excessive production of ROS plays a major role in UV-induced cutaneous and ocular damage via leading to various impairments including lipid peroxidation, protein oxidation, breakage of DNA strands, and also through depleting the endogenous enzymatic and non-enzymatic antioxidants (Darr & Fridovich, 1994; Shindo et al., 1993; Tülüce et al., 2012). ROS are believed to be critical mediators of the photoaging and photocarcinogenesis processes (Bowden, 2004; Droge, 2002). They can modify proteins, e.g., collagen cross-linking in tissue to form carbonyl derivatives, which accumulate in the papillary dermis of photo-damaged skin (Sander et al., 2002). In our study, while lipid peroxidation product MDA and protein oxidation product PC levels of the UVC group increased significantly compared with the control, their levels decreased markedly in combined treatment of the group HSCE with all ANTH doses in skin, lens, and retina tissues. Elevated MDA and PC levels show consistency with some previous studies (Anwar & Moustafa, 2001; Sander et al., 2002; Tülüce et al., 2012). Remarkably increased MDA and PC levels in the UVC group indicate excessive ROS in this group (Halliwell, 1990). In the presence of HSCE, the decreased levels of MDA and PC suggest that this extract could scavenge ROS resulting from UVC administration. Other biomarkers of oxidant status TOS and OSI which were monitored for the first time in our study showed a similar trend of higher level in the UVC group compared with controls. Their levels decreased in all HSCE-treated groups in line with MDA and PC.

There is an antioxidant system known as the GSH redox cycle. Impairment of this system appears to be closely associated with lipid peroxidation and protein oxidation (Yu et al., 2007). GSH is the most important cellular non-enzymatic antioxidant of this cycle. In the current study, its level diminished significantly in the UVC group compared with the control in all investigated tissues. A significant decrease of the skin GSH concentration is in accordance with Tülüce et al. (2012) whereas its concentration of lens tissue was slightly decreased in that study. GSH levels of the UVC group in the present study may have been depleted due to scavenging excessive ROS. Co-administration of HSCE with nearly each ANTH dose resulted in a significant increase of the GSH content indicating that the skin, lens, and retina enhanced their normal activity by the protective role of HSCE. This might be attributed to the ANTH content of HS that was demonstrated to be influential in scavenging ROS (Ademiluyi et al., 2013). TAS (an important biomarker of antioxidant capacity) which was determined for the first time in our study showed consistency with GSH in terms of its level in all groups. With regard to SOD, its activity diminished markedly in the UVC group compared with the control in all tissues, and rose again notably with all ANTH doses of HSCE in particularly lens tissue. Decrement of SOD activity in the UVC group is in line with some previous investigations (Anwar & Moustafa, 2001; Inal & Kahraman, 2000; Shindo et al., 1993; Tülüce et al., 2012). The SOD activity may have reduced because of the escalated production of ROS such as that lead to the inhibition of this enzyme (Karthikeyan et al., 2007). As ANTH of HS calyx are informed to be potent antioxidants and capable of inhibiting lipid peroxidation and scavenging ROS such as and , their co-administration with UVC may have protected SOD activity near control values in especially lens tissue (Ologundudu et al., 2010).

The results presented here led us to conclude that UVC exposure caused increment in levels of TOS, OSI, MDA, PC, serum enzymes, and renal function markers whereas decrement in TAS and GSH levels as well as SOD activity in rats. Co-administration of HSCE with three different doses of ANTH has a significant potential to counteract UVC-caused impairments probably owing to its ANTH contents which possess antioxidant and free radical defusing effects. Therefore, this extract could be useful in preventing the development of some cutaneous and ocular diseases in which UV and oxidative stress have a role in the etiology.

Declaration of interest

The authors report that there are no declarations of interest.