Original Papers
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Authors
Eman Al-Sayed 1*, Mohamed M. Abdel-Daim 2, 3*
Affiliations
1 2 3
Key words " Cupressus macrocarpa l " Cupressaceae l " hepatoprotective l " nephroprotective l " biflavones l " lipid peroxidation l
received revised accepted
Sept. 14, 2014 Sept. 24, 2014 Sept. 30, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383211 Published online October 22, 2014 Planta Med 2014; 80: 1665–1671 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Eman Al-Sayed Faculty of Pharmacy Ain-Shams University Department of Pharmacognosy Cairo, 11566 Egypt Phone: + 20 20 10 01 05 02 93 Fax: + 20 24 05 11 06 em_alsayed@ pharma.asu.edu.eg
Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt Department of Pharmacology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt United Graduate School of Drug Discovery and Medical Information Sciences, Department of Gene and Development, Graduate School of Medicine, Gifu University, Gifu, Japan
Abstract !
The hepatoprotective and nephroprotective activity of cupressuflavone isolated from Cupressus macrocarpa was investigated against CCl4-induced toxicity in mice. Cupressuflavone was administered (40, 80, and 160 mg/kg/day) for five days. CCl4 was administered (0.5 mL/kg intraperitoneally) at the end of the experiment. A substantial increase (p < 0.001) in the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, total bilirubin, cholesterol, creatinine, uric acid, urea, and malondialdehyde was observed in the CCl4treated group compared to the normal control group. In contrast, a significant reduction (p < 0.001) in glutathione and superoxide dismutase contents as well as the total protein level was evident in the CCl4-intoxicated mice. Cupressuflavone pretreatment markedly inhibited the CCl4induced increase in alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, cholesterol, creatinine, uric acid, urea, and malondialdehyde levels in a
Introduction !
The liver is the primary organ of metabolism, and the kidneys are the main organs involved in the excretion of xenobiotics; therefore, the toxic effects of chemicals appear primarily in the liver and kidney tissues [1]. Oxidative stress plays a prominent causative role in many diseases, including inflammation, liver damage, and carcinogenesis [2]. Oxidative stress is caused by the cellular excess of reactive oxygen species (ROS), which induces lipid peroxidation and cell injury [3]. Under normal states, the cells maintain the ROS
* Both of these authors contributed equally to this article.
dose-dependent manner (p < 0.001 at all the tested doses). In addition, a significant (p < 0.001) and dose-dependent decrease in the total bilirubin levels was evident by cupressuflavone pretreatment (80 and 160 mg/kg/day) when compared to the CCl4-intoxicated group. Furthermore, cupressuflavone administration significantly increased the activity of antioxidant parameters glutathione and superoxide dismutase as well as the serum protein levels (p < 0.001 at all the tested doses) in a dose-dependent manner. Histological observations confirmed the strong hepato- and nephroprotective activity. These findings suggest that cupressuflavone could exert a beneficial effect against oxidative stress by enhancing the antioxidant defense status, reducing lipid peroxidation, and protecting against the pathological changes induced by CCl4 in the liver and kidney tissues. The structure of cupressuflavone was identified by NMR, UV, and HRESI‑MS/MS spectral data. Supporting information available online at http://www.thieme-connect.de/products
levels with endogenous antioxidants [2]. The risk of cell injury may also be prevented by natural antioxidants, including dietary flavonoids [4]. Considerable evidence indicates that regular consumption of dietary flavonoids have beneficial effects on health, including protection against oxidative stress and various degenerative diseases [5, 6]. Flavonoids can restore the balance between the natural antioxidants and free radicals by direct scavenging of ROS, metal chelation, and the induction of natural antioxidant defenses, e.g., glutathione (GSH) and superoxide dismutase (SOD) [4, 5, 7]. The genus Cupressus (Cupressaceae) comprises twelve species. Different Cupressus species have been used in traditional medicine for the treat-
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Protective Role of Cupressuflavone from Cupressus macrocarpa against Carbon Tetrachloride-Induced Hepato- and Nephrotoxicity in Mice
Original Papers
ment of several ailments, including cough, inflammation, and infection [8]. Cupressus macrocarpa Hartw. ex Gord is a widely planted ornamental tree belonging to the family Cupressaceae [9]. Several contributions have been reported on the composition of C. macrocarpa essential oil and indicated that neral, hydroxy citronellal, geraniol, and piperitol are the main constituents of C. macrocarpa essential oil [9]. However, no pharmacological studies have been conducted on the nonvolatile constituents of C. macrocarpa. The main chemotaxonomic chemical components of the Cupressus genus are biflavonoids [10], for which a broad spectrum of biological activity has been reported [10, 11]. Cupressuflavone (CF), a biflavonoid isolated from Thuja orientalis, exhibited neutrophil elastase inhibitory activity in vitro [12]. Moreover, CF from C. sempervirens showed a potential osteoprotective effect [13]. Despite the diverse medicinal properties of biflavonoids, no comprehensive study, to the best of our knowl-
Fig. 1 Structure of cupressuflavone.
Fig. 2 Effect of cupressuflavone (CF) and silymarin on the hepatic function tests of CCl4-intoxicated mice. (CF 1) Group III (CCl4 + 40 mg/kg CF). (CF 2) Group IV (CCl4 + 80 mg/kg CF). (CF 3) Group V (CCl4 + 160 mg/kg CF). Data
Al-Sayed E, Abdel-Daim MM. Protective Role of … Planta Med 2014; 80: 1665–1671
edge, has so far been reported on the hepato- and nephroprotective activity of the isolated compound CF. Therefore, this study was designed to determine the hepato- and nephroprotective activity of CF isolated from C. macrocarpa against CCl4-induced toxicity in mice, along with possible mechanisms of hepato- and nephroprotective activity. A histopathological observation of liver and kidney sections was performed to confirm the hepato- and nephroprotective activity. The chemical structure of CF was identified by NMR, UV, and HRESI‑MS/MS data.
Results ! " Fig. 1) was elucidated based on NMR, UV, The structure of CF (l and HRESI‑MS/MS spectral data (Supporting Information), which are consistent with the data reported in the literature [10, 14, 15]. Based on the acute toxicity results, no adverse behavioral changes, toxicity symptoms, or mortality were observed in mice at doses up to 2000 mg/kg of CF (LD50 > 2000 mg/kg). Based on these findings, CF is considered safe in mice. A significant increase (p < 0.001) in the activity of the biomarkers of liver damage, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), was observed in the CCl4-intoxicated mice " Fig. 2). Pretreatment compared to the negative control group (l with CF produced a marked hepatoprotective effect and reduced the activity of all hepatic enzymes in a dose-dependent manner (p < 0.001 at all the tested doses). The percentage of decrease in the liver marker enzymes at the treatment doses (40, 80, and 160 mg/kg/day) was 21, 46, and 56% for ALT; 27, 52, and 63 % for AST; 23, 51, and 59 % for ALP; and 25, 50, and 62% for LDH, re-
are expressed as the means ± SEM (n = 8). Values having different superscripts are significantly different at p < 0.05.
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Table 1 Effect of cupressuflavone (CF) on biochemical parameters of CCl4-intoxicated mice. Animal groups Control CCl4 CCl4 + 40 mg/kg CF CCl4 + 80 mg/kg CF CCl4 + 160 mg/kg CF CCl4 + 200 mg/kg silymarin
Total bilirubin
Cholesterol
Total proteins
Creatinine
Uric acid
Urea
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
1.22 ± 0.05a 1.68 ± 0.04b 1.55 ± 0.02b (− 8%) 1.35 ± 0.04a (− 20%) 1.28 ± 0.04a (− 24%) 1.24 ± 0.04a (− 26%)
75.28 ± 3.40a 152.10 ± 4.70b 116.96 ± 4.95c (− 23%) 106.64 ± 4.05c (− 30%) 77.00 ± 3.54a (− 49%) 73.43 ± 3.18a (− 52%)
7.49 ± 0.16a 5.70 ± 0.13b 6.63 ± 0.10c (16%) 7.02 ± 0.13a, c (23%) 7.33 ± 0.08a (29%) 7.43 ± 0.14a (30%)
0.31 ± 0.03a 6.97 ± 0.43b 3.51 ± 0.26c (− 50%) 2.25 ± 0.29d (− 68%) 0.59 ± 0.07a (− 92%) 0.46 ± 0.06a (− 93%)
23.93 ± 0.99a 89.66 ± 4.58b 70.72 ± 2.67c (− 21%) 55.29 ± 3.27d (− 38%) 33.46 ± 1.86a (− 63%) 26.84 ± 1.87a (− 70%)
24.24 ± 1.13a 83.16 ± 4.37b 63.19 ± 2.85c (− 24%) 48.49 ± 3.47d (− 42%) 32.39 ± 1.64a (− 61%) 25.88 ± 1.52a (− 69%)
within the same column are significantly different at p < 0.05
spectively, compared to the CCl4-intoxicated group. Notably, CF pretreatment at a dose of 160 mg/kg markedly reduced the levels of ALT, AST, ALP, and LDH, which were comparable and nonsignificant to those in the normal and silymarin-treated groups " Fig. 2). Similarly, a marked and dose-dependent decrease in (l the serum bilirubin and cholesterol levels was observed in the " Table 1). In contrast, the total protein CF pretreated groups (l levels were significantly (p < 0.001) improved in all of the groups treated with CF in a dose-dependent manner. The total protein levels in the groups treated with 80 and 160 mg/kg of CF were comparable to those in the normal control and silymarin groups " Table 1). The biochemical markers of kidney damage, uric acid, (l urea, and creatinine were significantly (p < 0.001) reduced in all " TaCF-treated groups compared to the CCl4-intoxicated group (l ble 1). These findings suggest that CF effectively reduced the CCl4induced hepatorenal toxicity. A significant reduction (p < 0.001) in hepatic and renal GSH and SOD contents was observed in CCl4-intoxicated mice. In contrast, the malondialdehyde (MDA) levels were markedly increased (p < 0.001) compared with those in the normal control group " Fig. 3). Administration of CF at the treatment doses (40, 80, (l and 160 mg/kg/day) induced a marked increase in hepatic GSH (by 24, 61, and 89 %, respectively) when compared to the CCl4-intoxicated group (p < 0.001 for the groups treated with 80 and 160 mg/kg of CF). Also, the groups treated with CF showed a marked increase in renal GSH (by 31, 53, and 82%, respectively; p value < 0.05 for the group treated with 40 mg/kg, and p values < 0.001 for the groups treated with 80 and 160 mg/kg of CF). Moreover, pretreatment with CF, at all the tested doses, significantly (p < 0.001) improved the hepatic SOD (by 27, 69, and 113 %, respectively), and raised the activity of renal SOD (by 57, 84, and 103%, respectively) relative to the CCl4-intoxicated group. In addition, CF pretreatment, at all the tested doses, ameliorated the CCl4-induced increase in hepatic MDA by 24, 51, and 55 % and the renal MDA by 24, 37, and 53 % at the tested doses, respectively, when compared to the CCl4-intoxicated group (p < 0.001) " Fig. 3). Notably, the liver and kidney GSH contents in the (l groups treated with 160 mg/kg of CF were comparable and nonsignificant to those in the normal control and silymarin groups. CF pretreatment at a dose of 160 mg/kg was the most effective in reducing the hepatic and renal MDA levels. There was no significant difference between the MDA levels in the groups treated with 80 and 160 mg/kg of CF and those in the normal control or silymarin group. These results clearly indicated the strong in vivo antioxidant activity conferred by CF.
Liver sections of the normal control mice showed the preserved histological structure of the central vein surrounded by intact hepatocytes. CCl4 induced severe loss of hepatic architecture with ballooning degeneration in the hepatocytes all over the hepatic parenchyma together with multiple focal necroses. The pathological changes induced by CCl4 were markedly ameliorated in the groups treated with CF in a dose-dependent manner. Pretreatment with 160 mg/kg of CF conferred marked protection against liver damage as evidenced by the intact hepatic architecture. Mild ballooning degeneration in the hepatocytes was observed " Fig. 4). The in the groups treated with 40 and 80 mg/kg of CF (l histological examination of kidney sections of the normal control group revealed a normal histological structure of the glomeruli and tubules at the cortex with the absence of histopathological changes. CCl4 induced marked inflammatory cell aggregation in between the tubules, together with marked degeneration in the lining epithelium of all the tubules. The aforementioned renal histopathological changes induced by CCl4 were markedly reduced in the groups treated with CF at the three treatment doses. Notably, pretreatment with CF at a dose of 160 mg/kg preserved the normal histological structure of the kidneys and markedly reduced all the degenerative changes in the lining tubular epithelium. The group pretreated with 80 mg/kg of CF showed mild focal inflammatory cell infiltration in between the tubules. Mild degeneration in the lining epithelium of some of the tubules was " Fig. 5). observed in the group pretreated with 40 mg/kg of CF (l
Discussion !
Biflavonoids have been reported to have several pharmacological effects [10, 11]. Agathisflavone, a biflavone isolated from Canarium manii, exhibited a hepatoprotective activity against CCl4-induced hepatotoxicity in rats and mice [16]. Furthermore, amentoflavone, one of the major components of the Ginkgo biloba leaves, showed diverse pharmacological properties, including strong antioxidant activity, an inhibitory effect on lipid peroxidation, a neuroprotective effect, and anti-inflammatory activity [11]. The total extracts of C. sempervirens and Juniperus phoenicea showed hepatoprotective activity against CCl4-induced hepatotoxicity in rats [8, 17]. However, no study on the hepato- and nephroprotective activity of CF was reported. Therefore, this study was undertaken to determine the mechanistic basis for the hepato- and nephroprotective effects of CF.
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Data are expressed as the means ± SEM (n = 8). Numbers in parentheses represent the percentage of change from the CCl4-intoxicated group. Values having different superscripts
Original Papers
Fig. 3 Effect of cupressuflavone (CF) and silymarin on lipid peroxidation and antioxidant parameters in the liver and kidneys of CCl4-intoxicated mice. (CF 1) Group III (CCl4 + 40 mg/kg CF). (CF 2) Group IV (CCl4 + 80 mg/kg CF). (CF
Various xenobiotics, including CCl4, are known to cause hepatorenal toxicity. Liver damage is a major health problem which may develop into several liver diseases, including hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma [18]. CCl4 is a highly hepatotoxic chemical widely used in experimental animal models of liver and kidney damage that mimic human hepatorenal toxicity [3, 19]. The highly reactive trichloromethyl radicals (CCl3• and CCl3O2•) are formed from CCl4 by cytochrome P-450. These radicals initiate lipid peroxidation and necrosis of the liver. The trichloromethyl radicals also change the cellular antioxidant capacity by deactivating GSH and the antioxidant enzymes [3, 20]. In this study, administration of CCl4 to mice significantly increased the levels of AST, ALT, ALP, LDH, cholesterol, and total bilirubin. Furthermore, it reduced the total protein level, which indicated a severe loss of liver function. A histopathological examination revealed a severe loss of hepatic structure associated with multiple necroses and a marked ballooning degeneration of the hepatocytes. Moreover, marked inflammatory cell infiltration
Al-Sayed E, Abdel-Daim MM. Protective Role of … Planta Med 2014; 80: 1665–1671
3) Group V (CCl4 + 160 mg/kg CF). Data are expressed as the means ± SEM (n = 8). Values having different superscripts are significantly different at p < 0.05.
and degeneration of the kidney tissues were observed. CCl4 intoxication also induced severe oxidative stress as demonstrated by the marked increase in hepatic and renal MDA levels. The SOD and GSH levels in the liver and kidney tissues were markedly reduced in response to CCl4 intoxication. The results of the present study indicate that CF pretreatment reduced the increased MDA levels to their normal values. The inhibitory effect against lipid peroxidation suggests that CF could prevent the hepatorenal injury induced by CCl4 together with the subsequent pathological damage in the liver and kidney tissues. In contrast, pretreatment with CF markedly enhanced the GSH and SOD levels when compared to the CCl4-intoxicated mice. Modulation of these antioxidant enzymes clearly contributed to the hepatoand nephroprotective effect of CF. These findings suggest that the superior hepato- and nephroprotective activity of CF can be attributed to its ability to enhance the antioxidant defense status, to reduce the rate of lipid peroxidation, and to guard against the pathological changes induced by CCl4 intoxication in the liver and
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Fig. 5 Nephroprotective effect of cupressuflavone (CF) in CCl4-intoxicated mice. a Group I (normal control) showing a normal histological structure of the glomeruli (g) and tubules (t) at the cortex with the absence of histopathological changes. b Group II (CCl4-treated group) showing marked inflammatory cell aggregation (m) in between the tubules and marked degeneration (d) in the lining epithelium of all the tubules. c Group VI (CCl4 + 200 mg/kg silymarin) showing the absence of histopathological alterations. d Group V (CCl4 + 160 mg/kg CF) showing a normal histological structure. e Group IV (CCl4 + 80 mg/kg CF) showing focal inflammatory cell infiltration (m) in between the tubules. f Group III (CCl4 + 40 mg/kg CF) showing mild degeneration in the lining epithelium of some of the tubules (H&E, × 20). (Color figure available online only.)
kidney tissues. In conclusion, the protective effect of CF against oxidative stress associated with its protective effect against infil-
tration by inflammatory cells and other CCl4-induced pathological changes in the liver and kidneys indicated that CF has hepato-
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Fig. 4 Hepatoprotective effect of cupressuflavone (CF) in CCl4-intoxicated mice. a Group I (normal control) showing a normal histological structure of the central vein (cv) and intact hepatocytes. b Group II (CCl4-treated group) showing a severe loss of hepatic architecture with multiple focal necroses and ballooning degeneration in the hepatocytes (bd). c Group VI (CCl4 + 200 mg/kg silymarin) showing mild dilatation of the central vein. d, e and f Groups V, IV, and III (CCl4 + 160 mg/kg CF, CCl4 + 80 mg/kg CF, CCl4 + 40 mg/kg CF, respectively) showing mild ballooning degeneration (d) in the hepatocytes (H&E, × 20). (Color figure available online only.)
Original Papers
and nephroprotective therapeutic potential. The present study warrants further detailed studies using different animal models and controlled clinical trials on cupressuflavone as a promising hepato-nephroprotective agent for medicinal human use.
Materials and Methods !
General experimental procedures The NMR spectra were measured by a Bruker Ascend 400/R spectrometer (Bruker Avance BioSpin, Inc.). CD3OD was used as a solvent. Proton and carbon spectra were referenced internally to the TMS signal using a value of 0.00 ppm. The UV spectra were obtained with a Jasco V-630 UV/VIS spectrophotometer. HRESIMS was performed on a Bruker micrOTOF‑Q Daltonics time-of-flight mass spectrometer (Bruker Daltonics GmbH). The ionization technique was electrospray. The mass spectrometer was operated in the negative mode with the following: capillary voltage, 4000 V; end plate offset, − 500 V; drying gas (N2) flow rate, 8.4 L/ min; and temperature, 200 °C. For MS/MS measurements, argon was used as a collision gas and the voltage over the collision cell varied from 20 to 70 eV. The data were analyzed using Compass data analysis software (version 4.0 SP5; Bruker Daltonics). Analytical HPLC was performed using an Agilent HPLC‑DAD system consisting of a quaternary pump G1311A connected to a diode array and multiple wavelength detector G1315D, interface module D-7000, and autosampler G-1329A. Chromatographic separation was performed on a ZORBAX Eclipse XDB‑C18 column (4.6 × 150 mm; 5 µm; Agilent Technologies). The mobile phase consisted of acetonitrile (A) and 0.4 % phosphoric acid (B). The elution profile was 0–3 min, 100% B (isocratic); 3–30 min, 0– 30 % A in B; 30–35 min, 30–70 % A in B; 35–37 min, 70% A in B (isocratic) with a constant flow rate of 1 mL/min.
Chemicals CCl4 was purchased from El Nasr Pharmaceutical & Chemical Co. Silymarin (98% purity) was obtained from Sedico Pharmaceutical Co. All the assay kits were obtained from Biodiagnostics Co. The assay kit of LDH was purchased from Randox Laboratories Ltd. All other chemicals were of analytical grade. Sephadex LH-20 was purchased from Amersham Biosciences. RP‑C18 was obtained from Sigma-Aldrich GmbH, and precoated silica gel TLC GF254 was purchased from Riedel-De Häen-AG.
Plant material The leaves of C. macrocarpa were collected in August 2013 from El-Maryland Botanical Garden, Cairo, Egypt. The plant was botanically identified by Eng. Anour Ezeldein, the taxonomy specialist at the herbarium of El-Maryland Botanical Garden, Cairo, Egypt. A voucher specimen of C. macrocarpa was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU CMC2013).
Extract preparation and fractionation Air-dried powdered leaves of C. macrocarpa (2000 g) were extracted three times with MeOH. The total extract was concentrated under vacuum to obtain a gummy residue which was dissolved in 70 % MeOH and partitioned successively with hexane. The MeOH layer was then dried, redissolved in 50 % MeOH, and partitioned with CH2Cl2 to obtain two fractions. The 50 % MeOH fraction was concentrated and freeze-dried to obtain a dry powder (120 g). Column fractionation of part of this fraction (100 g)
Al-Sayed E, Abdel-Daim MM. Protective Role of … Planta Med 2014; 80: 1665–1671
was performed using Sephadex LH-20 (5 × 100 cm), eluted with H2O followed by H2O–MeOH mixtures of decreasing polarities (100 : 0–0 : 100, 1 L each). Fractions were combined based on their analytical HPLC profiles to afford ten major fractions. The fraction eluted with 40 % MeOH was concentrated and freezedried to obtain a dry powder (4 g) rich in CF. This fraction was then subjected to column fractionation on an RP-18 column (3 × 30 cm) with elution performed using H2O-MeOH mixtures (50 : 50–0 : 100, 50 mL each). Eight subfractions were combined based on their analytical HPLC profiles (tR of CF: 34.1 min). Elution with MeOH yielded CF (1.5 g, purity 88%).
Animals Male Swiss Albino mice, weighing 27.5 ± 2.5 g, were obtained from The Animal House Facility, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. The animals were housed in cages in a ventilated room under the controlled laboratory conditions of temperature (25 ± 2 °C) and under 12-h light/dark cycles. The mice were fed a standard rodent pellet chow, and water was provided ad libitum. The animals were acclimatized for one week before use. All the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH 1985) and approved by the ethical committee of the Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt (201406).
Acute toxicity study A group of normal male mice, weighing 27.5 ± 2.5 g, were used to determine the acute oral toxicity of CF according to a previous method [21]. The mice were divided into three groups (n = 8 per group). The subgroups were treated with 500, 1000, and 2000 mg/kg body weight p. o. of CF. The animals were observed for 24 h to record toxicity symptoms and mortality rates and for a further 14 days post-CF treatment to record possible toxicological or behavioral changes.
Experimental design Forty-eight Swiss Albino mice were divided into six groups (n = 8 per group). Group I was the normal control and received saline only. Group II was administered at a sublethal dose of CCl4 at a dose of 0.5 mL/kg body weight intraperitoneally (20% v/v in corn oil) at the end of the experiment and served as the positive control. The mice in groups III, IV, and V were treated orally with 40, 80, and 160 mg/kg body weight of CF, respectively. Group VI was treated with silymarin at a daily dose of 200 mg/kg [22]. All tested material and silymarin were administered orally, once daily, for five consecutive days. On the 5th day, a single i. p. injection of CCl4 (0.5 mL/kg body weight of 20 % v/v CCl4 solution in corn oil) was administered to all the groups, except the normal group, to induce hepatorenal injury [22]. Blood was collected by a direct cardiac puncture under diethyl ether anesthesia 24 h after the last treatment dose. The collected blood was left to clot at room temperature, and the sera were separated by centrifugation at 3000 rpm for 15 minutes and then stored at − 20 °C for biochemical analysis. The animals were then sacrificed by decapitation under diethyl ether anesthesia. The liver and kidney were removed rapidly, and a portion of each was homogenized for the estimation of GSH content, SOD activity, and lipid peroxidation. Another portion was preserved in 10 % formalin for the histopathology.
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Serum biochemical analysis Freshly separated sera were used for the estimation of liver biomarkers according to the manufacturer protocol and previously reported methods for determination of ALT, AST [23], ALP [24], LDH [25], total bilirubin [26], cholesterol [27, 28], and total proteins [29]. The biomarkers of kidney damage were estimated according to reported methods for determination of creatinine [30], urea [31], and uric acid [32].
Evaluation of tissue lipid peroxidation and antioxidant parameters Lipid peroxidation was estimated by measuring the MDA content in liver and kidney tissues [33]. The antioxidant parameters were assayed according to previous methods for evaluation of SOD activity [34] and GSH [35].
Histopathological examination Liver and kidney specimens from all experimental groups were fixed in 10% formol saline for 24 h, dehydrated in (50–100 %) ethanol, cleared in xylene, and then embedded in paraffin. Sections (4 µm thick) were prepared and then stained with hematoxylin and eosin. The liver and kidney sections were examined for pathological changes by a light microscope.
Statistical analysis All the data were expressed as the means ± SEM. The statistical analysis of the data was performed using the one-way ANOVA test followed by Tukeyʼs post hoc test to determine the difference between the means. All statistical analyses were performed using GraphPad InStat software (Version 3.06, La Jolla). P values < 0.05 were considered statistically significant.
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Supporting information NMR, UV, and HRESI‑MS/MS spectral data of the isolated compound, identifying it as CF, are available as Supporting Information.
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Conflict of Interest
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The authors have declared no conflicts of interest.
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Original Papers
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Authors
Eman Al-Sayed 1*, Mohamed M. Abdel-Daim 2, 3*
Affiliations
1 2 3
Key words " Cupressus macrocarpa l " Cupressaceae l " hepatoprotective l " nephroprotective l " biflavones l " lipid peroxidation l
received revised accepted
Sept. 14, 2014 Sept. 24, 2014 Sept. 30, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383211 Published online October 22, 2014 Planta Med 2014; 80: 1665–1671 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Eman Al-Sayed Faculty of Pharmacy Ain-Shams University Department of Pharmacognosy Cairo, 11566 Egypt Phone: + 20 20 10 01 05 02 93 Fax: + 20 24 05 11 06 em_alsayed@ pharma.asu.edu.eg
Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt Department of Pharmacology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt United Graduate School of Drug Discovery and Medical Information Sciences, Department of Gene and Development, Graduate School of Medicine, Gifu University, Gifu, Japan
Abstract !
The hepatoprotective and nephroprotective activity of cupressuflavone isolated from Cupressus macrocarpa was investigated against CCl4-induced toxicity in mice. Cupressuflavone was administered (40, 80, and 160 mg/kg/day) for five days. CCl4 was administered (0.5 mL/kg intraperitoneally) at the end of the experiment. A substantial increase (p < 0.001) in the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, total bilirubin, cholesterol, creatinine, uric acid, urea, and malondialdehyde was observed in the CCl4treated group compared to the normal control group. In contrast, a significant reduction (p < 0.001) in glutathione and superoxide dismutase contents as well as the total protein level was evident in the CCl4-intoxicated mice. Cupressuflavone pretreatment markedly inhibited the CCl4induced increase in alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, cholesterol, creatinine, uric acid, urea, and malondialdehyde levels in a
Introduction !
The liver is the primary organ of metabolism, and the kidneys are the main organs involved in the excretion of xenobiotics; therefore, the toxic effects of chemicals appear primarily in the liver and kidney tissues [1]. Oxidative stress plays a prominent causative role in many diseases, including inflammation, liver damage, and carcinogenesis [2]. Oxidative stress is caused by the cellular excess of reactive oxygen species (ROS), which induces lipid peroxidation and cell injury [3]. Under normal states, the cells maintain the ROS
* Both of these authors contributed equally to this article.
dose-dependent manner (p < 0.001 at all the tested doses). In addition, a significant (p < 0.001) and dose-dependent decrease in the total bilirubin levels was evident by cupressuflavone pretreatment (80 and 160 mg/kg/day) when compared to the CCl4-intoxicated group. Furthermore, cupressuflavone administration significantly increased the activity of antioxidant parameters glutathione and superoxide dismutase as well as the serum protein levels (p < 0.001 at all the tested doses) in a dose-dependent manner. Histological observations confirmed the strong hepato- and nephroprotective activity. These findings suggest that cupressuflavone could exert a beneficial effect against oxidative stress by enhancing the antioxidant defense status, reducing lipid peroxidation, and protecting against the pathological changes induced by CCl4 in the liver and kidney tissues. The structure of cupressuflavone was identified by NMR, UV, and HRESI‑MS/MS spectral data. Supporting information available online at http://www.thieme-connect.de/products
levels with endogenous antioxidants [2]. The risk of cell injury may also be prevented by natural antioxidants, including dietary flavonoids [4]. Considerable evidence indicates that regular consumption of dietary flavonoids have beneficial effects on health, including protection against oxidative stress and various degenerative diseases [5, 6]. Flavonoids can restore the balance between the natural antioxidants and free radicals by direct scavenging of ROS, metal chelation, and the induction of natural antioxidant defenses, e.g., glutathione (GSH) and superoxide dismutase (SOD) [4, 5, 7]. The genus Cupressus (Cupressaceae) comprises twelve species. Different Cupressus species have been used in traditional medicine for the treat-
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Protective Role of Cupressuflavone from Cupressus macrocarpa against Carbon Tetrachloride-Induced Hepato- and Nephrotoxicity in Mice
Original Papers
ment of several ailments, including cough, inflammation, and infection [8]. Cupressus macrocarpa Hartw. ex Gord is a widely planted ornamental tree belonging to the family Cupressaceae [9]. Several contributions have been reported on the composition of C. macrocarpa essential oil and indicated that neral, hydroxy citronellal, geraniol, and piperitol are the main constituents of C. macrocarpa essential oil [9]. However, no pharmacological studies have been conducted on the nonvolatile constituents of C. macrocarpa. The main chemotaxonomic chemical components of the Cupressus genus are biflavonoids [10], for which a broad spectrum of biological activity has been reported [10, 11]. Cupressuflavone (CF), a biflavonoid isolated from Thuja orientalis, exhibited neutrophil elastase inhibitory activity in vitro [12]. Moreover, CF from C. sempervirens showed a potential osteoprotective effect [13]. Despite the diverse medicinal properties of biflavonoids, no comprehensive study, to the best of our knowl-
Fig. 1 Structure of cupressuflavone.
Fig. 2 Effect of cupressuflavone (CF) and silymarin on the hepatic function tests of CCl4-intoxicated mice. (CF 1) Group III (CCl4 + 40 mg/kg CF). (CF 2) Group IV (CCl4 + 80 mg/kg CF). (CF 3) Group V (CCl4 + 160 mg/kg CF). Data
Al-Sayed E, Abdel-Daim MM. Protective Role of … Planta Med 2014; 80: 1665–1671
edge, has so far been reported on the hepato- and nephroprotective activity of the isolated compound CF. Therefore, this study was designed to determine the hepato- and nephroprotective activity of CF isolated from C. macrocarpa against CCl4-induced toxicity in mice, along with possible mechanisms of hepato- and nephroprotective activity. A histopathological observation of liver and kidney sections was performed to confirm the hepato- and nephroprotective activity. The chemical structure of CF was identified by NMR, UV, and HRESI‑MS/MS data.
Results ! " Fig. 1) was elucidated based on NMR, UV, The structure of CF (l and HRESI‑MS/MS spectral data (Supporting Information), which are consistent with the data reported in the literature [10, 14, 15]. Based on the acute toxicity results, no adverse behavioral changes, toxicity symptoms, or mortality were observed in mice at doses up to 2000 mg/kg of CF (LD50 > 2000 mg/kg). Based on these findings, CF is considered safe in mice. A significant increase (p < 0.001) in the activity of the biomarkers of liver damage, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), was observed in the CCl4-intoxicated mice " Fig. 2). Pretreatment compared to the negative control group (l with CF produced a marked hepatoprotective effect and reduced the activity of all hepatic enzymes in a dose-dependent manner (p < 0.001 at all the tested doses). The percentage of decrease in the liver marker enzymes at the treatment doses (40, 80, and 160 mg/kg/day) was 21, 46, and 56% for ALT; 27, 52, and 63 % for AST; 23, 51, and 59 % for ALP; and 25, 50, and 62% for LDH, re-
are expressed as the means ± SEM (n = 8). Values having different superscripts are significantly different at p < 0.05.
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Table 1 Effect of cupressuflavone (CF) on biochemical parameters of CCl4-intoxicated mice. Animal groups Control CCl4 CCl4 + 40 mg/kg CF CCl4 + 80 mg/kg CF CCl4 + 160 mg/kg CF CCl4 + 200 mg/kg silymarin
Total bilirubin
Cholesterol
Total proteins
Creatinine
Uric acid
Urea
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
1.22 ± 0.05a 1.68 ± 0.04b 1.55 ± 0.02b (− 8%) 1.35 ± 0.04a (− 20%) 1.28 ± 0.04a (− 24%) 1.24 ± 0.04a (− 26%)
75.28 ± 3.40a 152.10 ± 4.70b 116.96 ± 4.95c (− 23%) 106.64 ± 4.05c (− 30%) 77.00 ± 3.54a (− 49%) 73.43 ± 3.18a (− 52%)
7.49 ± 0.16a 5.70 ± 0.13b 6.63 ± 0.10c (16%) 7.02 ± 0.13a, c (23%) 7.33 ± 0.08a (29%) 7.43 ± 0.14a (30%)
0.31 ± 0.03a 6.97 ± 0.43b 3.51 ± 0.26c (− 50%) 2.25 ± 0.29d (− 68%) 0.59 ± 0.07a (− 92%) 0.46 ± 0.06a (− 93%)
23.93 ± 0.99a 89.66 ± 4.58b 70.72 ± 2.67c (− 21%) 55.29 ± 3.27d (− 38%) 33.46 ± 1.86a (− 63%) 26.84 ± 1.87a (− 70%)
24.24 ± 1.13a 83.16 ± 4.37b 63.19 ± 2.85c (− 24%) 48.49 ± 3.47d (− 42%) 32.39 ± 1.64a (− 61%) 25.88 ± 1.52a (− 69%)
within the same column are significantly different at p < 0.05
spectively, compared to the CCl4-intoxicated group. Notably, CF pretreatment at a dose of 160 mg/kg markedly reduced the levels of ALT, AST, ALP, and LDH, which were comparable and nonsignificant to those in the normal and silymarin-treated groups " Fig. 2). Similarly, a marked and dose-dependent decrease in (l the serum bilirubin and cholesterol levels was observed in the " Table 1). In contrast, the total protein CF pretreated groups (l levels were significantly (p < 0.001) improved in all of the groups treated with CF in a dose-dependent manner. The total protein levels in the groups treated with 80 and 160 mg/kg of CF were comparable to those in the normal control and silymarin groups " Table 1). The biochemical markers of kidney damage, uric acid, (l urea, and creatinine were significantly (p < 0.001) reduced in all " TaCF-treated groups compared to the CCl4-intoxicated group (l ble 1). These findings suggest that CF effectively reduced the CCl4induced hepatorenal toxicity. A significant reduction (p < 0.001) in hepatic and renal GSH and SOD contents was observed in CCl4-intoxicated mice. In contrast, the malondialdehyde (MDA) levels were markedly increased (p < 0.001) compared with those in the normal control group " Fig. 3). Administration of CF at the treatment doses (40, 80, (l and 160 mg/kg/day) induced a marked increase in hepatic GSH (by 24, 61, and 89 %, respectively) when compared to the CCl4-intoxicated group (p < 0.001 for the groups treated with 80 and 160 mg/kg of CF). Also, the groups treated with CF showed a marked increase in renal GSH (by 31, 53, and 82%, respectively; p value < 0.05 for the group treated with 40 mg/kg, and p values < 0.001 for the groups treated with 80 and 160 mg/kg of CF). Moreover, pretreatment with CF, at all the tested doses, significantly (p < 0.001) improved the hepatic SOD (by 27, 69, and 113 %, respectively), and raised the activity of renal SOD (by 57, 84, and 103%, respectively) relative to the CCl4-intoxicated group. In addition, CF pretreatment, at all the tested doses, ameliorated the CCl4-induced increase in hepatic MDA by 24, 51, and 55 % and the renal MDA by 24, 37, and 53 % at the tested doses, respectively, when compared to the CCl4-intoxicated group (p < 0.001) " Fig. 3). Notably, the liver and kidney GSH contents in the (l groups treated with 160 mg/kg of CF were comparable and nonsignificant to those in the normal control and silymarin groups. CF pretreatment at a dose of 160 mg/kg was the most effective in reducing the hepatic and renal MDA levels. There was no significant difference between the MDA levels in the groups treated with 80 and 160 mg/kg of CF and those in the normal control or silymarin group. These results clearly indicated the strong in vivo antioxidant activity conferred by CF.
Liver sections of the normal control mice showed the preserved histological structure of the central vein surrounded by intact hepatocytes. CCl4 induced severe loss of hepatic architecture with ballooning degeneration in the hepatocytes all over the hepatic parenchyma together with multiple focal necroses. The pathological changes induced by CCl4 were markedly ameliorated in the groups treated with CF in a dose-dependent manner. Pretreatment with 160 mg/kg of CF conferred marked protection against liver damage as evidenced by the intact hepatic architecture. Mild ballooning degeneration in the hepatocytes was observed " Fig. 4). The in the groups treated with 40 and 80 mg/kg of CF (l histological examination of kidney sections of the normal control group revealed a normal histological structure of the glomeruli and tubules at the cortex with the absence of histopathological changes. CCl4 induced marked inflammatory cell aggregation in between the tubules, together with marked degeneration in the lining epithelium of all the tubules. The aforementioned renal histopathological changes induced by CCl4 were markedly reduced in the groups treated with CF at the three treatment doses. Notably, pretreatment with CF at a dose of 160 mg/kg preserved the normal histological structure of the kidneys and markedly reduced all the degenerative changes in the lining tubular epithelium. The group pretreated with 80 mg/kg of CF showed mild focal inflammatory cell infiltration in between the tubules. Mild degeneration in the lining epithelium of some of the tubules was " Fig. 5). observed in the group pretreated with 40 mg/kg of CF (l
Discussion !
Biflavonoids have been reported to have several pharmacological effects [10, 11]. Agathisflavone, a biflavone isolated from Canarium manii, exhibited a hepatoprotective activity against CCl4-induced hepatotoxicity in rats and mice [16]. Furthermore, amentoflavone, one of the major components of the Ginkgo biloba leaves, showed diverse pharmacological properties, including strong antioxidant activity, an inhibitory effect on lipid peroxidation, a neuroprotective effect, and anti-inflammatory activity [11]. The total extracts of C. sempervirens and Juniperus phoenicea showed hepatoprotective activity against CCl4-induced hepatotoxicity in rats [8, 17]. However, no study on the hepato- and nephroprotective activity of CF was reported. Therefore, this study was undertaken to determine the mechanistic basis for the hepato- and nephroprotective effects of CF.
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Data are expressed as the means ± SEM (n = 8). Numbers in parentheses represent the percentage of change from the CCl4-intoxicated group. Values having different superscripts
Original Papers
Fig. 3 Effect of cupressuflavone (CF) and silymarin on lipid peroxidation and antioxidant parameters in the liver and kidneys of CCl4-intoxicated mice. (CF 1) Group III (CCl4 + 40 mg/kg CF). (CF 2) Group IV (CCl4 + 80 mg/kg CF). (CF
Various xenobiotics, including CCl4, are known to cause hepatorenal toxicity. Liver damage is a major health problem which may develop into several liver diseases, including hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma [18]. CCl4 is a highly hepatotoxic chemical widely used in experimental animal models of liver and kidney damage that mimic human hepatorenal toxicity [3, 19]. The highly reactive trichloromethyl radicals (CCl3• and CCl3O2•) are formed from CCl4 by cytochrome P-450. These radicals initiate lipid peroxidation and necrosis of the liver. The trichloromethyl radicals also change the cellular antioxidant capacity by deactivating GSH and the antioxidant enzymes [3, 20]. In this study, administration of CCl4 to mice significantly increased the levels of AST, ALT, ALP, LDH, cholesterol, and total bilirubin. Furthermore, it reduced the total protein level, which indicated a severe loss of liver function. A histopathological examination revealed a severe loss of hepatic structure associated with multiple necroses and a marked ballooning degeneration of the hepatocytes. Moreover, marked inflammatory cell infiltration
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3) Group V (CCl4 + 160 mg/kg CF). Data are expressed as the means ± SEM (n = 8). Values having different superscripts are significantly different at p < 0.05.
and degeneration of the kidney tissues were observed. CCl4 intoxication also induced severe oxidative stress as demonstrated by the marked increase in hepatic and renal MDA levels. The SOD and GSH levels in the liver and kidney tissues were markedly reduced in response to CCl4 intoxication. The results of the present study indicate that CF pretreatment reduced the increased MDA levels to their normal values. The inhibitory effect against lipid peroxidation suggests that CF could prevent the hepatorenal injury induced by CCl4 together with the subsequent pathological damage in the liver and kidney tissues. In contrast, pretreatment with CF markedly enhanced the GSH and SOD levels when compared to the CCl4-intoxicated mice. Modulation of these antioxidant enzymes clearly contributed to the hepatoand nephroprotective effect of CF. These findings suggest that the superior hepato- and nephroprotective activity of CF can be attributed to its ability to enhance the antioxidant defense status, to reduce the rate of lipid peroxidation, and to guard against the pathological changes induced by CCl4 intoxication in the liver and
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Fig. 5 Nephroprotective effect of cupressuflavone (CF) in CCl4-intoxicated mice. a Group I (normal control) showing a normal histological structure of the glomeruli (g) and tubules (t) at the cortex with the absence of histopathological changes. b Group II (CCl4-treated group) showing marked inflammatory cell aggregation (m) in between the tubules and marked degeneration (d) in the lining epithelium of all the tubules. c Group VI (CCl4 + 200 mg/kg silymarin) showing the absence of histopathological alterations. d Group V (CCl4 + 160 mg/kg CF) showing a normal histological structure. e Group IV (CCl4 + 80 mg/kg CF) showing focal inflammatory cell infiltration (m) in between the tubules. f Group III (CCl4 + 40 mg/kg CF) showing mild degeneration in the lining epithelium of some of the tubules (H&E, × 20). (Color figure available online only.)
kidney tissues. In conclusion, the protective effect of CF against oxidative stress associated with its protective effect against infil-
tration by inflammatory cells and other CCl4-induced pathological changes in the liver and kidneys indicated that CF has hepato-
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Fig. 4 Hepatoprotective effect of cupressuflavone (CF) in CCl4-intoxicated mice. a Group I (normal control) showing a normal histological structure of the central vein (cv) and intact hepatocytes. b Group II (CCl4-treated group) showing a severe loss of hepatic architecture with multiple focal necroses and ballooning degeneration in the hepatocytes (bd). c Group VI (CCl4 + 200 mg/kg silymarin) showing mild dilatation of the central vein. d, e and f Groups V, IV, and III (CCl4 + 160 mg/kg CF, CCl4 + 80 mg/kg CF, CCl4 + 40 mg/kg CF, respectively) showing mild ballooning degeneration (d) in the hepatocytes (H&E, × 20). (Color figure available online only.)
Original Papers
and nephroprotective therapeutic potential. The present study warrants further detailed studies using different animal models and controlled clinical trials on cupressuflavone as a promising hepato-nephroprotective agent for medicinal human use.
Materials and Methods !
General experimental procedures The NMR spectra were measured by a Bruker Ascend 400/R spectrometer (Bruker Avance BioSpin, Inc.). CD3OD was used as a solvent. Proton and carbon spectra were referenced internally to the TMS signal using a value of 0.00 ppm. The UV spectra were obtained with a Jasco V-630 UV/VIS spectrophotometer. HRESIMS was performed on a Bruker micrOTOF‑Q Daltonics time-of-flight mass spectrometer (Bruker Daltonics GmbH). The ionization technique was electrospray. The mass spectrometer was operated in the negative mode with the following: capillary voltage, 4000 V; end plate offset, − 500 V; drying gas (N2) flow rate, 8.4 L/ min; and temperature, 200 °C. For MS/MS measurements, argon was used as a collision gas and the voltage over the collision cell varied from 20 to 70 eV. The data were analyzed using Compass data analysis software (version 4.0 SP5; Bruker Daltonics). Analytical HPLC was performed using an Agilent HPLC‑DAD system consisting of a quaternary pump G1311A connected to a diode array and multiple wavelength detector G1315D, interface module D-7000, and autosampler G-1329A. Chromatographic separation was performed on a ZORBAX Eclipse XDB‑C18 column (4.6 × 150 mm; 5 µm; Agilent Technologies). The mobile phase consisted of acetonitrile (A) and 0.4 % phosphoric acid (B). The elution profile was 0–3 min, 100% B (isocratic); 3–30 min, 0– 30 % A in B; 30–35 min, 30–70 % A in B; 35–37 min, 70% A in B (isocratic) with a constant flow rate of 1 mL/min.
Chemicals CCl4 was purchased from El Nasr Pharmaceutical & Chemical Co. Silymarin (98% purity) was obtained from Sedico Pharmaceutical Co. All the assay kits were obtained from Biodiagnostics Co. The assay kit of LDH was purchased from Randox Laboratories Ltd. All other chemicals were of analytical grade. Sephadex LH-20 was purchased from Amersham Biosciences. RP‑C18 was obtained from Sigma-Aldrich GmbH, and precoated silica gel TLC GF254 was purchased from Riedel-De Häen-AG.
Plant material The leaves of C. macrocarpa were collected in August 2013 from El-Maryland Botanical Garden, Cairo, Egypt. The plant was botanically identified by Eng. Anour Ezeldein, the taxonomy specialist at the herbarium of El-Maryland Botanical Garden, Cairo, Egypt. A voucher specimen of C. macrocarpa was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU CMC2013).
Extract preparation and fractionation Air-dried powdered leaves of C. macrocarpa (2000 g) were extracted three times with MeOH. The total extract was concentrated under vacuum to obtain a gummy residue which was dissolved in 70 % MeOH and partitioned successively with hexane. The MeOH layer was then dried, redissolved in 50 % MeOH, and partitioned with CH2Cl2 to obtain two fractions. The 50 % MeOH fraction was concentrated and freeze-dried to obtain a dry powder (120 g). Column fractionation of part of this fraction (100 g)
Al-Sayed E, Abdel-Daim MM. Protective Role of … Planta Med 2014; 80: 1665–1671
was performed using Sephadex LH-20 (5 × 100 cm), eluted with H2O followed by H2O–MeOH mixtures of decreasing polarities (100 : 0–0 : 100, 1 L each). Fractions were combined based on their analytical HPLC profiles to afford ten major fractions. The fraction eluted with 40 % MeOH was concentrated and freezedried to obtain a dry powder (4 g) rich in CF. This fraction was then subjected to column fractionation on an RP-18 column (3 × 30 cm) with elution performed using H2O-MeOH mixtures (50 : 50–0 : 100, 50 mL each). Eight subfractions were combined based on their analytical HPLC profiles (tR of CF: 34.1 min). Elution with MeOH yielded CF (1.5 g, purity 88%).
Animals Male Swiss Albino mice, weighing 27.5 ± 2.5 g, were obtained from The Animal House Facility, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. The animals were housed in cages in a ventilated room under the controlled laboratory conditions of temperature (25 ± 2 °C) and under 12-h light/dark cycles. The mice were fed a standard rodent pellet chow, and water was provided ad libitum. The animals were acclimatized for one week before use. All the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH 1985) and approved by the ethical committee of the Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt (201406).
Acute toxicity study A group of normal male mice, weighing 27.5 ± 2.5 g, were used to determine the acute oral toxicity of CF according to a previous method [21]. The mice were divided into three groups (n = 8 per group). The subgroups were treated with 500, 1000, and 2000 mg/kg body weight p. o. of CF. The animals were observed for 24 h to record toxicity symptoms and mortality rates and for a further 14 days post-CF treatment to record possible toxicological or behavioral changes.
Experimental design Forty-eight Swiss Albino mice were divided into six groups (n = 8 per group). Group I was the normal control and received saline only. Group II was administered at a sublethal dose of CCl4 at a dose of 0.5 mL/kg body weight intraperitoneally (20% v/v in corn oil) at the end of the experiment and served as the positive control. The mice in groups III, IV, and V were treated orally with 40, 80, and 160 mg/kg body weight of CF, respectively. Group VI was treated with silymarin at a daily dose of 200 mg/kg [22]. All tested material and silymarin were administered orally, once daily, for five consecutive days. On the 5th day, a single i. p. injection of CCl4 (0.5 mL/kg body weight of 20 % v/v CCl4 solution in corn oil) was administered to all the groups, except the normal group, to induce hepatorenal injury [22]. Blood was collected by a direct cardiac puncture under diethyl ether anesthesia 24 h after the last treatment dose. The collected blood was left to clot at room temperature, and the sera were separated by centrifugation at 3000 rpm for 15 minutes and then stored at − 20 °C for biochemical analysis. The animals were then sacrificed by decapitation under diethyl ether anesthesia. The liver and kidney were removed rapidly, and a portion of each was homogenized for the estimation of GSH content, SOD activity, and lipid peroxidation. Another portion was preserved in 10 % formalin for the histopathology.
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Serum biochemical analysis Freshly separated sera were used for the estimation of liver biomarkers according to the manufacturer protocol and previously reported methods for determination of ALT, AST [23], ALP [24], LDH [25], total bilirubin [26], cholesterol [27, 28], and total proteins [29]. The biomarkers of kidney damage were estimated according to reported methods for determination of creatinine [30], urea [31], and uric acid [32].
Evaluation of tissue lipid peroxidation and antioxidant parameters Lipid peroxidation was estimated by measuring the MDA content in liver and kidney tissues [33]. The antioxidant parameters were assayed according to previous methods for evaluation of SOD activity [34] and GSH [35].
Histopathological examination Liver and kidney specimens from all experimental groups were fixed in 10% formol saline for 24 h, dehydrated in (50–100 %) ethanol, cleared in xylene, and then embedded in paraffin. Sections (4 µm thick) were prepared and then stained with hematoxylin and eosin. The liver and kidney sections were examined for pathological changes by a light microscope.
Statistical analysis All the data were expressed as the means ± SEM. The statistical analysis of the data was performed using the one-way ANOVA test followed by Tukeyʼs post hoc test to determine the difference between the means. All statistical analyses were performed using GraphPad InStat software (Version 3.06, La Jolla). P values < 0.05 were considered statistically significant.
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Supporting information NMR, UV, and HRESI‑MS/MS spectral data of the isolated compound, identifying it as CF, are available as Supporting Information.
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Conflict of Interest
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The authors have declared no conflicts of interest.
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