Ceratonia silique is a typical works of the Mediterranean country, which is chiefly used as animate being, human nutrient and in common people Tunisian traditional medical specialty. The hydroethanolic infusion obtained from C. silique leaves was fractionated by liquid-liquid divider utilizing n-hexane, trichloromethane and ethyl ethanoate in turn. Among the tried infusions, the ethyl ethanoate of C. silique ( EACS ) fraction exhibited the highest entire phenoplast and flavonoids content. The antioxidant activity using assorted in vitro systems showed a powerful free extremist scavenging activity of this infusion ( IC50 = 1.8 Aµg/ml ) than the other fractions. Six phenolic compounds were identified in the active ethyl ethanoate infusion by liquid chromatography electrospray ionisation tandem mass spectroscopy. They included ( 1 ) 1,6-Di-galloyl-glucose, ( 2 ) 1,2,6-Tri-galloyl-glucose, ( 3 ) Myricetin glucoside, ( 4 ) 1,2,3,6-Tetra-galloyl-glucose, ( 5 ) Myricetin rhamnoside and ( 6 ) Syringic acid. In the present survey, we focused on the protective consequence of Ceratonia silique ethyl ethanoate infusion in vivo against Carbon tetrachloride ( CCl4 ) induced hepato and nephrotoxicity in the experimental rats. The consequences showed that unwritten disposal of CCl4 caused increased degrees of hepatic and nephritic markers ( Alanine transaminase ( ALT ) , Aspartate transaminase ( AST ) , Alkaline phosphatase ( ALP ) , Lactate dehydrogenase ( LDH ) , I?-Glutamyl transpeptidase ( I?-GT ) , urea, creatinine and uric acid ) in the serum of experimental animate beings. It besides increased the oxidative emphasis ensuing in increased degrees of the lipid peroxidation with a attendant diminution in the degrees of enzymatic antioxidants ( Superoxide dismutase ( SOD ) , Catalase ( CAT ) , Glutathione peroxidase ( GPx ) ) in both liver and kidney. The pretreatment with ethyl acetate infusion of Ceratonia silique ( EACS ) ( 250 mg/kg b.w. ) by intraperitoneal injection for 8 yearss prevented CCl4 induced alterations in the degrees of hepatic and kidney diagnostic marker enzymes in the serum of experimental rats. It counteracted the CCl4 induced lipid peroxidation and maintained the hepatic and kidney antioxidant defence system at near normal in both liver and kidney. The biochemical alterations were consistent with histopathological observations proposing marked hepatoprotective and nephroprotective consequence of the EA leaves infusion. The antioxidant reactivity mediated by C. silique foliages may be anticipated to hold biological significance in extinguishing reactive free groups induced by CCl4 and the mechanism involved in the protection could be due to their antioxidant activities.
Keywords: Ceratonia silique, Carbon tetrachloride, oxidative emphasis, protective consequence, phenolic compounds.
Reactive O species ( ROS ) are assorted signifiers of activated O ( Yildirim et al. , 2000 ) . A disproportion of the reactive O species and the absence of their scavenge systems in cells lead to oxidative emphasis and increases the hazard of several human chronic diseases ( Sies et al. , 2005 ) . ROS contributes to the development of assorted diseases such as coronary artery disease, diabetes, malignant neoplastic disease, neurodegenerative diseases, liver cirrhosis and the ripening procedure ( Basaga, 1990 ) . The liver plays a cardinal function in the care of systemic lipid homeostasis and is particularly susceptible to ROS harm ( Hamelet et al. , 2007 ) . Assorted xenobiotics are known to do hepatotoxicity one among them is carbon tetrachloride ( CCl4 ) ( Kodavanti, Joshi, Young, Meydrech, & A ; Mehendale, 1989 ) . CCl4 is metabolized by cytochrome P4502E1 ( CYP2E1 ) to go trichloromethyl extremist ( .CCl3 ) and proxy trichloromethyl extremist ( .OOCCl3 ) , which are assumed to originate free radical-mediated lipid peroxidation taking to the accretion of lipid-derived oxidization merchandises that cause liver hurt ( Liao et al. , 2007 ; Shyur et al. , 2008 ) . Therefore, CCl4 is now of greatest concern as an environmental contamination ( ATSDR, 1994 ) . It was reported that CCl4 is one of the most normally used toxins in the experimental survey of liver diseases ( Wang et al. , 2007 ) . Previous surveies showed that both liver and kidneys are the mark variety meats of CCl4 ( David E et al. , 2010 ) . Abraham et al. , ( 1999 ) show that the nephrotoxic effects of CCl4 are besides associated with free extremist production ( Abraham, Wilfred and Cathrine, 1999 ) . CCl4 induces sublethal proximal cannular hurt in the kidney and focal changes in farinaceous pneumatocytes ( Rajesh and Latha, 2004 ) .
Hispathologically, exposure to CCl4 can consequences in hepatic steatosis, centrilobular mortification, and cirrhosis in the liver and acute cannular mortification in the kidney ( Brattin, Glende and Recknagel, 1985 ; Rush, Smith, Newton and Hook, 1984 ) .
To forestall the harm caused by ROS, populating beings have developed an antioxidant defence system that includes the presence of non ezymatic antioxidants and enzymes such as superoxide dismutase ( SOD ) , catalase ( CAT ) , and glutathione peroxidase ( GPx ) ( Valko et al. , 2007 ) . It has been anticipated that in add-on to these natural antioxidants, other man-made or natural ROS scavengers may cut down the incidence of free radical-mediated diseases. The usage of antioxidants in the bar and remedy of assorted diseases is escalating, and there is considerable involvement in the survey of the antioxidant activities of molecules such as works polyphenolic and carotenoid constituents ( Valko et al. , 2007 ; Fang et al. , 2002 ) . Antioxidants appear to move against disease procedures by promoting the degrees of endogenous antioxidant enzymes and diminishing lipid peroxidation ( Bansal et al. , 2005 ) .
A figure of surveies showed that assorted herbal infusions could protect liver and kidney against CCl4-induced oxidative emphasis by suppressing lipid peroxidation and heightening antioxidant enzyme activity ( Shahjahan, Sabitha, Jainu, & A ; Devi, 2004 ) .
Ceratonia silique ( C. silique ) , normally known as Carob, belongs to the household of Leguminosae. The tree attains a mature tallness and spread of 6-12 m, and sometimes more than 20 m, with subdivisions extended to anchor degree ( Shigenorik et al. , 2002 ) . The foliages and fruits of this works are used to handle a assortment of diseases. Carob cods have traditionally been used as animate being and human nutrient and presently the chief usage is the seed for gum extraction. Bark and foliages have been used in Tunisian common people medical specialty as laxative, diuretic, antidiarrheal and for the intervention of stomach flu of breastfeeding babes ( Kivcak et al. , 2002 ) . Furthermore, recent surveies have confirmed the presence of antioxidant, hypocholesterolemic activities in cods of C. silique ( Dimitris and Makris, 2004 ) and antiproliferative effects on T1 cell line and substances moving on peripheral benzodiazepine receptor in its foliages ( Avallone et al. , 2002 ; Corsi et al. , 2002 ) . From the experimental and clinical surveies performed on C. silique, it seems that most of its pharmacological actions are due to its antioxidant activity which is chiefly due to its ability to scavenge free groups and / or inhibit lipid peroxidation ( Kumazawa et al. , 2002 ) .
In our cognition, the usage of C. silique to relieve the oxidative harm induced by C tetrachloride has non antecedently examined. There are a few studies depicting its protective effects on the oxidative harm. However, several surveies have shown that the hepatoprotective consequence is associated with antioxidant rich works infusions ( De Shukla, Ravishankar, & A ; Bhavasar, 1996 ; Emmanuel, Amalaraj, & A ; Ignacimuthu, 2001 ) . In this survey, we report the antioxidant effects of different infusions of C. silique leaves in several in vitro systems: ( lipid peroxidation, DPPH and hydroxyl extremist scavenging activities ) and to find the possible protective effects of C. siliqua infusion against oxidative harm of the liver and kidney following orally disposal of CCl4, by finding oxidative emphasis profile and some serum biochemical parametric quantities.
Materials and Methods
2.2-diphenyl-l-picrylhydrazyl ( DPPH ) , butylated hydroxytoluene ( BHT ) , thiobarbituric acid ( TBA ) , glutathione ( GSH ) , oxidised glutathione ( GSSG ) , glutathione reductase ( GR ) , bovid serum albumen ( BSA ) and 2,4-dinitrophenyl hydrazine ( DNPH ) were purchased from Sigma Chemical France. Trichloroacetic acid ( TCA ) , hydrogen peroxide ( H2O2 ) , 5,5 ‘ dithiobis ( 2-nitrobenzoic acid ) ( DTNB ) , Folin-Ciocalteu reagent, Na carbonate ( Na2CO3 ) and other dissolvers, were of analytical class and were newly prepared in distilled H2O.
Preparation of C. siliqua foliage infusions
Fresh foliages of C. silique were collected from Chebba ( Mahdia, Tunisia, latitude 35.23A° and longitude. 11.11A° ) and the designation was performed harmonizing to the vegetation of Tunisia ( Chaieb & A ; Boukhris, 1998 ) and a verifier specimen ( LBPes C.S. 15.01 ) was deposited in the research lab of Biopesticides of the Centre of Biotechnology of Sfax. The dried foliages were land to ticket pulverization utilizing a bomber and the resulted stuff ( 500 g of pulverization ) was extracted by maceration in ethanol-water 80 % with shaking, at room temperature. 15 g of the dried hydroethanolic petroleum infusion was suspended in 300 milliliters distilled H2O and was consecutive partitioned with n-hexane ( 3 x 350 milliliter ) , methylene chloride ( 3 x 350 milliliter ) and ethyl ethanoate ( 3 x 350 milliliter ) . The ensuing three fractions were evaporated under vacuity to dryness to give the hexane ( thousand = 500 milligram ) , the methylene chloride ( thousand = 1.25 g ) and the ethyl ethanoate ( thousand = 10.5 g ) fractions. The staying aqueous bed was lyophilised to give H2O fraction ( thousand = 2.5 g ) . The stock solutions were kept at 4 A°C in the dark until farther analysis.
Entire phenolic content
Entire phenoplasts content was determined utilizing the Folin-Ciocalteu method ( Waterman & A ; Mole, 1994 ) adapted to a microscale. Briefly, 10 Aµl diluted extract solution was shaken for 5 min with 50 Aµl of Folin-Ciocalteau reagent. Then 150 Aµl of 20 % Na2CO3 was added and the mixture was shaken one time once more for 1 min. Finally, the solution was brought up to 790 Aµl by adding distilled H2O. After 90 min, the optical density at 760 nanometer was evaluated utilizing a spectrophotometer. Gallic acid was used as a criterion for the standardization curve. The phenolic content was expressed as milligram Gallic acerb equivalent/gram of dry infusion utilizing the additive equation based on the standardization curve.
Determination of entire flavonoids content
The flavonoids content in infusions was determined spectrophotometrically harmonizing to ( Quettier-Deleu et al. , 2000 ) , utilizing a method based on the formation of a complex flavonoid-aluminium, holding the maximal soaking up at 430A nanometer. The flavonoids content was expressed in milligram of quercetin tantamount per g of dry works infusion ( mg QE/g ) .
Antioxidant testing checks
DPPH extremist scavenging activity
Extremist scavenging activity of C. silique infusions was determined utilizing DPPH as a reagent harmonizing to the method of Kirby & A ; Schmidt ( 1997 ) with little alterations. Briefly, 1 milliliter of a 4 % ( w/v ) solution of DPPH extremist in methyl alcohol was assorted with 500 Aµl of sample solutions in ethyl alcohol at different concentrations. The mixture was incubated for 20 min in the dark at room temperature. The scavenging capacity was determined spectrophotometrically by supervising the lessening in optical density at 517 nanometers against a clean utilizing a spectrophotometer ( Bio-Rad SmartSpecTM plus ) . Lower optical density of the reaction mixture indicates higher free extremist scavenging activity. Ascorbic acid was used as positive control. The per centum DPPH scavenging consequence was calculated utilizing the undermentioned equation: DPPH scavenging consequence ( % ) = ( A control- A sample / A control ) A-100
Where Acontrol is the optical density of the control reaction incorporating all reagents except the tested compound. Asample is the optical density of the trial compound. Extract concentration supplying 50 % suppression ( IC50 ) was calculated from the graph plotting suppression per centum against extract concentration. Trials were carried out in triplicate.
I?-Carotene bleaching check
The antioxidant activity of the infusion was determined harmonizing to the ?-carotene bleaching method described by Pratt ( 1980 ) . A stock solution of ?-carotene-linoleic acerb mixture was prepared as follows: 0.5 milligram I?-carotene was dissolved in 1 milliliters trichloromethane ( HPLC class ) with 25 Aµl of linoleic acid and 200 milligram of Tween-20. Chloroform was wholly evaporated, utilizing a vacuity evaporator. Then, 100 milliliter of distilled H2O, saturated with O ( 30 min ) , were added with vigorous agitating. 4 milliliter of this reaction mixture were dispensed into trial tubings and 200 Aµl ( EACS ) fraction, prepared at different concentrations, were added and the emulsion system was incubated for 2 H at 500C. The same process was repeated with BHT as positive control, and a space as a negative control. After this incubation period, the optical density of each mixture was measured at 490 nanometer. Antioxidant activity in ?-carotene bleaching theoretical account in per centum ( AA % ) was calculated with the undermentioned equation: AA % = 1- ( A0 -At/A’0-A’t ) x100
Where A0 and A’0 are optical densities of the sample and the space, severally, measured at zero clip, and At and A’t are optical densities of the sample and the space, severally, measured after 2 h. All trials were carried out in triplicate.
HPLC-MS analysis of phenolic compounds
Reverse stage high public presentation liquid chromatography was used to analyze phenolic compounds present in the ethyl ethanoate fraction, utilizing the separation faculty ( Knawer Analogy ) equipped with a C18 column ( Zorbax, 2.6 ten 250 millimeter, 3.5 Aµm atom size, ) and a diode array sensor ( DAD ) . The samples were eluted with a gradient system dwelling of dissolver A ( H2O, 0.1 % formic acid ) and solvent B ( Acetonitrile, 0.1 % formic acid ) , used as the nomadic stage, with a flow rate of 500 Aµl/min. The temperature of the column was maintained at 250C and the injection volume was 10 Aµl. The gradient system started from 90 % A at 1 min, to 20 % B at 4 min, 80 % B at 30 min, 100 % B at 32 min, 100 % B at 36 min and 20 % B at 38 min. The extremums of the phenolic compounds were monitored at 280 nanometer. Electrospray ionization mass spectroscopic ( ESI-MS ) analysis of phenolic compounds in ethyl acetate fraction was performed utilizing an Applied Biosystems ( LC/MSD TRAP x CT ) . Mass spectra were achieved by electrospray ionization in both positive and negative manners. The capillaries 4500V ( negative ) and 3500V ( positive ) were used in this survey. The electrospray probe-flow was adjusted to 8 ml/min. Continuous mass spectra were obtained by scanning from 50 to 900 m/z. Designation of the phenolic compounds of each fraction was achieved by comparing with ESI-MS spectra comparings with literature studies.
Male albino Wistar rats ( weight of 125-135g ) were used in this survey. The animate beings were purchased from the Central Pharmacy of Tunisia ( SIPHAT, Tunisia ) . They were housed at 22 A±3 A°C with light: dark periods of 12h and minimal comparative humidness of 40 % . The rats were fed with a commercial balanced diet ( SICO, Sfax, Tunisia ) and imbibing H2O was offered ad libitum. All carnal experiments were conducted without anesthesia and harmonizing to the ethical Committee Guidelines for the attention and usage of research lab animate beings of our establishment.
After 2 hebdomads of acclimatisation, the rats were allocated indiscriminately to four experimental groups of eight animate beings each with free entree to nutrient and H2O.
Based on the preliminary experiments, the hepato and nephroprotective dosage of the EACS infusion of C. silique was decided. In multiple dose pretreatment experiment, EACS infusion was administered at 250 mg/kg bw by intraperitoneal injection.
– Group I: control rats received the vehicle ( olive oil, 1 mL/kg orally ) at twenty-four hours 8.
– Group II: received CCl4 in olive oil ( 0.5 mL/kg, i.p ) at twenty-four hours 8.
– Group III: received the ethyl ethanoate infusion ( EACS ) Ceratonia silique leaves ( 250 mg/kg BW ) daily by i.p injection for 8 yearss followed by a individual dosage of CCl4 ( ; ; ; ; ; ) ( reffff ) in olive oil at a dosage of 1ml/kg utilizing an intragastric tubing twenty-four-hours after the last dosing.
– Group IV: received Ceratonia siliqua leaves extract ( 250 mg/kg BW ) daily by i.p injection for 8 yearss.
The animate beings were killed on twenty-four hours 9 by cervical beheading. Blood samples were collected, allowed to coagulate at room temperature and serum separated by centrifugating at 4000 revolutions per minute for 15 min for assorted biochemical parametric quantities. The liver and the kidney were rapidly excised, minced with ice cold saline, blotted on filter paper and homogenized ( Ultra Turrax T25, Germany ) ( 1:2, w/v ) in 50 mmol/l phosphate buffer ( pH 7.4 ) . The supernatant and serum were frozen at -30A°C in aliquots until analysis.
Serum parametric quantities
Serum samples were obtained by the centrifugation of blood at 4000 revolutions per minute for 15 min at 4A°C, and were so divided into eppendorf tubings. Isolated sera were stored at – 30A°C until they were used for the analyses. The degrees of serum alanine transaminase ( ALT ) , aspartate transaminase ( AST ) , alkalic phosphate ( ALP ) , lactate dehydrogenase ( LDH ) , I?-Glutamyl transpeptidase ( I?-GT ) , uric acid, urea and creatinine were measured utilizing commercial kits harmonizing to the maker ‘s waies.
Oxidative emphasis analysis
Thiobarbituric acid reactive substances ( TBARS ) measurings
Lipid peroxidation in the tissue homogenate was estimated by mensurating thiobarbituric acid reactive substances ( TBARS ) and was expressed in footings of malondialdehyde ( MDA ) content which is the end merchandise of lipid peroxidation, harmonizing to Buege and Aust ( 1984 ) . In brief, 125 I?l of supernatants were homogenized by sonication with 50 I?l of TBS, 125 I?l of TCA-BHT in order to precipitate proteins and centrifuged ( 1000 g, 10 min, 4 a-¦C ) . 200 I?l of obtained supernatant were assorted with 40 I?l of HCl ( 0.6 M ) and 160 I?l of TBA dissolved in Tris and the mixture was heated at 80 A°C for 10 min. The optical density of the end point supernatant was read at 530 nanometer. The sum of TBARS was calculated by utilizing an extinction coefficient of 156 tens 10 5 mMa?’1 cma?’1.
Anitoxidant enzymes surveies
In liver and kidney tissues, SOD activity was determined harmonizing to the colorimetric method of Beyer and Fridovich ( 1987 ) utilizing the oxidising reaction of nitroblue tetrazolium ( NBT ) ; CAT activity was measured by the UV colorimetric method of Aebi ( 1984 ) utilizing H2O2 as substrate ; glutathione peroxidase ( GPx ) activity was measured by a alteration of the colorimetric method of Flohe and Gunzler ( 1984 ) utilizing H2O2 as substrate and the decreased GSH.
Pieces of liver and nephritic tissues were excised, washed with normal saline and processed individually for histopathological observation. The liver and kidney tissues were fixed in bouin solution, dehydrated in ranked ( 50-100 % ) intoxicant and embedded in paraffin. Thin subdivisions ( 4 – 5 Aµm ) were cut and stained with everyday hematoxylin-eosin ( H & A ; E ) . The subdivisions were examined microscopically for histopathology alterations, including cell mortification, fatty alteration, and ballooning devolution ( Gabe 1968 ) .
All values are expressed as average A± S.E.M. The consequences were analyzed by one-way analysis of discrepancy ( ANOVA ) followed by Tukey trial for multiple comparings utilizing SPSS for Windows ( version 11 ) . Differences were considered important at P & lt ; 0.05.
Entire phenoplasts ( TPC ) and flavonoids
C. silique foliages fractions were investigated for their entire phenolic content ( TPC ) by the Folin-Ciocalteu check and for their flavonoids by AlCl3 reagent. As shown in Table 1, the TPC values expressed as milligram Gallic acerb equivalents/g of dry infusion ( mg GAE/g ) of the consecutive C. silique leaves infusions ranged between 91.2 to 680 milligrams GAE/g and were in the undermentioned order: ethyl ethanoate fraction & gt ; dichloromethane fraction & gt ; H2O fraction & gt ; hexane fraction.
Ethyl acetate fraction of C. silique foliages ( EACS fraction ) have a entire flavonoid content of 193.3 milligram of quercetin equivalents/g of dried infusion, while the H2O fraction have the lowest concentration. It was observed that the sum of flavonoids in the analyzed works infusions showed a high correlativity with the entire sum of phenoplasts.
Antioxidant capacities of C. siliqua infusion
The consequence of the different C. silique fractions on DPPH extremist scavenging showed a dose-dependent activity that can be evaluated by the finding of the IC50 values matching to the sum of the fraction required to scavenge 50 % of DPPH groups nowadays in the reaction mixture. High IC50 values indicate low antioxidant activity. As shown in Table 1 the most powerful extremist scavenger infusion was ( EACS ) fraction ( IC50 = 1.8 Aµg/ml ) , followed by H2O fraction ( IC50 = 8.65 Aµg/ml ) and dichloromethane fraction ( IC50 = 41.01 Aµg/ml ) . Therefore, we can reason that these infusions were able to cut down the stable free extremist 2,2-diphenyl-1-picrylhydrazyl ( DPPH ) to the yellow-colored diphenylpicrylhydrazine. As shown in Fig. 1, ( EACS ) fraction exhibited a dose-dependent DPPH extremist scavenging activity. This latter was higher at lower concentrations of 1, 2.5 and 5 Aµg/ml than ascorbic acid used as positive control. In the i??-carotene bleaching method, the grade of linoleic acid oxidization is determined by mensurating oxidization merchandises ( lipid hydroperoxides, conjugated dienes and volatile byproducts ) of linoleic acid which at the same time attack i??-carotene, ensuing in bleaching of its characteristic xanthous colour in ethanolic solution ( Kumazawa et al. , 2002 ) . The antioxidant activity of C. silique was evaluated utilizing different concentrations of infusions and was compared with BHT used as mention. Consequences are presented in Table 2. The add-on of the ( EACS ) fraction and the BHT at a concentration of 5 and 20 Aµg/ml prevented the bleaching of I?-carotene with different grades. Antioxidant activities of 38.95 % and 75.79 % were obtained utilizing 20 Aµg/ml of the EACS fraction and BHT, severally. Hydroxyl extremist ( .OH ) can easy traverse cell membranes, and can readily respond with most biomolecules including saccharides, proteins, lipoids, and DNA in cells, and do tissue harm or cell decease. Therefore, taking.OH is of import for the protection of populating systems. As shown in Table 2, the ( EACS ) fraction exhibited a high hydroxyl extremist scavenging activity with an IC50 of 3.81 Aµg/ml than the Osmitrol ( IC50 = 203 Aµg/ml ) .
Designation of phenolic compounds
The ( EACS ) fraction was analyzed by HPLC-MS in order to place its chemical composing. The designation of the different extremums was based on their keeping times in HPLC, their characteristic UV/visible and mass spectra with positive and negative ionisation at different atomization electromotive forces, in comparing to reliable criterions. Fig. 3 showed the HPLC chromatogram of ( EACS ) fraction. HPLC elution of ( EACS ) fraction exhibits basically six major compounds. Close scrutiny of the MS and MS2 spectra obtained from the LC-UV profile of the ( EACS ) fraction confirmed the presence of single constituents from several categories: simple phenol, free flavonol, glycosylated flavonol and isoflavone ( Table 4 ) . By comparing mass spectra with those of literature informations given by Owen et al. , 2003, peak 1 ( tR= 10 min ) was identified as 1,6-Di-galloyl-glucose and the [ M-H ] + extremum was at m/z 484. The 2nd extremum ( tR = 12.4 min and [ M-H ] – of 635 ) , was identified as 1,2,6-Tri-galloyl-glucose. Top out 3 ( tR = 14.9min ) showed [ M-H ] – of 480 and was identified as Myricetin glucoside. The value of the characteristic fragment ion of the latter extremum, ( m/z =316 ) , was besides indistinguishable with the reported in the literature. The 4th extremum ( tR= 15.1min, [ M-H ] _ =787 and a fragment ion value of 617 ) was identified as 1,2,3,6-Tetra-galloyl-glucose which is the chief polyphenol. The 5th extremum ( tR = 16.2min, [ M-H ] – of 463 ) was identified as Myricetin rhamnoside. The value of its fragment ion, found to be of 316, was similar to that reported in algarroba bean fiber. The last extremum ( tR = 17.4min, [ M-H ] – of 197 ) was identified as the synergic acid. All these consequences are in understanding with those reported in the literature by Owen et al. , 2003 in Carob fiber.
Serum biochemical parametric quantities
The consequences of biochemical indexs of liver map are summarized in Table ; ; . The disposal of CCl4 caused terrible hepatotoxicity in the treated rats, as evidenced by the important lifts of serum ALT, AST, ALP, LDH and I?GT activities. However, pre- intervention with the ethyl acetate infusion of C. silique leaves significantly prevented CCl4-induced additions in the serum parametric quantities. Perceptibly, disposal of EACS at the dosage of 250 mg/kg bw/day about wholly recovered the impaired liver maps, ensuing from CCl4-induced toxicity. No important changes were seen in EACS entirely treated rats when compared to command rats.
Table ; ; ; depicts the degrees of nephritic markers such as uric acid, urea and creatinine in serum. A important addition in nephritic markers was observed in serum of CCl4-administered rats compared to controls. No important changes were seen in EACS entirely treated rats when compared to command rats, whereas intervention with EACS in CCl4 treated rats brought about important decrease in the degrees of nephritic markers compared to the CCl4 induced rats.
Effectss on lipid peroxidation
The repressive consequence of C. silique leaves extract on CCl4 induced peroxidative harm is shown in Fig. ; ; . The degrees of TBARS were significantly increased in both liver and kidney tissue of CCl4-treated animate beings when compared to command untreated rats. Treatment of C. silique leaves extract at a dose 250 mg/kg BW significantly inhibited the TBARS degrees ( Pa‰¤0.001 ) and reduced hepatic and kidney peroxidative emphasis as compared to CCl4 control group in the liver and kidney ( ; ; ; % ; ; ; % ) . No important difference in the values were observed in rats treated with C. siliqua leaves extract entirely compared to command rat values.
Effectss on antioxidant enzymes
Table ; ; illustrates the degrees of enzymatic antioxidants viz. superoxide dismutase ( SOD ) , catalase ( CAT ) and glutathione peroxidase ( GPx ) in the liver and kidney of control and experimental rats. A important lessening in the activities of hepatic and kidney enzymatic antioxidants ( SOD, CAT and GPx ) in CCl4-treated rats is seen. Treatment with C. silique leaves ( 250 mg/kg BW ) extract entirely did non impact any of the tried parametric quantities. However, in their combinations with CCl4, all antioxidant enzymes were normalized to their control values. The ethyl acetate infusion of C. siliqua leaves abolished the oxidative harm and toxicity induced by CCl4 accompanied by an betterment of antioxidant enzyme activities.
The liver showed the undermentioned histopathological alterations ( Fig. ; ; ; ) . The cardinal vena, hepatocyte cord, hepatocytes and portal countries were observed to be normal in the control group ( Fig. 3 ; ; ; ) . In the present survey, CCl4 application constituted histopathological alterations which caused terrible liver harm including sinusoidal dilation, fatty devolution, centrilobular, vacuolisation, mortification, inflammatory cell infiltration ( Fig. ; ; ; ; ) when compared with control liver ( Fig. ; ; ; ) . The above alterations were reduced in the liver of rats treated with C. silique leaves ( 250 mg/kg BW ) infusion and CCl4 ( Fig. 6 ; ; ; ) . The histological form was about normal in rats treated with C. siliqua leaves extract entirely ( Fig. 6 ; ; ; ; ) . Kidney subdivisions of normal histological visual aspect ( Fig. ; ; ; 3 ; ; ; ) and the CCl4 control group showed high grade of tubulointerstitial Bright’s disease ( Figaˆ¦. ) . The subdivisions of the animate beings belonging to the ethyl acetate infusion of C. silique leaves entirely and their combinations with CCl4 intervention groups showed normal histological visual aspect ( Figs ; ; ; ; ) . These findings clearly indicate that the liver and kidney tissues, which were damaged by CCl4 poisoning showed recovery with the ethyl acetate infusion of C. silique leaves intervention.
Harmonizing to the phenoplast and flavonoid sums, ethyl ethanoate was the better dissolver to pull out phenolic compounds of C. silique leaves compared to the other dissolvers due to their mutual oppositions and good solubility for phenolic constituents. In old survey, it was reported that algarroba bean cods contain 1.9 mg/g of entire polyphenols, 0.28 mg/g of proanthocyanidins, and 0.1 mg/kg of hydrolysable tannic acids ( gallo and ellagitannins ) , located chiefly in sources, whereas seeds contained merely hints of these constituents ( Dimitris & A ; Panagiotis, 2004 ) . The degree of the phenoplasts content was higher than that reported by Dutra et Al. ( 2008 ) for Pterodon emarginatus ( Leguminosae ) seeds which showed 8.52 milligram GAE/g. The IC50 of the DPPH trial of this fraction was better than the reported IC50 values obtained for Gallic acid ( IC50 = 64 Aµg/ml ) and BHA ( IC50 = 114 Aµg/ml ) ( Ozsoy et al. , 2009 ) . In old work, Shigenori et al. , ( 2002 ) demonstrated that the DPPH free extremist scavenging of petroleum cods polyphenols at 5 Aµg/ml was merely 13 % compared to 100 % in leaves infusions. The C. silique fraction was found to impede the extent of I?-carotene bleaching by slaking peroxide groups to end the peroxidation concatenation reaction. This fraction possessed better antioxidant activity than other infusions such as H2O infusion of chestnut fruit as described by Barreira et Al. ( 2008 ) . The suppression of lipid peroxidation by add-on of EACS leaves fraction can be used to better the quality and stableness of nutrient merchandises. At 10-20 mg/ml, the scavenging abilities of hot H2O infusions from Agaricus blazei, Agrocybe cylindracea and Boletus edulis were 36.1-49.6 % , 13.2-61.3 % , and 53.8-78.6 % , severally ( Tsai et al. , 2007 ) . Additionally, sing the concentrations and abilities to scavenge hydroxyl groups, it can be concluded that EACS fraction have better public presentation to suppress OH extremist formation than the Osmitrol used as antioxidant criterion.
The antioxidant activity of the EACS fraction could be attributed to its high entire phenolic content. The cardinal function of these compounds as scavengers of free groups was reported in several surveies ( Komali et al. , 1999 ; Moller et al. , 1999 ) . In the reviewed literature, it was found that flavonoid type and degree were different in infusions from C. silique cods, bean and leaves. It was demonstrated antecedently that myricetin, quercetin and kaempferol were detected in high sums with other minor flavonoids such as genistein, naringenin, luteolin, taxifolin, biochanin A and 7,3′,4′-trihydroxy-flavone in C. silique from the middle-East ( Vaya et al. , 2006 ) . Besides, a mixture of Gallic acid, ( – ) -epigallocatechin gallate, ( – ) -epicatechin gallate have been identified by Corsi et Al. ( 2002 ) from C. silique foliages.
As portion of our probe on the components of C. silique foliages, we have demonstrated that the actively ethyl acetate fraction contained chiefly syringic acid, myricetin glycosides and Gallic acids derived functions. Further, showing of the reported phenoplasts failed to place catechin, epicatechin, quercitin and kaempferol. Therefore, the proximate analysis of Tunisian C. silique components, compared to those obtained in old work, showed some fluctuation in the composing. This fluctuation in composing can be attributed to the diverseness of geographical environments ( dirt, sunshine, temperature, precipitation, etc. ) . Custodio et al. , ( 2009 ) have reported antecedently that gender significantly affected the phenolic profile with the intersexs being by and large richer in phenols.
Based on these consequences, we have chosen the EACS fraction rich in phenolic compounds, and to find the possible protective consequence of EACS infusion against oxidative harm of the liver and kidney following orally disposal of CCl4, by finding oxidative emphasis profile and some serum biochemical parametric quantities.
Liver hurt followed by CCl4 exposure is good established by the elevated degrees of serum hepatic marker enzymes bespeaking the cellular escape and loss of functional unity of hepatic membrane architecture. High degrees of ALT, AST, ALP, LDH and I?GT activities are sensitive indexs of liver cell hurt and are most helpful in acknowledging hepatic diseases ( Pradeep et al. , 2010 ) . CCl4 treated rats showed increased activities of these enzymes, reflecting the harm of the liver cells or alterations in the cell membrane permeableness taking to leakage of enzymes from cells to the circulation ( Botsoglou et al. , 2009 ) . Changes in the activities of these enzymes are liver specific and have been considered as a tool to analyze changing cell viability and cell membrane permeableness ( Dasguptaetal, 1996 ) . Serum LDH, a cytoplasmatic marker enzyme and I?GT, a membrane edge enzyme are the well-known indexs of cell and tissue harm by toxic substances and their degrees are besides well increased in CCl4- drunk rats. LDH is an intracellular enzyme, the addition of which in serum is an index of cell harm ( Kim et al. , 2001 ) . In the present survey increased degrees of serum hepatic markers suggested that an extended liver hurt was occasioned by CCl4 due to increased lipid peroxidation which have the ability to do membrane harm. CCl4 causes structural and functional hurt to the cell membrane and increased the membrane permeableness taking to the escape of hepatic enzymes into the blood. It is now by and large accepted that hepatotoxicity of CCl4 is the consequence of reductive dehalogenation, which is catalyzed by its specific isoenzyme of cytochrome P450 2E1, and which forms the extremely reactive free group. Hence, the suppression of P450 2E1 could ensue in decreased degrees of reactive metabolites, and therefore decreased tissue harm ( Zangar et al. , 2000 ; Weber et al. , 2003 ) . Administration of EACS at the dose degree ( 250 mg/kg BW ) attenuated the increased degrees of the serum enzymes ( ALT, AST, ALP, LDH and I?GT ) induced by CCl4 and caused a subsequent recovery towards standardization comparable to the control group. Nephritic failure has been reported with CCl4 poisoning. Significant addition in the degrees of serum uric acid, urea and creatinine in treated animate beings is declarative of kidney harm and a possible malfunction or failure of the kidneys. In rats pre-treated with ethyl acetate infusion of C. silique leaves at a dosage of 250 mg/kg BW, important lessenings serum urea, creatinine and uric acid degrees and suggest betterment in the kidney position were observed following CCl4 intervention as compared to CCl4-treated rats. These consequences lend acceptance to the usage of C. silique leaves extract as nephroprotective agent ( Kumazawa et al. , 2002 ) . Our consequences corroborated with surveies of Ahmed ( 2010 ) which denoted that algarroba bean polyphenols has nephroprotective consequence against cisplatin. Pre-treatment with ethyl acetate infusion of C. silique leaves attenuated the increased of these serum parametric quantities caused by CCl4. Previous surveies ( Kumazawa et al. , 2002 ) showed that algarroba bean has antioxidative activity, modulates blood lipid profile in worlds ( Gruendel et al. , 2007 ) and reduces growing of different cell lines ( Klenow et al. , 2008 ) . Reactive O species ( ROS ) are generated by many oxidation-reduction processes that usually occur in metamorphosis of aerophilic cells. If non eliminated, ROS can assail of import biological molecules, such as lipoids, proteins, DNA, enzymes, and RNA. Thus, ROS are involved in a figure of degenerative diseases such as malignant neoplastic disease, cirrhosis, diabetes, and Alzheimer ‘s disease ( Halliwell and Gutteridge, 1986 ) . In our survey, it has been reported that CCl4 induced an sweetening of the TBARS degrees in liver and kidney tissues of treated rats compared to controls. The lift of lipid peroxidation caused by CCl4 has been antecedently reported in rat theoretical account ( David E. et al. , 2010 ) , which is in understanding with the consequences obtained in this survey. Our consequences revealed that pre-treatment with EACS exhibited a important repressive function against TBARS formation in rats, and thereby diminished CCl4-induced hepatic and kidney harm. The bar of lipid peroxidation might, at least in portion, be derived from the capableness of EACS to scavenge ROS, which was supported by the observation that EACS reduced the degree of TBARS production. When the lipid peroxidation inhibitory potencies of the C. silicon oxide leaves extract were evaluated harmonizing to their repressive activities on the formation of TBARS produced during the oxidization procedure, EACS revealed a strong inhibitory ability towards lipid peroxidation. The addition of hepatic and kidney lipid peroxidation is in understanding with old surveies of David E. et Al. ( 2010 ) which demonstrated that CCl4 induced an sweetening of the rate of liver and kidney tissues lipid peroxidation in treated rats and, hence alleviated by methanol infusion of Alafia multiflora root bark. Take together, the consequences demonstrate that the EACS has considerable protective consequence on hepatic and nephritic amendss induced by CCl4. Furthermore, SOD, CAT and GPx activities were decreased in liver and kidney tissues of CCl4 induced treated rats. The decreased activity of SOD, CAT and GPx could be due to heighten lipid peroxidation or inactivation of the antioxidative enzymes. When rats treated with the ethyl acetate infusion of C. silicon oxide leaves prior to CCl4 disposal, the decrease of SOD, CAT and GPx activity was inhibited. Similar consequences were obtained when carob infusion administered to LT97 adenoma cells which protected against factors of oxidative emphasis, in peculiar H2O2 ( Klenow et al. , 2009 ) . In the present survey, it was noticed that the consequences of biochemical and oxidative profile of hepatic and kidney markers are in conformity with the earlier studies on the histoarchitecture of liver and kidney of treated and control rats. Assorted Studies demonstrated that liver is non the lone mark organ of CCl4 and it causes free extremist coevals in other tissues besides such as kidneys, bosom, lung, testicle, encephalon and blood ( Perez et al. , 2005 ) . In our survey, CCl4 selectively causes toxicity in liver and kidney of treated animate beings as compared to controls. In the present work, the hepatic histoarchitecture of the CCl4-treated rats resulted terrible necrotic alterations, inflammatory cell infiltration, fatty devolution and vacuolisation. It might be due to the formation of extremely reactive groups and subsequent lipid peroxidation induced by CCl4. The disposal of EACS reduced the histological changes provoked by CCl4 rather appreciable. It can be attributed to the antiradical/antioxidant and metal-chelating efficaciousness of EACS which significantly reduced the oxidative emphasis and Restoration of normal physiological province of an being. In position of the present survey, it can be concluded that EACS played a function of an antioxidant which includes free extremist scavenging and thereby improved the damaging province of liver cells which ragged its usage as a possible attenuating go-between in CCl4- induced hepatotoxicity. In fact, histological alterations, seen in the kidney of rats treated with CCl4 are characterized by a decrease archer ‘s infinite and devolution of cannular epithelial cells. Besides these, extended mortification in nephritic proximal tubules was observed. Our consequences confirmed old findings of Sulak et Al. ( 2005 ) and Kalender et Al. ( 2007 ) who had found degenerative alterations in kidney of rats exposed to methidathion and methyl parathion ( OPs pesticides ) . Result suggest that EACS intervention prior to CCl4 poisoning could forestall the CCl4-induced changes in kidney tissues of treated animate beings.
In drumhead, the above mentioned biochemical parametric quantities and histological groundss show that the pre-treatment with the ethyl acetate infusion of C. Silica leaves efficaciously protected rats against CCl4 induced hepatotoxicity and nephrotoxicity. The present work showed that the in vivo surveies and the in vitro antioxidant activity approved out before besides indicate that the ethyl acetate infusion of C. Silica leaves may be the active infusion. This therefore, suggests that the hepato and renoprotective effects of C. silicon oxide may be due to its antioxidant and free extremist scavenging belongingss. However, farther probes are indispensable to clarify the precise mechanism of active agents of C. silicon oxide protection against CCl4-induced hepatotoxicity and nephrotoxicity and it has to be tested against other biological parametric quantities.