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Phytochemical, antioxidant and hepatoprotective effects of Alnus nitida bark in carbon tetrachloride challenged Sprague Dawley rats
© The Author(s). 2016
Received: 3 March 2016
Accepted: 23 July 2016
Published: 3 August 2016
Alnus nitida (Spach) Endl. is traditionally used for inflammatory disorders. Diarylheptanoids constituents having diverse therapeutically importance including hepato-protective was reported in A. nitida. The aim of this study was to explore the antioxidant and hepato-protective profile of A. nitida stem bark’s crude methanol extract (ANM).
Crude methanol extract of A. nitida stem bark and its derived fractions were assessed for phytochemical classes and in vitro antioxidant profiling by multidimensional assays. Hepato-protective assessment of ANM was investigated on rats, which were made hepatotoxic using carbon tetrachloride (CCl4). Additionally HPLC-DAD analysis of ANM, and its derived ethyl acetate and aqueous fraction was carried out to determine the presence of active constituents.
Qualitative analysis of crude extract-and its fractions depicted the presence of terpenoids, saponins, coumarins, phenols and flavonoids. Maximum quantity of total phenolic content (TPC) and total flavonoid content (TFC) was recorded in ANM and its derived fractions; n-hexane (ANH), chloroform (ANC), ethyl acetate (ANE) and the residual aqueous (ANA). ANM exhibited the best total antioxidant capacity, total reducing power, and scavenging of DPPH and OH radicals. ANE and ANA exhibited strong scavenging potential for iron chelation, nitric oxide and β-carotene bleaching assay. ANM treatment converse the activities of serum-marker enzymes and lipid profile, altered by CCl4 treatment in rat. CCl4 induced hepatic-cirrhosis in rat resulted in decrease of antioxidant enzyme activities such as catalase, peroxidase, superoxide dismutase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase-which were restored towards the normal level with ANM. Similarly diminished level of reduced glutathione while enhanced level of lipid peroxides, hydrogen peroxide and nitrite in liver of cirrhotic rats was normalized by treatment of ANM. The histopathological studies of liver tissues also represented that ANM possessed the hepato-protective activity. HPLC-DAD analysis against eight known standards confirmed the presence of gallic acid, catechin and rutin in ANM and in ANA while in ANE gallic acid was only detected.
Based on the results of antioxidants, restoration of various antioxidant enzymes and histopathological studies, the recent study concludes that antioxidant potential of A. nitida bark might protect the liver damages.
KeywordsAlnus nitida Antioxidant Hepatoprotective HPLC Phenolics Flavonoids
Plants are currently recognized as an opulent source of antioxidant compounds (e.g., flavonoids, simple phenolics, stilbenes, anthocyanins), which are used chiefly in food industry [1, 2]. It is deliberated that utilization of plant-based antioxidants could be associated with reduced risk of occurrence of numerous human diseases narrated to the oxidative stress .
Antioxidants have long been known to protect the biological system through inhibition or deterrence of oxidation stress tempted by reactive oxygen substances produced during usual metabolic activities or environmental factors . Thus, the oxidative stress induced damages of DNA, lipids and proteins may cause various diseases .
Liver is an imperious organ in human body . Liver is subordinated with vital functions such as it sustains and normalizes the homeostasis in body. It plays an astounding role in human body as it mediates several biochemical pathways such as body defense against diseases, energy production, and for source of nutrition . Oxidative-stress plays a foremost role in the progress of liver diseases. The liver injury is recruited by the various noxious agents produced by chemicals, viruses or by their bio-activation to chemically reactive metabolites. These metabolites can be free radicals, which either prompts an immune response or directly affects the biochemistry of the cells by cooperating with cellular macromolecules. Even after the encroachment in modern system of medicine, there is deficiency of a trustworthy synthetic liver defensive drug. Hence, natural extracts /products from medicinal plants are considered to be effective for the management of liver disorders .
Experimental models of hepatotoxicity can be produced by alcohol, paracetamol, CCl4 etc. CCl4 is the extreme potent hepatotoxic agent practiced for experimental generation of liver fibrosis . CCl4 is metabolized by cytochrome P4502E1 to the trichloromethyl radical (•OCCl3) and peroxy trichloromethyl radical (•OOCCl3). It has been reported that one of the cause of CCl4-induced liver injury is lipid peroxidation, which is prompted and accelerated by free radical derivatives of CCl4 .
Alnus nitida (spach) Endl belongs to family Betulaceae, ordinarily known as Sharoliin Punjab and Seril iniKashmir, is a deciduousiwoody tree, distributed in westerniHimalayas from Yamuna westwards to Kashmir [11, 12]. Alnus species contained diarylheptanoids; a group of natural compounds having 1,7-diphenylheptane skeleton imparting diverse therapeutic effects. About 400 diarylheptanoids  have been isolated from different species of Alnus showing various pharmacological activities; anti-inflammatory [14, 15], anti-influenza , and hepato-protective . Various isolates or the derivatives of diarylheptanoids from A. japonica showed antioxidant effects during in vitro studies [18, 19]. The diarylheptanoids were also isolated from other medicinal sources such as Zingiber, Curcuma, Alpinia and Myrica. Two diarylheptanoids nitidoneiA and nitidone B have been reported from A. nitida found in Northern areas of Pakistan . Stem bark of A. nitida is pounded and paste is applied externally by the local communities as a remedy for swelling, injuries and pain. Its decoction is also used for internal injuries. Root or stem bark of A. nitida is blended with Urtica dioca, Rumex nepalensis, Zingiber officinale and paste is used in bone fractures . Extract of A. japonica have shown promising effects in alleviating the acetaminophen induced hepatic injury in rat . Hence, the current study was designed to scrutinize the preliminary phytochemical composition, as well as to estimate the antioxidant and hepato-protective activity of methanol extract of A. nitida stem bark against CCl4 induced hepatotoxicity in rat. The crude extract and its derived fractions; ethyl acetate and residual aqueous fractions exhibited significant antioxidant activity were subjected to HPLC-DAD analysis for the presence of polyphenolic constituents.
Reagents and chemicals
Analytical grade chemicals used: sodium carbonate, sodium nitrite, dosium dihydrogen, hydrogen peroxide, ferrous chloride, 2-deoxyribose, potassium ferricyanide, sulphuric acid were bought from Merck. 1,1-diphenyl-2-picryl-hydrazyl, potassium persulphate, 2-ethylbenzothiazoline sulfonic acid, rutin, nitro blue, Folin-Ciocalteu’s reagent, phenazine methosulphate, trichloroacetic-acid and tetrazolium were obtained from Sigma Chemicals Co., St. Louis USA. Oxidized glutathione (GSSG), (DTNB), glutathione (GSH),1,2-dithio-bis-nitroLbenzoic-acid (DTNB), glucose-6-phosphate, thiobarbituric acid (TBA), trichloroacetic acid (TCA), sodium tungstate, perchloric acid (PCA), 2,6-dichlorophenolindophenol, reduced glutathione (GSH), sodium hydroxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH), sodium tungstate, glucose-6-phosphate, rutin, catechin, gallic acid, caffeic acid, apigenin, quercetin, myricetin, and kampferol were bought from Sigma Chemicals Co., USA were used.
Alnus nitida stem bark was collected from Charbagh town of district Swat, Pakistan during March–April (2015.) The spot of collection was at 34.842727° north latitude and 72.431089° east longitude; at an elevation of 1000 m. Flora of Pakistan  was used for the identification of plant and further authenticated by Dr. Sumaira Sahreen, Associate Curator, Pakistan Museum of National History. The authenticated specimen (127963) was held at Pakistan Museum of National History.
At room-temperature for 2 weeks the bark of A. nitida was air-dried under-shade-and with the help of Willy Mill granulated to 80-mesh size. Bark powder (5 kg) was drenched in-15 L of 95 %-methanol and reprises-the soaking thrice-and filtered the-extract-with WhatmanLNo.L1 filter paper. Rotary evaporator was utilized to dry the filtrate (ANM) under vacuum. Partial purification or separation was done by solvent-solvent extraction in escalating polarity. In the distilled water the extract was suspended and the solvents i.e., n-hexane (ANH), chloroform (ANC) and ethyl acetate (ANE) were used in augmentation order of polarization and as an aqueous extract residual (ANA) was used. Rotary evaporator was used for the evaporation of solvents of all the fractions, and then preserved at 4 °C.
Different qualitative tests were employed to identify the phytochemical classes present in the crude methanol extract and various fractions of the stem bark of A. nitida.
Assessment of phenols
For the presence of phenols previously reported methodology was followed . Each sample (1 mg) was suspended in 2 ml of distilled water containing 10 % ferric chloride. The confirmation sign for the presence of phenol was the development of blue or green color.
Assessment of flavonoids
In order to investigate the presence of flavonoids in each sample Trease and Evans protocol  was employed. Briefly, 1 mg of every sample was allowed to react with 1 ml of 2 N sodium hydroxide. Appearance of yellow color was considered as the confirmation signal of flavonoid presence.
Assessment of coumarins
An amount of 1 mg of each sample was blended with 1 ml of 10 % sodium hydroxide. Appearance of yellow color in the test tube was the evidence of coumarins presence in the sample .
Assessment of saponins
Each sample (2 mg) was suspended in 2 ml of distilled water and vigorously mixed. The formation of a soapy layer of almost 1–2 cm was the indication of saponins presence .
Assessment of tannins
The confirmative signal of tannins presence was the development of dim blue or greenish dark shading on the mixing of 1 mg of every sample and 3 ml of 5 % ferric chloride .
Assessment of terpenoids
Each sample 0.5 mg was mixed with 3 ml of chloroform and 3 ml of concentrated-sulphuric acid. The appearance of red brown colored layer in the middle of two layers confirmed the existence of terpenoids .
Assessment of anthraquinones
Development of red color was considered as indication for the presence of anthraquinones after mixing of 1 mg of each sample with 2 ml of diluted 2 % hydrochloric acid .
Assessment of anthocyanins and betacyanins
Each sample (1 mg) was boiled for 10 min in 2 ml of 1 N sodium hydroxide. Formation of bluish green color was the sign of anthocyanin and yellow color formation of betacyanin presence .
Assessment of alkaloids
An amount of 2 mg of each sample was mixed with concentrated sulphuric acid. The reaction mixture was allowed to react with Mayer’s reagent. Appearance of green color or formation of white precipitates was the symbol of alkaloid presence .
Total phenolic as well as flavonoid contents were quantified by the following narrated procedures.
Total phenolic contents (TPC)
Spectrophotometric analysis was performed for the analysis of total phenolic content . Each sample (1 mg/ml) was briefly blended with 9 ml of distilled water and 2 ml of Folin Ciocalteu reagent. The acquired mixture was mixed vigorously for 10 min and 10 ml ofL 7 % Na2CO3 was further mixed in to the mixture. Mixture’s final volume was raised to 25 ml by adding distilled water and then placed into the incubator at room temperature for 60 min. Absorbance of the reaction mixture was measured at wavelength of 750 nm in triplicates for each sample. Gallic-acid was kept as standard, the estimation of TPC was done as mg of gallic acid equivalents (GAE) per gram of dryLextract/fraction.
Total flavonoid content (TFC)
For the TFC evaluation in the test samples, 0.3 ml of each sample was mixed with 0.25 M NaNO2 (0.25 ml) -followed by the addition of 0.1-ml of 0.3 MLAlCl3.6H2O, and 3.4 ml of-30 % methanol . After 5 min interval 2 ml aliquot of 1 M NaOH was added to it. At the wavelength of 506 nm the absorbance of reaction amalgam was measured against the reagent blank. Content of total flavonoid as-mg rutinLequivalentsLper gram of dryLextract/fraction was assessed by employing the calibration-curve of rutin.
High performance liquid chromatography (HPLC-DAD) analysis
HPLC analysis of ANM and selected plant fractions (ANE and ANA) was performed using HPLC-DAD (Agilent 1200, Germany) equipped with Zorbex RXC8 (Agilent, USA) analytical column with 5 μm particle size and 25 ml capacity using previously reported method . Each sample was diluted with HPLC grade methanol. Mobile phase was consisted of eluent A, (acetonitrile- methanol--water- acetic acid-/5: 10: 85: 1)-and eluent B (acetonitrile-methanol-acetic acid/40: 60:-1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50 % B), 21–25 min (50 to 100 % B), 26–30 min (100 % B) and 31–40 (100 to 0 % B) at flow rate ofL1 ml/min. The standards and samples were prepared in HPLC grade methanol (1 mg/ml), filtered through 0.45 μm-membrane filter andL20 μl was injected for the analysis. Among the standards rutin was investigated at 257 nm, catechin and gallic acid at 279 nm, caffeic acid and apigenin at 325 nm while quercetin, myricetin and kampferol were analyzed atL368 nm . The analysis was performed in triplicate and the column was reconditioned for 10 min after each run. Quantification was done by the integration of the peak by using the external standard method.
In vitro antioxidant assays
The in vitro antioxidant assays were carried out by preparing the plant samples (1 mg/ml) in 95 % methanol and then making its serial dilutions. The specific protocol was followed for finding specific scavenging activities of the plant samples.
DPPHL (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging assay
Nitric oxide scavenging assay
Nitric oxide scavenging activity of ANM and its derived fractions was estimated by using Griess reagent as the main ingredient . DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. For the development of Griess reagent, equimolar amount of 0.1 % napthylenediamine in distilled water and 1 % of sulphanilamide in 5 % phosphoric acid was added. An aliquot of 0.2 ml of sample was mixed with 0.2 ml of sodium-nitroprusside (10 mM) being formulated in saline phosphate buffer followed by addition of 2 ml aliquot of the Griess reagent to the reaction blend. For 3 h the reaction blend was incubated at room temperature and the absorbance was measured at the wavelength of 546 nm spectrophotometrically utilizing ascorbate as a positive control. For assessing the percentage inhibition of nitric oxide radical formation equation 1 was used.
Hydroxyl radical scavenging assay
β-Carotene bleaching assay
Chelating power assay
Reducing power assay
DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. Briefly, 4 ml of plant extract was taken and mixed with 4 ml of 0.3 M phosphate buffer (pH 6.8) and 4 ml of potassium ferricyanide (10 mg/l) and the reaction cocktail was placed in the incubator for 20 min at 80 °C for 10 min. After addition of 4 ml of trichloroacetic acid (200 mg/l) in the reaction cocktail, 2 ml was of it was diluted with 4 ml of distilled water and 0.6 ml of FeCl3 (0.2 %). Optical density of the reaction mixture was taken at 700 nm after 10 min of incubation. Gallic acid was appropriated as a standard .
The methodology of Prieto et al.  was used to assess the antioxidant capabilities of the plant samples. DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. Accordingly, 0.2 ml of the plant sample was mixed with 2 ml of the reagent solution (prepared by adding 28 mM Na3PO4 and 0.6 M H2SO4 with that of 4 mM ammonium molybdate). After incubation at 90 °C in a water bath for 80 min the reaction mixture was cooled down at room temperature and optical density was recorded at 765 nm.
In vivo CCl4 induced hepatotoxicity in rats
Six weeks old (180–200 g) Sprague–Dawley-male rats were acquired from the Animal House situated at the Quaid-i-Azam University Islamabad, Pakistan. The animals were maintained at 24 ± 3 °C with a 12 h dark/light cycle at Primate Facility of the Quaid-i-Azam University Islamabad, Pakistan. The animals were bred with basal diet with water ad libitum and were sustained in standard laboratory conditions. The basal diet was composed of 20 % protein (casein), 10 % sucrose, 5 % corn oil, 2 % choline chloride, 1 % vitamin mixture, 3.5 % salt mixture and 5 % fibers (cellulose). The remainder was corn starch up to 100 %. Prior to the research propagation ethical approval (Bch#0275) was obtained from Ethics Committee Quaid-i-Azam University Islamabad. Experiments on animals were performed in accordance with the guidelines of the institute of animal ethical committee, NIH, Islamabad.
Acute toxicity test
Six week old Male Sprague Dawley rats were kept in fasting conditions for overnight with just water availability. Three animals were intra-gastrically administered with dose-of 50 mg/kg bw and were monitored for mortality rate for 2 weeks. DMSO was used as a solvent for the preparation of extract/fraction samples. No initial progression of toxicity was observed, but the methodology was subsequently followed with augmented amount of doses i.e., 100,-200, 400, 1000, 2000, 3000 and 4000 mg/kg bw of the maximum dose of the extract. Three animals were used for each treatment. Mortality was not noticed at the highest dose of 4000 mg/kg, thus 200 and 400 mg/kg bw doses were selected for the evaluation of hepato-protective propagation activities .
Eight groups (each having 7 rats) of Sprague–Dawley male-rats were made to contemplate the hepatoprotective effects of ANM. Group I was taken as a control and remained untreated. Olive oil and DMSO (1:1; v/v) at a dose of 1 ml/kg bw was administrated to the Group II orally. Group III was intraperitoneally administered with CCl4 (1 ml/kg bw; CCl4:Olive oil; 2:8 v/v). An amount of 50 mg/kg bw of silymarin (in DMSO; w/v) as a reference chemical was given to Group IV after 24 h of CCl4 treatment. Group V and VI were initially treated with CCl4 and then after 24 h, they were orally administered with 200 and 400Lmg/kg bw (in DMSO) of ANM. Animals of Groups VII and VIII received only ANM in DMSO at a dose-of-200 and 400 mg/kg bw, respectively. Once the experiment was accomplished, weight of the animals was recorded. Then the animals were euthanized after ether anesthesia. Blood was collected from heart (atrium), immediately removed the liver and transferred to the chilled saline solution and weighted. One liver portion was prepared for-histopathalogical studies, while the second portion was preserved in liquid nitrogen-and stowed at −80 °C for-added enzymatic and DNA damage investigation.
Biochemical studies of serum
Different liver marker enzymes were used to perform the liver function tests such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and bilirubin in serum was evaluated by standard procedure of AMP Diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). Serum level of high density lipoproteins (HDL), low density lipoproteins (LDL), total cholesterol and triglycerides were estimated by following the procedures available on the kits.
Biochemical studies of liver
Small portion of liver was homogenized by the use of homogenizer. Volume of homogenate was noted and accordingly the 10× homogenate was prepared by mixing with EDTA (1 mM) and phosphate buffer (100 mM). The homogenate was placed for 20 min at 4 °C and then centrifuged at 12,000 × g in order to assemble the supernatant. The protein concentration in theLsupernatant was evaluated-according to the method of Lowry et al.  using BSA as standard.
Catalase assay (CAT)
On the basis of decomposition of hydrogen peroxide CAT actions were analyzed by following the method of Chance and Maehly . In short, 2.5 ml of phosphate buffer (50 mM; pH 6.6) as a solvent for of the supernatant (0.1 ml) and 0.4 ml of H2O2 (5.9 mM; pH 6.0), change in absorbance was noted for one minute at wavelength of 240 nm in a-spectrophotometer.-A change of 0.01 in-absorbance for one-minute was taken as one unit of CAT activity.
Activities of POD were evaluated based on guaiacol peroxidation . To find the POD activity of supernatant the reaction mixture was prepared by adding 0.3 ml of guaiacol (20 mM), 0.6 ml of H2O2l(40 mM), and 0.2 ml of the supernatant to 3.5 ml of phosphate buffer (50 mM, pH 5.6) serially. Change in absorbance of the reaction solution at 470 nm was observed for one minute. One unit of POD activity was defined as an absorbance-change-of 0.01 of the-solution-per minute.
Superoxide dismutase assay-(SOD)
For the assessment of hepatic SOD activity, to the reaction blend after the centrifugation (2500 × g for 20 min-followed by 10,000 × g for 20 min) 300 μl of supernatant along with 1.4 ml of sodium-pyrophosphate buffer (0.053 mM; pH 7.3), 0.2 ml of phenazine-methosulphateL(188 μM) were added to the reaction mixture. To initiate the reaction 0.4 ml of NADH (785 μM) was added and-then stopped after 3 min by adding 2 ml of glacial acetic acid. The color intensity was measured at wavelength of 560 nm .
Glutathione-S-transferase assay (GST)
The principle of this methodology is based on the interaction of GSH andL1-chloro-2,4-dinitrobenzeneL(CDNB)Land the subsequent conjugate made is measured with a spectrophotometer . Shortly, 1.575 ml of phosphate buffer (0.2 M, pH 6.8), 0.3 ml of GSH (2LmM), 0.028 ml of 1-chloro-2,4-dinitrobenzeneL(CDNB; 1LmM) were blended followed by 1.2 ml of supernatant. GST activity was estimated by noting-the change in absorbance at wavelength of 340 nm with a molar extinction-coefficient of 8.6 × 103 M−1 cm−1.
Glutathione reductase assay (GSR)
GSR analysis was done by following the method of Carlberg and Mannervik . Supernatant samples (0.2 ml) were amalgamated with 0.1 ml of EDTA (0.5 mM), 1.68 ml of phosphate buffer,-0.04 ml of oxidized glutathione (1 mM), and 0.1 ml of NADPH (0.1 mM). The OD was measured atL340 nm after blending. GST activity was estimated as nM NADPH oxidized/min/mg protein, using a molar extinction coefficient of 8.22 × 103 M−1 cm−1.
Glutathione peroxidase-assay (GSH-Px)
The activity of glutathione peroxidase was assayed as described earlier . Hepatic-supernatant (1.2 ml) of every rat was mixed with 1.46 ml phosphate buffer (0.2 M; pH 7.9), 0.1 ml of EDTA (2 mM), 0.2 ml of sodium azide (2 mM), 0.06 ml of glutathione reductase (1 IU/ml), 0.08 ml of GSH (2LmM), 0.2 ml of NADPH (0.4 mM),L0.01 ml of H2O2 (0.25 mM). The absorbance was noted at 340 nm, and GSH-Px activity was assessed by using a molar extinction coefficient of 6.23 × 103 M−1 cm−1.
Reduced glutathione assay (GSH)
The concentration of reduced glutathione was assessed by spectrophotometric technique . The basis of this method-is based on the breakdown-of 1,2-dithio-bis nitro-benzoic acid (DTNB) by sulfosalicylic acid, as a result yellow-color is produced. The yellow color produced was read immediately at 412 nm on a-spectrophotometer and was expressed as μM GSH/g tissue.
Estimation of lipid-peroxidation assay (TBARS)
The analysis of lipid peroxides as thiobarbituric acid reactive substances (TBARS) was done by following protocol of Iqbal and Wright . Shortly after the ascorbic acid and ferric chloride were mixed with the supernatant it was put in a shaking water bath at 37 °C for 1 h, after that trichloroacetic acid was added. 2.0 ml 0.69 % thiobarbituric acid was added and all the tubes were placed in a water bath to boil for 10 min, and then transferred to crushed ice bath before centrifuging at 2500 × g for 15 min. The quantity of TBARS manufactured in each of the samples was evaluated by measuring the OD of the supernatant atI535 nm against a reagent blank. The outcomes were expressed as nM TBARS/min/mg tissue-at 37 °C using a molar extinction coefficient of 2.58 × M−1 cm−1.
Hydrogen peroxide assay (H2O2)
Oxidation of phenol red in the presence of H2O2 mediated horseradish peroxidase was done to assay hydrogen peroxide (H2O2) . Horse radish peroxidase (8.5 units), phenol red (0.29 nM), dextrose (6.6 nM) and phosphate buffer (0.04 M; pH 8.0), comprising solution was used for the suspension of homogenate sample (3.0 ml) and incubation was done at 37 °C for 60 min. A volume of 0.01 ml of NaOH (1 N) was mixed following centrifugation at 800 × g for 5 min. Supernatant was checked for absorbance at 615 nm against a reagent blank. H2O2/min/mg tissue-based concentration was shown on the basis of H2O2 oxidized phenol red of standard curve.
Equal quantity i.e. 100 μl each of both 5 % ZnSO4 and 0.3 M NaOH was used to remove the proteins of tissue samples (100 mg), which were then centrifuged at 6400 g for 15–20 min. The supernatant was collected by centrifugation at 6400 × g for 20 min. To the cuvette Griess reagent (1.0 ml) was added, and the spectrophotometer was blanked at wavelength of 540 nm, then the supernatant was added to the cuvette having Griess reagent. Nitrite concentration was computed using a standard curve for sodium nitrite .
Tissue protein estimation
The method of Lowry et al.  was used to determine the total amount of soluble protein in tissue homogenate. To the tissue homogenate 200 μl of 1.1 M potassium phosphate buffer-(pH 8.0) was added to dilute the tissue sample. A volume of 1 ml of alkaline copper-solution was added to-this blend, and placed at room temperature. After incubation for 20 min, 200 μl of Folin-Ciocalteu phenol reagent was added. Reaction tubes containing the test mixtures were then vortexed and incubated again atL37°C for 20 min. At 650 nm optical density was measured spectrophotometrically. Total soluble proteins of the tissue samples-were then detected using standard curve of bovine serum-albumin.
Histopathological examination was performed for the hepatic injuries. Samples were paraffin embedded, after fixation in fixative solution consisting of: formaldehyde 20 %, absolute alcohol 70 %, glacial acetic acidL10 %. Sections were made at 4–5 μm, stained with hematoxylin/eosin and were examined under light-microscope-(DIALUX 20 EB).
The parametric data were expressed as the mean- ± SD for the 07 rats in each group. Statistix 8.1 software was used for statistical analysis. After estimating the normality test of all the groups for various parameters the data was analyzed for one way analysis of variance to compute the differences between groups. Post hoc testing was carried out for inter-group comparisons using the least significant difference (LSD) test at P > 0.01.
Qualitative phytochemical analysis
Phytochemical analysis of A. nitida bark methanol extract and derived fractions
Plant yield, total phenolics and flavonoid content
Estimation of plant extraction yield, total phenolics, flavonoids, antioxidant capacity and reducing power of Alnus nitida bark
Total phenolic contents expressed as gallic acid equivalents (mg/g of extract)
Total flavonoid contents expressed as rutin equivalents (mg/g of extract)
Total antioxidant capacity expressed as ascorbic acid equivalents (mg/g of extract)
Total reducing power expressed as ascorbic acid equivalents (mg/g of extract)
631.5 ± 1.7
221.5 ± 2.5
32.6 ± 1.2a
125.5 ± 5.0a,b
3.5 ± 0.5c
36.0 ± 2.6d
527 ± 3.1
98.6 ± 1.61
7.6 ± 0.9c
45.5 ± 3.3d
607 ± 1.97
211.2 ± 2.4
19.0 ± 0.6b
113.1 ± 2.7b,c
591.2 ± 1.3
114.8 ± 1.8
16.5 ± 1.1b
102.0 ± 3.3c
HPLC-DAD analysis of A. nitida stem bark
HPLC-DAD profile of A. nitida bark methanol extract, ANE and ANA
Polyphenolics (μg/g of extract)
In vitro antioxidant assessment
DPPH radical scavenging activity
IC50 values of different antioxidant activities of ANM and its fractions
Nitric oxide scavenging activity
β-carotene bleaching scavenging activity
Iron chelating assay
32.0 ± 1.8d
97.5 ± 2.9d
153.6 ± 4.0c
103.6 ± 4.2c
130.5 ± 4.1c
951.5 ± 4.3a
932.5 ± 3.3a
465.5 ± 2.8a
858.9 ± 3.8a
430.2 ± 3.9a
633.8 ± 3.9b
812.5 ± 2.9b
414.9 ± 2.8b
726.2 ± 4.7b
347.9 ± 2.2b
39.5 ± 2.1c,d
102.7 ± 3.3d
151.8 ± 3.1c
41.4 ± 1.9f
130.1 ± 0.8c
43.7 ± 2.9c
173.4 ± 2.0c
84.6 ± 3.7d
54.7 ± 4.4e
76.8 ± 4.5d
83.3 ± 4.2e
11.9 ± 0.5e
56.9 ± 2.5e
56.0 ± 1.7e
90.2 ± 2.3d
Correlation of IC50 values of different antioxidant activities with total phenolic and total flavonoid contents
DPPH scavenging activity
Nitric oxide scavenging activity
Hydroxyl radical scavenging activity
β-carotene bleaching scavenging activity
Iron chelating assay
Total antioxidant activity
Total reducing power Assay
Hydroxyl radical (•OH) scavenging assay
Hydroxyl radical scavenging capacity of an extract is directly linked to its antioxidant capacity. Figure 2b exhibited the radical scavenging ability of plant samples in the following order; ANM < ANE < ANA < ANC < ANH. Lowest IC50 values were recorded for crude extract (97.5 ± 2.9 μg/ml) followed by ANE (102.7 ± 3.3Lμg/ml) and ANA (173.4 ± 2.0 μg/ml) while ANC and ANH showed the higher IC50 values of (812.5 ± 2.9b μg/ml) and (932.5 ± 3.3 μg/ml) correspondingly. IC50 values of ANA, ANC and ANH were remarkably higher from rutin (83.3 ± 4.2 μg/ml). Significant correlation of IC50 values of hydroxyl radical scavenging was calculated for TPC (R2 = 0.841, P < 0.05) as well as for TFC (R 2 = 0.753, P < 0.05) (Table 4).
Nitric oxide (NO−) scavenging assay
Lowest IC50 value of (84.6 ± 3.7 μg/ml) was depicted by ANA comparative to standard ascorbic acid (56.9 ± 2.5 μg/ml). IC50 values of other extract/fractions were (151.8 ± 4.0 μg/ml), (153.6 ± 4.0 μg/ml), (414.9 ± 2.8 μg/ml) and (465.5 ± 2.8 μg/ml) for ANE, ANM, ANC and ANH respectively. Figure 2c illustrated the radical scavenging activity of different plant samples relative to standard ascorbic acid. IC50 values obtained for nitric oxide scavenging assay exhibited a significant correlation with TPC (R 2 = 0.77, P < 0.05) and non-significant correlation with TFC (R 2 = 0.55, P > 0.05) (Table 4).
Inhibition of β-carotene oxidation
The best results for inhibition of β-carotene oxidation was depicted by ANE and ANA with lowest IC50 values of (41.4 ± 1.9 μg/ml) and (54.7 ± 4.4 μg/ml) than that of standard catechin (90.2 ± 2.3 μg/ml). ANM, ANC and ANH showed the IC50 values of (103.6 ± 4.2 μg/ml), (726.2 ± 4.7 μg/ml) and (858.9 ± 3.8 μg/ml) respectively. Inhibition of β-carotene oxidation of ANM and its fractions at different concentrations are represented in Fig. 2d. The assay showed significant correlation of IC50 with both TPC (R 2 = 0.82, P < 0.05)-and TFC (R 2 = 0.68, P < 0.05) (Table 4).
Iron chelating activity
All the extract/fractions had high levels of ferrous ion chelating activity relative to standard EDTA (56.0 ± 1.7 μg/ml). Figure 2e indicated that the iron chelating activity of plant samples can be ranked as ANA, ANE, ANM, ANC and ANH. The lower IC50 values were recorded for ANA (76.8 ± 4.5 μg/ml), ANE (130.1 ± 0.8 μg/ml) and ANM (130.5 ± 0.8 μg/ml). TPC and TFC were significantly correlated (R 2 = 0.833, P < 0.05)-and (R 2 = 0.85, P < 0.05) with reducing power respectively (Table 4).
Reducing power assay
Crude extract (ANM) showed the highest reducing power with 125.5 ± 5.0 mg ascorbic acid equivalent/g followed by ANE (113.1 ± 2.7 mg ascorbic acid equivalents/g sample), ANA (102.0 mg ascorbic acid equivalents/g sample), ANC (45.5 ± 3.3 mg ascorbic acid equivalents/g sample) and ANH (36.0 ± 2.6 mg ascorbic acid equivalents/g sample). There was recorded a significant correlation between the reducing power and with both TPC (R 2 = 0.83, P < 0.05) and TFC (R 2 = 0.80, P < 0.05) shown in Table 2.
Total antioxidant capacity (phosphomolybdenum assay)
Total antioxidant capacity of various extracts was estimated by phosphomolybdate method and expressed as equivalents of ascorbic acid-(mg/g of extract). Total antioxidant capacity was found to decrease in the order, ANM > ANE > ANA > ANC > ANH. The assay showed significant correlation with TPC (R 2 = 10.74, P < 0.05) and TFC (R 2 = 0.79, P < i0.05) as shown in Table 2.
Estimation of acute toxicity
The plant crude methanol extract (ANM) and its derived fractions; ANH, ANC, ANE and ANA were found safe at all tested doses (up to 4000 mg/kg) and did not show any noxious symptom in rats like sedation, convulsions, diarrhea and irritation. During the two week assessment, no mortality was found.
Assessment of biochemical serum markers
Effect of different treatments of ANM on liver serum markers profile
45.0 ± 1.56e
40.3 ± 1.53e
63.7 ± 1.10d
0.59 ± 0.02d,e
40.8 ± 1.18e
39.4 ± 1.18e
60.4 ± 1.13d
0.61 ± 0.02d,e
CCl4 (1 ml/kg)
490.4 ± 5.19a
320.9 ± 3.68a
298.3 ± 3.29a
1.97 ± 0.01c
Silymarin + CCl4
96.7 ± 2.47d
96.9 ± 2.31d
108.2 ± 3.98c
0.73 ± 0.01c
ANM (200 mg/kg) + CCl4
137.2 ± 2.13b
166.8 ± 2.06b
196.7 ± 3.68b
1.02 ± 0.10b
ANM (400 mg/kg) + CCl4
101.4 ± 1.74c
121.4 ± 2.74c
168.6 ± 2.61c
0.66 ± 0.08c,d
ANM (200 mg/kg)
52.0 ± 3.99c,d
51.7 ± 2.81d
73.9 ± 2.75c
0.60 ± 0.03d,e
ANM (400 mg/kg)
41.24 ± 2.01e
37.2 ± 1.01e
62.2 ± 2.70d
0.59 ± 0.01e
Assessment of lipid profile of hepatic injury
Effect of different treatments of ANM on lipid profile
Total cholesterol (mg/dl)
83.6 ± 2.64e
99.4 ± 2.22f
46.1 ± 1.66e
32.1 ± 1.28d,e
89.5 ± 2.19e
90.3 ± 2.48f
43.14 ± 1.70e
30.4 ± 1.39e
CCl4 (1 ml/kg)
372.0 ± 5.63a
478.4 ± 5.96a
19.2 ± 1.02a
208.9 ± 2.89a
Silymarin + CCl4
120.8 ± 2.44d
162.0 ± 2.26d
40.0 ± 3.00e
43.4 ± 1.55c
ANM (200 mg/kg) + CCl4
132.8 ± 2.63b
187.8 ± 2.63b
39.3 ± 2.28c
44.1 ± 2.02b
ANM (400 mg/kg) + CCl4
124.1 ± 2.54c
152.1 ± 2.54c
41.19 ± 3.96d
40.02 ± 1.8b
ANM (200 mg/kg)
99.0 ± 3.98c,d
92.3 ± 2.98e
43.0 ± 2.98e
38.0 ± 1.98c
ANM (400 mg/kg)
86.7 ± 1.43e
87.2 ± 2.42f
47.4 ± 1.15f
35.1 ± 1.38d
Assessment of hepatic protein, TBARS,IH2O2,Initrite and GSH content
Effect of different treatments of A. nitida stem bark crude extract on liver tissue protein, TBARS, H2O2, nitrite and GSH content
Protein (μg/mg tissue)
H2O2 (nM/min/mg tissue)
Nitrite content ( μM/ml)
GSH (μM/g tissue)
6.41 ± 0.23a
1.52 ± 0.08d
6.38 ± 0.15g
61.5 ± 1.78d
47.0 ± 2.84a
6.14 ± 0.20a
1.73 ± 0.07c,d
6.77 ± 0.16g
64.2 ± 2.83d
45.65 ± 2.2a
CCl4 (1 ml/kg)
3.98 ± 0.16e
5.32 ± 0.06a
18.3 ± 0.26a
106.3 ± 4.67a
12.6 ± 1.23d
Silymarin + CCl4
5.16 ± 0.19b
2.11 ± 0.08b
8.37 ± 0.15e
70.4 ± 0.27c
46.7 ± 1.77a
ANM (200 mg/kg) + CCl4
4.42 ± 0.14d
2.11 ± 0.06b
12.2 ± 0.21b
90.2 ± 2.26b
30.74 ± 1.95c
ANM (400 mg/kg) + CCl4
5.02 ± 0.14c
2.02 ± 0.06b,c
11.1 ± 0.14c
71.1 ± 1.27c
41.3 ± 1.68b
ANM (200 mg/kg)
5.90 ± 0.16b,c
1.81 ± 0.39b
7.92 ± 0.29d
61.9 ± 2.75c
45.4 ± 1.48a,b
ANM (400 mg/kg)
6.48 ± 0.32a
1.51 ± 0.16d
7.50 ± 0.29f
60.0 ± 2.45d
49.1 ± 1.84a
Assessment of crude ANM by antioxidant enzymes
Effect of different treatments of A. nitida stem bark crude extract on liver tissues antioxidant enzymes
SOD (U/mg protein)
GST (nM/min/mg protein)
GPx (nM/min/mg protein)
GR (nM/min/mg protein)
6.68 ± 0.10a
10.39 ± 0.22a
5.54 ± 0.36a
139.8 ± 3.22a
92.8 ± 2.05a
137.2 ± 2.51a
6.35 ± 0.39a
10.06 ± 0.43a
5.05 ± 0.62a
139.05 ± 3.50a
93.3 ± 1.27a
133.1 ± 3.51b
CCl4 (1 ml/kg)
3.49 ± 0.09e
5.82 ± 0.12e
2.49 ± 0.29c
53.77 ± 1.61e
32.9 ± 1.79f
61.2 ± 1.33f
Silymarin + CCl4
5.29 ± 0.11c
8.57 ± 0.29b
4.65 ± 0.32ab
122.5 ± 2.41b
82.8 ± 1.74d
114.5 ± 2.58d
ANM (200 mg/kg) + CCl4
4.30 ± 0.15d
6.20 ± 0.31d
3.67 ± 0.27b
82.63 ± 1.61d
62.4 ± 1.77e
86.0 ± 3.05e
ANM (400 mg/kg) + CCl4
5.61 ± 0.19bc
7.57 ± 0.33d
4.61 ± 0.29a,b
103.8 ± 2.97c
85.9 ± 1.48c,d
119.19 ± 2.5c
ANM (200 mg/kg)
6.19 ± 0.20b
9.99 ± 0.24c
4.84 ± 0.36a,b
140.0 ± 3.18b
90.9 ± 2.82b,c
125.1 ± 3.31b
ANM (400 mg/kg)
6.53 ± 0.28a
10.14 ± 0.21b
5.73 ± 0.26a
141.3 ± 2.77a
91.0 ± 0.91a,b
136.2 ± 3.02a
The protective effects of ANM on GST, GPx and GR in liver tissue are shown in Table 9. Activity level of GST, GPx and GR enzymes was markedly decreased (P < 0.01) with CCl4 supplementation in liver tissue when compared to that of the control group. CCl4 toxicity was prevented noticeably with co-treatment of ANM in dose dependent manner. Concentration of GPx was found statistically similar to that of the control group at high dose of 400 mg/kg b.w. whereas concentration of GST and GR showed a significant difference. Activity level of GST, GPX and GR at maximum dose of ANM was statistically similar to the silymarin treated group.
Nature has served as opulent repository of medicinal plants for thousands of years and remarkable number of modern drugs has been isolated from natural sources, markedly from plant origin . In the present study, the complete antioxidant profiling of Alnus nitida stem bark was carried out in vitro and in vivo along with HPLC analysis. The preliminary phytochemical analysis of crude extract and its fractions exhibited a wide range of phytoconstituents such as alkaloids, anthraquinones, tannins, terpenoids, saponins, betacyanins, coumarins, flavonoids and phenols. Among the phytochemicals, phenolics and flavonoids have expanded a particular interest because of their wide ranging antioxidant activities. For this purpose polyphenols standards were selected on the basis of their reported medicinal properties for example; catechin and gallic acid have anticancer and antioxidant properties , caffeic acid exert the anticancer properties . Rutin has antioxidant, antiviral, antihypertensive and antiplatelet properties . In our analysis gallic acid was detected in crude extract as well as in ANE and ANA fractions. Therefore, it can be concluded that gallic acid is the chief phenolic compound donating to the antioxidant activity of the plant. Additionally, rutin and catechin were also detected in ANA which may be accountable for the increased antioxidant potential of the aqueous fraction. In our results significant positive correlation was found between TPC and TFC which also endorses the HPLC results.
Antioxidants especially phenolic and flavonoid contents have ability to scavenge free radicals such as superoxide and hydroxyl radicals . To investigate the complete antioxidant profile of the A. nitida bark five different in vitro bioassays (scavenging of DPPH, hydroxyl and nitric oxide, inhibition of β-carotene bleaching and iron chelating) were performed. DPPH radical scavenging is considered as a milestone for assessing the antioxidant potential. In this study ANM, depicted the maximum scavenging followed by ANE and ANA which is expected due to flavonoids which usually contain the high metal chelating activity [53, 54]. A. nitida stem bark extract was found to be a powerful scavenger of hydroxyl radicals which are responsible to react with an extensive range of molecules found in living cells . So, scavenging of hydroxyl radical is considered to be a potent antioxidant. In our results, highest activity was showed by the ANM which is coherence with Batool et al.  who reported that ethanol extract of Zanthoxylum alatum fruit as the most active to scavenge hydroxyl radicals. Nitric oxide has a compelling role in various inflammatory processes and continuous production of this radical is directly toxic to tissues . The results showed that the ANA (IC50 84.6 μg/ml) has highest nitric oxide scavenging activity which is comparable with the ascorbic acid (IC50 56.9 μg/ml). Similar behavior of activity was observed in iron chelating assay which showed the highest activity ANA (IC50 76.8 μg/ml) compared with EDTA (IC50 56.0 μg/ml). As we know that Fe (III) reduction is an indicator of electron donating activity, which is a vital mechanism of phenolic antioxidant action  so further study is required to identify the main agent responsible for such strong activity in two different assays. Oxidation of linoleic acid is considered to estimate the antioxidant potency of a plant crude extract and fractions which operates an antioxidant in the reaction mixture to check the consumption of β- carotene by acting on linoleic acid free radicals . The present study depicted the best β-Icarotene scavenging activity of ANE with lowest IC50 values even than that of standard catechin attributed to the greater amount of phenolics and flavonoid in ethyl acetate fraction.
Phosphomolybdenum and potassium ferricynide assays are quantitative method to evaluate total antioxidant capacity and total reducing power of the plant extracts respectively. The results of both assays showed similar pattern of contents with the highest values for the ANM followed by its fractions ANE > ANA > ANC > ANH. Our results have been supported by the previous study of Sikder et al.  who reported that crude methanol extract of plants exhibited highest value of antioxidant capacity and reducing power respectively. Moreover, all the antioxidant assays showed significant positive correlation with TFC and TPC except DPPH has non-significant correlation with TFC. Collectively, it can be proposed that he antioxidant activity of A. nitida may be accredited due to the existence of high content of phenolics and flavonoids which are well recognized as potential antioxidants and free radical scavengers and inhibit lipid peroxidation via the scavenging of radicals and metal chelation.
The antioxidant potential of A. nitida can also be described by the fact that the plants of this genus are endowed with terpenoids, flavonoids, diarylheptanoids, phenols, steroids and tannins. Diarylheptanoids are the dominant constituents within the genus Alnus, few of them exhibited antioxidant effects and inhibitory effects against nitric oxide and tumor necrosis factor a production . Two diarylheptanoids nitidone A and nitidone B have been reported from A. nitida found in Northern areas of Pakistan . About 400 diarylheptanoids  have been isolated from different species of Alnus showing various pharmacological activities; anti-inflammatory [14, 15], anti-influenza , hepatoprotective . Various isolates or the derivatives of diarylheptanoids from A. japonica showed antioxidant effects during in vitro studies [18, 19].
Liver acts as a center for metabolism of proteins, lipids, carbohydrates, and it also removes unwanted metabolites. Liver secretes a biochemical called bile, which plays an important role in digestion. Based on the significant in vitro antioxidant activity, bark of A. nitida was evaluated for the hepato-protective effect in CCl4 challenged rats. For this purpose, rats were intra-peritoneally administrated with CCl4 (1 ml/kg) followed by oral administration of crude extract of A. nitida bark with the concentration of 2001 and 400 mg/kg of rats body weight respectively. Administration of CCl4 causes hepatopathy which is indicated by elevation in AST, ALT, ALP and bilirubin in serum. Generally, CCl4 is metabolized by the liver into highly reactive metabolites which either directly or indirectly cause lipid peroxidation of the hepatocytes . Different cytosolic liver marker enzymes would then leaked out from these swollen and necrotic hepatocytes in to blood circulation and clearly elevated levels are obtained that is related with the immense centrilobular necrosis, ballooning, degeneration and cellular infiltration of the liver . The group administrated with CCl4 showed an increase in in serum level of triglycerides, total cholesterol, HDL and LDL (Table 7). The pathological changes in these rats indicated the potential damage in hepatic tissues prompted by CCl4 treatment (Fig. 2 and Tables 8 and 9). Our results displayed that treatment of rat with crude extract reduced the toxic effects of CCl4 and reestablished the level of above mentioned biochemical similar to untreated group which are supported by previous findings . Moreover, hepatic lesions are also minimized which may be due to presence of flavonoids and phenolics in favorable amount (Fig. 2). Crude extract possibly decreased the oxidative stress by scavenging of free radicals manufactured by CCl4 with subsequent renovation in enzymatic activities and oxidative stress indicators; TBARS,IH2O2, nitrite, protein concentration and GSH in hepatic homogenates. These results are in coherence with Sajid et al.  who reported the protective activity of Artemisia scoparia against CCl4 induced oxidative stress.
Naturally, antioxidant enzymes work in a harmonized fashion to avert the oxidative stress. The metabolic role of liver in detoxification of xenobiotics consequences in the production of ROS, where SOD, CAT, POD, GSH-Px, GST and GSR play a vital role in deterrence of oxidative stress in liver samples . For in vivo study, crude extract was selected because of high phenolics and flavonoids content, good antioxidant activity and noteworthy total antioxidant capacity and reducing power. In our study, activities of the antioxidant enzymes in hepatic samples were reduced by CCl4 and ameliorated by ANM. Similar protective results have been reported in cirrhotic animals .
The hepato-protective proficiencies of A. nitida obtained in this study might be also be contributed by other classes of chemicals. Buniatian et al. studied hepato-protective properties of altan (obtained on the basis of ellagitannins from the cones of black alder Alnus glutinosa) on the model of acute liver damage induced with CCl4. It was found that altan exhibits the hepato-protective activity even in a dose of 1 mg/kg which is ten-fold smaller than the dose of traditional flavonoid-based drugs . Antioxidant activity of diarylheptanoids by inhibition of lipid peroxidation has been reported showing the activity of diarylheptanoids more active than α-tocopherol . The anti-hepatotoxic effects of diarylheptanoids and related analogues were assessed utilizing CCl4 and galactosamine-induced cytotoxicity in primary cultured rat hepatocytes . So the diarylheptanoids might be the factor along with polyphenols contributing to the antioxidant activity of A. nitida.
Histopathological studies of ANM depicted that administration of CCl4 prompts extensive fatty change with white and yellow areas due to lipid peroxidation, congestion in blood vessels, cellular hypertrophy, necrotic foci, destruction of the lobular architecture, the development of septa and congested blood vessels with disturbed epithelium and nuclear degeneration in some areas which was significantly recovered by the crude extract (Fig. 2). The study revealed that the crude extract of A. nitida bark was comprised of polyphenol and terpenoids which show significant protective effect against hepatotoxicity induced by CCl4. Similar histological observation was found by various investigators  while assessing the protective effect of medicinal plant againstICCl4 and other drugs tempted hepatotoxicity in rats. Taken together our results showed the hepatoprotective effect of A. nitida bark against CCl4 induced toxicity both by in vitro and in vivo evaluation techniques.
Our study concluded that the hepatoprotective activity of A. nitida bark is likely due to the scavenging of free radicals and sustaining of endogenous antioxidant molecules. This effect appears to be facilitated by natural antioxidants in A. nitida bark, which remarkably diminished the oxidative stress and led to normal hepatic functions. Further exploration must be accompanied to reveal the mechanisms regarding the hepatoprotective effect of A. nitida bark at molecular level.
ALT, alanine transaminase; ANA, Alnus nitida soluble residual aqueous fraction of ANM; ANB, Alnus nitida butanol fraction of ANM; ANC, Alnus nitida chloroform fraction of ANM; ANE, Alnus nitida ethyl acetate fraction of ANM; ANH, Alnus nitida hexane fraction of ANM; ANM, Alnus nitida methanol extract of bark; AST: aspartate transaminase; BUN, blood urea nitrogen; CAT, catalase; CCl4, carbon tetrachloride; GSH, rerduced glutathione; GSR, glutathione reductase; GST, glutathione-S-transferase; POD, peroxidase; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances
MRK is intensely acknowledged for his kind supervision, expert guidance and substantial facilitations of all necessary materials and equipment. Dr. Bushra Mirza is also highly acknowledged for her assistance in HPLC.
The project was funded by the Department of Biochemistry Quaid-i-Azam University Islamabad Pakistan.
Availability of data and materials
All the data is contained in the manuscript.
MS made significant contribution to experimentation, acquisition and drafting of the manuscript. MRK has made substantial contribution to designing, analyzing and drafting of the manuscript. SAS, HI, TY and ZZ made a contribution in the experimentation and acquisition of the data. All authors read and approved the final manuscript.
MRK did his Diploma in Unani Medicine and Surgery (DUMS) and is a registered practitioner of the National Council for Tibb of Pakistan. He is working as Associate Professor at the Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan.
The authors declare that they have no competing interests.
Consent to publish
Ethics and consent to participate
This study makes use of rats and the experimental protocol for the use of animal was approved (Bch#0275) by the ethical board of Quaid-i-Azam University, Islamabad Pakistan.
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