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Total phenolic, flavonoid contents, in-vitro antioxidant activities and hepatoprotective effect of aqueous leaf extract of Atalantia ceylanica
© Fernando and Soysa; licensee BioMed Central Ltd. 2014
Received: 15 May 2014
Accepted: 6 October 2014
Published: 14 October 2014
Decoction prepared from leaves of Atalantia ceylanica is used in traditional medicine in Sri Lanka for the treatment of various liver ailments since ancient times. Lyophilized powder of the water extract of A. ceylanica leaves was investigated for its phytochemical constituents, antioxidant and hepatoprotective activity in-vitro.
The total phenolic and flavonoid contents were determined using Folin Ciocalteu method and aluminium chloride colorimetric assay respectively. The antioxidant activities of the decoction were investigated using 1,1-Diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical, nitric oxide scavenging assays and ferric ion reducing power assay. Hepatotoxicity was induced on porcine liver slices with ethanol to study hepatoprotective activity. Porcine liver slices were incubated at 37°C with different concentrations of the water extract of A. ceylanica in the presence of ethanol for 2 hours. The hepatoprotective effects were quantified by the leakage of alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) to the medium. Thiobarbituric acid reactive substances (TBARS) assay was performed to examine the anti-lipid peroxidation activity caused by the plant extract.
The mean ± SD (n =9) for the levels of total phenolics and flavonoids were 4.87 ± 0.89 w/w% of gallic acid equivalents and 16.48 ± 0.63 w/w% of (-)-Epigallocatechin gallate equivalents respectively. The decoction demonstrated high antioxidant activity. The mean ± SD values of EC50 were 131.2 ± 36.1, 48.4 ± 12.1, 263.5 ± 28.3 and 87.70 ± 6.06 μg/ml for DPPH, hydroxyl radical, nitric oxide scavenging assays and ferric ion reducing power assay respectively.
A significant decrease (p <0.05) was observed in ALT, AST and LDH release from porcine liver slices treated with A. ceylanica extract at a concentration of 2 mg/ml in the presence of ethanol (5 M) compared to that of ethanol (5 M) treated slices. Furthermore, a reduction in lipid peroxidation was also observed in liver slices treated with the leaf extract of A. ceylanica (2 mg/ml) compared to that of ethanol induced liver toxicity (p <0.05).
The results suggest that aqueous extract of A. ceylanica exerts hepatoprotective activity against ethanol induced liver toxicity of porcine liver slices which can be attributed to the antioxidant properties possessed by the plant material.
During hepatic drug biotransformation, free radicals are continuously generated. Free radicals are highly reactive, unstable molecules that react rapidly with adjacent molecules via a variety of reactions including: hydrogen abstraction, electron capturing and electron sharing leading to lipid peroxidation, protein oxidation, DNA strand breaks, and modulation of gene expression . Experimental evidence shows that these free radicals are involved in liver diseases and also lead to atherosclerosis, cancer, stroke, asthma, arthritis and other age related diseases [2, 3].
Plants as natural source of antioxidants have the potential to scavenge free radicals and inhibit their generation. It is a well established fact that the mechanism of hepatoprotective effects of certain drugs are related to their antioxidant capacity in scavenging free radicals and reactive oxygen species . Since, toxic effects of synthetic antioxidants have been reported, the interest for searching of natural antioxidants of plant origin has increased . Use of plant derived drugs in medical practice has shown that they are relatively non-toxic, safe and even free from serious side effects .
Atalantia ceylanica (Family: Rutaceae) locally known as “Yakinaran” is a dicotyledonous densely branched shrub up to 2.5 m height distributed in Sri Lanka and Southern India . The juice of the leaves is used in the preparation of pills administered for catarrh, bronchitis and other chest complaints . The root is used in the treatment of ague . Decoction prepared from leaves of Atalantia ceylanica is used in the treatment of liver diseases by traditional medical practitioners of Sri Lanka.
Due to the lack of scientific investigations carried out so far, the current research was launched to determine the phytochemical composition, antioxidant and hepatoprotective activity of the decoction prepared from Atalantia ceylanica.
Chemicals and equipment
The chemicals gallic acid, Folin Ciocalteu reagent, trichloroacetic acid, 2-deoxy-D-ribose and ethylenediamine tetra acetic acid (EDTA) were purchased from Sigma Chemicals Co. (P.O. Box 14508, St. Louis, MO 63178 USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical, (-)-epigallocatechin gallate, aluminium chloride and sulfanilamide were purchased by Fluka (Fluka chemie GmbH, CH-9471 Buchs). L-ascorbic acid, hydrogen peroxide, N-(1-naphthyl)-ethylene diamine dihydrochloride and ethanol were purchased from BDH Chemicals (BDH Chemicals Ltd, Poole, England). Sodium nitroprusside was purchased from Qualigens (A division of GlaxoSmith Kline Pharmaceuticals Ltd). Ferric chloride, potassium ferricyanide and sodium nitrite were purchased from Riedel De Haen Ag, Wunstorfer Strasse 40, SEELZE1, D3016, Germany.
Lactate dehydrogenase (LDH) enzyme assay kit was purchased from DiaSys (Alte Strasse 9, 65558, Holzheim, Germany). Alanine transaminase (ALT) and Aspartate transaminase (AST) enzyme assay kits were purchased from POINTE, SCIENTIFIC, INC (5449 Research Drive, Canton MI 48188, USA).
SHIMADZU UV 1601 UV Visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan) was used to read the absorbance. Deionized water was obtained from LABCONCO WATER PRO-PS UV ultra filtered water system (LABCONCO Corporation, Kansas city, Missouri).
The leaves of Atalantia ceylanica (Yakinaran) were collected from Nachchaduwa, Anuradhapura, Sri Lanka and was identified and confirmed by Department of Botany, Bandaranaike Memorial Ayurvedic Research Institute, Nawinna, Colombo, Sri Lanka. Voucher specimens are deposited at the above premises.
Preparation of the decoction
Washed plant leaves were dried until a constant weight was achieved. Dry weight of 30 g of plant material (n =3) was ground to a fine powder and boiled with 800 ml of deionized water until total volume reduced to 100 ml (1/8th of the original volume). The decoction was sonicated and filtered. The filtrate was centrifuged (2000 rpm, 10 min) and the supernatant was freeze dried. The freeze dried samples were weighed, and stored at -20°C in sterile tubes until further use. Lyophilized samples of A. ceylanica were prepared in triplicates and the yield was calculated as a percentage of dry weight.
Determination of total phenolic content
Total phenolic content of decoctions of A. ceylanica was determined by Folin Ciocalteu method . Calibration curve was constructed using gallic acid standards and the total phenolic content was expressed as w/w% gallic acid equivalents.
Determination of flavonoid content
The flavonoid content was measured by the aluminium chloride colorimetric assay . Calibration curve was plotted using (-)-epigallocatechin gallate (EGCG) standards and flavonoid content was expressed as w/w% EGCG equivalents.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity
L-Ascorbic acid was used as the reference standard antioxidant. The effective concentration needed to scavenge DPPH free radical by 50% (EC50) was calculated by regression analysis of the dose response curve plotted between percentage inhibition versus concentration of the test samples and the standard.
Hydroxyl radical (HO.) scavenging activity
Hydroxyl radical scavenging activity was measured based on the competition between deoxyribose and the test compound (the plant extract) to react with hydroxyl radical generated from Fe2+/Ascorbate/EDTA/H2O2 system according to the procedure as previously described with slight modification . Gallic acid was used as the reference standard antioxidant. The percentage scavenging of hydroxyl radical for A. ceylanica and the standard antioxidant was calculated according to equation 1. EC50 was calculated as described previously.
Nitric oxide radical (NO.) scavenging activity
NO. scavenging activity of the decoctions prepared was measured based on Griess - Ilosvay reaction with slight modification . The interference from the plant extract with the pink chromophore formed was minimized by background subtraction of absorbance for respective concentrations. L-Ascorbic acid was used as the reference standard antioxidant. The percentage scavenging of NO. for A. ceylanica and the standard antioxidant was calculated according to equation 1. EC50 was calculated as described previously.
Ferric ion reducing power assay
The ferric ion reducing power of the decoctions prepared was determined according to a method described previously . L-ascorbic acid was used as the reference standard antioxidant. Dose response curve was plotted between the absorbance versus concentrations of plant extracts or standards by linear regression analysis. EC50 was defined as the corresponding concentration of the plant extract which gives an absorbance value of 0.5.
Porcine liver tissue collection
Porcine liver tissue of either sex was obtained from the registered slaughter house in Dematagoda, Sri Lanka (See Additional file 1) with permission obtained from the chief municipal veterinary surgeon. A sample of liver tissue without distinction of lobes was excised using sterile scalpel blades and transferred immediately into ice cold sterile Krebs Ringers-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (KRHB) and transported to the laboratory on the day of the experiment within 5 minutes in a ice bath.
Preparation of medium
Tissue slices were prepared in sterile KRHB and maintained according to a method described previously [15, 16]. The buffer (KRHB) composition includes HEPES (2.5 mM), NaCl (118 mM), KCl (2.85 mM), CaCl2 (2.5 mM), KH2PO4 (1.5 mM), MgSO4 (1.18 mM) and glucose (4.0 mM). The pH was adjusted to 7.4 by 1 N NaOH. The medium was autoclaved for sterilization.
Hepatoprotective activity of A. ceylanica decoction in vitro
Where Total Enzyme activity = Enzyme activity in the medium + Enzyme activity in the tissue homogenate, Medium = Medium used for the incubation of liver tissue
The tissue homogenates were also assayed for total protein content and lipid peroxides formed [17, 18]. Standard curves were plotted using bovine serum albumin (BSA) and 1,1,3,3-Tetraethoxypropane (TEP) standards respectively and amount of lipid peroxides formed was expressed as micrograms of malondialdehyde (MDA) equivalents formed per gram of protein.
A minimum of three independent experiments were carried in triplicates unless otherwise specified. Students T test was performed for statistical analysis and results are presented as mean ± standard deviation (Mean ± SD). Value of p <0.05 was considered as significant. Regression analysis and statistical analysis were carried out using Microsoft Excel. Calibration curves of the standards were considered as linear if R2 > 0.99. EC50 values were calculated from either linear or logarithmic dose response curves where R2 > 0.90.
Results and discussion
Extraction yield, phenolic and flavonoid contents
The extraction yield, total phenolic content and flavonoid content obtained for A. ceylanica
Extraction yield (w/w% of dry weight)
8.56 ± 1.50 (n =3)
Phenolic content (w/w% Gallic acid equivalents)
4.87 ± 0.89 ( n =9)
Flavonoid content (w/w% (-)-Epigallocatechin gallate equivalents)
16.48 ± 0.63 (n =9)
Flavonoids are considered as potential antioxidants exerting their antioxidant activity by the mechanisms of radical scavenging and metal ion chelation to inhibit lipid peroxidation . Aluminium chloride colorimetric assay yielded total flavonoid content of 16.48 ± 0.63 w/w% (-)-Epigallocatechin gallate (EGCG) equivalents for A. ceylanica (Table 1). Methanolic extract of Aegle marmelos leaves which belongs to the same family of A.ceylanica exhibits a total phenolic content of 9.84 ± 0.023 mg/kg gallic acid equivalents and a total flavonoid content of 8.248 ± 0.029 mg/kg EGCG equivalents . This indicates that the aqueous extract of A. ceylanica possess a higher content of phytochemicals compared to Aegle marmelos.
DPPH radical scavenging activity
Hydroxyl radical (HO.) scavenging activity
Nitric oxide radical (NO.) scavenging activity
Ferric ion reducing power assay
The EC 50 values for antioxidant tests for aqueous leaf extract of A. ceylanica with respect to reference standard antioxidants
Ascorbic acid (μg/ml)
Gallic acid (μg/ml)
DPPH assay (n =9)
131.2 ± 36.1
3.30 ± 0.27
HO . scavenging (n =9)
48.4 ± 12.1
27.69 ± 4.14
NO . scavenging (n =3)
263.5 ± 28.3
276.3 ± 25.8
Fe 3+ reducing power (n =9)
87.70 ± 6.06
2.78 ± 0.30
Hepatoprotective activity of A. ceylanica aqueous extract
Liver slices have been successfully used for assaying hepatoprotective activity of compounds such as curcumin  and Pterocarpus marsupium extract . Further it has been found that, liver tissue collected from slaughter house is a useful model to study hepatotoxicity of different substances on the organ level . There is a similarity in liver specific metabolic activities with porcine liver cells with that of human liver cells . The present study was therefore carried out using liver slices prepared from fresh porcine liver obtained from the slaughter house.
A similar study carried out by Sinha et al. , on hepatoprotective activity of Picrorhiza kurroa Royle Ex. Benth aqueous root extract (Family: Scrophulariaceae) using mouse liver slice culture, the authors concluded that the high antioxidant activity of this plant is responsible for the suppression of alcohol induced toxicity. In a recent study carried out by Shokrzadeh and coauthors (2014), have found that ethanolic extract of Zataria multiflora can exert hepatoprotective activity against liver toxicity induced by cyclophosphamide (CP) in mice . Although CP is extensively used in chemotherapy, it possesses a wide spectrum of side effects including hepatotoxicity. Based on their results the authors concluded that Zataria multiflora, could be used concomitantly as a supplement agent against hepatotoxicity for the patients undergoing chemotherapy with CP.
The results obtained in this study indicate that the decoction prepared from A. ceylanica leaves possess effective hepatoprotective activity against ethanol induced toxicity in porcine liver slices. This can be attributed to the radical scavenging capacity of the plant extract hence justifying the use of this plant material in the treatment of various liver diseases in traditional medicine. The pharmacological profiles of this plant extract based on in vivo studies and clinical trials should be further investigated.
We acknowledge the financial assistance by National Science Foundation (Grant number RG/2005/HS/17) Sri Lanka and Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo. Authors particularly thank Ms. Sudeepa Sugathadasa and Ms. Pushpa Jeewandara, Department of Botany, Bandaranayake Memorial Ayurvedic Research Institute, Nawinna, Colombo, Sri Lanka, for the identification of the plant material. The technical assistance offered by Mr. Jayantha Weerasinghe, Mr. Thisira Andrahennadi, Mr. Saman Kolombage and Ms. Nilusha Rajapakse, Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo, is gratefully acknowledged.
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