Evaluation of Hepatoprotective activity of Eriocaulon quinquangulare in vitro using porcine liver slices against ethanol induced liver toxicity and free radical scavenging capacity
© Fernando and Soysa. 2016
Received: 24 September 2015
Accepted: 12 February 2016
Published: 24 February 2016
Production of reactive oxygen species is a common cause in alcohol induced liver diseases. Decoction prepared from the whole plant of Eriocaulon quinquingulare is prescribed to treat liver disorders. The aim of this study was to investigate the hepatoprotective activity and antioxidant capacity of the water extract of E. quinquangulare in vitro.
The aqueous extract of the whole plant of E. quinquangulare (AEQ) was investigated for its phytochemical constituents, antioxidant and membrane stabilization properties in-vitro. The antioxidant activities of AEQ were investigated using 1,1-Diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical, nitric oxide scavenging and ferric reducing antioxidant power (FRAP) assays. Membrane stabilizing effect of the extract was determined by hypotonic solution induced human erythrocyte hemolytic assay (HEHA). Further, hepatoprotective activity against ethanol induced hepatotoxicity was carried out using porcine liver slices.
The total phenolics and flavonoids were 10.3 ± 1.6 w/w % gallic acid equivalents and 45.6 ± 3.8 w/w % (−)-epigallocatechin gallate equivalents respectively. The values of EC50 for DPPH, hydroxyl radical and nitric oxide scavenging assays were 37.2 ± 1.7 μg/ml, 170.5 ± 6.6 μg/ml and 31.8 ± 2.2 μg/ml respectively. The reducing capability of AEQ was 6.9 ± 0.2 w/w % L-ascorbic acid equivalents in the FRAP assay. For hypotonic solution induced HEHA, the IC50 was 1.79 ± 0.04 mg/ml. A significant decrease (p < 0.05) was observed in ALT, AST and LDH release from the liver slices treated with AEQ compared to the ethanol treated liver slices. A significant reduction in lipid peroxidation (p < 0.05) was also observed in liver slices treated with the plant extract compared to that of the ethanol treated liver slices.
The results suggest AEQ possess hepatoprotective activity against ethanol induced liver toxicity of porcine liver slices which can be attributed to antioxidant properties and membrane stabilizing effects caused by the plant material.
KeywordsAntioxidant properties Eriocaulon quinquangulare Hepatoprotective activity Membrane stabilization activity
Reactive oxygen (ROS) and nitrogen species (RNS) are produced as byproducts during cellular metabolism or exposure to environment chemicals and radiation. Over production of ROS and RNS cause oxidative damage to biomolecules, provoke immune response, activate oncogenes and hasten the ageing process . Chronic alcoholism leads to the development of alcoholic liver disease and is a major health problem in the society. Metabolism of ethanol occurs in the liver, catalyzed by alcohol dehydrogenase, aldehyde dehydrogenase (ALDH), cytochrome P450 2E1 (CYP2E1), and catalase enzymes . These mechanisms result in the decrease of NAD+/NADH redox ratio and depletion of reduced glutathione (GSH) leading to oxidative stress [3, 4]. Several studies have shown that antioxidants including plant extracts protect against ethanol induced hepatotoxicity by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity . Use of plant derived drugs in medical practice has shown that they are relatively non-toxic, safe and free from serious side effects .
Eriocaulon quinquangulare (Family: Eriocaulaceae) locally known as “Heen kokmota” is a slender annual tuft. This monocotyledonous plant is distributed in lowlands in Sri Lanka . The total plant of Eriocaulon quinquangulare prepared as decoction is used to treat patients suffering from liver disorders, jaundice and splenomegaly in Sri Lanka .
The present study was carried out to determine the phytochemical composition, antioxidant, membrane stabilization and hepatoprotective activities of aqueous extract of E. quinquangulare (AEQ) to evaluate the scientific base of its application as a hepatoprotective drug.
Chemicals and equipment
The chemicals gallic acid, Folin ciocalteu reagent, trichloroacetic acid, sodium salicylate 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 (Flukachemie GmbH, CH-9471 Buchs). L-ascorbic acid, hydrogen peroxide, N-(1-naphthyl)-ethylenediamine dihydrochloride and ethanol were purchased from BDH Chemicals (BDH Chemicals Ltd, Poole, England). 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).
The whole plant of Ericaulon quinquangulare (Heen Kokmota) was collected from Kalutara District. This plant material was identified and confirmed by Department of Botany, Bandaranaike Memorial Ayurvedic Research Institute, Nawinna, Sri Lanka. A voucher specimen of the plant was deposited at the herbarium of the above Institute (acc number: 980c).
Preparation of the decoction
Decoction from total plant of Eriocaulon quinquangulare was prepared according to a procedure followed by Ayurvedic practitioners of Sri Lanka . Plant material was washed with deionized water and dried to achieve a constant weight at room temperature. Dried material was cut into small pieces and ground to a fine powder. A weight of 30 g was boiled with 800 ml of deionized water until its volume gets reduced to 100 ml (1/8th of the original volume). The decoction was sonicated and filtered. The filtrate was centrifuged (2000 rpm, 10 min). The supernatant was freeze dried and stored at −20 °C in sterile tubes until further use. The yield of the lyophilized powder was calculated as a percentage of the dry weight. The lyophilized powder was dissolved in deionized water or buffer for the experiments and concentration of the treated sample was calculated as μg/ml or mg/ml of the lyophilized sample.
The phytochemicals present in AEQ was determined according to a previously published method . The extract was screened for carbohydrates, tannins, phenols, phlobatannins, amino acids and proteins, saponins, flavonoids, sterols, terpenoids, cardiac glycosides, alkaloids, quinones and oxalates.
Determination of total phenolic content
Total phenolic content of AEQ was determined by Folin Ciocalteu method . Calibration curve was constructed using gallic acid standards and the total phenolic content was reported as w/w% gallic acid equivalents (GAE).
Determination of flavonoid content
The flavonoid content was quantified by the aluminium chloride colorimetric assay . Calibration curve was plotted using (−)-epigallocatechin gallate (EGCG) standards and flavonoid content was reported as w/w% EGCG equivalents.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity
L-Ascorbic acid was used as the positive control. 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 positive control.
Hydroxyl radical 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 radicals generated from Fe2+/Ascorbate/EDTA/H2O2 system according to the procedure as previously described with slight modifications . Gallic acid was used as the positive control. The percentage scavenging of hydroxyl radical for AEQ and the positive control was calculated according to equation 1. EC50 was calculated as described previously.
Nitric oxide radical (NO) scavenging activity
NO was generated from sodium nitroprusside (SNP) and NO scavenging activity of AEQ 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 positive control. The percentage scavenging of NO for AEQ and the positive control was calculated according to equation 1. EC50 was calculated as described previously.
Ferric reducing antioxidant power (FRAP) assay
The ferric ion reducing power of AEQ was determined according to a method described previously . L-ascorbic acid was used as the positive control. Dose response curve was plotted between the absorbance versus concentrations of plant extract or positive control. The ferric reducing antioxidant power of the decoction was expressed as w/w% L-ascorbic acid equivalents.
Porcine liver tissue collection
Porcine liver tissue of either sex was obtained from the registered slaughter house in Dematagoda, Sri Lanka with permission obtained from the chief municipal veterinary surgeon (Refer the Section III.24 of Additional file 1 regarding ethical approval).
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 min in an ice bath.
Preparation of medium
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 E. quinquangulare 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 [16, 17]. 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.
Membrane stabilization activity
The effective concentration needed to inhibit the lysis of human erythrocytes by 50 % compared to the control (IC50) was calculated by regression analysis of the dose response curves.
Triplicate measurements were obtained for each experiment 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, phytochemical constituents, phenolic and flavonoid contents
Phytochemical composition of AEQ
AEQ (n = 3)
Phenolic content (w/w% GAE)
10.3 ± 1.6
Flavonoid content (w/w % EGCG equivalents)
45.6 ± 3.8
Oxidative stress is caused due to a variety of endogenous ROS/RNS produced during metabolic processes including ethanol metabolism and is believed to contribute significantly in the development of a number of diseases [3, 4, 22]. Therefore total antioxidant capacity of the decoction prepared was evaluated against different radical systems which include DPPH, hydroxyl and nitric oxide as well as the ability to reduce ferric ions to achieve better understanding of the specific activities possessed by AEQ.
Antioxidant capacity of AEQ
EC50 for AEQ (n = 3)
EC50 for positive control
DPPH radical scavenging activity
37.2 ± 1.7 μg/ml
L- Ascorbic acid
3.3 ± 0.3 μg/ml (n = 9)
Hydroxyl radical scavenging activity
170.5 ± 6.6 μg/ml
27.7 ± 4.1 μg/ml (n = 9)
NO scavenging activity
31.8 ± 2.2 μg/ml
276.3 ± 25.8 μg/ml (n = 9)
FRAP assay (w/w % L-Ascorbic acid equivalents)
6.9 ± 0.2 (n = 3)
Nitric oxide is a small molecule that contains one unpaired electron. It is generated in biological systems by NADPH-dependent nitric oxide synthases. It acts as an important oxidative biological signaling molecule in a variety of physiological processes such as regulation of blood pressure and immune response, defense mechanism against pathogens, smooth muscle relaxation and neural signal transmission . Overproduction of NO may occur when the generation of nitrogen species supersedes the ability of the biological system to neutralize and eliminate them. Increased levels of NO may lead to nitrosylation reactions that can alter the structure of protein and inhibit their function. When super oxide reacts with NO, it produces strong oxidant molecule peroxynitrite ion (ONOO−), which causes DNA fragmentation and lipid peroxidation .
Hepatoprotective activity of AEQ in-vitro
Liver tissue obtained from slaughter house is a useful model to study hepatotoxicity of different substances in the organ level . Further, liver slices have been successfully used for assaying hepatoprotective activity of curcumin , stem bark extract of Pterocarpus marsupium  and leaf extract of Atalantia ceylanica . There is a similarity in liver specific metabolic activities between porcine and human liver cells . Hence liver slices prepared from fresh porcine liver obtained from the slaughter house were used in this study.
Membrane stabilization activity
Our findings suggest that the decoction prepared from E. quinquangulare has the potential to act as a strong antioxidant, hepatoprotective and a membrane stabilization agent. Mechanisms of membrane stabilization and free radical scavenging activity most possibly may contribute towards potent hepatoprotective activity possessed by the plant extract hence justifying its application in traditional medicinal system in Sri Lanka to treat liver ailments. However, further in vivo work including clinical trials is required to determine the synergistic and holistic effect caused by this decoction on the body.
The authors acknowledge the financial assistance by National Science Foundation Sri Lanka and Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo. Special thanks go to Ms. Sudeepa Sugathadasa and Ms. Pushpa Jeewandara, Department of Botany, Bandaranayake Memorial Ayurvedic Research Institute, Nawinna, Sri Lanka, for the identification of the plant material. We particularly thank Technical staff of Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo, for the assistance provided.
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