Scholarly article on topic 'Antioxidant and Hepatoprotective Effects of Murdannia bracteata Methanol Extract'

Antioxidant and Hepatoprotective Effects of Murdannia bracteata Methanol Extract Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Mun Fei Yam, Lee Fung Ang, Chung Pin Lim, Omar Ziad Ameer, Ibrahim Muhammad Salman, et al.

Abstract Murdannia bracteata (C. B. Clarke) is a local plant that is widely used in Malaysia as a traditional remedy for various diseases of the kidney and liver, including inflammation and cancer. In the present study, we investigated the antioxidant and hepatoprotective activities of M. bracteata methanol extract (MB). 2,2′-diphenyl-1-picrylhydrazyl radical scavenging activity, lipid peroxidation inhibition and trolox equivalent antioxidant capacity of MB were determined. The hepatoprotective activity of MB was studied using a CCl4-induced liver toxicity model in rats. The hepatoprotective effect was assessed by monitoring the plasma malondialdehyde level and serum alanine transaminase and aspartate transaminase activities. Histopathological changes of hepatic tissue were also investigated. The results indicated that MB possessed potential antioxidant, lipid peroxidation inhibition and free radical scavenging activities. Pretreatment of rats with MB (500 mg/kg and 1000 mg/kg per os) before induction of CCl4-induced hepatotoxicity showed a dose-dependent reduction in the necrotic changes in hepatic tissue. The increases in plasma malondialdehyde level, serum alanine transaminase and aspartate transaminase activities were also significantly inhibited by MB. The total phenolic content of MB determined using Folin-Ciocalteu assay was found to be 10%. The results of the present study indicated that the hepatoprotective effect of MB is most likely due to its antioxidant and free radical scavenging properties.

Academic research paper on topic "Antioxidant and Hepatoprotective Effects of Murdannia bracteata Methanol Extract"

J Acupunct Meridian Stud 2010;3(3):197-202

I RESEARCH ARTICLE I

ELSEVIER

Antioxidant and Hepatoprotective Effects of Murdannia bracteata Methanol Extract

Mun Fei Yam1,2*, Lee Fung Ang1, Chung Pin Lim1, Omar Ziad Ameer1, Ibrahim Muhammad Salman1, Mariam Ahmad1, Mahfoudh Al-musli Mohammed1, Mohd. Zaini Asmawi1, Muthanna Fawzy Abdulkarim1, Ghassan Zuhair Abdullah1

1School of Pharmaceutical Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysia 2Department of Human Anatomy, Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor, Malaysia

Received: May 15, 2009 Accepted: May 11, 2010

KEY WORDS:

free radical scavenging activity;

hepatoprotective; Murdannia bracteata

Abstract

Murdannia bracteata (C. B. Clarke) is a local plant that is widely used in Malaysia as a traditional remedy for various diseases of the kidney and liver, including inflammation and cancer. In the present study, we investigated the antioxidant and hepatoprotective activities of M. bracteata methanol extract (MB). 2,2'-diphenyl-1-picrylhydrazyl radical scavenging activity, lipid peroxidation inhibition and trolox equivalent antioxidant capacity of MB were determined. The hepatoprotective activity of MB was studied using a CCl4-induced liver toxicity model in rats. The hepatoprotective effect was assessed by monitoring the plasma malondialde-hyde level and serum alanine transaminase and aspartate transaminase activities. Histopathological changes of hepatic tissue were also investigated. The results indicated that MB possessed potential antioxidant, lipid peroxidation inhibition and free radical scavenging activities. Pretreatment of rats with MB (500 mg/kg and 1000 mg/kg per os) before induction of CCl4-induced hepatotoxicity showed a dose-dependent reduction in the necrotic changes in hepatic tissue. The increases in plasma malondialdehyde level, serum alanine transaminase and aspartate transaminase activities were also significantly inhibited by MB. The total phenolic content of MB determined using Folin-Ciocalteu assay was found to be 10%. The results of the present study indicated that the hepatoprotective effect of MB is most likely due to its antioxidant and free radical scavenging properties.

1. I introduction

Liver disease is a serious health problem. Unfortunately, conventional or synthetic drugs used in the treatment of liver disease are inadequate and sometimes cause serious side effects. This is one

reason why many people around Malaysia prefer traditional remedies for the treatment of liver ailments. It is, therefore, necessary to search for alternative drugs for the treatment of liver disease with more efficacy and safety and to improve, augment, or replace currently used drugs [1,2].

'Corresponding author. Department of Human Anatomy, Faculty of Medicine and Health Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. E-mail: yammunfei@yahoo.com

©2010 Korean Pharmacopuncture Institute

Murdannia bracteata (C. B. Clarke) is an annual evergreen shrub that is used in Asian countries, especially China, Malaysia, and Thailand, for the traditional treatment of inflammation, rash, liver disease, kidney disease and cancer [3,4]. However, the pharmacological study of M. bracteata has not been well documented. Therefore, studies were performed to test the antioxidant and hepatoprotec-tive effects of M. bracteata. Antioxidant properties were assayed in terms of inhibitory ability on scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'azino-bis(3-ethy)benz-thiazoline-6-sulfonic acid (ABTS). The present study was also undertaken to evaluate the protective effect of M. bracteata on CCl4-induced hepatotoxicity by analyzing alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, lipid peroxidation and the histopathology of liver damage. The total phenolic contents of methanolic M. bracteata were also determined using the Folin-Ciocalteu method.

2. Materials and Methods

2.1. Materials and chemicals

Rutin, quercetin, 1,1,3,3-tetraethoxypropan, butyla-ted hydroxytoluene (BHT), gallic acid, Folin-Ciocalteu reagent, DPPH, trichloroacetic acid (TCA), ABTS, phosphate buffered saline (PBS), ferric chloride (FeCl3), potassium persulfate and Harris hematoxylin were all purchased from Sigma Chemical Company (St. Louis, MO, USA). Potassium chloride (KCl) and sodium carbonate (Na2CO3) were provided by BDH Chemicals Ltd. (Poole, United Kingdom). Hydrochloric acid (HCl) and chloroform were obtained from R & M Marketing (Essex, United Kingdom). Methanol, absolute alcohol and eosin were purchased from Riedel-de Haen (Seelze, Germany). Xylene was purchased from Fisher Scientific (Loughborough, United Kingdom). Paraplast was purchased from Oxford Labware (St. Louis, MO, USA). Thiobarbituric acid (TBA) was purchased from Applichem (Darmstadt, Germany). Trolox was purchased from Calbiochem (Darmstadt, Germany). CCl4, methanol (HPLC grade), formalin and diethyl ether were purchased from Merck Co. (Darmstadt, Germany). Extra virgin olive oil was purchased from Unilever Bestfoods Italia (Inveruno, Italy).

2.2. Experimental animals

Male Sprague Dawley rats weighing 180-200 g were used throughout the study. The animals were obtained from the Animal House Facility, School of Pharmaceutical Sciences, Universiti Sains Malaysia (USM). They were maintained at a room temperature

of 28-30°C and allowed access to food (normal laboratory chow) and tap water ad libitum. The animals were acclimatized to laboratory conditions for 7 days before the commencement of experiments. All procedures in this study were performed in accordance with the Animal Ethics Committee Guidelines of USM.

2.3. Preparation of plant extract

M. bracteata was obtained from Permatang Damar Laut, Penang, Malaysia. The plant was identified by the Department of Herbarium and a voucher specimen No.10945 was deposited at the same institute in the School of Biological Sciences, USM. The plant material was dried in an oven at 45°C for 24 hours and pulverized into powder, after which it was put into a cellulose thimble and extracted via hot extraction with methanol and a Soxhlet apparatus (SCOTT, Duran, Germany). The obtained methanol extract of M. bracteata (MB) was concentrated using a rotary evaporator (Buchi RE121, Switzerland) under vacuum conditions and subsequently freeze dried (HETO Hetovac VR-1, Denmark). The yield obtained was 12.8%.

2.4. Assessment of total antioxidant and DPPH scavenging activities

The total antioxidant activity of MB was estimated using a trolox equivalent antioxidant capacity (TEAC) test [6]. ABTS was dissolved in deionized water to give a 7-mM solution. The ABTS radical cation (ABTS^+) was produced by reacting the ABTS stock solution with 2.45 mM potassium persulfate. The mixture was allowed to stand in the dark at room temperature (22-24°C) for 12-16 hours prior to use. The concentrated ABTS^+ solution was then diluted with PBS (pH 7.4) to a final concentration that would give an absorbance of 0.70 ± 0.02 at a 734 nm wavelength in an ambient temperature of 30°C. Stock solutions of trolox (0.5-4 mM) and MB (1 mg/mL) were prepared in deionized water. The spectrophotometer (Hitachi U-2000; Hitachi, Tokyo, Japan) was primarily blanked with PBS. Antioxidant containing solution (10 |L) was added to 2 mL of ABTS^+ solution. The decrease in absorbance was measured at 734 nm, 6 minutes after the addition of trolox and MB. Sample determinations were all performed in triplicate. The TEAC of MB was calculated by relating the decrease in absorbance to that of the trolox solution on a molar basis.

Free radical scavenging activity of MB was measured in terms of hydrogen donating or radical-scavenging ability using stable DPPH [6,7]. Rutin, quercetin or MB (0.1 mL) was added to 3 mL of 0.004% DPPH in methanol. Methanol (0.1 mL) was used as a control to MB. Absorbance at 517 nm was

determined spectrophotometrically after 30 minutes of incubation, and the percentage inhibition was calculated as [(A0 - A1)/40] x 100, where A0 is the ab-sorbance of the control, and A1 is the absorbance of the MB, rutin or quercetin. The free radical scavenging activity of MB, rutin and quercetin were expressed as 50% effective dose (EC50) in which the EC50 value was defined as the concentration (in mg/ mL) of sample that inhibited 50% of the formation of DPPH radical.

2.5. Assessment of hepatoprotective activity

2.5.1. CCl4-induced liver damage

Rats were randomly divided into five experimental groups with six rats per group. The first group, which was not treated with CCl4, received distilled water (10 mL/kg per os [p.o.]) and served as the control group. The other five groups received the following treatments: Group 1: distilled water (10 mL/kg p.o.) + CCl4; Group 2: MB (250 mg/kg p.o.)+CCl4; Group 3: MB (500 mg/kg p.o.) + CCl4; Group 4: MB (1000 mg/kg p.o.)+CCl4; Group 5: Sylimarin (100 mg/ kg p.o.). The distilled water and MB were administered orally once daily for 7 days by gastric gavage needle. Sixty minutes after the administration of the last dose on the seventh day, CCl4 diluted in olive oil (1:1) was administered at 1 mL/kg orally to all groups except control. Twenty-four hours after CCl4 administration, blood was collected from all rats by cardiac puncture while the animals were under general anesthesia using inhalation of diethyl ether. The animals were finally sacrificed by cervical dislocation. Plasma was prepared in heparinized tubes by centrifugation at 3500 rpm and 4°C for 10 minutes for lipid peroxidation analysis. Serum was prepared by clotting the blood in blank test tubes at room temperature, and was subsequently separated from the blood cells through centrifugation at 3500 rpm and 4°C for 15 minutes. The serum was analyzed for ALT and AST activities [8]. AST and ALT were analyzed at the Department of Biochemistry, Pathology Laboratory, Lam Wah Ee Hospital, Penang, by using COBAS Integra 800 (Hoffmann-La Roche Ltd., Basel, Switzerland).

2.5.2. Lipid peroxidation in plasma

The methodology described by Kurtel et al was used [9]. Plasma sample (1 mL) was mixed thoroughly with 2.0 mL TCA (15% w/v)-TBA (0.375%)-0.25N HCl and centrifuged at 10,000 rpm for 5 minutes. The supernatant was mixed with 20 |L of BHT (0.02% in 95% ethanol, w/v) to prevent further oxidation and heated for 15 minutes in a boiling water bath. After

cooling under running water, the flocculent precipitate was removed by centrifugation at 10,000 rpm for 5 minutes. The absorbance of the sample was measured at 532 nm against a blank that contained all the reagents except plasma. 1,1,3,3-tetraethoxy-propan was used as standard for calibration of the curve [9].

2.5.3. Assessment of in vitro lipid peroxidation inhibition

Adult male rats (n=3; 180-220 g) were used in this study. The animals were terminally anaesthetized with diethyl ether before being sacrificed by cervical dislocation. The liver was removed via abdominal dissection, and 5% (w/v) liver homogenate was prepared in 0.15 M potassium chloride (KCl) using a homoge-nizer (Homogeniser; MSE Scientific Instruments, Crawley, England) under ice-cold (0-4°C) conditions. The homogenate was then centrifuged (Eppendorf 5403, Germany) at 3000 rpm for 15 minutes at 4°C and the resultant supernatant was separated for analysis. 100 |L of MB (0.156-10 mg/mL in distilled water) was dispensed into test tubes followed by 0.5 mL of supernatant and 1 mL of 0.15 M KCl. Peroxidation was initiated by the addition of 100 |L of 0.2 mM FeCl3. The mixture was incubated at 37°C for 30 minutes after which the reaction was stopped by adding 2 mL of ice-cold thiobarbituric acid TBA-TCA-HCl-BHT solution. The TBA-TCA-HCl was prepared by dissolving 1.68 g TCA and 41.60 mg TBA in 10 mL 0.125 M HCl. One mL of BHT-solution (1.5 mg/mL eth-anol) was added to 10 mL TBA-TCA-HCl solution. The reaction mixture was heated for 60 minutes at 90°C, and then cooled on ice (4°C) before finally being centrifuged at 3000 rpm. Supernatants were removed and the color absorbency was measured in a spectro-photometer at 532 nm wavelength [10,11]. A control experiment was performed in the presence of distilled water without MB. The percentage inhibition of lipid peroxidation in the samples was calculated using the following formula:

Inhibition of lipid peroxidation (%)=[(A0 -A-|)/A0] x 100,

where A0 is the absorbance of the control (100 |L of distilled water), and A1 is the absorbance of sample containing MB. The lipid peroxidation inhibition of MB was expressed as EC50.

2.5.4. Histopathological studies

Immediately after cardiac puncture, animals were sacrificed and the liver of each rat was removed and fixed in 10% formalin before being processed using a Citadel 1000 histokinette (Shandon Scientific Ltd., Cheshire, UK). After processing, the tissues were

embedded in paraffin with Histo-Center II-N (Barn-stead/Thermolyne, Dubuque, IA, USA) and sectioned into 5-|m thicknesses using Histocut 820 (Cambridge Instrument GmbH, Nussloch, Germany). The tissue sections were then stained with hematoxylin and eosin for microscopic observation of cell necrosis, fatty changes and infiltration of Kupffer cells and lymphocytes [11]. The extent of pathological changes in rat liver was graded as normal (0), mild (+), moderate (++), and severe (+++) [12].

2.6. Determination of total phenolic contents of MB

The concentration of total phenolic contents of MB was determined using a modified Folin-Ciocalteu phenol method described by Slinkard and Singleton and an external calibration with gallic acid [13]. Extract solution (1 mg/mL in methanol; 100 |L) and each of the various concentrations of gallic acid (1, 0.5, 0.25, 0.125 and 0.0625 mg/mL in methanol) were pipetted into separate test tubes. Distilled water (2 mL) was added to one of the test tubes and 0.2 mL of Folin-Ciocalteu reagent was added to the rest of each tube and mixed thoroughly. After 4 minutes of incubation, 1 mL of 15% sodium carbonate was added, and the mixture was allowed to stand for 2 hours at room temperature (24-26°C). The absorbance of the mixture was then measured using a spectrophotometer at 760-nm wavelengths. The coefficient of regression, R2 was found to be 0.9997. The concentration of the total phenolic compounds in MB was determined as milligrams of gallic acid equivalent by using an equation obtained from a standard gallic acid curve given as y=2.2257x -0.0106, where y is the absorbance unit and x is the concentration of phenolic compound.

2.7. Statistical analysis

Statistical analysis involved the use of SPSS (SPSS Inc., Chicago, IL, USA). Data were indicated as the

mean ± standard error of the mean and were processed using two-way analysis of variance. Significant differences between the means of the groups were determined using a least significant difference multiple comparisons test. Three levels of probability values were chosen to indicate statistical significance: p < 0.05, p < 0.01 and p < 0.01.

3. Results

3.1. Total antioxidant, DPPH scavenging activities and phenolic content

The TEAC assay was applied to assess the total amount of ABTS radical that can be scavenged by MB. The total antioxidant activity of MB was expressed as mM trolox equivalents. The total antioxidant activity of 1 mg/mL MB was 0.90 mM trolox equivalents. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction in DPPH radical was determined by the decrease of its ab-sorbance at 517 nm by antioxidants. The MB showed a concentration-dependent antiradical activity. EC50 of MB, rutin and quercetin were 2.82 ± 0.019 mg/mL, 0.156 ±0.002 mg/mL and 0.09 ±0.002 mg/mL, respectively. MB contains 10% gallic acid equivalent of phenolic compounds.

3.2. Hepatoprotective activity

CCl4-induced hepatic injury is a commonly used model for hepatoprotective drug screening. Rats treated with a single dose of CCl4 developed significant hepatic damage as assessed from the elevated serum levels of ALT and AST (Table 1). In contrast, groups treated with 1000 mg/kg of MB showed a significant inhibition in the elevation of ALT and AST levels induced by CCl4 induction. The latter result was comparable to that of distilled water plus CCl4 group (p < 0.05 and p< 0.01, respectively). This

Table 1 Effects of different doses of Murdannia bracteata methanol extract (MB) and silymarin on alanine transaminase

(ALT), aspartate transaminase (AST) and malondialdehyde (MDA) levels after challenge with CCl4*

Test sample Dose (mg/kg) Serum ALT level (U/L) Serum AST level (U/L) Plasma MDA (nmol/mL)

Normal 45.3 + 5.6* 139.1 ± 20.8* 1.37+0.08*

Distilled water+CCl4 1195.7+49.3* 1617.3 + 113.4* 2.36+0.21*

Silymarin+CCl4 100 232+38.3*s 565.3 + 39.5*11 1.49+0.15*

MB+CCl4 250 1210.3 + 115.8* 1565.2+79.9* 2.19+0.16s

MB+CCl4 500 828.6+ 130.3* 1295.7+78.3* 1.81+ 0.17s11

MB + CCl4 1000 701.2+ 195.5*# 950.4+ 191.3s1 1.68+0.23#

*Data represented as mean ± standard error of mean; < 0.001 compared to the distilled water and CCl4-treated group; < 0.001

compared to the normal group; §p < 0.01 compared to the normal group; "p < 0.05 compared to the normal group; ^p < 0.01 com-

pared to the distilled water and CCl4-treated group; #p < 0.01 compared to the distilled water and CCl4 treated group.

Table 2 Histopathological effect of Murdannia bracteata methanol extract (MB) and silymarin against CCl4-induced hepatotoxicity

Microscopic observation*

Groups Dose (mg/kg) Necrosis Fatty changes Infiltration of lymphocyte and Kupffer cell

Normal Distilled water+CCl4 Silymarin+CCl4 MB+CCl4 MB + CCl4 MB + CCl4 100 250 500 1000 0 +++ 0 +++ ++ + 0 +++ + +++ ++ + 0 +++ + +++ + +

*0 indicates normal, + indicates mild degree of damage, ++ indicates moderate degree of damage and +++ indicates severe degree of damage.

may suggest that treatment with MB accorded a protection against CCl4-induced increase in serum AST and ALT levels in a dose-dependent manner. The degree of hepatoprotection seemed to increase with the dose of MB (Table 1).

Histopathological observations (Table 2) performed in this study supported the results obtained from the serum enzyme assays. Sinusoidal congestion, broad infiltration of Kupffer cells, ballooning lymphocytic degeneration and loss of cell boundaries were observed in sections from liver of animals administered CCl4 at a dose of 1 mL/kg p.o. Liver necrosis was especially observed in the centrilobular regions. Necrosis bridges to other centrilobular veins were also observed in the CCl4-treated group. Only slight necrosis was observed in the liver tissue of rats treated with MB (500 mg/kg and 1000 mg/kg p.o.) and silymarin (100 mg/kg p.o.) plus CCl4 without extension of necrosis bridges. There was also less infiltration of Kupffer cells and lymphocytes with a reduced degree of ballooning degeneration and fatty accumulation in MB (500 mg/kg and 1000 mg/kg p.o.) and silymarin (100 mg/kg p.o.) plus CCl4 groups.

3.3. Lipid peroxidation inhibition

The results indicated that administration of MB possessed a dose-dependent antilipid-peroxidase activity in plasma (Table 1) and liver tissue. MB inhibited both lipid peroxidation in a concentration-dependent manner. EC50 of MB on lipid peroxidation in liver tissue was 1.785 mg/mL ± 0.01 mg/mL.

4. Discussion

CCl4 is a potent hepatotoxin that produces centrilobular hepatic necrosis and subsequent liver injury. CCl4-induced hepatic injury is commonly used as an experimental method for the study of

hepatoprotective effects of various drugs or medicinal plant extracts [14-16]. CCl4-induced liver injury depends on a toxic agent that has to be metabolized by the liver NADPH-cytochrome P450 enzyme system to a highly reactive intermediate. It has been suggested that this toxic intermediate is the trichlo-romethyl radical (CCl3^) that produces damage to the liver [17,18]. This free radical attacks lipids on the membrane of endoplasmic reticulum, initiating lipid peroxidation, and eventually leads to liver damage [19-22]. Hepatocellular necrosis caused by CCl4 leads to the elevation of serum marker enzymes, namely ALT and AST, which are released from the liver into blood [23-25]. The increased levels of ALT and AST are conventional indicators of liver injury. The present study showed a significant increase in serum ALT and AST levels after exposure to CCl4, thus indicating the likely occurrence of liver damage. The results obtained from the study indicate that MB exhibited hepatoprotective effects against CCl4-induced liver damage in a dose-dependent manner by inhibiting the elevation of hepatic enzymes (namely AST and ALT).

Histopathological observations also support the hepatoprotective potential of MB. In this study, MB exhibited a remarkable concentration-dependent inhibition of lipid peroxidation. MB was further confirmed to have free radical scavenging and antioxidant activities in a concentration-dependent manner. It appears, therefore, that the hepatoprotective activity of MB against CCl4-induced hepatic damage is likely to be due to its antioxidant capacity and free radical scavenging activity.

The hepatoprotective activity of many plants has been attributed to their high phenolic contents [26]. Our spectrophotometrical study revealed that the total phenolic contents of MB were approximately 10%. The phenolic compounds from MB may have contributed towards the antioxidant and hepatoprotective effects of the extract because other

studies have demonstrated that various phenolic compounds contain significant antioxidant and hepa-toprotective activities [26].

In conclusion, we have shown that oral administration of MB was able to inhibit CCl4-induced liver damage in rats. Thus, the present study reveals that MB possesses antioxidant and free radical scavenging activities and has a protective effect against CCl4-induced hepatic damage.

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