Scholarly article on topic 'Antioxidant and hepatoprotective activity of Cordia macleodii leaves'

Antioxidant and hepatoprotective activity of Cordia macleodii leaves Academic research paper on "Chemical sciences"

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{Antioxidant / Hepatoprotective / " Cordia macleodii " / DPPH / "Nitric oxide"}

Abstract of research paper on Chemical sciences, author of scientific article — Naseem N. Qureshi, Bhanudansh S. Kuchekar, Nadeem A. Logade, Majid A. Haleem

Abstract This investigation was undertaken to evaluate ethanolic extract of Cordia macleodii leaves for possible antioxidant and hepatoprotective potential. Antioxidant activity of the extracts was evaluated by four established, in vitro methods viz. 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging method, nitric oxide (NO) radical scavenging method, iron chelation method and reducing power method. The extract demonstrated a significant dose dependent antioxidant activity comparable with ascorbic acid. The extract was also evaluated for hepatoprotective activity by carbon tetrachloride (CCl4) induced liver damage model in rats. CCl4 produced a significant increase in levels of serum glutamate pyruvate transaminase (GPT), serum glutamate oxaloacetate transaminase (GOT), Alkaline Phosphatase (ALP) and total bilirubin. Pretreatment of the rats with ethanolic extract of C. macleodii (100, 200 and 400mg/kg po) inhibited the increase in levels of GPT, GOT, ALP and total bilirubin and the inhibition was comparable with Silymarin (100mg/kg po). The present study revealed that C. macleodii leaves have significant radical scavenging and hepatoprotective activities.

Academic research paper on topic "Antioxidant and hepatoprotective activity of Cordia macleodii leaves"

Saudi Pharmaceutical Journal (2009) 17, 299- 302

SHORT COMMUNICATION

Antioxidant and hepatoprotective activity of Cordia macleodii leaves

Naseem N. Qureshi a,% Bhanudansh S. Kuchekar b, Nadeem A. Logade a, Majid A. Haleem a

a Govt. Aided AIT Institute of Pharmacy, P.O. Box 123, Survey 100, Pharmacy Nagar, Malegaon, Nasik District, MS 423203, India b Govt. College of Pharmacy, Karad Satara District, MS, India

Received 1 February 2009; accepted 23 May 2009 Available online 10 November 2009

KEYWORDS

Antioxidant; Hepatoprotective; Cordia macleodii; DPPH; Nitric oxide

Abstract This investigation was undertaken to evaluate ethanolic extract of Cordia macleodii leaves for possible antioxidant and hepatoprotective potential. Antioxidant activity of the extracts was evaluated by four established, in vitro methods viz. 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging method, nitric oxide (NO) radical scavenging method, iron chelation method and reducing power method. The extract demonstrated a significant dose dependent antioxidant activity comparable with ascorbic acid. The extract was also evaluated for hepatoprotective activity by carbon tetrachloride (CCl4) induced liver damage model in rats. CCl4 produced a significant increase in levels of serum glutamate pyruvate transaminase (GPT), serum glutamate oxaloacetate transaminase (GOT), Alkaline Phosphatase (ALP) and total bilirubin. Pretreatment of the rats with ethanolic extract of C. macleodii (100, 200 and 400 mg/kg po) inhibited the increase in levels of GPT, GOT, ALP and total bilirubin and the inhibition was comparable with Silymarin (100 mg/kg po). The present study revealed that C. macleodii leaves have significant radical scavenging and hepatoprotective activities.

© 2009 King Saud University. All rights reserved.

1. Introduction

Reactive oxygen species (ROS) including free radicals such as superoxide anion radicals (Oj'), hydroxyl radicals (OH')

* Corresponding author.

E-mail address: prin_nqureshi@rediffmail.com (N.N. Qureshi).

1319-0164 © 2009 King Saud University. All rights reserved. Peerreview under responsibility of King Saud University. doi:10.1016/j.jsps.2009.10.007

non-free radicals such as H2O2, Singlet Oxygen (1O2) along with various forms of active oxygen are involved in various physicochemical processes in the body and aging (Finkel and Holbrook, 2000). Free radicals are implicated in a large number of chronic degenerative diseases, inflammation, cataract, atherosclerosis, rheumatism, arthritis, ischemia, etc. (Droge, 2002). Free radical mechanism is one of the most important mechanisms of liver damage.

Metabolism of certain drugs like paracetamol, produce free radicals, which cause liver damage.

Antioxidants may offer resistance against oxidative stress by scavenging free radicals, inhibiting lipid peroxidation and by other mechanisms and thus prevent disease (Miller and Rice-Evans, 1997).

Cordia macleodii (Boraginaceae) commonly known as Dah-iphalas (Hindi) is a tree having broad ovate, glabrous green leaves with crenate margins. Preliminary phytochemical analysis of C. macleodii leaves indicated the presence of relatively high levels of flavonoids. Several flavonoids are reported to possess antioxidant and hepatoprotective properties (Alan and Miller, 1996), hence the present investigation was undertaken to determine the antioxidant and hepatoprotective potential of C. macleodii leaves.

2. Materials and methods

Fresh leaves of C. macleodii were collected in Nandurbar district, Maharashtra, India and were authenticated by Botanical Survey of India, Pune. The leaves were shade dried and crushed to make coarse powder. The powder (250 g) was extracted with 3 l of ethanol (95%v/v) by continuous extraction method for 48 h. Solvent was distilled off and the extract was concentrated and dried under reduced pressure, which yielded a brownish green mass. The extract was preserved at 2-4 0C. The extract and Silymarin were suspended in distilled water with 1% Tween 80 and administered to albino rats in different doses.

Healthy albino rats (120-150 g) of either sex were procured from National Toxicology Center, Pune MS, India and were kept in standard plastic animal cages in groups of five animals with 12 h of light and dark cycle. The rats were fed on standard rat feed and provided water ad libitum. Prior to initialization of experimentation the animals were acclimatized to laboratory conditions for a week. The experiments were carried out according to guidelines of 'Committee for Prevention and Control of Scientific Experimentation on Animals' (CPCSEA) New Delhi, and the procedures were approved by Institutional Animal Ethics Committee (IAEC), Govt. College of Pharmacy, Karad, Maharashtra, India.

2.1. Antioxidant activity

The antioxidant activity was determined by four established methods. Diphenyl picryl hydrazyl (DPPH), radical scavenging method, nitric oxide (NO) radical scavenging method, iron chelation method and reducing power method.

DPPH radical scavenging activity was measured by spectro-photometric method (Blois, 1958). One milliliter of ethanolic solution of the extract of various concentrations (25-800 ig/ ml) were mixed with 1 ml of ethanolic solution of DPPH (200 iM). Similarly 1 ml ethanolic solutions of ascorbic acid of various concentrations (25-400 ig/ml) were mixed with 1 ml of DPPH solution. A mixture of 1 ml of ethanol and 1 ml of ethanolic solution of DPPH (200 iM) served as control. After mixing, all the solutions were incubated in dark for 20 min and then absorbance was measured at 517 nm. The experiments were performed in triplicate and % scavenging activity was calculated by using the formula:

% Scavenging =

absorbance of control — absorbance of test absorbance of control

Nitric oxide radical scavenging activity was measured by using Griess' reagent (Green et al., 1982). Different concentrations of the ethanolic extract (25-800 ig/ml) in standard

phosphate buffer solution (pH 7.4) were incubated with an equal volume of sodium nitroprusside solution (5 mM) in standard phosphate buffer (pH 7.4) at 25 0C for 5 h. In an identical manner solutions of different concentrations of ascorbic acid (25-400 ig/ml) in standard phosphate buffer (pH 7.4) were also incubated with an equal volume of sodium nitroprusside solution (5 mM) in standard phosphate buffer (pH 7.4). Control experiments without the test compound but with equivalent amount of buffer were also conducted. After incubation, 0.5 ml of the incubation mixture was mixed with 0.5 ml of Griess' reagent (sulphanilamide 1%, o-phos-phoric acid 2% and naphthyl ethylene diamine dihydrochlo-ride 0.1%) and the absorbance was measured at 546 nm. From the absorbance the % scavenging activity was calculated using the above formula. The experiments were performed in triplicate.

Iron chelating activity is also a measure of antioxidant activity (Benzie and Strain, 1996). Solutions of extract (2 ml in 5% v/v methanol) of different concentrations (25-800 ig/ ml) and solutions of ascorbic acid (2 ml in 5% v/v methanol) of different concentrations (25-400 ig/ml) were incubated with methanolic o-phenanthroline solution (1 ml, 0.05%) and ferric chloride solution (2 ml, 200 iM) at ambient temperature for 10 min. After incubation, the absorbance of solutions was measured at 510 nm. The experiments were performed in triplicate.

Reducing power of the extract was also evaluated by Oyai-zu method (Oyaizu, 1986). 2.5 ml of solutions of different concentrations of the extract (25-800 ig/ml) in standard phosphate buffer solution (pH 6.6) were incubated with 2.5 ml of potassium ferricyanide solution (1% w/v) at 50 0C for 20 min. In an identical manner solutions of different concentrations of ascorbic acid (25-400 ig/ml) were also incubated. After incubation, 2.5 ml of 10% trichloro acetic acid solution was added to each of the tubes and the mixture was centrifuged at 650 rpm for 10 min. Five milliliters of the upper layer solution was mixed with 5 ml of deionized water and 1 ml of ferric chloride solution (1% w/v) and the absorbance was measured at 700 nm.

Hepatoprotective activity: the hepatoprotective activity of C. macleodii leaves extract was determined by using carbon tetrachloride induced heptotoxic rat model (Baheti et al., 2006). Rats were divided in six groups each comprising of five rats. Before treatment, the rats were fasted overnight with free access to water. Group I served as vehicle control and received Tween 80 in distilled water 1% v/v (5 ml/kg, po) for 7 days. Group II served as toxic control and was administered vehicle (5 ml/kg po) daily and carbon tetrachloride in olive oil 1:1 v/v (0.7 ml/kg, ip) on alternate days for 7 days. Group III served as standard control and was administered Silymarin (100 mg/ kg, po, daily) along with carbon tetrachloride in olive oil 1:1 v/v (0.7 ml/kg, ip) on alternate days for 7 days. Groups IV, V and VI were, respectively, administered ethanolic extract of C. macleodii (100, 200 and 400 mg/kg, po) daily and carbon tetrachloride in olive oil 1:1 v/v (0.7 ml/kg, ip) was administered on alternate days for 7 days. At the end of treatment, blood was withdrawn under light ether anesthesia by retro orbital cannulation and the rats were dissected to isolate liver. The blood samples after coagulation were centrifuged and the sera isolated were used for estimation of the biochemical markers of liver damage viz. GOT, GPT, ALP and bilirubin levels.

Antioxidant and hepatoprotective activity of Cordia macleodii leaves

2.2. Statistical analysis

All data obtained were analyzed by ANOVA followed by Students' t test. Values at P < 0.001 were considered significant.

3. Results

Alcoholic extract of C. macleodii leaves in graded concentrations was tested for antioxidant activity in four different in vitro models. It was observed that the test compounds scavenged free radicals in a concentration dependent manner in the models studied. Maximum percentage inhibition of DPPH by the extract was 81.20% at 800 ig/ml concentration (Table 1). Standard drug ascorbic acid showed 90.91% inhibition of the DPPH radical at 400 ig/ml.

In the nitric oxide model, the maximum percentage inhibition of nitric oxide radicals by C. macleodii was 72.70% at 800 ig/ml (Table 1). Ascorbic acid at 200 ig caused 94.97% inhibition.

The reducing power of C. macleodii extract was dose dependent and is shown in Table 1. The maximum absorbance of C. macleodii extract at 800 ig/ml is comparable with ascorbic acid 200 ig/ml.

The iron chelation of C. macleodii extract was also dose dependent and is shown in Table 1. C. macleodii at 800 ig/ ml produced maximum absorbance of 0.433 while ascorbic acid at 400 ig/ml produced maximum absorbance of 0.330.

In the hepatoprotective study, after treatment with CCl4, a significant increase in levels of GOT, GPT, ALP, bilirubin and also an increase in liver weight as compared to the normal control were observed suggesting the liver toxicity. While in the groups of rats pretreated with Silymarin the levels of GOT, GPT, ALP and Bilirubin were significantly lower. The groups of rats pretreated with C. macleodii extract, demonstrated dose dependent inhibition of elevation of the biochemical parameters. C. macleodii at a dose of 400 mg/kg showed inhibition of elevation of the biochemical parameters comparable with Silymarin (100 mg/kg). Also the liver weight was significantly reduced in Silymarin and C. macleodii treated groups (Table 2).

4. Discussion

In the present study four methods were used for evaluation of antioxidant activity. The first two methods were for direct

measurement of radical scavenging activity and the remaining two evaluated the reducing power and iron chelation activity. C. macleodii demonstrated good radical quenching activity against both DPPH and the nitric oxide radicals. DPPH is a relatively stable free radical which when encounters proton donors such as antioxidants, it gets quenched and the absorbance decreases (Wu et al., 2003). Results indicated definite scavenging activity of the extract towards DPPH radicals in comparison with ascorbic acid.

Nitric oxide is a free radical produced in the mammalian cells and is involved in regulation of various physiological processes. However, excess production of nitric oxide is associated with several diseases like adjuvant arthritis, cancer etc. (Gibanananda and Sayed, 2002; Ialenti et al., 1993). NO free radical scavenging activity of the extracts was studied by using Griess reagent. C. macleodii ethanolic extract was found to scavenge the NO free radical dose dependently.

Reducing power and iron chelation methods indirectly evaluate the antioxidant activity. Iron chelation method evaluates the reducing power. o-Phenanthroline is a selective chelating agent for ferrous ion. It is used for determination of extent of reduction of ferric ions to ferrous ions by antioxidants. C. macleodii extract showed dose dependent increase in absor-bance, which indicates conversion of ferric ions to ferrous ions. In reducing power method as well, a dose dependent increase in absorbance is indicative that the extract is capable of donating hydrogen atoms.

C. macleodii demonstrated dose dependent anti-oxidant activity comparable with ascorbic acid. In all the methods, maximum antioxidant activity was found at the dose of 800 ig/ml of C. macleodii extract.

CCl4 is one of the most commonly used hepatotoxin in the experimental study of liver disease. The hepatotoxic effects of CCl4 are largely due to generation of free radicals (Shenoy et al., 2001). CCl4 is biotransformed by the cytochrome P450 system to produce the trichloromethyl free radicals, which in turn covalently bind to cell membranes and organelles to elicit lipid peroxidation (Recknagel et al., 1989). Several plants, e.g. Foeniculum vulgare (Ozbek et al., 2003) and Panax notoginseng (Yoshikawa et al., 2003) have been tested for their efficacy in controlling the CCl4 induced liver damage. Further it has been evident that several phytoconstituents have the ability to induce microsomal enzymes either by accelerating the excretion of CCl4 or by inhibition of lipid peroxidation induced by

Table 1 Antioxidant activity of ethanolic extract of C. macleodii and ascorbic acid.

Treatment group % Inhibition (mean ± SEM) Absorbance (mean ± SEM)

DPPH NO Iron chelation Reducing power

C. macleodii (800 ig/ml) 81.20 ± 1.31 72.70 ± 0.44 0.433 ± 0.017 1.53 ± 0.02

C. macleodii (400 ig/ml) 72.73 ± 1.41 37.69 ± 0.87 0.263 ± 0.009 0.80 ± 0.01

C. macleodii (200 ig/ml) 51.20 ± 1.97 17.92 ± 0.60 0.153 ± 0.003 0.40 ± 0.003

C. macleodii (100 ig/ml) 45.47 ± 1.83 07.33 ± 0.44 0.086 ± 0.003 0.20 ± 0.006

C. macleodii (50 ig/ml) 39.40 ± 2.34 02.70 ± 0.17 0.057 ± 0.009 0.073 ± 0.007

C. macleodii (25 ig/ml) 36.67 ± 1.11 01.16 ± 0.44 0.027 ± 0.003 0.026 ± 0.007

Ascorbic acid (400 ig/ml) 90.91 ± 0.53 - 0.330 ± 0.026 -

Ascorbic acid (200 ig/ml) 87.57 ± 1.98 94.97 ± 1.90 0.153 ± 0.007 1.51 ± 0.032

Ascorbic acid (100 ig/ml) 81.52 ± 1.61 54.27 ± 0.77 0.11 ± 0.006 0.66 ± 0.030

Ascorbic acid (50 ig/ml) 68.18 ± 2.93 29.48 ± 0.73 0.063 ± 0.003 0.29 ± 0.01

Ascorbic acid (25 ig/ml) 47.88 ± 2.64 18.43 ± 0.89 0.033 ± 0.003 0.123 ± 0.009

Values represent the mean ± SEM; number of readings in each group = 3.

Table 2 Effect of ethanolic extract of C. macleodii and Silymarin rats. on serum biochemical parameters in CCl4 induced liver damage in

Treatment Serum biochemical parameters Liver weight (g)

GPT (U/l) GOT (U/l) ALP (U/l) Bilirubin (mg/dl)

Vehicle CCl4 Silymarin C. macleodii 100 C. macleodii 200 C. macleodii 400 22 ± 1.30 241 ± 18.93* 43 ± 1.82** 77.6 ± 3.77** 54.2 ± 4.54** 23.2 ± 2.87** 31.2 ± 1.39 294 ± 8.86* 39.6 ± 3.97** 73.8 ± 4.91** 44.6 ± 2.4** 25 ± 1.90** 161.4 ± 3.53 456.6 ± 20.05* 180 ± 23.73** 218.6 ± 12.63** 167.6 ± 9.19** 81 ± 9.69 ** 0.58 ± 0.04 1.28 ± 04* 0.74 ± 0.05** 0.82 ± 0.04** 0.76 ± 0.05** 0.48 ± 0.04** 5.96 ± 0.18 10.68 ± 0.32* 6.86 ± 0.44** 8.52 ± 0.44 7.3 ± 0.44** 6.38 ± 0.32**

Values represent the compared with CCl4 mean ± SEM; number of mice used in each group treated group. = 5; *P < 0.001 compared with the vehicle treated group, **P < 0.001

CCl4. Phytoconstituents like flavonoids and triterpenoids are known to possess hepatoprotective activity (Baek et al., 1996).

Damage of liver cell is reflected by an increase in the levels of hepatospecific enzymes, these are cytoplasmic and are released in to circulation after cellular damage (Sallie et al., 1991). In this study significant increase in the total bilirubin content and in the GOT, GPT and ALP activities in the CCl4 treated group could be taken as an index of liver damage. Treatment with C. macleodii extract inhibited CCl4 induced increase in total bilirubin and GOT, GPT and ALP activities as compared with CCl4 treated group. The CCl4 induced a significant increase in liver weight, which is due to blocking of secretion of hepatic triglycerides in plasma (Aniya et al., 2005). Silymarin and the extract prevented the increase of liver weight in rats.

In this study the alcoholic extract of C. macleodii was found to have antioxidant and hepatoprotective activities. It is reported that flavonoids and triterpenoids have antioxidant and hepatoprotective actions (Alan and Miller, 1996; Xiong et al., 2003). Chemical examination of the extract of C. macleodii showed the presence of flavonoids and triterpenoids. These suggest that the antioxidant activity and protective effects against CCl4 induced hepatic cell injury of the extract could be due to the flavonoids and triterpenoids present in C. macle-odii. Further studies are required to establish the phytoconstit-uents responsible for the antioxidant and hepatoprotective actions.

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