Scholarly article on topic 'In vitro antioxidant and hepatoprotective potential of chenopodium album extract'

In vitro antioxidant and hepatoprotective potential of chenopodium album extract Academic research paper on "Chemical sciences"

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Academic research paper on topic "In vitro antioxidant and hepatoprotective potential of chenopodium album extract"

3 In vitro antioxidant and hepatoprotective potential ^ of chenopodium album extract

Ashish Baldi, Naveen Kumar choudhary

Department of Phytopharmacology, Mandsaur Institute of Pharmacy, Mandsaur, Madhya Pradesh, India

Introduction: Chenopodium album (Bathua), a native Indian herb, has been used for treatment of abdominal pain, eye disease, throat troubles and cardiovascular disorders. The present study was carried out to explore antioxidant and hepatoprotective efficacy of C. album extract against carbon tetrachloride (CCl4) induced hepatotoxicity in rats. Materials and Methods: The ethanolic extract of C. album seeds was evaluated for in vitro antioxidant activity using three established models (diphenyl picryl hydrazyl radical scavenging method, 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical cation decolorization assay and nitric oxide radical scavenging method) and hepatoprotective activity was also assessed against CCl4 induced hepatic damage in rats at the doses of 300 mg/kg and 450 mg/kg. Results: C. album extract was found to exhibit excellent antioxidant and free radical scavenging activity, when compared with ascorbic acid during in vitro studies. C. album extract at a dose of 450 mg/kg showed inhibition of elevation of the biochemical parameters comparable with silymarin (100 mg/kg) and hence shown remarkable protection in hepatic injury induced by free radicals generated due to administration of CCl4 as toxicant. The observations were also supplemented with histopathological examination of rat liver sections. Conclusion: This study establish scientific evidences to folklore use of C. album in treatment of hepatic disorders and also suggests that free radical scavenging and antioxidant activities of phytoconstituents may be the possible mechanisms of its hepatoprotective potential.

Key words: 2,2' - azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), carbon tetrachloride, diphenyl picryl hydrazyl, free radical scavenging, nitric oxide, silymarin


The importance of free radicals and reactive oxygen species (ROS) has attracted increasing attention of researchers involved in the field of medical science. Production of ROS is normal but essential process during cell metabolism to carry out important physiological functions.[1] Yet, overproduction and imbalance between production and utilization of free radicals results in oxidative stress. ROS including free radicals such as superoxide anion, hydroxyl, non-free radicals like H2O2 and singlet oxygen along with various forms of free oxygen species are involved in hepatocellular injury and related disorders, chronic degenerative diseases, inflammation, cataract, atherosclerosis, rheumatism, arthritis and ischemia etc.[2,3]

commonly used medicines like paracetamol, diclofenac, etc., alcoholism, and certain disease state have been reported to affect liver functioning. The major clinical manifestation of liver disorder is jaundice. Despite of the excellent regeneration capacity of this organ, a slight injury or toxicity may lead to fatal complications. Therefore, being a vital organ, its protection has a special status in therapeutics.[4]

Liver, an important organ actively involved in metabolic functions, is a frequent target of number of toxicants.[5] Modern allopathic treatment does not hold promise to cure liver disease perfectly. Several synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene are available, but are quite unsafe and their toxicity is a problem of concern.[6] However, traditional system of Indian medicines, i.e., Ayurveda recommended a number of medicinal preparations for the treatment of liver disorders based on indigenous plants and their extracts.[7] Therefore, natural products and their active principles as sources for new drug discovery and treatment of diseases have attracted attention. Foods rich in antioxidants offer resistance against oxidative stress by scavenging free radicals, inhibiting lipid peroxidation and by other mechanisms and thus prevent and cure liver damage.[8] Thus, the efficacy of the drug would be preventive and passive for defending against damages.[9]

Prolonged exposure to certain xenobiotic, pollutants, long term drug therapy, excessive use of some of the

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Address for correspondence: Dr. Ashish Baldi, Mandsaur Institute of Pharmacy, Rewas Dewra Road, Mandsaur - 458 001, Madhya Pradesh, India. E-mail: Received: 26-12-2011; Accepted: 15-02-2013

Carbon tetrachloride (CCl4) is a widely used experimental hepatotoxicant to study hepatoprotective potential of test drugs/preparation as the changes associated with CCl4 induced liver damage has been appeared very similar to the acute viral hepatitis.[10] The principle causes of CCl4 induced hepatic damage are lipid peroxidation and decreased activities of antioxidant enzymes and generation of free radicals.[1112] The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against hepatic damage.[9]

In recent years, a number of plants have been shown to possess hepatoprotective property by improving antioxidant status but absence of detailed and systematic scientific studies for scientific validation of their therapeutic potential remains a major area of concern for their acceptance by pharmaceutical industries.[13] Therefore, present study was carried out to explore antioxidant and hepatoprotective efficacy of Chenopodium album extract against CCl4 induced hepatotoxicity in animal model.

C. album, commonly known as Bathua or Goose foot, is well distributed in India and usually found as weed in early grain fields in Madhya Pradesh, Rajasthan, Punjab, Kashmir, Sikkim, Bengal, and Mumbai.[14] The plant mainly contains flavonoids, alkaloids and tannins and is reported to improves appetite, have laxative, diuretic, anthelmintic effect. The plant is also known to be useful in abdominal pain, eye disease, throat troubles and cardiovascular disorders.[15] Several flavonoids and alkaloids are reported to possess antioxidant and hepatoprotective properties.[16] Hence, present investigation was undertaken to determine the antioxidant and hepatoprotective potential of C. album seeds.


Plant Materials

The seeds of C. album were collected from local field of Mandsaur (M.P.) and authenticated by Dr. Rakesh Gupta, Department of Dravyaguna, Smt. Dhairya Prabha Devi Sojatia Ayurved Medical College, Bhanpura. Voucher specimen was deposited to herbarium of SDPS Ayurved Medical College, Bhanpura vide specimen no. SDPS/09/ PS/114.


The air dried seeds were powdered in an end runner mil for 18 h. The powder (250 g) was passed through 100 mesh size sieve and defatted with petroleum ether (60-80°) for 48 h. The defatted powder was allowed to dry at 25 ± 2°C in order to remove traces of petroleum ether. The defatted seeds powder was then subjected to continuous extraction using soxhlet apparatus with ethanol (95% v/v) as a menstrum. After which, ethanol was distilled off and the extract

so obtained was concentrated and dried under reduced pressure. The dried extract was 5.8% w/w.

Phytochemical Study

The extract was analyzed for presence of various phytochemicals viz. carbohydrates, alkaloids, glycosides, phenolics and flavanoids by performing qualitative analysis.[17]

Animal Studies

Institutional Animal Ethical Committee (IAEC) of the institute approved the study protocol (Mandsaur Institute of Pharmacy/IAEC/11/1019/C/06/010) and was in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals. Three month old Wistar Albino rats (150-200 g) of either sex were obtained from Animal House, Mandsaur Institute of Pharmacy, Mandsaur (M.P.). They were maintained under standard laboratory conditions at 25 ± 2°C, relative humidity (50 ± 15%) and normal photoperiod (12-h light/ dark cycle) were used for the experiment. Commercial pellet diet (Nav Maharastra Chakan Oil Mills Ltd., New Delhi, India) and water were provided ad libitum throughout the course of study.

For induction of hepatic injury, CCl4 was obtained from Suvidhinath Lab, Baroda and standard hepatoprotective drug, silymarin, was procured from Microlabs Ltd., Bengaluru, Karnataka.

Antioxidant Activity

The antioxidant activity was determined by three established methods, diphenyl picryl hydrazyl (DPPH) radical scavenging method, ABTS radical cation decolorization assay and nitric oxide (NO) radical scavenging method.

DPPH radical scavenging activity was measured by spectrophotometric method.[18] For the present study, the samples were prepared in different concentrations, i.e., 5-100 |ig/ml in methanol. The ethanolic extract of C. album (samples) at above concentrations was mixed with 3 ml of 100 |iM DPPH prepared in methanol and final volume was made up to 4 ml with methanol. The absorbance of the resulting solutions was recorded in triplicate after 20 min at 25 ± 2oC against ascorbic acid. The disappearance of color was read spectrophotometrically at 517 nm using a Shimadzu visible spectrophotometer. Percent scavenging was calculated by following equation:

% Scavenging = 100 x [Absorbance (Blank) -Absorbance (Sample)/Absorbance (Blank)]

From obtained Radical Scavenging Capacity (RSC) values, the half maximal inhibiting concentration (IC50) were

calculated, which represents concentration of scavenging compound that caused 50% neutralization.

ABTS radical cation (ABTS+) was produced by reacting ABTS solution (7 mM) with 2.45 mM ammonium persulfate and the mixture was allowed to stand in dark at 25 ± 2°C for 12-16 h before use. For this study, different concentrations (5-100 |ig/ml) of the ethanolic extracts (2 ml) were added to 1.2 ml of ABTS solution and final volume was made up with ethanol to 4 ml. The absorbance was read at 745 nm and the experiment was performed in triplicate.[19]

NO was generated from sodium nitroprusside and measured by Griess' reaction.[20,21] Sodium nitroprusside (5 mM) in standard phosphate buffer saline solution (0.025 M, pH: 7.4) was incubated with different concentrations (5-100 |ig/ml) of ethanolic extracts dissolved in phosphate buffer saline and the tubes were incubated at 25 ± 2°C for 5 h. Control experiments without the test compounds but with equivalent amounts of buffer were conducted in an identical manner. After 5 h, 0.5 ml of incubation solution was removed and diluted with 0.5 ml of Griess' reagent (1% sulphanilamide, 2% O-phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride). The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and its subsequent coupling with napthyl ethylenediamine was recorded at 546 nm. The experiment was repeated in triplicate.[22]

Hepatoprotective Activity

The CCl4 induced hepatotoxic rat model was used to determine the hepatoprotective activity of C. album extract.[23] The rats were divided into the following five groups each containing 6 rats (n = 6): Group I: Control rats: Tween 80 in distilled water

1% v/v (5 ml/kg, p.o.) for 7 days Group II: Toxic control rats: Administered vehicle (5 ml/kg p.o.) daily + CCl4 in olive oil 1:1 v/v (0.7 ml/kg, ip) on alternate days for 7 days Group III: Reference rats: Treated with silymarin 100 mg/kg + CCl4 in olive oil 1:1 v/v (0.7 ml/kg, ip) on alternate days for 7 days Group IV: C. album treated rats: Received ethanolic extract of C. album seeds 300 mg/kg body weight

p.o. + CCl4 in olive oil 1:1 v/v (0.7 ml/kg, ip) on alternate days for 7 days Group V: C. album treated rats: Treated with ethanolic extract of C. album seeds 450 mg/kg body weight p.o. + CCl4 in olive oil 1:1 v/v (0.7 ml/kg, ip) on alternate days for 7 days.

After 24 h of last treatment, rats were anesthetized with ether and blood samples from each animal of all groups were collected by retro-orbital plexus puncture in sterilized centrifuge tubes and the rats were then dissected to isolate liver. The blood samples were then allowed to coagulate at 30°C for 45 min and serum portion was separated from each sample by centrifugation at 25,000 g at 30°C for 10 min and subjected to biochemical investigation to assess liver function on the basis of total bilirubin, serum aminotransferase (alanine and aspartate) and alkaline phosphatase.[24] Total protein was estimated as per the method of Lowry et al.[25]

Statistical Analysis

The results are expressed as means ± standard deviation (SD) and values were calculated for each group. A one way analysis of variance (ANOVA) followed by Dunnet's test for significance analysis using Graph Pad Prism software. The minimum level of significance was set of P < 0.05.


The results of preliminary phytochemical screening revealed the presence of glycosides, phenolic compounds and flavonoids.

Alcoholic extract of C. album seeds in graded concentrations (5, 25, 50, 75 and 100 |ig/ml) was tested for antioxidant activity in three 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 38.78% at 100 |ig/ml concentration [Table 1]. Standard drug, i.e., ascorbic acid showed 48.59% inhibition of the DPPH radical at 50 ^g/ml.

In ABTS radical cation method, maximum absorbance of C. album extract at 100 ^g/ml level was comparable

Table 1: Antioxidant activity of ethanolic extract of Chenopodium album'*

Concentration (Mg/ml) DPPH ABTS NO

C. album Ascorbic acid C. album Ascorbic acid C. album Ascorbic acid

5 8.23±1.12 22.32±1.43 9.54±0.79 12.97±0.99 10.79±0.69 22.11±0.23

25 14.14± 1.19 36.71±1.31 26.43±1.17 38.72±1.38 13.22±0.87 30.29±0.39

50 24.67±0.98 48.59±1.09 34.32±2.12 49.49±1.33 20.43±0.56 45.89±0.56

75 32.32±1.65 54.65±1.72 51.77±1.75 65.89±1.61 27.21±0.99 55.76±0.59

100 38.78±1.25 58.21±1.11 63.34±1.43 76.97±1.32 35.96±0.74 63.67±0.69

*The values given are mean of triplicate readings in different experiments, "All results are statistically significant with P<0.05 and values represents the mean±SEM; DPPH - Diphenyl picryl hydrazyl; NO - Nitric oxide; C. album - Chenopodium album; ABTS -2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)

with ascorbic acid (75 ^g/ml). The extract shows free radical scavenging activity in dose dependent manner as shown in Table 1. In NO model, maximum percentage inhibition of NO radicals by C. album extract was 35.96% at a concentration of 100 |ig/ml [Table 1]. Ascorbic acid at 50 |ig/ml level caused 45.89% inhibition.

In the hepatoprotective study, after treatment with CCl4, a significant increase in levels of AST, ALT, ALP, total bilirubin and in liver weight, as compared to the normal control, were observed suggesting the liver toxicity and the decrease in total protein confirmed the liver toxicity. Levels of biochemical parameters were significantly lower in the rats pre-treated with silymarin. The groups of rats pre-treated with C. album extract, demonstrated dose dependent inhibition of elevation of the biochemical parameters. C. album extract, at a dose of 450 mg/kg, showed comparable inhibition of increase in biochemical parameters with reference to silymarin (100 mg/kg). Also the liver weight was significantly reduced in silymarin and C. album treated groups [Table 2].

Histopathological Studies

The involvement of free radicals in the pathogenesis of liver injury has been investigated for many years by using acute poisoning with CCl4. CCl4 an extensively studied liver toxicant, and its metabolites such as trichloromethyl peroxy radical (CCl4.O2) are known to be involved in the pathogenesis of liver damage. It is bio-transformed by the cytochrome P-450 system to produce the trichloromethyl free radical, which in turn covalently binds to cell membranes and organelles to elicit lipid peroxidation, disturb Ca2+ haemostasis and finally result in cell death.[26]

The effect of C. album (300 and 450 mg/kg) and silymarin on liver histopathology of CCl4 treated rats are presented in Figure 1. As seen in Figure 1a, liver section of normal rat showed distinct hepatic cells with well-preserved cytoplasm, prominent nucleus and nucleolus. CCl4 induced liver damage can be observed directly in Figure 1b, where the section showed massive fatty change, necrosis, lymphocyte infiltration, the loss of cellular boundaries,

and join together of nucleus. Figure 1c presents the rat liver section treated with silymarin and CCl4. In 300 mg/kg C. album and CCl4 treated group, section suggested moderate degree of damage, with some fatty change, necrosis, and lymphocyte infiltration [Figure 1d]. Figure 1e shows the section of group treated with high dose C. album (450 mg/kg) and CCl4, sections of these two groups were nearly comparable to the normal group, with no obvious necrosis was observed.


In the present study, three widely used methods for evaluation of antioxidant activity viz. DPPH, ABTS and NO assays were applied. C. album showed good radical scavenging activity against both DPPH and ABTS radicals. DPPH is a relatively stable free radical which when encounters proton donors such as antioxidants, it gets quenched and the absorbance decreases.[27] Results indicated definite scavenging activity of the extract towards DPPH radicals in comparison with ascorbic acid. The ABTS decolourization assay is based on the principle of inhibition/ decrease in the absorbance of the radical cation (ABTS+). This chemical reaction results in direct generation of ABTS radical mono cation prior to addition of antioxidant components instead of in presence of antioxidant.

NO is a free radical produced in the mammalian cells and is involved in regulation of various physiological processes. However, excess production of NO is associated with several diseases like adjuvant arthritis, cancer etc.[2829] NO free radical scavenging activity of the extracts was studied by using Griess reagent. C. album ethanolic extract was found to scavenge the NO free radical dose dependently.

C. album demonstrated dose dependent anti-oxidant activity comparable with ascorbic acid. Furthermore the results were presented in terms of % scavenging capacity as compared to initial concentration taken of free radicals. As the tests were carried out to investigate antioxidant potential of the extract at preliminary levels, i.e., maximally up to 100 |ig/ml, increasing the concentration would surely resulted in

Table 2: Effect of ethanolic extract of Chenopodium album on biochemical parameters

Groups Biochemical parameters Liver

Aspartate Alanine Alkaline Total bilirubin Total protein weight (g)

aminotransferase (IU/l) aminotransferase (IU/l) phosphatase (IU/l) (mg/dl) (gm/dl)

Control 45.20±1.24 144.6±0.42 156.2±1.58 0.53±0.05 7.37±0.18 5.96±0.18

Toxic control, carbon 243.5±2.70* 396.8±0.43* 388.4±9.4* 3.60±0.15* 4.49±0.09 9.62±0.32


Reference, silymarin 109.9±1.11# 204.2±1.27# 164.8±1.24# 1.15±0.08# 7.08±0.23 6.35±0.27

(100 mg/Kg)

C. album, 300 mg/Kg 134.5±3.6# 258.2±1.46# 202.2±6.2# 2.6±0.17# 5.56±0.12 8.06±0.28

C. album, 450 mg/Kg 114.0±0.99# 230.5±2.12# 170.6±2.2# 1.62±0.04# 6.30±0.16 7.54±0.17

n=6; *P<0.05 compared to normal; #P<0.05 compared to toxic control treated group; C. album - Chenopodium album

Figure 1: Effect of C. album and silymarin on the liver histopathology of the carbon tetrachloride treated rat. (a) Liver section of control rat; (b) Liver section of carbon tetrachloride treated rat, toxic control; (c) Liver section of silymarin (100 mg/kg) treated rat; (d) Liver section of rat treated with C. album (300 mg/kg) and (e) Liver section of rat treated with C. album (450 mg/kg)

greater scavenging potential. C. album extract used in present study had shown antioxidant potential in order of ABTS > DPPH > NO. The scavenging capacity of the extract was compared to ascorbic acid as standard. It was found that the IC50 values of C. album extract was 72.46 |ig/ml and 143.63 |ig/ml for ABTS and DPPH models, which were quite comparable with ascorbic acid having respective value of 50.77 |ig/ml and 54.41 |ig/ml, respectively.

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.[30] CCl4 is metabolized to trichloromethyl free radicals by the cytochrome P450 system.[31] These free radicals then covalently bind to macromolecules of cell membranes and organelles to elicit lipid peroxidation, which will cause the loss of integrity of cell membrane.[32] Several plants, e.g. Cordia macleodii[16] and Panax notoginseng[33] have been tested for their efficacy in controlling the CCl4 induced liver damage.

Liver damage is always associated with cellular necrosis, increase in tissue lipid peroxidation and deplection in the tissue glutathione (GSH) levels. In addition, serum levels of many biochemical markers like serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), cholesterol, bilirubin, alkaline phosphate are elevated. One of the

earlier study on Launaea intybacea plant also revealed that aqueous extract significantly reduced serum bilirubin, SGOT, SGPT and alkaline phosphatase (ALP) levels and liver homogenates lipid peroxidase (LPO), superoxide dismutase (SOD), catalasae (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and increases GSH levels, suggesting hepatoprotective activity. Phytoconstituents like flavonoids and triterpenoids are known to possess hepatoprotective activity.[34] Our phytochemical study also reveals the presence of glycosides, flavonoids and phenolics. These suggest that the antioxidant activity and protective effects against CCl4 induced hepatic cell injury of the extract could be due to the flavonoids, glycosides and phenolics present in C. album. Liver damage can be assessed by biochemical studies. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are present in high concentration in hepatic cells, which get released out into circulation due to the hepatic injury.[35] In present study, significant increase in the total bilirubin, AST, ALT and ALP activities in the CCl4 treated group could be taken as an index of liver damage. Treatment with C. album extract inhibited CCl4 induced liver damage by increase in total bilirubin, AST, ALT 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.[36] Silymarin and the extract prevented the increase of liver weight in rats.

As observed in our experiment, administration of CCl4 led to the elevation of total bilurubin, AST, ALT and ALP levels in serum and increase in weight of liver while decrease in total protein content, indicating significant liver damage. Pre-treatment with C. album had reversed these trends towards normalizations, reflecting that C. album possesses potent hepatoprotective activity in vivo. Polyphenols and glycosides present in the ethanolic extract of C. album were reported for excellent antioxidant potential.1371 A glycoside namely chenoalbuside from alcoholic extract of C. album was also reported to exhibit antioxidant potential.1381 The antioxidant activity of these phytoconstituents are mainly owing to their redox properties, i.e., the ability to act as reducing agents, hydrogen donors and singlet oxygen quenchers, and to some extent, could also be due to their metal chelation potential.1391 Phytoconstituents like flavonoids and phenolics are already known to possess hepatoprotective potential by inhibiting xenobiotic induced hepatotoxicity mainly due to their antioxidant or free radical scavenging activities.139-421

Furthermore, silymarin, a flavonoid from Silybum marianum was used as standard to study the hepatoprotective potential. The mechanism of action of this phytocompound is based on its antioxidant potential to a variety of free radicals. C. album extract used in present study had shown hepatoprotective potential as compared with silymarin on the basis of restoration of biochemical parameters such as AST, ALT, ALP, bilirubin and total protein content. On the basis of this and antioxidant potential of C. album extract against different kinds of free radicals as established during in vitro assays, it was presumed that the mechanism of hepato-protection was largely due to antioxidant potential of C. album extract. Histopathological observation of the liver tissue had directly supported this conclusion. Therefore, present work provides a conclusive scientific evidence for traditional use of C. album in treatment of hepatic disorders.


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How to cite this article: Baldi A, Choudhary NK. In vitro antioxidant and hepatoprotective potential of chenopodium album extract. Int J Green Pharm 2013;7:50-6.

Source of Support: Nil, Conflict of Interest: None declared.

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