Asian Pacific Journal of Tropical Biomedicine (2012)1-4
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Document heading
Comparison of antioxidant activity and total phenolic content of Amaranthus tristis and Celosia argentea var spicata IswaryaVelu, Ashwini Ravi, Divya Gopalakrishnan, Bhuvaneshwari Manivannan, Mythili Sathiavelu and Sathiavelu * Arunachalam School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu - 632 014, India
ARTICLE INFO
ABSTRACT
Article history: Received 15 April 2011 Received in revised form 27 April Accepted 28 June 2011 Available online 28 June 2011
Objective: To compare the antioxidant activity and total phenolic content of the plants Amaranthus tristis and Celosia argentea var spicata which belongs to the family Amaranthaceae. Methods: Methanol and aqueous extracts of the plants were chosen for this study. Total phenolic content was estimated by Folin-Ciocalteau assay and antioxidant potential by 2, 2-diphenyl-1picrylhydrazyl radical scavenging assays. Results: Results reported that methanol and aqueous extract of Celosia spicata had maximum scavenging activity of 38.12% and 14.01% with gallic acid equivalence of 12.24 mg/g and 12.82 mg/g respectively at the concentration of 100 毺g/mL. Methanolic extract of Amaranthus tristis was found to have maximum scavenging activity of 52.10% at 100 毺g/mL and total phenolic content of 10.82 mg/g at 100 毺g/mL where aqueous extract showed 4.92% activity with phenolic content of 10.23 mg/g at 100 毺g/mL. Conclusions: Thus these results concluded that aqueous extract of Celosia spicata have more scavenging activity and higher phenolic content than Amaranthus tristis. It was also found that the methanolic extracts showed their maximum activity than aqueous extracts. Plant derived antioxidants, especially phenols and flavonoids, have been described to have various properties like anticancer, antiaging and prevention of cardiovascular diseases. Further, separation and characterization of the bioactive compound from the plant is to be evaluated and reported in near future.
Keywords: Antioxidant
2011
2, 2-diphenyl-1-picrylhydrazyl
Phenolic content Gallic acid equivalence
1. Introduction Free radicals (FR) can be defined as any chemical species that are capable of existing with one or more unpaired outer shell electrons. They are extremely reactive and generally highly unstable which are produced endogenously during cellular metabolism in all forms of aerobic living system, in addition to exogenous sources such as environmental pollutants, drug, radiation, and pathogens[1]. Their by-products are generally reactive oxygen species ( ROS ) such as super oxide anion, hydroxyl radical and hydrogen peroxide which are generated by normal physiological processes and various exogenous factors initiate peroxidation of membrane lipids as well as a wide range of other biological molecules. At low or moderate concentrations, ROS exert beneficial effects on cellular responses and immune function but at high levels, FR and oxidants generates oxidative stress, a deleterious process that can damage cell structures, including lipids, proteins, *Corresponding author: Dr. Sathiavelu Arunachalam, Assistant professor (Sr.), School of Bio Sciences and Technology, VIT University, Vellore - 632 014, Tamil Nadu, India. Tel: + 91- 9443457024 E-mail:
[email protected]
and DNA. Several experimental investigations suggest that FR and ROS may be related to high number of diseases[2]. Oxidative stress plays a major role in the development of chronic and degenerative ailments such as cancer, autoimmune disorders, rheumatoid arthritis, cataract, aging, cardiovascular and neurodegenerative diseases[2-4]. The human body has developed several mechanisms in order to counteract oxidative stress by producing antioxidants. Antioxidants are either naturally produced in the body, or externally supplied through foods and/or supplements[4]. A ntioxidants are compounds which can delay or inhibit the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative chain reactions[5,6]. The major action of antioxidants in cells is to prevent damage due to the action of ROS[6]. Polyphenolic compounds belong to the category of secondary plant metabolites which are widespread in the plant kingdom and have multiple applications in food, cosmetic and pharmaceutical industries[7]. The antioxidative effect is mainly due to phenolic components, such as flavonoids, phenolic acids, and phenolic diterpenes[4]. The antioxidant capacity possessed by phenolic compounds is mainly due to their redox properties, which permit them to act as reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators[5,7,8]. Antioxidants act as free radical scavengers and thereby enhance the immune defense and lower the
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risk of cancer and degenerative diseases[4]. Besides their roles as antioxidants, these compounds also exhibits a wide spectrum of medicinal properties, such as antiallergic, anti-inflammatory, anti-microbial, anti-thrombotic, cardio-protective and vasodilatory effects[7,1]. Now a days there was an increasing interest in finding antioxidant phytochemicals, because they can inhibit the propagation of free radical reactions, thereby protecting the human body from diseases. The most effective components seem to be flavonoids and phenolic compounds of many plant raw materials, particularly in herbs, seeds, and fruits showing their metal-chelating capabilities and radical-scavenging properties which enabled phenolic compounds to be thought of as effective free radical scavengers and inhibitors of lipid peroxidation[4]. Plant derived antioxidants exert their effects by enhancing the level of antioxidant enzymes such as superoxide dismutase or by lowering the levels of lipid peroxides in the blood or liver[1]. As plants produce a lot of antioxidants to control the oxidative stress caused by sunbeams and oxygen, they can represent a source of new compounds with antioxidant activity. The intake of natural antioxidants has been associated with reduced risks of cancer, cardiovascular disease, diabetes, and other diseases associated with ageing[5,2]. In traditional societies, nutrition and health care are strongly interconnected and many plants have been consumed both as food and for medicinal purposes [6]. Among the various medicinal and culinary herbs, some endemic species are of particular interest because they may be used for the production of raw materials or preparations containing phytochemicals with significant antioxidant capacities and health benefits. Many medicinal plants contain large amounts of antioxidants other than vitamin C, vitamin E, and carotenoids[5]. However, during recent years people have been more concerned about the safety of their food and the potential effect of synthetic additives on their health. The two most commonly used synthetic antioxidants; Butylated hydroxyl anisole (BHA) and butylated hydroxytoluene (BHT) have begun to be restricted because of their toxicity and DNA damage induction[1,3]. Therefore, natural antioxidants from plant extracts have attracted more due to their safety. Recent researchers have been interested in finding novel antioxidants to combat and/or prevent ROSmediated diseases[3]. Amaranthus tristis L inn belongs to the family Amaranthaceae which is a most commonly cultivated plant throughout India. In English it is known as Chinese S pinach, G arden A maranth, and F ountain P lant and in Ayurveda as Maarisha-rakta (red var.). In Tamil, it is called as Arai-keerai. It is used as an astringent in menorrhagia, leucorrhoea, dysentery, diarrhea, hemorrhagic colitis and also used in cough, bronchitis and consumption; externally emollient[9] and also as a diuretic[10]. The plant contains amarantin, isoamarantin, betaine, amino acids, sterols which plays their role in their activities. It is used as an edible plant and also an ornamental plant[9]. Celosia argentea var spicata belongs to the family Amaranthaceae which is a common weed occurring throughout India[9]. It is a common weed found in cultivable lands[11]. It is also known as Silver cockscomb in English[12]. I n A yurvedic it is called as S hitivaaraka, V itunna. I t was also known as Pannaikeerai in Tamil and in folk as Shveta-murga, Sarvari, Sarvali, and Surali. It is an erect, glabrous profusely branched annual herb and commonly found in hills, in arable lands, as a weed of cultivation, in
harvested fields, less in plains. Tender shoots and leaves are cooked and eaten[13]. Its flowers were used in treatment of menorrhagia, blood-dysentery where seeds in the treatment of anti diarrhoeal, also used in stomatitis. As a whole plant, they act as antibacterial, antiscorbutic and cooling agent. Their tender leaves are rich in potassium and in vitamin B1 and B6. An alcoholic extract of the leaves and its flavanoids showed antibacterial activity, which was comparable to ampicillin and streptomycin. The seeds contain 11.6%-17% of protein and 6.4%-10.9% of fatty oil. The seeds and roots yield triterpenoid saponins. An alcoholic extract of the seeds possess significant diuretic activity[9]. T he purpose of the study is to compare the radical scavenging activity and the total phenolic content of the plants Amaranthus tristis and Celosia spicata belonging to the same family Amaranthaceae and to determine the relation between the antioxidant activity and their phenolic content. 2. Materials and methods 2.1. Sample collection Amaranthus tristis and Celosia spicata leaves were collected from the fields of Vellore district, Tamilnadu, India in a sterile bag and transferred to a laboratory. 2.2. Preparation of the extract
Plant material was thoroughly washed with water and kept in 1% HgCl2 for 5 min. It was then washed with sterile distilled water and kept overnight at room temperature. Then the plant material was ground into fine powder using dry mixer. Ten gram of the respective powder was extracted sequentially with methanol and distilled water respectively. These mixtures were then incubated for 24 h with occasional shaking and filtered with Whatman filter paper to obtain filtrate which was further evaporated to obtain the extract. Stock solutions of aqueous and methanol extracts of 1 mg/mL was prepared[6].
2.3. DPPH radical scavenging assay
2 mL of extract solution (10, 20, 40, 60, 80 and 100 毺g/mL) was made in methanol. To the extract solution, 1 mL of DPPH ( 2 , 2 -diphenyl- 1 -picrylhydrazyl ) solution of 0 . 2 mM/mL methanol was added and mixed vigorously. Then the mixture was incubated in darkness for 40 min. Absorbance was measured at 517 nm using a UV-Vis spectrophotometer ( D ouble beam 1 - 2902 , L asang ) with methanol as blank. Ascorbic acid was used as positive control[15]. The level of percentage scavenging of DPPH by the extracts was calculated according to the following formula: % DPPH radical scavenging =
Absorbance of the control - Absorbance of the sample
--------------------------------------× 100 Absorbance of the control
2.4. Estimation of total phenolic content Total phenolic contents of methanol and aqueous extracts of the plants Amaranthus tristis and Celosia spicata was determined using Folin-Ciocalteau method[14]. The crude
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methanol extracts were diluted in methanol and crude aqueous extracts were mixed with deionised water to obtain different concentrations 25 , 50 , 75 and 100 毺 g. To the 50 毺L of each extract, 2.5 mL of Folin-Ciocalteau reagent was added and 2 mL of 7.5% Na2CO3 was also added. The mixture was then incubated at 45 °C for 15 min. The absorbance was measured after incubation at 765 nm. Na2CO3 solution was used as blank. The results were expressed as Gallic acid equivalence in mg. 3. Results
aqueous extracts of the plant Celosia spicata was found to have maximum GAE of 12.24 and 12.82 mg/g respectively at the concentration of 100 毺g/mL. Total phenolic content of the aqueous extracts of both plants showed dose dependent increase.
% Inhibition
100
GAF (ug)
I n this study, the radical scavenging activity was determined by DPPH radical scavenging assay and the results were reported in Figure 1 and 2. The methanol extract of the plant Amaranthus tristis was found to have maximum radical scavenging activity of 52.10% at the concentration of 100 毺g/mL and the aqueous extract was found to have 4.92% at the concentration of 100 毺g/mL. Whereas the methanol and the aqueous extract of Celosia spicata was found to have maximum scavenging activity of 38.12% and 14.01% respectively at the concentration of 100 毺g/mL. Antioxidant activity of the aqueous extracts of both plants showed a dose dependent increase in radical scavenging activity.
10
5 0
0
25
50
Concentration (毺g/mL)
75
100
Amaranthus tristis Celosia spicata Figure 3. Total phenolic content of the methanolic extract of the Amaranthus tristis and Celosia spicata. 15
10
GAF (ug)
50
5
0
20
40
60
Concentration (毺g/mL)
80
100
Amaranthus tristis Celosia spicata Ascorbic acid Figure 1. DPPH radical scavenging activity of the methanolic extracts of the Amaranthus tristis and Celosia spicata.
0
0
25
50
75
100
Amaranthus tristis (毺g/mL)Celosia spicata Concentrion Figure 4. Total phenolic content of the aqueous extract of the Amaranthus tristis and Celosia spicata.
4. Discussion
15
% Inhibition
10
5 0
extract of Amaranthus tristis was found to have maximum
GAE of 10.23 mg/g at the concentration of 100 毺g/mL where the aqueous extract was found to have maximum GAE of 8.82 mg/g at the concentration of 100 毺g/mL. The methanolic and
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3.1. DPPH radical scavenging assay
0
of the Amaranthus tristis and Celosia spicata was measured.
Results are expressed as Gallic acid equivalence (GAE) in mg and reported in the Figure 3 and Figure 4. The methanolic
0
20
40 60 Concentration (毺g/mL)
Amaranthus tristis
80
100
Celosia spicata
Figure 2. DPPH radical scavenging activity of the aqueous extracts of the Amaranthus tristis and Celosia spicata.
3.2. Estimation of total phenolic content
P henolic compounds are the major chemical which possesses high antioxidant potential. In the current study, total phenolic content of the methanol and aqueous extracts
In plants, phenolics such as phenolic acids and flavonoids were considered as an important compounds which plays an important role in determining the antioxidant activity. Phenolic acids have been repeatedly implicated as natural antioxidants in fruits, vegetables and other plants. For example, caffeic acid, ferulic acid, and vanillic acid are widely distributed in the plant kingdom. Rosmarinic acid was an important phytochemical found to be a potent active substance against human immunodeficiency virus type 1 (HIV-1)[5]. Phenolics are secondary metabolites that are ubiquitously present in plant metabolites. Plants phenolics are biosynthesized through different pathways, especially the shikimic acid pathway-the most important biosynthetic pathway. P henolics were considered as an important compound which plays a vital role in different biological activities such as antioxidant activity etc. Plant phenolics have high levels of antioxidant activities[18]. Plant phenolics are one of the major groups of compounds acting as primary antioxidants or free radical terminators, so it was necessary
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IswaryaVelu et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-4
to determine their total amount in the selected plant extracts[17]. DPPH has been extensively used as a free radical to evaluate reducing substances and as a reagent for investigating the free radical scavenging activities of compounds[18]. DPPH has the advantage of being unaffected by certain side reactions, such as metal ion chelation and enzyme inhibition, unlike other FR such as the hydroxyl radical and super oxide anion and the reduction can be measured directly in the reaction medium using a continuous spectrophotometric assay. A freshly prepared DPPH solution exhibits a deep purple colour with maximum absorption at 517 nm. When an antioxidant is present in the medium, the purple colour generally fades or disappears. Antioxidant molecule quenches DPPH FR by providing hydrogen atoms or by electron donation, via a free radical attack on the DPPH molecule and convert them to a colorless compound which results in decrease in the absorbance at 516 nm. Hence, the more rapidly the absorbance decreases, the more potent the antioxidant activity of the extract. This test is a commonly employed assay in antioxidant studies of specific compounds or extracts across a short time scale[16]. FR are the major substances which involved in many disorders like neurodegenerative diseases, cancer and AIDS . Antioxidants are useful for the management and the treatment of those diseases due to their scavenging activity [17]. Amaranthus tristis contains about 0.44 g/L of carotenes than other green leafy vegetables. It was also reported that many green leafy vegetables of Indian regions are rich sources of antioxidants[6]. Another report reveals that Amaranthus tristis showed highest absorbance i.e high reducing power as compared to plants Phyllanthus niruri and Basella alba. Amaranthus tristis showed highest %inhibition among other plants. Its methanolic and aqueous extracts showed 31% and 32% inhibition respectively[19]. The radical scavenging activity and the phenolic activity of Amaranthus tristis and Celosia spicata were compared. From the results, it was concluded that Celosia spicata had maximum radical scavenging activity and higher phenolic content than Amaranthus tristis. Each plant has different type of compounds which plays a major role in their pharmacological activities such as antioxidant activity etc. Therefore these results show the necessity to analyze the antioxidants present in the several medicinal plants. This might be useful to treat several diseases where FR play a major role in causing the disease. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements The authors are thankful to VIT University for providing infrastructure, constant support and encouragement.
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