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Chinese Journal of Natural Medicines 2014, 12(11): 08270832

Chinese Journal of Natural Medicines

Antioxidant activity and functional group analysis of Evolvulus alsinoides

Duraisamy Gomathi 1, Ganesan Ravikumar 2, Manokaran Kalaiselvi 3, Kanakasabapathi Devaki 4, Chandrasekar Uma 5*

1 Food Analyst, Post Harvest Technology Centre, Tamil Nadu Agricultural University, Coimbatore;

2 Research Associate, Dalmia Centre for Research and Development, Coimbatore; 3 Assistant Professor, Departments of Biotechnology, Kongunadu Arts and Science College, Coimbatore;

4 Department of Biochemistry, Karpagam University, Coimbatore 641021;

5 Department of Biochemistry, College of Medicine & Health Science, Hawassa University, Hawassa, Ethiopia

Available online 20 Nov. 2014

[ABSTRACT] AIM: Many oxidative stress-related diseases occur as a result of the accumulation of free radicals in the body. Free radicals are generated by various endogenous systems, exposure to different physiochemical conditions, or pathological states. A balance be-tween free radicals and antioxidants is necessary for appropriate physiological function. A lot of studies are going on worldwide directed towards finding natural antioxidants of plant origin. FTIR spectroscopy is used to develop a rapid and effective analytical method for studying the main constituents in medicinal plants. The chemical constituents in the plants were identified and moni-tored for their medicinal properties. The aim of this study was to evaluate the in vitro antioxidant activities and FTIR spectroscopic analysis of the ethanolic extract of Evolvulus alsinoides. METHOD: Free radical scavenging activity of ethanolic extract of the whole plant of E. alsinoides was evaluated by in vitro methods, including total antioxidant assay (FRAP method) and hydrogen peroxide scavenging activity using ascorbic acid as a standard. The degree of lipid peroxidation was examined by estimating the thiobarbituric acid reactive substances (TBARS) using standard methods and the functional groups were analyzed using FTIR spectroscopy. The IR spectrum in the mid-infrared region 4 000–400 cm–1 was used for discriminatio and to identify various functional groups present in E. alsinoides. RESULTS: The findings indicated the presence of amino acids, amides, amines, carboxylic acids, carbonyl compounds, organic hydrocarbons, and halogens in the ethanolic extract of E. alsinoides, and the antioxidant activities were significantly increased, when compared with the standard antioxidant ascorbic acid, in a dose-dependent manner. CONCLUSION: The findings indicated promising antioxidant activity of the crude extract of E. alsinoides, and needs further ex-ploration for their potential effective use.

[KEY WORDS] Evolvulus alsinoides; Convolvulaceae; Ethanolic extract; FT-IR; Free radicals; Ascorbic acid

[CLC Number] R965; R917 [Document code] A [Article ID] 2095-6975(2014)11-0827-06

Introduction

Free radicals and other reactive species produced during

aerobic metabolism in the body can cause oxidative damage

of amino acids, lipids, proteins, and DNA. It has been estab-

lished that oxidative stress is among the major causative fac-

[Received on] 31-Nov.-2013

[*Corresponding author] Chandrasekar Uma: Associate Prof.,

E-mail: umachandrasekaran29@gmail.com These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

tors in the induction of many chronic and degenerative dis-

eases including atherosclerosis, ischemic heart disease, ageing,

diabetes mellitus, cancer, immunosuppression, neurodegenerative

diseases, and others [1-2]. The most effective way to eliminate

free radicals which cause the oxidative stress is with the help

of antioxidants. Antioxidants, both exogenous and endogenous,

whether synthetic or natural, can be effective in preventing

free radical formation by scavenging them or promoting their

decomposition and suppressing such disorders [3-4].

A potent broad spectrum scavenger of these species may

serve as a possible preventive intervention for free radical-

mediated cellular damage and diseases. Antioxidant-based

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drugs and formulations for the prevention and treatment of

complex diseases, like Alzheimer’s disease and cancer, have

appeared during the last three decades [5]. Recent studies have

shown that a number of plant products, including polyphenols,

terpenes, and various plant extracts exert an antioxidant action.

There is also a considerable amount of evidence revealing an

association between individuals who have a diet rich in fresh

fruits and vegetables and the decreased risk of cardiovascular

diseases and certain forms of cancer.

There is currently immense interest in natural anti-

oxidants and their role in human health and nutrition.

Considerable amount of data have been generated on the

antioxidant properties of food plants around the globe [6].

However, traditionally used medicinal plants await such

screening. On the other hand, the medicinal properties of

plants have also been investigated due to their potent

pharmacological activities, low toxicity, and economic

viability [7]. Many plants synthesize substances that are

useful to the maintenance of health in humans and other

animals. The use of plants as medicines predates written

human history. Medicinal plants are in many ways having

their own immense importance in current era. All plants

produce chemical compounds as part of their normal

metabolic activities. These metabolic activities can be

measured using HPLC, FTIR, etc.

Fourier Transform Infrared Spectroscopy (FTIR) is one

of the most widely used methods to assist in the identification

of the chemical constituents and elucidate the compounds

structures, and has been used as a requisite method to identify

medicines in pharmacopoeias of many countries. Owing to

the fingerprint characteristics and extensive applicability to

the samples, FTIR has played an important role in pharma-

ceutical analysis in recent years [8-10]. Several medicinal plants

(Rasayana) have also been extensively used in the Indian

traditional (Ayurveda) system of medicine for the treatment of

various diseases. Some of these plants have shown potent

antioxidant activity [11]. However, the majority of plants

have not been screened for such activity. In order to con-

tribute further to the knowledge of Indian traditional plants,

the present study is focused on Evolvulus alsinoides (L.) L.

(Convolvulaceae) to determine the free radical scavenging

properties and the identification of various chemical groups

present in the ethanolic extract of plant material. Evolvulus

alsinoides (L.) L. (Convolvulaceae), commonly known as

‘vishnukranti’. It is used to cure fever, cough, cold, venereal

diseases, azoospermia, adenitis and dementia. It has a known

nootropic and anti-inflammatory activity [12-13]. In early stud-

ies, researchers reported the presence of chemical constitu-

ents like triacontane, pentatriacontane, evolvine, β-sitosterol,

two alkaloids betaine and shankpushpin, four unidentified

alkaloids A, B, C, caffeic acid, 6-methoxy-7-O-b-glu-

copyranoside coumarin, 2-C-methyl erythritol, kaempferol-7-

O-b-glucopyranoside, kaempferol-3-O-b-glucopyranoside and

kaempferol-3-O-b-glucopyranoside and quecet-

ine-3-O-b-glucopyra-noside in this species [14] .

Materials and Methods

Collection of plant material The whole plant of Evolvulus alsinoides (L.) L. used for

the investigation was obtained from Coimbatore District, Tamilnadu, India. The plant was authenticated by Dr. P. Sat-yanarayana, Botanical Survey of India, TNAU Campus, Coimbatore. The voucher number is BSI/SRC/5/23/2011- 12/Tech.-514. Fresh plant material was washed under running tap water, air dried, and powdered. Sample extraction

One hundred grams of dried plant powder was extracted in ethanol (500 mL) in an orbitory shaker for 72 h. Repeatedly extraction was done with the same solvent until a clear color-less solvent is obtained. The combined extracts were evapo-rated to dryness using a rotary evaporator at 40–50 °C. One gram of extract was obtained for each 100 g of dried plant ma-terial and was stored at 0–4 °C in an air-tight container. Total antioxidant activity (FRAP Assay)

A modified method [15] was adopted for the FRAP assay. The stock solutions included 300 mmol·L−1 acetate buffer (3.1 g C2H3NaO2·3H2O and 16 mL C2H4O2), pH 3.6, 10 mmol·L−1 TPTZ (2,4,6‐tripyridyl‐S‐triazine) solution in 40 mmol·L−1 HCl, and 20 mmol·L−1 FeCl3·6H2O solution. The fresh working solu-tion was prepared by mixing acetate buffer (25 mL), TPTZ (2.5 mL), and FeCl3·6H2O (2.5 mL). The temperature of the solution was increased to 37 °C prior to use. Plant extract (150 μL) was allowed to react with FRAP solution (2 850 μL) for 30 min in the dark. Readings of the colored product (ferrous tripyridyltri-azine complex) were taken at 593 nm. Hydrogen peroxide scavenging activity

Hydrogen peroxide scavenging activity of the extract was estimated by replacement titration. The assay was performed by adding hydrogen peroxide (1.0 mL, 0.1 mmol·L−1) and 1 mL of various concentrations of extracts were mixed, fol-lowed by 3% ammonium molybdate (2 drops), sulfuric acid (10 mL, 2 mol·L−1), and potassium iodide (7 mL, 1.8 mol·L−1). The mixed solution was titrated with 5.09 mmol·L−1 sodium thiosulfate until the yellow color disap-peared [16]. The percentage of scavenging of hydrogen perox-ide was calculated as:

H2O2 Scavenged (%) = cont test

cont100

A A

A

where, Acont was volume of sodium thiosulfate used to titrate the control sample in the presence of hydrogen peroxide (without extract), Atest was the volume of sodium thiosulfate solution used in the presence of extract. In vitro lipid peroxidation activity of the plant extract in the rat liver homogenate

Malondialdehyde has been identified as the product of li-pid peroxidation that reacts with thiobarbituric acid to give a red color absorbing at 535 nm [17]. Rat livers were processed

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to obtain a 10% homogenate in cold phosphate buffered saline, at pH 7.4 using a homogenizer, and centrifuged. The degree of lipid peroxidation was assayed by estimating the thiobar-bituric acid reactive substances (TBARS) using the standard methods with minor modifications. Different concentrations of the ethanolic extract of the whole plant of E. alsinoides were added to the liver homogenate. Lipid peroxidation was initiated by adding 15 mmol·L−1 ferrous sulfate solution (100 µL) to the tissue homogenate (3.0 mL). After 30 min, this reac-tion mixture (100 µL) was mixed with 10% TCA (1.5 mL, tricholoroacetic acid). After 10 min, the tubes were centrifuged and supernatant separated and mixed with 0.67% TBA (1.5 mL, thiobarbituric acid) in 50% acetic acid. The mixture was heated for 30 min in a boiling water bath. The intensity of the pink colored complex formed was measured at 535 nm. The results were expressed as nmoles of MDA formed/mg protein. FTIR spectrum analysis

The evaporated ethanol extract of E. alsinoides was ground into a fine powder using an agate mortar and a stan-dard KBr pellet was examined with the FT-IR spectrometer in the region 4 000–400 cm−1.

Results

The extracts from number of medicinal plants which are known to have some biologically active principles are used in Ayurvedic preparations and these extracts are prepared in bulk for commercial purposes. In this study, the antioxidant activ-ity of E. alsinoides was evaluated employing in vitro assay methods, such as scavenging activity of FRAP, hydrogen peroxide, and inhibition of lipid peroxidation, and the func-tional groups present in the ethanolic extract were identified through FTIR technique.

Fig. 1 Ferric reducing antioxidant power assay of the etha-nolic extract of Evolvulus alsinoides FRAP assay of the ethanolic extract of Evolvulus alsinoides. Concentration of sample 200–1 000 µg·mL−1. Ascorbic acid was used as standard

The FRAP scavenging capacity of the ethanol extract of E. alsinoides at five different concentrations (200–1 000 μg·mL−1) exhibited optical densities of 0.907, 1.309, 1.397, 1.561, and 1.825, respectively at 595 nm, as depicted in Fig. 1. Since the FRAP assay is easily reproducible and linearly related to molar

concentration of the antioxidant present, it can be inferred that the extract of E. alsinoides may act as a free radical scavenger.

The whole plant ethanolic extract of E. alsinoides demonstrated hydrogen peroxide scavenging activity in a concentration dependent manner with an IC50 of 460 μg·mL−1, while the IC50 of the standard (ascorbic acid) was 740 μg·mL−1 (Fig. 2). In vitro lipid peroxidation was as-sessed using an assay which determines the production of malondialdehyde and related compounds in rat liver [18].

Fig. 2 Hydrogen peroxide radical scavenging activity of ethanolic extract of Evolvulus alsinoides Hydrogen peroxide radical scavenging activity of Evolvulus alsinoides. Concentration of sample 200–1 000 µg·mL−1. Ascorbic acid is used as standard

Fig. 3 In vitro lipid peroxidation activity of ethanolic extract of Evolvulus alsinoides Lipid peroxidation activity of Evolvulus alsinoides. Sample concentration: 200–1 000 µg·mL−1. Ascorbic acid is used as standard

homogenate. Malondialdehyde (MDA) is one of the major degradative products of lipid peroxidation and serves as a marker for oxidative stress.

The percentage inhibition of lipid peroxidation produced by the ethanolic extract of E. alsinoides is depicted in Fig. 3. The standard ascorbic acid showed high inhibitory activity of lipid peroxidation followed by E. alsinoides. At 1 000 µg·mL−1 of the extract, the percentage inhibition obtained by ascorbic acid and E. alsinoides was found to be 87% and 70%, respectively.

The FTIR analysis was done to identify the functional groups present in the ethanolic extract of E. alsinoides. The

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FTIR spectroscopic studies revealed the existence of various functional groups in the whole plant extract of E. alsinoides.

The absorption bands and wave numbers (cm–1) of dominant peaks obtained from absorption spectra are shown in Table 1.

Table 1 Assignment of IR absorption bands in the spectra of the medicinal plants

Functional groups Frequency range (cm–1) Intensity

Aminoacids

C-N Stretching 844.82 (s), (m)

(Aliphatic & Aromatic nitro group)

NH3+ Stretching 2 011.76, 2 038.76, 2 061.9, 2 304.94 (s), (m)

(Overtones & combination) 2 391.73, 2 318.44

NH3+ Bending 1 516.05, 1 639.49 (m), (w)

(NH3+ asym & sym bending)

Co2- Stretching 1 161.15, 1 247.94 (w)

(NH3+ in-plane bending)

Nitro compounds 1 375.25 (s)

(N02 Sym stretching)

Nitroso compounds 1 247.94, 1 516.05 (s)

(N=O and C-N stretching)

Nitrate 1 375.25 (s)

(O-N02 stretching asymm.)

Nitrite

(N-O stretching & O-N=O bending) 1 639.49, 590.22 -

Nitrosamine

(N=O stretching & N-N stretching) 1 448.54, 1 062.78 (s), (m)

Oxime

OH stretching 3 402.43 -

Hydroxyl compounds

(OH stretching) 3 402.43 (s)

(Conjugate chelation) 2 864.29, 2 927.94 (w) broad

(OH in-plane bending) 1 375.25, 1 448.54 (v) broad

(O-H bending) 723.31 (m) very broad

Ethers

(C-O-C stretching) 1 062.78, 1 247.94, 844.82 (s), (m), (w)

Carbonyl compounds (ketones)

(C-H stretching) 2 864.29, 2 927.94 (w), (m)

(C=O stretching) 1 639, 1 720 (s)

Carboxylic acid

(OH stretching) 2 864, 2 927 (m)

(C=O stretching) 1 720 (s)

(C-O stretching and O-H in plane bending (coupled)) 1 161, 1 375 (m)

(OH bending) 908 (m) broad

Aldehydes

(C=O stretching) 1 720 (s)

(C-CHO skeletal) 1 062, 1 161, 1 247, 1 375 (v)

(C-H bending) 844, 908, 47 (w)

Amide

(N-H stretching) 3 402 (m)

(C=O Stretch) 1 639, 1 720 (s)

(N-H in-plane bending) 1 516 (s)

(OCN formation) 534, 551, 590, 723 (m), (s)

Halogen compounds

(Fluoride) 1 062, 1 161, 1 247, 1 375 (vs)

(Chloride) 590, 723 (s)

(Bromide) 534, 551, 590, 723 (s)

(Iodide) 534, 551, 590 (s)

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Discussion

Free radicals are involved in many disorders, including neurodegenerative diseases and cancer Plant-based drugs containing radical scavengers are gaining importance in the prevention and treatment of such diseases [19]. In the present investigation the ethanolic extract of E. alsinoides used to study the free radical scavenging activity, In vitro lipid per-oxidation and FTIR analysis. The ability of plant extract to reduce ferric ions was determined in FRAP assay. The results showed that very good antioxidant power compared to that of control ascorbic acid which was supported by Elangovan et al., 2013 [20] who reported that the Evolvulus alsinoides has significant antioxidant activity.

Hydrogen peroxide is a kind of reactive oxygen species (ROS) which has been given much attention during the last decades because of its ability to penetrate biological mem-branes. However, it may be toxic if converted to hydroxyl radical in the cell [2]. Once inside the cell, hydrogen peroxide can probably react with Fe2+ and possibly Cu2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is al-lowed to accumulate [21-22]. Hydrogen peroxide scavenging activity showed a very good scavenging activity which is comparable to standard ascorbic acid. Scavenging of H2O2 by this plant extract may be attributed to the phenolics in the ethanolic extract [23], which donate electron to H2O2, thus reducing it to water.

Free radicals induce lipid peroxidation in polyunsaturated

lipid rich areas like brain and liver [24]. In this study, in vitro

lipid peroxidation was induced to rat liver by using ferrous

sulfate. Based on the results, E. alsinoides inhibits lipids per-

oxidation in a concentration-dependent manner. The secon-

dary stage of linoleic acid oxidation is the formation of

malondialdehyde. The thiobarbituric acid reaction with MDA

is generally considered to be an indicator of the secondary

breakdown products of oxidized polyunsaturated fatty acids [25]. The antioxidant activity of this plant may be due to the

presence of antioxidant phytochemicals like polyphenolics,

steroids, and triterpenes [23, 26].

Antioxidant activity of phytocompounds was correlated to their chemical structures. Analysis of medicinal plant ma-terial by using FTIR, we can obtain a large number of macro-scopic structures information and holistic variation rules of chemical constituents in medicinal materials. Hence, the FT-IR analysis was done and the results showed the presence of various functional groups in the ethanolic extract of plant material. The results showed the presence of O–H stretching vibration, C-N stretching and C=O stretching vibration means that some carbonyl compounds existed in this plant. So, de-pending on the fingerprint characters of the peaks positions, shapes and intensities, the fundamental components in the

materials can be seen clearly [27]. The peaks at 1 600–800 cm–1 attributed to C=O stretching (lipids) and 1 600–720 cm–1 attributed to the region of amide I band of tissue protein [28]. Structure-activity relationship of some phenolic compounds (e.g. flavonoids, phenolic acids, tannins) has been studied [29]. The free radical scavenging and antioxidant activity of pheno-lics mainly depends on the number and position of hydro-gen-donating hydroxyl groups on the aromatic ring of the phenolic molecules [30]. The functional groups like O-H pre-sent in all phenolic compounds and C-N is common to all alkaloids. The presence of O-H and C-N groups confirmed the presence of phenolic compounds and alkaloids in ethano-lic extract of E. alsinoides. Hence it can serve as a good anti-oxidant.

The more intense bands occurring at different frequen-cies indicats the presence of O–H/N–H, C–H and C-CHO skeletal vibrations which denotes the presence of carboxylic acid, amino acids, alkenes, nitrates, ethers, organic halogen compounds and carbohydrates in plant material [10, 31-32]. The presence of carboxylic acid in ethanolic extract of Evolvulus alsinoides served as a main pharmaceutical product in curing ulcers, jaundice, head ache, fever, pain in liver, treatment of edema and rheumatic joint pains. The results represented in table 1 showed that the plant extract is rich in amides and amino acids which is the main group of protein synthesis and the presence of halogens in plant extract may served as a disinfectant. There is no absorbance in between the region 2 220−2 260 cm–1 indicates that no cyanide groups in our plant extract [32].

Conclusion

In conclusion, the findings of this study support this view that some medicinal plants are promising sources of potential antioxidants The providing data can just enrich the existing comprehensive data of antioxidant activity of plant material and the FTIR results revealed the presence of amino acids, carboxylic acids, aldehydes, ethers, hydroxyl groups and am-ides in the ethanolic extract of E. alsinoides which may be efficient as preventive agents in the pathogenesis of some diseases.

Acknowledgement

The authors thank the Chancellor, Advisor, Vice Chan-cellor, and Registrar of Karpagam University for providing facilities and encouragement.

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Cite this article as: Duraisamy Gomathi, Ganesan Ravikumar, Manokaran Kalaiselvi, Kanakasabapathi Devaki, Chandrasekar Uma. Antioxidant activity and functional group analysis of Evolvulus alsinoides [J]. Chinese Journal of Natural Medicines, 2014, 12 (11): 827-832