DOI: 10.1002/elan.201400555

Calligonum polygonoides Linnaeus Extract: HPLC-ECand Total Antioxidant Capacity EvaluationSara M. C. Gomes,[a] Isabel P. G. Fernandes,[a] Narpat Singh Shekhawat,[b] Sunita Kumbhat,[c] andAna Maria Oliveira-Brett*[a]

1 Introduction

Phenolic compounds constitute one of the most numerousgroups of plant secondary metabolites. The interest in thebioactivity of phenolic compounds is due to the potentialhealth benefits of these polyphenolic components andtheir involvement in important biological and industrialprocesses. Flavonoids can be found in fruits, vegetables,tea, wine and chocolate. The basic structure of flavonoidsconsists on the tricyclic C6C3C6. Their benefits topeople are anti-inflammatory, hormonal, anti-bleeding,anti-allergic and even prevention of the cancer onset. It isalso attributed to flavonoids the improvement in hair andnails condition since they promote the vitamin C absorp-tion. However, the most important is their antioxidantproperty, which fights free radicals in the human body,detoxifying the body, and making better use of nutrients.The consumption of fruits and vegetables has long beenassociated with health benefits due to their high antioxi-dants concentration.

The genus Calligonum (family polygonaceae) comprisesabout 80 species, has numerous therapeutic benefits al-ready known and studied, and are distributed throughoutnorthern Africa, southern Europe and western Asia. Cal-ligonum polygonoides Linnaeus, locally known as Phog orPhogra, is a common woody shrub grown naturally insand dunes of the desert of Rajasthan, in India [1] and itis well known for its adaptability in extreme xeric condi-tions. The plant is ecologically valuable as it stabilizes thesand dunes and prevents soil erosion, and is also an im-portant source of food [1, 2]. It has leafless fleshy greensmooth branches and white stem and produce small nu-merous flower buds and succulent flowers that are con-verted in hairy/spiny fruits. The over exploitation of the

plant has made it vulnerable and endangered species inits habitat [2].

Calligonum polygonoides Linnaeus is known for its me-dicinal properties apart from being traditionally used asfood during frequent famines, Figure 1 [36]. Theseplants produce numerous flower buds during March andApril. The local people harvest branches bearing onopened but still green flower buds, sun-dry, harvest andstore them, as valuable condiment/vegetable, phogflowers or phogla. The vegetable phogla is used tomake delicious heat relieving Raiyta (a curd/yogurtpreparation) by locals. The flowers of the plants areknown to contain a high amount of protein, possess tonicand digestive properties, being also useful against asthma,cough and cold. The juice is applied to the eye as an anti-dote to scorpion sting, the latex is used to treat eczema,bite dogs and abortion, and the roots decoction mixedwith catechu is used as gargle for sore gum. Phytochemi-cal screening of Calligonum polygonoides shows the pres-ence of flavonoids, alkaloids, proteins, tannins, steroids,phenols, carbohydrates and terpenoids in flower buds,flowers, seeds, and stems [711].

Thus, owing to the health properties associated withthese shrubs, in which the phenolic antioxidants play

[a] S. M. C. Gomes, I. P. G. Fernandes, A. M. Oliveira-BrettDepartment of Chemistry, University of Coimbra3004-535 Coimbra, Portugal*e-mail: brett@ci.uc.pt

[b] N. S. ShekhawatDepartment of Botany, Jai Narain Vyas (J.N.V.) UniversityJodhpur-342033, India

[c] S. KumbhatDepartment of Chemistry, Jai Narain Vyas (J.N.V.)UniversityJodhpur-342033, India

Abstract : Flavonoids in Calligonum polygonoides Lin-naeus extract were separated, detected, and identified byreverse-phase high-performance liquid chromatography(RP-HPLC) with electrochemical detection (EC) in com-bined isocratic and gradient elution using a glassy carbonor a boron doped diamond electrode. Ultrasonicationcoupled with a microwave-assisted technique was devel-oped to optimize the extraction of the phenolic com-

pounds. The total antioxidant capacity was quantifiedusing the DPPHC method and voltammetry. The RP-HPLC-EC led to the detection of nine different flavo-noids: catechin, delphinidin, fisetin, myricetin, epicate-chin, kuromanin, rutin, callistephin and procyanidin A2,in a single run by direct injection of the sample extractsolution.

Keywords: Calligonum polygonoides Linnaeus DPPHC method Electrochemical detection Flavonoids Reverse-phase high-performance liquid chromatography

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a crucial role, it is indispensable to have reliable methodscapable of measuring their antioxidant activity.

Many different methods can be used to measure thetotal antioxidant activity, the DPPHC (1,1-diphenyl-2-pic-rylhydrazine) method measures the radical scavenging ac-tivity [1215], presenting advantages when compared toother spectrophotometric methods such as ABTS(2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) method[1618], because the DPPHC does not require the genera-tion of the radical. In fact, the DPPHC is a commercialradical that can be reduced by antioxidant molecules,changing its color from purple to yellow after the reac-tion, and causing the absorbance decrease at the wave-length l516 nm.

The electrochemical techniques, mostly voltammetrictechniques, are applied to the study of electroactive spe-cies, including the antioxidant compounds, in order toevaluate the antioxidant activity of plant extracts, herbalproducts or other compounds [1922] presenting manyadvantages such as speed of analysis, low cost, simplicityand low consumption of reagents when compared toother methods [23]. Its important to note that the vol-tammetric techniques depend only on the inherent elec-trochemical properties of antioxidants present in thesample.

Antioxidants can act as reduction agents and tend tobe easily oxidized at inert electrodes, enabling the estab-lishment of a relationship between electrochemical be-havior and antioxidant power, the lower the oxidationpotential the greater the antioxidant power [24].

The presence of voltammetric signals at low anodic po-tentials indicates the presence of polyphenolics of highantioxidant capacity, whereas the polyphenolics with lowantioxidant activity show electrochemical activity athigher oxidation potentials [25].

The electrochemical behavior of flavonoids, such as cat-echin (flavan-3-ols) and epicatechin (flavonols), that ex-hibit an identical oxidation potential [24,26], are verygood antioxidant electrochemical standards.

The electrochemical index (EI) was defined as the totalphenolics concentration obtained using electrochemicaltechniques for predominant and representative phenolicclasses, taking into account the peak potential (Eap) andpeak current (Iap) [23,24, 26].

The lower peak potential corresponds to greater elec-tron donor ability and higher peak current, and the EI iscalculated using the equation [23]:

EI Iap1=Eap1 Iap2=Eap2 ... Iapn=Eapn

Extraction of phytochemicals from plant materials isthe most important step before further analysis, and it hasbeen accomplished by traditional extraction processes,such as solid-liquid extraction, using solvents such asmethanol, ethanol and acetone, and also through steamdistillation. The separation process consists in the distri-bution of analyte between two immiscible phases. It is im-portant that the extraction processes occurs with selectiveseparation of the target components from the sample atmaximum amount and/or interferences elimination.

Fig. 1. Calligonum polygonoides Linneaus: (A) plant (B) flowering plant (C) Dried flower buds.

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One of the simplest extraction techniques is the ultra-sonic extraction that is easy to perform in an ultrasonicbath. In this method, the crushed sample is mixed witha suitable solvent and placed into the ultrasonic bathwhere the working temperature and extraction time areset [2729]. Extraction of the flavonoids from seeds hasbeen performed by ultrasonication, using hydrochloricacid in methanol as extraction solvent (MeOH/HCl(99 :1) v/v) [28,30]. In order to increase the efficiency ofthe extraction procedure, microwave radiation has alsobeen used before ultrasonication [31].

Reverse-phase high-performance liquid chromatogra-phy (RP-HPLC) methods with electrochemical (EC)glassy carbon detectors have been widely used for theseparation, detection and identification of polyphenols indifferent extracts and plant materials [30,32, 33]. Theelectrochemical detectors enable highly selective and sen-sitive results, and it is possible to modify the experimentalconditions, such as the electrolyte ionic strength, bufferand pH, in order to maximize the sensitivity.

The boron doped diamond electrode (BDDE) electro-chemical properties are different from other electrodematerials (glassy carbon, pyrolytic graphite and carbonpaste): a wide working potential window in aqueous solu-tions, low and stable background current, long term sta-bility and low sensitivity to dissolved oxygen, good re-sponse for several redox analytes without any convention-al pretreatment, weak adsorption of polar molecules andoptical transparency in the UV/Vis and IR regions of theelectromagnetic spectrum, useful properties for spectroe-lectrochemical measurements [34].

Polarographic and spectrophotometric assays andHPLC with UV detection, since polyphenols absorb UVand visible light, have also been used [27,35].

The aim o