Comparison of Extraction Methods for the Determination of Antioxidant Activity in Extracts of Hippophae Rhamnoides L. and Lippia Citriodora. The Effect of Seasonal Collection

Abstract

In this work, the presence of natural antioxidants has been studied by measuring the antioxidant activity, of two selected samples sea buckthorn (Hippophae rhamnoides L.) and verbena (Lippia Citriodora). Leaves and fruits of two varieties (Leikora and Hergo) of sea buckthorn which are grown in different regions of Greece and verbena leaves grown in the same environment with the variety of sea buckthorn (Leikora) commercially sold as mixtures of the two plants to produce a specific beverage were selected and studied. easonal collection (Leikora variety) of two different crops was studied, in order to investigate the change in the content of antioxidant compounds in the same area at every year of cultivation. Specifically, the aim of this study was to determine the concentration of Total Phenolic Compounds (TPC) in the samples and the assessment of their antioxidant action by different spectrophotometric methods. Extracts were studied using different extraction solvents (methanol/ water 80/20 v/v and water to create beverages). Different extraction methods were compared (Ultrasound Assisted Extraction versus a conventional method) in order to determine the best method of extraction along with the extraction solvent suited for the quantitative release of phenolic components. The total phenolic content was determined by the Folin-Ciocalteu method using gallic acid as the standard and the antioxidant capacity of the plant extracts was measured by their ability to scavenge commercially available free radicals such as (a) DPPH (2,2-diphenyl-1-picrylhydrazyl) and, (b) ABTS (2,2'-azinobis-(3- ethylbenzothiaziline-6- sulfonate) using L- ascorbic acid and Trolox as standards respectively.

Keywords: Antioxidants, Hippophae rhamnoides L; Extraction comparison; Lippia citriodora; Seasonal collection

Abbreviations

UAE: Ultrasound-Assisted Extraction; DPPH: 2,2-Diphenyl- 1-Picrylhydrazyl; ABTS: 2,2’-Azino-Bis(3-ethylbenzothiazoline-6- Sulphonic acid); FC: Folin-Ciocalteu; GAE: Gallic Acid Equivalents; AAE: Ascorbic Acid Equivalents; TE: Trolox Equivalents; TPC: Total Phenolic Content

Introduction

In an effort to minimize the undesirable effects of synthetic food preservatives in human health, food industries and scientists have recently turned their interest to new preservatives. Sea buckthorn and verbena contain antioxidants which are known to prevent rancidity of fats in foods and dietary antioxidants that reduce the adverse effects of reactive species such as free radicals of oxygen and nitrogen, in the normal functioning of the human body. Sea Buckthorn (Hippophae L.) is a deciduous shrub that belongs to the family of Elaiagnoeidon and considered according to several studies, an important ally of our health [1]. The common species of sea Buckthorn (Hippophae rhamnoides L.) is by far the most widespread and contains a number of compounds including carotenoids, tocopherols, sterols, flavonoids, lipids, ascorbic acid, tannins. Such compounds are of interest not only from chemical point of view, but also because many of them have biological and therapeutic effects including antioxidant properties [2,3].

The official name of verbena is lippia citriodora and is a deciduous shrub belonging to the family of vervenakia and comprises about 200 species of herbs, shrubs and small trees [4]. Verbena contains active substances such as vervenalin, berberine, essential oils and tannin strange that unlike conventional tannins have not yet been investigated. These substances confer to the diuretic-decongestant action, antimicrobial, astringent, soothing and relaxing, anticonvulsive properties of the plant [5,6]. The chemical composition of the essential oils of many types of Lippia has been investigated by using several chromatographic techniques. Elements found in higher frequency in these essential oils are: limonene, caryophyllene, p-cymene, camphor, linalool, pinene and thymol.

Phytochemicals are a heterogeneous group of substances found in all plant products and are an important part of human diet. In the category of phytochemicals widespread redox secondary metabolites of phenolic nature can be found. Polyphenols give color to stem, leaves, flowers and fruits, and anthocyanins are the color component of most red and blue parts of the plant [7,8]. They occur very frequently in young leaves, which probably exert repulsive action in herbivorous insects. It is well known that the antioxidant and pharmacological properties of the plants are associated with the presence of phenolic compounds. These phytochemical compounds are derived from phenylalanine and tyrosine and do not contain any nitrogenous functional group. The structure varies from low molecular weight compounds with a single aromatic ring to large and complex tannins, polyphenols and phenolic molecules havingat least one aromatic ring with one or more hydroxyl groups attached. Some phenolic compounds can be classified according to the number of phenolic compounds and their components [7-9].

Ultrasound-Assisted Extraction (UAE) is a hight energy extraction technique. The ultrasonic radiation, which has frequencies higher than 20 kHz, facilitates the extraction of organic and inorganic compounds from solid substrates using liquid solvents. Sonication is the production of sound waves that generate bubbles close to the tissue sample, which are cleaved and disrupt the cell walls, thereby releasing the contents of the cells [10,11]. A suitable solvent is mixed with a sample and sonication is performed under controlled temperature for a specified period. The quality of the extract is influenced not only by the sonication time, temperature and choice of solvent, and the ultrasonic wave frequency and wavelength distribution [12]. The two most common ways of applying ultrasonic wave’s extraction samples are that of a probe and bath [13-15].

The antiradical activity characterizes the ability of compounds to react with a radical, while the in vitro antioxidant action is the ability of compounds to inhibit the oxidation process in a system in vivo. Through spectrophotometry it was possible to determine the concentration of total phenols in the extracts of the sea buckthorn and verbena samples for both crops (2012-2013), as well as extracts ability to bind two different free radicals (DPPH, ABTS) [16].

A method for the assessment of antioxidant activity is referred to as a discoloration test, as for lipophilic and hydrophilic antioxidants, including the carotenoids, and antioxidants in plasma is the method with the use of ABTS free radical. First, the process comprises the direct production of the ABTS free radical without involving an intermediate radical. Secondly, is a discoloration assay thus radical cation pre–formed prior to addition of the antioxidant system, instead of the radical produced continuously in the presence of antioxidant [17]. By the presence of molecules which are hydrogen donors, the radical ABTS reduced quantitatively depending on the activity of the hydrogen donor. The scanning of the radical ABTS either by transferring a hydrogen or by transfer of an electron from a compound antiradical [18].

ABTS•+ (blue-green) + ?? —> ABTS (colorless) + [??]•+

ABTS• (blue-green ) + ?? —> ABTS-? (colorless) + ?•

The (DPPH •) test is often employed as the ABTS method as well, to characterize the scavenging ability of free radicals of the phenolic compounds (AH). Detailed published protocols differ in more than one experimental conditions and results for the relative order or magnitude of activity is often contradictory. The DPPH • do not dimerize, remains in its monomeric form in solution, exhibits a stable absorption over a wide pH range, and it resists oxidation. Moreover, the reaction conditions are mild and the results provide basic information about the activity of the compounds in relation to their structure. The free radical has a maximum absorbance at 516 nm is purple. Presence of a scanner free radical root receives an electron or a hydrogen molecule and so is diamagnetic and purple color gradually becomes yellow [19,20].

DPPH• (purple)+ AH DPPH-H (yellow)+ A• →

The total phenolic content of the extracts from samples of sea buckthorn and verbena were measured by using the Folin-Ciocalteu (FC) reagent. The FC reagent consists of salts of molybdenum (Mo) and tungsten (W). In an alkaline environment, the phenolic compound is oxidized and the reagent is reduced to oxides having the characteristic blue color of pentavalent molybdenum. The color intensity is proportional to the phenolic content, the concentration of which is expressed in equivalents of a selected model in this case was chosen gallic acid [21].

Materials and Methods

Plant materials and reagents

To conduct the experiments two sea buckthorn varieties (Hippophaë rhamnoides L.) and verbena leaves were selected. Buckthorn mixture of ramnoeides and Leikora (Hippophae rhamnoides-Leikora) namely: sea buckthorn leaves, sea buckthorn dried fruit, sea buckthorn fresh fruit from Konitsa region Ioannina (Greece) and verbena leaves from the same domain. (Samples September 2012 and September 2013). Buckthorn ramnoeides ?ergo (Hippophae rhamnoides -Hergo) namely: sea buckthorn leaves, sea buckthorn dried fruit, sea buckthorn fresh fruit from Nea Gonia Chalkidiki in Northenn Greece (Samples September 2013). All samples were analyzed within three months of collection.

Sodium Carbonate anhydrous (?a₂C?₃) is purchased from Carlo Erba agents, Italy. Gallic acid (3,4,5-Trihydroxybenzoic acid anhydrous) and DPPH radical (2,2-D?phenyl-1-picrylhydrazyl) were purchased from Alfa Aesar GmbH &Co KG, Germany and the Folin-Ciocalteu reagent was purchased from Merck (KGaA, Germany). ABTS: ABTS 2,2’-Azino-b?s(3-ethylbenzoth?azol?ne- d-sulfon?c acid ammonium salt ?) was purchased from Chemical Industry Co. L?D, Japan. L-Ascorbic acid, analytical reagent grade, was purchased from Fischer Chem?cal, UK and Trolox: 6-Hydroxy- 2,5,7,8-tetramethylchroman-2-carboxyl?c acid 97%, was from Sigma- Aldr?ch, Germany.

Quantification was done via calibration curve with standards (external standard method). All standards were prepared as stock solutions in methanol (L-ascorbic acid), in water (Gallic acid) and in ethanol (Trolox). Stock/working solutions of the standards were stored in darkness. All solvents and reagents from various suppliers were of the highest purity needed for each application.

Plant preparation

All samples were processed as quickly as possible and in a shady and cool atmosphere, where fresh fruit frozen, dried leaves and fruits were stored in a cool, shady place. Samples from 2012 crop variety Leikora used for comparing extraction methods (classical extraction, UAE) in different parts of sea buckthorn plant. Both extracts were prepared from the dried fruit and of dried leaves, as well as extracts prepared from dried verbena leaves. Samples of the crop variety Leikora 2013 and samples of the variety Hergo were used for comparing the two varieties. Also all sample extracts were prepared by using the conventional extraction procedure. It should be mentioned that decoctions were also prepared from the above samples. Also, fresh and dried fruits from the two varieties were used to estimate the difference in concentration of antioxidants between fresh and dried fruit.

Samples of fresh fruit were lyophilized (freeze drying) for better maintenance as the absence of water suspends microbial and enzyme activity. Also, the dry sample is easier to be pulverized compared to the fresh sample, thus increasing the surface of the material and thereby increasing the extraction process [22,23].

Extraction Procedure

Ultrasound-Assisted Extraction was the first extraction method applied. In a special oval bottle each time, 1 g of powdered sample was placed and 50 mL of methanol:water, 80:20 (v/v) was added, followed by adjusting the ultrasound generating device. Also, the flask was immersed in ice, so that the temperature during extraction did not exceed 35°C. Specifically, the power of ultrasound ranged in percentage 80 % and the function of pulses (Pulse mode) adjusted to 10 sec On then 5 sec Off (power intensity device probe 750Watt). Overall, the time for each extraction was 15 minutes. Upon completion of the extraction, filtration was followed by using Buchner funnel, to remove solids and to have clear extracts, while the final volume was 50 mL.

For conventional extraction method, 1 g of each powdered material was placed in separate beakers, then 50 mL of methanol:water, 80:20 (v/v) was added for the extraction of the phenolic content by diffusion having samples shaken at regular intervals. After 48 h in the dark and at ambient temperature extraction is completed, followed by filtration using Buchner funnel, to remove solids and the clarified extract diluted to a final volume of 50 mL. The extracts were kept frozen at -200C in sealed containers.

For the preparation of decoctions, 1 g of each powdered material was used. Then 50 mL of deionized water at 80 °C was added. The samples were left to extract components for 3min, as it is normally performed for infusion preparation, and then filtered and diluted to a final volume of 50 mL. The spectrophotometric analyses were performed within 1h after preparation of infusions which were freshly prepared daily.

Determination of Total Phenolics

The same procedure was applied for all extracts to determine the total phenolic content using the same quantities of reagents. Initially, a solution of saturated sodium hydrogen carbonate Na₂C0₃ was prepared as follows: 20 g anhydrous sodium carbonate (Na₂C0₃) were dissolved in 80 mL of boiling distilled H₂0. After the solution of sodium carbonate reached ambient temperature, 8 g of excess Na₂C0₃, was added and the solution was left for 24 h in the dark and well sealed. Finally, the solution was filtered through a fluted filter and diluted to 100 mL with distilled H₂0 to a volumetric flask. This solution is stable and suitable to use over a long period. Gallic acid (GA) was used as the standard phenolic substance to compare with extracts. Originally a stock solution of GA (stock) concentration 5g GA / L was prepared from which aqueous solutions were prepared with concentration of 25 to 600 mg GA / L. In plastic cuvettes of 2,5 mL, 20.0 ML standard or diluted sample were placed using electronic pipettes, plus 1500 mL distilled H₂0 and 100 mL of Folin-Ciocalteu reagent (commercially available). Following vigorous stirring and after waiting for 8 min, 300 mL of saturated Na₂C0₃ solution were added and the mixture was sealed with parafilm followed by vigorous shake again. Then, the cuvettes were placed for 30 min in a water bath at 40 °C. Then the absorbance was measured at 750 nm (A₇₅₀) for each sample or standard. The error correction in the value of the absorption due to the solvent of samples and standards is the “blank” sample. The experimental procedure and calculations were made in triplicate for each sample or standard solution, while different series of experiments were performed in the same day, but on different days as well. Final results are expressed in mg GAE g^-1.

Determination of Antioxidant Activity Using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Method

Initially the DPPH radical was dissolved in methanol to prepare a concentrated (stock) solution of 0.001M from which a diluted solution of 100 μM was prepared, which is later used for measuring the antiradical activity of samples. 20.0 μL of diluted samples were placed in plastic cuvettes together with 1500.0 μL of DPPH 100 μM solution, left in the dark for 1 min and then the absorbance was measured at 516 nm (A516) every 10 min until the absorbance was stabilized in a minimum point at a plateau time.24 The time for the reaction to reach this point, denoted plateau depends on the type of sample, the concentration, the ambient conditions (temperature and light), and obviously the concentration of DPPH. At the same time, the absorbance of DPPH solution used was measured so as to calculate the percentage of the halting at the plateau time. Also measurements of the blank were made to correct the error caused by the solvent.

L-ascorbic acid (AA), was used as standard compound to prepare the standard curve for quantification because ascorbic acid reacts rapidly and completely with DPPH radical. A stock solution of 1 mg / mL was prepared by dissolving 0,1 g of ascorbic acid in 100 mL of methanol. Then the preparation of dilute solutions of ascorbic acid was followed with concentrations ranged between 800 to 1800 μg AA / mL in order for the preparation of standard curve. The solutions of ascorbic acid were prepared on each day of conducting the experiment. The experimental procedure and calculations were made in triplicate for each sample or standard solution. Final results are expressed in mg AAE g-1.

Determination of Antioxidant Activity Using the ABTS Free Radical Scavenging Method

The first step was to prepare the ABTS • ⁺radical. To do so, an aqueous solution containing the dissolved substance in concentration of 7 mM ABTS was prepared, which is the concentrated solution of the radical (stock solution). Also sodium persulfate (Na₂S₂0₈) 2,45 mM was also prepared. The mixture of the aforementioned solution was left in darkness for 16h at room temperature. Oxidation of ABTS by the persulfate ions starts directly, but the stoichiometry of the reaction is 1: 0.5, whereby the oxidation is incomplete. The radical in this form is stable for more than 2 up to 4 days in the dark, hence solution was stored in the refrigerator. Before using the ABTS+ for estimating antiradical capacity of the sample solution, the radical solution was appropriately diluted with ethanol to give absorbance between 0,70 ± 0,02. As standard compound Trolox was chosen, which is prepared as ethanolic stock solution of 0.006 M, from which solutions with concentration between 0.20 to 1,50 mM were prepared for conducting the standard curve for quantification. In plastic cuvettes of 2,5 mL, 15.0 μL standard or diluted sample using electronic pipettes were placed, plus 1500 μl of the diluted radical solution of ABTS⁺. The cells are sealed with parafilm and stirred vigorously prior to storage in dark. Measurements were performed at 734nm. The error correction in the value of the absorption due to the solvent for samples and standard was deducted. The experimental procedure and calculations were made in triplicate for each sample or standard solution. Final results are expressed in mg Trolox Equivalents (TE) g^-1.

Results and Discussion

In Table 1 below, the abbreviations for each of the samples studied are given for the two different crop seasons of collection.