Comparison Between the Phytochemical and Antioxidant Properties of Plants Used in Plant Infusions for Medicinal Purposes

It has already been acknowledged among the medical community that plant based treatments represent an interesting contribution to modern therapeutics due to the presence in their composition of molecules with pharmacological and antioxidant action. The aim of this study was to evaluate the contents of total phenolics, flavonoids, and caffeine in six plants used traditionally by healers in Portugal and usually consumed as tea or infusion namely: Camellia sinensis, Melissa officinalis, Lippia citriodora, Cymbopogon citratus, Matricaria chamomilla, and Tilia cordata. Total phenolics ranged from 32.05 mg GAE/100g for aqueous extracts obtained from leaves of L. citriodora to 145.28 mg GAE/100g for aqueous extracts of C. sinensis. Significant variations in the flavonoid content were also found among analyzed plants and depending on the nature of the extract, with C. sinensis standing out again with the highest values (78.31 mg CE/100g) and the ethanolic extract obtained from the flowers of T. cordata exhibiting the lowest content (25.15 mg CE/100g). The concentration of caffeine was also very diverse and followed the sequence M. officinalis < T. cordata < C. citratus < M. chamomilla < L. citriodora < C. sinensis. The antioxidant activity of each plant was evaluated in vitro using a standard model system, the DPPH assay, and was found to vary according to C. citratus (90.9%) > C. sinensis (87.8%) > M. officinalis (50.7%) > M. chamomilla (45.3%) > T. cordata (32.2%) > L. citriodora (28.0%). The aqueous extracts presented lower antioxidant activity than the corresponding ethanolic ones.


Introduction
The health benefits associated with the consumption of tea and herbal infusions have resulted in their recognition as botanical dietary supplements, which are widely consumed nowadays as adjuvants for complementary and alternative medicines (Monbaliu et al., 2010).Many of these health benefits are due to the action of antioxidant molecules such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), carotenoids, polyphenols (a wide class of components including phenolic acids, catechins, flavonols and anthocyanins) and caffeine, in which plants are naturally rich (Giovanelli & Buratti, 2009;Jehad, 2009).The current state of medical knowledge attributes to antioxidant molecules a crucial role in counteracting the effects of oxidative stress caused by Reactive Oxygen Species (ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl, nitric oxide and peroxynitrite radicals.It is now recognized that oxidative stress is related to the pathogenesis of several diseases such as metabolic syndrome, cancer and degenerative disorders (Alfadda & Sallam, 2012;Brieger et al., 2012;Finkel, 2000).The oxidation of lipids, proteins, DNA, carbohydrates, and other biological molecules by toxic ROS may cause DNA mutations and/or damage target cells and tissues, and this often results in cell senescence and death.This lead many clinicians and researchers to advocate as a strategy for inhibiting, delaying or even reversing the carcinogenesis process and preventing cardiovascular disease the chemoprevention through the intake of natural antioxidants (Ferri & Grassi, 2010;Grassi et al., 2009;Valko et al., 2007).The antioxidants obtained from plants do not induce the negative side effects provoked by synthetic ones (Ndhlala, Moyo, & Staden, 2010).For example one of the most frequently used synthetic antioxidants in food industry at high doses, butylated hydroxyanisole (BHA), is known to exhibit genotoxic and carcinogenic effects, while butylated hydroxytoluene (BHT) was proven to cause hemorrhaging (Ndhlala et al., 2010;Kahl & Kappus, 1993;Chen, Pearson & Gray, 1992;Ito et al., 1986).These facts have prompted many researchers to quest for potent and cost-effective antioxidants from various plant sources.
The aim of this study was to determine the contents of phenolics, flavonoids and caffeine in extracts obtained from six plants, usually consumed in Portugal as tea or infusions for medicinal purposes namely: C. sinensis, M. officinalis, L. citriodora, C. citratus, and flowers of M. chamomilla, and T. cordata (Table 1).

Plant Material
Fresh herbal plant materials were purchased from a reputable medicinal plant producer located in the North of Portugal (41º 52′ 26″ N; 8º 50′ 26″ W, average altitude: 16 m), characterized by a Mediterranean climate.Six medicinal plants were studied: Camellia sinensis, Melissa officinalis, Matricaria chamomilla, Tilia cordata, Cymbopogon citratus and Lippia citriodora (Table 1).The collected plant material was air-dried at room temperature (20±2ºC) and protected from light.Dried plant material was cut and stored in tightly sealed dark containers until needed.

Preparation of Plant Extracts
Medicinal plants can be used fresh or dried.Drying is the most common method for post-harvest preservation, and must be accomplished as soon as possible after harvesting, to maintain the quality and to prevent any contamination and phytochemical deterioration.The dried plant material was coarsely crushed in small pieces of 2-5 mm using a cylindrical crusher and extracted with water or ethanol.Each extract was obtained by mixing 5.0 g of dried plant in 200.0 ml of water or ethanol at approximately 100ºC for 10 min.The extracts were filtered through a paper filter (Whatman, No. 1) and stored under refrigeration in glass flasks tapered with screw plastic lid.This procedure has been described by several authors as efficient conditions to extract phytochemicals, such as phenolic and flavonoid compounds from plant material (Katalinic et al., 2006;Su et al., 2006).

Determination of Caffeine by UV/Vis Spectrophotometry
Total caffeine content was determined using a portion of 0.25 g of each plant that was dissolved in distilled water.20 mL of sample solution were pipetted to a 250 mL flask to which 10 mL of 0.01 mol/L hydrochloric acid and 2 mL lead acetate solution were added together with distilled water to complete the volume.The flask contents were shaken up and filtered.50 mL of filtered solution were then mixed with 0.2 mL of 4.5 mol sulfuric acid and the necessary volume of distilled water to complete the volume of a 100 mL flask.The resultant solution was shaken and filtered.The absorbance of the working standards and samples were measured on a UV/Vis spectrophotometer (Shimadzu, model UV-1800) at 274 nm using a 10 mm quartz cuvette.The caffeine concentration in the samples was calculated from the calibration curve obtained.

Total Phenolic Compounds Analysis
Total phenolics were determined colorimetrically using the Folin-Ciocalteau reagent (Velioglu et al., 1998) procedure with minor modifications.Briefly, 200 μL of each extract (aqueous and ethanolic, respectively) was mixed with 1.5 mL of Folin-Ciocalteau reagent (previously diluted 10-fold with distilled water), and left to stand at 22±1ºC for five minutes.1.5 mL sodium bicarbonate solution (8%) was added to the mixture.After 90 min of incubation, the absorbance was measured at 725 nm.Total phenolics were quantified from the calibration curve obtained from measuring the absorbance of a known concentration of gallic acid standards (15-300 mg/ml).The concentrations were expressed as mg of gallic acid equivalents (GAE) per 100g of dry weight.The total phenolic assay was measured six times for each plant extract.

Total Flavonoids Assay
Total flavonoid content was evaluated by the aluminum chloride assay, previously described (Zhishen et al., 1999).An aliquot (1 mL) of aqueous, ethanolic extract or standard solution of catechin [50,100,150,200,250 and 300 mg/mL] was added to a 10 ml volumetric flask containing 4 mL of distilled water.To this flask was also added 0.3 mL of NaNO 3 (5%).After 5 min of incubation, 0.3 mL of 10% AlCl 3 was added, and at the 6 th min, 2 mL of NaOH (1 M).The mixture was completed up to 10 mL.The final solution was mixed and the absorbance was measured against a reagent blank at 510 nm.Total flavonoid content was expressed as mg catechin equivalents (CE)/100g dry weight (DW).The total flavonoid assay was measured six times for each plant extract.

Evaluation of Antioxidant Activity
When the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) reacts with an antioxidant compound, capable of donating hydrogen, it is reduced.The extent of this reaction is a measure of the antioxidant capacity.The reaction mixture in each one of the 96-wells of an ELX80 Microplate Reader (Bio-Tek) consisted of one of the different concentration plant extracts (20 μL) and an aqueous/methanolic solution (80:20 v/v, 280 μL) containing DPPH radicals (6x10 -5 mol/L).The changes in color (from deep-violet to light-yellow) were measured at 517 nm, according to the method previously reported (Shah et al., 2011).Results are expressed as percentage of radical scavenging activity (% RSA), which is defined by the following expression: % RSA = [(A DPPH-A S )/A DPPH ] X 100, where, A s is the absorbance of the solution with the sample extracts and A DPPH the absorbance of the DPPH • solution.All determinations were performed in triplicate.The EC 50 , i.e., the concentration necessary to inhibit 50% of DPPH • radical, was determined according to published procedures (Kulisic et al., 2006;Cheel et al., 2005).Sample concentration corresponding to 50% of DPPH • absorbance was calculated graphically.Trolox was used as standard.

Statistical Analysis
Data are reported as mean±standard deviation obtained from six measurements.Statistical analyses were performed using the statistical package SPSS v 20.0 (SPSS for Windows; SPSS Inc., Chicago, IL).One-way ANOVA was employed to compare three or more groups using post-hoc Dunnett's test for multiple comparisons.Independent samples t-test was used to compare the extracts obtained from the same plant.Differences at p < 0.05 (95% confidence level) were considered to be significant.Herbal medicine or phytotherapy, referring to the medical use of plant organs (leaves, stems, roots, flowers, fruits and seeds) for curative purposes helped mankind since immemorial times.It is now time to use the scientific and technical knowledge at our disposal, to unravel the scientific basis of the therapeutic action of plants, especially its relation to their chemical composition.

Results and Discussion
The caffeine content, in the part used for medicinal purposes, of the six plants studied is presented in Table 2.
Caffeine, an alkaloid of the methylxanthine family, is mainly found in plant leaves as a secondary metabolite, it is a pharmacologically active substance and, depending on the consumed dose, may be a mild central nervous system stimulant because it acts as an adenosine-receptor antagonist (Schellack, 2012).In particular, caffeine is a nonspecific, competitive blocker of adenosine A1 and A2A receptors, distributed throughout the central nervous system (Cauli & Morelli, 2005).Binding of adenosine to target receptors has general depressant effects, slowing heart rate and lowering blood pressure.A recent study revealed a new face to caffeine: its potential as antioxidant, by demonstrating that caffeine has the ability to directly binding to the hydroxyl radical thus neutralizing it (Carmona & Galano, 2011).This means that caffeine is not only a metabolic stimulant but also a protective antioxidant.The results presented in Table 2 reveal that caffeine amounts are quite diverse among the six plants consumed as infusions, ranging from 717.79 mg/100g (M.officinalis) to 3195.47 mg/100g (C.sinensis).The fact that C. sinensis presented the highest caffeine content, agrees with the data previously published by some investigators (Schellack, 2012;Friedman et al., 2005;Henning et al., 2003) on commercial teas consumed in the United States.The lower caffeine values exhibited by M. officinalis, T. cordata and C. citratus may explain why these are traditionally used as calmative infusions (Shah et al., 2011;Moradkhani et al., 2010;Cotrim et al., 1999).
The variability in the levels of caffeine among the teas was, however, much narrower than that of catechins found in the generality of teas obtained from medicinal plants (Dadáková et al., 2010;Velayutham et al., 2008).
Total phenolic and flavonoid contents of the six Portuguese medicinal infusions and teas are shown in Table 3.In our experiments two different extracts where studied: aqueous and ethanolic.The tea or infusion may be considered a sort of water extract; however, in order to quantify the liposoluble components it is necessary to use organic solvents.The extraction of bioactive compounds from plant materials is the first step in the utilization of phytochemicals in preparation of dietary supplements or nutraceuticals, food ingredients, pharmaceutical and cosmetic products (Vinha et al., 2013;Dai & Mumper, 2010).
The first aspect that stands out from the results presented in Caffeine is a central nervous system and metabolic stimulant and also an antioxidant.Therefore, it is fair to assume that part of the beneficial effects, traditionally attributed to the plants studied here, result from the action of these compounds.Accordingly the plant extracts demonstrated a high capacity to scavenge free radicals, i.e. antioxidant capacity.This ability followed the sequence C. citratus > C. sinensis > M. officinalis > M. chamomilla > T. cordata > L. citriodora.To the best of our knowledge, this is the first study demonstrating the antioxidant potential of these medicinal plants cultivated in Portugal.The results indicate that, to a certain extent, plant composition fits the medical purposes for which it is traditionally used.Interestingly the plants presenting higher contents of phytochemicals (C.sinensis, C. citratus and M. officinalis) are the ones that are traditionally used to treat the widest range of ailments.

Table 1 .
Botanical designation and popular Portuguese name of the plants studied, together with the part of the plant used for medicinal purposes

Table 2 .
Caffeine concentration (mg/100g dry weight) of the six plants studied d All values are expressed as means ± S.D. obtained from six measurements.Different superscript letters indicate statistically different caffeine contents (p < 0.05).

Table 3
. Phytochemicals contents determined in aqueous and ethanolic extracts obtained from the six medicinal plants: The phenolics concentration is expressed in (mg GAE/100g sample) and that of flavonoids in ( Table 3 is the richness in phenolics and flavonoids exhibited by the plants studied, superior or comparable to many autochthonous fruits and vegetables for example