JOANNA KIELAR

II MSU

MWB UG I GUMed

REPORT

ERASMUS INTERNSHIP

3 MONTH PROGRAMME

FARO, 30.06 – 30.09.2014

Fatty acids contents, biophenols composition and antioxidant activity of roots and leaves of Cymodocea nodosa

Luísa Custódio^1, Joanna Kielar, Hugo Pereira, Maria João Rodrigues¹, Catarina Vizetto-Duarte¹, Luísa Barreira¹ and João Varela¹

¹Centre of Marine Sciences, University of Algarve, Faculty of Sciences and Technology, Ed. 7, Campus of Gambelas, 8005-139 Faro, Portugal.

Abstract

The presence of natural antioxidant in plants is well known. This paper reports the antioxidative activities of methanolic and hexane extracts of of marine spieces - C. nodosa.The antioxidative activity of phenolic extracts from C. nodosa (roots and leaves) was examined using 6 different antioxidant activity assays ( determination of antioxidant activity by metal-related methods and determination of antioxidant activity by radical-based methods). The content of total phenolics in the extracts was determined spectrometrically according to the Folin-Ciocalteu procedure and calculated as gallic acid equivalents (GAE). All the extracts exhibited hight total phenolic content and at least 3 assays proved high antioxidative activity. Among, all extracts remarkable high antioxidant activity and high total phenolic content (GAE > 35 mg GAE/g, DW) was found in methanol extract of leaves. Based on IC50 calculations, C. nodosa is a promising source of phenolic compounds that may be used in pharmaceutical and food industries.To utilize this significant source of natural antioxidants, further characterization of the phenolic composition is needed using HPLC in order to indentify the main phenolic compounds of the extracts.

Key words: Antioxidative activities, C. nodosa, total phenolic content, IC50

1. Introduction

Plant secondary metabolites are economically important in the field of food additives, nutraceuticals, and drugs. This is especially the case for phenolic acids. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. In addition, they have a metal chelation potential (Rice-Evans et al., 1995).

Phenolic compounds exhibit a wide range of physiological properties, such as anti-allergenic, anti-artherogenic, anti-inflammatory, anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilatory effects (Benavente-Garcia et al., 1997, Manach, Mazur, et al., 2005, Middleton et al., 2000,Puupponen-Pimiä et al., 2001 and Samman et al., 1998).

Phenolic compounds have been associated with the health benefits derived from consuming high levels of fruits and vegetables (Hertog, Feskens, et al., 1993 and Parr and Bolwell, 2000). However, the identification of novel sources of phenolic acids has become of scientific and economic interest, and marine ecosystems can offer a great potential in this field (e.g., Guinea et al. 2012).

The oceans are a potential source for a wide variety of non-drug nutritional natural products. Several species of seaweeds are used as human food or as raw material for the production of compounds of nutritional interest (Cardozo et al. 2007). Compared with algae, seagrasses remain very little exploited despite the tremendous opportunities they offer to find new commercially valuable phytochemicals (Achamlale et al. 2009, Nuissier et al. 2010).

Seagrass beds generate considerable standing biomass. Storms and autumn leaf drop result in large accumulations of detritus along shorelines, which could be exploited as raw material. Local managers are under great public pressure to remove this material whenever it accumulates on beaches and shorelines used for recreational purposes. In most cases, the collected biomass is disposed of in waste disposal sites; however, according to the European Community guidelines on environmental protection, land filling is no longer possible without prior treatment. It has therefore become necessary to recycle the seagrass detritus. Considering the economic potential of phenolic acids within the pharmaceutical, cosmetic, and food industries, it appears of interest to reconsider the content and antioxidant properties of C. Nodosa . This work report s the antioxidant activity of C. nodosa’s hexane and methanol extracts of roots and leaves which was evaluated separetly using 6 different antioxidant activity assays and total polyphenolic content, flavonoids and tannins content.

2. Materials and methods

2.1. Plant material

Samples from Cymodocea nodosa were collected in the South of Portugal in Olhaõ. The taxonomical classification was confirmed by Dr. Aschwin H. Engelen (CCMAR, University of Algarve) and a voucher specimen is kept in the herbarium . The collected plants were separated into leaves and rhizomes, rinsed with distilled water, dried at 40°C for one week, powdered and stored at -20°C.

2.2. Extraction of polyphenols

Biophenols were sequentially extracted according to Falleh et al., (2011) and Grignon-Dubois (2013), with some modifications. Dried samples (10g) were first extracted with hexane overnight (100 mL) to remove lipids, carotenoids and chlorophylls, and centrifuged. The pellet was then extracted overnight with 50% aqueous methanol (1:40, w/v), with strirring, The process was repeated, and the obtained extracts were pooled, filtered (Whatmann No. 4), resuspended in DMSO at the concentration of 20mg/mL and stored at -20°C.

2.3. Determination of different phenolic groups

2.3.1. Total phenolics content (TPC)

The TPC of the extracts was determined by the F-C assay according to Velioglu et al. [58]. The extracts (5 µL at the concentration of 10 mg/mL) were mixed with 10-fold diluted F-C reagent in distilled water (100 µL) and incubated at RT for 5 min. Then, 100 µL of sodium carbonate (Na2CO3, 75 g/L, w/v) were added, samples were incubated for 90 minutes at RT, and the absorbance measured at 725 nm, on a microplate reader (Biotek Synergy 4). Results were expressed as gallic acid equivalents using a calibration curve of gallic acid standard solutions, in milligrams per gram of extract (mg GAE/g, DW).

2.3.2. Total flavonoids content (TFC)

TFC was estimated by the AlCl3 colorimetric method adapted to 96-well microplates [59]. The extracts (30 µL at the concentration of 1 mg/mL) were mixed in 96-well plates with 180 µL of distilled water and 10 µL of 5% NaNO2, and incubated for 6 min. Then, 20 µL of 10% of AlCl3 (in methanol) was added. After 6 min. 60 µL of 4% NaOH was added and the plates further incubated for 15 minutes. Absorbance was measured at 510 nm in a microplate reader (Biotek Synergy 4). Different concentrations of rutin were used as standard compound for the quantification of TFC. Results were expressed as milligrams of rutin equivalents per gram of extract (mg RE g-1, DW).

2.3.3. Condensed tannins content (CTC)

CTC was evaluated by the 4-dimethylaminocinnamaldehyde-hipocloric acid (DMACA-HCl) colorimetric method adapted to 96-well microplates [59]. Aliquots of the extracts (10 µL at 1 mg/mL) were mixed with 200 µL of 1% of DCAMA (in methanol), and 100 µL of 37% HCl. After 15 minutes of incubation, absorbance was measured at 640 nm in a microplate reader (Biotek Synergy 4). CTC was calculated based in a standard curve of different concentrations of catechin and the results were expressed as milligram of catechin equivalents per gram of dry weight (mg CE g-1 DW).

2.3.4. Hydroxycinnamic acid derivatives

Hydroxycinnamic acid derivatives were detected by spectrophotometry according to the method described in Obied et al. [60], modified to 96-well microplates. The extracts (20 µL at the concentration of 1 mg/mL) were placed in 96-well plates, diluted with 20 µL of an aqueous ethanol solution (95%, v/v) containing 0.1% hydrochloric acid and mixed with 160 µL of 2% hydrochloric acid. The absorbance was measured at 320 nm (Biotek Synergy 4), and results were expressed as caffeic acid equivalents milligrams per gram of extract (mg CAE g-1, DW) using a calibration curve of caffeic acid.

2.3.5. Flavone and flavonol content

Flavone and flavonol content was quantified according to the method described by Ahn et al. [61], with modifications. Briefly, 50 µL of 2% AlCl3-ethanol solution was added to 50 µL of the extracts at the concentration of 1 mg/mL or quercetin as standard. After 1 h at RT, the absorbance was measured at 420 nm (Biotek Synergy 4). Results were expressed as quercetin equivalents in milligrams per gram of extract (mg QE g-1, DW).

2.3.6. Hydroxycinnamic acid derivatives

Hydroxycinnamic acid derivatives were detected by spectrophotometry according to the method described in Obied et al. [60], modified to 96-well microplates. The extracts (20 µL at the concentration of 1 mg/mL) were placed in 96-well plates, diluted with 20 µL of an aqueous ethanol solution (95%, v/v) containing 0.1% hydrochloric acid and mixed with 160 µL of 2% hydrochloric acid. The absorbance was measured at 320 nm (Biotek Synergy 4), and results were expressed as caffeic acid equivalents milligrams per gram of extract (mg CAE g^-1, DW) using a calibration curve of caffeic acid.

2.4. Determination of antioxidant activity by radical-based methods

2.4.1. Radical scavenging activity (RSA) on DPPH radical

RSA on the DPPH free radical was evaluated according to Brand-Williams et al., (1995) adapted to a 96-well microplate scale (Moreno et al., 2006). Samples (22 µl at concentrations ranging from 0.25 to 10 mg/ml) were mixed with 200 µl of DPPH solution (120 µM) in methanol in 96-well flat bottom microtitration plates, and incubated in darkness at room temperature (RT) for 30 min. The absorbance was measured at 515 nm and RSA was expressed as percent inhibition, relative to a control containing DMSO in place of the sample. Butylated hydroxytoluene (BHT) was used as a positive control at the same concentrations of the extracts.

2.4.2. Radical scavenging activity (RSA) on ABTS radical

The RSA on ABTS radical was evaluated by the method described by Wang et al. (2007). A stock solution of ABTS•+ (7.4 mM) was generated by reacting equal amounts of ABTS with potassium persulfate (2.6 mM) for 16 h in the dark at RT. The ABTS•+ solution was diluted with ethanol to obtain an absorbance of at least 0.7 at 734 nm (Biotek Synergy 4). The samples (10 μL at concentrations of ranging from 0.25 to 10 mg/ml) were mixed in 96-well microplates with 190 μL of ABTS•+ solution. After a period of incubation of 6 min the absorbance was measured at 734 nm (Biotek Synergy 4). Results were expressed as RSA (%) relative to a control containing DMSO. BHT was used as the positive control at the same concentrations of the extracts.

2.4.3. Nitric oxide (NO) scavenging activity

The NO scavenging activity was evaluated according to Ho et al (2010). The extracts (50 µl at the concentrations of 1, 5 and 10 mg/ml) were mixed in 96 well plates with 50 µl of 10 mM sodium nitroprusside in phosphate buffer (PBS) and incubated in the light for 90 min at RT. Then, 50 µl of Griess reagent (1% of sulphanilamide and 0.1% of naphthylethylenediamine in 2.5% HPO3 were added and absorbances were read at 546 nm. Ascorbic acid was used as the positive control at the concentration of 1 mg/ml.

2.5. Antioxidant activity by metal-related methods

2.5.1. Reducing power activity (iron(III) to iron(II) reduction)

The ability of the extracts to reduce Fe3+ was assayed by the method of Oyaizu (1986), and modified by Megías et al (2009). Samples (50 µl at the concentrations of 1, 5 and 10 mg/ml), distilled water (50 µl) and 1 % potassium ferricyanide (50 µl) were mixed and incubated in at 50 ºC for 20 min. Then, 50 µl of 10 % trichloroacetic acid (w/v) and ferric chloride solution (0.1 %, w/v) were added, and the absorbances were measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. BHT was used as a positive control at the concentration of 1 mg/ml.

2.5.2. Metal chelating activity on copper

The Cu2+ chelating activity was determined using pyrocatechol violet (PV) as described by Megías et al. (2009). Samples (30 µl at the concentration of 1, 5 and 10 mg/ml) were mixed in 96-well microplates with 200 µl of 50 mM Na acetate buffer (pH 6), 6 µl 4 mM PV in above buffer and 100 µl of CuSo4•5H20). The change in color of the solution will be measured at 632 nm using a microplate reader. The synthetic metal chelator EDTA was be used as a positive control at the concentration of 1 mg/ml.

2.5.3. Metal chelating activity on iron

The Fe2+ chelating activity was determined by measuring the formation of the Fe2+ ferrozine complex according to Carter et al. (2002) and Megías et al (2009), with some modifications. Samples (30 µl at the concentration of 1, 5 and 10 mg/ml) were mixed in 96-well microplates with 200 µl dH20 and 30 µl of a FeCl2 solution (0.1 mg/ml in water). After 30 min, 12.5 µl of ferrozine solution (40 mM in water) was added. Change in color was measured in a microplate reader at 562 nm. The synthetic metal chelator EDTA was used as a positive control at the concentration of 1 mg/ml.

3. Results and Discussion

   1. The Extraction yield and phenolic content of the extracts

EXTRACT	SOLVENT	YIELDS OF EXTRACT (g)	YIELDS OF EXTRACT (%)	TPC (mg GAE/g DW)
ROOTS	HEXANE	0,0366	0,36	15,51±0,54
	MeOH	1,6415	16,39	25,01±1,20
LEAVES	HEXANE	0,1526	1,52	24,55±0,76
	MeOH	2,0702	20,68	36,4±0,95

Table 1. Extraction yields and total phenolic content of plant extracts.

The yield of plant hexane and methanolic extracts and the concentration of total phenolic content for both roots and leaves (mg/1 g dry weight) are shown in (Table 1, Figure 1). TPC was estimated by usin Folic- Ciocalteu metod. Total phenolic content of the different extracts was solvent dependent and expressed as milligrams of gallic acid equivalents (GAE) equivalent. From the table, C.nodosa show the highest extraction yields (20,68%) and total phenolic content (36,42%) for methanolic extract of leaves among the samples. The efficiency of extraction yields of hexane extracts were lower than the methanolic ones. Based on Herode_ et al. (2003), the percentage of extraction yields will increase with the particle size of sample, temperature extraction and the ratio of solvent and sample extraction. From the analysis, it also showed that hexane extract of roots had the lowest total phenolic content. Different levels reported in these study may be attributed to the different solvent used for extraction, procedures and standards used to express as total phenolic contents. Moreover various phenolic compounds have different response to this assay (Singleton and Rossi, 1965). However, the measurement of colour changes after 90 minutes incubation could be used to determine the existence of phenolics in samples. This may due to the antioxidant properties of plant extract that react as reductant agent which known as redox action. Further investigation of the phenolic composition of the extracts in order to identify the main compounds is needed.

Figure 1. Total phenolic content.

1. Total flavonoids content

The content of flavonoids expressed as rutin equivalents, varied from 289,22 to 531,18 mg rutin equivalent/g extract (Figure 3, Table 2). The hexane extract from leaves shown the highest amount of flavonoids contents.

Figure 3. Total flavonoids content (TFC). Results were expressed as milligrams of rutin equivalents per gram of extract (mg RE g-1, DW).

Extract	Solvent	TPC	TFC	CTC	Hydroxycinnamic Acid deriviates content
Roots	hexane	15,51±0,54	338,63±118,49	-	90,09±6,29
	MeOH	25,01±1,20	289,21±231,56	32,54±1,81	-
Leaves	hexane	24,55±0,76	531,18±149,89	-	43,72±1,93
	MeOH	36,4±0,95	429,80±188,79	-	26,52±2,19

Table 2. Total phenolics, flavonoids, tannins and hydroxicynnamic acid deriviatesld of hexane and methanol extract of leaves and roots of C. nodosa. Each value in the table is represented as mean ± SD (n = 3).

1. Condensed tannins content (CTC)

Tannins were recorded only in hexane extract of leaves (Figure 4, Table 2.). The results show that hexane is the best solvent for extracting tannins, while in methanol extracts condensed tannins content was under detectable level. The fact that tannins were detected only In hexane extract of leaves suggest that hexane is a better solvent for extraction of tannins than methanol. Furthermore the presence of tannins In leaves can be explained by the fact that tannins are especially common in leaves.

Figure 4. Condensed tannins content calculated based in a standard curve of different concentrations of catechin and the results were expressed as milligram of catechin equivalents per gram of dry weight (mg CE g-1 DW).

1. Hydroxycinnamic acid deriviates

The phenolic acids values were expressed as caffeic acid equivalent (mg CAE/1 g DW). The results are presented in Table 2 and Figure 5. The highest amount of the phenolic acids was determined in hexane extract of roots (90,09±6,29 mg CAE/1 g DW).

Figure 5. Results were expressed as caffeic acid equivalents milligrams per gram of extract (mg CAE g-1, DW) using a calibration curve of caffeic acid.

1. Antioxidant activity evaluation. (IC50 )values of antioxidant activities

The proton radical scavenging action is known to be one of the various mechanisms for measuring antioxidant activity. Table 3 shows the IC50 values of DPPH and ABTS radical scavenging activity of the hexane and methanolic extracts of C. nodosa roots and leaves. The DPPH test provides information about the activity of test compounds with stable free radicals and its effect is thought to be due to their hydrogen donating ability. For the DPPH radical, methanolic extract of leaves showed lowest DPPH based IC50 (0,99 mg/ml) while hexene extract of roots (4,89 mg/ml) had the highest IC50 value (Table 3). Higher the IC50 value signifies less antioxidant activity and vice-versa.

For the ABTS radical, the methanolic extract of leaves showed the highest activity with the IC50 value of 1,26 mg/ml while values observed for other samples were slightly higher and were given in the Fig. 3. Apparently, the antioxidant activities of other samples showed very similar trend as observed with DPPH method. The DPPH activity of all the samples studied were nearly twice lower than their ABTS values (Table 3). Factors like stereo-selectivity of the radicals or the solubility of the extract in different testing systems have been reported to affect the capacity of extracts to react and quench different radicals (Yu et al., 2002). Wang et al. (1998) found that some compounds which have ABTS scavenging activity did not show DPPH scavenging activity. This further showed the capability of the extracts to scavenge different free radicals in different systems, indicating that they may be useful therapeutic agents for treating radical-related pathological damage. Nitric oxide (NO) scavenging activity assay didn’t indicate any activity for all the extracts.

Reducing power is to measure the reductive ability of antioxidant, and it is evaluated by the transformation of Fe (III) to Fe (II) in the presence of the sample extracts. The ability to reduce Fe (III) may be attributed from hydrogen donation from phenolic compounds (Shimada et al., 1992) which is also related to presence of reductant agent (Duh, 1998). In addition, the number and position of hydroxyl group of phenolic compounds also play a role in their antioxidant activity (Rice-Evans et al.,1995). In this assay hexane extract of roots didn’t show any activity. That may be due to the presence of different phenolic compounds extracted in hexane that may be influencing the reaction. For the hexane leaves extract as well as methanolic extracts of leaves and roots the antioxidant activity was very similar and the IC50 value varies from 4,38 mg/ml for leaves methanolic extract to 6,04 mg/ml for roots methanolic extract (Table 3).

The iron chelating activity assay indicated the antioxidant activity only for the hexane extract of leaves (Table 3), while the copper chelating activity assay shown activity for all the extracts apart from the hexane roots one. The values of the IC50 for metal chelating and reducing power activity assays are significantly higher then the ones obtained for radical – based methods what means in this case that the antioxidant activity is much lower.

Table 3. IC50 values of antioxidant activities.All the calculations were performed using GraphPad Prism 6.

Extracts of C.nodosa in methanol were more effective than those in hexane and the efficiency of the extraction in methanol was significantly higher. In addition, the leaves extracts had greater activity than at the roots extracts. Hight content of the phenolics like flavonoids and hydrixycinnamic acid deriviates, evaluated during this study in the extracts, may contribute to the high antioxidant activitiy of seagrass. These results indicate that extraction of bioactive molecules from natural sources such as halophyte species, with appropriate solvents, can provide fractions with high biological activity that could be used as preservatives in food or pharmaceuticals. Antioxidant activity proved by at least 3 different assays for roots and leaves in two diferent solevent and the values of IC50 makes the C. nodasa a potential candidate of new sources of phenolics, thus antioxidant (Table 3, Figure 6,7,8,9).

Figure 6. IC50 for hexane root extract evaluated for 6 different assays.

Figure 7. IC50 for hexane leaves extract evaluated for 6 different assays.

Figure 8. IC50 for methanol root extract evaluated for 6 different assays.

Figure 9. IC50 for hexane root extract evaluated for 6 different assays.

ADDITIONAL PROJECT

After I evaluated the antioxidant activity of C. nodosa I was asked to check the antioxidant activity of Sabella spallanzanii. It is a species of marine polychaete worms in the family Sabellidae and the most common names include the the Mediterranean fanworm and the feather duster worm. European fan worms grow to a total length of 9 to 40 cm (4 to 16 in) and are usually larger in deep water. They have stiff sandy tubes formed from hardened mucus secreted by the worm which protrude from the sand, and a two-layered crown of feeding tentacles which can be retracted into the tube. One of the layers forms a distinct spiral. The colour of the tentacles is variable but they are usually banded in orange, purple and white or they may be a uniform pale grey. Various epiphytic organisms settle and grow on the tubes which may be rather wrinkled near their bases.I prepared acetone and DCM extracts of both parts- the worm and the tube in order to evaluate the antioxidant activity using the same 6 different assays as for the seagrass. The total phenolic content was estimated using Folin - Ciocalteu method. The publications is in preparation and the results I obtained are shown in Table A1 and Figures A1,A2,A3,A4,A5.

	IC50
	DCM
	TUBE
DPPH	0
ABTS	9,2417
IRON_CHELATING	1,3740
COPPER CHELATING	4,2357
IRON REDUCING	2,5123
NO ASSAY	0

Table A1. IC50 values of antioxidant activities.All the calculations were performed using GraphPad Prism 6.

Figure A1. Total phenolic content of S. Spallanzanii extracts.