Determining Phenolic Compounds in Fruit and Vegetables

Table of contents

1. Introduction
   1. antioxidants
   2. antioxidants and health
   3. antioxidants in vegetables
   4. antioxidants in fruit
   5. phenolics
   6. carotenoids
   7. Food Synergy
   8. Objectives
2. Materials and Methods
   1. Introduction
   2. Methanolic extraction of antioxidant compounds
   3. Determination of Total Phenolic content.
   4. Ferric Reducing Antioxidant Power (FRAP) assay.
   5. Trolox Equivalent Antioxidant capacity (TEAC) assay.
   6. Determination of Total Carotenoids.
3. Results and Discussion
   1. Introduction
   2. Total Phenolic Content
   3. FRAP assay
   4. TEAC assay
   5. Total Carotenoid Content
   6. Conclusion
4. Bibliography

1. Introduction
   1. Antioxidants:

Antioxidants are often defined as “any substance that, when present at low concentration compared with that of an oxidizable substrate, significantly delays or prevents oxidation of that substrate”,(Halliwell 1999). They are a group of compounds, available in a number of different chemical forms, all having the ability to counteract the harmful effects of free radicals. (Anbudhasan et al. 2014) Antioxidants scavenge the body for harmful species responsible for oxidative stress in the body, such as free radicals, and neutralise them by donating an electron, while remaining stable themselves (Sarangarajan et al. 2017). There are produced naturally in the body but are also occur naturally in foods such as fruit, vegetables, cereals, seeds, herbs, spices and certain beverages. (Xu et al. 2017). There are many different classifications of antioxidants, such as synthetic antioxidants, e.g Butylated hydroxyanisole (BHT), as well as enzymes with antioxidant properties such as superoxide dismutase (SOD), synthesised naturally in the body from certain proteins and minerals attained from certain foods. Vitamins such as A, E, C and beta carotene are often referred to as low molecular weight antioxidants, and cannot be synthesised in the body, therefore must be acquired from a healthy and balanced diet. Plant derived antioxidants or Phytochemicals, are a vast group of antioxidants predominately found in fruits and vegetables, and are the antioxidants of interest in this study. Phytochemicals can be divided into a number of different groups depending on their structure and function, some groups include polyphenols, carotenoids and flavonoids. Interest in antioxidants has grown greatly since the 19^th and 20^th centuries, where studies on antioxidants focused on the uses of antioxidants in industry, to prevent corrosion of metal and vulcanization of rubber, as well as methods of preservation, in particular preventing the peroxidation of lipids (Lobo et al. 2010; Cömert and Gökmen 2018). It has been discovered that antioxidants not only inhibit oxidation in foods but also in humans (Cömert and Gökmen 2018), and it is now known that antioxidants help protect the body from the harmful effects of oxidative stress, the number one precursor of a number of chronic diseases. A number of epidemiological studies have been carried out which demonstrate this, such as (Vauzour et al. 2010), which investigates the role in which antioxidant phytochemicals found in fruit and vegetables, mainly, and how they play in the prevention of cancer, CVD and neurodegeneration. It is now understood that antioxidants have a more significant role with regard to human health, predominately in the prevention of a number of chronic diseases.

1.2 Antioxidants and Health:

Free radicals are molecular species which contain an unpaired electron, making them very unstable and highly reactive (Lobo et al. 2010). These free radicals attack healthy body cells, in an attempt to stabilise themselves, resulting in damage to these cells, a common precursor in the onset of aging and certain diseases such as cancer and heart disease (Harman 1992). Free radicals are formed naturally in the body as a by-product of a number of biological processes, such as respiration. Substances such as fried foods, cigarette smoke and alcohol, due to poor diet and unhealthy lifestyle can also be responsible for ingestion of reactive nitrogen species (RNS) and reactive oxygen species (ROS), both common forms of oxidants, found in the body (Lobo et al. 2010). Oxidative stress occurs when an imbalance between oxidants and antioxidant defences occurs in the body, damaging the structure of cells and their contents, lipids, nucleic acids, proteins etc., forming abnormal cells or even killing healthy cells (Anbudhasan et al. 2014). Oxidative stress is understood to be a major contributing factor to the onset of many chronic diseases such as cardiovascular disease, diabetes, and certain forms of cancer (Lobo et al. 2010; Rahal et al. 2014) (how do antioxidants counteract oxidative stress?; Mediterranean diet?)

1.3 Antioxidants in Fruit and vegetables

A number of well-established health organisations promote eating a diet rich in fruit and vegetables, even suggesting that half our meal is composed of either fresh fruit or vegetables.(Septembre-Malaterre et al. 2017). This is mainly due to the great health benefits which can be gained from both fruit and vegetables. Fruit and vegetables are an excellent source of dietary antioxidants, for example; water soluble Vitamin C and phenolic compounds, in addition lipid soluble Vitamin E and carotenoids. Working for the body’s first defence line, carotenoids and Vitamin E, quench singlet oxygen, helping protect against oxidative stress. Whereas vitamin C and Flavonoids also contribute to reducing oxidative stress but have also shown protective activity against the α-tocopherol in human LDL, as well as regenerating Vitamin E from the α- chromanoxy radical (Podsędek 2007). A number of studies, especially on the Mediterranean diet, which has a high intake of fruits and vegetables, correlate the protection from oxidative damage in the cells to the prevention of a number of chronic diseases such as obesity, diabetes and cancer, all of which are widespread in countries following the western diet where intake of vegetables is not as high (Blasa et al. 2010).  Green leafy vegetables and brassica vegetables according to studies have the highest antioxidant capacity, followed by root crops (Lako et al. 2007). Generally raw fruit and vegetables have a higher antioxidant content, than vegetables which have being subject to processing, either cutting, freezing or cooking (Podsędek 2007).

1.4 Phenolics

Phenolics are compounds that come from a diverse group of secondary plant metabolites (Haminiuk et al. 2012). They are made naturally in plants but can also be synthesized in times of stress, such as wounding, infection and UV radiation to name a few. They have a vast range of structures, varying from that of a simple phenolic acid molecule, to more complex structures such as that of flavonoids, and even polymerised compounds such as tannins and lignins (Naczk and Shahidi 2004). Phenolic compounds contain an aromatic ring, characteristic to their structure. This aromatic ring contains one or more hydroxyl group, and in some cases phenol subunits, accounting for the variation among Phenolic compounds (Stalikas 2007). Phenolic compounds are considered antioxidant, due to a number of different chemical events they carry out, which result in antioxidant actions such as; enzyme inhibition, metal chelation, hydrogen donation from suitable groups and oxidation to non-propagating power (Parr and Bolwell 2000). Due to the free radical scavenging power of phenolic compounds, which is known to reduce oxidative stress, the risk of developing certain diseases such as cancer, autoimmune diseases and many more, is significantly reduced in a diet rich in Phenolics. (Haminiuk et al. 2012).

1.6 Carotenoids

Carotenoids are a structurally diverse group of natural pigments, synthesised by photosynthetic organisms. Carotenoids are tetraterpenes, and have structures based on a 40-carbon polyene chain containing conjugated double bounds. To date more than 750 naturally occurring carotenoids have been discovered, and classified into Xanthophyll’s, a groups of oxygenated carotenoids and carotenes, non-oxygenated carotenoids. Carotenoids are responsible for the bright colours of a number of fruit and vegetables. Such as their light harvesting role in chloroplasts, and antioxidant benefits such as antioxidant protection against ROS (Esteban et al. 2015).  Carotenoids are known as lipophilic antioxidant vitamins, and are precursors of vitamin A in the diet of humans (Bahonar et al.). Carotenoids have a number of functions in both plants and humans. The positive health effects associated with carotenoids antioxidant activity is mainly due to the ability of the conjugated double bond structure to delocalise unpaired electrons, which results in the quenching of singlet oxygen and terminate reactive free radicals formed in tissues (Jomova and Valko 2013).   Cancer studies and yellow coloured foods

1.7 synergistic, additive and antagonistic effects.

There are a number of studies with evidence to support that consistent consumption of fruit and vegetables is associated with the reduction in the risk of chronic diseases. However, after testing many studies suggest that the antioxidant levels of individual fruit and vegetables alone doesn’t correlate to such levels of health benefits (Liu 2004). In recent years, research has focused on combinations of fruits and vegetables together, and have discovered that the preventative effect of antioxidants increases greatly when fruits and vegetables are paired together, suggesting a synergistic or additive effect occurs. A lot of researchers now believe that interactions between certain phytochemicals of both fruit and vegetables are responsible for the powerful antioxidant activities and health benefits associated with a diet of varied fruits and vegetables. However, this is not always the case and it has also been observed in some studies that some combinations can have a negative impact on the antioxidant effect, referred to as antagonism. The three different interactions observed can be defined as; additive, the combination results in an antioxidant potential which is equal to the sum of the two individual fruits or vegetables. Synergistic and antagonistic interactions are associated when the result of the combined effect is greater or less than the sum of the two individual effects (Phan et al. 2016).

1.8 Objectives

2. Materials and Methods:

2.4 Ferric Reducing Antioxidant Power (FRAP) assay:

The ferric reducing antioxidant power of each sample was determined following a modified version of the method described by (Benzie and Strain 1996). The FRAP assay involves the reduction of ferric tripyridyltriazine, at a low pH, to the more complex ferrous-(2,4,6,-tripyridly-s-triazine)₂, resulting in a colour change, colourless [Fe (III)] to blue [Fe (II)]. The ferric reducing power of the different samples are measured by using the antioxidants of the various samples as reductants, and measuring the absorbance at 593 nm(Payne et al. 2013).  In order to carry out the assay, two different FRAP reagents were prepared, 10:1:1 (acetate buffer: TPTZ: FeCl₃.6H₂O) for use on the samples and 10:1:1 (acetate buffer: TPTZ: H₂O) for the construction of the standard curve. The reference standard curve was prepared using varying concentrations of FeSO₄.7 H₂O standard, prepared using different quantities from a 1mM stock solution, combined with water and the appropriate FRAP reagent as per the table below. The standards were then left to stand for 30 minutes in the dark before reading the absorbance’s at 593 nm. For preparation of the samples, added to a cuvette was 90µl of sample extract, 2 ml of appropriate FRAP reagent and 1 ml water. Before measuring the absorbance’s, the spectrometer was measured using a blank consisting of 1 ml of water and 2 ml FRAP reagent. FRAP values were then calculated and expressed as µM FeSO₄ 100g sample.

1: Quantities used for preparation of the FRAP assay standard curve.

	1	2	3	4	5	6	7	8
H2O (µl)	1000	985	970	940	880	820	760	700
FRAP (ml)	2	2	2	2	2	2	2	2
1mM Std. (µl)	0	15	30	60	120	180	240	300
Std conc (µM)	0	5	10	20	40	60	80	100

 

2.5 Trolox Equivalent Antioxidant capacity (TEAC) assay.

The Trolox equivalent antioxidant capacity (TEAC) assay is a measure of the total antioxidants present in the fruit and vegetable individually and pairing samples. A modified version of the 2, 2-diphenyl-1- picrylhydrazyl (DPPH) radical scavenging assay described by(Goupy et al. 1999) was used.

A stock solution was prepared by dissolving 0.024g of DPPH (Sigma-Aldrich Ireland Ltd) in 100 ml of methanol in darkness and left for one hour to allow for solution to stabilise.A working solution was prepared from this stock solution by diluting DPPH stock solution and methanol 1:5. It was mandatory that absorbance of this solution read between 1.2 and 1.5 at 515 nm in order to be suitable for testing.

A standard curve was prepared for this procedure using known concentrations of the antioxidant Trolox (Sigma-Aldrich Ireland Ltd). The stock solution consisted of 0.0516g of Trolox in 100 ml methanol and diluted 1:10 giving a 0.2 Mm Trolox solution. Working solutions were made using this stock solution (Table 1).The absorbance was measured at 515 nm.

All fruit and vegetable samples were tested in duplicate. 0.7 ml suitably diluted fruit and vegetable individual or pairing samples were added to Eppendorf tubes along with 0.7 ml of DPPH stock solution.  All samples were vortexed and incubated at room temperature for 30 minutes in darkness before testing at 515 nm.

Five reagent blanks consisting of 0.7 ml DPPH stock solution and 0.7 ml methanol were prepared. An average blank value was obtained from these samples. This absorbance value was halved to give a target absorbance reading. Samples were diluted to obtain a value above and below this target reading for TEAC calculations. A sample blank for each fruit and vegetable individual and pairing samples consisted of 0.7 ml of suitably diluted sample and 0.7 ml of methanol.1.4 ml methanol was used as a blank to zero the spectrophotometer. TEAC values were expressed as TEAC μmol/100g. The inverse of the concentration of the sample required to decrease the initial DPPH concentration by 50% (IC50) was also calculated. This is known as antiradical power (ARP) and is expressed as gram of Fresh Weight/L.

Trolox Solutions	Volume 0.2 Mm Trolox(ml)	Volume MeOH(ml)	Dilution
0.01	0.5	9.5	1:20
0.02	1	9	1:10
0.03	1.5	8.5	1:6:66
0.04	2	8	1:5
0.05	2.5	7.5	1:4

 

2.6 Determination of total carotenoids:

Determination of total carotenoid (TC) content required an acetone extraction to be carried on fruit samples (Biehler et al. 2010). Fruit and vegetable samples were tested individually and in pairings.7g of fresh fruit and vegetables samples were ground in a mortar and pestle with 10 ml of acetone containing Magnesium carbonate. The samples were filtered through Whatman No.4 filter paper (55.5 mm) in darkness. The resulting residue of the chopped sample was then re-extracted with 10 ml of extraction solvent each time until it ran clear. After the final extraction, the combined filtrate volume was brought up to 40 ml with acetone. Centrifugation was carried out at 14000 rpm for 5 minutes at room temperature. The samples were then placed in glass cuvettes and their absorbance read using a spectrophotometer (Shimadzu Corp, serial no. A11634900311, model UV-1800 240V IVDD) at three different wavelengths: 470 nm, 662 nm and 645 nm. Samples were re-diluted if absorbance read greater than 0.8. A blank of acetone was used to zero the spectrometer prior to use. The total carotenoid concentration was calculated by the following equations and expressed as μg/g fresh weight:

Chlorophyll A (Ca) (μg/ml extract) = (11.75)(A662) – (2.35)(A645)

Chlorophyll A (Cb) (μg/ml extract) = (18.61)(A645) – (3.96)(A662)

Total Carotenoids (μg/ml extract) = (1000)(A470) – (2.27) (Ca) – (81.4) (Cb) /227

2.7 Separation, characterisation and quantification of phenolic antioxidants by GC-MS:

In order to identify and quantify the different phenolic compounds in the fruit and vegetable samples, a modified version of the method described by (Zuo et al. 2002)  was followed. Firstly, the free phenolic acids of the fruit and vegetable samples were extracted. 120g of each fresh fruit/vegetable sample was finely chopped and homogenised with 400 ml of distilled water using an Ultra-Turrax T-25 tissue homogeniser. The sample was then filtered under vacuum and 20 ml of the obtained filtrate was acidified to pH 2, using 1N HCL. To isolate the phenolic acids from other phenolic moieties which may be present in the samples, a solvent extraction was carried out. The acidified filtrate was extracted twice with 20 ml of diethyl ether. The collected ethereal phase was then extracted twice with 20 ml of 5% NaHCO₃. The alkali aqueous solution was retained and acidified to pH 2, with 1N HCL, and then re-extracted with 20ml of diethyl ether twice.

The ethereal extracts were evaporated until dry using a rotary evaporator, under reduced pressure conditions at 35˚C. The phenolic acids were removed from the round bottomed flask to an amber GC vial using 1-2 ml of diethyl ether and a glass pipette. The diethyl ether was then evaporated using a nitrogen evaporator. The vials were sealed and stored at -80˚C until ready for derivation.

The samples were then derived by adding 50 µl of BSTFA + TMCS derivatization reagent and heating to 60 ˚C silylated for 30 minutes in a sealed mini-vial. The silylated samples were then analysed using GC-MS, following the same GC-MS conditions used in the (Zuo et al. 2002) method.

References :

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Payne, A.C., Mazzer, A., Clarkson, G.J.J. and Taylor, G. (2013) ‘Antioxidant assays – consistent findings from FRAP and ORAC reveal a negative impact of organic cultivation on antioxidant potential in spinach but not watercress or rocket leaves’, Food Science & Nutrition, 1(6), 439-444, available: http://dx.doi.org/10.1002/fsn3.71.

Zuo, Y., Wang, C. and Zhan, J. (2002) ‘Separation, Characterization, and Quantitation of Benzoic and Phenolic Antioxidants in American Cranberry Fruit by GC−MS’, Journal of Agricultural and Food Chemistry, 50(13), 3789-3794, available: http://dx.doi.org/10.1021/jf020055f.

 

4.0 Bibliography

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Zuo, Y., Wang, C. and Zhan, J. (2002) ‘Separation, Characterization, and Quantitation of Benzoic and Phenolic Antioxidants in American Cranberry Fruit by GC−MS’, Journal of Agricultural and Food Chemistry, 50(13), 3789-3794, available: http://dx.doi.org/10.1021/jf020055f.