pHD thesechapter 2 amended

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pHD thesechapter 2 amendedpHD

    Chapter 2 Study of the properties of functional


2.1 Introduction

    2.1.1 Blackcurrant extract

    Anthocyanins in blackcurrant extract (BCE) are responsible not only for the attractive colour, but also health benefits, mainly associated with their antioxidant property (Delgado-Vargas and Paredes-López, 2003). Organic acid, phenolics and sugars were in a crude extract, which was further isolated and purified using various column chromatographic methods to get high purity anthocyanins (> 99%)(Kähönen et al., 2003; Longo and Vasapollo, 2006). The composition of BCE may vary because of different methods of extraction and variety of blackcurrant berries cultivars. The BCE used in the current study is not specified by the supplier; consequently, characterization of the blackcurrant extract was performed.

2.1.2 Methods of characterization of blackcurrant extract

    High-performance liquid chromatography (HPLC) with UV detection has been used extensively for separation and quantification of individual anthocyanins (Garzón and Wrolstad, 2001; Matsumoto et al., 2001b; Nielsen et al., 2003). Generally, a C18 column with gradient elution was used. The elution solvents consisted of an acidic solution and an organic solvent. Either 9% formic acid or 0.5% phosphoric acid or 0.1% trifluoroacetic acid can be used as the acidic solution. The organic solvent can be methanol or acetonitrile. Individual anthocyanins were detected and quantified at 520 nm. In this study, formic acid (9%) and acetonitrile were used as elution solvents. HPLC for analysis of individual anthocyanins in blackcurrant extract was performed on Phenomenex LUNA C18column; its performance was stable within an extended pH

    range from 1.5 to 10. High stability of the column in low pH conditions facilitates analysis of anthocyanins in acidic condition.

    Identification was facilitated by using on-line liquid chromatography-mass spectrometry (LC-MS) techniques (Kähönen et al, 2003; Longo and Vasapollo, 2006; Nielsen et al., 2003). LC/QTOF-MS (liquid chromatography/quadruple time of flight MS/MS) equipped with an electrospray interface was used for identification of the individual


    anthocyanins in the current study. The electrospray interface provides gentle ionization by disintegration of the aerosol solution of a sample with a countercurrent of dry heated

    gas (70ºC) into smaller charged droplets at a low voltage. As solvent vaporises, the N2

    intact molecular ions were produced in the gas-phase, then introduced into a quadrupole time of flight MS, in which the mass of the parent ions of anthocyanins were determined (Chernuchevich et al. 1997). The parent ions were then further fragmented by collision with argon gas, and the mass of the fragment ions was detected in a second MS. In the case of anthocyanins, the parent and fragment ions correspond to the flavylium ions and aglycon ions, respectively, due to loss of the neutral sugar moiety from the parent ions (McGhie et al., 2003).










    Figure 2.1 The skeleton structures of flavanone, phenolic acids, flavone, flavonol, chalcone and anthocyanidin.

    Anthocyanins and phenolics including flavanone, phenolic acids, flavone and flavonol are flavonoid compounds that naturally occur in berries. The skeleton structures of flavanone, phenolic acids, flavone, flavonol, chalcone and anthocyanidin are shown in Figure 2.1. In acid conditions, anthocyanins have the anthocyanidin structure, but


    transform into the chalcone structure in neutral and alkaline solution ( Brouillard and Dubois 1977)

    High levels of both anthocyanins (410 mg/100g of berries) and ascorbic acid (180 mg/100g of berries) in blackcurrant were reported in Anderson and Vang-Pedersen’s

    study (1993) (as cited in Iversen, 1999). Consequently, ascorbic acid is present naturally in crude BCE if the extract is not further purified. HPLC is the technique of choice for analysis of ascorbic acid in food products. (Iversen, 1999; Rodrígez-ComesaƄa et al.,

    2002; Fontannaz et al., 2006). Isocratic elution on reversed-phase columns like C18 with acidic solution such as acetic acid or phosphoric acid has been frequently used. The absorbance at 250 nm was monitored during the analysis of ascorbic acid.

2.1.3 Determination of pKa of blackcurrant anthocyanins (BCA)

    The stability and colour of anthocyanins are highly dependent on pH. In acidic solution

    +, (pH 16), BCA are in equilibrium between the three species: the flavylium cation AHcarbinol B and the quinonoidal base A. The structure of the three forms is shown in Figure 2.2.

    Anthocyanin structural transformation in acidic media is thought to follow the mechanism: (Brouillard and Dubois 1977)



    In low pH conditions (pH 1 to pH 3), BCA exists predominantly in the flavylium form, which exhibits a red colour. The proportion of colourless carbinol B increases with increasing pH to pH 5.0. The flavylium cation-carbinol equilibrium is shown below:


    The equilibrium expression is:



     ++so that on a graph of lg ([B]/[AH]) against pH, when [B]/[AH] =1 the value of pH is

    equal to pK a.



    OHOH+AHFlavylium cationBCarbinol base





    quinonoidal base or anhydro baseA

    +Figure 2.2 Fast equilibria between the flavylium cation AH, the carbinol pseudobase B and quinonoidal base A.

The pK of BCA can be determined spectrophotometrically. BCA are prepared in a

    +different pH buffer solutions. In low pH such as ~ pH=1.1, BCA exist as AH form. The absorbance of the BCA is obtained using the Beer-Lambert Law:

    ++A= εb C AH AH T

    ++where εis the molar absorptivity of AH at λ, b is the cell path length and C is AH maxT

    total BCA concentration. At pH approximately 5.0, BCA is converted totally to carbinol

    base B form. The absorbance at the same wavelength is given by

    A = εb C BB T

    where εis the molar absorptivity of B at λ. At intermediate pH the absorbance is A = B max

    +++εb C + εb C For a given C at each pH buffer, C + C= Ctherefore AH AHB B.TAHB T,

    +++[B]/[AH] = C /C = (AA)/ (A ? A) (Harris, 2003) BAHAHB


2.1.4 Stability of blackcurrant anthocyanins

    Reported studies (Cabrita et al., 2000; Brouillard, 1981; Fossen et al., 1998; Torskangerpoll and Andersen, 2005) show that the stability of anthocyanins and their colours are highly dependent on pH, due to changes in concentration of the four species: flavylium cation, quinonoidal base, pseudobase or carbinol and chalcone. Conversion of one species to another is typically accompanied by dramatic changes in colour and stability. Among the four species, the red flavylium cation present at pH 1.0?2.4 is the

    most stable (Cabrita et al., 2000). Other factors that affect the rate of anthocyanin degradation include temperature, oxygen, enzymes, light, acylation, co-pigments and metal ions (Delgado-Vargas and Paredes-López, 2003; Mazza and Brouillard, 1987). An

    acylation reaction may occur between C3 sugar residues and aromatic acids such as p-coumaric acid o, ferulic acid, or aliphatic acids such as acetic acid, by esterification, frequently occurring at the 6-OH or less frequently the 4-OH group (Giusti and Wrolstad, 2003). Acylated anthocyanins are more stable than corresponding non-acylated forms (Hoshino et al., 1980; Mazza and Brouillard, 1987, Torskangerpoll and Andersen, 2005).

    2+, Hoshino and coworkers (Hoshino et al., 1980) reported that metal ions such as Mg3++Fe and K stabilize acylated anthocyanins due to formation of anthocyanin-metal complexes. By contrast, Mazza and Brouillard, (1987) suggested that metal-anthocyanin complexes did not contribute to the stability of anthocyanins, due to the decomposition of the complexes with time. Since traces of ferric and ferrous ions from industrial processing equipment could find their way into anthocyanin solutions, the effects of ferrous and ferric ions on anthocyanins were investigated in the present work. Moreover, the changes in BCA in response to a range of physical and chemical stimuli including pH and temperature were determined in this study.

2.1.5 Determination of antioxidant activity of blackcurrant extract

    A number of assays such as oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox equivalent antioxidative capacity (TEAC) and ferric reducing antioxidant power (FRAP) that are being used to evaluate antioxidant activity depend on different mechanisms (Prior et al., 2005). The mechanism of these assays is briefly described below.

    In ORAC assay, an antioxidant is added to a mixed solution of 2,2’-azobis(2-

    amidinopropane) dihydrochloride (AAPH) with a fluorescent compound (FL) ( 9-(O-


    is generated by AAPH. The carboxyphenyl)-6-hydroxy-3-isoxanthenone). ROO

    reactions in the mixture solution are shown below:

    ROO+ FL ? ROOH + oxidised FL (loss of fluorescence).

    Ou et al., (2001) proposed the oxidation pathway of FL in presence of AAPH. In the absence of a radical scavenger, the fluorescent intensity of FL decays continually over time, giving an area under the curve (AUC). In the presence of antioxidant, blank

    ROO+ AH (antioxidant) ? ROOH + A, ROO+ A? ROOA

    and the loss of the fluorescence of FL will be retarded, giving a longer decay time of fluorescence determined as AUC. Therefore, the antioxidant capacity of the antioxidant

    antioxidant as a radical quencher can be assessed by the difference between AUC antioxidant

    and AUC(?AUC). A higher value of ?AUC indicates higher peroxyl radical blank,

    quenching capacity of the antioxidant. ORAC assay is performed at pH 7.4 (phosphate buffer).

DPPH is relatively stable free radical, whose structure is shown in Figure 2.3.


    ONNN 2


Figure 2.3 Chemical structure of 2,2-diphenyl-1-picrylhydrazyl (DPPH).

    DPPH easily dissolves in methanol or ethanol and has a purple colour with maximum absorbance at 514 nm. DPPH free radical can accept an electron or hydrogen radical. The main reaction of DPPH with phenolic compounds is:

    DPPH?+PheOH ? DPPHH + PheO

    and the resulting?decrease in concentration of DPPH? can be measured using electron spin resonance (Calliste et al., 2001; Ahn et al., 2004) or determined by the decrease in


    absorbance at 514 nm using a spectrophotometer (Espín et al., 2000; Kähkönen and Heinonen, 2003 ).

    The TEAC assay is based on the reaction between antioxidants and radical cation of 2,2′

    ?+). The decrease in the intensity of azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS

    ?+colour of ABTS reflects the scavenging ability of antioxidants to the radical cation.

    FRAP assay determines the ferric reducing ability of a sample. The FRAP reaction between antioxidants and FRAP agent prepared using acetic acid (pH 3.6) is:

    3+2+ [Fe(III)(TPTZ)] + antioxidant ? [Fe(II)(TPTZ)]22

    2+where TPTZ is 2,4,6-Tripyridyl-s-triazine, [Fe(II)(TPTZ)] which has an intense blue 2

    colour and an absorbance maximum at 593 nm. The FRAP reaction takes account of only the ferric reducing ability of a sample. Consequently, it can be argued that the ferric reducing ability has little relationship with the free radical scavenging ability of an antioxidant. However, Nielsen et al (2003) reported that FRAP assay and TEAC assay correlated well for the antioxidant capacity of black currant juices. Cao and Prior (1998) also reported that there are significant linear relationships between ORAC assay and FRAP assay in assessment of antioxidant capacity of human serum especially when protein was removed in the ORAC assay. FRAP assay has good response to BCE and is simple and easy to perform for large amount of sample.

    In summary, ORAC assay, performed at pH 7.4 phosphate buffer, evaluates peroxyl radical quenching ability of anthocyanins. DPPH assay is performed in methanol or ethanol medium for measurement of H-atom donor capacity of anthocyanins, while the FRAP assay at pH 3.6 (acetate buffer), determines ferric reducing capability of antioxidants. The structure of anthocyanins changes with pH, and so will their antioxidant activities. Combination of these assays can properly evaluate overall antioxidant capacity of BCA in biological systems and food systems. Consequently, FRAP assay was chosen to investigate the change in antioxidant activity of BCE during heating at a range of pHs. ORAC assay and DPPH assay were performed to assess the free radical scavenging ability of blackcurrant extract.


    2.1.6 Investigation of acylated anthocyanins using Fourier-transform infrared spectroscopy (FTIR)

    An FTIR spectrum provides information on functional groups and chemical bonds in

    -1) together with the organic compounds. The functional group region (3000 -1500 cm

    -1fingerprint region (900-650 cm), which is characteristic of the bending regions of the

    functional group, is of great assistance in investigation if a sample undergoes a chemical reaction and if the interaction happens between an encapsulated sample and a polymer carrier. Disappearance, appearance, shifts and broadening of bands indicate changes in structure of organic compounds (Socrates, 1998).

     by HPLC due to change in polarity of Acylated anthocyanins may be determined

    anthocyanins by esterification, resulting increase or decrease in the elution time compared to non-acylated anthocyanins (Wulf and Nagel, 1978; Hong and Wrolstad, 1990). However, the peak representing acylated anthocyanins with acetic acid was postulated and not confirmed. Hong and Wrolstad (1990) developed a simple saponifiction experiment to determine acylated pigments. The determination depends on the disappearance of the acylated anthocyanins peak after alkaline hydrolysis. However, this method only works well with the anthocyanins that are not alkaline labile (Hong and Wrolstad, 1990). Their study also reported that the relative peak area of cyanidin 3-glucoside decreased after treatment with alkaline borate buffer. According to our study, delphinidin 3-O-sugars are more labile to alkaline treatment than corresponding cyanindin 3-O-sugars. Therefore, investigation of acylated anthocyanins being produced during heating in acetic buffer was performed using FTIR. A new band

    - 1at 1740 cm(ester C=O stretching vibration) for the anthocyanins suggests that some acylated pigments were produced during heating at pH 3.6.

2.1.7 Folic acid

    The folic acid group belongs to the class of water soluble B-vitamins that are essential nutrients for cell replication as coenzymes and prevent neural tube defects (Daly et al., 1995; Berry et al., 1998). The structure of folic acid is shown in Figure 1.3.

Methods for analysis of folic acid: Although the microbiological assay procedures

    employing Lactobacillus casei, Pedicoccus cerevisiae and Streptococcus faecalis are

    sensitive for determining folate, these assays are lengthy and lack reproducibility (Day


    and Gregory, 1981). HPLC-based methods have been reported to work well for quantification of natural and added folate in foods, beverages and tablets (Saxby et al., 1983; Nguyen et al., 2003; Höller et al., 2003; Pérez Prieto et al., 2006). Most developed methods are based on solid phase extraction, clean-up and pre-treatment, followed by HPLC separation, then analysis using a UV absorbance or fluorescence detector. Isocratic elution with methanol and phosphate buffer (pH 6.8) with addition of tetrabutylammonium dihydrogen phosphate as ion-pair (Osseyi et al., 1998), and a gradient elution using a mixture of acetonitrile or methanol with phosphate buffer (pH 2.152.8) (Nguyen et al., 2003; Höller et al., 2003) or methanol and ammonium acetate (8 mM) (Pérez Prieto et al., 2006) were used to get good resolution with a C18 column.

    In this study, a large number of folic samples in pH buffers simulating gastric and enteric juices were collected during during a study of the release profile of folic acid, and it was necessary to develop a reliable and relatively simple method to provide adequate resolution from buffer interference and to maintain stability of folic acid in the samples during analysis especially using the autosample HPLC.

    Stability of folic acid: The degradation products of folic acid lack the bioactivity of folic acid. A number of factors such as temperature (Chen and Cooper, 1979; Day and Gregory, 1983; Nguyen et al., 2003), pH and buffer ions (Paine-Wilson and Chen, 1979; O’Broin et

    al.,1975) , oxygen (Chen and Cooper, 1979) and light (Thomas et al., 2000) affect the stability of folic acid. Aerobic hydrolysis of folic acid in acid conditions (Saxby et al.,1983) oxidative cleavage of folic acid with alkaline potassium permangnate (Saxby et al.,1983) and photolysis of folic acid (Akhtar et al., 1997; Thomas et al., 2000) were reported to occur by cleavage of the C?N bond between the pteridine ring and p-aminobenzoyl,

    resulting in pterin-6-carboxylic acid and p-aminobenzoylglutaminc acid. There is general

    agreement that folic acid is stable in the pH range 5.0?10.0 and increasely unstable with

    decrease in pH beyond pH 5.0. O’Broin et al. (1975) first reported that the stability of folic

    acid decreased dramatically in phosphate buffer at pH 6.0, 7.0 and 8.0. Some experimental data published shows that a lower stability form of folic acid was also observed at pH ~ 3.0 compared to that at pH 1.0 and pH 2.0 (O’Broin et al. 1975), and at pH 2.6 below

    100ºC (Paine-Wilson and Chen, 1979).


2.2 Experimental

    2.2.1 Materials

    Dried blackcurrant extract was supplied by New Zealand Pharmaceuticals Ltd (Nelson, New Zealand) and (1?3, 1?4)β-D-glucan by Gracelink Ltd. (Wellington, New

    Zealand). 2,4,6-Tripyridyl-s-triazine (TPTZ) and DMSO-d were obtained from Aldrich 6

    Chemicals (Milwaukee, WI), formic acid, ferric chloride (FeCl.6HO), ethanol and 32

    ferrous sulphate (FeSO.7HO) were Analar grade materials from BDH Chemicals 42

    (Poole,UK). Acetonitrile (HPLC grade) was from Scharlau (Barcelona, Spain). 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), 6-Hydroxy-2,5,7,8-

    tetramethychroman-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Folic acid (purity ~ 98%), tetrabutylammonium dihydrogenphosphate, and ascorbic acid were purchased from Sigma (St. Louis, MO). 9-(O-Carboxyphenyl)-6-hydroxy-3-isoxanthenone (Fluorescein) was purchased from Riedel-De Haen AG (Seelze-Hannover, Germany); sodium hydroxide was obtained from May & Baker Ltd (Dagenham, England).

    All chemicals used for preparation of buffer solutions such as acetic acid and citric acid were from BDH (Poole, UK). Ondina oil 68 (white mineral oil) was supplied by The Shell Company (Melbourne, Australia) and soybean oil was purchased in a supermarket.

2.2.2 Characterisation of blackcurrant extract (BCE)

    Identification of blackcurrant anthocyanins. Identification of the component

    anthocyanins in BCE was accomplished using an online photodiode array (PDA) together with LC/QTOF-MS (Thermo Electron Corporation, Framington, MA), and a Phenomenex LUNA C18column (4.6 mm x 250 mm, i.d. 5 μm) (Torrance, CA)

    attached to a Finnigan HPLC system (Thermo Electron Corporation). The mobile phase for LC/QTOF-MS analysis was 2.2 M formic acid (solvent A) and acetonitrile (solvent

    -1B). Elution was performed at a flow rate of 1 mL min using a gradient elution starting

    with 1% B for 0.5 min, to 7% B at 1 min; 7% B, 1.0-7.0 min; to 10% B at 9.5 min; 10% B, 9.5-13 min; to 13% B at 15 min; 13% B, 15-18 min; to 40% at 20 min; to 1% B at

    -121 min; 1% B, 21-27 min. After the sample (10 mg mL) prepared in 2.2 M formic acid

    (5?25 μL) was introduced into the HPLC column, the PDA was monitored at 520 nm.

    The elution profile for the LC/QTOF-MS system was slightly different from that for the


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