Feverfew as a source of bioactives for functional foods: Storage stability in model beverages

JOURNAL OF FUNCTIONAL FOODS

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Feverfew as a source of bioactives for functional foods: Storage stability in model beverages Eunice N. Marete, Jean-Christophe Jacquier, Dolores O’Riordan* Institute of Food and Health, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

A R T I C L E I N F O

A B S T R A C T

Article history:

The potential of feverfew infusions, used traditionally for the treatment of various ailments

Received 15 June 2010

associated with pain and inflammation, was investigated as a source of nutraceuticals in

Received in revised form

the manufacture of beverages with anti-inflammatory properties. Acidified feverfew model

10 January 2011

beverages (2.9 6 pH 6 6) were stored at 5 and 22 C in order to assess the degradation of one

Accepted 14 January 2011

of the key bioactives, parthenolide and their colour stability. Parthenolide degradation fol-

Available online 22 February 2011

lowed pseudo-first order kinetics. The acidic infusions (pH 6 4.6) exhibited good colour stability at both 5 and 22 C, but the rate of parthenolide hydrolysis increased dramatically

Keywords:

with decreasing pH. In contrast, more neutral infusions (pH 6.0) exhibited higher partheno-

Feverfew infusions

lide stability, but displayed progressive browning possibly due to enzymatic oxidation of

Parthenolide

phenols. Therefore, refrigerated storage of mildly acidic infusions (pH 4.6) was found to

Colour

be the optimum for colour retention, phenolic and parthenolide content with a shelf-life

Stability Beverages

1.

of approximately 4 months.

Introduction

The incorporation of bioactive compounds into functional foods is a rapidly growing market (Bech-Larsen & Scholderer, 2007; Maria, 2006). In the functional food category, functional beverages are the fastest growing segment with strong consumer interest in foods with energy enhancing, anti-ageing, relaxing and general well being properties. There is also increased consumer interest in traditional products with preference for organic plant ingredients (Gruenwald, 2009). As a result, recent studies have focused on the potential of phytochemicals as natural sources of health promoting ingredients for functional foods and beverages (Butt, Nazir, Sultan, & Schroen, 2008; Harbourne, Jacquier, & O’Riordan, 2008, 2009; Rupasinghe, Wang, Huber, & Pitts, 2008). Feverfew (Tanacetum parthenium) is a medicinal plant that has been used traditionally to treat various conditions including prophylaxis of migraine headaches, relief of pain and inflammation from arthritis. Pharmacological studies indi-

 2011 Elsevier Ltd. All rights reserved.

cate that a sesquiterpene lactone, parthenolide (Fig. 1) is responsible for the biological activity of feverfew preparations (Kang, Chung, & Kim, 2001; Miglietta, Bozzo, Gabriel, & Bocca, 2004; Won, Ong, Shi, & Shen, 2004). In addition, feverfew contains phenolic compounds which have been reported to possess anti-inflammatory activity (Williams, Harborne, Geiger, & Hoult, 1999). The medicinal property associated with this plant is an indication that it may provide opportunities as a source of bioactives in the development of beverages with anti-inflammatory properties. Recent studies in our laboratory have shown that the extraction temperature had a significant effect on the colour and consequently on the phenolic content of aqueous feverfew infusions. Low extraction temperature of 20–70 C resulted in dark coloured infusions with low concentration of phenols due to enzymatic activity. Infusions prepared at higher temperatures (P80 C) were light coloured and yielded higher amounts of phenols and parthenolide, therefore providing a good condition for the preparation of infusions suitable for

* Corresponding author: Tel.: +353 1 7167016; fax: +353 1 7161147. E-mail address: [email protected] (D. O’Riordan). 1756-4646/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2011.01.004

JOURNAL OF FUNCTIONAL FOODS

CH3

H3C

O

CH2 O O

Fig. 1 – Structure of parthenolide.

possible incorporation into functional beverages (Marete, Jacquier, & O’Riordan, 2009). Nevertheless, prior to incorporation into an acidic food product such as a functional juice drink, the stability and other attributes of the feverfew infusions like the colour need to be examined as they may affect the potency and consumer acceptability of the final product. Previous studies on the stability of parthenolide in feverfew solutions within a pH range of 2.4–7.2 at 20–25 C have shown that parthenolide degraded faster at acidic pH compared to neutral pH (Fonseca, Rushing, Thomas, Riley, & Rajapakse, 2006). However, the exact degradation kinetics of parthenolide was not assessed. These authors also mentioned a rapid discolouration of the feverfew solutions at pH greater than five; however, they did not study the effect of storage time or other storage temperatures on the colour of the infusions. Other authors have examined the degradation kinetics of parthenolide (pH 1–9) at ambient temperature (Jin, Madieh, & Augsburger, 2007), albeit in alcoholic solutions (50% ethanol medium). Although parthenolide was found to be more stable in the pH range 5–7, this study did not consider the colour stability of the infusions as their analysis was focused on the stability of the compound in a medium suitable for pharmacological preparations. Furthermore, these studies did not examine the stability of the active ingredients at lower temperatures (e.g., refrigerated temperatures) which are commonly used for storage and distribution of food and beverage products. Therefore, the objectives of this study were first to evaluate the effect of pH on the degradation kinetics of parthenolide with storage temperature (5 and 22 C) in acidified aqueous solutions. The pH range studied was representative of that found in non-alcoholic beverages. Secondly, this study intended to determine the optimum storage conditions in order to preserve the colour of these infusions without affecting their potency.

2.

Materials and methods

2.1.

Plant material

Organically grown feverfew was harvested in Roscommon, Ireland. The aerial parts were frozen at 20 C and subsequently dried for 72 h using a freeze dryer (Edwards Super Modulyo, Davidson and Hardy Ltd., Belfast, UK). The dried samples were then ground into a moderately fine powder (180–355 lm) ( WHO, 1998) using a lab mill with a sieve size of 3 mm (Christy and Norris Ltd., Ipswich, UK).

2.2.

Extraction

Feverfew powder (5.0 g) was extracted in 200 ml of distilled water at 100 C for 10 min as described by Marete et al. (2009), with the aim of maximising the bioactive constituents

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and minimising polyphenol oxidase (PPO) activity. Extraction was carried out in triplicate. The pH of the infusions was then adjusted using various concentrations of citric acid (Reagent grade, Fisher Scientific, Leicestershire, UK) and sodium citrate (Reagent grade, Hopkin and Williams, Nottinghamshire, England) to achieve a pH of 2.9, 3.7, 4.6 and 6.0 with a final citric/ citrate concentration of 0.06 M to mimic the citric content in fruit beverages. To minimise microbial degradation, 300 ppm of potassium sorbate (Reagent grade, Hoechst, Frankfurt, Germany) and 250 ppm of benzoic acid (Reagent grade, Sigma-Aldrich, Munich, Germany) were added as preservatives. These preservatives did not have a significant effect on the total phenol content. Additional filtration using Acrodisc 0.2 lm GHP (hydrophilic polypropylene) membrane (Pall Life Sciences, Hampshire, UK) was carried out to prepare samples for HPLC analysis.

2.3.

Determination of the total phenolic content

The total phenolic content of the feverfew infusions was measured spectrophotometrically at 760 nm (UV-1240, Shimadzu, Kyoto, Japan) after reaction with the Folin-Ciocalteu reagent (Reagent grade, Sigma-Aldrich, Munich, Germany), as described by Marete et al. (2009). Results were expressed as mg of gallic acid equivalents (GAE) per ml of the infusion.

2.4.

Colour changes of feverfew infusions during storage

Colour of feverfew infusions was determined using a Chroma metre (CR-300, Minolta Ltd., Milton Keynes, Buckinghamshire, UK). The Hunter Lab scale was used with L*, a* and b* coordinates expressing the lightness, redness-greenness and blueness-yellowness, respectively. The coordinates a* and b* can be used to express the hue angle (h) and chroma (C*) according to McGuire (1992). Hue angle is defined as a colour wheel with red-purple at an angle of 0, yellow at 90, bluish-green at 180 and blue at 270 whereas chroma is the measure of colour saturation.

2.5. Separation and quantification of parthenolide in feverfew infusions Analysis of parthenolide content in the feverfew infusions was carried out using an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA) as described in detail in Marete et al. (2009). Reversed phase chromatography was performed using an Agilent ZORBAX Eclipse XDB-C18 column (150 mm · 4.6 mm i.d.; 5 lm), mobile phase of acetonitrile (HPLC grade, Sigma-Aldrich):water (55:45 v/v), flow rate of 1 ml/min and UV detection at 210 nm. The injection volume was 20 ll. Parthenolide (HPLC grade) purchased from Extrasynthese, Genay, France was used as the standard. To determine the stability of parthenolide with storage temperature, the infusions at each pH level (2.9, 3.7, 4.6 and 6.0) were divided into two aliquots and kept at 5 ± 1 or 22 ± 2 C in clear vials. The residual parthenolide content in the samples was analysed with storage time. To determine the kinetic parameters, a first order Eq. (1) was used.

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ð1Þ

where C is the parthenolide concentration at time t (mg/ml), Co is the initial parthenolide concentration (mg/ml), k is the first order rate constant (h1) and t is the storage time (h). In addition, parthenolide shelf-life (t0.1, time required to cause a 10% degradation of the total parthenolide content) was calculated according to Eq. (2). t0:1 ¼

 lnð0:9Þ k

ð2Þ

58 56 54

L* values

C ¼ Co expðktÞ

3 ( 2 0 1 1 ) 3 8 –4 3

52 50 48

2.6.

Data analysis

46

All measurements were carried out on three independently prepared infusions at each pH level. Statistical analysis was performed using SAS software (SAS Institute, Cary, NC, USA) version 9.1.3. Tukey pairwise comparison test was used for comparison of means.

0

The colour of the infusions at acidic pH levels (2.9, 3.7 and 4.6) did not change significantly with time (up to 9 months) at both storage temperatures of 5 and 22 C. However, the storage temperature had a major influence on the colour evolution of infusions at pH 6.0 with storage time. As shown in Fig. 2a, the lightness of these infusions decreased from 56 ± 1 to 47 ± 0.1 after 56 days of storage at 22 C whereas the lightness of the infusions stored at 5 C only changed from 56 ± 1 to 52 ± 0.3. The storage temperature also had a major effect on the chroma (saturation) of these infusions. Storage of the infusions at 22 C caused a significant decrease in the chroma from 11.5 ± 0.8 to 1.0 ± 0.1 after 56 days of storage whereas storage of the infusions at 5 C caused only a slight decrease from 11.5 ± 0.8 to 8.7 ± 0.6. The effect of storage temperature on the hue angle of infusions at pH 6.0 is shown in Fig. 2b. Infusions stored at 22 C were initially yellowish in colour with a hue angle of 52 ± 2 but progressively changed to reddish-brown with a hue angle of 330 ± 5 after 56 days of storage. Storage of these infusions at 5 C did not have a significant effect on the hue angle. Interestingly, it seems that the lightness, chroma and hue angle values of these infusions stored for 56 days at 22 C are similar to those observed for infusions prepared using lower range extraction temperatures of 20–70 C (Marete et al., 2009). In this study it was shown that the change in colour was due to phenol oxidation. Therefore, the total phenol content of these infusions was measured in order to assess the cause of browning upon storage.

3.2. Effect of pH and storage temperature on the phenolic content of feverfew infusions The phenolic content of feverfew infusions at pH 2.9–6.0 stored at 5 C was not significantly affected after 56 days of storage (Fig. 3a). In contrast, the phenolic content of the infusions (pH 6.0) stored at 22 C showed a significant (p 6 0.05)

20

30

40

50

60

50

60

90

Results and discussion

3.1. Effect of pH and storage temperature on the colour of feverfew infusions

10

Storage time (days)

45

Hue angle (hº)

3.

44

0

315

270 0

10

20

30

40

Storage time (days)

Fig. 2 – Colour of feverfew infusions at pH 6.0 with storage at 5 C (s) and 22 C (d), (a) L* values and (b) hue angle. (The broken lines are visual fit only.) decrease from 73.1 ± 2.9 to 43.6 ± 4.1 mg/100 ml GAE after 56 days of storage (Fig. 3b). There was a smaller but nonetheless significant decrease in the phenolic content of the lower pH infusions (pH 2.9 and 4.6) at 22 C observed after 56 days of storage (Fig. 3b). Browning of the infusions (pH 6.0) with storage at 22 C coincided with significant phenolic degradation of 40% which might have occurred as a result of residual enzymatic activity. Marete et al. (2009) have shown that the presence of the enzyme, polyphenol oxidase caused browning of feverfew infusions at extraction temperatures of 20–70 C. The optimum pH for polyphenol oxidase activity in plants varies but is generally at or near neutral pH values (Gundogmaz, Dogan, & Arslan, 2003; Yoruk & Marshall, 2003). Therefore, there is a likelihood that feverfew infusions would exhibit a residual enzymatic activity at a pH of around 6.0 even though the extractions were performed at the optimum temperature of 100 C for 10 min. Acidification of the infusions below pH 5 seems to inhibit enzymatic activity and result in stable infusions both in terms of colour and phenolic content. Nevertheless, as Fonseca et al. (2006) and Jin et al. (2007) have shown that this acidification could potentially impact negatively on the parthenolide content, the next part of this study dealt with the degradation kinetics of parthenolide at various pH levels.

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600

a a

b b

c c

d d

500

60 400

mAU

Total phenols (mg/100ml GAE)

80

40

300

200

Parthenolide

20 100

0

0 pH 6.0

pH 4.6

pH 3.7

pH 2.9

0

2

pH of the infusions

4

6

8

Time (minutes)

Fig. 4 – Sample HPLC chromatogram of feverfew infusion.

a

c d

e

f e

g

60

5

b Parthenolide (mg/100ml)

Total phenols (mg/100ml GAE)

80

40

20

0 pH 6.0

pH 4.6

pH 3.7

pH 2.9

4

3

2

1

pH of the infusions

Fig. 3 – Effect of pH and storage time on the total phenol content at (a) 5 C and (b) 22 C at day 0 (j) and 56 days (h) of storage. Bars with same letters are not significantly different at p P 0.05.

3.3. Effect of pH, storage temperature and time on the stability of parthenolide in feverfew infusions A sample HPLC chromatogram of feverfew infusion showing parthenolide is shown in Fig. 4. Storage temperature and pH of feverfew infusions had a significant effect on the degradation of parthenolide. The effect of pH on the parthenolide degradation with storage at 22 C is shown in Fig. 5. The relationship between the residual concentration of parthenolide and the storage time followed a pseudo-first order kinetics. The degradation rate constants at different pH levels were determined by non-linear regression analysis (Table 1) with excellent correlation coefficient (r2 > 0.99). A similar trend on parthenolide degradation was observed in the samples stored at 5 C. From both Fig. 5 and Table 1, it is clear that parthenolide in lower pH samples degraded faster than at more neutral pH levels. These observations are in agreement with Fonseca et al. (2006) who reported that parthenolide degraded faster in low pH aqueous solutions than at neutral pH, although the authors did not determine the degradation kinetics. Similarly, Jin et al. (2007) reported first order degradation kinetics of parthenolide in feverfew alcoholic solutions (pH 1–9) kept

0 0

1000

2000

3000

4000

Time (hours)

Fig. 5 – Parthenolide degradation in feverfew infusions at pH 2.9 (d), 3.7 (m), 4.6 (j) and 6.0 () at 22 C.

at room temperature. The authors found that parthenolide was stable in the pH range of 5–7, becoming unstable when the pH was below three or above seven. Degradation of parthenolide at low pH (Jacobsson, Kumar, & Saminathan, 1995) has been shown to be due to the hydrolysis of the epoxide ring to give trans diol derivatives (Smith, 1994). The pH-rate profiles of parthenolide hydrolysis at both 5 and 22 C are shown in Fig. 6. It is evident that the pH-rate profile is not temperature dependent, with just a shift towards higher rates at higher temperature. These profiles illustrate the relative stability of parthenolide at neutral pH values compared to acidic pH levels where a marked increase in the rate of hydrolysis is noticed. These pH-rate profiles were modelled according to Eq. (3). k ¼ ko þ A½H3 Oþ 

ð3Þ

where ko is the first order rate constant for decomposition at neutral pH and A is a constant which indicate the pH dependency of the degradation rate. As can be seen on Fig. 6, this model fits the experimental rate values very well (r2 > 0.996) at both temperatures. Combining Eqs. (3) and (1) will give Eq. (4):

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Table 1 – Kinetic parameters for the parthenolide degradation in feverfew infusions stored at 5 or 22 C at different pH values. 5 C 3

22 C

1

pH

k · 10

6.0 4.6 3.7 2.9

0.027 ± 0.005 0.038 ± 0.001 0.10 ± 0.01 0.73 ± 0.1

(h )

2

t0.1 (days)

3

r

162.1 116.2 43.9 6.0

k · 10

0.907 0.956 0.993 0.98

1

(h )

t0.1 (days)

0.20 ± 0.01 0.40 ± 0.01 1.2 ± 0.1 7.8 ± 0.4

22 11 3.7 0.6

r2 0.994 0.997 0.995 0.994

1.0

-1

0.8

Parthenolide (C/Co)

log(k)

-2

-3

-4

0.6

0.4

0.2

-5 2

3

4

5

6

7

pH

ð4Þ

which allows the prediction of the relative amount of residual parthenolide (C/Co) as a function of both time and pH. For example, one can estimate the storage time required to degrade 10% parthenolide at pH 4 (130 ± 10 h) or the minimum pH to ensure less than 20% degradation over 30 days (pH > 4.74). The implementation of this model on feverfew infusions at 22 C and reference pH value of four is shown in Fig. 7, by combining all experimental points of parthenolide contents at all pH values studied with a very good correlation (r2 > 0.991). This model may be useful to estimate the optimum storage parameters of functional beverages fortified with feverfew infusions.

4.

0

500

1000

1500

2000

Time equivalent pH 4 (hour)

Fig. 6 – pH-rate profile for the degradation of parthenolide in feverfew infusions at 5 C (s) and 22 C (d).   
 C ¼ exp  ko þ A H3 Oþ t Co

0.0

Conclusion

These results provide very important information for the development of functional beverages enriched with feverfew infusions. The storage temperature and pH of the feverfew infusions greatly influenced the kinetics of degradation of parthenolide, with samples near neutral pH exhibiting more stability than those at acidic pH levels. Infusions at acidic pH levels showed good colour stability with storage temperature (5 or 22 C) for more than 60 days but storage of feverfew infusions (pH 6.0) resulted in progressive browning which is due to degradation of phenols. To prepare infusions rich in phenols and with a suitable colour for incorporation into bev-

Fig. 7 – Parthenolide degradation in feverfew infusions at pH 2.9 (d), 3.7 (m), 4.6 (j) and 6.0 () at 22 C and reference pH 4. erages, feverfew infusions may be acidified to a pH value of 4.6. In addition refrigerated storage of these infusions will retain 90% of parthenolide for approximately 4 months.

Acknowledgements This work was funded by the Food Institutional Research Measure (FIRM) administered by the Department of Agriculture, Fisheries and Food, Republic of Ireland.

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