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Food Research International 35 (2002) 351–356

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Anthocyanin and antioxidant capacity in Roselle


(Hibiscus Sabdariffa L.) extract
Pi-Jen Tsaia, John McIntoshb, Philip Pearceb, Blake Camdenb, Brian R. Jordanc,*
a
Department of Food Science, National Pingtung University of Technology and Science, Taiwan 900, Republic of China
b
Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, Private Bag 11 222, New Zealand
c
Division of Soil, Plant and Ecological Sciences, PO Box 84, Lincoln University, Canterbury, New Zealand

Received 8 February 2001; received in revised form 12 July 2001; accepted 16 July 2001

Abstract
The relation between antioxidant activity and anthocyanin was determined in Roselle (Hibiscus sabdariffa L.) petals. Petals from
Roselle, cultivar F141, were collected and dried in Taitung, Taiwan. Roselle extract was prepared by extracting dried Roselle petals
in boiling water. The relation between the anthocyanin color and antioxidant capacity was elucidated by comparing absorbance at
520 nm, with ferric reducing ability of plasma (FRAP), oxygen radical absorbance capacity (ORAC) and total antioxidant status
(TAS) antioxidant assays. The results showed that the antioxidant capacity of Roselle extract increased when extraction time or
weight of petals increased. The FRAP assay showed a linear relationship with anthocyanin as determined at 520 nm. Comparisons
between FRAP and ORAC or FRAP and TAS assays gave a linear relation. These results suggest that anthocyanin is the major
source of antioxidant capacity in Roselle extract. Further purification using Amberlite XAD-2 and HPLC indicated that antho-
cyanin and a brown pigment in the extract account for about 51 and 24% of the antioxidant capacity, respectively. Under different
processing temperatures and storage periods, anthocyanin content declines. However, other phenolic compounds increase and
overall there is only a relatively small decrease in total phenolic compounds and antioxidant activity. # 2002 Elsevier Science Ltd.
All rights reserved.
Keywords: Anthocyanin; Antioxidant capacity; Roselle (Hibiscus sabdariffa L.)

1. Introduction gami, 1994) and the biological effect of anthocyanin in


low density lipoprotein and lecithin-liposome systems
Roselle (Hibiscus sabdariffa L.), an annual shrub, is (Meyer, Yi, Pearson, Waterhouse, & Frankel, 1997;
commonly used to make jellies, jams and beverages. The Satué-Gracia, Heinonen, & Frankel, 1997). Anthocya-
brilliant red color and unique flavor make it a valuable nins were also found to have many times more activity
food product. The anthocyanin pigments that create the than common antioxidants such as ascorbate (Wang,
color (Tsai & Ou, 1996) are responsible for the wide Cao, & Prior, 1997). Overall, there is now increasing
range of coloring in many foods. Recently, the biologi- evidence that antioxidants in the human diet are of
cal activities of anthocyanin, such as antioxidant activ- major benefit for health and well-being.
ity, protection from atherosclerosis and anti- Being high in anthocyanin, Roselle petal is both a
carcinogenic activity have been investigated, and shown good colorant and potentially a good source of anti-
to have some beneficial effects in the treatment of dis- oxidants. However, owing to the high reactivity of
eases. For instance, there have been reports on the anthocyanin during processing and storage, more
antioxidant activity in grape anthocyanin (Igarashi, information is needed to clarify the relation between the
Takanashi, Makino, & Yusui, 1989; Tamura & Yama- color and antioxidant activity. The focus of this
research is to determine the level of antioxidant activity
* Corresponding author. Tel.: +64-3-325-2811; fax: +64-3-325-
in Roselle extracts and how it relates to the anthocyanin
3843. content through different antioxidant assays. As part of
E-mail address: jordanb@lincoln.ac.nz (B.R. Jordan). this study, we have determined the contribution of the
0963-9969/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0963-9969(01)00129-6
352 P.-J. Tsai et al. / Food Research International 35 (2002) 351–356

ml of water. Other samples were taken from commer-


Nomenclature cially available beverages. These drink samples were
obtained from a local supermarket.
FRAP: ferric reducing ability of plasma
ORAC: oxygen radical absorbance capacity 2.2. Analysis of anthocyanin, phenols and antioxidant
TAS: total antioxidant status activity
GAE: gallic acid equivalent
CCE: catechin equivalent The filtrate was passed through an Amberlite XAD-2
CFAE: caffeic acid equivalent polymeric resin column. The column was washed with
RUE: rutin equivalent water and the adsorbed material eluted with methanol
DEL: delphinidin-3-sambubiose (Pouget, Vennat, Lejuene, & Pourrat, 1990). The eluate
A520: absorbance at 520 nm was concentrated on a rotary evaporator under reduced
pressure at 45  C. The concentrate was applied to either
a LiChroprep RP-8 or LiChrosorb RP C18 HPLC col-
anthocyanin to the total antioxidant activity through umn (Merck, Darmstadt, Germany) and the anthocya-
liquid chromatography and HPLC. In addition, we nin and other phenolic pigments eluted with a gradient
have characterised changes in anthocyanins, anti- mixture of 5% acetic acid and acetonitrile (Tsai & Ou,
oxidants and phenolic compounds during processing 1996). Fractions, 1 ml, were collected from the columns.
and storage. Anthocyanin pigments were measured by their absorp-
tion at 520 nm (Hong & Wrolstad, 1990) using a spec-
trophotometer (Ultrospec 2000, Biochrom Ltd,
2. Materials and methods Cambridge, UK), and antioxidants measured by ferric
reducing ability of plasma (FRAP), total antioxidant
2.1. Sample preparation status (TAS) and oxygen radical absorbance capacity
(ORAC) methods. On the basis of spectral identifica-
To prepare Roselle extract, the petals of Hibiscus tion, the phenol compounds were divided into four
sabdariffa, cultivar F141, were collected and dried in classes: hydroxycinnamates as caffeic acid equivalents,
Taitung, Taiwan. They were dried at 50  C for 36 h and peak 316 nm; anthocyanins as delphinidin, peak 520
stored at 25  C before extraction. Preliminary experi- nm; flavan-3-ols as catechin equivalents, peak 280 nm;
ments were carried out to establish the best extraction flavonols as rutin equivalents, peak 365 nm and hydro-
procedures to give accurate and consistent results. Sub- xybenzoates as gallic acid equivalents, peak 280 nm.
sequently a single protocol was used to study anti-
oxidant levels. The extract was routinely prepared by 2.3. Antioxidant assays
boiling 3 g of dried Roselle petals for 3 min with 300 ml
of water. The extract was rapidly filtered through a The antioxidant concentration of the Roselle extract
Buchner funnel and then chilled on ice and kept at 4  C was routinely measured by the FRAP method using a
before assay. For comparison, several samples of the Roche Cobas Fara II analyser (Roche, Basel, Switzer-
following drinks were also analyzed, 17 samples of land; Benzie & Strain, 1996). ORAC was also measured
black teas, three samples of green teas, nine samples of as it is frequently used as an international standard
red wine and nine samples of white wine. Tea and coffee (Cao & Prior, 1999). TAS was determined using a kit
samples were prepared by boiling 1 g for 3 min with 300 supplied by Randox Laboratories Ltd (Co. Antrim,

Fig. 1. Effect of (a) extraction time and (b) sample weight on the FRAP of Roselle petals.
P.-J. Tsai et al. / Food Research International 35 (2002) 351–356 353

Fig. 2. Correlation of FRAP and TAS measurement of antioxidant


Fig. 3. Correlation of FRAP and ORAC measurement of antioxidant
activity in Roselle extract.
activity in Roselle extract.

UK.). Both ORAC and TAS were also measured on a


Roche Cobas Fara II analyser.

2.4. Total phenolic compounds assay

Total soluble phenolics in the Roselle extract were


determined with Folin-Ciocalteu’s reagent by the
method of Slinkard and Singleton (1977) using gallic
acid as a standard.

3. Results and discussion

Preliminary studies were carried out to determine the


conditions needed for the extraction of antioxidant Fig. 4. FRAP activity as mean and S.D. in various beverages. *BlT,
black tea: n=17 (1 g was extracted for 3 min in 300 ml at 100  C);
activity. The antioxidant activity was low over the first *GrT, green tea: n=3 (1 g was extracted for 3 min in 300 ml at 100  C);
minute of extraction, but then increased rapidly and *CfB, coffee: n=3 (1 g was extracted for 3 min in 300 ml at 100  C);
remained at the same level for 3–5 min (Fig. 1A). This RdW, red wine: n=9; WhW, white wine: n=9; RoW, rose wine: n=3;
indicates that an incubation of 3 min gives essentially **OrJ, orange juice:n=3 (Bottled); *RoX, roselle extract: n=9 (1 g
complete extraction. It was also shown that antioxidant was extracted for 3 min in 300 ml at 100  C).
activity increased proportionally to the weight of
Roselle petals used (Fig. 1B). For a series of standard
samples, a linear relationship existed between FRAP sulphite or vitamin C. Metabisulphite is routinely used
and TAS (R2=0.9411, Fig. 2) or ORAC (R2=0.9288, in wine production and many juices are supplemented
Fig. 3). This suggests that any of these methods will give with vitamin C.
an accurate measurement of antioxidant status. The relation between anthocyanin content and anti-
To ascertain the relative level of antioxidants that oxidant capacity was investigated by measuring Roselle
were present in the Roselle extract, comparisons were extracts over a wide range of absorbance values at 520
made with other beverages. Fig. 4 gives representative nm and for antioxidant activity by the FRAP assay.
data of antioxidants in a variety of selected drinks. Red There was a linear relationship between FRAP activity
wine has previously been shown to have a higher anti- and A520 as shown in Fig. 5. The correlation coefficient
oxidant capacity than black tea and orange juice (Benzie was 0.8375. This is slightly higher than the result that
& Szeto, 1999). In the present study red wine had a Prior et al. (1998) found in berries using ORAC to
substantially higher antioxidant level than other bev- measure antioxidant activity. Our results also agree with
erages. Green teas were routinely highest in antioxidant the findings that wine made from grapes with a higher
activity in all the teas tested. Roselle extract was anthocyanin content had a higher antioxidant capacity
approximately 16–25% as active as green tea. The high (Kähkönen et al., 1999).
antioxidant activity in the commercial wine and juice The Roselle extract was fractionated to find out which
sample may partially arise from the presence of metabi- compounds were responsible for the antioxidant activity.
354 P.-J. Tsai et al. / Food Research International 35 (2002) 351–356

Fig. 6. The A520 and FRAP contributions of Roselle fractions after


column chromatography on XAD-2 and LiChroprep RP-8. *Fr A,
Fig. 5. Correlation of antioxidant activity (FRAP) and anthocyanin
Water eluate from XAD-2; Fr B, MeOH eluate from XAD-2; Fr B1,
content (A520) of Roselle extract.
Red fraction eluted by HOAc:acetonitrile (delphinidin 3-sambubio-
side); Fr B2, pink fraction eluted by HOAc, acetonitrile (cyanidin 3-
sambubioside); Fr B3, brown fraction eluted by HOAc:acetonitrile.

Table 1
Distribution of anthocyanin and antioxidant activity in Roselle infusion

Fraction A520 FRAP Weight Total


phenolics

Aqueous Roselle extract 100% 100% 100% 100%


Fraction A (Water elutate from XAD-2 column) 2% 3% 70% 5%
Fraction B (MeOH eluate from XAD-2 column) 86% 75% 17% 81%
Fraction B1 Red (RP-8 column)Del-3-sambubiosidea 73% 48% – 49%
Fraction B2 Pink (RP-8 column)Cy-3-sambubiosideb 13% 3% – 9%
Fraction B3 Brown (RP-8 column) 0.5% 24% – 23%
a
Delphinidin 3-sambubioside.
b
Cyanidin 3-sambubioside.

The extract was passed through an Amberlite XAD-2 tion with Ciocalteu’s reagent. The amount of phenolic
column where the pigments were adsorbed. The column compounds in the Roselle extract (23 mg/g dry weight)
was initially washed with water to give the non-absor- is similar to that found by Mazza, Fukumoto, Delaquis,
bed fraction ‘‘A’’. This fraction contained 70% (by Givard, and Ewert (1999) in various fruits such as
weight) of the extracted material but only 3% of the strawberries and currants.
antioxidant activity. The pigments were eluted with The effects on composition of drying and storage at
methanol, giving Fraction ‘‘B’’ which contained 86% of different temperatures were investigated. After drying
the total pigments as estimated by A520. This fraction was and storage at 20  C for 15 weeks, 90% of the total
further separated into three sub-fractions on a LiChro- phenolic compounds remained. Storage at 40  C only
prep RP-8 column with an acetic acid: acetonitrile gra- decreased the phenolic compounds by a few percent.
dient: sub-fraction B1 was red, B2 was pink and B3 was Even after drying at 75  C and storage for 15 weeks at
brown. These data are summarised in Table 1 and Fig. 6. 40  C, 85% of the total phenolics remained. The effects
After comparison with authentic standards (pur- on the percentage composition of drying and storage at
chased from Polyphenols Laboratories, Sandnes, Nor- different temperatures are summarised in Table 2.
way) in the HPLC system, the red pigment was However, under these conditions, the anthocyanins
identified as delphinidin 3-sambubioside (85% of the decreased from about 80% of the total phenolics to
anthocyanin) and the pink pigment was provisionally about 50%. There was a corresponding increase in the
identified as cyanidin 3-sambubioside (Tsai & Ou, percentage of the other phenolics (Fig. 7). These appar-
1996). These two pigments account for 51% of the total ently contradictory results are consistent with the con-
FRAP activity. The remainder of the activity (24%), version of some monomeric anthocyanins into
was due to the brown pigment which was composed of polymerised phenolics during storage.
phenolic compounds. This conclusion is based on the Measurement of antioxidant activity under these
absorbance spectra of the brown fraction and its reac- conditions showed a strong correlation to anthocyanin
P.-J. Tsai et al. / Food Research International 35 (2002) 351–356 355

Table 2
Effect of drying and storage temperatures on the percentage of total phenolic compounds found in Roselle

Drying temp Storage temp Storage time GAE CCE CFAE RUE DEL Total phenolics
( C) ( C) (weeks) (%) (%) (%) (%) (%) (%)

25 20 0 2.62 5.60 2.97 2.61 86.20 100


4 2.39 6.59 2.84 2.60 85.58 100
15 2.85 8.09 3.06 6.25 79.75 100

25 40 0 2.62 5.60 2.97 2.61 86.20 100


4 2.69 5.69 3.09 2.75 85.78 100
15 3.11 9.23 6.37 11.65 69.04 100

50 20 0 4.25 7.48 3.20 4.80 80.27 100


4 3.76 7.85 2.78 6.30 79.31 100
15 1.53 8.35 7.46 13.08 69.58 100

50 40 0 4.25 7.48 3.20 4.80 80.27 100


4 5.02 12.28 5.21 6.95 70.54 100
15 5.61 19.82 5.35 11.81 57.41 100

75 20 0 6.57 4.74 10.83 5.65 72.21 100


4 12.12 5.93 5.10 6.05 70.80 100
15 12.10 12.46 9.83 10.79 54.82 100

75 40 0 6.57 4.74 10.83 5.65 72.21 100


4 6.25 12.63 15.04 5.08 61.00 100
15 9.26 23.91 9.13 7.74 49.96 100

3-sambubioside and cyanidin-3-sambubioside are rapidly


absorbed from the gastrointestinal tract when fed to rats.
Further studies are underway to confirm this finding.

Acknowledgements

We are grateful for the research support provided


from the National Scientific Council Association of the
Government of the Republic of China.

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