Introduction

Physico-chemical Roselle (Hibiscus sabdariffa L.) is currently an

characteristics of important cash crop grown in the East Coast
roselle (Hibiscus of Malaysia especially in Terengganu and
Kelantan. It is locally known as ``Asam
sabdariffa L.) Kumbang'', ``Asam susur'' and ``Asam paya''.
Roselle produces red edible calyxes from
Peng-Kong Wong which many edible products can be produced.
Salmah Yusof In countries like India, Tropical Africa,
Philippines and Indonesia, roselle has been
H.M. Ghazali and utilised for a long time in producing
Y.B. Che Man refreshing beverages, jellies, jams, sauces and
preserves (Esselen and Sammy, 1973;
Clydesdale et al., 1979). In Malaysia,
utilisation of roselle for such products is
The authors considered new.
Anthocyanins are water-soluble flavonoid
Peng-Kong Wong is a Student, Salmah Yusof is a
pigments, which are responsible for the most
Lecturer, H.M. Ghazali and Y.B. Che Man are Vice-
spectacular red, blue and purple colours in
Deans and Lecturers, all at the Universiti Putra Malaysia,
many fruits and vegetables. The brilliant red
Serdang, Selangor, Malaysia.
pigments contained in the red calyxes of
roselle are anthocyanins (Du and Francis,
Keywords 1973). Natural anthocyanins are considered
Food, Nutrition, Research to be a suitable alternative for artificial food
dyes in many applications, especially in the
Abstract food industry. Therefore, a detailed
understanding of individual anthocyanin may
The physico-chemical characteristics of roselle from
contribute to the use of roselle as a natural
Malaysia were studied. The parameters analysed included
food colorant in the future.
size, weight, pH, titratable acidity, soluble solids,
Recently, many reports have remarked on
anthocyanin contents, organic acids and sugars. Succinic
the positive beneficial health effects of
and oxalic acids were the predominant organic acids
found in roselle while glucose was the major sugar
anthocyanin, ascorbic acid and carotenoids.
present. The present study was also conducted to
Anthocyanin has been correlated with their
determine and quantify the most biologically effective
antioxidant property in the role of reduction
natural antioxidants present in roselle. Ascorbic acid,
of coronary heart disease and cancer (Bridle
-carotene and lycopene, were separated, identified and
and Timberlake, 1997; Delgado-Vargas et al.,
quantified by HPLC. The amounts of ascorbic acid, 2000). A large number of epidemiological
-carotene and lycopene contents were 141.09mg/100g, studies have also reported the use of
1.88mg/100g and 164.34g/100g, respectively. The antioxidant vitamins such as ascorbic acid and
individual anthocyanin in roselle was characterised by TLC carotenoids and suggested their possible links
and HPLC. Delphinidin-3-sambubioside and cyanidin-3- to inhibit the development of cancer (Ziegler,
sambubioside were the main anthocyanin present in 1989; Krinsky, 1989), coronary heart disease
roselle. (Gerster, 1991; Kritchevsky, 1992) and
atherosclerosis (Mezzetti et al., 1995). This
has attracted many researchers and scientists
Electronic access
to characterise these specific components of
The current issue and full text archive of this journal is fruits and vegetables. It has also been
available at demonstrated that fresh or well-processed
http://www.emeraldinsight.com/0034-6659.htm plant-derived foods are the best sources of
antioxidant vitamins. However, very little
information has been generated on roselle
fruit. Undoubtedly, this question can be
Nutrition & Food Science
answered by gaining an in-depth knowledge
Volume 32 . Number 2 . 2002 . pp. 68±73
# MCB UP Limited . ISSN 0034-6659 of roselle. Since information on local roselle is
DOI 10.1108/00346650210416994 limited, the objectives of this study were to
68
Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.) Nutrition & Food Science
Peng-Kong Wong et al. Volume 32 . Number 2 . 2002 . 68±73

determine physico-chemical characteristics Total soluble solids content was determined

and identify the major anthocyanins present by using a digital refractometer (Atago,
in our local product. Tokyo) with a scale of 0-32 Brix. Total
titratable acidity was determined by titrating
10mL of the extract with 0.1N NaOH until
Materials and methods pH 8.1 and the results were expressed as per
cent malic acid.
Raw materials
Fresh roselle fruits at commercial maturity Determination of total anthocyanins
(20-30 days after flower bloom) were Total anthocyanins were determined
obtained from the Department of Agriculture, according to the methods developed by
Terengganu, Malaysia. To prevent
Wrolstad et al. (1982). An amount of 20g of
deterioration in quality parameters, decored
roselle calyxes was added to 80mL water and
calyxes were frozen at 20 C before use.
blended at full speed. The filtered extraction
was used for analysis. Total anthocyanins
Chemicals
were expressed as g delphinidin-3-glucoside/
High performance liquid chromatography
100g calyxes.
(HPLC) grade solvents such as acetonitrile,
chloroform and phosphoric acid were
Determination of sugar
obtained from BDH (Poole, UK). -carotene
Sugar was analysed using HPLC previously
and lycopene standards were obtained from
described by Hunt et al. (1977). A Waters
Sigma Chemical Co. (St Louis, Missouri).
(Milford, Massachusetts) chromatography
Ascorbic acid standard was purchased from
system, which consisted of a Waters 410 RI
Hamburg Chemicals (Hamburg, Germany).
detector, coupled with a Waters 600
All other solvents and chemicals used were
controller and the data were processed by
analytical grade purchased from Merck
means of Millennium 2010 chromatography
(Darmstadt, Germany).
manager software. The column used was
Waters Bondapak NH2 (3:9 300mm,
Determination of weight, diameter,
125A Ê , 10m). The eluent used was a mixture
length, per cent calyxes weight and
per cent seed weight of acetonitrile and distilled water (80:20,
The diameter and the length of the fruit were vol/vol) at a flow rate of 1mL/min. The
measured using vernier caliper (Mitutoyo, injection volume was 20 L. Glucose,
USA). The diameter at the largest rounded fructose and sucrose were used as calibration
portion of the fruit was measured. For standards.
weight determination, the fruit was weighted
using Denver electronic balance (model Determination of organic acid
AA-200, Denver Instrument Company, Organic acid was determined using HPLC
USA). method by Jinap and Dimick (1990). The
organic acid was separated by HPLC with a
Determination of pH, total soluble solids Waters (Milford, Massachusetts)
and titratable acidity chromatography system, consisting of a
The pH, total soluble solids and titratable Waters 486 Tunable Absorbance Detector,
acidity values were determined according to coupled with a Waters 600 controller and a
Ranggana (1977). An amount of 10g of software interface (Millennium 2010
roselle calyxes was blended with 50mL of Chromatography Manager Software;
distilled water. The mixture was boiled for Millipore Co., Milford, Massachusetts) for
one hour and the water lost by evaporation processing the data. The ion exchange
was replaced. After boiling, the mixture was column used was Bio-Rad Aminex HPX-87H
cooled and transferred to 100mL volumetric (300 7:8mm). Isocratic elution using 0.01N
flask and made up to the volume. This extract sulphuric acid in distilled water (vol/vol) as
was filtered and aliquots were used for mobile phase was carried out at a flow rate of
analysis. The pH was determined by a glass- 1mL/min and detected at 214nm, 20
electrode pH meter (Cyberscan 500, microlitre of samples were injected. Oxalic,
EUTECH Instrument, Singapore) using citric, tartaric, malic and succinic acids were
buffers of pH 4.0 and 7.0 for calibration. used as calibration standards.
69
Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.) Nutrition & Food Science
Peng-Kong Wong et al. Volume 32 . Number 2 . 2002 . 68±73

Ascorbic acid extraction and HPLC acetonitrile/chloroform (92:8, vol/vol) as

separation mobile phase.
The extraction method and HPLC analysis of
ascorbic acid was modified from the Anthocyanin extraction and purification
procedure of Wimalasiri and Wills (1982). The sample preparation for HPLC analysis of
The reversed-phase column used was Waters anthocyanin was carried out according to the
Nova Pak C18 (3:9 300mm, 60A Ê , 4m). procedure previously described by De Ancos
Isocratic elution acetonitrile/water (80:20, et al. (2000).
vol/vol) as mobile phase was carried out at a
flow rate of 1mL/min and detected at 254nm, HPLC separation of anthocyanin
the UV absorbance maximum for ascorbic The separation and identification of
acid. A total of 5g of roselle calyxes was anthocyanin was performed according to the
homogenised with 200mL of 3 per cent citric procedure described by De Ancos et al.
acid solution and filtered through Whatman (2000). The anthocyanins were also separated
no. 1 filter paper. The aqueous solution was by HPLC with a Waters (Milford,
purified by passing through a C18 Sep-Pak Massachusetts) chromatography system. The
cartridge (Water Associates, Milford, method used a reversed-phase column
-Bondapack C18 (3:9 300mm, 125A Ê,
Massachusetts), and 0.45 m membrane
filter. Twenty microlitre of sample was 10m). The mobile phase was composed of
injected. 4 per cent phosphoric acid (solvent A) and
100 per cent acetonitril (solvent B). The
-carotene and lycopene extraction solvent program was isocratic elution with
-carotene and lycopene sample preparation 6 per cent B from 0 to 10 min, linear gradient
were modified from the work of Tee and Lim to 20 per cent B from 10 to 55 min and
(1991). A total of 20g of roselle sample was isocratic elution at 20 per cent B from 50 to
homogenised with 80mL 95 per cent ethanol 60 min. The flow rate was 1mL/min and
and placed in 250 boiling flask. Twenty detection was carried out at 520nm.
microlitre of 100 per cent potassium
Identification of anthocyanins by thin
hydroxide was added and the mixture was
layer chromatography (TLC)
refluxed for 30 minutes. The extract was
Anthocyanins were extracted according to the
hydrolysated three times with 50mL hexane.
procedure of De Ancos et al. (2000).
All extracts were pooled and further washed
Individual anthocyanins were isolated by
with distilled water until free of alkali (test
TLC using Merck F254 60 silica gel plates
with phenolphthalein). The hexane extract
(0.2mm). For detection of anthocyanins the
was passed through anhydrous sodium
plates were developed using a mixture
sulphate. The hexane solution was evaporated
containing n-butanol, acetic acid and water in
to dryness in a rotary evaporator, dissolved in
the ratio of 40:10:20.
mobile phase, and filtered through a 0.45m
filter. Twenty microlitre of the sample was
then injected into the HPLC system.
Results and discussion
HPLC separation of -carotene and
Table I shows the physical properties of
lycopene
roselle fruit used in the present study. Roselle
The HPLC separation and identification of
-carotene and lycopene were carried out Table I Physical characteristics of roselle fruit
according to the procedure described by
Roselle Measurement
Hsieh and Karel (1983). The -carotene and
lycopene were also separated by HPLC with a Calyxes (g) 6.12 0.65
Waters (Milford, Massachusetts) Seeds (g) 5.18 0.06
Calyxes (%) 54.11 2.34
chromatography system. Column effluents
Seeds (%) 45.89 2.34
were monitored at 450nm for -carotene and
Diameter (cm) 3.12 0.33
lycopene. The reversed-phase column used
Ê, Length (cm) 4.28 0.31
was -Bondapack C18 (3:9 300mm, 125A
10m). Isocratic elution was carried out at a Note: Each value in the table represents the mean
standard deviation of six analyses for three replications
flow rate of 1mL/min with a mixture of
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Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.) Nutrition & Food Science
Peng-Kong Wong et al. Volume 32 . Number 2 . 2002 . 68±73

fruits are dark red in colour. The fruit consists Organic acids present in the roselle samples
of two major parts namely the calyx and the were quantified using oxalic, citric, tartaric,
seed portion. Each fruit contains five calyxes. malic and succinic acids as calibration
These calyxes had an average measurement of standards. However, only oxalic, tartaric,
4.28cm long and 3.12cm in diameter. The malic and succinic acids were identified from
weight of the calyxes and seed are 6.12 and roselle. The values for the individual organic
5.18g, respectively, giving an average weight acids are shown in Table II. The total amount
of a fruit of about 11.30g. of organic acid present was 1.23g/100g.
The values for pH, titratable acidity, total Succinic and oxalic acid were the two
soluble solids and anthocyanin contents of predominant organic acids in roselle, giving a
roselle are as shown in Table II. Results total average of 76 per cent. The remaining
showed that roselle contains high acid and organic acids were tartaric acid and malic acid,
low total soluble solids with pH, titratable which contribute to an average portion of 14
acidity value and total soluble solids at 2.49, per cent and 10 per cent, respectively. There is
2.42g/100g malic acid and 3.30 Brix, little information in the literature to compare
respectively. The anthocyanin contents of with current findings. Earlier workers Esselen
roselle were relatively high with a total and Sammy (1973) had reported that d-malic
amount of 2.52g/100g. Meanwhile, the total acid was detected in roselle.
sugar contents for roselle was 3.28 per cent. Roselle was analysed for its ascorbic acid, -
They are made up of glucose, fructose and carotene and lycopene contents by HPLC
sucrose. The relative proportions of glucose, (Table II). The most conventional method of
fructose and sucrose in roselle are listed in
determining ascorbic acid content is using the
Table II. Glucose was found to be the major
2,6-dichlorophenol indophenol dye titration
sugar for roselle followed by sucrose and
method. However, this method could not be
fructose. Generally the total sugar value in
applied to all kinds of fruits and vegetables,
roselle was lower compared to blackcurrants,
especially highly coloured samples that
raspberries and strawberries (6.3 per cent, 4.5
contain anthocyanins and carotenoids.
per cent and 5.6 per cent, respectively) (Kirk
Metaphosphoric acid was the common
and Sawyer, 1997). Roselle is thus
medium used in the extraction of ascorbic
characterised as a highly acidic fruit with very
acid. However, citric acid was also found to
low sugar contents.
be suitable as extraction medium. Besides,
Table II Chemical characteristics of roselle fruit metaphosphoric acid was found to interfere
with the analyses as it had a similar retention
Roselle Measurement
time to dehydroascorbic acid, which absorbed
pH 2.49 0.00 strongly at 214nm (Wimalasiri and Wills,
Titrability acidity (g/100g malic 2.42 0.03 1982). The value obtained suggested that
acid) roselle provides about 235 per cent RDA for
Total soluble solid ( Brix) 3.30 0.12 ascorbic acid per 100g of edible portion and
Total anthocyanin (g/100g 2.52 0.05 about 32 per cent RDA for vitamin A per
expressed as delphinidin-3- 100g of edible roselle calyxes. Current RDA
glucoside) for ascorbic acid and vitamin A are 60mg and
Sugars (g/100g) 1,000 RE per day, respectively. Roselle was
Glucose 1.29 0.15 found to contain higher amounts of ascorbic
Fructose 1.12 0.26 acid compared to oranges and mango (Kirk
Sucrose 0.87 0.21 and Sawyer, 1997) and it is also a good source
of -carotene compared to cherries and
Organic acid (g/100g)
papayas (Holden et al., 1999).
Succinic acid 0.51 0.08
Previous workers, Du and Francis (1973)
Oxalic acid 0.43 0.05
characterised the anthocyanins of roselle and
Tartaric acid 0.17 0.03
found that it contained delphinidin-3-
Malic acid 0.12 0.03
sambubioside, cyanidin-3-sambubioside,
Ascorbic acid (mg/100g) 141.09 22.54
cyanidin-3-glucoside and delphinidin-3-
-carotene (mg/100g) 1.88 0.31
glucoside. Hong and Wrolstad (1990)
Lycopene (g/100g) 164.34 70.10
separated and identified four of these
Note: Each value in the Table represents the mean
anthocyanins using HPLC. In this study, only
standard deviation of six analyses for three replications
three out of four anthocyanins were detected.
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Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.) Nutrition & Food Science
Peng-Kong Wong et al. Volume 32 . Number 2 . 2002 . 68±73

Figure 1 illustrates the distinct peak of TLC plate representing the two main
monomeric anthocyanin of roselle by HPLC anthocyanins in roselle. The Rf values and
chromatogram. Anthocyanins a-c were visible maximal of each peaks were confirmed
identified by matching their retention times to by comparison of their values from the
those of the anthocyanins present in an literature (Pouget et al., 1990; Du and
authentic sample of roselle juice, as decribed Francis, 1973). Pigments a and b were
by Hong and Wrolstad (1990). The identified as delphinidin-3-sambubioside and
anthocyanin was expressed as percentage cyanidin-3-sambubioside, respectively
based on total peak area. The relative (Table III). Delphinidin-3-sambubioside was
percentages distributed by delphinidin-3-
blue-mauve in colour meanwhile cyanidin-3-
sambubioside, cyanidin-3-sambubioside and
sambubioside was pink-mauve in colour. The
delphinidin-3-glucoside in roselle were 71.40
isolated pigments were then chromatographed
per cent, 26.60 per cent and 2.00 per cent,
by HPLC.
respectively. Results of this study showed that
delphinidin-3-sambubioside, cyanidin-3-
sambubioside were the major anthocyanins
present and this corresponded to those Conclusion
reported earlier (Hong and Wrolstad, 1990).
This study represents an initial attempt to
However, cyanidin-3-glucoside was
undetected. The percentages for the major characterise the physical and chemical
anthocyanins were relatively higher than those properties of our local product roselle, which
reported by Hong and Wrolstad (1990). has not been thoroughly studied. Succinic acid
Spectral information and Rf values of the and oxalic acids were the predominant organic
individual peaks separated by TLC is given in acids present in roselle. Its high titratable
Table III. Two spots were separated from the acidity and low contents of sucrose, fructose
and glucose categorised it as a high acid
Figure 1 HPLC chromatogram of roselle anthocyanins product. The present study also showed that
roselle could be considered to be a significant
source of vitamin C and also a
fair source of vitamin A. A 100g serving of
frozen roselle or less would supply 100 per cent
of current RDA (60mg/day) of ascorbic acid.

` ... the consumption of roselle should be

encouraged since it has beneficial
health effects... '

Roselle is rich in anthocyanins. Two major

anthocyanins present were namely
delphinidin-3-sambubioside and cyanidin-3-
sambubioside. Knowing these characteristics,
the consumption of roselle should be
encouraged since it has beneficial health
effects. Its high contents of oxalic acid, natural
vitamins and antioxidants are valuable in
meeting the current trends of food intake.

Table III Spectral and Rf value characterisation of major anthocyanins found in roselle
Visible Rf 100
Peak no. Identification Maxima (nm) (TLC) Reference
a Delphinidin-3-sambubioside 542a 37 Du and Francis (1973)
b Cyanidin-3-sambubioside 528a 44 Du and Francis (1973)
a
Note: 0.01% HCL in MeOH

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Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.) Nutrition & Food Science
Peng-Kong Wong et al. Volume 32 . Number 2 . 2002 . 68±73

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