Introduction

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

characteristics of
roselle (Hibiscus
sabdariffa L.)
Peng-Kong Wong
Salmah Yusof
H.M. Ghazali and
Y.B. Che Man
The authors
Peng-Kong Wong is a Student, Salmah Yusof is a
Lecturer, H.M. Ghazali and Y.B. Che Man are Vice-
Deans and Lecturers, all at the Universiti Putra Malaysia,
Serdang, Selangor, Malaysia.
Keywords
Food, Nutrition, Research
Abstract
The physico-chemical characteristics of roselle from
Malaysia were studied. The parameters analysed included
size, weight, pH, titratable acidity, soluble solids,
anthocyanin contents, organic acids and sugars. Succinic
and oxalic acids were the predominant organic acids
found in roselle while glucose was the major sugar
present. The present study was also conducted to
determine and quantify the most biologically effective
natural antioxidants present in roselle. Ascorbic acid,
important cash crop grown in the East Coast
of Malaysia especially in Terengganu and
Kelantan. It is locally known as ``Asam
Kumbang'', ``Asam susur'' and ``Asam paya''.
Roselle produces red edible calyxes from
which many edible products can be produced.
In countries like India, Tropical Africa,
Philippines and Indonesia, roselle has been
utilised for a long time in producing
refreshing beverages, jellies, jams, sauces and
preserves (Esselen and Sammy, 1973;
Clydesdale et al., 1979). In Malaysia,
utilisation of roselle for such products is
considered new.
Anthocyanins are water-soluble flavonoid
pigments, which are responsible for the most
spectacular red, blue and purple colours in
many fruits and vegetables. The brilliant red
pigments contained in the red calyxes of
roselle are anthocyanins (Du and Francis,
1973). Natural anthocyanins are considered
to be a suitable alternative for artificial food
dyes in many applications, especially in the
food industry. Therefore, a detailed
understanding of individual anthocyanin may
contribute to the use of roselle as a natural
food colorant in the future.
Recently, many reports have remarked on
the positive beneficial health effects of
anthocyanin, ascorbic acid and carotenoids.
Anthocyanin has been correlated with their
antioxidant property in the role of reduction
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,
1.88mg/100g and 164.34g/100g, respectively. The
individual anthocyanin in roselle was characterised by TLC
and HPLC. Delphinidin-3-sambubioside and cyanidin-3-
sambubioside were the main anthocyanin present in
roselle.
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Nutrition & Food Science
Volume 32 . Number 2 . 2002 . pp. 68±73
# MCB UP Limited . ISSN 0034-6659
DOI 10.1108/00346650210416994
68
studies have also reported the use of
antioxidant vitamins such as ascorbic acid and
carotenoids and suggested their possible links
to inhibit the development of cancer (Ziegler,
1989; Krinsky, 1989), coronary heart disease
(Gerster, 1991; Kritchevsky, 1992) and
atherosclerosis (Mezzetti et al., 1995). This
has attracted many researchers and scientists
to characterise these specific components of
fruits and vegetables. It has also been
demonstrated that fresh or well-processed
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
answered by gaining an in-depth knowledge
of roselle. Since information on local roselle is
limited, the objectives of this study were to
 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
and lycopene standards were obtained from
Sigma Chemical Co. (St Louis, Missouri).
Ascorbic acid standard was purchased from
Hamburg Chemicals (Hamburg, Germany).
All other solvents and chemicals used were
analytical grade purchased from Merck
(Darmstadt, Germany).
Determination of weight, diameter,
length, per cent calyxes weight and
per cent seed weight
The diameter and the length of the fruit were
measured using vernier caliper (Mitutoyo,
USA). The diameter at the largest rounded
portion of the fruit was measured. For
weight determination, the fruit was weighted
using Denver electronic balance (model
AA-200, Denver Instrument Company,
USA).
Determination of pH, total soluble solids
and titratable acidity
The pH, total soluble solids and titratable
acidity values were determined according to
Ranggana (1977). An amount of 10g of
roselle calyxes was blended with 50mL of
distilled water. The mixture was boiled for
one hour and the water lost by evaporation
was replaced. After boiling, the mixture was
cooled and transferred to 100mL volumetric
flask and made up to the volume. This extract
was filtered and aliquots were used for
analysis. The pH was determined by a glass-
electrode pH meter (Cyberscan 500,
EUTECH Instrument, Singapore) using
buffers of pH 4.0 and 7.0 for calibration.
69
Sugar was analysed using HPLC previously
described by Hunt et al. (1977). A Waters
(Milford, Massachusetts) chromatography
system, which consisted of a Waters 410 RI
detector, coupled with a Waters 600
controller and the data were processed by
means of Millennium 2010 chromatography
manager software. The column used was
Waters  Bondapak NH2 (3:9  300mm,
125A Ê , 10m). The eluent used was a mixture
of acetonitrile and distilled water (80:20,
vol/vol) at a flow rate of 1mL/min. The
injection volume was 20 L. Glucose,
fructose and sucrose were used as calibration
standards.
Determination of organic acid
Organic acid was determined using HPLC
method by Jinap and Dimick (1990). The
organic acid was separated by HPLC with a
Waters (Milford, Massachusetts)
chromatography system, consisting of a
Waters 486 Tunable Absorbance Detector,
coupled with a Waters 600 controller and a
software interface (Millennium 2010
Chromatography Manager Software;
Millipore Co., Milford, Massachusetts) for
processing the data. The ion exchange
column used was Bio-Rad Aminex HPX-87H
(300  7:8mm). Isocratic elution using 0.01N
sulphuric acid in distilled water (vol/vol) as
mobile phase was carried out at a flow rate of
1mL/min and detected at 214nm, 20
microlitre of samples were injected. Oxalic,
citric, tartaric, malic and succinic acids were
used as calibration standards.
 Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.)
Peng-Kong Wong et al.
Ascorbic acid extraction and HPLC
separation
The extraction method and HPLC analysis of
ascorbic acid was modified from the
procedure of Wimalasiri and Wills (1982).
The reversed-phase column used was Waters
Nova Pak C18 (3:9  300mm, 60A Ê , 4m).
Isocratic elution acetonitrile/water (80:20,
vol/vol) as mobile phase was carried out at a
flow rate of 1mL/min and detected at 254nm,
the UV absorbance maximum for ascorbic
acid. A total of 5g of roselle calyxes was
homogenised with 200mL of 3 per cent citric
acid solution and filtered through Whatman
no. 1 filter paper. The aqueous solution was
purified by passing through a C18 Sep-Pak
cartridge (Water Associates, Milford,
Massachusetts), and 0.45 m membrane
filter. Twenty microlitre of sample was
injected.
Nutrition & Food Science
Volume 32 . Number 2 . 2002 . 68±73
acetonitrile/chloroform (92:8, vol/vol) as
mobile phase.
Anthocyanin extraction and purification
The sample preparation for HPLC analysis of
anthocyanin was carried out according to the
procedure previously described by De Ancos
et al. (2000).
HPLC separation of anthocyanin
The separation and identification of
anthocyanin was performed according to the
procedure described by De Ancos et al.
(2000). The anthocyanins were also separated
by HPLC with a Waters (Milford,
Massachusetts) chromatography system. The
method used a reversed-phase column
-Bondapack C18 (3:9  300mm, 125A Ê,
10m). The mobile phase was composed of
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
70
 Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.)
Peng-Kong Wong et al.
fruits are dark red in colour. The fruit consists
of two major parts namely the calyx and the
seed portion. Each fruit contains five calyxes.
These calyxes had an average measurement of
4.28cm long and 3.12cm in diameter. The
weight of the calyxes and seed are 6.12 and
5.18g, respectively, giving an average weight
of a fruit of about 11.30g.
The values for pH, titratable acidity, total
soluble solids and anthocyanin contents of
roselle are as shown in Table II. Results
showed that roselle contains high acid and
low total soluble solids with pH, titratable
acidity value and total soluble solids at 2.49,
2.42g/100g malic acid and 3.30 Brix,
respectively. The anthocyanin contents of
roselle were relatively high with a total
amount of 2.52g/100g. Meanwhile, the total
Nutrition & Food Science
Volume 32 . Number 2 . 2002 . 68±73
Organic acids present in the roselle samples
were quantified using oxalic, citric, tartaric,
malic and succinic acids as calibration
standards. However, only oxalic, tartaric,
malic and succinic acids were identified from
roselle. The values for the individual organic
acids are shown in Table II. The total amount
of organic acid present was 1.23g/100g.
Succinic and oxalic acid were the two
predominant organic acids in roselle, giving a
total average of 76 per cent. The remaining
organic acids were tartaric acid and malic acid,
which contribute to an average portion of 14
per cent and 10 per cent, respectively. There is
little information in the literature to compare
with current findings. Earlier workers Esselen
and Sammy (1973) had reported that d-malic
acid was detected in roselle.
sugar contents for roselle was 3.28 per cent.
They are made up of glucose, fructose and
sucrose. The relative proportions of glucose,
fructose and sucrose in roselle are listed in
Table II. Glucose was found to be the major
sugar for roselle followed by sucrose and
fructose. Generally the total sugar value in
roselle was lower compared to blackcurrants,
raspberries and strawberries (6.3 per cent, 4.5
per cent and 5.6 per cent, respectively) (Kirk
and Sawyer, 1997). Roselle is thus
characterised as a highly acidic fruit with very
low sugar contents.
Table II Chemical characteristics of roselle fruit
Roselle
pH
Titrability acidity (g/100g malic
acid)
Total soluble solid ( Brix)
Total anthocyanin (g/100g
expressed as delphinidin-3-
glucoside)
Sugars (g/100g)
Glucose
Fructose
Sucrose
Roselle was analysed for its ascorbic acid, -
carotene and lycopene contents by HPLC
(Table II). The most conventional method of
determining ascorbic acid content is using the
2,6-dichlorophenol indophenol dye titration
method. However, this method could not be
applied to all kinds of fruits and vegetables,
especially highly coloured samples that
contain anthocyanins and carotenoids.
Metaphosphoric acid was the common
medium used in the extraction of ascorbic
acid. However, citric acid was also found to
be suitable as extraction medium. Besides,
metaphosphoric acid was found to interfere
with the analyses as it had a similar retention
Measurement
time to dehydroascorbic acid, which absorbed
2.49  0.00 strongly at 214nm (Wimalasiri and Wills,
2.42  0.03 1982). The value obtained suggested that
roselle provides about 235 per cent RDA for
3.30  0.12 ascorbic acid per 100g of edible portion and
2.52  0.05 about 32 per cent RDA for vitamin A per
100g of edible roselle calyxes. Current RDA
for ascorbic acid and vitamin A are 60mg and
1,000 RE per day, respectively. Roselle was
1.29  0.15 found to contain higher amounts of ascorbic
1.12  0.26 acid compared to oranges and mango (Kirk
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.
71
 Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.)
Peng-Kong Wong et al.
Figure 1 illustrates the distinct peak of
monomeric anthocyanin of roselle by HPLC
chromatogram. Anthocyanins a-c were
identified by matching their retention times to
those of the anthocyanins present in an
authentic sample of roselle juice, as decribed
by Hong and Wrolstad (1990). The
anthocyanin was expressed as percentage
based on total peak area. The relative
percentages distributed by delphinidin-3-
sambubioside, cyanidin-3-sambubioside and
delphinidin-3-glucoside in roselle were 71.40
per cent, 26.60 per cent and 2.00 per cent,
respectively. Results of this study showed that
delphinidin-3-sambubioside, cyanidin-3-
sambubioside were the major anthocyanins
present and this corresponded to those
reported earlier (Hong and Wrolstad, 1990).
However, cyanidin-3-glucoside was
undetected. The percentages for the major
anthocyanins were relatively higher than those
reported by Hong and Wrolstad (1990).
Spectral information and Rf values of the
individual peaks separated by TLC is given in
Table III. Two spots were separated from the
Figure 1 HPLC chromatogram of roselle anthocyanins
Table III Spectral and Rf value characterisation of major anthocyanins found in roselle
Peak no. Identification
a Delphinidin-3-sambubioside
b Cyanidin-3-sambubioside
a
Note: 0.01% HCL in MeOH
Nutrition & Food Science
Volume 32 . Number 2 . 2002 . 68±73
TLC plate representing the two main
anthocyanins in roselle. The Rf values and
visible maximal of each peaks were confirmed
by comparison of their values from the
literature (Pouget et al., 1990; Du and
Francis, 1973). Pigments a and b were
identified as delphinidin-3-sambubioside and
cyanidin-3-sambubioside, respectively
(Table III). Delphinidin-3-sambubioside was
blue-mauve in colour meanwhile cyanidin-3-
sambubioside was pink-mauve in colour. The
isolated pigments were then chromatographed
by HPLC.
Conclusion
This study represents an initial attempt to
characterise the physical and chemical
properties of our local product roselle, which
has not been thoroughly studied. Succinic acid
and oxalic acids were the predominant organic
acids present in roselle. Its high titratable
acidity and low contents of sucrose, fructose
and glucose categorised it as a high acid
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.
Visible Rf  100
Maxima (nm) (TLC) Reference
542a 37 Du and Francis (1973)
528a 44 Du and Francis (1973)
72
 Physico-chemical characteristics of roselle (Hibiscus sabdariffa L.)
Peng-Kong Wong et al.
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