African Journal of Food Science Vol. 6(21) pp.

506-511, 15 November, 2012

Available online at http://www.academicjournals.org/AJFS
DOI: 10.5897/AJFS12.099
ISSN 1996-0794 2012 Academic Journals

Full Length Research Paper

Antioxidant and antibacterial activities of

Hibiscus sabdariffa L. extracts
Alaa G. Al-Hashimi
Food Science and Biotechnology Department, Agriculture College, Basrah University, Iraq.
E-mail: dr.alaagh@yahoo.co.uk.
Accepted 19 September, 2012

The present study investigated the total content of phenolic compounds, antioxidant activity, reducing
power and chelating of ferrous ion in aqueous and alcoholic extracts of Hibiscus sabdariffa L. The total
phenolic content was 77.2 mg/g and 87.7 mg/g for the aqueous and alcoholic extracts, respectively. The
antioxidant activity was equal in rates for both the roselle alcoholic extract and artificial antioxidant
BHT (75.67%), alcoholic extract showed the highest reducing ability with rate 222.60%, the chelating of
ferrous ion in both aqueous and alcoholic extracts were 73.97 and 32.29% at 5 mg/ml concentration.
The antibacterial activity of roselle extracts against Escherichia coli, Staphylococcus aureus,
Streptococcus mutans and Pseudomonas aeruginosa, showed varying degrees of inhibition on the
tested organisms.

Key words: Hibiscus sabdariffa, antioxidant, antimicrobial.

INTRODUCTION

Roselle (Hibiscus sabdariffa) is an edible plant used in 2002). Tsai et al. (2002) suggest that anthocyanin is the
various applications including foods. Among them, the major source of antioxidant capacity in roselle petale
most popular are the fleshy red calyces used for making extract. Previous study reported that the aqueous extract
wine, juice, jam, syrup, pudding, cakes, ice cream or tea. of this plant could inhibit several nosocomial infectious
Roselle flower and calyces is also known for its bacteria such as methicillin-resistant S. aureus and
antiseptic, diuretic, antioxidant and antimutagenic Klebsiella pneumoniae (Liu et al., 2005). However, it is
properties (Salleh et al., 2002). The dried flowers of this uncertain whether roselle calyx aqueous extract could
plant contain gossipetine and hibiscin (anthocyanins); the inhibit the growth of Salmonella typhimurium DT104, E.
petals yield glucoside hibiscritin (flavanol); and the coli O157:H7, Listeria monocytogenes, Staphylococcus
calyces are rich in riboflavin, ascorbic acid, niacin, aureus and Bacillus cereus. On the other hand, the
carotene, calcium and iron (Duke, 1983). protocatechuic acid, a polyphenol which is containing a
Roselle is an important source of vitamins, minerals, 3,4-dihydroxy substructure, is a compound that naturally
and bioactive compounds, such as organic acids, occurs in roselle calyx. Several in vitro studies have
phytosterols, and polyphenols, some of them with indicated that this compound can inhibit the growth of E.
antioxidant properties. The phenolic content in the plant coli and fungi (Fernandez et al., 1996; Aziz et al., 1998).
consists mainly of anthocyanins like delphinidin-3- Aqueous-methanolic extract of H. sabdariffa L. calyces
glucoside, sambubioside, and cyanidin-3-sambubioside; have been found to exhibit antibacterial activities against
other flavonoids like gossypetin, hibiscetin, and their S. aureus, Bacillus stearothemophilus, Micrococcus
respective glycosides; protocatechuic acid, eugenol, and luteus, Serratia mascences, Clostridium sporogenes, E.
sterols like -sitoesterol and ergoesterol (Ali-Bradeldin et coli, K. pneumonae, B. cereus and Pseudomonas
al., 2005). Roselle calyx extract is a good source of fluorescence (Olaleye, 2007). Antibacterial effects of this
antioxidants from its anthocyanins (Ajiboye et al., 2011). plant extract against Escherichia coli, P. aeruginosa and
Anthocyanin is one type of flavonoid component that S. aureus suggest that they may possess remarkable
can be in Roselle calyces (Wang et al., 2002; Tsai et al., therapeutic action in the treatment of gastrointestinal
 Al-Hashimi 507

infection and diarrhea in man and skin diseases (Rogger 500 nm. The level of lipid peroxidation inhibition by each fraction
et al., 1990). was calculated from the absorbance ratio to that of a blank without
any sample.
The aim of this study was to evaluate the antioxidative
and antimicrobial activity of aqueous and alcoholic
extracts of calyx, and to evaluate the relationship Determination of reducing power
between the antioxidative activity and total phenolic
content of the roselle. The reducing power of extracts from Roselle sample was
determined according to the method of Oyaizu (1986). Extracts
solution in methanol and water at different amounts (0.2 to 1 mg)
were mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5
MATERIALS AND METHODS ml of potassium ferricyanide (1%). The mixture was incubated at
50C for 20 min. After 2.5 ml of TCA (10%) was added, the mixture
Plant was centrifuged at 3000 rpm for 10 min. Supernatant (2.5 ml) was
mixed with distilled water (2.5 ml) and 0.5 ml of ferric chloride
The dried red roselle (Hibiscus sabdariffa L.) calyces were (0.1%) and the absorbance was measured at 700 nm. Higher
purchased from Basrah local market. 2 kg of calyx were grinded by absorbance of the reaction mixture indicates greater reducing
electric grinder, and then samples were sieved and kept in power.
polyethylene bags at 5C in refrigerator.

Chelating of ferrous Ion

Preparation of extracts
The published method by Decker and Welch (1990) was used to
Alcoholic extraction investigate the ferrous ion chelating ability of different Roselle
sample fractions. A 5 ml amount of each Roselle sample fraction
Prepared according to Harbone (1973), 5 g of powdered material was mixed with 0.1 ml of 2 mM FeCl2 and 0.2 ml of 5 mM ferrozine
along with 100 ml of alcohol was shaken well occasionally for the solutions. The absorbance at 562 nm was determined after reaction
first 6 h and kept undisturbed for 18 h. The liquefied extract thus for 10 min. A complex of Fe2%/ferrozine showed strong absorbance
obtained was concentrated in a vacuum pump and the percentage at 562 nm
was calculated with the weight of the roselle powder taken.

Antibacterial activity assay

Aqueous extraction
The antibacterial activity of the extracts was determined using the
For preparation of water extraction, 25 g sample of roselle was agar cup diffusion as described by Adeniyi et al. (1996). A 1 ml of
added to 250 ml distilled water and the mixture was boiled for 10 an overnight culture of each bacterial isolate (equivalent to 107 to
min while stirring with a magnetic stirrer. Then, the extract was 108 cfu ml-1) was used to seed sensitivity test agar plates
filtered from filter paper (Glin et al., 2004). maintained at 45C. The seeded plates were allowed to set, and a
sterile cork borer of 8 mm diameter was used to cut equidistant
wells on the surface of the agar. The wells were filled with 0.1 ml
Determination of total phenolic solution of each extract reconstituted with methanol at a
concentration of 10 mg ml-1. Gentamycin at 5 g ml-1 was included
Total phenolic constituents of plant extracts were performed as positive control. The plates were incubated at 37C for 24 h after
employing the literature methods involving Folin-Ciocalteu reagent which the diameter of zones of inhibition were measured.
and gallic acid as standard (Slinkard and Singleton, 1977). Extract
solution (0.1 ml) containing 1000 g extract was taken in a
volumetric flask, 46 ml distilled water and 1 ml Folin-Ciocalteu Statistical analysis
reagent were added and flask was shaken thoroughly. After 3 min,
3 ml of solution 2% Na2CO3 was added and the mixture was All analysis were conducted in triplicate (n = 3), and an ANOVA test
allowed to stand for 2 h with intermittent shaking. Absorbance was (using SPSS statistical software) was used to compare the mean
measured at 760 nm. The same procedure was repeated to all values of each treatment. Significant differences between the
standard gallic acid solutions (0 to 100 mg, 0.1 ml-1) and standard means of parameters were determined by using the Duncan test (p
curve was obtained. < 0.05).

Antioxidant activity assay RESULTS

The antioxidant activity analysis using ferric thiocyanate was
Over the years, the study on medicinal plants to reveal
performed according to the method reported by Osawa and Namiki
(1981). 0.6 g of Roselle sample was dissolved in 0.12 ml of 98% the mechanism of action and to justify their claims by
ethanol, and 2.88 ml of a 2.51% linoleic acid solution in ethanol and traditional healers has been increased. An angle of this
9 ml of a 40 mM phosphate buffer (pH 7.0) were added. The research has been the study of bioactive components
mixture was incubated at 40C in a stoppered test tube in the dark and antioxidant properties of the H. sabdariffa L. The total
for 3 days (72 h). During the incubation, a 0.1 ml aliquot was taken phenolic compounds found in alcoholic and aqueous
from the mixture, and diluted with 9.7 ml of 75% ethanol, followed
by the addition of 0.1 ml of 30% ammonium thiocyanate. Precisely
extracts of H. sabdariffa L. calyx were shown in Figure 1.
3 min after adding the 0.1 ml of 20 mM ferrous chloride in 3.5% The figure illustrates that there is a plain rise in the
hydrochloric acid, the absorbance of the red color was measured at amount of phenolic compound extracted by ethanol,
508 Afr. J. Food Sci.

Figure 1. Total phenolic content of roselle extracts.

Table 1. Antioxidant activity of aqueous and alcoholic H. sabdariffa L. extracts.

Antioxidant activity (%)

Concentrate mg/ml
Aqueous extract Alcoholic extract BHT -Tocopherol
20 14.020.00 19.50.00 11.890.01 29.720.01
40 27.440.01 31.870.01 40.010.00 32.430.01
60 35.850.01 56.870.01 52.430.01 42.700.00
80 46.830.01 72.780.00 67.560.01 62.160.01
100 62.550.00 75.670.01 75.670.01 69.720.02

Table 2. Reducing power of aqueous and alcoholic H. sabdariffa L. extracts.

Reducing power (%)

Concentrate mg/ml
Aqueous extract Alcoholic extract BHT -Tocopherol Ascorbic acid Citric acid
20 49.650.01 71.300.00 55.220.00 27.670.00 59.320.02 23.020.00
40 65.730.001 110.340.01 81.250.02 36.360.00 148.370.00 67.760.01
60 118.180.00 187.820.01 85.840.01 44.60.01 173.750.00 140.780.00
80 141.950.00 195.650.00 144.640.00 83.030.00 180.80.00 172.360.01
100 160.830.03 222.600.00 156.250.00 92.110.00 218.220.01 188.810.00

which was (87.7 mg/g) than the amount of phenolic that the reducing ability of all samples increased when
compound extracted by water which was (72.22 mg/g). the concentration of extracts increased. Alcoholic extract
The antioxidative activities of H. sabdariffa L. calyx showed that the highest reducing ability was 222.60% at
extracted by ethanol and water comparing with artificial the concentration 5 mg/ml, which approaches to the rate
antioxidant BHT, -tocopherol is presented in Table 1. of reducing power of ascorbic acid (218.22%) followed by
The antioxidative activities of ethanolic extraction and citric acid (188.81%), the reducing ability of aqueous
BHT was equal in rates (75.67%) followed by - extract was (160.83%) which was higher than -
tocopherol (69.72%) and roselle aqueous extraction tocopherol (92.11%) and BHT (156.25%) at the same
(62.55%) at the concentrate 5 mg/ml. concentration.
The reducing power of ethanolic and water H. The ferrous ion chelating ability of H. sabdariffa L. calyx
sabdariffa L. calyx extracts are summarized in Table 2. alcoholic and aqueous extracts is shown in Table 3;
The reducing power increased significantly (P < 0.05) EDTA hardly carried the ferrous ion chelating ability due
with increasing of extracts concentration. The data shows to its chemical structure properties which was (95.74%)
 Al-Hashimi 509

Table 3. Chelating ferrous ion of aqueous and alcoholic H. sabdariffa L.

Chelating ferrous ion (%)

Concentrate mg/ml
Aqueous extract Alcoholic extract EDTA Citric acid
20 27.850.00 25.750.001 23.180.01 9,120.00
40 48.130.01 26.160.00 34.900.00 17,020.01
60 51.700.01 28.750.00 94.830.03 26.570.01
80 71.030.00 31.750.001 95.130.01 34.830.00
100 73.970.01 32.290.001 95.740.00 52.920.00

Table 4. Antibacterial activity of water and ethanolic extracts of H. sabdariffa exhibited.

Hibiscus sabdariffa Zones of inhibition of organism (mm)

extracts E. coli S. aureus Streptococcus mutans P. aeruginosa
Water 40 40 28 27
Ethanolic 46 20 30 17

at the concentration 5 mg/ml. The aqueous extract with results found by Christian and Jackson (2009) and
performed the best ferrous ion chelating ability (up to Anokwuru et al. (2011) which were 5.25 and 27.6 mg/g of
73.97%) followed by citric acid (52.92%). However, a ethanol extracts of H. sabdariffa L. calyx, respectively.
decrease in ferrous ion chelating ability was observed in However, a research conducted by Koffi et al. (2010)
the alcoholic extracts (32.29%). Antibacterial activity test indicated that ethanol was the best solvent for the
showed that aqueous and alcoholic extract of H. extraction of phenol in the Ivorian plant.
sabdariffa L. had growth inhibitory effect on several Antioxidants are significant in the prevention of human
tested microorganism. Inhibition zone was wide against illness and may function as free radical scavengers,
E. coli, S. aureus, Str. mutans, and P. aeruginosa for complexes of pro-oxidant metals, reducing agents and
both extracts as shown in Table 4. The aqueous extract quencher of singlet oxygen formation (Andlauer and
of H. sabdariffa showed equal inhibition ability against E. Furst, 1998). The statistical analysis results presented in
coli and S. aureus (40 mm) which was highest than Str. Table 1 shows significant differences (P < 0.05) between
mutans (28 mm) and P. aeruginosa (27 mm). Alcoholic roselle extracts and the artificial antioxidant, as well as
extract exhibited higher inhibition ability against E. coli the rate of antioxidant activity increases as the
(47 mm) than against S. aureus (20 mm), Str. mutans (30 concentrate of roselle extract increased. Plants are
mm) and P. aeruginosa (17 mm). potential sources of natural antioxidants. It produces
various antioxidative compounds to counteract reactive
oxygen species (ROS) in order to survive (Lu and Foo,
DISCUSSION 1995).
Antioxidant activity of the Roselle extract correlated
The chemical components contained in the flowers of H. strongly to its anthocyanin content (Tsai et al., 2002).
sabdariffa include anthocyanins, flavonoids and According to Duh and Yen (1997), the roselle extract is
polyphenols (Tzu-Lilin et al., 2007). The petals are an electron donor and can react with free radicals to
potentially a good source of antioxidant agents as convert them into more stable products and terminate
anthocyanins and ascorbic acid (Prenesti et al., 2007). radical chain reactions. Falade et al. (2005) indicated that
The results of the current study clearly demonstrated that the extract of roselle was found to be very high in
alcoholic extracts of H. sabdariffa L. contain highest ascorbic acid content or ascorbate, which is a well-known
phenolic compounds than aqueous extract as shown in natural antioxidant and excellent reducing agent
Figure 1. This result indicates that alcohol is a better (Buettner and Jurkiewicz, 1996). The reducing power has
solvent than water for extraction of phenol from H. been used as one of the antioxidant capability indicators
sabdariffa L. calyx and this result agrees with Anokwuru of medicinal herbs (Duh and Yen, 1997). Reducing power
et al. (2011). The solubility of phenolic compounds is is to measure the reductive ability of antioxidant, and it is
governed by the chemical nature of the plant, as well as evaluated by the transformation of Fe(III) to Fe(II) in the
the polarity of the used solvents. Ethanol is a good presence of the sample (Glin et al., 2003).
solvent for polyphenol extraction and is safe for human The data presented in Table 2 shows that the reducing
consumption (Shi et al., 2005). The current study agrees ability of all samples increased when the concentration of
510 Afr. J. Food Sci.

extracts was increased, and this may be because of the compounds and/or volatile oils are known to inhibit a wide
increment of the phenolic amount at the high range of organisms (Cheesbrough, 1984). Antibacterial
concentration. This result is similar to that reported by activity of gossypetin isolated from H. sabdariffa was
Glin et al. (2003). Among all the reducing power tests, investigated and the activity may be due to polyphenolic
alcoholic extract of H. sabdariffa L. had the highest nature of the flavonoid gossyypetin (Mounnissamy et al.,
reducing ability which may be because of the use of an 2002).
alcoholic solution which provides satisfactory result for
the extraction process (Koffi et al., 2010). The results are
in agreement with those reported by Shil et al. (2005), Conclusion
that the ethanol is a good solvent for polyphenol
extraction and is safe for human. The present study indicated that both aqueous and
Ferrous ion, commonly found in food systems, is well ethanol extracts from the calyx H. sabdariffa L. have
known as an effective pro-oxidant (Hsu et al., 2003). significant natural phenols content and antioxidant
Polyphenols can chelate pro-oxidant metal ions, such as activity. As compared to the artificial antioxidant BHT and
iron and copper, thus preventing free radical formation -tocophero, the aqueous and alcoholic extracts possess
from these pro-oxidants (Kris-Etherton et al., 2002). The strong reducing power and high ferrous ion chelating
ferrous ion chelating effect of H. sabdariffa L. extract is ability. Roselle calyx aqueous and alcoholic extracts of H.
presented in Table 3. Aqueous extract had high ferrous sabdariffa L. effectively and dose-dependently inhibited
ion chelating capability comparing with EDTA. An the growth of E. coli, S. aureus, Str. Mutans and and P.
effective ferrous ion chelator affords protection against aeruginosa.
oxidative damage by removing iron that may otherwise

participate in HO generation through the fenton type
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