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Ethnobotanical
Leaflets 14: 781-96 , 2010. Evaluation
of Antioxidant, Anti-tyrosinase and Antibacterial Activities of Selected Hibiscus Species S.K.
Wong1, Y.Y. Lim1 and E.W.C. Chan2* 1School of Science, 46150 Petaling Jaya, Selangor, Malaysia Corresponding Author: Email: erchan@yahoo.com Issued:
July 01, 2010 Abstract Leaves and flowers of selected Hibiscus
species, used in traditional medicine, were evaluated for antioxidant,
antityrosinase and antibacterial activities. Information on these species is
meagre and this study would contribute new and additional knowledge on the
bioactivities of the genus. Antioxidant properties (AOP) of six species
assessed were total phenolic content (TPC), total anthocyanin content (TAC),
ascorbic acid equivalent antioxidant capacity (AEAC), ferric reducing power
(FRP), ferrous ion chelating (FIC) ability and lipid peroxidation inhibition
(LPI) activity. Antityrosinase and antibacterial activities of four species
were assessed using the modified dopachrome and disc diffusion methods,
respectively. Leaves and flowers of Hibiscus
tiliaceus showed outstanding AOP. Leaves of species with high TPC and AEAC had low FIC ability and vice versa. Red flowers which yielded
the highest TAC also displayed high
FIC ability and LPI activity. Leaves of H. tiliaceus had the strongest
antityrosinase (AT) activity. With very strong AOP and AT activity, leaves
of H. tiliaceus have potentials to be developed into functional food
and skin care products. At 1 mg extract/disc, leaves of Hibiscus sabdariffa were found to
inhibit Gram-positive bacteria of Bacillus
cereus, Micrococcus luteus and Staphylococcus aureus. At 2 mg extract/disc, leaves of H. sabdariffa inhibited both
Gram-positive and Gram-negative bacteria of Escherichia coli, Pseudomonas aeruginosa and Salmonella choleraesuis.
This is the first report of leaf
extracts of H. sabdariffa inhibiting Gram-negative bacteria. Adding
1 mM of ethylenediamine
tetraacetic acid (EDTA) to the agar slightly enhanced
the antibacterial activity of leaves of H.
sabdariffa on Gram-negative bacteria. With a
wide spectrum of inhibition against Gram-positive and Gram-negative bacteria,
leaves of H. sabdariffa are worthy of further investigation as a
natural wide spectrum antibacterial agent. Keywords: Hibiscus, antioxidant, antityrosinase, antibacterial, leaves, flowers. Introduction The genus Hibiscus (Malvaceae) comprises about
275 species in the tropics and sub-tropics (Dasuki,
2001). Within the Malesian region, 43 species are found. Most Hibiscus
species have a remarkable colour pattern with the base of corolla forming a
deep-coloured heart (Lowry, 1976). Another feature is flower colour change
among species of which the most spectacular is in flowers of Hibiscus mutabilis L.
Leaves of Hibiscus are simple, lobed, alternate or spiral and have
paired stipules (Ng, 2006). Flowers are radially symmetrical with cup-shaped calyx, five petals
joined at the base, style bearing many stamens and stigma with five hairy
lobes. With
attractive and colourful flowers, plants of Hibiscus are widely planted as
ornamentals and are used in traditional medicine. Of the species studied,
leaves and flowers of H. mutabilis,
believed to have emollient and cooling effect, are used to relieve swellings
and skin infections (Dasuki, 2001). Leaves and flowers of Hibiscus
rosa-sinensis L. are used as an
antiseptic for boils and ulcers. The sap from
flowers is used as colouring agent. Leaves of Hibiscus
sabdariffa L.
are used as poultice for abscesses and ulcers. Young shoots and leaves are
eaten raw or cooked as vegetable. The red fleshy calyces are widely used to
make beverages and jams. Stems
and roots of Hibiscus taiwanensis
Hu have been used as anti-inflammatory, antifungal,
antipyretic, and antihelminthic agents (Wu et al., 2005). Flowers of Hibiscus tiliaceus L. are
widely used for birth control and for treating skin infections (Rosa et al., 2006). Leaves and flowers of selected Hibiscus species are used in traditional medicine. Information on
their antioxidant, antityrosinase and antibacterial activities is meagre.
This study would contribute new and additional knowledge on the bioactivities
of the genus. Materials and Methods Plant
materials Hibiscus species studied were H.
mutabilis, H. rosa-sinensis, H.
sabdariffa, Hibiscus schizopetalus (Dyer)
Hook. f., H. taiwanensis and H. tiliaceus (Fig. 1). Voucher specimens of species studied were deposited in
the herbarium of Monash University Sunway Campus in
Fig. 1. Hibiscus species studied. Extraction For analysis
of bioactivities, plant materials (1 g) of fresh leaves and petals were powdered with liquid nitrogen in a mortar and extracted using methanol
(50 ml), with continuous swirling for 1 h at room
temperature with an orbital shaker. Extracts were filtered under suction and stored at -20°C for further use. Antioxidant properties Antioxidant properties assessed were total phenolic content, total anthocyanin content, radical scavenging activity, ferric reducing power, ferrous ion chelating ability and lipid peroxidation inhibition activity. Total phenolic content (TPC) was determined using the
Folin-Ciocalteu assay (Kähkönen et al., 1999). Extracts (300 μl;
triplicate) were introduced into test tubes followed by 1.5 ml of
Folin-Ciocalteu’s reagent (10 times dilution) and 1.2 ml of sodium carbonate
(7.5% w/v). The tubes were allowed to stand for 30 min before absorbance at 765 nm was measured. TPC was expressed as
gallic acid equivalent (GAE) in mg per 100 g of material. The calibration
equation for gallic acid was y = 0.0111x - 0.0148 (R2 =
0.9998), where y is absorbance and x is concentration of gallic acid in
mg/l. Total anthocyanin content (TAC) was determined by the pH differential method (Teow et al., 2007). Potassium chloride solution (2 ml, 1 M and pH 1.0) was added to 1 ml of extract in triplicate. Measurements were blanked against sodium acetate buffer (2 ml, 1 M and pH 4.5) with the same amount of extract. Absorbance was measured at 520 nm and 700 nm. TAC was expressed as cyanidin-3-glucoside equivalent (CGE) in mg per 100 g of sample. The molar extinction coefficient of cyanidin-3-glucoside was 26 900. Radical
scavenging activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Miliauskas et al., 2004). Different dilutions of extract (1 ml) were added
to 2 ml of DPPH (5.9 mg/100 ml methanol). Absorbance was measured at 517 nm
after 30 min. Radical-scavenging was calculated as IC50 and
expressed as ascorbic acid equivalent antioxidant capacity (AEAC) in mg AA/100 g = IC50(ascorbate)/IC50(sample)
x 105. The IC50 of ascorbic acid used for calculation
of AEAC was 0.00387 mg/ml. Ferric reducing power (FRP) was assessed using the potassium ferricyanide assay (Chu et al., 2000). Different dilutions of extract (1 ml) were added to 2.5 ml phosphate buffer (0.2 M and pH 6.6) and 2.5 ml of potassium ferricyanide (1% w/v). The mixture was incubated at 50 oC for 20 min. Trichloroacetic acid solution (2.5 ml and 10% w/v) was added to stop the reaction. The mixture was then separated into aliquots of 2.5 ml and diluted with 2.5 ml of water. To each diluted aliquot, 0.5 ml of ferric chloride solution (0.1% w/v) was added. After 30 min, absorbance was measured at 700 nm. FRP was expressed as mg GAE/g. The calibration equation for gallic acid was y = 16.767x (R2 = 0.9974), where y is absorbance and x is concentration of gallic acid in mg/ml. Ferrous ion chelating (FIC)
ability was assessed using the ferrous-ferrozine assay (Singh and Rajini, 2004). Solutions of 2 mM FeSO4 and 5 mM ferrozine were diluted 20 times. FeSO4 (1 ml) was
mixed with different dilutions of extract (1 ml), followed by
ferrozine (1 ml). Absorbance (A) was
measured at 562 nm after 10 min.
Chelating ability = (1 – Asample/Acontrol) x 100%. Lipid peroxidation inhibition (LPI) activity was determined using the β-carotene bleaching assay (Kumazawa et al., 2002). Emulsion of β-carotene and linoleic acid was prepared by adding 3 ml of β-carotene (5 mg in 50 ml chloroform) to 40 mg of linoleic acid and 400 mg of Tween 40. Chloroform was evaporated under vacuum and oxygenated ultra-pure water (100 ml) was added and mixed well. Initial absorbance (A) of the emulsion was measured at 470 nm. Aliquots of the emulsion (3 ml) were mixed with 10, 50 and 100 ml of extracts and incubated in a water bath at 50 °C for 1 h. Bleaching rate of β-carotene was measured at 470 and 700 nm. Measurement at 700 nm was needed to correct for the presence of haze. LPI expressed as antioxidant activity (AOA) was calculated as bleaching rate (BR) of β-carotene = ln(Ainitial/Asample)/60 and AOA = (1 - BR.sample/BRcontrol) x 100% where Ainitial and Asample are absorbance of the emulsion before and 1 h after incubation, and BRsample and BRcontrol are bleaching rates of the sample and negative control, respectively. Antityrosinase
activity Antityrosinase (AT) activity was determined using the
modified dopachrome method with
L-3,4-dihydroxyphenylalanine (L-DOPA) as
substrate (Masuda et al., 2005). Assays were conducted in a 96-well
microtitre plate and a plate reader was used to measure absorbance at 475 nm
with 700 nm as reference. Samples were dissolved in 50% dimethylsulphoxide
(DMSO). Each well contained 40 µl of sample with 80 µl of phosphate buffer (0.1 M, pH 6.8), 40 µl of tyrosinase (31 units/ml) and 40 µl of
L-DOPA (2.5 mM). Each sample was accompanied by a blank that had all the
components except L-DOPA. Results were compared with a control consisting of
50% DMSO in place of sample. AT activity was calculated as (Acontrol - Asample)/Acontrol x 100%. Leaves
of Psidium guajava L. (Guava) were
used as positive control as they have very high AT activity (Vimala et al.,
2006). Antibacterial
activity The
disc diffusion method (Chung et al., 2004) was used to screen for antibacterial activity of
leaf extracts. Agar cultures of Gram-positive bacteria of Bacillus cereus, Micrococcus luteus and Staphylococcus
aureus, and Gram-negative bacteria of Escherichia
coli, Pseudomonas aeruginosa
and Salmonella choleraesuis were
prepared. Suspensions of bacteria (100 µl) were spread evenly onto 20 ml
Mueller-Hinton agar preset in 90 mm Petri dishes. Paper discs (6 mm diameter)
were impregnated with 1 mg of extract dissolved in 100 µl solvent, and
transferred onto the inoculated agar. Streptomycin susceptibility discs (10
µg) and methanol impregnated disc were used as positive and negative
controls, respectively. After incubation overnight at 37 oC,
inhibition zones were measured and recorded as mean diameter (mm). Antibacterial activity was also
expressed as inhibition percentage of streptomycin and arbitrarily classified
as strong for inhibition of ≥ 70%, moderate for inhibition 50 < 70%,
and weak for inhibition < 50% (Chan et
al., 2007). Antibacterial activity was also
determined using the minimum inhibitory dose (MID) method (Mackeen et al., 2000). For species with antibacterial activity, the disc diffusion method
was repeated using diluted extracts and loaded onto paper discs. The MID is
the minimum concentration of extract in mg/disc that showed positive
inhibition of test bacteria. Two
approaches were adopted to enhance the efficacy of extracts to inhibit
Gram-negative bacteria (Wong, 2008). The first approach
was to repeat the disc diffusion method by increasing the extract
concentration from 1 to 2 mg extract/disc. The second approach was to add 1 mM of ethylenediamine tetraacetic acid
(EDTA) to the agar before culturing the bacteria.
The diffusion method was repeated using 1 and 2 mg extract/disc. Results and Discussion Description of species Leaves of H.
mutabilis (Confederate rose) are broadly ovate
with mostly five triangular lobes. Flowers are white in the morning, turning
pink in the afternoon, and red in the evening. Leaves H. rosa-sinensis (China
rose) are ovate with serrated margins.
Flowers are red with a long and slender style, anthers yellow and
stigma red. Leaves of H. sabdariffa ( Antioxidant
properties Leaves
of H. tiliaceus showed outstanding
antioxidant properties (AOP) with TPC and AEAC values of 2080 mg GAE/100 g and 2370 mg
AA/100 g, respectively (Table 1). Values were 2.4 and 2.7 times
higher than those of H. mutabilis which
ranked second. Based on TPC and AEAC, ranking was: H. tiliaceus > H. mutabilis > H.
sabdariffa > H. taiwanensis >
H. schizopetalus ~ H. rosa-sinensis. Table 1. Total phenolic content (TPC) and ascorbic acid equivalent antioxidant capacity (AEAC) of leaves of Hibiscus species.
Values of TPC and
AEAC are means ± SD (n = 3). For each
column, values followed by the same letter (a-e) are not statistically
different at P < 0.05 as
measured by the Tukey HSD test. For each species, samples were collected from
the same location. Abbreviations: GAE = gallic acid equivalent and AA = ascorbic
acid. Leaves of H. schizopetalus, H. sabdariffa and H. rosa-sinensis had better FIC ability than those of H. mutabilis, H. tiliaceus and H. taiwanensis (Fig. 2). Leaves of species with higher TPC and AEAC had lower FIC ability for H. tiliaceus and H. mutabilis, and vice versa for H. schizopetalus and H. rosa-sinensis. This suggests the presence of compounds in leaves of H. schizopetalus and H. rosa-sinensis with relatively weak radical scavenging activity but good metal chelating ability that could prevent the generation of hydroxyl radicals via Fenton’s reaction.
Fig. 2. Ferrous ion chelating ability of leaves of Hibiscus species Flowers
of H. tiliaceus with TPC, AEAC and
FRP values of 2420 mg GAE/100 g, 3180
mg AA/100 g and 14 mg GAE/g, respectively, were significantly higher than all other species (Table 2). Based
on TPC, ranking was: H. tiliaceus > H. rosa-sinensis > H. taiwanensis ~ H.
schizopetalus ~ H. mutabilis > H. sabdariffa. The red flowers of H. rosa-sinensis and H.
schizopetalus, which yielded the highest TAC, displayed high FIC ability and LPI activity (Fig. 3). Species with low TAC such as H.
mutabilis and H. sabdariffa
displayed low or no FIC ability and LPI activity. TAC appears to be
positively correlated with FIC ability and LPI activity in flowers of Hibiscus species. Anthocyanins with potent metal-chelating activity have been reported in peels of the egg plant Solanum melongena L.
(Noda et al., 2000) and leaves of Perilla
pankinensis Decne (Gülcin et al., 2005). Table 2. Total phenolic content (TPC), total anthocyanin content (TAC), ascorbic acid equivalent antioxidant capacity (AEAC) and ferric reducing power (FRP) of flowers of Hibiscus species
Values of TPC, TAC,
AEAC and FRP are means ± SD (n = 3).
For each column, values followed by the same letter (a-f) are not
statistically different at P <
0.05 as measured by the Tukey HSD test. For each species, samples were
collected from the same location. Abbreviations: GAE = gallic acid
equivalent, AA = ascorbic acid and CGE = cyanidin-3-glucoside equivalent. Of the
six Hibiscus species, leaves and
flowers of H. tiliaceus had the
strongest AOP. A likely explanation is that H. tiliaceus is the only indigenous tree species while the other
species are exotic shrubs and herbs. Being long-lived, trees have to produce a wide range of chemical
defenses against herbivores and infections. Higher
antioxidant activity of trees than shrubs and herbs has been reported (McCune and Johns,
2007). Based on AOP of leaves and flowers, the six Hibiscus species can be divided into three categories (Wong et al., 2009). They are species with comparable values in leaves and flowers (H. tiliaceus), species with significantly higher values in leaves than flowers (H. mutabilis and H. sabdariffa), and species with significantly higher values in flowers than leaves (H. taiwanensis, H. rosa-sinensis and H. schizopetalus). Antityrosinase activity Of four species of Hibiscus
tested, leaves of H. tiliaceus (42%)
had the strongest AT activity (Table 3). Values
were comparable to leaves of P. guajava (41%) as
positive control. Ranking of AT activity was: H. tiliaceus > H.
mutabilis > H. rosa-sinensis
~ H. sabdariffa. Leaves of H. tiliaceus displayed the highest tyrosinase
inhibition among 39 tropical plant species screened by Masuda et al.
(2005).
a Fig. 3. Ferrous ion chelating (FIC) ability
(a) and lipid peroxidation Antibacterial
activity At 1 mg extract/disc, leaves of H. tiliaceus and H. sabdariffa were found to inhibit Gram-positive bacteria of M. luteus, S. aureus and B. cereus (Table 4). Leaves of H. tiliaceus exhibited moderate antibacterial activity against all three Gram-positive bacteria. Leaves of H. sabdariffa weakly inhibited M. luteus and B. cereus. Mean diameter zone of inhibition of streptomycin was 20 mm for M. luteus, 18 mm for S. aureus and 18 mm for B. cereus. Leaves of all four Hibiscus species showed no antibacterial activity on Gram-negative bacteria of P. aeruginosa, S. choleraesuis and E. coli. Table
3.
Antityrosinase (AT) activity of leaves of Hibiscus
species
Values of AT activity are means ± SD (n = 3). Values followed by the same letter (a-c) are not statistically different at P < 0.05 as measured by the Tukey
HSD test. Concentration of extracts used was 0.5
mg/ml. Leaves of P. guajava were
used as positive control. Table
4. Antibacterial activity of
leaves of Hibiscus species at 1 mg
extract/disc
Antibacterial
activity is categorized as moderate ++ for inhibition 50 < 70% or weak + for inhibition < 50%
based on inhibition percentage compared to streptomycin. Chung et al. (2004) reported that none of
191 plant extracts from 30 families inhibited Gram-negative bacteria.
Similarly, screening of extracts of
26 edible plant species by Alzoreky and Nakahara (2003) showed that
Gram-negative bacteria were not susceptible to plant extracts. Screening of
five different extracts from five ethnomedicinal plant species also showed
that most of the antibacterial activity detected was against Gram-positive
bacteria (Murugan et al., 2008). Gram-negative bacteria are
generally less susceptible to plant extracts than Gram-positive bacteria due
to their outer membrane of lipopolysaccharide and lipoprotein, which is
resistant towards antibacterial substances (Chopra and Greenwood, 2001;
Alzoreky and Nakahara, 2003). For Gram-positive bacteria, MID of leaf extracts of H. sabdariffa was 1 mg/disc for M. luteus and B. cereus, and 2 mg/disc for S.
aureus (Table 5). MID of leaf extracts of H.
tiliaceus was 0.5 mg/disc for M.
luteus, 0.25 mg/disc for S. aureus
and 1 mg/disc for B. cereus. Table
5. Minimum inhibitory dose (MID) of
leaves of Hibiscus sabdariffa and H. tiliaceus
Antibacterial
activity is categorized as moderate ++ for
inhibition 50 < 70% or weak + for inhibition < 50%
based on inhibition percentage compared to streptomycin. Screening for antibacterial activity
of plant extracts is normally done using the disc diffusion method at
concentration of 1 mg extract/disc. By increasing the concentration to 2 mg
extract/disc, leaves of H. sabdariffa
displayed moderate inhibition on all six Gram-positive and Gram-negative bacteria (Table 6). Gram-negative
bacteria were not inhibited at 1 mg extract/disc. Results indicated that
phenolic compounds in leaves of H.
sabdariffa might be bactericidal and the mode of action might be dose
dependent. At 2 mg extract/disc, antibacterial activity of leaves of H. tiliaceus remained the same as 1 mg
extract/disc. Inhibition was moderate on S.
aureus, and weak on M. luteus and B. cereus with no inhibition on the three Gram-negative bacteria. Results indicated that the inhibitive
properties of phenolic compounds in leaves of H. tiliaceus might be non-dose dependent. This is the first report of leaf extracts of H.
sabdariffa inhibiting Gram-negative bacteria of P. aeruginosa, S.
choleraesuis and E. coli. None of plant extracts from 50 species reported by Wiart et al.
(2004) inhibited E. coli. With a wide spectrum of inhibition against
Gram-positive and Gram-negative bacteria, leaves of H. sabdariffa are
worthy of further investigation as a natural
wide spectrum antibacterial agent. Similar wide spectrum of
antibacterial activity has been reported in extracts of Andrographis paniculata (Burm. F.) Nees (Sule et al., 2010). Table
6. Antibacterial activity of
leaves of Hibiscus sabdariffa and Hibiscus tiliaceus at 2 mg extract/disc
Antibacterial
activity is categorized as moderate ++ for inhibition 50 < 70% or weak + for inhibition < 50%
based on inhibition percentage compared to streptomycin. Adding 1 mM of EDTA to the agar slightly enhanced the antibacterial activity of leaves of H. sabdariffa on Gram-negative bacteria (Table 7). At 2 mg/disc, extracts of H. sabdariffa moderately inhibited E. coli and weakly inhibited P. aeruginosa. At 1 mg/disc, leaves of H. sabdariffa weakly inhibited P. aeruginosa which showed no inhibition without EDTA. Leaves of H. tiliaceus showed no activity on Gram-negative bacteria at extract concentrations of 1 and 2 mg/disc. This study also yielded other interesting results. EDTA adversely affected the bactericidal activity of streptomycin. With S. choleraesuis, the antibiotic showed no zones of inhibition. In the absence of EDTA, the antibiotic yielded zones of inhibition of 13 mm. With P. aeruginosa, EDTA enhanced the bactericidal effect of streptomycin as the zone of inhibition was enlarged to 22 mm compared to 14 mm without EDTA. Haque and Russell (1974) have reported that EDTA can permeabilise the outer membrane of P. aeruginosa, making the bacteria susceptible to antibiotics and certain antiseptic agents. Sensitivity of Gram-negative bacteria to antibacterial agents is mainly attributed to increasing permeability of the outer membrane and releasing endogenous phospholipases degrading membrane lipids (Alzoreky and Nakahara, 2003). In this study, adding 1 mM of EDTA to the agar rendered streptomycin ineffective against S. choleraesuis but enhanced the efficiency of the antibiotic against P. aeruginosa. This observation has not been reported before. Alzoreky and Nakahara (2003) had reported that EDTA inhibited the growth of B. cereus while S. aureus grew prolifically. Table 7.
Antibacterial activity of leaves of Hibiscus sabdariffa and
Hibiscus tiliaceus on Gram-negative
bacteria with 1 mM of EDTA added to the agar (fresh weight).
Based on inhibition percentages compared to
streptomycin, antibacterial activity is categorized as moderate ++ for inhibition 50 < 70%,
or weak + for inhibition < 50%. Conclusion Of six Hibiscus
species screened, leaves and flowers of H.
tiliaceus showed outstanding AOP. Leaves of species with high TPC, AEAC and FRP had low FIC ability. Red flowers with high TAC is positively correlated with FIC
ability and LPI activity. Based on AOP
of leaves and flowers, the six species screened can be categorized into
species with comparable values in leaves and flowers, species with
significantly higher values in leaves than flowers, and species with
significantly higher values in flowers than leaves. Of four species of Hibiscus tested, leaves of H. tiliaceus had the strongest AT
activity. With
strong AOP and AT activity, leaves of H. tiliaceus have potentials to
be developed into functional food and skin care products. At 1 mg
extract/disc, leaves of H. sabdariffa were
found to inhibit Gram-positive bacteria of M. luteus, S. aureus and B. cereus but not Gram-negative
bacteria of P. aeruginosa, E. coli and S.
choleraesuis. At 2 mg
extract/disc, leaves displayed inhibition on all six Gram-positive and
Gram-negative bacteria. Adding 1 mM of EDTA to the agar slightly enhanced the
antibacterial activity of leaves against Gram-negative bacteria. This is the first report of leaf extracts
of H. sabdariffa inhibiting Gram-negative bacteria. With a wide
spectrum of inhibition against both Gram-positive and Gram-negative bacteria,
leaves of H. sabdariffa are worthy of further investigation as a
natural wide spectrum antibacterial agent. Acknowledgement The authors are thankful to Monash
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