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International Journal of Food Science and Nutrition
ISSN: 2455-4898
Impact Factor: RJIF 5.14
www.foodsciencejournal.com
Volume 2; Issue 5; September 2017; Page No. 01-06
Studies on the therapeutic properties of Roselle (Hibiscus sabdariffa) calyx: A popular ingredient in
the cuisine of North East India
*1 Kekungu-u Puro, 2 Chubasenla Aochen, 3 Sandeep Ghatak, 4 Samir Das, 5 Rajkumari Sanjukta, 6 Kamal Prasad
Mahapatra, 7 Anjani Kumar Jha, 8 Ingudam Shakuntala, 9 Arnab Sen
1, 3, 4, 5, 8, 9 Animal Health Division, ICAR-Research Complex for North Eastern Hill Region, Umiam, Meghalaya, India
2 Biotechnology Division, ICAR-Research Complex for North Eastern Hill Region, Umiam, Meghalaya, India
6 Agroforestry Division, ICAR-Research Complex for North Eastern Hill Region, Umiam, Meghalaya, India
7 Horticulture Division, ICAR-Research Complex for North Eastern Hill Region, Umiam, Meghalaya, India
Abstract
Roselle plant (Hibiscus sabdariffa) of Malvaceae family finds its mention in folk medicine of North East India for its use in
various ailments. In the present study, the proximate value and nutritional quality, antioxidant activity, antimicrobial properties,
anti-proliferative activity and apoptosis-inducing capacity of roselle found in North East India was evaluated. Nutritional
evaluation of the calyx establishes it as a good source of dietary antioxidants and ascorbic acid. Antimicrobial activity of the
aqueous extract upto 25mg/ml concentration showed complete growth inhibition of both gram positive (Staph. aureus) and gram
negative (E. coli, K. pneumoniae) bacteria while antioxidant activity with 100 mg/ml concentration was high (90% scavenging
activity) that gradually decreases with dilution in concentration. The antiproliferative and apoptotic activities were evaluated in
cervical (HeLa) cancer cell lines. It exhibited anti-proliferative activity in a concentration-dependent manner; the concentration
@100mg/ml can induce upto 51.8% apoptosis. The studies indicated its potential benefits for health which can further be tested
and validated for use as supplement in the food.
Keywords: Hibiscus sabdariffa, cancer cells, antioxidant, antimicrobial activity
Introduction
The roselle plant, with an attractive flower believed to be
native to Africa, is cultivated in Sudan, India, Malaysia and
Taiwan. It is also one of the signature food ingredients of
indigenous delicacies such as Jingtah jajewsaw in Meghalaya,
Masor tenga in Assam, Gakro ghapha in Nagaland, Silok
sougri Mapan Metpa in Manipur etc. The flowers are also
used as a dye by virtue of its bright red colour. In addition to
their use in food, various parts of the roselle plant have been
used in traditional medicine for the prevention of diseases. It
has been used in herbal tea to treat hypertension, pyrexia and
liver damage although the pharmaceutical components are
poorly defined [1].Various workers reported the roselle calyx
possesses various beneficial effects in human health as
mediated through its antioxidant property [2, 3].
Supplementation of roselle calyx extract in ex vivo murine
hematopoietic stem cells culture increased the survivability of
cells [4]. It also had an inhibitory effect on biofilm formation
of oral pathogenic bacteria [5].
The calyces are also rich in vitamins, especially ascorbic acid
[6]. Ascorbic acid is an essential dietary component. Various
workers have reported variable content suggesting the type of
soil influences its ash and mineral content causing variations
within the same species [7, 8, 9, 10]. The antimicrobial activity,
antibacterial and cytotoxicity studies in dose-dependent
manner have also been studied [11, 12]. Moreover, the
antimicrobial activity on isolates from food, veterinary, and
clinical samples have indicated that roselle extract contain
potential antimicrobials in foods [13].Antioxidative activity of
the extract was also reported in cancerous cell lines while the
antioxidant potential in the form of scavenging free radical
and inhibitory effect on XO activity has been evaluated [14, 15].
The effects of roselle-anthocyanins (HA) on human cancer
cells (HL-60) showed apoptosis of cells in a dose and time-
dependent manner [16]. It has also been reported to induce
apoptosis in leukemic cells via reduction of retinoblastoma
phosphorylation and Bcl2 expression [17].
In the present study, the nutritional constituents, antioxidant
activity, antimicrobial properties, antiproliferative activity and
apoptotic potential of locally available roselle plant,
commonly used in the indigenous cuisines, is evaluated to
determine its therapeutic benefits.
Materials and Method
Sample collection and preparation
The roselle calyces (Figure 1) were harvested between
November-December from Bhoirymbong village in Ri-Bhoi
district, located between 25°40´ to 25°21´N latitude and
90°55´15´´ to 91°16´E longitude in the state of Meghalaya,
India. The calyces were dried and powdered for proximate
analysis of nutrient contents and others. The extracts @100
mg/ml (w/v) concentration with water was prepared,
homogenized and clarified by centrifugation at 10000 rpm for
30 min at 4°C. The aqueous extract was collected and filtered
International Journal of Food Science and Nutrition
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with 0.45 µm syringe filter for further in vitro studies. One
gram of the powder was extracted overnight in 80% methanol
and filtered. The filtered extract was used for estimation of
Total antioxidant capacity (TAC) and phenols, respectively.
Fig 1: Roselle plant (a) flowering stage and (b) calyces
Tables and Figures
Estimation of Proximate analysis
The proximate analysis of roselle calyces was done as per the
method prescribed by AOAC for estimating moisture content,
dry matter (DM), total ash (TA), crude protein (CP), ether
extract (EE) and crude fibre (CF) [18]. The nitrogenous free
extract (NFE) was calculated from 100 - (% moisture + % CF
+ % CP + % EE + % TA).
Total phenols content
Phenol content was determined with Folin-Ciocalteau reagent
by the method described by Singleton et al. [19]. 0.5 ml of
extract was initially made up to 3 ml with 80% methanol. 1 ml
of DMSO and 1 ml of 10% Folin-Ciocalteau reagent was
added and mixed. 3 ml of 1% Na2CO3 was finally added after
3 minutes, and the tubes were incubated for 2 h at room
temperature. A standard curve was plotted using different
concentrations of gallic acid (50-300 μg/ml). Absorbance was
measured at 760 nm. The phenolic contents were calculated
on the basis of the calibration curve of gallic acid (50-300
ug/ml) and expressed as gallic acid equivalents (GAE), in
milligrams per gram of dry weight.
Ascorbic Acid content
Vitamin C content was determined by the method described
by Sadasivam and Manickam [20]. Two gram of the powder
was extracted with 4% oxalic acid overnight. The extract was
filtered and used for the analysis. To 10 ml of the extract, a
few drops of bromine water was added until the solution
became coloured, confirming the completion of the oxidation
of ascorbic acid to dehydroascorbic acid. The final volume
was then made to 50 ml with 4% oxalic acid. 2 ml of sample
aliquots was made up to 3 ml of distilled water. To this 1 ml
of 2, 4-Dinitrophenylhydrazine reagent was added and 1-2
drops of 10% thiourea was added to remove excess bromine.
The reaction mixture was mixed and incubated at 37oC for 3
h. 5 ml of 80% sulphuric acid was then added to dissolve the
osazone crystals formed. A standard curve was plotted using
different concentrations of Ascorbic acid (20-120 μg/ml).
Absorbance was measured at 540 nm. The total phenolic
content was expressed in milligrams per gram of dry weight.
Total Antioxidant Capacity
Total antioxidant capacity was estimated with
phosphomolybdenum reagent by the method described by
Prieto et al. [21]. To 0.3 ml of sample extract, 3 ml of
phosphomolybdenum reagent solution (0.6 M sulphuric acid,
28 mM potassium phosphate and 4 mM ammonium
molybdate) was added. The tubes were then incubated at 95oC
for 1 h. After cooling at room temperature the absorbance was
measured at 695 nm. A standard curve of ascorbic acid was
plotted (20-100 µg/ml) to calculate the values. Total
antioxidant capacity was expressed as Ascorbic acid
equivalents (AAE), in milligrams per gram of dry weight.
Free radical scavenging activity assay
Antioxidants are compounds with free radical scavenging
activity and its activity in the extract was measured using 2, 2-
Diphenyl-1-picrylhydrazyl (DPPH) assay as per the method
described by Yamasaki et al. [22]. 20 µl of plant extracts (five
serial dilutions RD1-RD5 of 100, 50, 25, 12.5 and 6.25
mg/ml) or standard solution (BHT) is added to 200 µl of
DPPH reagent in 96 well plates in triplicates. Absolute
methanol is used as reagent blank. All reagents is mixed and
incubated for 30 min at room temperature, protected from
light. The absorbance is measured at 490 nm with an ELISA
reader (Lab systems Multiskan Plus, Thermo Fisher Scientific,
USA).The percentage of DPPH free radical scavenging
activity is calculated as % inhibition = 100 - [(As-Ab)/Ac x
100] Where As is the absorption of extract, Ab is the
absorption of the blank sample and Ac is the absorption of the
control.
Antimicrobial assay
The activity of antimicrobial properties was studied using agar
dilution method [23]. One ml of 0.5 McFarland microbial
inoculum preparation by direct colony suspension (CLSI
guidelines) of Escherichia coli (E.coli) -ATCC 25922,
Staphylococcus aureus (Staph. aureus) -ATCC 25923 and
Klebseilla pneumoniae (K. pneumoniae) - ATCC 700603
having approximately 106 bacterial/ml was added to 1 ml of
serial two-fold dilution (100, 50, 25, 12.5, 6.25, 3.125 mg) of
aqueous extract of roselle in tubes and incubated at 37°C for
24 h. The incubated tubes were poured into agar plates in
duplicate and incubated at 37°C for 24-48 h. The minimum
inhibitory concentration (MIC) endpoint is recorded as the
lowest concentration of roselle extract that completely inhibits
growth under suitable incubation conditions using methanol
and non-treated control.
Anti-proliferative assay
Two-fold serial dilution of the aqueous extract was prepared
and upto five dilutions (100, 50, 25, 12.6 and 6.25 mg/ml)
were used for studying the anti-proliferative effects in HeLa
cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide (MTT) assay as per Mosmann with slight
modification [24]. The cells @1x106/ml in Earle’s Minimum
Essential Medium (EMEM), (Sigma-Aldrich, USA) with 10%
FBS were suspended in a 96 well plate @100 µl/well and 100
µl of each dilution were added in triplicates with mitogen-
Concanavalin A (ConA) and Phytohaemagglutinin A (PHA)
@ 10mg/ml as positive control. The cells were incubated at
International Journal of Food Science and Nutrition
3
37°C with 5% CO2 for 72 h. 10 µg MTT dye was then added
to each well and further incubated for 4 h. The supernatants
were removed and dimethyl sulfoxide (DMSO) @150 µl was
added to each well and incubated at room temperature by
gentle shaking for 30 min. The supernatants were collected
and the absorbance was measured at 570 nm with an ELISA
reader (Lab systems Multiskan Plus, Thermo Fisher Scientific,
USA).
Apoptosis assay
The apoptosis assay was done using Annexin V- FITC
apoptosis detection kit (Calbiochem, Germany) in flow
cytometer (BD – LSR Fortessa, USA). Two-fold five serial
dilution (100, 50, 25, 12.6 and 6.25 mg/ml) of the aqueous
extract was prepared for studying apoptosis in HeLa cells
along with non-treated control. The cells @1x106/ml in
Earle’s Minimum Essential Medium (EMEM), (Sigma-
Aldrich, USA) with 10% FBS were suspended in a 96 well
plate @100 µl/well and 100 µl of each dilution were added in
triplicates. The control wells were treated with 100 ml PBS
(pH 7.2). The cells were incubated at 37°C with 5% CO2 for
24 h. The cells were then processed using apoptosis detection
kit following manufacturer’s instructions. The percentage of
apoptosis was measured in flow cytometer with setting of
unstained cells and cells stained with isotype control. Samples
were acquired taking 10000 cell counts.
Statistical analysis
The experiment were done in triplicates and the results were
expressed as mean ± SD. Statistical difference compared
between various treated and untreated groups were analyzed
by one way analysis of variance (ANOVA) followed by
Tukey test for significance. Statistical analysis was considered
significant if P< 0.05.
Results and Discussion
Nutritive value
Proximate composition of dried calyces is presented in Table
1. Dry matter, crude fibre, crude protein, ash and moisture
content were 40.45, 20.83, 27.32, 4.67 and 6.79%,
respectively. The NFE was calculated as 64.14% which
represent the soluble carbohydrate and other easily utilizable
non-nitrogenous components. The analyses present a
comparable proximate composition. The nutritional content of
the calyx was in the range as reported in three varieties found
in Sudan with the moisture content range from 6.19 -12.07%,
CP between 5.5 to 9.76%, CF from 10.74 and 12.17% and
carbohydrate between 60.43 and 61.76% [25]. Carbohydrate
content as high as 68.7% followed by crude fibre (14.6%) and
ash content (12.2%) has also been reported [26].
Table 1: Proximate composition of roselle calyces
Content
Values (%)
Moisture
09.53 ± 1.796
Dry matter (DM)
90.47 ± 1.796
Crude protein (CP)
08.31 ± 0.360
Crude fiber (CF)
11.53 ± 0.608
Ether extract (EE)
00.80 ± 0.035
Total ash (TA)
05.69 ± 0.431
Nitrogen free extract (NFE)
64.14 ± 1.294
Values are mean of triplicates + SD
Biochemical properties
Table 2 presents the biochemical properties of the calyx
analyzed. The total phenol content of methanolic extract was
measured at 7.12 mg GAEg-1, and total antioxidant capacity at
3.39 mg AAE g-1. A lower value of 2.91 mg GAE g-1 has also
been reported [27]. The presence of polyphenols in the plants in
itself is indicative of the presence of antioxidative function,
due to their inherent high redox potentials which make them
efficient reducing agents, hydrogen donors and singlet oxygen
quenchers [28]. The total antioxidant capacity measured in the
methanolic extract was 3.39 + 0.25 mg AAE g-1. Clear
correlations have also been established between antioxidant
activity and phenolic content in plant extracts [29]. The results,
therefore, reinforce the potential of Roselle as an important
source of natural dietary antioxidants. Roselle calyces in the
study exhibited an Ascorbic acid content of 0.801 mg g-1; the
dietary conversion is 80 mg/100 g edible portion. Duke and
Atchley had reported an ascorbic acid content of 14 mg/100g
in fresh calyces6. Tee et al. also established that roselle
calyces had higher ascorbic acid content than orange [30]. The
calyces in this study exhibited a dietary ascorbic acid value of
80 mg/100 g; this value is higher than the established average
values of 42.7 and 43 mg/100 g for orange and papaya,
respectively [31]. Roselle calyces are, thereby a nutritionally
viable source of food and condiment because of its high
antioxidant capacity and an appreciable amount of ascorbic
acid content.
Table 2: Biochemical Properties of calyces
Parameter
Value
Total Phenol content (mg GAEg-1)
7.12 + 0.20
Total Antioxidant capacity (mg AAEg-1)
3.39 +0.25
Total Ascorbic Acid content (mg g-1)
0.801 + 0.05
Values are mean of triplicates + SD
Free radical scavenging assay
The scavenging activity ranged from 89.7% in RD1 to 57.02%
in RD5 indicating the concentration-dependent inhibition
(Table 3). The standard BHT showed 99.56% inhibition.
Antioxidant activity of roselle also represents the free radical
scavenging activity which was found to be dose-dependent.
The 100 mg/ml concentration of roselle extract exhibited
89.70% scavenging activity compared to 99% standard BHT,
thereby indicating high antioxidant potentials. The ethanol
crude extract from the dried flowers have capacity of
quenching free radical and inhibiting xanthine oxidase (XO)
International Journal of Food Science and Nutrition
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activity [32]; in animal models, calyces extracts have
demonstrated hypocholesterolemic and antihypertensive
properties [33].
Table 3: Inhibition/scavenging activity of serial dilution RD1-RD5
of roselle extract, C – Standard BHT contral (n=3)
Dilution
Conc (mg/ml)
% inhibition/scavenging activity
RD1
100
89.70 ± 0.639
RD2
50
63.56 ± 2.132
RD3
25
60.69 ± 1.350
RD4
12.6
58.38 ± 2.286
RD5
6.25
57.01 ± 4.045
C
Std BHT
99.56 ± 0.638
Values are mean of triplicates + SD
Antimicrobial assay
The MIC of two-fold dilution exhibited a complete growth
inhibition of both gram positive (Staph. aureus) and gram
negative (E. coli, K. pneumonia) bacteria up to 2nd dilution (25
mg/ml). Numerous colonies (> 300 colonies) were observed
from the 3rd dilution onwards indicating non-inhibition of
growth (Table 4). There is no growth in methanol control
plates while non-treated control plates have numerous
colonies present. The antimicrobial effect on both gram
positive and gram negative bacteria was dose-dependent as
observed by Chau et al. against food spoilage bacteria using
ethanolic extract [12]. The antimicrobial activity was reported
by other workers on isolates from food, veterinary, and
clinical samples and suggested that it might be potent agents
as food additives to prevent contamination from these bacteria
[13].
Table 4: Antimicrobial pattern of various concentration of roselle
(n=3)
Concentration (mg/ml)
Staph. aureus
E. coli
K. pneumoniae
100
-
-
-
50
-
-
-
25
-
-
-
12.5
+
+
+
6.25
+
+
+
MC
-
-
-
NTC
+
+
+
+ = growth present, - = no growth. MC = methanol control, NTC =
non-treated control
Anti-proliferative assay
The anti-proliferative effect of the extract on the cells was
visualized by microscope at high power magnification. The
formation of formazan crystal on adding MTT dye was
observed (Figure 2). The stimulation index (SI) of roselle-
treated cells was calculated (Figure 3). A gradual decrease in
SI up to third dilution (@ 25mg/ml) was first observed, which
then increased with dilutions. The initial decrease and
subsequent increase of SI perhaps correspond to the apoptosis
of cells in the different concentration of extract compared to
SI of non-treated control which showed non-inhibition of cell
growth while the standard mitogen ConA show higher
stimulation than PHA. The antiproliferative activities of
roselle extract on cervical (HeLa) cancer cell lines showed
dose dependent inhibitory effects. Similar observations was
reported on its activities on different cell lines like ovarian
(Caov-3), breast (MCF-7, MDA-MB-231) and cervical
(HeLa) cancer cell lines found that it exhibit the strongest
anti-proliferative potency towards the MCF-7 cancer cells [14].
Fig 2: Photograph showing (a) inhibition of HeLa cell growth and (b)
formazan crystal formation (20 X)
Fig 3: Stimulation index of antiproliferative assay with serial dilution
(RD1-RD5) of roselle extract in HeLa cells. C - Non-treated control,
Mitogen-Con A and PHA @10mg/ml. (n=3)
Apoptosis in HeLa cells
The fractions of the cells as early apoptosis, late apoptosis,
necrotic and healthy cells were calculated in percentage from
the total population of cells counted. The result showed the
induction of apoptosis is concentration dependent (Table 5)
ranges from 51.8% to 9.4 % in decreasing order of
concentration in roselle-treated HeLa cells. The apoptotic
activity of roselle was found to be concentration dependent in
cervical (HeLa) cancer cell lines. Similar report on effects in
human cancer cells (HL-60) using roselle-anthocyanins (HA)
show apoptosis of cells in a dose- and time-dependent manner
[16]. The 6.3% apoptosis in non-treated control cells probably
represent the percentage of necrotic cell death. Therefore, the
use of roselle calyces in the dietary habits definitely offers
benefits in terms of its antioxidant properties, antimicrobial
effects, antiproliferative and apoptotic activities against
cancerous cells. The other parts of the plant, viz. leaves and
seeds needs further investigation in this regard.
Table 5: Apoptosis of HeLa cells treated with various concentration
of rosells extract (n=3)
Concentration (mg/ml)
Apoptosis (%)
100
51.8 ± 0.6364
50
25.8 ± 0.3536
25
20.5 ± 0.2121
12.5
10.6 ± 0.5657
6.25
9.4 ± 0.1414
Non-treated control
6.3 ± 0.0707
International Journal of Food Science and Nutrition
5
Conclusions
The oral indigenous traditional knowledge on therapeutic uses
of traditional foods ingredient have been supported by
research studies and documentation. Besides the regular
dietary use of roselle in popular delicacies in North east region
of India, the current study also reiterates roselle as a source of
natural antioxidants and a good and cheaper source of
Ascorbic acid, more than most fruits and vegetables. The
promising preliminary results with its extracts on cancerous
cells studies also provide new avenues for natural therapeutic
research on cancer. Therefore, with many enriching chemical-
biological knowledge from animal and human models using
plant extracts, future focused studies with greater scientific
robustness in terms of standardization of dose for its
effectiveness, safety and tolerability will permit the
formulation of safe, effective therapeutic herbal formulations
which can be used as an acceptable source for curing many
health issues and restoring general health.
Acknowledgement
The research work is done under the project “Investigation
and evaluation of ethno-veterinary medicines through
biological activity screening of medicinal plants” Code no.
IXX08847 of the institute. The authors thank the Director,
ICAR-RC for NEH Region and Umiam for providing
necessary facilities to carry out the work.
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