Antiviral activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against dengue virus type 2 (GenBank accession no. MH488959)

Abstract

This study investigated the antiviral activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against dengue virus type 2 (DENV-2). The pulp of red pitahaya and the leaves of red spinach were extracted using
methanol followed by sub-fractionation and Amberlite XAD16N column chromatography to obtain betacyanin fractions. The
half maximum cytotoxicity concentration for betacyanin fractions from red pitahaya and red spinach on Vero cells were 4.346
and 2.287 mg ml−1, respectively. The half-maximal inhibitory concentration (IC50) of betacyanin fraction from red pitahaya was
125.8 μg ml−1 with selectivity index (SI) of 5.8. For betacyanin fraction from red spinach, the IC50 value was 14.62 μg ml−1 with
SI of 28.51. Using the maximum non-toxic betacyanin concentration, direct virucidal effect against DENV-2 was obtained from
betacyanin fraction from red pitahaya (IC50 of 126.70 μg ml−1; 95.0% virus inhibition) and red spinach (IC50 value of 106.80 μg
ml−1; 65.9% of virus inhibition). Betacyanin fractions from red pitahaya and red spinach inhibited DENV-2 in vitro.

Full text

1
Antiviral activity of betacyanins from red pitahaya (Hylocereus
polyrhizus) and red spinach (Amaranthus dubius) against dengue
virus type 2 (GenBank accession no.MH488959)
Ying JunChang1, Lian YihPong2,3, Sharifah S.Hassan2,3 and Wee SimChoo1,*
SHORT COMMUNICATION
Chang etal., Access Microbiology
DOI 10.1099/acmi.0.000073
Received 25 April 2019; Accepted 03 October 2019; Published 31 October 2019
Author aliations: 1School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia; 2Jerey Cheah
School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia; 3Infectious
Diseases and Health Cluster, Tropical Medicine and Biology Platform, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway,
Selangor, Malaysia.
*Correspondence: Wee Sim Choo, choo. wee. sim@ monash. edu
Keywords: Betalain; cytotoxicity; viral infectivity; pathogenic; red dragon fruit; viral replication.
Abbreviations: ATCC, American Type Culture Collection; BCIP, 5- bromo-4- chloro-3’-indolyphosphate p- toluidine salt; BSA, Bovine serum albumin;
CC50, Half maximum cytotoxicity concentration; CMC, Carboxymethyl cellulose; CO2, Carbon dioxide; DENV-2, Dengue virus serotype 2; DENV, Dengue
virus; DHF, Dengue hemorrhagic fever; DSS, Dengue shock syndrome; ESI, Electrospray ionization; FBS, Fetal bovine serum; FFU, Foci- forming
units; HCl, Hydrochloric acid; HEPES, 4-(2- hydroxyethyl)-1- piperazineethanesulfonic acid; HPLC, High performance liquid chromatography; IC50, Half
maximal inhibitory concentration; LC- MS/MS, Liquid chromatography- Mass spectrometry/Mass spectrometry; MEM, Minimum essential medium;
MOI, Multiplicity of infection; MTT, 3-(4,5- dimethylthiazol-2- yl)-2,5- diphenyltetrazolium bromide; NBT, Nitrotetrazolium blue chloride; PBS, Phosphate
buer saline; PRNT, Plaque reduction neutralisation test; Q- TOF LC/MS, Quadrupole time- of- flight- Liquid chromatography/Mass spectrometry; SDS,
Sodium dodecyl sulfate; SI, Selectivity index; TBS, Tris- buered saline; TBST, Tris- buered saline containing Tween 20.
000073 © 2019 The Authors
This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Abstract
This study investigated the antiviral activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaran-
thus dubius) against dengue virus type 2 (DENV-2). The pulp of red pitahaya and the leaves of red spinach were extracted using
methanol followed by sub- fractionation and Amberlite XAD16N column chromatography to obtain betacyanin fractions. The
half maximum cytotoxicity concentration for betacyanin fractions from red pitahaya and red spinach on Vero cells were 4.346
and 2.287 mg ml−1, respectively. The half- maximal inhibitory concentration (IC50) of betacyanin fraction from red pitahaya was
125.8 µg ml−1 with selectivity index (SI) of 5.8. For betacyanin fraction from red spinach, the IC50 value was 14.62 µg ml−1 with
SI of 28.51. Using the maximum non- toxic betacyanin concentration, direct virucidal eect against DENV-2 was obtained from
betacyanin fraction from red pitahaya (IC50 of 126.70 µg ml−1; 95.0 % virus inhibition) and red spinach (IC50 value of 106.80 µg
ml−1; 65.9 % of virus inhibition). Betacyanin fractions from red pitahaya and red spinach inhibited DENV-2 in vitro.
INTRODUCTION
Dengue fever is an infectious disease caused by dengue virus
(DENV). DENV is an arthropod- borne virus that is commonly
distributed across the tropical and subtropical regions of
the world and is transmitted to humans mainly by female
Aedes aegypti mosquitoes [1]. ere are four antigenically
related, but serologically distinct types of DENV (DENV-1,
2, 3, 4), with DENV-2 being the most lethal serotype [2, 3].
Heterotypic secondary infection by a dierent serotype of
DENV is thought to be associated with the elevated virus
titre, due to antibody- dependent enhancement that can cause
severe dengue diseases such as dengue haemorrhagic fever
(DHF) or dengue shock syndrome (DSS) characterized by
plasma leakage, thrombocytopenia, severe bleeding and can
ultimately lead to organ failure and death [4]. Many studies
focused on the control of DENV by combating mosquito
vector, which is almost exclusively responsible for the trans-
mission of DENV [5]. Currently, there is no antiviral therapy
available for dengue infection and the only licensed dengue
vaccine, namely Dengvaxia showed inconsistent ecacy
across people [6]. e development of antiviral therapy is
equally important in the control of dengue infection as well
as preventing the progression of patient from dengue fever to
DHF or DSS. Natural compounds as antiviral agents can be
used in tandem with vaccine in controlling DENV infection.
Betacyanin, a red- violet pigment belongs to a class of plant
pigment called betalain [7]. Betacyanin extracts from dierent
sources of plant have a wide range of biological activities such
as antioxidant, anticancer, antilipidemic and antimicrobial
activities [8]. e antiviral activity of betacyanin was only
reported against tobacco mosaic virus in plant [9], hence
there is a research gap for the antiviral activity of betacyanin

2
Chang etal., Access Microbiology 2019
against human pathogenic viruses. Red pitahaya (Hylocereus
polyrhizus) and red spinach (Amaranthus dubius) are two
prolic crops commonly found in the tropical and sub-
tropical countries where dengue is endemic. Both plants
have multiple types of betacyanin and dierent betacyanin
composition, hence they were selected as the sources of
betacyanin. e objectives of this study were to investigate
the cytotoxicity eect of betacyanin fractions from red pita-
haya and red spinach on Vero cells, followed by investigating
the antiviral activity of betacyanin fractions against dengue
virus serotype 2 (DENV-2) via virus yield inhibition assay
and virucidal assay. Only DENV-2 was investigated because
it is the most lethal among four serotypes [3] and it has low
ecacy for Dengvaxia vaccine especially for seronegative
individuals [10].
METHODS
Preparation and characterization of betacyanin
fractions
Red pitahaya and red spinach were purchased from a super-
market (Bandar Sunway, Malaysia). Only fruits with 13–15
°Brix measured using a refractometer (Atago, Minato- ku,
Japan) were used for the extraction to ensure the fruits
were within the same range of ripeness. °Brix is a unit of
measurement for total soluble solids and in this case, mainly
sugar. is range of °Brix corresponds to a suitable degree
of ripeness of the fruits as sugar content is correlated with
degree of ripeness. Leaves of the red spinach with similar
length (7–9 cm) were picked, washed and spin in a salad
spinner to dry. is range of length is the typical length of
the red spinach leaves. e pulp of red pitahaya and the red
part of the leaves of red spinach were used for extraction.
e sub- fractionated extracts were prepared according to a
previous study [11]. e dried red pitahaya and red spinach
crude extracts were dissolved in 100 ml distilled water and
were adjusted to pH 2.0 by HCl. e extracts were then
partitioned with equal volume of ethyl acetate. is step was
repeated three times. e aqueous phase was collected and
concentrated with the addition of ethanol by evaporation
under low pressure at 40 °C. e sub- fractionated extracts
were freeze dried and semi- puried using polyaromatic
adsorbent resin Amberlite XAD16N (Sigma- Aldrich, St.
Louis, USA). Briey, the resin was soaked overnight in
distilled water at 4 °C. Before packing into a glass column,
the resin was rinsed with distilled water for three–four
times until the supernatant was clear. Acidied water was
prepared by adding triuoroacetic acid to distilled water
until pH 3 was reached. e packed column was rinsed with
1 l of distilled water and activated with 0.5 l of 2 % sodium
hydroxide solution. e resin was then neutralized by
rinsing with 0.5 l of distilled water, followed by conditioning
to pH 3 with acidied water. e dried sub- fractionated red
pitahaya (5.0 g) or red spinach (0.25 g) extracts was dissolved
in 10 ml of distilled water, followed by loading into the
column containing Amberlite XAD16N. e resin was then
desalted by rinsing with 0.5 l of acidied water at a ow rate
of 10 ml min−1 before eluting with acidied methanol (95 : 5,
methanol: acidied water). e semi- puried extracts were
then concentrated using rotary- evaporator at 40 °C, freeze
dried and stored at −80 °C until analyses. ese extracts were
termed betacyanin fractions. e betacyanin content for
dried red pitahaya and red spinach fractions were calculated
[12] and HPLC analysis was carried out [13]. Agilent 6520
Accurate Mass Q- TOF LC/MS (Agilent Technologies, Santa
Clara, USA) with dual ESI source operating in a positive
ionization mode were used to perform liquid chromatog-
raphy (LC)- MS/MS analysis [14].
Host cell cytotoxicity assay
Vero cells (African green monkey kidney; ATCC CCL-18)
were seeded on 96- well plates at 1×104 cells ml−1 1 day prior
to conducting the assay. e conuent cell were treated with
growth medium [1×minimum essential medium (MEM)
containing 2 % fetal bovine serum (FBS), 1 % HEPES and
1 % penicillin- streptomycin antibiotic] containing various
concentrations of betacyanin fractions from red pitahaya
and red spinach for 48 h at 37 °C in a humidied incubator
with 5 % CO
2
. e cells were washed once with PBS before
adding 10 µl of 5 mg ml−1 of 3-(4,5- dimethylthiazol-2- yl)−2,
5- diphenyltetrazolium bromide (MTT) reagent to each well
containing 90 µl of PBS and incubated at 37 °C, 5 % CO2 for
4 h. en 100 µl of solubilization solution (10 % SDS in 0.01 N
HCl) was added and plate was further incubated overnight.
e absorbance was measured using a microplate reader (Bio-
Rad, Hercules, USA) at 560 nm (OD560). e cell viability (%)
was calculated and the half maximum cytotoxicity concentra-
tion (CC50) was deduced from the dose- response curve.
Virus yield inhibition assay
Plaque reduction neutralisation test (PRNT) was rst carried
out to determine virus yield inhibition. DENV-2 was isolated
from a dengue- infec ted patient serum and conrmed by whole
genome sequencing (GenBank accession no. MH488959).
Table 1. Betacyanins characterized by HPLC and LC- MS/MS in
betacyanin fractions from red pitahaya and red spinach
Compound Molecular
weight
(g mol−1)
[M+H]+ m/z MS2
m/z
Percentage
(%)
Red pitahaya
Betanin 550 551 389 28.98±0.62
Isobetanin 550 551 390 5.57±0.47
Phyllocactin 636 637 594, 390 51.3±0.12
Hylocerenin 695 696 598, 387 14.12±0.69
Red spinach
Amaranthine 726 727 679, 540,
390
70.27±0.11
Decarboxy-
amaranthine
682 683 594, 508 346 21.57±0.29
Betanin 550 551 389 8.15±0.26

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Chang etal., Access Microbiology 2019
Vero cells were seeded on a 96- well plate and incubated at
37 °C with 5 % CO2 until 90–95 % conuency was reached.
e cells were then infected with DENV-2 at an m.o.i. of 0.5
[15] for 1 h at 37 °C with 5 % CO2. Various concentrations
of betacyanin fractions from red pitahaya and red spinach
were prepared using milli- Q water. Aer 1 h of infection, the
inoculum was removed and cells were washed once with PBS
before incubating with growth medium containing diluted
extracts for 48 h at 37 °C with 5 % CO2. A virus control
consisting of infected cells without the addition of betacyanin
fraction and a cell control were included as assay controls. e
supernatants of infected cells were harvested and subjected to
focus formation assay in duplicate aer 48 h of post- infection.
Focus forming assay [16] with some modications was
performed to allow quantication of the virus yield. Vero cells
were seeded on 24- well plates at 5×104 cells ml−1 a day before
the assay. e cells were inoculated with 100 µl of supernatant,
which was serially diluted in tenfold, and incubated at 37 °C
with 5 % CO2 for 1 h. e plate was gently rocked every 12 min
during 1 h of infection. e inoculum was then removed and
cells were overlaid with 2 % carboxymethyl cellulose (CMC)
overlay medium, followed by incubating at 37 °C with 5 % CO
2
for 4 days. e overlay medium was removed and cells were
washed twice with 1×Tris- buered saline (TBS) containing
0.1 % Tween 20 (TBST). e cells were then xed with cold
80 % acetone for 10 min at room temperature followed by
washing. Aer the incubation with blocking buer [1 %
bovine serum albumin (BSA), 0.5 % Triton X-100 in TBST]
at 37 °C with 5 % CO2 for 45 min, the cells were incubated with
mouse monoclonal dengue virus type 1, 2, 3 and 4 antibody
[D1-11(3)] (GeneTex) at 37 °C for 1 h. Aer three times of
washing for 5 min, the cells were incubated with alkaline
phosphatase- conjugated goat antimouse IgG (GeneTex) at
37 °C for 1 h followed by washing. e cells were then incu-
bated with a mixture of NBT (nitrotetrazolium blue chloride)
and BCIP (5- bromo-4- chloro-3’-indolyphosphate p- toluidine
salt) substrates (Bio Basic) for 10 min. e immunostained
plates were rinsed under running water and allowed to dry
before counting the foci. e percentage of focus inhibition
was then calculated. e half- maximal inhibitory concentra-
tion, IC
50
, was determined as the concentration of betacyanin
fraction, which causes 50 % reduction in virus yield.
Virucidal assay
Vero cells were seeded on 24- well plates at 5×10
4
cells ml
−1
a day before virucidal assay. In a 96- well plate, various
concentrations of betacyanin fractions prepared in milli- Q
water were incubated with 30 foci- forming units (f.f.u.) of
DENV-2 at an equal volume for 1 h at 37 °C with 5 % CO
2
before being transferred onto cells in the 24- well plate. FFA
was then performed as described above. e IC50 values were
determined as mentioned above.
Statistical analysis
GraphPad Prism soware (version 7.04) was used for the
statistical analyses, determination of CC50 and IC50, and
graphical illustrations of antiviral eects of betacyanin frac-
tions from red pitahaya and red spinach. e virus yield inhi-
bition assay and virucidal assay were performed in duplicate
(two technical replicates) using two independent red pitahaya
and red spinach extracts (two independent extraction).
RESULTS AND DISCUSSION
Betalains are water- soluble nitrogen- containing pigments,
which can exist as the red- violet betacyanins or yellow
betaxanthins. e aglycon betanidin is the backbone of all
betacyanins in which the occurrence of dierent betacyanin
structures is due to the glycosylation and acylation of the 5- O-
or 6- O- glucosides [7]. e most abundant betacyanin found
in betacyanin fraction from red pitahaya was phyllocactin,
followed by betanin, hylocerenin and isobetanin (Table1).
In betacyanin fraction from red spinach, the most abundant
Fig. 1. Cytotoxicity eect of betacyanin fraction from (a) red pitahaya and (b) red spinach on Vero cells. Values represent the mean± of
assay performed in duplicate. The half maximum cytotoxicity concentration (CC50) values were obtained using GraphPad Prism software.

4
Chang etal., Access Microbiology 2019
betacyanin found was amaranthine, followed by decarboxy-
amaranthine and betanin. e betacyanin composition of
betacyanin fractions from red pitahaya and red spinach in this
study is similar to a previous study [13]. e purity of beta-
cyanins in these fractions were higher than those of previous
studies [11, 13] as column chromatography was employed to
further purify the extracts from red pitahaya and red spinach.
e bioactivity of the betacyanin in these fractions can only
be conrmed by isolating individual betacyanin and deter-
mine its bioactivity to prove that any eect is really due to
the predominant betacyanins present and not due to some
minor constituents. Amaranthine was previously isolated
from red spinach extract using preparative HPLC and proven
to possess antibiolm activity similar to its extract [11].
e half maximum cytotoxicity concentration (CC
50
), which
is the concentration required to reduce cell viability by 50%,
was at 4.38 mg ml−1 (betacyanin content: 664.73 μg ml−1) for
betacyanin fraction from red pitahaya (Fig.1a) and at 2.42
mg ml−1 (betacyanin content: 416.83 μg ml−1) for betacyanin
fraction from red spinach (Fig.1b). e betacyanin frac-
tions from red pitahaya and red spinach were considered
to be non- cytotoxic to Vero cells at the extract concentra-
tions below 2.50 mg ml−1 (betacyanin content: 379.50 μg
ml−1) and 1.25 mg ml−1 (betacyanin content: 215.75 µg ml−1),
respectively. e antiviral assays were then conducted using
these ranges of non- cytotoxic concentrations. ere was an
increase in the cell viability at the non- toxic concentrations.
is may be due to hormesis in the Vero cells whereby a
dose- response relationship is exhibited showing low- dose
stimulation and high- dose inhibition [17]. In addition, the
large error bars at these concentrations could be reduced
with larger replicates. ere was only one study reporting
on the cytotoxicity eect of betacyanin extract from red
pitahaya on human embryonic kidney (HEK-293) cells and
human monocytes (THP-1) [18]. is study is the rst to
report of the cytotoxicity of betacyanin fractions from red
pitahaya and red spinach on Vero cells. It was suggested that
a daily intake of not more than 100 mg of betanin (a type of
Fig. 2. Antiviral eect of betacyanin fractions from (a) red pitahaya and (b) red spinach. The IC50 values were obtained using GraphPad
Prism software. (c) The representative focus formation of DENV-2 in Vero cells treated with betacyanin fractions from red pitahaya and
red spinach at the maximum non- toxic betacyanin concentration.

5
Chang etal., Access Microbiology 2019
betacyanin) in puried form can be used as colourants and
providing eective bioactivity [19].
Betacyanin fractions from red pitahaya and red spinach exhib-
ited antiviral activity against DENV-2 aer virus adsorption to
the host cells in a dose- dependent manner (Fig.2). e IC50 of
betacyanin fraction from red pitahaya was 125.8 μg ml
−1
, with
selectivity index (SI) of 5.28. SI was calculated by dividing
CC
50
over IC
50
. For betacyanin fractions from red spinach, the
IC50 value was 14.62 μg ml−1, with a SI of 28.51. ese results
suggest that the infectivity of DENV-2 was suppressed by the
betacyanins from red pitahaya and red spinach. However,
further investigation on measuring the cellular processes of
DENV-2 aected by incubation with betacyanins should be
carried out in the future to determine whether betacyanins
interfere with the cellular processes used by the virus in its life
cycle. To evaluate the virucidal eect of betacyanin fractions
on DENV-2, various non- toxic concentrations of betacyanin
fraction were incubated with DENV-2 directly and the virus
infectivity was assessed via focus formation assay. Betacyanin
fraction from red pitahaya demonstrated direct virucidal
eect against DENV-2 with an IC
50
of 126.70 μg ml
−1
and with
95.0 % of virus inhibition (Fig.3a) at the maximum non- toxic
betacyanin concentration (379.5 μg ml−1). As for betacyanin
fraction from red spinach, the IC50 value was 106.8 μg ml−1
and the virus inhibition was 65.9 % (Fig.3b) at the maximum
non- toxic betacyanin concentration (172.6 μg ml−1). ese
results of virucidal assay imply that betacyanin fractions from
red pitahaya and red spinach may be able to inactivate the
extracellular DENV-2 particles. e betacyanins most prob-
ably bind to the virus particles, thus inactivating the DENV-2
from initiating the virus infection. Previous studies showed
that natural compounds, which can inhibit DENV replication
show interactions with the non- structural proteins [20, 21].
us, it is deemed that betacyanin might interact with the
non- structural protein especially the envelope (E) protein of
DENV-2, which is crucial for virus attachment, suppressing
the infectivity of DENV-2 by interfering the attachment of
virus to the host cell [22, 23]. However, the mechanisms
of inhibition by the betacyanins on DENV-2 need to be
conrmed by further investigation.
For both dose- dependent antiviral assay and virucidal assay,
betacyanin fraction from red spinach seem to have a better
antiviral eect against DENV-2 than red pitahaya, whereby
the IC50 values of betacyanin fraction from red spinach were
lower than those of betacyanin fraction from red pitahaya.
In addition, the higher SI value of betacyanin fraction from
red spinach than that of red pitahaya also suggests the better
antiviral activity of betacyanin fraction from red spinach.
Individual betacyanin should be isolated to identify the
candidate exerting the highest antiviral eect. e IC50 values
of betacyanin fraction from red spinach (14.6 μg ml
−1
) for the
inhibition DENV-2 is also lower when compared with some
other natural compounds such as baicalein rich Scutellaria
baicalensis extract (86.59 to 95.19 μg ml−1) and quercetin
(28.90 μg ml−1) [24, 25], indicating a better antiviral property
of red spinach. Both compounds belong to the avonoid
group of polyphenols. e ndings in this study suggest that
the betacyanin fractions from red pitahaya and red spinach
can be potentially to be developed as antiviral agents as part
of antiviral therapy against DENV-2. To further investigate
the eectiveness of these betacyanin fractions in inhibiting
the DENV infection, the determination of mode of action
of these extracts and in vivo study are needed to be carried
out in future.
Funding information
This project was supported by the School of Science and the Infectious
Diseases and Health cluster of the Tropical Medicine and Biology Plat-
form (grant number 5140762), Monash University Malaysia.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Fig. 3. Virucidal eect of betacyanin fractions from red pitahaya and red spinach. The dose- response of plaque inhibition (%) against
betacyanin content (µg ml−1) of betacyanin fraction from (a) red pitahaya and (b) red spinach against DENV-2. The IC50 values were
obtained using GraphPad Prism software.

6
Chang etal., Access Microbiology 2019
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