Screening of Carica papaya L. and Psidium guajava Leaves Fraction as Antiviral Drug to Dengue Virus
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ISSN: 03875547
Volume 44, Issue 02, February, 2021
Screening of Carica papaya L. and Psidium guajava
Leaves Fraction as Antiviral Drug to Dengue Virus
Siti Ma’rufah1, Leli Saptawati2, Yuliasari3, Fachira Rachel4, Debita Istifadah4, T. Mirawati Sudiro5, Beti
Ernawati Dewi5,6, Betty Suryawati2*
Department of Pharmacology, Faculty of Medicine, Sebelas Maret University, Surakarta, Central Java 1
Department of Microbiology, Faculty of Medicine, Sebelas Maret University, Surakarta Central Java2
Department of Biomedic, Faculty of Medicine, Sebelas Maret University, Surakarta, Central Java 3
Undergraduate Faculty of Medicine, Sebelas Maret University, Surakarta, Central Java 4
Department of Microbiology, Faculty of Medicine, University of Indonesia/Cipto Mangunkusumo General
Hospital, Jakarta5
Cluster of Immunology and Infecrious Disease, Indonesian Medical Education and Research Institute,
Faculty of Medicine, University of Indonesia/Cipto Mangunkusumo General Hospital, Jakarta 6
Corresponding author: 2*
Keywords: ABSTRACT
Papaya leaves (Carica papaya Up to now, there is no specific antiviral drug to dengue virus (DENV).
L.) Guajava leaves (Psidium Medicinal plants have been developing in order to combat DENV
guajava), Dengue virus,
DENV-2 NGC infection. The crude extract of Carica papaya L and Psidium guajava
leaves showed antiviral activity to DENV in vitro. The aim of this study
to screen antiviral activity to DENV among Carica papaya L and
Psidium guajava leaves fractions. This research is a laboratory
experimental study. The study was conducted at the Pharmacology
laboratory, Faculty of Medicine, Sebelas Maret University and Virology
and Molecular Biology Laboratory of the Department of Microbiology,
Faculty of Medicine, The University of Indonesia. DENV at an MOI of
0.5 FFU/cell was treated with 20 ug/mL of Carica papaya L and Psidium
guajava leaves fractions and was infected to Vero cell. The affectivity
and safety of DENV antiviral drug was determined by focus assay and
MTT assay respectively. Data from the subsequent research were
processed using the one-way ANOVA method. From MTT results, we
found that all fractions of papaya were classified as non-toxic to Vero
cell with the lower toxicity after treated with chloroform and water
fraction. Similar results were found from Vero cell treated with guajava
leave fraction with viability above 100%. Based on the Focu Forming
Units (FFU) assay, the highest inhibition percentage was n-hexane
fraction of papaya leave. The percentage of inhibition of benzene and n
hexane guajava leaves fraction were effective as antiviral drug to DENV
with the inhibition percentage values of 27.4 % and 33.1%, respectively
For the conclusion, the n-hexane fraction of papaya leaves and guajava
leaves have potential as DENV antiviral agents in vitro.
This work is licensed under a Creative Commons Attribution Non-Commercial 4.0
International License.
667Ma’rufah, et.al, 2021 Teikyo Medical Journal
1. INTRODUCTION
Dengue hemorrhagic fever (DHF) is a disease that is transmitted to humans through the bites of the
mosquitoes Aedes aegypti and Aedes albopictus [1]. The host of DHF is human and the agent is dengue
virus, which belongs to the Flaviviridae family and the genus Flavivirus. Dengue virus consists of 4
serotypes, namely DENV-1 (Dengue Virus - 1), DENV-2. DENV-3, and DENV-4 [2]. Dengue virus has
been reported to have infected more than 100 countries, especially in densely populated urban areas and
settlements in Brazil and other parts of South America, the Caribbean, Southeast Asia, and India. The
number of people infected is estimated at between 50 and 100 million, half of whom are hospitalized and
result in 22,000 deaths each year; an estimated 2.5 billion people or nearly 40 percent of the world's
population. Until now, there is no vaccine to prevent DHF and there are no specific drugs for its cure, only
supportive therapy is the main principle [3].
1.1 The main objective of the study
The aim of this study is to find out the most effective among the leaves fractions as a DENV antiviral drug.
This study specifically examines the n-hexane, benzene, the chloroform, n-butanol, and water fraction of
papaya and guava leaves.
1.2 Pervious works
Papaya leaves (Carica papaya L.) and guava leaves (Psidium guajava) have been shown to have potential
antiviral for dengue. [4] Papaya leaves contain active substances including alkaloids, saponins, flavonoids,
and proteolytic enzymes such as papain and chymopapain, [5] while guava contains carotenoids and
flavonoids. [6] The quercetin flavonoids in papaya and guava leaves are formed bonds that can damage the
structure of the NS2B-NS3 protease in dengue virus 2 (DENV-2). The function of this enzyme are for the
replication of the dengue virus. Therefore, destruction of thins enzyme structure, will disrupt viral
replication and reduce the amount of dengue virus in the body. [7]
1.3 Methods
This research is a laboratory experimental study with a posttest only control group design that looks at the
effectiveness of the extract fraction of natural papaya and guava leaves as an in vitro antiviral for dengue.
We used DENV serotype 2 strain of New Guinea C (DENV2 NGC) and Vero cells. DENV treated with
water fraction, n-hexane fraction, benzene fraction, chloroform fraction, and n-butanol fraction from papaya
and guava leaf extract at a concentration of 20 µg / ml. Leaf extract is an extract of active compounds made
from papaya and guava leaves dissolved in 70% ethanol and processed through the maceration method. The
leaf extract was then fractionated stratified, using the liquid-liquid fractionation method. The solvents used
were n-hexane, benzene, chloroform, n-butanol, and water, which sequentially had polarity values of 0.009;
0.111; 0.259; 0.506; 1.00. The dependent variable in this study is the effectiveness of dengue antiviral,
which can be seen from two factors, the percentage of inhibition, and the percentage of viability. To find,
how much inhibition of the extracts of natural ingredients on the growth of the dengue virus, a focus
forming unit test was conducted. In this test, the percentage of fraction inhibition will be obtained extract
natural ingredients against the growth of the dengue virus. Herbal extracts to be effective in inhibiting
dengue virus replication if the percentage value of inhibition is> 20%. To determine the toxicity of natural
extract fractions to Vero cells, it can be done using the MTT test. In this study, dimethyl sulfoxide (DMSO)
was used as a control. The absorbance check was checked at a wavelength of 490 nm. Herbal extracts to be
safe for cells if the percentage value of cell viability is> 50%. The data on the percentage of viability and
percentage of inhibition, as well as the normality, homogeneity, and post hoc tests were analyzed using the
one-way ANOVA test, using SPSS version 22.
668ISSN: 03875547
Volume 44, Issue 02, February, 2021
2. FINDINGS
2.1 Normality Test and Homogeneity Test
Based on the results of the normality test, it was found that all fractions had Sig. > 0.05, so it can be
concluded that the data is normally distributed. The homogeneity test method used is the Levene’s Test.
The data are concluded to have the same variance if the Sig. > 0.05 and has different variances if the Sig.
20%.
Table 1. Percentage of viability of papaya (Carica papaya L.) fraction and leaves extract against Vero cells
95% Confidence
Average
Std. Interval for Mean
Sample Viability
Deviation Lower Upper
(%)
Bound Bound
Papaya The n-hexane fraction 148.3 32.8 66,728 229,872
Benzene fraction 102.4 16.9 60,340 144,526
Chloroform fraction 98.1 20.4 47,265 148,869
N-butanol fraction 125.3 8.2 104,803 145,731
Water fraction 93.5 14.8 56,763 130,170
Guava The n-hexane fraction 115.6 5.7 101.598 129.669
Benzene fraction 114.0 16.7 72.417 155.583
Chloroform fraction 114.0 1.7 109.808 118.259
N-butanol fraction 138.0 14.6 101.765 174.235
Water fraction 101.4 14.8 64.687 138.046
Table 2. The percentage of inhibition of papaya and guava leaf fraction.
95% Confidence
Average
669Ma’rufah, et.al, 2021 Teikyo Medical Journal
Std. Interval for Mean
Sample Inhibition
Deviation Lower Upper
(%)
Bound Bound
Papaya n-hexane fraction 63.2 3.8 53,721 72,679
Benzene fraction 5,2 13.9 -29,420 39,754
Chloroform fraction -15.8 6.3 -31,561 0.027
N-butanol fraction 10.7 11.8 -18,565 39,965
Water fraction 22.2 9,2 -0,767 45,167
Guava The n-hexane fraction 27.4 2.3 21.635 33.098
Benzene fraction 33,2 4.2 22.703 43.631
Chloroform fraction 9.6 5.6 -4.371 23.571
N-butanol fraction 19.5 11.6 -9.350 48.283
Water fraction 9.0 1.1 6.234 11.766
3. The effectiveness fractions of natural ingredients as dengue antiviral
The effectiveness fractions of natural ingredients as dengue antiviral was assessed based on the toxicity of
fractions and extracts of natural ingredients against Vero cells (can be seen through the percentage of
viability values) and the concentrations needed by extracts and fractions to inhibit DENV replication (can
be seen through the percentage value of inhibition). Research conducted by [8] reported that herbal extracts
that had a cell viability percentage value> 50% indicated that the extract was non-toxic to host cells. Herbal
extracts to be effective in inhibiting dengue virus replication if the percentage value of inhibition is> 20%.
[9]
3.1 Viability Percentage Value
This present study shows that the papaya and guava leaf all fractions are classified as non-toxic against
Vero cells, because the percentage value of viability is> 50%. The results of this study are supported by
research conducted by [10] which states the value of CC50(Cytotoxic concentration 50%) of papaya extract
against Vero cells is 104,37µg/ml, that there is no in vitro toxicity to Vero cells. Previous research by [4]
also stated that papaya leaf extract (Carica papaya L.) was not toxic to Huh7it-1 cells, with a CC50 244.76
µg/ml. Research by [11] also stated that chloroform extract from papaya (Carica papaya L.) leaves was not
toxic to LLC-MK2 cells in vitro. This is because papaya leaves have many active ingredients that act as
antioxidants, namely flavonoids, alkaloids, amino acids, phenols, tannins, and saponins [12], [3].
Flavonoids have the ability as antioxidants that are able to transfer an electron or a hydrogen atom to free
radical compounds, so that they can inhibit lipid peroxidation, suppress tissue damage by free radicals and
inhibit some enzymes. Saponins are known to be able to scavenge H2O2, thus avoiding the cells from
possible oxidative stress, [14] while amino acids indicate the formation of glutathione, which has an active
inhibiting protein. free radicals, thereby reducing oxidative stress on cells and preventing cells from lysis
[5]. The results of this study are supported by research conducted by [6] who extracted essential oils from
guava leaves showing no in vitro toxicity to Vero cells. Other studies that utilize the ethanol extract of
guava leaves (Psidium guajava) as a candidate for cancer therapy have also shown no toxicity to HeLa cells.
[17] The maceration mechanism using other solvents also showed good toxicity results from guava leaf
extract against MDA-MB-231 cells. (18) Research conducted by [19] also showed that the ethanol extract
and fractions of guava leaves were cytoprotective against cells. This may be due to the antioxidant effect of
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secondary metabolites primarily flavonoids.
3.2 Inhibition Percentage Value
The n-hexane fraction from papaya leaf extract (Carica papaya L.) is effective as a dengue antiviral
according to the research. This was also confirmed by the results of the Post Hoc Bonferroni test which
stated that the mean percentage inhibition of the n-hexane fraction had a statistically significant difference
with the mean percentage of inhibition of the benzene fraction (Sig. 0,000), the chloroform fraction (Sig.
0,000), and the n-butanol fraction (Sig. 0,000), where the three fractions were declared ineffective to have
inhibition against DENV-2. Compared to water extract and ethanol extract from papaya leaves (Carica
papaya L.), n-hexane extract has the highest saponin content. Compared to 56 other phytochemicals found
in papaya leaves, saponins had the best binding affinity for Dengue Virus (DENV) NS1, NS3 Helicase, and
NS5. Saponins work effectively to inhibit DENV replication by binding to NS1 (nonstructural protein 1)
and inhibiting NS3 (nonstructural protein 3) helicase, thereby inhibiting RNA in DENV replication.
Saponins also inhibit the activity of 2'Omethyltransferase-NS5 domain (NS5MTase (DENV)) which then
induces viral RNA methylation [20], [21]. Where the three fractions were declared ineffective had an
inhibitory power against DENV-2. Papaya leaf extract (Carica papaya L.) contains many active substances
including alkaloids, saponins, flavonoids, and proteolytic enzymes such as papain and chymopapain [11].
The water fraction of papaya leaf extract (Carica papaya L.) has an average inhibition percentage value of
22.2 ± 9.2% which is said to be effective as a dengue antiviral, according to the study [22]. However, when
compared with the percentage value of inhibition of other samples, the average percentage of inhibition of
the water fraction was still lower than the average percentage of inhibition of n-hexane (63.2 ± 3.8%) and
papaya leaf extract (95.8 ± 1)., 9%). Based on the Post Hoc Bonferroni test, the mean percentage of
inhibition of the water fraction was not statistically significant difference with the mean percentage of
inhibition of the benzene fraction (Sig. 0.561) and the n-butanol fraction (Sig. 1,000), where the two
fractions were declared not effective in inhibiting DENV 2. Water fraction from papaya leaf extract (Carica
papaya L.) contains active substances including flavonoids, alkaloids, saponins, phenols, betasianin, and
amino acids. [20] Flavonoids, saponins, and alkaloids are known to play a role in inhibiting viral replication
and viral RNA methylation by binding to NS1, NS2B-NS3 protease, NS3 helicase, and NS5MTase (DENV)
[21]. The n-butanol fraction from papaya leaf extract (Carica papaya L.) has an average inhibition
percentage value of 10.7 ± 11.8%. effective as a dengue antivirus. The n-butanol fraction contains active
compounds, namely flavonoids, saponins, anthraquinones, terpenoids, glycosides, steroids, and
carbohydrates [23]. However, although it contains flavonoids and saponins which are proven to be able to
inhibit DENV2 replication, the carbohydrate content in the n-butanol fraction is thought to cause this
fraction to be ineffective, because carbohydrates act as a mediator for the process of DENV entry into host
cells [24].
The benzene fraction from papaya leaf extract (Carica papaya L.) has an average inhibition percentage
value of 5.2 ± 13.9%, said to be ineffective as a dengue antiviral. Benzene from papaya leaves contains
active flavonoids, which according to the literature are known to be able to inhibit the replication of the
dengue virus. [25] However, the phytocomponents in the benzene fraction were among the smallest
compared to 9 other samples, so they are not effective as dengue antivirals [26]. The chloroform fraction
from papaya leaf extract (Carica papaya L.) has an average inhibition percentage value of -15.8 ± 6.3% is
said to be ineffective as a dengue antiviral, contrary to the research of [27], [28] but according to the
research of Joseph [11] the chloroform fraction contains active substances, namely flavonoids, alkaloids,
tannins, and phenols which are known to be able to inhibit DENV replication [27]. However, the
concentration of the active substance in the fraction [11]. In the screening that has been carried out, the
percentage of infectivity of the benzene fraction was 66.83 ± 4.21% and the n-hexane fraction 72.63 ±
671Ma’rufah, et.al, 2021 Teikyo Medical Journal 2.31%. From the percentage of infectivity, it can be determined the percentage of inhibition of the benzene fraction 33.17 ± 4.21% and the n-hexane fraction 27.37 ± 2.31%, while the other extract fractions show the inhibition percentage results
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Volume 44, Issue 02, February, 2021
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