Background
Dengue fever is a viral disease transmitted by mosquitoes in the genus
Aedes, the principal species are
A. aegypti and
A. albopictus. The infection occurs mainly in tropical and subtropical regions of the planet. The number of cases in the past 30 years has increased considerably, this disease affects more than 100 countries around the world with 100 million cases each year, 500 thousand requiring hospitalization, and approximately 25,000 resulting in death each year [
1‐
3].
Dengue virus (DENV), a member of the
Flaviviridae family, is an enveloped virus containing a ~ 11 kb genome of positive single-stranded RNA which encodes three structural proteins (C, pr-M, E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) [
4]. Four serotypes of dengue virus (DENV1, DENV2, DENV3 and DENV4) cause dengue fever (DF) and more severe manifestations like dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [
1].
Currently there are no specific antiviral compounds for the treatment, however, several research groups have sought antiviral compounds by molecular docking [
5,
6] or using medicinal plants to inhibit a viral target [
7,
8]. Current treatment to severe dengue is supportive fluid therapy under medical supervision [
9]. Having no specific antiviral therapy or an antiviral agent for dengue treatment, different methods for prevention have been established by controlling the mosquito reproduction or spread [
10,
11]. Due to the lack of new molecules, some clinical researches have proposed the repurposing of well-known drugs such as chloroquine, prednisolone, balapiravir, celgosivir, and lovastatin, however, although those drugs are safe, they have not been successful at decreasing viral load, antigenemia, fever or inducing a beneficial effect to dengue patients [
12].
Ethnopharmacology has contributed significantly to the discovery of new drugs [
13,
14]. In recent years, the focus on medicinal plants widely used in traditional systems has increased worldwide [
15]. Based on ethnobotanical information some studies have demonstrated several compounds with anti-dengue potential activity such as, 7-0-methyl-glabranine [
16], baicalein [
17], catanospermine [
7], quercetin and fisetin [
18,
19].
Within the Mexican population, dengue is commonly known as “five-day fever”, “breakbone fever” or simply “breaker”. For the treatment of this disease there are therapeutic resources of traditional medicine, whose information has been collected over many years in important databases such as the Medicinal Herbarium of the Mexican Social Security Institute (Herbarium IMSS-M) [
20]. In this collection, multiple uses of medicinal plants native to Mexico are referenced, including some that have traditionally been used against dengue, such as
Taraxacum officinale, Urtica dioica, Calea integrifolia and
Caesalpinia pulcherrima.
These plants are widely used in traditional medicine around the world for the treatment of many illnesses.
U. dioica has great medicinal potential, its extracts have been used for the treatment of eczema, digestion, pain, anemia, arthritis, rheumatism [
21], and it inhibits inflammatory processes caused by seasonal allergies [
22].
T. officinale is used in the treatment of anemia, liver cirrhosis, rheumatoid arthritis and also it has been reported with anti-inflammatory, anti-oxidative, anti-carcinogenic, analgesic, anti-hyperglycemic, laxative and diuretic activities and also as stimulating for the digestive system [
23]. It was reported that
T. officinale has inhibitory potential against HIV and its reverse transcriptase [
24].
C. pulcherrima is used in the treatment of cough [
25], contains flavonoids and some reports show that aqueous extracts of flowers, leaves and stem, have inhibitory effect on several viruses, including herpes (HSV1-2) and adenoviruses (ADV-3, ADV-8, ADV-11) [
26]. Meanwhile,
C. integrifolia has been reported with antihyperglycemic activity and used in the treatment of diabetes [
27,
28].
Of these plant species, only
U.dioica has been reported as a source of an anti-dengue constituent, since the N-Acetyl-D-Glucosamine-specific lectin of this plant (UDA) has proved effective at reducing the viral infection of the four dengue serotypes [
29]. However, other compounds, distinct from proteins have not been studied. Therefore, in this work it was analyzed if the aqueous and methanolic extracts of these four plants have inhibitory activity on DENV2 replication.
Methods
Reagents
Solvents methanol, ethyl acetate, formic acid and dimethyl sulfoxide (DMSO) reagent or HPLC grade and silica gel (Kiselgel 69) were obtained from Merck KGaA (Darmstadt, Germany). Media and supplements for cell culture were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA) and molecular grade agarose was obtained from Promega (Madison, USA). All other reagents used in analytical methods were purchased from Sigma-Aldrich Chemicals, except where otherwise indicated.
Cells
C6/36 cell line (
A. albopictus) was maintained in minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin. Cell line BHK-21 (hamster kidney neonate) was cultured in MEM supplemented with 5% FBS and antibiotics. Cells were incubated at 37 °C in 5% CO
2. As is recommended for this type of experiments,
Aedes albopictus C6/36 cells (ATCC: CRL-1660) were used for viral propagation [
30] and baby hamster kidney BHK-21 cells (ATCC: CCL-10) were used to quantify the virus by plaque reducing assays [
7,
31].
Virus
Dengue virus serotype 2 (DENV2) strain Thailand/16681/1984 used in this study was kindly provided by Dr. Alvaro Aguilar-Setien (IMSS, Mexico City, Mexico). To obtain the inoculum used in all experiments, dengue virus was replicated in C6/36 cells for 5 days. Subsequently the viral supernatant was centrifuged for 5 min at 10,000 rpm to remove the cell debris. Aliquots were stored at − 70 °C until use [
32,
33].
Viral titration
Infection of the cells by DENV2 was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using reported specific primers [
34] and by immunofluorescence using an antibody against the viral prM protein, as previously reported by our group [
32,
33]. Quantification of DENV2 was performed by the lytic plaque assay in BHK-21 cells on six wells plates; after 24 h, the cells were washed with PBS and infected with 10-fold serial dilutions of the virus inoculum. After 1 h, the not absorbed virus was removed, cells were washed with PBS and then 0.35% agarose and DMEM were added. Plates were incubated for 72 h at 4 °C and 5% CO
2. After that, 5% trichloroacetic acid was used to fix the cells and subsequently they were stained with 0.05% crystal violet in 20% ethanol [
32,
33,
35].
Selection and collection of medicinal plants
The plant species T. officinale, U. dioica, C. integrifolia and C. pulcherrima used in this study were identified as likely sources of active compounds against dengue virus from ethnobotanical information obtained from Herbarium IMSS-M (Catalogs: 1988, 1990, 1994; available in paper format only), which concerns its use in syndromes or diseases which are probably dengue, such as inflammatory muscle pain. All the plant specimens were collected in the state of Puebla, Mexico, in the municipalities of San Jeronimo Tecuanipan (19° 00′ 00″ North, 98° 24′ 54″ West) and in Atlixco (18° 54′ 45″ North, 98° 25′ 40″ West). The aerial part of the plants was cropped with contaminant-free cutter and transported to the laboratory. The vegetal material was taxonomically identified by experts of the Herbarium IMSS-M at Mexico City and reference vouchers of the plants material T. officinale, U. dioica, C. integrifolia and C. pulcherrima were deposited with the codes IMSS-M 164320, 164340, 17080 and 17120, respectively.
Processing of plants and obtaining of crude extracts
Leaves of each plant were separated, washed and air-dried at room temperature (26 °C) for 2 weeks, after which they were grinded to a uniform powder in a blender (Nutribullet LLC, Pacoima, CA, USA). The extraction from pulverized plants was conducted in a Soxtherm® system (Gerhardt, Königswinter, Germany) at 60, 85 and 120 °C using methanol or water as solvents. In the extraction process the variables were the solvents and the extraction temperatures, but the extraction time (60 min), the reduction range (30 s) and the pulse reduction (2 s) remained constant. Methanolic and aqueous extracts were dried using a rotary evaporator (Heidolph Instruments Gmbh & Co. KG, Germany) at 60 °C or 90 °C, respectively. The dried residue was weighed, dissolved in DMSO [
17] and used for cytotoxicity and inhibition of viral replication tests.
Determination of cytotoxicity
The cytotoxic concentration 50 (CC
50) was obtained in BHK-21 cells with the conventional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay by serial dilutions (between 0.1 and 1200 μg/ml) of the extracts in DMSO, used as vehicle to all extracts or fractions. After 3 days of incubation at 37 °C and 5% CO
2, the culture medium was removed, the cells were washed twice with PBS and the standard MTT protocol was performed [
36]. Experiments were carried out in triplicate and done twice.
Viral inhibition assays
BHK-21 cells were seeded in 6-wells plates during 24 h. The infection was performed with 0.5 ml of DENV inoculum in a dilution to produce approximately 100 lytic plaques per well (a 1:3100 dilution from a titrated viral inoculum at 3.1X10
6 PFU/ml). After 1 h incubation, the viral inoculum was removed and washed with PBS twice. Immediately, different concentrations of each of the extracts were added. As control for inhibition the study used: a) cells without extract and without infection, b) infected cells without extract and, c) cells without infection and with extract [
37]. Subsequently molecular biology grade agarose was added to a final concentration of 0.35% in DMEM with 2.5% FBS. After 3 days, the cells were fixed with 10% trichloroacetic acid for 10 min and stained with 0.1% crystal violet for 3 min. Results of the inhibition of viral replication were reported as a percentage of the formed lytic plaques compared to the control infection assay (100%), which was not treated with plant extracts [
32,
33]. Experiments were carried out in triplicate and done twice.
Chromatographic fractionation
T. officinale and U. dioica extracts were fractionated. The extracts were suspended in deionized water and partitioned twice with dichloromethane and the solvents were exhaustively removed to obtain dichloromethane fraction. This fraction was subjected to silica gel column (10 × 30 cm) and eluted with a gradient of n-hexane-ethyl acetate (9:1, 8:2,…1:1) to yield 17 fractions. These fractions were evaluated in the same biologic assays.
Identification of compounds by HPLC-DAD-ESI/MSn
For the chemical analysis, 100 mg of sample were mixed with 1.5 mL of methanol:water:formic acid (25:24:1, v:v:v) extractant, then vortexed and sonicated in an ultrasonic bath for 60 min at room temperature. The samples were kept at 4 °C overnight and sonicated again for 60 min. A centrifugation was performed for 10 min at 10,000 rpm to separate the supernatant from the solid residue. The supernatant was filtered through a 0.22 μm PVDF filter before analysis (Millipore, MA, USA). The chromatographic analyses for identification of compounds were carried out on a Luna C18 column (250 × 4.6 mm, 5 μm particle size; Phenomenex, Macclesfield, UK). Water/formic acid (99:1, v/v) and acetonitrile were used as the mobile phases A and B, respectively, with a flow rate of 1 mL/min. The linear gradient started with 8% solvent B, reaching 15% solvent B at 25 min, 22% at 55 min, and 40% at 60 min, which was maintained to 70 min. The injection volume was 20 μL, and the analyses were carried out using an Agilent HPLC 1100 series model equipped with a photodiode array detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany). The equipment consisted of a binary pump (model G1312A), an autosampler (model G1313A), a degasser (model G1322A), and a photodiode array detector (model G1315B). The HPLC system was controlled by ChemStation software (Agilent, version 08.03). The mass detector was an ion trap spectrometer (model G2445A) equipped with an electrospray ionization interface, and was controlled by LCMSD software (Agilent, version 4.1). The ionization conditions were 350 °C and 4 kV, for capillary temperature and voltage, respectively. The nebulizer pressure and nitrogen flow rate were 65.0 psi and 11 L/min, respectively. The full-scan mass covered the range of m/z from 100 to 1200. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 to 2 V. The mass spectrometry data were acquired in the positive ionization mode for anthocyanins and in the negative ionization mode for other flavonoids. The MSn was carried out in the automatic mode on the more-abundant fragment ion in MS (n-1).
Statistical analysis
To calculate cytotoxicity (CC50) and lytic plaques reduction (IC50) the GraphPadPrism program (Software, San Diego, CA.) was used. The selectivity index (SI) was calculated by the ratio of the value of CC50/IC50.
Discussion
Multiple studies have reported the discovery or obtaining of compounds with anti-dengue properties [
8,
16‐
18,
29,
42‐
47], however, up to now there are no commercially available drugs for treatment of dengue.
In the present study we included plants widely used in traditional medicine in many countries for the treatment of many diseases or organic disorders [
48,
49]. Extracts from our selected plants presented different activities per species, temperature and solvent used. The lesser toxic extract was
T. officinale obtained with methanol and the more toxic was the methanolic extract obtained from
C. pulcherrima.
We performed a protocol in which the cells were infected and subsequently, the plant extract was added as possible inhibitor, trying to simulate a system with an active DENV infection, like it occurs in a natural infection. Therefore, this protocol suggests that the inhibitory effect of virus replication is obstructed in an intermediate step in the replication cycle, similar to what has been reported in other experimental inhibition of DENV [
18], possibly by interaction of flavonoids with viral enzymes involved in the ARN synthesis or maturation of polyprotein.
By comparing the extracts obtained at three different temperatures, we observed a higher inhibition of DENV2 replication with those obtained at 60 °C (Fig.
1a), suggesting the selection and conservation of compounds with anti-dengue properties. The time and temperature of extraction of compounds with pharmacological potential are important parameters to optimize production and lower costs for the overall process. Different authors propose that increasing the temperature in the extraction process promotes increased solubility and the diffusion coefficient of phenolic compounds, provided they do not cause denaturation [
50,
51].
A similar process occurs with
C. pulcherrima, the aqueous extracts of this plant contain compounds like quercetin with a broad inhibitory activity on adenovirus 8 and 3 and on herpesviruses [
26]. It is possible that the reason for the activity is not a single molecule, but a mixture of them and that could have an effect on different viruses through different mechanisms.
An important parameter to consider is the selectivity index (SI), which ranged from 5.59 and 9.43 for the extracts or fractions. These values are similar to other reports of natural products with some anti-dengue activity [
18,
19,
47,
52,
53]. SI represents the relative effectiveness of a product in inhibiting viral replication compared to inducing cell citotoxicity. High therapeutic index represents a relative low cytotoxicity and high antiviral activity. This parameter is used for in vitro tests, unlike the therapeutic index which is a measurement with a similar meaning, but performed in vivo [
54,
55].
In our work the aqueous extracts showed less inhibition compared with extracts obtained with methanol, as it was reported previously [
42] to assess the inhibitory effect of aqueous and methanol extracts of
Hydrocotyle sibthorpioides on the replication of DENV.
Through HPLC-DPA-ESI-MS/MS we identified 11 compounds in the
T. officinale F9 and 6 in the
U. dioica F7. In both fractions, quercetin derivatives were found. Several studies report that quercetin have anti-dengue activity [
18,
43,
44], although the specific mechanisms remain undetermined. However, the interaction of quercetin or its derivatives with the NS2B-NS3 protease [
45,
56], the envelope (E) protein [
57,
58] and the NS5 polymerase [
59] have been predicted by in silico methods.
On the other hand, luteolin derivatives were found in the
T. officinale fraction. Luteolin has been reported to have inhibitory activity on several viruses such as enterovirus 71, Coxsackievirus A16 [
60] and chikungunya virus [
61]. In recent articles, the interaction of one of the identified compounds, luteolin-7-O-glucoside, and other luteolin derivatives, has been predicted to interact with the NS2/NS3 protease using in silico analysis [
46,
56]. In addition, a very recent study showed that luteolin reduces DENV infection through the inhibition of human furin, which is an enzyme involved in the maturation of the virions, whereby viral particles with a low infectious capacity are produced [
53].
Other compounds identified in
T. officinale F9 were caffeic acid and caffeoylquinic acid derivatives. These molecules have been shown as inhibitors of some important viruses such as hepatitis B [
62], influenza A [
63], herpes simplex [
64] and more recently, dengue virus [
47]. In this last study, Zanello et al., show the effect of two derivatives on the four DENV serotypes.
Conclusions
It is interesting that analyzed fractions of
T. officinale and
U. dioica have several molecules with demonstrated antiviral activity. Recently, an antiretroviral activity (HIV-1) in aqueous extracts of
T. officinale was identified [
24], while other studies have identified an inhibitory effect against yellow fever virus (prototype of
Flaviviridae) [
65]. Is it possible that different compounds in this plant could have effects against different viruses?
The study of the recognized components present in our factions is determinant to identify the probable anti-dengue molecule and/or their effects on other viruses, including the recent mosquito-borne emerging viruses chikungunya and Zika. It is also necessary to study a formulation of several active compounds in these fractions, searching a probable synergism in the process of inhibition of virus infection. Our group is currently performing these studies.