Abstract
Aims
The aim of this work was to evaluate the antiviral activities of Baccharis dracunculifolia (extract and essential oil), propolis and some isolated compounds (caffeic and cinnamic acids) against poliovirus type 1 (PV1) replication in HEp-2 cells.Method
Three different protocols (pre-, simultaneous and post-treatments) were used to verify the effect of addition time of the variables on PV1 replication by crystal violet method and relative viral RNA quantification by real-time PCR for analysing in which step of virus replication the variables could interfere.Conclusions
Data revealed that the B. dracunculifolia showed the best antiviral activity percentage in the simultaneous treatment, as well as lower relative viral quantification by real-time PCR. Variables might block partially the viral entry within cells, affect the steps of viral cycle replication into cells, or lead to RNA degradation before the virus entry into cells or after their release to the supernatant.Significance and impact of the study
Baccharis dracunculifolia is the most important botanical source of the south-eastern Brazilian propolis, and its potential for the development of new phytotherapeutic medicines has been investigated. Propolis is commonly used for its antimicrobial and immunomodulatory activities. Nevertheless, B. dracunculifolia and propolis effects on PV1 have not been investigated yet.Free full text
Anti‐poliovirus activity of Baccharis dracunculifolia and propolis by cell viability determination and real‐time PCR
Abstract
Aims: The aim of this work was to evaluate the antiviral activities of Baccharis dracunculifolia (extract and essential oil), propolis and some isolated compounds (caffeic and cinnamic acids) against poliovirus type 1 (PV1) replication in HEp‐2 cells.
Method: Three different protocols (pre‐, simultaneous and post‐treatments) were used to verify the effect of addition time of the variables on PV1 replication by crystal violet method and relative viral RNA quantification by real‐time PCR for analysing in which step of virus replication the variables could interfere.
Conclusions: Data revealed that the B. dracunculifolia showed the best antiviral activity percentage in the simultaneous treatment, as well as lower relative viral quantification by real‐time PCR. Variables might block partially the viral entry within cells, affect the steps of viral cycle replication into cells, or lead to RNA degradation before the virus entry into cells or after their release to the supernatant.
Significance and Impact of the Study:
Baccharis dracunculifolia is the most important botanical source of the south‐eastern Brazilian propolis, and its potential for the development of new phytotherapeutic medicines has been investigated. Propolis is commonly used for its antimicrobial and immunomodulatory activities. Nevertheless, B. dracunculifolia and propolis effects on PV1 have not been investigated yet.
Background
A variety of natural products and their derivatives have been considered as potential candidates for the treatment of human viral diseases (Huleihel and Isanu 2002; Gekker et al. 2005). Recently, the inhibitory effects of medicinal plants extracts on the replication of several viruses have been reported. Herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis B virus (HBV) and severe acute respiratory syndrome (SARS) virus were strongly inhibited by various plants extracts (Mukhtar et al. 2008).
Poliovirus (PV) is a single‐stranded RNA, nonenveloped virus, belonging to the family Picornaviridae and genus Enterovirus. It is the aetiological agent of poliomyelitis, and once it reaches the central nervous system, one may develop paralytic poliomyelitis – a disease characterized by a classic manifestation of flaccid paralysis. The incidence of paralytic poliomyelitis has been reduced over the last decades, especially by the systematic use of vaccines; however, this disease is still endemic in Asia and Africa (Felipe et al. 2006). Because of its replication in several types of cultured cells, PV is one of the most studied and understood viral models (Faccin et al. 2007).
As to PV, extracts of Tridax procumbens, Carissa carandas, Mallotus philippensis, Agaricus brasiliensis and Euphorbia grantii among others were found to be efficient against the replication of poliovirus type 1 (PV1) (Semple et al. 2001; Felipe et al. 2006). A potent activity against HIV and HSV in vitro was observed using extracts of Baccharis trinervis (Palomino et al. 2002). In this study, we evaluated the effect of Baccharis dracunculifolia against PV1 replication.
Baccharis dracunculifolia is used in folk medicine as an anti‐inflammatory agent and for the treatment of gastrointestinal diseases. This plant is the most important botanical origin of Brazilian propolis, called green propolis because of its colour (Bankova et al. 1999; Da Silva Filho et al. 2004).
Propolis is the generic name for the resinous substance produced by honeybees and commonly used to improve health and to prevent several diseases. It has been used for medicinal purposes since ancient times, and its antimicrobial, antitumoural and immunomodulatory activities have been reported (Sforcin 2007). With regard to antiviral properties of propolis, its inhibitory effect on several viruses including influenza, HIV, HSV, adenovirus and vesicular stomatitis virus has been well documented (Ito et al. 2001; Gekker et al. 2005; Moreno et al. 2005). These findings have indicated the potential of propolis as a possible antiviral drug. However, antiviral effects of B. dracunculifolia have not been investigated yet.
In this study, the antiviral activity of B. dracunculifolia (extract and essential oil), propolis and isolated compounds (caffeic and cinnamic acids) was assessed using PV1. The variables were incubated with HEp‐2 cells prior, simultaneously or subsequently, to PV1 addition to the cell cultures in order to understand their mechanism of action.
Materials and methods
Plant material, oil and extract isolation and major compounds
Baccharis dracunculifolia leaves were collected in ten different regions of Brazil (São Paulo, Minas Gerais and Paraná State) in their natural habitat, between May 2004 and April 2005. Plants were identified by Dr Nelson Ivo Matzenbacher (Department of Bioscience, PUC/RS, Brazil), and voucher specimens (n=
1298) were deposited in the Herbarium of the Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA), University of Campinas, São Paulo, Brazil.
Extracts of B. dracunculifolia were obtained from dried and powdered leaves samples (500mg), dissolved in 20
ml of 90% ethanol in 125
ml Erlenmeyer flasks, which were stirred at 40°C and 170
rev
min−1 on a shaker (Innova 4300; New Brunswick Scientific, Edison, NJ). After 2
h, flasks were cooled down to room temperature and filtered using analytical filter papers. Aliquots (5
ml) of the hydroalcoholic extracts were transferred to an appropriate vial and dried under air circulation (40°C). The yielding of crude extracts from B. dracunculifolia (90
mg) was of 18%.
The essential oil of the dried leaves (500g) was extracted by hydro‐distillation using a Clevenger‐type apparatus. After extraction, the volume of essential oil obtained was measured, and the essential oil was stored in hermetically sealed glass containers with rubber lids, covered with aluminium foil to protect the contents from light and kept under refrigeration at 8°C. The oil yield was 0·6% based on the dry weight of the plant. High performance liquid chromatography (HPLC) profile of B. dracunculifolia extract and flame ionization detector (FID) capillary gas chromatogram of the essential oil were carried out (Sousa et al. 2007).
Caffeic and cinnamic acids were purchased from Acros Organics (Morris Plains, NJ, USA). Extract, caffeic and cinnamic acids were diluted in 100μl of 70% ethanol and subsequently diluted in minimum essential medium (MEM; GIBCO, NY, USA) supplemented with 0·1
g
l−1 of l‐glutamine, 2·2
g
l−1 sodium bicarbonate, 10
ml
l−1 nonessential amino acids and 10% foetal calf serum (LGC, Biotecnologia LTDA., Cotia, Brazil). Essential oil (1
ml) was diluted in culture medium containing 0·2% dimethylsulfoxide (DMSO; Sigma‐Aldrich, USA).
Specific dilutions of each variable (Baccharis extract and essential oil, caffeic and cinnamic acids) were prepared for each assay in order to achieve 5, 10, 25, 50 and 100μg per 100
μl. The same procedure was carried out with 0·2% DMSO (essential oil solvent) and 70% ethanol (extract and isolated compounds solvent) to obtain the respective concentrations of DMSO in the essential oil, and of alcohol in the extract, caffeic and cinnamic acids.
Propolis sample
Propolis was collected in the Beekeeping Section, UNESP, Campus of Botucatu, Brazil. Propolis was ground, and 30% ethanolic extracts of propolis were prepared (30g of propolis, completing the volume to 100
ml with 70% ethanol), in the absence of bright light, at room temperature with moderate shaking. After a week, extracts were filtered, and the dry weight of the extracts was calculated (120
mg
ml−1). Propolis chemical composition was investigated using thin layer chromatography (TLC), gas chromatography (GC) and gas chromatography–mass spectrometry (GC‐MS) analyses (Bankova et al. 1998). Propolis was diluted in MEM, and specific dilutions were prepared to achieve 5, 10, 25, 50 and 100
μg per 100
μl. The same procedure was carried out with 70% ethanol (propolis solvent).
Cells and virus
HEp‐2 cells (human laryngeal epidermoid carcinoma cells) were stored in liquid nitrogen. Afterwards, cells were cultured in flasks with MEM supplemented with 10% foetal calf serum and, before use, 2ml of trypsin (0·2% trypsin in 5% EDTA) was added to each flask until cells detachment. Cells were counted using a haemocytometer and cultured in a 96‐well flat‐bottomed plate (Corning, NY, USA), at a final concentration of 2
×
105
cells per well.
PV1 (vaccinal strain Sabin I, stored at −80°C in our laboratory) was propagated in HEp‐2 cell cultures and maintained at −80°C. The virus titre was determined according to Reed and Muench method (Lennette 1995) and expressed in 50% tissue culture infectious dose (TCID50).
Cytotoxic assay
Prior to antiviral assays, cell viability after incubation with each variable in different concentrations was assessed in order to carry out the assays only with noncytotoxic concentrations. The evaluation of cytotoxicity was carried out by crystal violet method (Ait‐Mbarek et al. 2007).
HEp‐2 cells grown in 96‐well flat‐bottomed microplates were incubated with different concentrations (5, 10, 25, 50 and 100μg per 100
μl) of B. dracunculifolia (extract and essential oil), propolis, caffeic and cinnamic acids, as well as with 70% ethanol and 0·2% DMSO. Control cells were incubated with medium alone. The final volume in each well was 100
μl.
Cell viability was determined after 48‐h incubation at 37°C and 5% CO2. The medium was removed, and 100μl of 0·5% crystal violet solution was added to the cells. After 10
min of incubation at room temperature, plates were washed, and viable crystal violet‐stained cells were lysed with 1% sodium dodecyl sulphate (SDS). Optical densities (OD) were read at 492
nm in an ELISA reader, and the percentage of cell viability was calculated using the formula: (OD test/OD control)
×
100. Assays were carried out in triplicate.
Antiviral assays
The antiviral effect of the variables on PV1 replication was determined according to Faccin et al. (2007) with minor modifications, in three different protocols, as follows:
(i) Pretreatment: HEp‐2 cells were resuspended at a concentration of 2×
106
cells
ml−1 in MEM, cultured in 96‐well flat‐bottomed plates and incubated with the variables at noncytotoxic concentrations: B. dracunculifolia (extract and essential oil), caffeic and cinnamic acids
=
5, 10 and 25
μg per 100
μl; propolis
=
5 and 10
μg per 100
μl. After 2
h, the medium was removed, and 100 TCID50 per 100
μl (using the dilution of 10−2·5 of virus stock) were added and incubated for 48
h at 37°C and 5% CO2.
(ii) Simultaneous treatment: HEp‐2 cells (2×
106
cells
ml−1) were incubated simultaneously with noncytotoxic concentrations of each variable and 100 TCID50 per 100
μl (10−2·5) of virus suspension for 48
h.
(iii) Post‐treatment: HEp‐2 cells (2×
106
cells
ml−1) were incubated with 100 TCID50 per 100
μl (10−2·5) of virus suspension for 2
h for virus adsorption. After incubation, the medium was removed, and variables at noncytotoxic concentrations were added and incubated for 48
h.
Cells with virus but without variables were considered as a positive control. Each variable was evaluated in triplicate, and assays were repeated three times. The percentage of antiviral activity, corresponding to viable HEp‐2 cells, was assessed by crystal violet method as described above.
In these same conditions, other plates were prepared for relative viral RNA quantification by real‐time PCR in order to verify in which step of virus replication the variables could interfere.
After incubation with B. dracunculifolia (extract and essential oil), propolis, caffeic and cinnamic acids, 100μl of supernatant were collected and centrifuged at 400
g for 15
min to remove cellular debris. Afterwards, the cell monolayer was treated with 100
μl of lysis buffer of the RNAspin Mini RNA Isolation kit (GE Healthcare, NJ, USA), and the lysate was collected. Samples of supernatant and cell lysates were submitted to total RNA extraction.
Extraction of total RNA and cDNA synthesis
Total RNA was extracted using the RNAspin Mini RNA Isolation kit (GE Healthcare), according to the manufacturer’s instructions. Extracted RNA preparations were stored at −80°C.
Total RNA extracted (4μl) was reverse transcribed with 1
μl of random primer (250
ng
μl−1), and the mixture was incubated for 5
min at 70°C. For each sample, the master mix was prepared with 4
μl of reaction buffer (Improm II 5×; Promega, WI, USA), 2·4
μl of MgCl2 (25
mmol
l−1), 1
μl of RNase out (10
units), 0·5
μl of dNTP (20
mmol
l−1), 0·5
μl Improm RT II (Promega) and 6·6
μl of free nuclease water. Afterwards, samples were cooled down to 4°C, and 15
μl of master mix were added, incubating for 5
min at 25°C, 60
min at 42°C and for 15
min at 70°C. Each cDNA was stored at −20°C.
Real‐time PCR
Each tube of reaction contained 4μl of PV cDNA template, 10
μl of the master mix SYBR Green I (Invitrogen, CA), 0·4
μl of each primer (final concentration 200
nmol
l−1), 0·4
μl ROX reference dye and 5·2
μl of PCR‐grade water. Specific primers for PV capsid gene (VP1–VP4) were 5′‐AGT TTC ACC GAA GGC GGA‐3′ (F) and 5′‐CGC TGA CAC AAA ACC AAG GA‐3′ (R) (GeneBank accession no. AY184219), resulting in a 102‐bp amplified product. The PCR programme consisted of the following steps: 95°C for 10
min for initial denaturation, amplification for 40 cycles (95°C for 15
s for denaturation, 60°C for 1
min for annealing and extension), and to confirm the PCR product one cycle of melting curve analysis at 95°C for 15
s, 60°C for 30
s and 95°C for 15
s. Fluorescence data were collected during each annealing/extension step and analysed using ABI PRISM® 7300 Sequence Detector (Applied Biosystems, CA, USA) and sds ver. 1.2.3 (Sequence Detection Systems 1.2.3, 7300 Real Time PCR System; Applied Biosystems) software.
In every PCR run, negative (no template) and positive PV controls were processed as a routine quality control of the assay. Assays were carried out in duplicate, and the RNA quantification reduction percentage in the supernatant and lysate samples was calculated considering the positive control as 100%, using the formula: [1−
(RNA samples/RNA control)]
×
100.
Standard curve
The standard curve was generated by performing serial dilutions of the PV1 RNA extracted of cell culture. To the smallest dilution of RNA standard, it was given the relative value 100 and, following the same reason of dilution, the other 3 points were 50, 25 and 12·5. The virus quantity in the samples was expressed in relation to the standard curve.
Statistics
Wilcoxon test was used to detect differences between propolis concentrations, and Friedman test to the other variables, with the significant level at P<
0·05 (Zar 1999).
Results
Baccharis dracunculifolia (extract and essential oil) and propolis chemical composition
The chromatographic profiles of B. dracunculifolia leaves (extract and essential oil) are showed in in1,1, ,2,2, respectively. From HPLC analysis and comparatives studies, involving authentic standard and the samples investigated here, it was possible to carry out the characterization of the extract of this plant. According to Fig. 1, caffeic acid, ferulic acid, aromadendrin‐4′‐methyl ether and artepillin C were the main phenolics detected in the plant hydroalcoholic extract (Sousa et al. 2007). GC‐MS and FID capillary gas chromatography analysis of the essential oil of B. dracunculifolia revealed that the main chemical components were α‐pinene, β‐pinene, limonene, trans‐caryophyllene, aromadendrene, α‐humulene, germacrene D, bicyclogermacrene, δ‐cadinene, nerolidol, spathulenol, viridiflorol, guaiol and α‐muurolol (Fig. 2).
High performance liquid chromatography profile of Baccharis dracunculifolia extract: caffeic acid (1), ferulic acid (2), aromadendrin‐4′‐methyl ether (3) and artepellin C (4), internal standard (is) (veratraldehyde).
FID capillary gas chromatogram of essential oil of Baccharis dracunculifolia: α‐pinene (1), β‐pinene (2), limonene (3), trans‐caryophyllene, (4), aromadendrene (5), α‐humulene (6), germacrene D (7), bicyclogermacrene (8), δ‐cadinene (9), nerolidol (10), spathulenol (11), viridiflorol (12), guaiol (13), α‐muurolol (14) and internal standard (is) (piperonal).
The main constituents of our propolis sample, investigated by TLC, GC and GC‐MS analyses, were flavonoids (kaempferid, 5,6,7‐trihydroxy‐3,4′‐dimethoxyflavone, aromadendrine‐4′‐methyl ether), a prenylated p‐coumaric acid and two benzopyranes (E and Z 2,2‐dimethyl‐6‐carboxyethenyl‐8‐prenyl‐2H‐benzopyranes), essential oils (spathulenol, (2Z,6E)‐farnesol, benzyl benzoate and prenylated acetophenones), aromatic acids (dihydrocinnamic acid, p‐coumaric acid, ferulic acid, caffeic acid, 3,5‐diprenyl‐p‐coumaric acid, 2,2‐dimethyl‐6‐carboxy‐ethenyl‐8‐prenyl‐2H‐1‐benzo‐pyran), di‐ and tri‐terpenes, among others (Bankova et al. 1998).
Cytotoxic assay
Data showed that the noncytotoxic concentrations for each variable were B. dracunculifolia (extract and essential oil), caffeic and cinnamic acids=
5, 10 and 25
μg per 100
μl, and propolis
=
5 and 10
μg per 100
μl. Ethanol and DMSO had no effects in cell viability (Búfalo et al. in press).
Antiviral assay
For the antiviral assays, noncytotoxic concentrations of each variable were used. In the pretreatment, B. dracunculifolia extract showed the most efficient antiviral action (31·0%), followed by B. dracunculifolia oil (28·9%), propolis (10·9%), cinnamic acid (10·5%) and caffeic acid (8·5%). With regard to the concentrations of each variable, the most efficient concentration for the plant extract and essential oil was 25μg per 100
μl in comparison with the other concentrations for the same variable (P
<
0·05). No significant differences were seen between the different concentrations of propolis, caffeic and cinnamic acids (P
>
0·05) ((3,3, ,4,4, ,5,5, ,6,6, ,77).
Antiviral activity percentage and relative RNA viral quantification (supernatant and cell lysate) of the extract. (a): Pretreatment, (b): simultaneous treatment, (c): post‐treatment. Friedman test (P
<
0·05). (
) 0
μg per 100
μl; (
) 5
μg per 100
μl; (
) 10
μg per 100
μl and (
) 25
μg per 100
μl.
Antiviral activity percentage and relative RNA viral quantification (supernatant and cell lysate) of the essential oil. (a): Pretreatment, (b): simultaneous treatment, (c): post‐treatment. Friedman test (P
<
0·05). (
) 0
μg per 100
μl; (
) 5
μg per 100
μl; (
) 10
μg per 100
μl and (
) 25
μg per 100
μl.
Antiviral activity percentage and relative RNA viral quantification (supernatant and cell lysate) of the propolis. (a): Pretreatment, (b): simultaneous treatment, (c): post‐treatment. Wilcoxon test (P
<
0·05). (
) 0
μg per 100
μl; (
) 5
μg per 100
μl and (
) 10
μg per 100
μl.
Antiviral activity percentage and relative RNA viral quantification (supernatant and cell lysate) of the caffeic acid. (a): Pretreatment, (b): simultaneous treatment, (c): post‐treatment. Friedman test (P
<
0·05). (
) 0
μg per 100
μl; (
) 5
μg per 100
μl; (
) 10
μg per 100
μl and (
) 25
μg per 100
μl.
Antiviral activity percentage and relative RNA viral quantification (supernatant and cell lysate) of the cinnamic acid. (a): Pretreatment, (b): simultaneous treatment, (c): post‐treatment. Friedman test (P
<
0·05). (
) 0
μg per 100
μl; (
) 5
μg per 100
μl; (
) 10
μg per 100
μl and (
) 25
μg per 100
μl.
In the simultaneous treatment, the highest inhibition of virus replication was achieved by Baccharis extract (74·0%), followed by propolis (52·2%), essential oil (33·3%), cinnamic acid (29·8%), and caffeic acid (26·7%). The most efficient concentration for Baccharis extract, caffeic and cinnamic acids was 25μg per 100
μl, followed by propolis (10
μg per 100
μl) (P
<
0·05). There were no significant differences between the different concentrations of Baccharis essential oil (P
>
0·05) ((3,3, ,4,4, ,5,5, ,6,6, ,77).
Data from the post‐treatment showed that the most efficient variables were Baccharis essential oil (49·6%), propolis (39·1%), Baccharis extract (36·1%), caffeic acid (22·3%) and cinnamic acid (7·9%). As to the concentrations, Baccharis (extract and essential oil) and caffeic acid (25μg per 100
μl) and propolis (10
μg per 100
μl) inhibited significantly the PV1 replication (P
<
0·05). No significant differences were seen between the concentrations of the cinnamic acid (P
>
0·05) ((3,3, ,4,4, ,5,5, ,6,6, ,77).
A comparison between the variables regarding their concentrations and antiviral action in all protocols (pre‐, simultaneous and post‐treatments) revealed that Baccharis extract (25μg per 100
μl) showed the best antiviral activity (74·0%) in the simultaneous treatment (P
<
0·05) (Table 1).
Table 1
Comparison between the protocols (pre‐, simultaneous and post‐treatments) with regard to the antiviral activity of each variable
| Variables | 5 | 10 | 25 | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Pretreatment | Simultaneous treatment | Posttreatment | Pretreatment | Simultaneous treatment | Posttreatment | Pretreatment | Simultaneous treatment | Posttreatment | |
| Propolis | b | ab | a | b | a | ab | – | – | – |
| Extract | b | a | ab | b | a | ab | b | a (74·0%) | ab |
| Essential oil | a | a | a | b | ab | a | b | ab | a |
| Caffeic acid | a | a | a | b | a | ab | b | a | ab |
| Cinnamic acid | a | a | a | b | a | b | ab | a | b |
Different small letters indicate significant differences between the treatments in the same concentration by Friedman test (P<
0·05). The best antiviral activity is shown in bold, and the percentage is between parentheses.
Relative quantification of viral RNA
PCR efficiency (78·1%) was determined using the slope of the standard curves, and the real‐time PCR reproducibility was represented by the correlation coefficient (R=
0·99). Amplicons specificity was confirmed by melting curve analysis. A single melting peak at 80°C to specific amplicon of 102
bp was obtained, indicating that the primers used in this study were specific for PV1 (data not shown).
In the pretreatment, the highest RNA quantification reduction percentage was seen in the supernatant samples after incubation with B. dracunculifolia extract, caffeic and cinnamic acids, in comparison with the positive controls (94%, 90% and 89% respectively), using 25μg per 100
μl. These same variables also showed higher RNA reduction percentage than the essential oil and propolis, using 10 and 5
μg per 100
μl (Baccharis extract
=
74% and 70%; caffeic acid
=
79% and 73%; cinnamic acid
=
78% and 65% respectively). Baccharis essential oil showed the following RNA reduction percentages 66%, 64% and 63%, using 25, 10 and 5
μg per 100
μl. For propolis, the reduction percentages were 60% and 41%, using 10 and 5
μg per 100
μl ((3,3, ,4,4, ,5,5, ,6,6, ,77).
In cell lysates, B. dracunculifolia extract showed the best RNA reduction percentage in all concentrations (97%, 93% and 89%), followed by the essential oil, propolis, caffeic and cinnamic acids ((3,3, ,4,4, ,5,5, ,6,6, ,77).
In the simultaneous treatment, the highest RNA quantification reduction percentage was seen in the supernatant samples after incubation with B. dracunculifolia extract (99%, 96% and 89%, respectively to 25, 10 and 5μg per 100
μl), followed by cinnamic acid (93%, 88% and 72%), caffeic acid (93%, 80% and 72%) and Baccharis essential oil (88%, 81% and 77%). For propolis, the RNA reduction percentages were 80% and 30%, using 10 and 5
μg per 100
μl respectively ((3,3, ,4,4, ,5,5, ,6,6, ,77).
In cell lysates, Baccharis extract led to decreased RNA quantification (RNA reduction percentages: 97%, 96% and 95%, using 25, 10 and 5μg per 100
μl). The RNA reduction percentages for the other variables were: Baccharis essential oil
=91%, 69% and 61%; cinnamic acid
=
85%, 46% and 18%; caffeic acid
=
85%, 45% and 23%; and propolis
=
77% and 68% ((3,3, ,4,4, ,5,5, ,6,6, ,77).
In the post‐treatment, caffeic acid led to the lowest relative viral RNA in the supernatant (RNA reduction percentage: 76%, 51% and 44%), followed by the other variables (cinnamic acid=
72%, 48% and 40%; propolis
=
53% and 20%; essential oil
=
46%, 39% and 18%; Baccharis extract
=
26%, 25% and 10%) ((3,3, ,4,4, ,5,5, ,6,6, ,77).
In cell lysates, the RNA reduction percentages were: Baccharis extract=
77%, 48% and 41%; cinnamic acid
=
58%, 15% and 14%; propolis
=
35% and 3%; essential oil
=
26%, 8% and 5%, and caffeic acid
=
18%, 12% and 13% ((3,3, ,4,4, ,5,5, ,6,6, ,77).
The best results were obtained in the simultaneous treatment, followed by the pre‐ and post‐treatments for B. dracunculifolia extract, caffeic and cinnamic acids, Baccharis essential oil and propolis, either in the supernatants or in cells lysates. Higher relative viral RNA quantification was observed in the cell lysate in comparison with the supernatants, in all protocols and variables ((3,3, ,4,4, ,5,5, ,6,6, ,77).
One may verify that real‐time PCR data are in agreement with the Table 1, and an association between the highest antiviral activities of the variables in the crystal violet method could be established with the lowest relative viral quantification in the real‐time PCR.
Discussion and conclusions
Medicinal plants have been widely used to treat a variety of infectious and noninfectious diseases, and 25% of the commonly used medicines contain compounds isolated from plants (Mukhtar et al. 2008). The investigation of natural products with antiviral action has attracted the researchers’ interest; nevertheless, no articles are found in the literature dealing with antiviral activity of B. dracunculifolia. In this study, we wish to report for the first time the antiviral activity of this plant on PV1 replication.
With regard to the antiviral assays, the highest percentage of viral inhibition and consequently smallest relative RNA viral quantification were obtained with 25μg per 100
μl of B. dracunculifolia extract in the simultaneous treatment. Baccharis dracunculifolia essential oil showed a lower antiviral activity compared to the extract.
When PV1 was added to the cells simultaneously with the extract or propolis, there was a decreased RNA quantification in cell lysate as well as in the supernatant samples, however, higher amounts of RNA were found in the lysate in comparison with the supernatant. Although the viral entry into cells could have been inhibited, B. dracunculifolia and propolis exerted their antiviral activity probably when PV1 was within cells, affecting the cycle replication.
In the pretreatment, variables were removed before adding the virus, what could lead cells more resistant to virus attack. However, there was a higher viral entry into cells, in comparison with the simultaneous treatment, discarding our hypothesis. On the other hand, in the post‐treatment, variables were added after the virus, and the RNA viral quantification was higher than that in the simultaneous treatment. One may speculate that variables interfered in virus output by infected cells, or led to RNA degradation in the supernatant after virus output.
Extracts of B. trinervis showed inhibitory effects on HSV and HIV replication when added simultaneously to the virus, suggesting that the extracts inhibited the virus‐cell attachment, virus‐cell fusion and cell‐to‐cell fusion (Palomino et al. 2002).
As to propolis, Amoros et al. (1992) verified the effect in vitro of alcoholic extract of propolis against several viruses of DNA and RNA, including HSV, adenovirus, and vesicular stomach virus, showing that propolis reduced HSV titres; but the other viruses were less susceptible to its action.
Serkedjieva et al. (1992) reported that the pretreatment of canine kidney epithelial (MDCK) cells with propolis had no effect on influenza virus replication. However, a reduced viral infectivity was seen adding propolis simultaneously with the virus or immediately after virus adsorption, suggesting that adsorption and the penetration of virus were inhibited in the initial stage of replication cycle.
Ito et al. (2001) verified the efficient activity of propolis anti‐HIV in vitro. Gekker et al. (2005), using microglial cell cultures, showed propolis activity against HIV in a concentration‐dependent manner, suggesting that this effect may have occurred because of caffeic acid phenethyl ester (CAPE) action, involving, in part, the inhibition of viral entry into cells.
According to Huleihel and Isanu (2002), propolis caused 50% inhibition of HSV infection, and indirect evidence pointed out to a strong interaction between propolis and the surface of Vero cells, but not with HSV particles. Administration of propolis before or simultaneously to infection yielded the most significant inhibitory effect, suggesting that this effect were because of propolis blockage of the cell membrane receptors for HSV or because of changes inside the cells, which could in turn affect the virus replication cycle.
Huleihel and Ishano (2001) and Matsuo et al. (2005) suggested that the antiviral activity of propolis might be attributed to flavonoids action, which play a significant role in the antiviral process. Tait et al. (2006) related that natural and synthetic flavonoids might interfere with picornavirus replication preventing the decapsidation of viral particles and RNA release within cells or blocking viral RNA synthesis.
Isolated compounds are important to understand the possible mechanism of action of propolis and its vegetal source. In our study, caffeic and cinnamic acids showed a lower antiviral activity when compared to B. dracunculifolia and propolis, suggesting that these acids may be involved in B. dracunculifolia and propolis’ antiviral effects. It has been reported that the potential of propolis and Baccharis are due to a natural mixture of its components, and that a single constituents would not have an activity greater than that the total extract (Kujumgiev et al. 1999).
The real‐time PCR has been described as a quantitative detection method for nucleic acids (Min et al. 2006). This method provides higher sensitivity and specificity to quantify viral nucleic acids, and an association between the concentrations of viral nucleic acid and cell culture infectivity by PV1 could be established herein.
In our study, B. dracunculifolia (25μg per 100
μl) showed an efficient percentage of antiviral activity evidenced by the crystal violet method (74·0%) in the simultaneous treatment, which was associated to the lowest relative viral quantification in the real‐time PCR. Some explanations to these effects may be raised: (i) variables might block partially the viral entry within cells, (ii) variables could have affected the steps of viral cycle replication into cells and (iii) RNA degradation before the virus entry into cells or after virus release to the supernatant. However, more investigations are still needed in order to explore the potential of these variables as antiviral agents and to understand their mechanisms of action against PV1 cycle replication.
Acknowledgement
Authors thank CNPq, CAPES and FAPESP for the financial support, and Dr Lídia Raquel de Carvalho, IB, UNESP, Campus of Botucatu, for the statistical analysis.
References
- Ait‐Mbarek, L. , Ait‐Mouse, H. , Elabbadi, N. , Bensalah, M. , Gamouh, A. , Aboufatima, R. , Benharref, A. , Chait, A. et al. (2007) Anti‐tumor properties of blackseed (Nigella sativa L.) extracts. Braz J Med Biol Res 40, 839–847. [Abstract] [Google Scholar]
- Amoros, M. , Sauvager, F. , Girrel, C. and Cormier, M. (1992) In vitro antiviral activity of propolis. Apidologie 23, 231–240. [Google Scholar]
- Bankova, V.S. , Popov, S.S. and Marekov, N.L. (1998) On the chemical composition of some propolis fractions with antiviral action. Acta Microbiol Bulg 23, 52–57. [Abstract] [Google Scholar]
- Bankova, V.S. , Boudourova‐Krasteva, G. , Sforcin, J.M. , Frete, X. , Kujumgiev, A. , Maimoni‐Rodella, R. and Popov, J. (1999) Phytochemical evidence for the plant origin of Brazilian propolis from São Paulo state. Z Naturforsch 54, 401–405. [Abstract] [Google Scholar]
- Búfalo, M.C. , Candeias, J.M.G. , Sousa, J.P.B. , Bastos, J.K. and Sforcin, J.M. (in press) In vitro cytotoxic activity of Baccharis dracunculifolia and propolis against HEp‐2 cells. Nat Prod Res, in press. [Abstract] [Google Scholar]
- Da Silva Filho, A.A. , Bueno, P.C.P. , Gregório, L.E. , Andrade e Silva, M.L. , Albuquerque, S. and Bastos, J.K. (2004) In‐vitro trypanocidal activity evaluation of crude extract and isolated compounds from Baccharis dracunculifolia D. C. (Asteraceae). J Pharm Pharmacol 56, 1195–1199. [Abstract] [Google Scholar]
- Faccin, L.C. , Benati, F. , Rincão, V.P. , Mantovani, M.S. , Soares, S.A. , Gonzaga, M.L. , Nozawa, C.P. , Linhares, R.E.C. (2007) Antiviral activity of aqueous and ethanol extracts and of an isolated polysaccharide from Agaricus brasiliensis against poliovirus type 1. Lett Appl Microbiol 45, 24–28. [Abstract] [Google Scholar]
- Felipe, A.M.M. , Rincão, V.P. et al. (2006) Antiviral effect of Guazuma ulmifolia and Stryphnodendron adstringens on Poliovirus and Bovine Herpesvirus. Biol Pharm Bull 29, 1092–1095. [Abstract] [Google Scholar]
- Gekker, G. , Hu, S. , Spivak, M. , Lokensgard, J.R. , Peterson, P.K. (2005) Anti‐HIV‐ 1 activity of propolis in CD4+ lymphocyte and microglial cell cultures. J Ethnopharmacol 102, 158–163. [Abstract] [Google Scholar]
- Huleihel, M. and Isanu, V. (2002) Anti‐herpes simplex virus effect of an aqueous extract of propolis. Isr Med Assoc J 4, 923–927. [Abstract] [Google Scholar]
- Huleihel, M. and Ishano, V. (2001) Effect of propolis extract on malignant cell transformation by moloney murine sarcoma virus. Arch Virol 146, 1517–1526. [Abstract] [Google Scholar]
- Ito, J. , Chang, F.R. , Wang, H.K. , Park, Y.K. , Ikegaki, M. , Kilgore, N. and Lee, K.H. (2001) Anti‐HIV activity of moronic acid derivates and the new melliferone‐related triterpenoid isolated from Brazilian propolis. J Nat Prod 64, 1278–1281. [Abstract] [Google Scholar]
- Kujumgiev, A. , Tsvetkova, I. , Serkedjieva, Y. , Bankova, V. , Christov, R. and Popov, S. (1999) Antibacterial, antifungal and antiviral activity of propolis of different geographic origin. J Ethnopharmacol 64, 235–240. [Abstract] [Google Scholar]
- Lennette, D.A. (1995) General principles for laboratory diagnosis of viral, rickettsial, and chlamydial infections In Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections ed. Lennette E.H., Lennette D.A. and Lennette E.T. pp. 3–25. Washington: American Public Health Association. [Google Scholar]
- Matsuo, M. , Sasaki, N. , Saga, K. and Kaneko, T. (2005) Citotoxicity of flanonoids toward cultured normal human cells. Biol Pharm Bull 28, 253–259. [Abstract] [Google Scholar]
- Min, B.S. , Noh, Y.J. , Shin, J.H. , Balk, S.Y. , Min, K.I. , Ryu, S.R. , Kim, B.G. , Park, M.K. (2006) Assessment of the quantitative real‐time polymerase chain reaction using cDNA standard for human group A rotavirus. J Virol Methods 137, 280–286. [Abstract] [Google Scholar]
- Moreno, M.I.N. , Zampini, I.C. , Ordonez, R.M. , Jaime, G.S. , Vattuone, M.A. and Isla, M.I. (2005) Evaluation of the cytotoxicity, genotoxicity, mutagenicity and antimutagenicity of propolis from Tucuman, Argentina. J Agric Food Chem 53, 8957–8962. [Abstract] [Google Scholar]
- Mukhtar, M. , Arshad, M. , Ahmad, M. , Pomerantz, R.J. , Wigdahl, B. and Parveen, Z. (2008) Antiviral potentials of medicinal plants. Virus Res 131, 111–120. [Europe PMC free article] [Abstract] [Google Scholar]
- Palomino, S.S. , Abad, M.J. , Bedoya, L.M. , Garcia, J. , Gonzales, E. , Chiriboga, X. , Bermejo, P. , Alcami, J. (2002) Screening of South American plants against human immunodeficiency vírus: preliminary fractionation of aqueous extract from Baccharis trinervis . Biol Pharm Bull 25, 1147–1150. [Abstract] [Google Scholar]
- Semple, S.J. , Pyke, S.M. , Reynolds, G.D. and Flower, R.L.P. (2001) In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus. Antiviral Res 49, 169–178. [Abstract] [Google Scholar]
- Serkedjieva, J. , Manolova, N. and Bankova, V. (1992) Anti‐influenza virus effect of some propolis constituents and their analogues (esters of substituted cinnamic acids). J Nat Prod 55, 294–297. [Abstract] [Google Scholar]
- Sforcin, J.M. (2007) Propolis and the immune system: a review. J Ethnopharmacol 113, 1–14. [Abstract] [Google Scholar]
- Sousa, J.P.B. , Bueno, P.C.P. , Gregório, L.E. , Da Silva Filho, A.A. , Furtado, N.A.J.C. , De Sousa, M.L. and Bastos, J.K. (2007) A reliable quantitative method for the analysis of phenolic compounds in Brazilian propolis by reverse phase high performance liquid chromatography. J Sep Sci 30, 2656–2665. [Abstract] [Google Scholar]
- Tait, S. , Salvati, A.L. , Desideri, N. and Fiore, L. (2006) Antiviral activity of substituted homoisoflavonoids on enterovirus. Antiviral Res 72, 252–255. [Abstract] [Google Scholar]
- Zar, J.H. (1999) Biostatistical Analysis, 4th edn New Jersey: Pretince Hall. [Google Scholar]
Full text links
Read article at publisher's site: https://doi.org/10.1111/j.1365-2672.2009.04354.x
Read article for free, from open access legal sources, via Unpaywall:
https://europepmc.org/articles/pmc7197736?pdf=render
Citations & impact
Impact metrics
Citations of article over time
Alternative metrics
Article citations
Antiviral action of aqueous extracts of propolis from Scaptotrigona aff. postica (Hymenoptera; Apidae) against Zica, Chikungunya, and Mayaro virus.
Sci Rep, 14(1):15289, 03 Jul 2024
Cited by: 2 articles | PMID: 38961137 | PMCID: PMC11222429
Anti-Biofilm Perspectives of Propolis against Staphylococcus epidermidis Infections.
Biomolecules, 14(7):779, 29 Jun 2024
Cited by: 1 article | PMID: 39062493 | PMCID: PMC11274400
Hive Products: Composition, Pharmacological Properties, and Therapeutic Applications.
Pharmaceuticals (Basel), 17(5):646, 16 May 2024
Cited by: 0 articles | PMID: 38794216 | PMCID: PMC11124102
Review
Free full text in Europe PMC
Antiviral Potential of Specially Selected Bulgarian Propolis Extracts: In Vitro Activity against Structurally Different Viruses.
Life (Basel), 13(7):1611, 23 Jul 2023
Cited by: 2 articles | PMID: 37511986 | PMCID: PMC10381642
Virucidal Activity of Lemon Essential Oil against Feline Calicivirus Used as Surrogate for Norovirus.
Antibiotics (Basel), 12(2):322, 03 Feb 2023
Cited by: 4 articles | PMID: 36830233 | PMCID: PMC9952628
Go to all (44) article citations
Data
Data behind the article
This data has been text mined from the article, or deposited into data resources.
BioStudies: supplemental material and supporting data
Nucleotide Sequences
- (1 citation) ENA - AY184219
Similar Articles
To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.
In vitro cytotoxic activity of Baccharis dracunculifolia and propolis against HEp-2 cells.
Nat Prod Res, 24(18):1710-1718, 01 Nov 2010
Cited by: 12 articles | PMID: 20419558
Baccharis dracunculifolia, the main botanical source of Brazilian green propolis, displays antiulcer activity.
J Pharm Pharmacol, 59(4):603-608, 01 Apr 2007
Cited by: 39 articles | PMID: 17430646
Artepillin C and phenolic compounds responsible for antimicrobial and antioxidant activity of green propolis and Baccharis dracunculifolia DC.
J Appl Microbiol, 122(4):911-920, 01 Apr 2017
Cited by: 52 articles | PMID: 28066967
Ethnomedicinal, phytochemical and pharmacological investigations of Baccharis dracunculifolia DC. (ASTERACEAE).
Front Pharmacol, 13:1048688, 28 Nov 2022
Cited by: 5 articles | PMID: 36518668 | PMCID: PMC9742423
Review