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African Journal of Pig Farming ISSN: 2375-0731 Vol. 11 (3), pp. 001-010, March, 2023. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 
 
 

 

Full Length Research Paper 

 

In vitro antiviral activity of Brazilian Cerrado plant 
extracts against animal and human herpesviruses 

 
Marina A. Padilla1,2, Isabela C. Simoni1*, Verônica Moreira H. Hoe1, Maria Judite 
B. Fernandes1, Clarice W. Arns2, Juliana R. Brito3 and João Henrique G. Lago4

 
 

1
Centro de Pesquisa e Desenvolvimento de Sanidade Animal, Instituto Biológico, Av. Cons. Rodrigues Alves, 1252, 

CEP 04014-900, São Paulo, SP, Brasil. 
2
Laboratório de Virologia Animal, Universidade Estadual de Campinas, Campinas, SP, Brasil. 

3
Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, São Paulo, Brazil. 

4
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Paulo, Brazil. 

 
Accepted 16 January, 2023  

 
The Brazilian savanna known as “Cerrado” is very rich in medicinal plants that are used by the local 
population for treatment of several illnesses. The herpesvirus is a serious problem worldwide, and 
affects both animal and human health. This work aimed to study the antiviral activity of eight extracts 
from plants natives of “Cerrado” region against human (HSV-1), equine (EqHV-1) and swine 
herpesviruses (SuHV-1). The results showed that all plant extracts: Banisteriopsis variabilis, Byrsonima 
intermedia, Campomanesia xanthocarpa, Erythroxilum deciduum, Lacistema hasslerianum, Ocotea 
pulchella, Stryphodendron adstringens and Xylopia aromatica presented antiviral activity against at 
least one herpesvirus. Furthermore, it was observed a direct anti-herpes effect of extracts from B. 
variabilis and B. intermedia in non-toxic concentrations against all herpesviruses. B. intermedia crude 
aqueous extract showed the most promising results with selective index values of the 41.76 ± 0.04; 4.12  
± 0.1 and 193.97 ± 0.09 respectively against HSV-1, EqHV-1 and SuHV-1. Due to this, B. intermedia 
extract was also analyzed by HPLC/MS allowing for the identification of gallic acid and quercetin as 
main compounds. 

 
Key words: Brazilian savanna, Cerrado, Byrsonima intermedia, antiviral, herpesvirus, gallic acid, quercetin.  

 
 
INTRODUCTION 

 
The herpes viruses are a serious worldwide problem that 
can affect the health of both animals and human beings. 
The herpes simplex virus (HSV-1) is pathogenic to 
humans (Wyler et al., 2017), whereas equine (EqHV-1) 
and swine herpesviruses (SuHV-1), are responsible for 
causing serious diseases in horses and pigs respectively, 

 
 
 
 

 
resulting in large economic losses (Wernike et al., 2013; 
Gulati et al., 2016). These viruses belong to the subfamily 
Alphaherpesvirinae, characterized by a rapid lytic viral 
cycle and the establishment of neuronal latency, which 
can be reactivated (Riaz et al., 2017). The emerging of 
resistant virus strains to available drugs against human  
  

*Corresponding author. E-mail: isabelasimoni@gmail.com, simoni@biologico.sp.gov.br. 

 
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons 

Attribution License 4.0 International License 



2 

 

 
 
 

 

herpesvirus is a growing problem, particularly in 
immunocompromised patients (Vadlapudi et al., 2013). 
More recently, outbreaks of equine herpesvirus have had 
an impact on the equine industry, and stimulated interest 
in antiherpetic interventions (Vissani et al., 2016). The 
SuHV-1, also known as pseudorabies virus, is the 
causative agent of Aujeszky‟s disease affecting other 
mammals besides pigs, is still present in wild boar and 
there are no antiviral drugs approved for veterinary use 
(Zouharova et al., 2016). Therefore, it is necessary to find 
new alternative drugs against these diseases that affect 
both humans and animals (Martinez et al., 2015; Nocchi 
et al., 2016). Medicinal plants are natural resources, 
leading to valuable herbal products often used to treat a 
large number of various diseases in traditional medicine 
(Katiyar et al., 2012; Wachtel-Galor and Benzie, 2011).  

The South American flora represents one of the world‟s 
richest sources of material with pharmacological activity, 
and Brazil has the biggest vegetal diversity (Dutra et al., 
2016). One of them is the “Cerrado” (Brazilian Savannah) 

covering more than 2 million km
2
 and land representing 

25% of the area of the country (Sawyer, 2016). This 
vegetation consists of more than 6,000 vegetal species, 
having many compounds such as saponins, tannis, 
steroids, and others (Bessa et al., 2013), with biological 
activities including antifungal, antibacterial, antiprotozoal 
and antiviral ones (Alves et al., 2000; Mesquita et al., 
2005, 2007; Silva Junior et al., 2009; Brandão et al., 
2010).  

Many plant extracts have been described for antiviral 
activity against herpesviruses in vitro such as 
Conocephalum conicum, Polypodium glycyrrhiza and the 
water-soluble extract from the narcissus bulb Flos 
verbasci, showed anti-HSV activity. The aqueous extracts 
of Helichrysum aureonitens (Asteraceae) shoots inhibited 
HSV-1 (Chattopadhyay and Khan, 2008). Compounds in 
green tea extract could also inhibit HSV-1 and black tea 
extract enriched with theaflavins has the potential to 
prevent the spread of HSV-1 (Cantatore et al., 2013). 
Moreover, Lithraea molleiodes, Sebastiana brasiliensis 
and Sebastiania klotzschiana used against infectious 
diseases showed antiherpetic activity with no cytotoxicity, 
as did the aqueous extract of Beta vulgaris (Betancour-
Galvis et al., 1999). The aqueous extracts from B. 
vulgaris and S. brasiliensis also exhibited antiviral activity 
against swine herpesvirus (Koseki et al., 1990; Simoni et 
al., 2014) and Cecropia pachystachya, Melochia villosa 
and P. acuminatum presented the most relevant results 
against bovine and swine herpesviruses (Simoni et al., 
2014).  

Simoni et al. (2007) reported on the in vitro research for 
potential antiviral properties of the 16 species collected 
from the Brazilian Cerrado against bovine herpesvirus 
(BoHV-1), infectious bursal disease virus (IBDV) and 
avian reovirus. They found 8 plant extracts active against 
BoHV-1 including Byrsonima intermedia, Banisteriopsis 
variabilis, Stryphodendron adstringens and Xylopia 

  
  

 
 

 

aromatica.  
Species of Banisteriopsis are described as having 

antioxidant and antibacterial properties, while also 
inhibiting the activity of monoamine oxidases. 
Banisteriopsis caapi is the most studied species of this 
genus because it is used by some religious groups in 
Brazil as an ingredient of the drink locally known as 
ayahuasca. Chemical compounds identified in this plant 
species include alkaloids, harmine and harmaline (Wang 
et al., 2010; Santos et al., 2017). B. intermedia belonging 
to the Malpighiaceae family popularly known as “murici” 
has been used in folk medicine to treat fever, as diuretic, 
for skin infections and ulcers. B. intermedia is described 
mainly as being antimicrobial and as anti-inflammatory 
(Moreira et al., 2011; Sannomiya et. al., 2007). S. 
adstringens is one of the most widely used medicinal 
species by the Brazilian population. The stem bark is 
usually used to treat leucorrhea, gonorrhea, vulvo-vaginal 
candidiasis, gastritis, sore throat, diarrhea and bleeding 
(Costa et al., 2010). Extracts from the leaves present 
trypanocidal activity associated with the chemical 
compound tannin (Ishida et al., 2009). Extracts from X. 
aromatica have been described for antibacterial, 
antifungal, insecticidal, antimicrobial, and antiparasitic 
activities. Many compounds in this genus have been 
identified, but the triterpenoids are present in higher 
concentrations (Stashenko et al., 2004; Costa et al., 
2013).  

The aim of this work was continue the search with 
these same eight positive plant extracts to evaluate the 
antiviral activity against equine, swine and human 
herpesviruses together with additional studies of antiviral 
and virucidal potential. Additionally, the phytochemical 
profile of the extract from B. intermedia, which displayed 
higher antiviral activity, was performed by HPLC/MS 
analysis. 
 
 
MATERIALS AND METHODS 
 
Plant materials 
 
The “Cerrado” plant species B. variabilis B. Gates, B. intermedia A. 
Juss., Campomanesia xanthocarpa O. Berg, Erythroxylum 
deciduum A. St.-Hil., Lacistema hasslerianum Chodat, Ocotea 
pulchella (Nees) Mez, S. adstringens (Mart.) Coville, and X. 
aromatica (Lam.) Mart were collected in Mogi-Guaçu (22º18‟S and 
47º20‟W), São Paulo State, Brazil. The specimens were 
authenticated by a taxonomist in the Herbarium of the Instituto de 
Botânica, São Paulo, Dr. Eduardo Luis Martins Catharino. 

 

Preparation of crude extracts 
 
Crude aqueous extracts were prepared by grinding dried leaves 
with de-ionized distilled water (10%, w/v) in a mixer and 
maintenance at 4ºC overnight. The aqueous extracts were filtered 
on Whatman-1 filter paper and freeze-dried in Flex-dry MP FTS 
systems. Lyophilized extracts were dissolved in equal parts of 
sterile de-ionized distilled water and Eagle minimum essential 
medium (MEM 2x) at a final concentration of 10,000, 4,000 or 2,000 



3 

 

 
 
 

 
µg/mL. The extracts were centrifuged at 2,500 g/10 min in a Hettich 
Zentrifugen model Rotina R and sterilized by filtration (0.22 µm 
filter). 

 

HPLC/MS analysis 
 
Crude aqueous extract from B. intermedia was filtered on a Sep-
Pak column using MeOH as eluent. Samples containing 1 ml of the 
crude extract was analyzed by HPLC using a Luna C-18 
(Phenomenex) column (5 mm, 250 X 4 mm), with a gradient from 
MeOH:H2O:HCCOH 1% 75:25 (0 min) to MeOH 100% (30 min), 
flow rate 1.0 ml/min and detection at 254 nm. HPLC/MS (negative 
form) were acquired on a Bruker micrOTOF-QII coupled to an 
Apollo ion source set as follows: dry temperature at 180°C and 
voltage at 4.5 kV. The mass/charge ratios were detected in scan 
(m/z 100–1200 Da) and product ion scan (m/z 50–1200 Da) modes, 
using the same chromatographic method described above. 

 

Cell cultures and viruses 
 
The equine herpesvirus 1 (EHV-1), strain A4/72 (Moreira et al., 
1998) and the herpes simplex virus 1 (HSV-1), strain KOS (Silva et 
al., 2010) were propagated in Vero (African Green monkey – ATCC 
CCL 81). The swine herpesvirus 1 (SuHV-1) strain NP (Nova Prata) 
(Fonseca et al., 2010) was propagated in MDBK (Mardin and Darby 
Bovine Kidney – ATCC CCL 22). All cells were grown in MEM with 
10% fetal bovine serum (FBS) (Simoni et al, 2007). 

 

Cytotoxicity assay 
 
The assays were performed using 96- well microtiter plates with 
30,000 cell/well. After 24 h of incubation at 37ºC in a humidified 5% 
CO2 atmosphere, each cell type was exposed to decreasing 
concentrations of plant extract in triplicate. Any cell morphology 
alteration was observed at light microscopy during the next 3 days 
to determine the maximum non-cytotoxic concentration (MNCC). 
Monolayers of cells incubated only with MEM were used as a 
control. 

 

Antiviral assay 
 
For the assays, MDBK or Vero cells were prepared in 96-well 

microplates at density of 3 × 10
4
 cells per well and incubated for 24 

h at 37ºC with 5% CO2. After 24 h of incubation, the medium was 
poured off and 100 µL of extracts at dilutions corresponding at 
MNCC were added. The cells were incubated for 1 h and after this 
period, 50 µL of logarithmic dilutions of viruses were inoculated for 
96 h. MDBK cells were infected with SuHV-1 and Vero cells were 
infected with EHV-1 or HSV-1. Controls consisted of untreated 
infected (virus titer), treated non-infected (extract control), untreated 
non-infected (cell control) cells. In addition, to verify if the antiviral 
activity of the extract remained below its MNCC, a dose-response 
experiment was carried out using it at range of 250 until 1.9 µg/mL. 

 

Direct anti-herpes assay 
 
The assay was described previously by Simoni et al. (1996). Briefly, 
100 µL of each extract at MNCC was mixed with 100 µL of 
logarithmic dilutions of each virus, incubated at 37°C in 5% CO2 for 
1 h. Then, 50 µL of mixture was inoculated at the monolayers in 
each well of a 96-well plate. In addition, to verify if the direct anti-
herpes effect of the extract remained below its MNCC, a dose-
response experiment was carried out using it at range of 250 until 

 
 
 
 

 
1.9 µg/mL. 

 

MTT assay 
 
The MTT assay was performed to confirm qualitatively the MNCC 
and the antiviral activity of the extracts that presented PI% more 
than 97%. Cells in a 96-well microplate were incubated with 100 
μL/well of increasing concentrations of crude extracts in 
quadruplicate with at least 5 different concentrations. Thereafter, 
100 TCID of virus was added. After incubation for 72 h, the medium 
was removed and added on cell monolayer 50 µL of MTT (1 
mg/mL) to each well. The microplate was then incubated at 37ºC for 
4 h. The supernatant was then removed from each well without 
disturbing the cell clusters containing formazan crystals. To 
solubilize the formazan crystal, 100 µL of SDS was added and the 
microplate was incubated again overnight. The absorbance of the 
wells was read in a computer-controlled microplate reader (Spectra 
Max Plus 384) at 540 nm wavelengths. The 50% cytotoxic 
concentration (CC50) of the test compound was defined as the 
concentration that reduced the absorbance of mock-infected cells 
by 50% of that of the control. The 50% antiviral effective 
concentration (IC50) was expressed as the concentration that 
achieved 50% protection of virus- infected cells from the HSV-1, 
EHV-1 and SuHV-1 induced destruction (Takeuchi et al., 1991). 

 

Data and statistical analysis 
 
The antiviral and virucidal activities were based on reduction of viral 
titers using CPE criteria. Values were expressed as titer (TCID 50 
µL) (Reed and Muench, 1938) and viral inhibition index (VII) 
calculated as the difference of virus titer between treated and 
untreated infected control cultures. The VII was considered positive 
when ≥ 1.5 (Barros et al., 2012).  

The 50% cytotoxic (CC50) and 50% inhibition (IC50) 
concentrations were calculated from concentration-effect curves 
obtained from nonlinear regression analysis of concentration-effect 
curves by the GraphPad Prism 5 Demo program. The results were 
obtained from triplicate independent assays. The percentage of 
cytotoxicity was calculated as [(A – B)/A] × 100, where A and B are 
the OD540 of untreated and of treated cells, respectively. The 
percentages of protection were calculated as [(A − B) × 100/(C − 
B)], where A, B and C indicate the absorbance of the 
extracts/fractions, virus and cell controls, respectively. Each 

obtained EC50 value was defined as the effective concentration that 
reduced the absorbance of infected cells to 50% when compared 
with cell and virus controls. The selectivity index (SI) was 

determined by the ratio of CC50 to EC50 and expressed as mean ± 
s.e.m. The statistically different effects of tested extracts on the 
inhibition of virus replication were compared with the control group 
using the Student‟s t-test with p≤0.05 for significant result. 
 

 

RESULTS 

 

Cytotoxicity 

 

The MNCCs of the extracts were determined in two 
different cell lines, MDBK and Vero and the 
concentrations used in bioassays are presented in Table  
1. In Vero cells, the CC50 was lower than that one 
observed in MDBK cells. X. aromatica presented the 
lowest cytotoxic to Vero and MDBK cells with MNCC of 
1250 and 625 µg/mL, respectively. The extract from O. 
pulchella was the most cytotoxic for both cell lines with 



4 

 

         

 Table 1. Antiviral activity of extracts at MNCC and CC50 against HSV-1, EHV-1 and SuHV-1.     
         

 Plant extract MNCC
a
 Vero CC50

b
 HSV-1

c
 EHV-1 MNCC MDBK CC50 SuHV-1 

 Banisteriopsis variabilis 625 435.8 2.25 3.75 312 >2,000  3.24 

 Byrsonima intermedia 31.2 141.1 3.25 4.87 250 782.1  5.45 

 Campomanesia xanthocarpa 62.5 n.t 0 0.62 250 1,299  2.0 

 Erythroxilum deciduum 125 n.t. 1.0 0.26 31.2 n.t.  1.16 

 Lacistema hasslerianum 62.5 n.t. 0.5 0.26 62.5 821  2.0 

 Ocotea pulchella 31.2 n.t. 0 0.26 31.2 290.9  1.76 

 Stryphodendron adstringens 625 n.t. 2.75 1.0 62.5 >2,000  2.0 

 Xylopia aromatica 1250 203.1 1.5 1.76 625 >2000  3.24 
 

a: MNCC: Maximum non-cytotoxic concentration; HSV-1: Human Herpesvirus type 1 SuHV-1: Swine Herpesvirus type 1; EHV-1: Equid 

Herpesvirus type 1; b: 50% cytotoxic concentration (μg/mL); c: VII: Viral inhibition index; n.t.: CC50 not tested. 

 
 

 
Table 2. Direct anti-herpes effect of extracts at MNCC against herpes simplex 
virus, equine herpesvirus and swine herpesvirus in cell cultures. 

 

 
Plant extract 

VII
a
 VII VII 

 

 

HSV-1
b
 EHV-1

c
 SuHV-1

d
 

 

  
 

 Banisteriopsis variabilis 3.0 3.67 3.34 
 

 Byrsonima intermedia 4.5 4.0 5.22 
 

 Stryphodendron adstringens n.t. n.t. 0.75 
 

 Xylopia aromatica 1.5 0.84 1.67 
 

 
a: VII: Viral inhibition index; b: Human Herpesvirus type 1; c: Equid Herpesvirus type 
1; d: Swine Herpesvirus type 1; n.t.: Not tested. MNCC: Maximum non-cytotoxic 
concentration. 

 
 

 

MNCC of 31.2 µg/mL. The other extracts showed MNCC 
ranging between these values for both cells. 

 
 
 

 

for SuHV-1and HSV-1, while the extracts from S. 

adstringens had action only on HSV-1. 
 

 

Antiviral activity 

 

Results are presented in Table 1. The extracts from B. 
variabilis, B. intermedia, S. adstringens and X. aromatica 
presented VII greater than or equal to 1.5 for HSV-1. The 
extracts of B. variabilis, B. intermedia and X. aromatica 
were positive for EHV-1 while the extracts B. variabilis, B. 
intermedia, C. xanthocarpa, L. hasslerianum, O. 
pulchella, S. adstringens and X. aromatica were effective 
for SuHV-1. 
 

 

Direct anti-herpes effect 

 

Assays to study the direct anti-herpes effect were done 
only with the extracts that were effective for at least two 
of the viruses tested in the antiviral assays. These results 
are presented in Table 2.  

The extracts of B. variabilis and B. intermedia, 
presented an inhibition against the three viruses with VII 
greater or equal to 3.0. X. aromatica was only effective 

 
 

Additional studies with Byrsonima intermedia 

 

B. intermedia was selected for additional studies and to 
characterize its antiviral properties because it showed the 
highest values of VII against all herpesviruses.  

Table 3 shows the results of the cytotoxic, antiviral 
activities and selectivity index (SI) obtained from B. 
intermedia extracts. The MNCC of B. intermedia extracts 
that did not cause alterations in the morphology of the 
cells visible under the optical microscope (Table 2) were 

lower than that obtained with CC50 of 141.1 for Vero cells 
and 782.1 for MDBK cells. B. intermedia presented SI 
values of 41.76 ± 0.04; 4.12 ± 0.1 and 193.97 ± 0.09 
respectively against HSV-1, EqHV-1 and SuHV-1.  

Figure 1 shows the antiviral activity of extract from B. 
intermedia at 8 set concentrations against the three 
viruses showing that treatment with B. intermedia extract 
resulted in reduced viral titers in dose-response curves. 
At 15.6 µg/mL, the initial reduction occurred firstly with 
SuHV-1 and then with EHV-1 and HSV-1 at 62.5 µg/mL. 
At 250 µg/mL, the greater VII was also obtained with 



5 

 

           
 

  Table  3.  Selectivity  index,  cytotoxic  and  antiviral  activities  from  B.  intermedia  aqueous  extract  against  swine 
 

  herpesvirus, equine herpesvirus and herpes simplex virus by the MTT assay.    
 

           
 

  
Plant extract 

Vero HSV-1 
SI

c
 

EqHV-1 
SI 

MDBK  SuHV-1 
 

  CC50
a
 IC50

b
 IC50 CC50 IC50 SI  

     
 

  Byrsonima intermedia 141.1 3.37 41.76 ± 0.04 34.24 4.12 ± 0.1 782.1 4.03 193.97 ± 0.09 
  

a: 50% cytotoxic concentration (μg/mL); b: 50% inhibitory concentration (μg/mL); c: SI - Seletivity Index - ratio of CC50 to IC50. 

SuHV-1: Swine Herpesvirus type 1; EHV-1: Equid Herpesvirus type 1; HSV-1: Human Herpesvirus type 1.  
 
 

 

7        

6        

5        

4        
VII        

3        

2        

1        

0        
1.9 3.9 7.8 15.6 31.2 62.5 125 250  

 
 
 
 
 
 
 
 
 

 
 SuHV-1 

 
 EqHV-1  

 
HSV-1 

 

 
Concentration (µg/mL) 

 
Figure 1. Antiviral activity of different concentration of extract from B. intermedia against Swine Herpesvirus type 1 (SuHV-1); Equine 
Herpesvirus type 1 (EHV-1); Human Herpesvirus type 1 (HSV-1); VII: Viral Inhibition Index. 

 
 

 

SuHV-1 followed by EHV-1 and HSV.  
Likewise, Figure 2 shows the direct anti-herpes effect 

against SuHV-1, EHV-1 and HSV-1 showing that 
treatment of virions with this various concentrations of 
extracts for one hour resulted in reduced viral titers. The 
extracts were active at the concentrations range 250 to 
31.2 µg/mL. At 3.9 µg/mL, the reduction occurred firstly 
with SuHV-1 and then with EHV-1 and HSV-1 at 7.8 
µg/mL. At 250 µg/mL, the greater VII was obtained for 
SuHV-1 and EHV-1 followed by HSV. 
 

 

Phytochemical profile of Byrsonima intermedia 

 
As follow-up, the B. intermedia extract was subjected in 
the current study to HPLC/MS analysis (Figure 3). The 

peak at Rt = 10.2 min corresponds to a [M – H]
-
 quasi-

molecular ion peak at m/z 169, suggesting the 
occurrence of gallic acid (A). The second peak, detected 

at Rt = 13.6 min corresponds to a [M – H]
-
 quasi-

molecular ion peak at m/z 301, suggesting the presence 
of flavonoid quercetin (B). 

 
 
 

 

DISCUSSION 

 

Cytotoxicity 

 

The cytotoxic assay at the first stage of screening is very 
important to determine the maximum concentration of the 
aqueous extract that did not induce changes in cell 
morphology. This step is critical because the virus is an 
intracellular parasite and uses the cell machinery to 
replicate, so it must be ensured that the virus have the 
ideal conditions for their growth (Cos et al., 2006).  

The study of cytotoxicity in different cell lines is 
important because one of the inherent drawbacks of in 
vitro antiviral testing is the environmental sensitivity of 
animal cells in culture, although the in vitro methodology 
is faster and less costly (McCutcheon et al., 1995). 
 

 

Antiviral activity 

 

Brazilian Cerrado plants may lead to the study of a broad 
source of new natural compounds; however, most are 



6 

 

   
 
 

 

 7        

 6        

 5        

VII 4        

 3        

 2        

 1        

 0        
 1.9 3.9 7.8 15.6 31.2 62.5 125 250  

 
 
 
 
 
 
 
 
 
 

HSV-1  
 

 SuHV-1 
 

 EqHV-1 

 

 
Concentration (µg/mL) 

 
Figure 2. Direct anti-herpes effect of different concentrations of extract from B. intermedia against Swine Herpesvirus type 1 (SuHV-1); 
Equine Herpesvirus type 1 (EHV-1); Human Herpesvirus type 1 (HSV-1); VII: Viral Inhibition Index. 

 
 

 

being developed for use as antibiotics more than antiviral 
drugs. This is because the viruses present certain 
characteristics among others of replicating within the cell 
and persisting in the host. Their combat must be more 
rational by the choice of species of plants that have 
already been used by the population to treat the 
diseases.  

Many Brazilian Cerrado species have already been 
described as having antiviral activity. Chattopadhyay et 
al. (2015) showed that Byrsonima verbascifolia had 
strong anti-HSV activities, whereas Maldini et al. (2011) 
found that B. crassifolia used as anti-inflammatory has 
been described as having compounds that also display 
antiviral activity against HSV-1 and HIV.  

In the present study, eight extracts reduced at least one 
of the viral titers, as compared to untreated control cells. 
B. variabilis, B. intermedia and X. aromatica species were 
active against all these herpesviruses. Furthermore, a 
strong inhibition was obtained from B. variabilis and B. 
intermedia. S. adstringens extract also showed an 
effective inhibition against SuHV-1 and HSV-1. Extract 
from X. aromatica presented the highest values of VII 
against SuHV-1. Antiviral activity was also observed for 
extracts from C. xanthocarpa and O. pulchella only 
against SuHV-1.  

Frias et al. (2012) isolated from metanolic extract of B. 
variabilis, the flavonoids quercetin, rutin, and apigenin. 
Quercetin is found in a wide variety of plants and studies 
showed that it could reduce infectivity of target cells and 
replication against herpesviruses. The results of our work 
indicated that the extracts of B. variablis showed 
inhibitory effect against the three herpesviruses and 
probably this inhibitory effect is due to presence of 

 
 
 

 

these flavonoids.  
Felipe et al. (2006) described the inhibition BoHV-1 

replication of aqueous extracts from S. adstringens stem 
bark. Simoni et al. (2007) showed that the leaves 
presented moderate activity against BoHV-1. In the 
present work, leaves extracts from S. adstringens also 
presented moderate activity against EHV-1 but strong 
inhibition against HSV-1 and SuHV-1.  

Antiviral drugs have been reported for herpetic 
infections for both human and animal herpesviruses. 
Although the regular use of vaccines helps in 
improvement of animal herds and avoids viral shedding, 
treatment of herpesvirus infections is not completely 
prevented by immunization (Gulati et al., 2016; Maxwell, 
2017). Thus, antiviral agents can be useful not only as 
preventives but also as therapeutic in herpesvirus 
infections which leads to a great interest in discovering 
new effective and safe drugs. The results of this present 
work suggest the importance of searching for new 
compounds for veterinary and human medicine in 
Brazilian Cerrado plants because they have shown 
potential for developing new antivirals.  

Several plant extracts with promising results have been 
described for their antiviral potential against diverse 
herpes viruses. For example, Cardamine angulata, 
Conocephalum conicum and Polypodium glycyrrhiza 
showed activity against bovine herpesvirus and HSV-1 
(McCutcheon et al., 1995). Aqueous extract from 
Guettarda angelica seeds exhibited strong inhibition for 
three animal herpesviruses including bovine, swine and 
equine herpesviruses 1 (Barros et al., 2012).  

The present results were promising especially for B. 
intermedia and indicating that materials derived from their 



7 

 

  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. HPLC analysis of crude aqueous extract from B. intermedia leaves and identified 
compounds gallic acid (A) and quercetin (B). 

 
 

 

leaves exhibited antiviral activity and selectivity toward 
three herpesviruses HSV-1, EqHV-1 and SuHV-1. In 
2007, Simoni et al. (2007) described the antiviral activity 
of the B. intermedia against bovine herpesvirus (BoHV-1) 
and against avian reovirus indicating a broad spectrum of 
action of this plant species. 
 

 

Direct anti-herpes effect 

 

To verify if the mechanism of action of extracts is also 
due to inhibition on the viruses in extracellular conditions, 
the selected extracts showing antiviral activity were 
studied.  

Many plants may exhibit different mechanisms of viral 
inhibition acting on more than one target during viral 
replication. The Opuntia streptacantha inhibited virus 
replication and inactivated extracellular virus, such as 
HSV, equine herpes virus, pseudorabies virus (Jassim 

 
 
 

 

and Naji, 2003). Bidens pilosa extract was effective 
against HSV-1 and HSV-2 with potent virucidal activity 
(Vadlapudi et al., 2013). Virucidal activity has also been 
described in crude hydroethanolic extract from the stem 
bark of S. terebinthifolia and was effective against HVS-1 
in the attachment and penetration stages (Nocchi et al., 
2016). Moreover, hot water extract of B. pilosa showed 
potent inhibitory activity against HSV-1 and HSV-2 in 
Vero cells. The authors suggest multiple targets of activity 
because this extract showed virucidal activity to block the 
binding of virus to host cells and viral cell penetration. 
The B. pilosa extract also inhibits virus adsorption to cells 
and affects some intracellular steps of viral replication 
(Nakama et al., 2012). These findings are in agreement 
with those observed in this study. 

 

Additional studies with Byrsonima intermedia 
 
The extracts from B. intermedia  were  tested  in  their 



8 

 

 
 
 

 

maximum non-toxic concentrations in which no alteration 

of normal cell morphology was observed, and the CC50 

for both MDBK and Vero cells was obtained. Therefore, 
the extract showed no toxicity even when used at high 
concentrations in the reduction of viral titers assay and 
direct anti-herpes assay. Further, B. intermedia extracts 
showed anti-herpes activity in a concentration-dependent 
manner in both assays, suggesting that this effect may 
have occurred either acting directly on the viral particle or 
in other stages of the replicative cycle.  

Additional studies should be done to study the 
mechanism of action of extracts of B. intermedia against 
those herpesviruses indicating at which stage the extract 
has activity against viruses. 
 

 

Phytochemical profile 

 

Using dereplication procedures by HPLC/MS, the 
occurrence of gallic acid and quercetin in the active 
extracts from B. intermedia was identified. As previously 
reported, these compounds were detected in the MeOH 
leaves extract from B. intermedia which displayed 
mutagenic activity (Sannomiya et al., 2007).  

Gallic acid is a phenolic compound present in many 
plants, fruits and vegetables. Studies have demonstrated 
that gallic acid exhibits a strong inhibitory effect against 
several viruses such as the herpes simplex virus, hepatite 
C virus, HIV and enterovirus 71. Therefore, the potential 
activity observed by GA suggests that this activity is due 
to the hydrophobic interaction between the functional 
group, (hydroxyl) and virion components of enveloped 
herpesvirus. In addition, gallic acid showed an inhibitory 
effect in a time-dependent manner, notably similar to the 
current study (Govea‑ Salas et al., 2016). Quercetin, a 
flavonoid found in several plant species, including fruits 
and edible vegetables, displayed several biological 
potentials (Sharma et al., 2018), including antiviral 
against HSV-1 (Garrett et al., 2012). Additionally, Choi et 
al. (2009) described that quercetin-3-rhamnoside from 
Houttynia cordata extract inhibit influenza A replication in 
the initial stage of virus infection by indirect interaction 
with virus particles.  

New studies are needed to bring the active substances 
present in B. intermedia extract and to identify in detail 
the ways in which they act. The other extracts that can be 
considered promising must also pass through the 
processes of fractionation and isolation of substances in 
order to verify the potential of this activity. All of them 
here have shown their potential to lead to the 
development of an alternative therapy for the treatment of 
such important infections worldwide. 

 

Conclusion 
 
This study has demonstrated a broad antiviral activity of 
Brazilian Cerrado plants and indicated that they can be 

  
  

 
 

 

effective potential candidates for the development of new 
strategies to treat viral infections. B. intermedia was 
chosen for further research as well as gallic acid and 
quercetin all necessary to elucidate the mechanism of 
action of the extract or compounds both on herpesviruses 
replication and on direct antiviral effect. 
 

 

CONFLICT OF INTERESTS 

 

The authors have not declared any conflict of interests. 
 

 

ACKNOWLEDGEMENTS 

 

The authors are thankful to the Enrica G. D‟Alessandro 
for reviewing English. 
 

 
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