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1
IVENS CAMARGO FILHO
Atividade Antiviral e Modo de Ação de um Peptídeo Isolado de
Sorghum bicolor (L.)
MARINGÁ 2005
UNIVERSIDADE ESTADUAL DE MARINGÁ
DEPARTAMENTO DE FARMÁCIA E FARMACOLOGIA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS
Livros Grátis
http://www.livrosgratis.com.br
Milhares de livros grátis para download.
2
UNIVERSIDADE ESTADUAL DE MARINGÁ
DEPARTAMENTO DE FARMÁCIA E FARMACOLOGIA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS
IVENS CAMARGO FILHO
Atividade Antiviral e Modo de Ação de um Peptídeo Isolado de
Sorghum bicolor (L.)
Dissertação apresentada ao Programa de Pós-Graduação
em Ciências Farmacêuticas da Universidade Estadual de
Maringá como requisito parcial para obtenção do título
de Mestre em Ciências Farmacêuticas.
Orientador: Prof. Dr. Benedito Prado Dias Filho
Co-orientador: Prof. Dra. Tânia Ueda Nakamura
MARINGÁ 2005
3
IVENS CAMARGO FILHO
Atividade Antiviral e Modo de Ação de um Peptídeo Isolado de
Sorghum bicolor (L.)
Dissertação apresentada ao Programa de Pós-Graduação em Ciências Farmacêuticas da Universidade Estadual de Maringá como requisito parcial para obtenção do título de Mestre em Ciências Farmacêuticas.
Aprovada em 16 de novembro de 2005
BANCA EXAMINADORA
Prof. Dr. Benedito Prado Dias Filho
Universidade Estadual de Maringá – UEM
Prof. Dr. Diógenes Aparício Garcia Cortez
Universidade Estadual de Maringá – UEM
Profª Drª. Jacinta Sanchez Pelayo
Universidade Estadual de Londrina – UEL
4
Este estudo foi desenvolvido no laboratório de Microbiologia Básica
Aplicada a Produtos Naturais e Sintéticos do Departamento de Análises Clínicas,
no laboratório de Farmacognosia do Departamento de Farmácia e Farmacologia,
no laboratório de Organização Funcional do Núcleo, Departamento de Biologia
Celular.
5
Aos meus pais, Ivens Camargo e
Célia Regina, pelo amor, orações
e apoio em todas as etapas de
minha vida.
Ao meu primeiro e único amor,
Priscila, que esteve comigo todos
os momentos mesmo quando
estava longe.
6
AGRADECIMENTOS
Ao Prof. Benedito Prado Dias Filho, pela enorme paciência, compreensão, amizade
e aprendizado que adquiri com a sua orientação.
Ao professor Dr Celso Vataru Nakamura e pelo apoio e incentivo e a Professora
Tânia Ueda Nakamura pela co-orientação principalmente em relação a cultura de
célula.
Aos Professores Lourdes Botelho Garcia, Maria Cristina Bronharo Tognim e
Benício Alves Abreu Filho, pelo incentivo em todos os momentos.
A Marinete Martinez pela amizade, ensinamentos e a colaboração para este
trabalho.
As minhas amigas Marie Eliza Zamberlan da Silva, Kelly Ishida, Denise de
Oliveira Scoaris, Raíssa Pedroso Bocchi, que desde a iniciação científica estiveram
comigo dividindo as alegrias e as dificuldades da pesquisa.
A Adriana Valente Teixeira Volpe pela companhia,
As amigas Heloísa Bressan Gonçalves, Érika Ravazzi Franco, Thelma Onozato,
Cecília Truite, Michele Vendramento, Eliana Harue Endo, Simone Hernandez,
Amanda Bortolucci, Andrea Koroishi, Nilza Bittencourt, Jean Colocite, Rafael
7
Yamamoto, Aline Facchin, Adriana Santos, Paula Gaudino Carvalho, Vanessa Ido,
Patrícia Honda, Patrícia Santos.
Aos colegas de trabalho da Microbiologia Básica: Márcio Guilhermetti, Rosana
Ferreira Carli, Adriana Rossetti Barrivieira, Maria Aparecida Manzotti,
Prisciliana Carvalho, Zelita Rodrigues Souza.
Ao corpo técnico dp Programa de Pós-Graduação em Ciências Farmacêuticas
desta Universidade, em especial a Helena e a Sônia do Departamento de Farmácia
e Farmacologia.
Às instituições financiadoras: Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) e ao Programa de Pós –Graduação em Ciências
Farmacêuticas desta Universidade.
Aos meus irmãos Ivania e Caio Fábio pela amizade e companheirismo, e aos meus
amigos que sempre estiveram comigo.
A DEUS por me capacitar e guiar meus passos.
A todos que de alguma forma contribuíram para a realização deste trabalho.
8
SUMÁRIO
INTRODUÇÃO...................................................................................................................
09
REFERÊNCIAS..................................................................................................................
27
ANEXO
Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor
Abstract...................................................................................................................
.01
1. Introduction...................................................................................................................
03
2. MATERIALS AND
METHODS.................................................................................................05
2.1. Antiviral-Guided
Isolation.......................................................................................05
2.2. Cells and
Viruses.....................................................................................................06
2.3. Inhibition of Virus-Induced Cytophatic
Effect........................................................06
9
2.4. Citotoxicity
Assay....................................................................................................08
2.5. Antibacterial
Activity...............................................................................................08
3. RESULTS AND
DISCUSSION..................................................................................................09
4. References.....................................................................................................................
.14
5. Legends.........................................................................................................................
..29
10
Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor
Ivens Camargo Filhoa, Diógenes Aparício Garcia Cortezb, Tânia Ueda-Nakamurac,
Celso Vataru Nakamurac, Benedito Prado Dias Filhoc*
aPrograma de Pós-graduação em Ciências Farmacêuticas.
bDepartamento de Farmácia e Farmacologia.
cDepartamento de Análises Clínicas, Universidade Estadual de Maringá, Av. Colombo,
5790, 87020-900 Maringá, PR
Corresponding author
Phone: + 55 44 3261 4429. Fax: + 55-44-32614860, e-mail address: [email protected]
11
Abstract
In this paper we described the purification of an antiviral peptide from seeds of
Sorghum bicolor (L.) by a procedure that includes gel filtration, ion exchange, and high-
performance liquid chromatography (HPLC) in a reverse-phase column. Its molecular
weight, determined by chromatographic mobility on the Shim-pack Diol-150 gel
permeation column in HPLC, was found to be of 2 000 Da. The peptide designated 2 kD
peptide inhibited strongly the replication of herpes simplex virus type 1 (HSV-1) dose-
dependently by 40-90% of the control level, after incubations with 20-100 µg/ml of the
peptide, with EC50 of 12.5 µg/ml. The concentration of peptide with 50% cytotoxicity
on Vero cells was higher than 200 µg/ml. Pre-incubation of HSV-1 with various
concentrations of the 2 kD peptide showed dose-dependent cytopathic effects (CPE)
reduction patterns at the concentration ranging from 12.5 to 100 µg/ml. The presence of
2 kD peptide before HSV-1 infections shown moderate inhibition of virus-induced CPE
as compared to during or after infections, with EC50 of 25, 12.5 and 12,5 µg/ml,
respectively. Similar results were observed when 2 kD peptide was assayed against
bovine herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, 2 kD
peptide failed to inhibit polio vaccine virus, a non-enveloped virus (Fig. 10). Hence,
these results being taken together, it is conceivable that 2 kD peptide was able not only
to inhibit the initiation and the spread of infection by it also had a protective effect on
the cells rendering them resistant to virus infection.
Keywords: Sorghum seeds, Chromatographic techniques, Antiviral peptide,
Cytotoxicity.
12
1. Introduction
Currently, 40 antiviral chemotherapeutic agents have been approved for use in the
treatment of individuals infected with a variety of different viruses (De Clercq, 2004).
Most of the approved drug date from the last 5 years, and at least half of them are used
for treatment of human immunodeficiency virus (HIV) infection. The others are used in
the treatment of herpesvirus (e.g. herpes simplex virus, varicella zoster virus and
cytomegalo virus), hepatitis B virus, hepatitis C virus or influenza virus infections. The
majority of the approved antiviral agents are nucleoside analogs which act by inhibiting
viral DNA synthesis (herpesvirus) or viral reverse transcription (HIV).
The emergence of drug-resistant viral strains in individuals who required chronic
therapy for effective clinical management of their infection, the adverse side effects and
the suboptimal pharmacokinetics of the drugs currently available encourage the use of
naturally occurring antiviral proteins and synthetic derivatives with potential promise
for clinical use. For this reason, many investigators have attempted to search for new,
effective and inexpensive antiviral drug from natural sources. It has been reported in
vitro and in vivo activity against selected sexually transmitted (Becker, 1980; Harmsen
et al., 1995; Logu et al., 2000).
In recent years, a large number of antimicrobial proteins have been discovered in
animals, insects, and plants. These molecules, which are either constitutive or inducible,
are recognized as important components of the innate defense system (Boman, 2000).
These proteins are termed antimicrobial because they have an unusually broad spectrum
of activity. This may include an ability to kill or neutralize bacteria, fungi (including
yeast), parasites, and even enveloped viruses such as HIV and the herpes simplex virus.
13
Foregoing studies have reported three proteins of 18, 26 and 30 kDa isolated from
sorghum endosperm, which affected hyphal growth of Fusarium moniliforme (Kumari
and Chandrashekar, 1994; Kumari et al., 1994). The 18 kDa antifungal protein removes
cell wall polysaccharides, while the 26 and 30 kDa protein fraction caused leakage of
cytoplamatic contents. More recently, Mincoff et al, (2005) have reported an antifungal
protein that strongly inhibited the growth of species of Candida.
Our research approach is to discover novel plant-derived natural product as new
lead, which could be developed for the treatment of infectious diseases. In the course of
screening plants for antiviral proteins, we examined the inhibitory effects of a protein
extract of sorghum against HSV-1. Using antiviral-guided fractionation, we have
isolated and characterized an antiviral peptide from seeds of Sorghum bicolor L.
14
2. Materials and methods
2.1. Antiviral-guided isolation
Sorghum seeds were obtained from Embrapa Milho e Sorgo – Sete Lagoas, Minas
Gerais, Brazil. The seeds (200 g) were ground in a coffee mill, and the resulting meal
was homogenized in 1 l buffer (10 mM sodium dibasic phosphate, 15 mM sodium
monobasic phosphate, 100 mM KCl, and 1.5% EDTA) for 2 h at 4°C. The homogenate
was squeezed through cheesecloth and clarified by centrifugation (5 min at 7000 g). A
protein extract was prepared by the addition of a solution of 50% ethanol / 3.3%
trifluoroacetic acid (TFA), followed by stirring for 60 min at 4°C in order to extract the
soluble proteins. The preparation was then centrifuged at 30,000 g for 60 min at 4°C
and the supernatant lyophilized. The dried material was dissolved in 4 - (2 -
hydroxyethyl) - 1 - piperazineethanesulfonic acid (HEPES) buffer (20 mM), and
neutralized with 5 M NaOH before final centrifugation at 30,000 g for 30 min at 4°C;
the result was termed the crude extract (Mincoff et al, 2005). The crude extract was
applied to a Shim-pack DIOL 150 (Shimadzu Co. Tokyo, Japan) column (7.9 mmID x
25 cm) previously equilibrated with 0.2 M sodium sulfate in 0.01 M phosphate buffer,
pH 7.0. The column was eluted with the same buffer at a flow rate of 60 ml/h, and the
elution was monitored at 280 nm. The fractions with antiviral activity were pooled and
loaded onto a Shim-pack PA-DEAE-01 (Shimadzu Co. Tokyo, Japan) anion-exchange
column (8 mm ID x5ml) equilibrated with 14 mM Tris-HCl, pH 8.2 (eluent A). The
column was eluted with eluent B (A + 0.5 M sodium chloride) 60-min linear gradient
from 0-100% B, at a flow rate of 60 ml/h. The elution was monitored at 280 nm. The
active fraction was collected and rechromatographed under the same conditions until a
15
single antiviral activity peak appeared during elution. The single antiviral peak was
applied in a reverse-phase column Microsorb MV 100-5 C-18 (250 mm x 4.6 mm)
equilibrated with 0.1% TFA in water. An elution gradient (0-60% acetonitrile in 0.1%
TFA in water from 0-95 min) was employed to elute the protein. A single peak of
antiviral activity was also applied to a column of Shim-pack DIOL and the molecular
weight was estimated using a least-square plot constructed for a range of proteins of
known molecular weight: bovine serum albumin (66 kD); ovalbumin (45 kD), carbonic
anhydrase (29 kD); Trypsin inhibitor (21 kD); B12 vitamin (1.3 kD).
2.2. Cells and viruses
The herpes simplex virus type 1 (HSV-1) and bovine herpes virus (BHV) and
Poliovirus type 1 (ATCC-VR58) were a gift from Dra. Rosa Elisa Linhares,
Microbiology Department, State University of Londrina. Vero cells, used to measure the
antiviral activity against HSV-1 and BSV, were originally purchased from ATCC. Vero
cells were grown in Dulbecco’s Modified Eagle medium [DMEM (Gibco Grand Island,
NY, USA)] supplemented with 10% foetal calf serum (FCS, Gibco), 100 U/ml
penicillin and 100 µg/ml streptomycin (Gibco). The viruses were titrated by inoculation
of cells with 10-fold dilutions using the endpoint dilution technique.
2.3. Inhibition of virus-induced cytopathic effect
Effect of test samples before virus infection. Confluent Vero cells in 96-well tissue
culture plates (Nunc) were washed with PBS. One hundred microlitres culture medium
containing different concentration of test compound were added to each well and cells
were incubated for 1 h at 37ºC and 5% CO2. After removal of the test compound, the
16
cells were washed with PBS and then infected with 103 TCID80/well of HSV-1. After 1
h incubation the unadsorbed virus was removed, the cell monolayer was washed with
PBS and further incubated in DMEM for 72 h. At that time, medium culture was then
removed, monolayer fixed with 10% trichloroacetic acid for 1 h at 4°C, and
subsequently washed 5 times with deionized water. Microplates were then left to dry at
room temperature for at least 1 h, and then stained for 30 min with 0.4%
sulforhodamine B (SRB) in 1% acetic acid. After this time, microplates were washed 4
times with 1% acetic acid. Bound SRB was solubilised with a 150 µl 10 mM unbuffered
Tris-base solution and the plates were left on a plate shaker for at least 15 min.
Absorbance was read in a 96-well plate reader at 530 nm. The virus-induced CPE of the
tests was expressed as a percentage of the optical density in comparison with the
parallel virus control and cell control. (Papazisis et al.,1997). The concentration that
reduce 50% of CPE in respect to that of virus control was estimated from the plots of
the data and was defined as 50% inhibitory concentration (IC50)
Effect of test samples during the infection. The assay was performed as described
above, with the exception that the test compound was added together with the virus.
After 1 h incubation the solution containing unadsorbed virus was removed, the cell
monolayer was washed with PBS and further incubated in DMEM for 72 h. The virus-
induced CPE of the tests was expressed as described above.
Effect of test samples on infected cells. Confluent Vero cells were washed with PBS
and infected with 103 TCID80/well of HSV-1. After 1 h incubation the unadsorbed virus
was removed, the cell monolayer was washed with PBS and then incubated with
increasing concentration of test samples in DMEM for 72 h. At that time, medium
culture was then removed and assayed as described above.
17
2.4. Cytotoxicity assay
The cytotoxicity assay was carried out, with some modifications, as previously
described (Skehan et al., 1990). Briefly, confluent Vero cell monolayers grown in 96-
well cell culture plates were incubated with a ten-fold serial dilution of the test samples
starting with a concentration of 200 µg/ml – for 48 h at 37°C and 5% CO2. At that time,
cultures fixed with 10% trichloroacetic acid for 1 h at 4°C, washed 5 times with
deionized water. Microplates were then left to dry at room temperature and stained for
30 min with 0.4% sulforhodamine B (SRB) in 1% acetic acid as describes in 2.3. The
cytotoxicity was expressed as a percentage of the optical density of the control.
2.5. Antibacterial activity
The antibacterial activity was determined by microdilution techniques in Mueller-
Hinton broth (Merck) according to NCCLS (2001). Inoculates were prepared in the
same medium at a density adjusted to a 0.5 McFarland turbidity standard [108 colony-
forming units (CFU)/ml] and diluted 1:10 for the broth microdilution procedure.
Microtiter plates were incubated at 37ºC and the MICs were recorded after 24 h of
incubation. Two susceptibility endpoints were recorded for each isolated. The MIC was
defined as the lowest concentration of compounds at which the microorganism tested
did not demonstrate visible growth. Minimal bactericidal concentration (MBC) was
determined by subculturing 10 µl fro each negative well from the positive growth
control. MBC was defined as the lowest concentration yielding negative subcultures or
only one colony.
18
3. Results and discussion
The starting material for the isolation of antiviral peptide from Sorghum bicolor was
the acid-soluble protein extract obtained from the seeds. Bioassay-guided fractionation
of crude protein extract was carried out by chromatographic procedures, whereby the
eluates were monitored by absorbance determination at 280 nm and assayed for antiviral
activity against HSV-1. Upon fractionation by gel filtration on Shim-pack DIOL, the
mixture resolved into three peaks, with the antiviral activity coeluting with the second
peak (Fig. 1). In the second step, the protein fraction as isolated by passage over a
Shim-pack PA-CM/SP cation-exchange column in HPLC (data not shown). The
proteins not retained by the column contained all the antiviral activity and were further
separated in a third step by a anion-exchange chromatography at pH 8.2 on a Shim-pack
PA-DEAE-01 anion-exchange column in HPLC. Elution of the column with a linear
gradient from 0-500 mM sodium chloride yielded three distinct peaks (Fig. 2). The
active fraction was purified in the final step by reverse-phase chromatography on a
Microsorb MV 100-5 C-18 column (Fig. 3). After three cycles of reverse-phase
chromatography, the elution of a single peak of antiviral activity was achieved (data not
shown). A single peak of antiviral activity was then applied to a column of Shim-pack
DIOL. On the basis of the chromatographic mobility of the purified antiviral peptide on
molecular exclusion column in HPLC a molecular weight of 2,000 was estimated using
a least-square plot constructed for a range of proteins of known molecular weight (Fig.
4).
The antiviral activities of crude extract, fractions and the purified peptide (termed 2
kD peptide) against HSV-1 were examined in susceptible cells that were infected with
103 TCID80/well of HSV-1. After incubating at 37ºC for 1 h, the unadsorbed virus was
19
removed, the cell monolayer was washed with PBS and then incubated with increasing
concentration of test samples. Antiviral activity was then determined by inhibition of
virus-induced cytopathic effect and the EC50 are reported in Table 1. It was considered
that if the extract, fractions, or isolated peptide displayed an EC50 less than 15 µg/ml,
the antiviral activity was strong; from 15 to 50 µg/ml the antiviral activity was
moderate, from 50 to 100 µg/ml the antiviral activity was weak, over 100 µg/ml they
were considered inactive. The 2 kD peptide showed strong activity against HSV-1 with
EC50 of 12.5 µg/ml. Before testing their antiviral activity, the cellular toxicity of test
samples was determined. As measured by sulforhodasmine B (SRB) colorimetric assay,
the concentration of 2 kD peptide with 50% cytotoxicity on Vero Cell (CC50) was
higher than >200 µg/ml. Therefore, the selective index (SI) of 2 kD peptide against
HSV-1, calculated by dividing the CC50 by the EC50 was higher than 16 µg/ml.
An antiviral compound could protect cells against virus infection in several ways: by
directly inactivation of the virus or by interfering with the replication cycle. Therefore,
2 kD peptide was tested for its virucidal effect and antiviral activity before, during or
after virus infections by cytopathic effect inhibition assay. Pre-incubation of HSV-1
with various concentrations of the 2 kD peptide showed dose-dependent CPE reduction
patterns at the concentration ranging from 6 to 100 µg/ml (Fig. 5). In attempt to find out
whether 2 kD peptide can be internalized into cells or bound to the cellular membrane to
exert antiviral effects, confluent Vero cells were incubated with different concentrations
of peptide, which were then removed before infection (Fig. 6); different concentrations
of peptide added together with the virus (Fig. 7); infected with the virus and then
incubated with different concentrations of peptide (Fig. 8). The presence of 2 kD
peptide before HSV-1 infections shown moderate inhibition of virus-induced cytopathic
20
effect (EC50= 25µg/ml) as compared to during or after infections (EC50= 12.5 µg/ml)
(Fig. 9).
Under certain conditions, herpetic lesion might be complicated by secondary
bacterial infections. Therefore, it was investigated whether the 2 kD peptide exerts, in
addition to its antiviral effects, a significant antibacterial activity against gram-negative
and gram-positive bacteria (Table 2). The antibacterial activities of acid-soluble crude
extract and purified peptide are reported in Table 1. The 2 kD peptide presented
significant activity on both Staphylococcus aureus and Bacillus subtilis with MIC of
180 µg/ml. It is interesting to note that the acid-soluble protein extract shown good
activity against S. aureus and B. subtilis with MIC of 75 µg/ml. In contrast to the
relative low MIC for gram-positive bacteria, gram-negative bacteria were not inhibited
by both protein extract and 2 kD peptide at concentration ≤ 600 and ≤180 µg/ml,
respectively. This is to be expected because the outer membrane of gram-negative
bacteria is known to present barrier to penetration of numerous antibiotic molecules,
and the periplasmic space contains enzymes which are able of breaking down molecules
introduced from outside.
The use and search for drugs and dietary supplements derived from plants have
accelerated in recent years. Ethnopharmacologists, botanists, microbiologists, and
natural-products chemists are combing the Earth for phytochemical and “leads” which
could be developed for the treatment of infectious diseases. According to this author,
while 25 to 50% of current pharmaceuticals are derived from plants, none are used as
antimicrobial. Plants produce very bioactive molecules that allows to them to interact
with other organisms in their environment. Many of these substances are important in
21
the defense against herbivores and contribute to the resistance to diseases (Cowan
1999). Plants, therefore, can be promising sources of antimicrobial agents.
Recently, Kan et al. (2005) reviewed anti-HSV substances from natural sources,
including both extracts and pure compound from herbal medicines, reported in studies
from several laboratories. The role of traditional medicine for the development of anti-
HSV compounds was also discussed. According to theses author, a large number of
small molecules like phenolics, polyphenols, terpenes, flavonoids, sugar-containing,
were found to be promising anti-herpetic agents.
Several peptide antibiotics (also known as antimicrobial peptides or natural
antibiotics), in particular, defensins, have also been show to display in vitro antiviral
(Bastian and Schafer, 2001, Lehrer, 2004, Matanic et al., 2004). In some cases, the
binding of the peptides to viral glucoproteins (lectin-like behavior) has been implicated
as the potential mechanism of antiviral action. These peptides are among the main
effector molecules in host innate immunity and act on a variety of tumor cells as well as
a broad spectrum of microbes such as bacteria, fungi, protozoa, and enveloped viruses.
Features common to all the peptide antibiotics are small size (12 to 100 amino acid
residues), polycationic charge, and amphipathic structure having associated -helices or
ß-pleated sheets. The currently proposed antimicrobial mechanism of this class of agent
is direct electrostatic interaction with negatively charged microbial cell membranes,
followed by physical disruption (for reviews see Lehrer, 1989; Oren and Shai, 1998
Boman, 1995; 2000).
Herpesviruses are frequently cited as examples of viruses that enter cells by fusion
of the virion envelope with a cell membrane, often the plasma membrane. Several
different cellular molecules can function in HSV entry. HSV primarily uses heparan
22
sulfate for initial attachment, but other glycosaminoglycans, such as dextran or
dermatam sulfate, can substitute in its absence (Deepak and Spear 2001). The essential
gD-binding receptors include a diverse array of molecules including protein members of
immunoglobulin and tumor necrosis factor receptor families, as well as modified forms
of heparan sulfate (Campadelli-Fiume et al., 2000; Spear et al., 2000). Once HSV has
bound to the cell surface, the cellular factors that determine whether it fuses directly or
enters via endocytosis are not known.
The virucidal activity may be caused by the disintegration of the entire HSV
particles, the solubilization of the virus envelope, or the chemical modification,
degradation, or masking of some of the essential envelope proteins (Zhu et al., 2004).
The 2 kD peptide at the concentration of 25 µg/ml could directly inactivate 80% of
HSV-1. Similar results were observed when 2 kD peptide was assayed against bovine
herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, 2 kD peptide
failed to inhibit polio vaccine virus, a non-enveloped virus (Fig. 10). Hence, these
results taken together, it is conceivable that 2 kD peptide was able not only to inhibit the
initiation and the spread of infection by it also had a protective effect on the cells
rendering them resistant to virus infection.
Acknowledgements
This study was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Capacitação e Aperfeiçoamento de Pessoal de Nível Superior,
(Capes), Fundação Araucária, and Programa de Pós-graduação em Ciências
Farmacêuticas da Universidade Estadual de Maringá.
23
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26
Table 1. Antiviral activities of acid-soluble protein extract and fractions on herpes simplex
virus (HSV-1) by inhibition of virus-induced cytopathic effect.
Purification step CC50 (µg/ml) EC50 (µg/ml) SIa
Acid-soluble protein extract 210 72 2.9
DIOL HPLC - - -
DEAE HPLC (first) 255 19 13
DEAE HPLC (second) - - -
C18 HPLC >200 12.5 >16
aSI (Selective index) = CC50/EC50; - not determined
27
Table 2. Antibacterial activity of crude extract and 2 kD peptide isolated from Sorghum
seeds
Bacteria MIC(MBC) µg/ml
Acid-soluble protein extract 2 kD peptide
Gram-positive
Staphylococcus aureus 75(>600) 180(180)
Bacillus subtilis 75(150) 180(>180)
Gram-negative
Escherichia coli >600 >180
Pseudomonas aeruginosa >600 >180
28
Fig. 1
Minutes0 2 4 6 8 10 12 14
mA
U
0
20
40
60
80
100
Active fraction
29
Fig. 2
Minutes
0 1 2 3 4 5 6 7 8 9 10
mA
U
0
5
10
15
20
% N
aCl 0
.5 M
0
5
10
15
20
Active fraction
30
Fig. 3
Minutes0 1 2 3 4 5 6 7 8 9 10
mA
U
0
10
20
30
40
% A
ceto
nitr
ile
0
2
4
6
8
10Active fraction
31
Fig. 4
Minutes0 2 4 6 8 10 12 14
mA
U
0
5
10
15
20
25
30
35
1
10
100
1000
1 1.5 2 2.5
Ve / Vo
Mol
ecu
lar
wei
gh
t (x
100)
Active fraction
32
Fig. 5
C
B
D 0
20
40
60
80
100
120
140
1 10 100
2 kD peptide (µ g/ml)
% C
PE
re
du
ctio
n
A
33
Fig. 6
B
C
D 0
20
40
60
80
100
120
140
1 10 100
2 kD peptide (µg/ml)
% C
PE
re
du
ctio
n
A
34
Fig.7
C
B
D 0
20
40
60
80
100
120
140
1 10 100
2 kD peptide (µg/ml)
% C
PE
re
du
ctio
n
A
35
Fig.8
B
C
D 0
20
40
60
80
100
120
140
1 10 100
2 kD peptide (µg/ml)
% C
PE
re
du
ctio
n
A
36
Fig. 9
0
20
40
60
80
100
1 10 100
2 kD peptide (µg/ml)
CP
E (%
of c
ontro
l)
virucidal
before
during
after
37
Fig. 10
0
20
40
60
80
100
120
0 50 100 150
2 kD peptide (µg/ml)
% C
PE
red
uct
ion
HSV-1
BHV
Poliovirus
38
Legends
Figure 1. (A) HPLC gel filtration on Shim-pack DIOL. The crude extract was applied to
a Shim-pack DIOL column, previously equilibrated with 0.2M sodium sulfate in 0.01M
phosphate buffer, pH 7.0, eluted with the same buffer at a flow rate of 60 ml/h, and 1-ml
fractions were collected.
Figure 2. HPLC ion-exchange resin PA-DEAE. The active fraction from gel filtration
was loaded onto a Shim-pack PA-DEAE-01 anion-exchange column (8mm∅ ml x 5 ml)
equilibrated with eluent A (14 mM Tris-HCl, pH 8.2). The column was eluted with
eluent B (A + 0.5M sodium chloride) 60-min linear gradient from 0 to 100% B, at a
flow rate of 60 ml/h. Two cycles of ion-exchange chromatography of the active fraction
led to the elution of a single peak containing the antiviral activity (data not shown).
Fractions eluting with 10% of 0.5 M NaCl showed antiviral activity. Data correspond to
one representative experiment out of three.
Figure 3. HPLC on reverse-phase resin Microsorb C-18. The fraction with antiviral
activity was applied in a reverse-phase column Microsorb- MV 100-5 C-18 (250 x 4.6)
equilibrated with 0.1% TFA in water. An elution gradient (0-60% acetonitrile in 0.1%
TFA in water from 0-95 min) was employed to elute the protein. Fractions eluting with
5 % of Acetonitrile showed antiviral activity. Data correspond to one representative
experiment out of three.
Figure 4. HPLC gel filtration on Shim-pack DIOL-150. Active fraction from Microsorb
C-18 column was applied to a column of Shim-pack DIOL-150 (7.8mm∅ x 25cm)
previously equilibrated with 0.2M sodium sulfated in 0.01M phosphate buffer, pH 7.0.
The column as eluted with the same buffer at a flow of 60 ml/h, and 1-ml fractions were
collected. [• 2 kD antiviral protein and molecular-weight standards: 1 bovine serum
albumin (66 kD); ovalbumin (45 kD), carbonic anhydrase (29 kD); Trypsin inhibitor(21
39
kD); B12 vitamin (1.3 kD). Data correspond to one representative experiment out of
three.
Figure 5. Direct virucidal effect of 2 kD peptide on HSV-1. Viral suspension was pre-
incubated with different concentration of 2 kD peptide at 37 ºC for 1h. The mixture as
then used to infect Vero cells. The inhibition of viral infectivity was determined by
virus-induced cytopathic effect assay and expressed as % of the control. The results
represent mean values ± S.E. for at least three separate experiments. Magnification of
visual observation of control (B) and treatment (C) =: 200 x. Bar = 100 µm.
Figure 6. The antiviral activity of 2 kD peptide on HSV-1 determined by pre-treatment
of Vero cells with test compound. Different concentrations of 3kD peptide were added
to the cell monolayer and incubated for 1 h at 37ºC before HSV-1 infection. The
antiviral effect was determined by CPE reduction assay and expressed as % of the
control. The results represent mean values ± S.E. for at least three separate experiments.
Magnification of visual observation of control (B) and treatment (C) =: 200 x. Bar =
100 µm.
Figure 7. The antiviral activity of 2 kD peptide on HSV-1 determined by pre-mixing
virus with different concentrations test compound. After 1 h incubation, the solution
containing unadsorbed virus was removed, the cell monolayer was washed with PBS
and further incubated in DMEM for 72 h. The antiviral effect was determined by CPE
reduction assay and expressed as % of the control. The results represent mean values ±
S.E. for at least three separate experiments. Magnification of visual observation of
control (B) and treatment (C) =: 200 x. Bar = 100 µm.
Figure 8. The antiviral activity of 2 kD peptide on HSV-1 determined by treatment of
virus infected cells with test compound. Confluent Vero cells were infected with HSV-
1. After 1 h incubation the unadsorbed virus was removed and the cell monolayer was
40
then incubated with different concentrations of test compound. The antiviral effect was
determined by CPE reduction assay and expressed as % of the control. The results
represent mean values ± S.E. for at least three separate experiments. Magnification of
visual observation of control (B) and treatment (C) =: 200 x. Bar = 100 µm.
Figure 9. Dose response curves of 2 kD peptide on HSV-1 determined by virus-induced
cytopathic effect assay in Vero cells. Legend: virucidal, pre-treatment of virus with test
compound; before, pre-treatment of cells with test compound; during, pre-mixing virus
with test compound; after, treatment of virus infected cells with test compound.
Figure 10. The antiviral activity of 2 kD peptide on HSV-1, BHV, and Poliovirus
determined by pre-mixing virus with different concentrations test compound. After 1 h
incubation, the solution containing unadsorbed virus was removed, the cell monolayer
was washed with PBS and further incubated in DMEM for 72 h. The antiviral effect was
determined by CPE reduction assay and expressed as % of the control. The results
represent mean values ± S.E. for at least three separate experiments.
41
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