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: bpdfilho@uem.br

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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25

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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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