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Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor.

Introduction

Currently, 40 antiviral chemotherapeutic agents have been approved for use in the treatment of individuals infected with a variety of viruses (De Clercq, 2004). Most of the approved drugs 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 herpes virus (e.g., herpes simplex virus, varicella zoster virus, and cytomegalovirus), 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 (herpes virus) or viral reverse transcription (HIV).

The emergence of drug-resistant viral strains in individuals who require chronic therapy for effective clinical management of their infection, adverse side-effects, and the suboptimal pharmacokinetics of the drugs currently available encourage the study 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 drugs from natural sources. Both in vitro and in vivo activity of such compounds against selected sexually transmitted viruses has been reported (Becker, 1980; Harmsen et al., 1995; Logu et al., 2000).

In recent years, many 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.

Three proteins of 18, 26, and 30 kDa have been isolated previously 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 fractions cause leakage of cytoplasmic contents. More recently, Mincoff et al. (2005) reported an antifungal protein that strongly inhibits the growth of species of Candida.

Our research approach is to discover novel plant-derived natural products as new leads, 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 herpes simplex virus type 1 (HSV-1). Using antiviral-guided fractionation, we isolated and characterized an antiviral peptide from seeds of Sorghum bicolor L.

Materials and methods

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 11 buffer (10 mM sodium dibasic phosphate, 15 mM sodium monobasic phosphate, 100 mM KC1, and 1.5% EDTA) for 2 h at 4 [degrees]C. The homogenate was squeezed through cheesecloth and clarified by centrifugation (5 min at 7000g). A protein extract was prepared by addition of a solution of 50% ethanol/3.3% trifluoroacetic acid (TFA), followed by stirring for 60 min at 4 [degrees]C in order to extract the soluble proteins. The preparation was then centrifuged at 30,000g for 60 min at 4 [degrees]C and the supernatant lyophilized. The dried material was dissolved in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (20 mM), and neutralized with 5 M NaOH before final centrifugation at 30,000g for 30 min at 4 [degrees]C; the result was termed the crude extract. The crude extract was applied to a Shim-pack DIOL-150 (Shimadzu Co., Tokyo, Japan) column (7.9 mm ID 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 x 5 ml) equilibrated with 14 mM Tris-HC1, pH 8.2 (eluent A). The column was eluted with eluent B (A + 0.5 M sodium chloride) 60-min linear gradient from 0% to 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 single antiviral activity peak appeared during elution. The single antiviral peak was applied in a reversed-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 to 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), and vitamin B12 (1.3 kD).

Cells and viruses

The HSV-1, bovine herpes virus 1 (BHV-1) and polio vaccine virus were provided by Dr. Rosa Elisa Linhares, Microbiology Department, State University of Londrina. Vero cells, used to measure the antiviral activity against HSV-1 and bovine herpes virus (BHV), were originally purchased from ATCC. Vero cells were grown in Dulbecco's Modified Eagle medium [DMEM (Gibco, Grand Island, NY, USA)] supplemented with 10% fetal calf serum (FCS, Gibco), 100 U/ml penicillin and 100 [micro]g/ml streptomycin (Gibco). The viruses were titrated by inoculation of cells with 10-fold dilutions using the endpoint dilution technique (De Clercq, 1982).

Antiviral assay

Effect of test samples before virus infection

Confluent Vero cells in 96-well tissue culture plates (Nunc) were washed with PBS. One hundred microliters of culture medium containing different concentrations of test compound were added to each well, and the cells were incubated for 1 h at 37 [degrees]C and 5% C[O.sub.2]. After removal of the test compound, the cells were washed with PBS and then infected with [10.sup.3] [TCID.sub.80]/well of HSV-1. After 1 h incubation, the cells were washed with PBS twice to remove any unabsorbed virus, and further incubated in DMEM for 72 h. Next, the culture medium was removed, and the monolayer was fixed with 10% trichloroacetic acid for 1 h at 4 [degrees]C, and subsequently washed 5 times with deionized water. The microplates were 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. Next, the microplates were washed 4 times with 1% acetic acid. Bound SRB was solubilized with a 150 [micro]l 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 cytopathic effect (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 reduced 50% of CPE with respect to that of virus control was estimated from the plots of the data, and was defined as the [EC.sub.50] value. Acyclovir (Sigma, St. Louis) was used as a positive control drug.

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 unabsorbed virus was removed, and 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 [10.sup.3] [TCID.sub.80]/well of HSV-1. After 1 h incubation, the unabsorbed virus was removed, and the cell monolayer was washed with PBS and then incubated with increasing concentrations of test compound in DMEM for 72 h. The culture medium was then removed and assayed as described above.

Direct virucidal effect of 3 kD peptide on HSV-1

Viral suspension was pre-incubated with a different concentration of 2 kD peptide at 37 [degrees]C for 1 h. The mixture was then used to infect Vero cells. The inhibition of viral infectivity was determined by virus-induced CPE assay and expressed as % of control.

Cytotoxicity assay

The cytotoxicity assay was carried out, with some modifications, as described previously (Skehan et al., 1990). Briefly, confluent Vero cell monolayers grown in 96-well cell culture plates were incubated with a 10-fold serial dilution of the test compound, starting with a concentration of 500 [micro]M--for 72 h at 37 [degrees]C and 5% C[O.sub.2]. After 48 h, cultures fixed with 10% trichloroacetic acid for 1 h at 4 [degrees]C were stained for 30 m with 0.4% SRB in 1% acetic acid, and subsequently washed 5 times with deionized water. Bound SRB was solubilized with a 200 [micro]l 10 mM unbuffered Tris-base solution. Absorbance was read in a 96-well plate reader. The dye was removed by four washes with 1% acetic acid. Bound peptide was extracted with 10 mM Tris. The cytotoxicity was expressed as a percentage of the optical density of the control.

Antibacterial assay

The antibacterial activity was determined by the microdilution technique, in Mueller-Hinton broth (Merck) according to NCCLS (2001). The following bacteria were used: Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6623, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 15442. Cultures of these microorganisms were grown in nutrient broth (Difco Laboratories, Detroit, MI) at 37 [degrees]C and maintained on nutrient agar slants at 4 [degrees]C. Inoculates were prepared in this medium at a density adjusted to a 0.5 McFarland turbidity standard [[10.sup.8] colony-forming units/ml] and diluted 1:10. Microtiter plates were incubated at 37 [degrees]C and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. 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 [micro]l from each negative well from the positive growth control. MBC was defined as the lowest concentration yielding negative subcultures or only one colony.

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 the crude protein extract was carried out by chromatographic procedures, in which 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. 1A). In the second step, the protein fraction was isolated by passage over a Shim-pack PA-CM/SP cation-exchange column in high-performance liquid chromatography (HPLC) (data not shown). The proteins not retained by the column contained all the antiviral activity. In a third step, they were further separated by 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 to 500 mM sodium chloride yielded three distinct peaks (Fig. 1B). In the final step, the active fraction was purified by reversed-phase chromatography on a Microsorb MV 100-5 C-18 column (Fig. 1C). After three cycles of reversed-phase chromatography, elution of a single peak of antiviral activity was achieved (data not shown). This 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 a molecular exclusion column in HPLC (Fig. 2A), a molecular weight of 2000 was estimated using a least-square plot constructed for a range of proteins of known molecular weight (Fig. 2B).

[FIGURE 1 OMITTED]

The antiviral activities of the crude extract, the fractions, and the purified peptide (termed 2 kD peptide) against HSV-1 were examined in susceptible cells that were infected with [10.sup.3] [TCID.sub.80]/well of HSV-1. After incubating at 37 [degrees]C for 1 h, the unabsorbed virus was removed, and the cell monolayer was washed with PBS and then incubated with increasing concentrations of test samples. Antiviral activity was then determined by inhibition of the virus-induced CPE; the [EC.sub.50] values are reported in Table 1. We considered that if the extract, fractions, or isolated peptide displayed an [EC.sub.50] value less than 10 [micro]M, the antiviral activity was strong; from 10 to 50 [micro]M the antiviral activity was moderate; antiviral activity was weak from 50 to 100 [micro]M; and at over 100 [micro]M, was considered inactive. The 2 kD peptide showed strong activity against HSV-1 with [EC.sub.50] and [EC.sub.90] values of 6.25 and 15.25 [micro]M. respectively. In these experiments, acyclovir was used as the positive control compound, and the [EC.sub.50] values ranged from 0.1 to 1 [micro]M. Before we tested their antiviral activity, we determined the cellular toxicity of the test samples. As measured by an SRB colorimetric assay, the concentration of 2 kD peptide with 50% cytotoxicity on Vero Cell ([CC.sub.50] value) was 250 [micro]M. thus 40 times exceeding its [EC.sub.50] value.

[FIGURE 2 OMITTED]

An antiviral compound could protect cells against virus infection in several ways, by directly inactivating the virus or by interfering with the replication cycle. Therefore, the 2 kD peptide was tested for its virucidal effect and antiviral activity before, during, or after virus infections by CPE inhibition assay. Pre-incubation of HSV-1 with various concentrations of the 2 kD peptide showed dose-dependent CPE reduction patterns at concentrations from 10 to 50 [micro]M (Fig. 3). In attempts to determine whether the 2 kD peptide can be internalized into cells or bound to the cellular membrane to exert antiviral effects, confluent Vero cells were incubated with (i) different concentrations of peptide, which were then removed before infection; (ii) different concentrations of peptide added together with the virus; or (iii) infected with the virus and then incubated with different concentrations of peptide. The presence of 2 kD peptide before HSV-1 infections showed moderate inhibition of the virus-induced CPE ([EC.sub.50] value = 12 [micro]M), as compared to during or after infections ([EC.sub.50] value = 6.25 [micro]M) (Fig. 3). The mechanism of this inhibition remains to be investigated.

Under certain conditions, herpetic lesions can be complicated by secondary bacterial infections. Therefore, we investigated whether the 2 kD peptide exerts, in addition to its antiviral effects, a significant antibacterial activity against Gram-negative and Gram-positive bacteria. The 2 kD peptide showed moderate activity on both Staphylococcus aureus and Bacillus subtilis with an MIC value of 90 [micro]M (data not shown). In contrast to the relatively low MIC for Gram-positive bacteria. Gram-negative bacteria were not inhibited by either the protein extract or the 2 kD peptide at concentrations [less than or equal to]300 and [less than or equal to]100 [micro]M, respectively. This was to be expected, because the outer membrane of Gram-negative bacteria is known to present a barrier to the penetration of numerous antibiotic molecules, and the periplasmic space contains enzymes that are capable of breaking down molecules introduced from outside.

[FIGURE 3 OMITTED]

The search for and use of 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 substances and "leads" which could be developed for the treatment of infectious diseases (Cowan, 1999). According to this author, although 25-50% of current pharmaceuticals are derived from plants, none of them are used as an antimicrobial. Plants produce very bioactive molecules that allow them to interact with other organisms in their environment. Many of these substances are important in the defense against herbivores, and contribute to disease resistance. Plants, therefore, may be promising sources of antimicrobial agents.

Recently, Khan et al. (2005) reviewed anti-HSV substances from natural sources, including both extracts and pure compounds from herbal medicines, reported in studies from several laboratories. They also discussed the role of traditional medicine in the development of anti-HSV compounds. According to Khan et al. (2005), a large number of small molecules, such as phenolics, polyphenols, terpenes, flavonoids, and sugar-containing have been found to be promising antiherpetic agents.

Several peptide antibiotics (also known as antimicrobial peptides or natural antibiotics), in particular defensins, have also been shown to display in vitro antiviral activity (Bastian and Schafer, 2001; Lehrer, 2004; Matanic and Castilla, 2004). In some cases, the binding of the peptides to viral glucoproteins (lectin-like behavior) has been suggested 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. The features common to all the peptide antibiotics are a small size (12-100 amino acid residues), a polycationic charge, and an amphipathic structure having associated [alpha]-helices or [beta]-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 et al., 1989; Oren and Shai, 1998; Boman, 1995, 2000).

In the study by Matanic and Castilla (2004), the in vitro antiviral activity of antimicrobial cationic peptides cecropin A, melittin, magainin I and II, and indolicidin against the arenavirus Junin virus (JV), and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) was evaluated. According to these authors, the [EC.sub.50] value of cecropin A against JV was 3.24 [micro]M and virus yield was inhibited by more than 90% in cultures treated with 40 [micro]M of cecropin A. In contrast, this peptide did not exhibit inhibitory effect on the production of HSV-1 and HSV-2 infectious particles, even at a concentration of 40 [micro]M. Melittin showed inhibitory action against the three viruses assayed at a concentration of 3 [micro]M. On the other hand, magainins I and II, that produced a dose-dependent inhibition of HSV-1 and HSV-2 multiplication with similar [EC.sub.50] values for both viruses, were inactive against JV. At the highest concentration tested (50 [micro]M), indolicidin reduced HSV-1 and HSV-2 yields by 99%.

Herpesviruses are frequently cited as examples of viruses that enter cells via 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 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.

[FIGURE 4 OMITTED]

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 a concentration of 12.5 [micro]M, could directly inactivate 80% of HSV-1. Similar results were observed when the 2 kD peptide was assayed against the BHV, an enveloped virus like HSV-1. On the other hand, the 2 kD peptide showed weak activity against the polio vaccine virus, a non-enveloped virus (Fig. 4). The 40% inhibitory activity against poliovirus was achieved with 25 [micro]M. This inhibitory activity was not affected by increasing the 2 kD peptide concentration to 50 [micro]M. Thus, the anti-HSV-1 action of the 2 kD was not only potent but also specific. Taking these results together, it is conceivable that the 2 kD peptide was able not only to inhibit the initiation and the spread of infection, but it also had an in vitro prophylactic effect on HSV-1 infection.

Acknowledgments

This study was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Capacitacao e Aperfeicoamento de Pessoal de Nivel Superior, (Capes), Fundacao Araucaria, and Programa de Pos-graduacao em Ciencias Farmaceuticas da Universidade Estadual de Maringa. The authors would like to thank Marinete Martinez Vicentin for skillful technical assistance.

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I. Camargo Filho (a), D.A.G. Cortez (b), T. Ueda-Nakamura (c), C.V. Nakamura (c), B.P. Dias Filho (c,*)

(a) Programa de Pos-graduacao em Ciencias Farmaceuticas, Brazil

(b) Departamento de Farmacia e Farmacologia, Brazil

(c) Departamento de Analises Clinicas, Universidade Estadual de Maringa, Av. Colombo, 5790, 87020-900 Maringa, PR, Brazil

*Corresponding author. Tel.: + 55 44 3261 4955; fax: + 55 44 3261 4860.

E-mail address: bpdfilho@uem.br (B.P. Dias Filho).
Table 1. Antiviral activities of acid-soluble protein extract and
fractions on herpes simplex virus (HSV-1) by inhibition of virus-induced
cytopathic effect

 [CC.sub.50] [EC.sub.50]
Purification step ([micro]M) ([micro]M) SI (a)

Acid-soluble protein extract 105 36 2.9
DEAE HPLC (first) 127 9.5 13
[C.sub.18] HPLC 250 6.25 40

(a) SI (selective index) = [CC.sub.50]/[EC.sub.50] aciclovir (positive
control) [EC.sub.50] = 0.1-1.0 mM.
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Author:Filho, I. Camargo; Cortez, D.A.G.; Ueda-Nakamura, T.; Nakamura, C.V.; Filho, B.P. Dias
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Mar 1, 2008
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