Anti-herpes simplex virus effect of a seed extract from the tropical plant Licania tomentosa (Benth.) Fritsch (Chrysobalanaceae).
Incubation of acyclovir-resistant herpes simplex virus type 1 (ACVr-HSV1), during infection of the HEp-2 cell culture, with an extract prepared from the seeds of Licania tomentosa (Benth.) Fritsch (Chrysobalanaceae) species impaired the productive replication of this virus in a concentration-dependent manner. The extract was able to inhibit extracellular virus (virucidal effect) and also interfered with a very early event of cell infection, at a non-cytotoxic concentration.
Key words: Chrysobalanaceae, HSV, antiviral activity, Licania tomentosa
Because the plant kingdom is rich in bioactive compounds (Cox and Balick, 1994) plant extracts and their natural derivatives can be considered as potential sources of new drugs active against herpes simplex virus (HSV) infection. The Brazilian flora is a great reserve of medicinal plants with marked biological activity, used commonly as infusions or decoctions by native populations (Seidl, 1999). In a previous work, as part of a project to investigate the antiviral effect of some Brazilian plants, we described the anti-HSV activity of extracts of Alternanthera brasiliana (Lagrota et al., 1994, 1995), Persea americana (Miranda et al., 1997) and Vitex polygama (Goncalves et al., 2001).
In this work, we describe the anti-HSV-1 activity of extracts of Licania tomentosa (Benth.) Fritsch (Chrysobalanaceae), a native plant from the Mata Atlantica region and an abundant ornamental tree in Rio de Janeiro City, Brazil. Although there are no data regarding the popular medicinal use of this species, it was included in the study because six other Venezuelan species from the family Chrysobalanaceae have been reported to contain bioactive compounds (Bilia et al. 1999).
Here, we report the inhibitory activity of the seed crude extract of L. tomentosa against acyclovir-resistant herpes simplex virus type 1 (ACVr-HSV1).
Material and Methods
L. tomentosa seeds were collected in the summer season at Tijuca District, Rio de Janeiro City (Brazil). Voucher specimens were authenticated botanically by Dr. Regina Ejzemberg and deposited at the Instituto de Microbiologia Professor Paulo de G6es (IMPPG), Universidade Federal do Rio de Janeiro (UFRJ), Brazil.
The viable seeds were dried for 48 h at 37[degrees]C. After that, the dried seed power (20 g) was added to 100 ml of 0.85% NaCl solution to achieve a concentration of 200 mg/ml. The mixture was allowed to stand for 2 h at room temperature and overnight for 4 [degrees]C, then filtered through Whatman paper n[degrees] 1. The filtrate was added to an equal volume of glycerol and was kept at -20 [degrees]C. The final concentration of L. tomentosa dried seed was 100 mg/ml (wt/vol) in NaCl:glycerol (1:1). Before carrying out the tests, the extract was diluted in Eagle's minimum essential medium (MEM-Eagle) and sterilized by filtration using a 0.22 [micro]m Millipore membrane filter.
Mannose (M4625), galactose (G0625), N-acetylglucosamine (A8625) and galactosamine (G0264) were purchased from Sigma Chemical Company (USA).
Cell culture and virus
HEp-2 (human larynx epidermoid tumor) cells were cultivated in MEM-Eagle, supplemented with 5% fetal bovine serum (FBS), gentamicin (50 [micro]g/ml) and fungizon (2.5 [micro]g/ml), in a humidified 5% [CO.sub.2] atmosphere at 37 [degrees]C. The HSV-1 was isolated from a typical lip lesion in our laboratory and identified as an acyclovir-resistant (ACVr-HSV-1) mutant (Miranda et al., 1997).
Starting from an extract concentration of 10 mg/ml in MEM-Eagle, two-fold serial dilutions were added to triplicate HEp-2 cell monolayers cultivated in 96-well microplates. The treated cells were then incubated at 37 [degrees]C for 48 h. The cytotoxicity was first evaluated by the observation of any alteration in the cell morphology, in order to determine the maximum non-toxic concentration (MNTC) (Miranda et al., 1997). Afterward, the same microplates were also evaluated using the neutral red capture method (Neyndorff et al., 1990). Briefly, a neutral red solution in phosphate saline buffer (50 [micro]g/ml) was added to the cells for 3 h at 37 [degrees]C. The cells were then fixed with 4% formalin in 0.85% NaCl and the dye was extracted from the cells using a mixture of methanol:acetic acid:water (50:1:49). The optical density value of the cells treated with the different concentrations of the extract was read at 490 nm using an automatic spectrophotometer (Espectra 1 vision SLT -- Lab Instruments gls m.b.H. ). Considering the OD in the cell control as 100%, the cytotoxic concentration ([CC.sub.50] value) of the extract was determined as the graphic (Fig. 1A).
Antiviral activity assay
Anti-HSV-1 activity was evaluated by the reduction of the virus titers ([[TCID].sub.50]) using the end-point method (Reed and Muench, 1938). A half plate of HEp-2 confluent cell monolayers cultivated in 96-well microtiter plates was treated with the extract at the MNTC and the other half was treated with MEM-Eagle. Immediately after, ten-fold serial dilutions of ACVr-HSV-1 ([10.sup.6.0] [[TCID].sub.50]/ml) were added to treated and untreated cell cultures. After incubation for 48 h at 37 [degrees]C in a humidified atmosphere of 5% [CO.sub.2] the virus titers in treated and untreated cells were determined. The antiviral activity was expressed as percentage of inhibition (PI) of virus replication and calculated according the formula: 1 - (antilogarithmic test value / antilogarithmic control value) x 100 (Miranda et al., 1997).
Determination of virucidal effect
The ability of the seed extract to directly inactivate the virus particle (virucidal effect) was investigated by adding 100 [micro]l of ACVr-HSV-1 preparation ([10.sup.6.0][TCID.sub.50]/ml) to 900 [micro]l of extract at the MNTC or MEM-Eagle for 2 h at 37 [degrees]C. After incubation, 10-fold dilutions of each mixture (virus + extract or virus + MEM-Eagle) were added to HEp-2 cells cultivated in microplates. After 48 h at 37 [degrees]C, the virus titers were calculated using the end-point determination (Reed and Muench, 1938). The virucidal index (VI) was determined using the formula: VI = virus control titer / virus test titer (Miranda et al., 1997). Indexes lower or equal to 1 were not considered significant.
Determination of virucidal index reduction (VIR)
The ability of the sugars to inhibit the seed extract is direct inactivation of the virus particle was investigated. Seed extract at concentration of 625 [micro]g/ml was added to mannose, galactose, N-acetylglucosamine or galactosamine solutions (1 mM) in phosphate buffer saline (PBS) for 1 h of incubation at 37 [degrees]C. A control without sugar was included. Afterwards, 100 [micro]l of ACVr-HSV-1 preparation ([10.sup.6.0][TCID.sub.50]/ml) was added to 900 [micro]l of each mixture of extract-sugar and incubated for 2 h at 37 [degrees]C. Then, each 10-fold dilution of each mixture was added to confluent HEp-2 cell monolayers cultivated in microplates. After 48 h at 37 [degrees]C, the virus titers were calculated using end-point determination (Reed and Muench, 1938). The virucidal index reduction (VIR) was calculated using the formula VIR = VI control -- VI sugar test.
Effect of extract on cell pre-treatment
A half-microplate containing confluent HEp-2 cell monolayers was treated with the extract at the MNTC (test) and the other half with MEM-Eagle (control). The cells were then incubated at different temperatures and times of incubation. Afterwards, the cells were washed three times and inoculated with 10-fold dilutions of ACVr-HSV-1. The microplates were then incubated for 1 h at 4 [degrees]C and the cells were again washed three times, replaced by MEM-Eagle and incubated for up to 48 h at 37 [degrees]C. The virus titers in the control and the test solutions were calculated and the antiviral activity was expressed as described in the antiviral activity assay section.
Intracellular inhibition assay
In one experiment, HEp-2 cells were seeded in a 96-well microplate until confluence, infected with 10-fold serial dilutions of ACVr-HSV-1 and incubated for 1 h at 4 [degrees]C. Soon after, the cells were washed three times with cold MEM-Eagle solution. A half-microplate was replaced with the extract at the MNTC and the other half with MEM-Eagle. The monolayers were then incubated for 2 h at 37 [degrees]C. The cells were again washed three times, replaced with MEM-Eagle, and incubated for up to 48 h at 37 [degrees]C, when the virus titers in the control and the test solutions were calculated and the antiviral activity was expressed as described in the antiviral activity assay section.
In another experiment, HEp-2 cell cultures were infected with 10-fold dilutions of ACVr-HSV-1 and incubated immediately for 2 h at 37 [degrees]C. Afterwards, the extract at the MNTC or MEM-Eagle was each added to a half-microplate and the cells were re-incubated for up to 18 h. The cells were then washed three times with MEM-Eagle and re-incubated for up to 48 h at 37 [degrees]C, when the virus titers were calculated and the antiviral activity was expressed as described above.
The MNTC of the seed extract was 625 [micro]g/ml and the [CC.sub.50] value was 7.66 mg/ml (Fig. 1A). Starting from the MNTC, the virus inhibition was dose-dependent (Fig. 1B), exhibiting 50% inhibition ([ED.sub.50] value) at concentration of 9 [micro]g/ml. Based on the ratio [CC.sub.50] value/[ED.sub.50] value, the selectivity index (SI) was 851. In elucidating the mechanism of inhibition of the extract on the ACVr-HSV-1 replication, L. tomentosa seed extract was submitted to different conditions. When the virus suspension was placed in direct contact with the extract at the MNTC, for 2 h at 37 [degrees]C, inactivation of the virus particles occurred (VI = 2.46). Nevertheless, if the virus suspension was treated with the extract plus mannose or galactosamine, the VI was reduced to 1 (VIR = 1.46) and 0.75 (VIR = 1.74), respectively. If the virus suspension was treated with the extract plus galactose or Nacetylglucosamine, however, these sugars inhibited the virus inactivation completely (VIR = 2.46).
To verify the effect of the extract on pre-treatment of the HEp-2 cells, the cells were incubated with L. to-mentosa seed extract at temperatures of 4 [degrees]C, 20 [degrees]C and 37 [degrees]C and incubated for 1, 2, 3 and 4 h, before the virus infection. It was verified that no inhibition occurred if the cells were pre-treated with the extract for 1 h or 2 h, before the virus infection, at temperatures of 2 [degrees]C and 37 [degrees]C (Fig. 1). Nevertheless, the extract had an unexpected cytotoxicity at 4 [degrees]C, at all temperatures. On the other hand, if the extract was placed in contact with the HEp-2 for 3 h before virus infection, 69.1% and 90% inhibition occurred at 20 [degrees]C and 37 [degrees]C, respectively. After 4 h, 88.0% and 90.2 % inhibition occurred at 20 [degrees]C and 37 [degrees]C, respectively. The best inhibition occurred at a temperature of 37 [degrees]C (Fig. 2).
When the extract was added immediately after the virus attachment and removed completely soon after completing 2 h of virus infection, the PI was 92.7% (data not shown). If the extract was added 2 h after cell infection, however, the inhibition was not so evident (74.4%).
The cellular toxicity of the seed extract of Licania tomentosa was verified by morphological changes in HEp-2 cell cultures and the determination of cell viability by neutral red dye uptake. These methods are used currently as an indirect measure of cell viability (Borenfreund and Shopsis, 1985, Neyndorff, et al., 1990, Hall et al., 1998, de la Iglesias et al., 1998). Our results showed that even at concentrations above the MNTC, the cells with morphologic alterations were still alive, allowing us to find a high selectivity index.
The herpes simplex virus replication cycle is completed around 16-18 h and the immediate-early (JE) proteins of the whole viral synthesis are made at 2 to around 4 h (Whitley et al., 1998). In order to determine whether the L. tomentosa seed extract had antiviral activity in the initial step of virus replication, the extract was added as soon as possible after the virus adsorbed to the cells and removed completely before beginning the virus synthesis in the cell nucleus. In this step, the inhibition of virus replication was 92.7%. If the extract was added after 2 h of infection, the P1 was 74.4%, indicating that the preferential mechanism of antiviral inhibition may occur in the early phase of cell infection, between virus step adsorption and the synthesis of initial viral proteins. The preferential, however, mechanism of the antiviral action of the extract was observed when the virus suspension was placed in direct contact with the extract at MNTC, suggesting the presence of lectin-like substances able to bi nd to the virus particles. HSV-1 glycoproteins consist of an N-linked glycosylation composed of a pentasaccharide core [[(mannose).sub.3] - [(N-acetylglucosamine).sub.2]] and a number of side-chains with the composition sialic acid-galactose-glucosamine, or a O-linked glycosylation less important to virus adsorption, composed of a core of N-acetylgalactosamine and a side-chain of N-acetylglucosamine, fucose and sialic acid (Roizman and Sears, 1996). As lectins are found in many plant seeds, they could bind to glycoproteins present in the viral envelope. Furthermore, the crude seed extract agglutinated different types of human and sheep erythrocytes and this hemagglutination was inhibited by the addition of mannose, galactose, and N-acetylglucosamine (data not shown). The inhibition of virucidal activity also occurred when these sugars were added to the extract.
Marchetti et al. (1995) investigated eleven lectins for their inhibitory activity towards herpes simplex virus type 1 and observed that the virus adsorption as well as the virus replication were inhibited if lectins were present in infected cells. Ito et al. (1978) also demonstrated the antiviral effect of phytohemagglutin (PHA) on the pre-treatment of cells.
Our results indicate that the extract has a protective effect on HEp-2 cells that is dependent on the temperature and time of contact. The extract, however, had an unexpected cytotoxicity (cell lysis) at 4 [degrees]C at all times of incubation. This effect could be attributed to a phospholipase D (PLD activity). We were not successful in demonstrating this enzyme activity via two different methods: fluorimetric (Cholifa-Caspi et al., 1998) and periodide determination, using L-[alpha] phosphatidyl choline (SIGMA- L4129) as substrate (Kates et al., 1972). On the other hand, Zwaal et al. (1975), investigating eight purified phospholipases on intact erythrocytes, found neither induction of hemolysis nor hydrolysis of phospholipids in intact cells. Only PLC of Clostridium welchii, which was not detectable in plants, had lipophilic effects.
From our results, we could say that the extract protects from virus infection, but that cell lysis occurs by a mechanism until not elucidated. In conclusion, the L. tomentosa seed extract is a potent inhibitor of ACVr-HSV-1 replication in vitro at non-toxic concentrations; and has a virucidal effect.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
We thank Soluza Goncalves dos Santos for techinical assistance and Luciana Nagaroli Cavalcanti for the fluorimetric analysis. This work was supported by the Conselho Nacional de Desenvolvimento Cientifico (CNPq), Coordenacao de Aperfeicoamento de Pessoal do Ensino Superior (CAPES), Fundacao de Amparo a Pesquisa do Rio de Janeiro (FAPERJ), Financiadora de Estudos e Projetos (FINEP) and Fundacao Jose Bonifacio (FUJB-UFRJ).
Bilia AR, Braca A, Mendez J, Morelli I (1999) Molluscicidal and piscicidal activities of Venezuelan Chrysobalanaceae plants. Life Sci 66: PL53-59
Borenfreund E, Shopsis C (1985) Toxicity monitored with a correlated set of cell-culture assays. Xenobiotica 15: 705-711
Cholifa-Caspi V, Yona E, Discovitch M (1998) Kinetic analyses in mixed micelles of partially purified rat brain phospholipase D activity and its inactivation by phosphatidylinositol 4,5-bisphosphate. Neurochem Res 23: 589-599
Cox PA, Balick MJ (1994) The ethnobotanical approach to drug discovery. Scientific Amer June: 60-65
de Ia Iglesia P, Melon S, Lopez B, Rodriguez M, Blanco MI, Mellado P, de Ona M (1998) Rapid screening tests for determining in vitro susceptibility of herpes simplex virus clinical isolates. J Clin Microbiol 36: 2389-2391
Goncalves JLS, Leitao SG, Miranda MMFS, Santos MGM, Romanos MTV, Wigg MD (2001) In vitro antiviral effect of flavonoid-rich extracts of Vitex polygama (Verbenaceae) against acyclovir-resistant herpes simplex virus type 1. Phytomedicine 8: 477-480
Hall JO, Novakofski JE, Beasley VR (1998) Neutral red assay modification to prevent cytotoxicity and improve reproducibility using E-63 rat skeletal muscle cells. Biotech Histochem 73: 211-221
Ito M, Girvin L, Barron AL (1978) Inactivation of human cytomegalovirus by phytohemagglutinins. Arch. Virol. 57: 97-105
Kates M (1972) In: Techniques in lipidology. Isolation, analyses and identification of lipids. Laboratory techniques in biochemestry and molecular biology (eds Work TS, Work E), North-Holland/American Elsevier. 369
Lagrota MHC, Wigg MD, Santos MMG, Miranda MMFS, Camara FP, Couceiro JNSS, Costa SS (1994) Inhibitory activity of extracts of Althernantera brasiliana (Amaranthaceae) against the herpes simplex virus. Phytother Res 8: 358-361
Lagrota MHC, Wigg MD, Miranda MMFS, Santos MGM, Costa SS (1995) Inhibiton of herpes simplex virus replication by different extracts of Caryophyllales. Biomed Letters 51: 127-135
Marchetti M, Mastromarino P, Rieti S, Seganti L, Orsi N (1995) Inhibition of herpes simplex, rabies and rubella viruses by lectins with different specificities. Res Virol 146: 211-215
Miranda MMFS, Almeida AP, Costa SS, Santos MGM, Lagrota MHC, Wigg MD (1997) In vitro activity of extracts of Persea americana leaves on acyclovir-resistant and phosphonoacetic resistant herpes simplex virus. Phytomedicine 4: 347-352
Neyndorff HC, Bartel DL, Tufaro F, Levy JG (1990) Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 30: 485-490
Reed LJ, Muench H (1938) A simple method of estimating fifty percents endpoints. Am J Hyg 27: 493-497
Roizman B, Sears AE (1990) Herpes simplex viruses and their replication. In: Virology 3rd (ed Fields BN, et al): Raven Press, Ltd. New York, 2231-2295
Seidl PR (1999) Prospects for Brazilian natural products. Ann Acad Bras Cienc 71: 239-247
Whitley RJ, Kimberlin DW, Roizman B (1998) Herpes simplex viruses. Clin Infect Dis 26: 541-555
Zwaal RF, Roelofsen B, Comfurius P, van Deenen LL (1975) Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases. Biochim Biophys Acta 406: 83-96
M.M.F.S. Miranda (1), J.L.S. Goncalves (1), M.T.V. Romanos (1), F.P. Silva (1), L. Pinto (2), M.H. Silva (2), R. Ejzemberg (2), L.F.Z. Granja (2), and M.D. Wigg (1)
(1.) Departmento de Virologia Prof. Paulo de Goes Instituto de Microbiologia, Universidade Federal do Rio de Janeiro (UFRJ), CCS, Bloco I, Rio de Janeiro, Brasil
(2.) Departmento de Imunologia do Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro (UFRJ), CCS, Bloco I, Rio de Janeiro, Brasil
M.M.F.S. Miranda, Departamento de Virologia do Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro (UFRJ), CCS, Bloco I, Caixa Postal 68040, CEP 21941-590, Rio de Janeiro, Brazil Tel.: ++55-2125626749; Fax: ++55-2125608344; e-mail: firstname.lastname@example.org.