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Journal of the Chilean Chemical Society

versão On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.49 n.2 Concepción jun. 2004

http://dx.doi.org/10.4067/S0717-97072004000200007 

 

J. Chil. Chem. Soc., 49, N 2 (2004), pags.:143-145

ANTIVIRAL ACTIVITY ON HANTAVIRUS AND APOPTOSIS OF VERO CELLS OF NATURAL AND SEMI-SYNTHETIC COMPOUNDS FROM HELIOTROPIUM FILIFOLIUM RESIN.

 

B. Modak 1*, H. Galeno 2 and R. Torres 1.

1Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile.
2Instituto de Salud Pública de Chile, Sección Virología, Santiago, Chile.

Dirección para correspondencia


ABSTRACT

The antiviral activity on Hantavirus Andes variety of resinous exudate from Heliotropim filifolium and its pure compounds was studied. From filifolinol 2, the main compound of the resins, filifolinoic acid 1 was obtained. The resin was the most active. Furthermore, we observed cytostatic effect and apoptosis on Vero E6 cells with 3-O-methylgalangine 5.

Keywords: Heliotropium filifolium, resinous exudate, antiviral properties, Hantavirus, cytostatic effect, apoptosis.


INTRODUCTION

At present, the virus and bacteria are charging relentlessly more than 16 million lives per year every where, and many countries, especially the poorest, constitutes the main cause of death among children and adult youths [1].

A genus of virus that at present has generated special attention, with an extensive repercussion as well as in the scientific community, as in the communication media, are the Hantavirus. In the spring of 1993 it was observed in the southwestern region of North America, the apparition of an epidemic bud of a new and serious respiratory illness that was caused by a pertinent virus to genus Hantavirus, that was called "Hantavirus Pulmonary Syndrome " (HPS) [2]. The responsible virus of this illness, although belonging to a known kind, they had not been detected before, in other other part of the world. The ubiquity of the Hantavirus and their potential to cause severe illness in the man, determine that these virus represent an important sanitary problem [3].

Hantavirus have its natural reservoir in the rodents and from them can infect to man. In Chile, the HPS is an endemic illness, that affects preferably to the young adult population residents in rural sectors. In 2003, 29 cases have been detected, where the 31% caused death [4]. This infection does not have specific treatment and there are not specific know effective medicines to do prophylaxis in persons exposed to risk of infection.

Previous studies carried out in our laboratory showed that some compounds obtained from the vegetable specie Heliotropium filifolium presented interesting results as antiviral of the human virus Polio stump sabin, Herpes simplex 1, Herpes simplex 2, Junin and Respiratory syncytial virus [5]. This vegetal is found in desert environments and faces to extremes climatic conditions, which has carried it to produce an resinous exudate that covers its foliar surface and the stem, as a mechanism of protection against the adverse conditions in which its developed. The secondary metabolites that compose the resin consist of a mixture of flavonoids and geranyl aromatic derivatives with spiranic ring [6,7].

In the search of medicines for the infectious processing as the HPS, we have studied the inhibitory effect on Hantavirus of geranyl aromatic derivatives and flavonoids obtained from Heliotropium filifolium by a viral focus formation inhibition test.

EXPERIMENTAL

Isolation of tested compounds: the natural compounds, filifolinol 2 and their ester 3, and the flavonoids ayanine 4 and 3-O-methylgalangine 5, were obtained from resinous exudate of Heliotropium filifolium (Miers) Reiche (ST-2214 SSUC). This specie was collected during the flowering season, October 1997, in the north of Vallenar (IV region, Chile, 28°45´S,7049W).Voucher specimens were deposited in the Herbarium of the Faculty of Biological Science of Catholic University of Chile. The resinous exudate was extracted by immersion of the fresh plant material in CH2Cl2, for 30 s at room temperature. The extract was concentrated to a sticky residue (6.3 % w/w). The extract was purified by CC (silica gel) using a hexane-EtOAc step gradient. The compounds were identified by 1H-NMR and 13C-NMR, IR, UV and mass spectrometry [6,7].

Filifolinoic acid 1: Filifolinol (1.36 g) was dissolved in distilled water (10 mL) and methanol (10 mL) and was added NaOH(1,Og). The mixture was left at room temperature for 24 hours. Subsequently added HCl 12 M until pH 1. The compound was purified by CC (silica gel) using hexane-EtOAc step gradient to afford 625 mg of 1 as a white solid , mp: 156-158C. [a]24d = -10,11° . IR band (KBr): 3350, 2955, 1666 cm-1. 1H- RMN ( 400 MHz, CDCl3 ) 0.78 ppm (3H,d,J=7 Hz,H-4'), 1.0 ppm (3H,s,H-10'), 1.15 ppm (3H,s,H-9'), 1.53 ppm (1H,m,H-5'), 1.60 ppm (1H,m,H-5'), 1.63 ppm (1H,m,H-6'), 1.70 ppm (1H,d,J=3,5 Hz,OH de C-7'), 1.95 ppm (1H,m,H-6'), 2.30 ppm (1H,m,H-3'), 3.01 ppm (1H,d,J=17,2 Hz,H-1'), 3.31 ppm (1H,m,H-7'), 3.65 ppm (1H,d,J=17,1 Hz,H-1'), 6.71ppm (1H,d, J=8,0 Hz, H-2), 7.81 ppm (1H,d,J=1,15 Hz,H-5), 7.91 ppm (1H,dd,H-3).

Cytotoxicity test: Before the assay with Hantavirus and to establish the most appropriate concentration of work, the cytotoxicity of the solvent was analyzed as well as the pure compounds and the resin. The test compounds were dissolved in broth of cell (EMEM supplemented with 10% SFB) and added to monolayers Vero E6 cells. The cells were observed for 5 days.

Inoculation of the cells with Hantavirus: the monolayers cells were inoculated with a suspension of Andes CHI 99-7913 virus to a dilution to produce between 40 to 60 viral focus of growth by plate for the experiment with resin and filifolinol, and 400 to 600 viral focus for the experiment with the other compounds. For this purpose were added 200 and 2000 mL respectively of dilution virus to the plates. They were incubated during 4 hours at 37C to produce the viral adsorption on the cells.

Incubation of cells with the test compounds: the medium with the viral dilution was withdrawn and the cells were washed with a new medium. Then 500 µL of the different concentrations of the test compounds (stock solution 10 mg/mL) in the EMEM-methyl cellulose-SFB medium were added to the respective plates. The control plates were covered with EMEM-methyl cellulose-SFB medium without compound. Finally the plates were incubated for 7 days at 37°C in atmosphere of CO2.

Developing of the viral focus of growth:

a. 0000Fixing: the semisolid medium that covers the cells was aspired and the monolayers were washed 3 times with PBS-BSA at 1%, dried to air and fix with methanol-acetone (1:1)mixture added to H2O2 0.2% by 15 minutes at 4C. When finalized the fixing the plates were air dried and then covered with 200 µL of PBS-BSA until subsequent dyed with immunoperoxidase.

b. 0000 To dye: the plates were covered with 200 µL of rabbit serum antinuclear protein of Hantavirus to a dilution 1:2000 in PBS-BSA for 2 hours in a Rocket shaker at room temperature. The solution was removed and the plates washed 3 times with PBS-BSA. Then were added 200 µL of conjugated peroxidase Ac anti IgG of rabbit to a dilution 1:1000 incubated for 2 hours at room temperature in shaker. The solution was aspired and the plates washed 3 times with PBS-BSA. Finally they were added 200 µL of DAB Metal substrate and the plates were maintained 2 hours at room temperature, after which, the solution was replaced for PBS.

c. 0000 Reading: the development viral growth focus were examined under microscope with increase of 25 X. The quantity developed focus was registered as well as an estimation of its size and intensity.

RESULTS AND DISCUSSION

Filifolinac acid 1 was obtained by basic hydrolysis of filifolinol. The spectroscopy results were expected for the structure, in basis of the compounds 2 and 3 [6,7]. The RMN1H spectra was similar to filifolinol, except for the absence of signal at 3.89 ppm for protons on C-8. This confirm the hydrolysis.

The effect was established on the replication of Hantavirus variety Andes of the resin of Heliotropium filifolium , filifolinoic acid 1, filifolinol 2, filifolinyl senecionate 3, ayanine 4 and 3-O-methylgalangine 5. Prior to the assay with Hantavirus, the toxicity of solvent toxicity, as well as that of the respective pure compounds (table 1).


Table 1: Cytopathic effect in Vero E6 cells.

µg/mL
(dilution)
Observation
day
Solvent
Ethanol
Compound
1
Compound
2
Compound
3
Compound
4
Compound
5
Resin

1.25 3 - - - - - - -
(1:8000) 5 - - - - - - -
2.5 3 - - - - - - -
(1:4000) 5 - - - - - - +/-
5 3 - - - +/- - - +/-
(1:2000) 5 - - - + - - +
10 3 - - - +/ - +/ +
(1:1000) 5 - - +/- +++ - +/- ++
20 3 - - +/- + - ++ ++
(1:500) 5 - - + ++++ - ++++ ++++

  Scale:  
  -
+/-
+
++
+++
++++
: absence of effect
: very light
: light
: moderate
: intense
: total loss of monolayer cells

The results obtained for the antiviral activity on Hantavirus Andes variety, of the compounds and the resin, were expressed in percentage of inhibition of the viral replication regarding the control (table 2).


Table 2: Effect of the test compounds on the replication of Hantavirus.

Test Compounds
(dilution)
µg/mL % of inhibition

Control - 0.0
Resin 10 (1:1000) 36.0
1 5 (1:2000) 0.9
2 5 (1:2000) 27.5
3 5 (1:2000) -1.9
4 5 (1:2000) 2.4
5 5 (1:2000) -9.8

It was established the rank of action of the different compounds over witch no cytotoxic effect is observed that would interfere in the viral replication.

The results indicate that the resin and its main component, filifolinol, possess some degree of antiviral activity, where the most significant was with the resin to a concentration of 10 µg/mL, being achieved a 36% of inhibition of the viral development, also it was observed a reduction of the focus size. Nevertheless, to this dilution a light effect of toxicity is observed. Based on these results we are testing the antiviral effect on Hantavirus with other resins of species of the genus Heliotropium. On the other hand, ilifolinyl senecionate and 3-O-methylgalangine promote the of the virus growth in the cells.

The cytotoxicity assays, allowed to establish that filifolinyl senecionate and 3-O-methylgalangine, present interesting activities on the Vero E6 cells. These compounds to concentration 10 µg/mL stimulated the acidification of the medium. This fact has been interpreted like a stimulatory property of the cell metabolism. It was observed besides, that these compounds present a cytostatic action, inducing the cells to proliferative rest (table 3). These results have carried us to study a possible anticancer activity.


Table 3: Cytopathic effect of test compounds on Vero E6 cells.

Test
compounds
µg/mL
(dilution)
Observation
day
Apoptosis Acidification
of the
medium
Inhibition of the
replication

Control 5 (1:2000) 5 - - -
  10 (1:1000) 5 - - -
  20 (1:500) 5 - - -
Resin 5 (1:2000) 5 - - -
  10 (1:1000) 5 - - -
  20 (1:500) 5 + + +
1 5 (1:2000) 5 - - -
10 (1:1000) 5 - - -
20 (1:500) 5 - - -
2 5 (1:2000) 5 - - -
10 (1:1000) 5 - - -
20 (1:500) 5 - - -
3 5 (1:2000) 5 - - -
10 (1:1000) 5 + + +
20 (1:500) 5 + + +
4 5 (1:2000) 5 - - -
10 (1:1000) 5 - - -
20 (1:500) 5 - - -
5 5 (1:2000) 5 - - -
10 (1:1000) 5 + + +
20 (1:500) 5 + + +

  Scale:  
  -
+
: absence of effect
: presence of effect

On the other hand, 3-O-methylgalangine seems to favor the cytopathic effect, since in preinfected cells with Hantavirus, induced the premature formation of round cells that are removed of the monolayers. When dyeing these cells with antibody monoclonal antinucleus protein of Hantavirus and its subsequent revealed by immunofluorescence , appear with great deposit of viral antigen.


 
Fig. 1: Test compounds

 

ACKNOWLEDGEMENTS

The authors would like to thank DICYT Universidad de Santiago.

REFERENCES

1. J.Randal, Cuestiones Mundiales, Publicaciones Electrónicas del Servicio Informativo y Cultural de los Estados Unidos (USIS),1,17, (1996).         [ Links ]

2. J.Duchin, F.Koster, C.Peters, G.Simpson, B.Tempest, S.Zaki, T.Ksiazck, P.Rollin, S.Nichol, E.Umland, R.Moolenaar, S.Reef, K.Nolte, M.Gallear, J.Butler and R.Breiman, New England J. Med., 330, 949, (1994).         [ Links ]

3. C.Schmaljohn and B.Hjelle, Emerg.Infect Dis., 3, 95, (1997).         [ Links ]

4. Informe situación epidemeológica de Hantavirus, Departamento de Epidemiología, Ministerio de Salud de Chile, Mayo 2003.         [ Links ]

5. R.Torres, B.Modak, A.Urzúa, F.Delle Monache, E.Damonte and C.Pujol, Bol. Soc. Chil. Quim., 47, 259, (2002).         [ Links ]

6. R.Torres, B.Modak, A.Urzúa, F.Delle Monache, G.Delle Monache and E.Gacs-Baitz, Phytochemistry, 36, 249, (1994).         [ Links ]

7. A.Urzúa, B.Modak and R.Torres, Bol. Soc. Chil. Quim., 46,175, (2001).         [ Links ]

 

Correspondencia a: e-mail: bmodak@lauca.usach.cl.

Received: November 7, 2003 ­ Accepted: December 30, 2003

 

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