A phytochemical study of
Viral infections represent a current problem accounting for severe damage to human health and economic losses in livestock [
Dengue virus belonging to the Flaviviridae family,
HSV belongs to the family Herpesviridae and the subfamily Alphaherpesvirinae and is characterized by neurovirulence, latency, and reactivation. The prevalence of HSV infection has increased in recent years, making it a highly relevant public health issue. Early detection and treatment are of paramount importance for disease control [
Encephalomyocarditis virus (EMCV) family Picornaviridae, genus
Vaccinia (VACV) is a virus of the genus
As part of a bioprospecting project, whose main goal is to discover potential antiviral natural products of plants from Brazilian Cerrado and Atlantic Forest biomes, we have screened several species of plants collected in the state of Minas Gerais [
After drying in air circulating oven at 40°C for 72 h, the plant material—136.3 g of leaves, 461.5 g of stems, and 13.8 g fruits—were ground and extracted by percolation with 96% EtOH at room temperature. The solvent was removed in a rotary evaporator under reduced pressure at 50°C, leaving dark residues—EEFFL, 34.2 g, EEFFS, 60.0 g, and EEFFF, 2.5 g—for leaves, stems, and fruits, respectively, which were kept in a vacuum desiccator until constant weight.
In the HPLC analyses, an exploratory gradient elution was used [
To a portion of EEFFL (10.0 g), MeOH was added, and an insoluble precipitate was separated by filtration through sintered glass funnel and washed thoroughly with MeOH yielding 2.8 g. The precipitate was recrystallized out from methanol/water (1 : 1) giving 1.9 g of compound
To a portion of EEFFS (10.0 g), cold MeOH was added giving a precipitate which was separated by filtration on sintered glass funnel and washed thoroughly with MeOH yielding 2.43 g. The precipitate was recrystallized out twice from MeOH to give 405.0 mg of compound
To a portion of EEFFF (1.0 g), MeOH was added and an insoluble precipitate was separated by filtration on sintered glass funnel and washed thoroughly with methanol. The precipitate was subjected to recrystallization to give compound
Vero cells (ATCC CCL-81) and LLCMK2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Cultilab, Campinas, SP, Brazil) at 37°C, in 5% CO2 atmosphere, supplemented with 5% fetal bovine serum, 50
HSV-1 was obtained from the collection of Laboratório de Virus, UFMG, Belo Horizonte, Brazil. DENV-2, EMCV, and VACV-WR were kindly donated by Dr. L. Figueiredo (USP, Ribeirão Preto, Brazil), Dr. I. Kerr (London Research Institute, London, UK), and Dr. C. Jungwirth (University of Würzburg, Würzburg, Germany), respectively. The viruses were titrated by TCID50 in Vero cells [
Vero and LLCMK2 cells were exposed to different concentrations of extracts/fractions/compounds for 48 and 72 h [
The antiviral activity (EC50) of extracts∖fractions∖compounds was evaluated by the MTT assay [
The compounds isolated were identified on the basis of spectral analyses and comparison with literature data. 1D and 2D 1H and 13C-NMR spectra such as COSY, HSQC, and HMBC were obtained on a Bruker Avance DRX400 instrument in DMSO-d6 with TMS as internal standard. Chemical shifts are given as
HPLC-DAD analyses allowed identifying xanthones as major constituents in all the extracts, as inferred from their UV spectra that were registered online (Figure
RP-HPLC-DAD fingerprints for the crude ethanol extracts from (a) ethanol extract from
Bioguided fractionation of
Chemical structures of mangiferin (
Additional quantity of mangiferin (
Finally, column chromatographic fractionation of the ethanol fruit extract afforded four C-glucosylxanthones which were shown to be identical to those obtained from the leaves extract, mangiferin (
Confirming previously published results [
Cytotoxicity (CC50, Vero, and LLCMK2 cells), in vitro antiviral activity (EC50), and selectivity index (SI) for ethanol extracts from
Extracts/fractions/compound | Vero |
LLCMK2 CC50 |
|
SI |
|
SI |
|
SI |
|
SI |
---|---|---|---|---|---|---|---|---|---|---|
EEFFS | >500 | 173.9 ± 9.8 | 93.2 ± 5.4 | >5.4 | 59.2 ± 2.4 | >8.4 | 322.5 ± 14.4 | >1.5 | 42.6 ± 2.3 | 4.1 |
EEFFF | >500 | >500 | 147.8 ± 2.4 | >3.4 | 252.7 ± 3.9 | >2.0 | 134.4 ± 5.9 | >3.7 | 13.1 ± 1.6 | >38.2 |
EEAFL | >500 | >500 | 85.6 ± 4.1 | >5.8 | 83.7 ± 3.1 | >6.0 | 199.4 ± 13.8 | >2.5 | 16.3 ± 6.8 | >30.7 |
FFHDF (1 : 1) | 222.0 ± 7.3 | 86.7 ± 8.5 | NA | NA | NA | NA | ||||
FFDF | 263.3 ± 13.9 | 95.1 ± 9.3 | NA | NA | NA | NA | ||||
FFDEF (1 : 1) | 50.7 ± 2.5 | 13.8 ± 2.1 | NA | NA | NA | 3.9 ± 0.36 | 3.5 | |||
FFEF | >500 | >500 | NA | NA | NA | NA | ||||
FFEMF (1 : 1) | >500 | >500 | 169.7 ± 21.0 | >2.9 | 182.9 ± 11.4 | >2.7 | 190.5 ± 14.7 | >2.6 | 31.8 ± 5.7 | >15.7 |
FFMF | >500 | >500 | 50.3 ± 2.8 | >9.9 | NA | NA | 41.8 ± 5.6 | >12.0 | ||
FFMWF (2 : 1) | >500 | >500 | 35.7 ± 2.0 | >14.0 | NA | NA | 22.8 ± 0.8 | >21.9 | ||
FFMWF (1 : 2) | >500 | >500 | NA | NA | NA | NA | ||||
Mangiferin ( |
>500 | >500 | 267.9 ± 6.7 |
>1.9 | 182.7 ± 14.3 |
>2.7 | NA | 265.5 ± 14.0 |
>1.9 | |
2′- |
>500 | >500 | 4.6 ± 1.5 |
>108.7 | 23.8 ± 1.0 |
>21.0 | NT | 4.1 ± 0.4 |
>121.9 | |
2′- |
>500 | >500 | 47.4 ± 6.1 |
>10.5 | NA | 241.0 ± 31.8 |
>2.1 | 40.4 ± 4.2 |
>12.4 | |
Chrysin ( |
>500 | >500 | 146.3 ± 15.9 |
>3.4 | 123.5 ± 10.5 |
>4.0 | NA | NA | ||
2′- |
>500 | >500 | 77.4 ± 4.3 |
>6.5 | NA | NT | 3.5 ± 0.5 |
>148.9 | ||
|
>1000 | >1000 |
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SI, selectivity index;
Dose-response curves for antiviral activity of ethanol extracts from
An aliquot of the leaves extract (EEFFL, 10.0 g) was submitted to bioguided fractionation. Initially, addition of cold methanol to EEFFL led to mangiferin (
Dose-response curves for antiviral activity of mangiferin (
EEFFS (10.0 g) was also submitted to bioguided fractionation. Initially, addition of cold methanol to the crude ethanol extract led to the separation of mangiferin (
Finally, bioguided fractionation of EEFFS (10.0 g) by extraction with cold methanol led to mangiferin (
Phytochemical investigation of EtOH extracts of leaves, stems, and fruits from
As shown in Figure
Mangiferin (
Recent data on biological activity of mangiferin cinnamoyl esters showed that they are antioxidant agents similar to mangiferin and are antiplasmodial with moderate activity in vitro against
Recently, a total of twenty xanthones were isolated from
Our results reveal that
The authors declare no conflicts of interest.
Thanks are due to Dr. J. A. Lombardi, Departamento de Botânica, Instituto de Biociências, UNESP, Rio Claro, Brazil, for collection and taxonomical determination