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Investigation of some Hypericum species native to Southern of Brazil for antiviral activity
Journal of Ethnopharmacology 77 (2001) 239– 245 www.elsevier.com/locate/jethpharm
Investigation of some Hypericum species native to Southern of Brazil for antiviral activity A.C. Schmitt a,b, A.P. Ravazzolo a,b, G.L. von Poser c,* a
Departamento de Patologia Clı´nica Veterina´ria, Laborato´rio de Virologia Veterina´ria, Uni6ersidade Federal do Rio Grande do Sul, 90540 -000, Porto Alegre, RS, Brazil b Centro de Biotecnologia, Laborato´rio de Virologia Molecular, Uni6ersidade Federal do Rio Grande do Sul, 91501 -970, Porto Alegre, RS, Brazil c Laboratorio de Farmacognosia, Faculdade de Farma´cia, Uni6ersidade Federal do Rio Grande do Sul, Programa de po´s-graduac¸a´o em Cieˆncı`as Farmaceˆuticas, A6. Ipiranga, 2752, 90610 -000 Porto Alegre, RS, Brazil Received 13 February 2001; received in revised form 11 July 2001; accepted 17 July 2001
Abstract Three plant species, Hypericum connatum, Hypericum caprifoliatum, Hypericum polyanthemum (Guttiferae), growing in Southern of Brazil were chemically investigated and tested for their antiviral activity against feline immunodeficiency virus (FIV). The chemical analysis revealed the presence of polyphenolic compounds such as tannins and flavonoids. Hypericin was not detected in these species. The aqueous extract (AE), the aqueous extract with low tannin concentration (LTCAE) and the methanolic extract (ME) were tested for their cytotoxic properties in concentrations of 50 – 150 mg/ml. AE was toxic to CRFK for the three species in all concentrations. LTCAE and ME varied between different concentrations being not toxic or allowing 80% of cell growth. LTCAE and ME (10–50 mg/ml) were analyzed for antiviral activity by inhibition of CPE and measuring FIV genome from cell culture supernatant. LTCAE of all species in this work did not cause any inhibition of FIV. Although no difference was seen in CPE, a lower number of viral particles in the supernatant was observed when FIV infected cells were treated with ME of H. connatum. These results suggest that some plants of the genus Hypericum from Southern Brazil contain compounds with potential antiviral activity against lentiviruses. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Antiviral; Feline immunodeficiency virus; Guttiferae, Hypericum spp; Plant extracts
1. Introduction The control of the human immunodeficiency virus (HIV-1) infection, responsible for the acquired immunodeficiency syndrome (AIDS), has been considered to be a major goal of public health. The therapeutic utility of anti-HIV-1 compounds is comprised of the appearance of severe side effects and the emergence of drug-resistant isolates of HIV-1 (Vandamme et al., 1998; Colgrove and Japour, 1999). Therefore, it is very important to identify new drugs that treat the human immunodeficiency virus (HIV-1) infection. Feline immunodeficiency virus (FIV) is a causative agent of acquired immunodeficiency syndrome (AIDS)like disease in cats. Pathogenesis of FIV infection is * Corresponding author. E-mail address: [email protected] (G.L. von Poser).
thought to be similar to that of HIV infection (Pedersen et al., 1987; Ishida and Tomoda, 1990). Likewise to HIV, FIV is a RNA enveloped virus belonging to the lenti6irus genus of the Retroviridae family, which replicates in lymphocytes, monocyte/macrophage lineage and dendritic cells (Toyosaki et al., 1993; Cadore´ et al., 1997; Hartmann, 1998), and causes the progressive CD4+ cell decrease leading to immune dysfunction (Ackley et al., 1990). Analogous to the HIV infection in man, the clinical course of FIV infection is defined in five stages: acute, asymptomatic carrier, persistent generalized lymphadenopathy, AIDS related complex phase, and AIDS (Ishida and Tomoda, 1990). The FIV is also an attractive model for AIDS research due to its similarity to HIV-1 in many molecular and biochemical properties. This virus has been used to study vaccine strategies, pharmacokinetics and pharmacodynamics of antiviral compounds against HIV-1
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(Bendinelli et al., 1995; North and La Casse, 1995; Zhu et al., 1996; Lee et al., 1998). Efforts have been made to evaluate the antiviral activity of a wide array of natural products, including plants, in order to isolate and characterize novel compounds which could inhibit virus replication and/or treat viral infection, or even serve as models for new molecules (Hamburger and Hostettmann, 1991; Lednicer and Narayanan, 1993; Elisabetsky et al., 1994; Vlietinck and Vanden Berghe, 1998; Simo˜ es et al., 1999). Members of the Guttiferae family, for example, have been used in traditional medicine to treat wounds, lymphatitis, parotitis, hepatitis, gastrointestinal disorders and tumors, which could be related to viral agents (Andrade et al., 1998; Ishiguro et al., 1998; Kosin et al., 1998; Likhitwitayawuid et al., 1998; Ali et al., 1990). Many compounds have been isolated from plants of this family and have had their antiviral activity studied. The USA National Cancer Institute has done extensive research in this family and some substances as benzophenones and pyranocoumarins have demonstrated an excellent anti-retroviral activity (Mckee et al., 1998; Fuller et al., 1999a,b). Calanolide-A, a coumarin isolated from tropical plants of the genus Calophyllum, has exhibited both in vitro and in vivo activity against HIV-1 and is under clinical investigation (Kashman et al., 1992; Currens et al., 1996; Xu et al., 1998, 1999). Hypericin and pseudohypericin, isolated from plants of the genus Hypericum also received attention due to the antiviral action on lipid enveloped and non-enveloped DNA and RNA viruses. These substances are highly effective in preventing viral-induced manifestations that follow infection with a variety of viruses in vitro and in vivo, and reduce the spread of HIV-1 in vitro (Meruelo et al., 1998; Vlietinck et al., 1998). Considering the presence of antiviral compounds in the Guttiferae family and the long traditional reputation of Hypericum genus as a medicinal plant for the treatment of a variety of conditions, three species of this genus were chosen and collected in the Southern region of Brazil. One of these species, Hypericum connatum, known as ‘orelha-de-gato’ (cat’s ear), is used in traditional medicine as tonic, astringent, and to treat mouth wounds (Correˆ a, 1984). In the search for bioactive compounds from natural sources we have examined the antiviral action of aqueous and methanolic extracts of Hypericum spp. against FIV, as an experimental model for HIV-1.
2. Methodology
2.1. Plant collection All the plants were collected in Rio Grande do Sul State, Southern Brazil. The aerial parts of Hypericum
caprifoliatum Cham. and Schltdl. were collected in the ‘Morro Santana’, Porto Alegre in May 1998. Hypericum polyanthemum Klotzsch ex Reichardt was collected in Cac¸ apava do Sul, in August 1998, and H. connatum Lam. was collected in Capa˜ o do Lea˜ o in January 1999. The voucher specimens have been deposited in the Herbarium of the Federal University of Rio Grande do Sul (ICN/UFRGS) (H. caprifoliatum, Bordignon, 1400; H. connatum, Bordignon and Salazar, 1527; H. polyanthemum, Bordignon et al., 1429).
2.2. Extracts preparation A 10 g sample of dried and powdered plant material was macerated in 100 ml methanol for 24 h. The sample was then filtered through Whatman number 1 filter paper and the marc was washed with another 100 ml of methanol. Both methanolic solutions were combined and evaporated to dryness under reduced pressure. Aqueous extracts (AE) were obtained by macerating dried and powdered plant material in boiled distilled water for 20 min. As methanolic extracts (ME), aqueous extracts were filtrated and evaporated to dryness under reduced pressure. Low tannin concentration aqueous extracts (LTCAE) were obtained by reducing tannins concentration from AE after an addition of 1 ml of 1% protein (gelatin) and centrifugation (3000×g; 5 min) for three times. The extracts were dissolved in Dulbecco’s modified Eagle medium (D-MEM) (Gibco-BRL) to a final concentration of 10 mg of crude plant extract per ml (10 mg/ml). The ME extracts were additionally dissolved in 0.2% dimethyl sulfoxide (DMSO) and sonicated. Then, the extracts were filtrated in a 0.22 mm membrane and stored at −20 °C until tested. DMSO was diluted at 1, 0.5, 0.25, and 0.125% and also analyzed for its cytotoxicity in CRFK cells.
2.3. Cell culture and cytotoxicity assays Crandell feline kidney cells (CRFK/ATCC —CCL94) were propagated in D-MEM medium with 5% fetal bovine serum (FBS) and 200 000 IU/ml penicillin and 200 mg/ml streptomycin. To test for cytotoxicity, CRFKs monolayers (4×104 cells per well) were grown in 96-well microtiter plates, and exposed to serial dilutions of the extracts, ranging from 50 to 150 mg/ml, as described elsewhere (Axarlis et al., 1998). Parallel control groups received the vehicle DMSO (0.2%) diluted in the same way as the extracts. DMSO (1, 0.5, 0.25, and 0.125%) dilutions were tested. The cells were kept at 37 °C for 72 h and after that time were stained by the May–Gru¨ nwald–Giemsa
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(MGG) technique. Cells were examined microscopically for the presence of cytotoxic effects.
2.4. Anti6iral assay A full-length clone 34TF10 of the Petaluma strain was transfected into CRFK cells by liposome method to produce viral stock. The supernatant was monitored for the presence of virus by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (Caldas et al., 2000), and cells were microscopically observed for the presence of cytopathic effect (CPE) characterized by syncytia and cell death. As described by Tozzini et al. (1992), CRFK cultures may present multinucleated cells without virus infection. To avoid this misinterpretation of viral CPE, we develop a method of viral detection in the supernatant of transfected cells (Caldas et al., 2000). Briefly, three primers were designed to detect viral genomic RNA in the supernatant of transfected and infected cells: one reverse primer and two forward primers specific to gag gene. The viral RNA was extracted from the supernatant, submitted to a reverse transcription reaction with the reverse primer and finally to a hemi-nested PCR. The viral stocks were prepared from medium containing extra cellular virus released from transfected CRFK cells. Aliquots were stored frozen at − 80 °C. To screen for antiviral activity, confluent 1-day-old CRFK monolayers were grown in 24-well plates and cultures of cells (4× 104 cells per well) were infected with 3-fold diluted FIV stock. After 2 h of adsorption, the supernatant was removed and the extracts were added in different concentrations (10– 50 mg/ml). Controls consisted of cells only and infected cells without extracts. After an incubation time of 6 days, the medium was collected and stored at − 80 °C and cells were stained by the MGG technique. The syncytia and cell death were observed. The presence of FIV in the supernatant was monitored by measuring virus genome using the RT-PCR and RT-‘hemi-nested’-PCR techniques and are described above (Caldas et al., 2000). All the tests were performed in quadruplicate, and the test for H. connatum ME was repeated three times. To analyze viral RNAs, these RNAs were extracted from supernatant with Trizol (Gibco-BRL) reagent according to manufacturer’s instructions. The RT-PCR and RT-‘hemi-nested’-PCR techniques were used to amplify a central region of the genome containing the gag gene of FIV. To measure FIV genome in supernatant, 6 ml of the PCR reactions were loaded into 2% agarose gel and the gel photograph was analyzed using Scion Image software (Scion Corporation). This analysis allows a semiquantification of viral genomic RNA by comparison of a known amount of DNA (Low Mass Ladder, GibcoBRL) to the DNA fragments obtained by PCR.
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2.5. Phytochemical analyses The chemical analysis (flavonoids, tannins, anthraquinones, alkaloids, saponins, etc) of the three species was performed using methods described by Harborne (1984). Chromatographic analyses were done from methanolic extracts for the presence of hypericin and pseudohypericin and flavonoids according to methods described by Wagner et al. (1984). Thin layer chromatography (TLC) for hypericin and derivatives was performed on silica gel GF254 using as eluent toluene: ethylformiate: formic acid (50:40:10). The chromatogram was sprayed with pyridine and visualized at a wavelength of 365 nm. Hypericin and derivatives appear as light red spots. TLC for flavonoids and phenolic acids (Wagner et al., 1984) was carried out on silica gel GF254 using as eluent ethylacetate: acetic acid: formic acid: water (100:11:11:27). Chromatograms were sprayed with Natural Reagent/PEG 4000 and visualized at a wavelength of 365 nm. The flavonoids (rutine, quercitrin, isoquercitrin, and hyperoside) and the phenolic acids (chlorogenic and caffeic acids) used as reference substances, appear as orange-yellow and blue spots, respectively.
3. Results The cytotoxicity of the extracts of the three plants in CRFK was determined by microscopic examination of cell death and integrity after an incubation time of 72 h (Table 1). The H. caprifoliatum, H. polyanthemum and H. connatum AE extracts were toxic to CRFK in all concentrations. Based on these results, AE of the three species were not used for antiviral test. LTCAE and ME extracts varied between different concentrations being non-toxic or allowing 80% of cell growth. The ME extracts of the three plants were highly toxic to CRFK in dilutions ranging from 75 to 150 mg/ml. At 50 mg/ml, H. polyanthemum and H. connatum ME extracts allowed 80% of cell growth, but at the same dilution H. caprifoliatum ME extract caused cell death. LTCAE and DMSO dilutions were non-toxic to CRFKs in this study. The results of the antiviral activity of the extracts were expressed by CPE identification and virus detection in supernatant (Table 2). On day 6, after infection and exposure to plant extracts, it was observed that LTCAE of the three species and ME of H. polyanthemum did not affect the viral CPE and were not active against FIV (Table 2). Conversely, no inhibition was seen in CPE although ME of H. connatum was highly active against FIV in supernatant, indicating a putative antiviral activity of this extract. It was not possible to detect FIV genome by RT-PCR in supernatant treated
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with ME extract of H. polyanthemum and LTCAE extract of H. connatum, but amounts of FIV could be measured when RT-‘hemi-nested’-PCR was performed (Caldas et al., 2000). The RT-‘hemi-nested’-PCR technique was performed to evaluate the degree of inhibition. The results indicate that there was no difference between FIV measured in FIV infected cells supernatant treated with LTCAE extract of the three species studied here when compared with virus control, which did not receive any extract treatment. Nevertheless, when FIV infected cells were treated with ME extract of H. connatum, the quantity of virus was dramatically lower than the virus control (Fig. 1). The phytochemical analyses revealed substances like tannins and flavonoids, including anthocyanins, but there was no evidence of the presence of saponins, coumarins, or quinones, such as hypericin and derivatives. The chromatographic analysis of the flavonoids showed the presence of quercitrin in the three plants. Isoquercitrin was found in H. polyanthemum and H. connatum; and hyperoside was not found in H. connatum, being the main flavonoid in H. caprifoliatum and H. polyanthemum. Very small amounts of rutin were found in H. caprifoliatum and H. connatum. In addition, chlorogenic acid was found in the latter.
4. Discussion and conclusions The therapeutic properties of plants have been attributed to crude extracts or isolated compounds reflecting, in many times, their role in traditional medicine. Hypericum perforatum, the most studied species of the Guttiferae family, has a good reputation of an useful medicinal herb. Moreover, by studying its compounds, researchers have confirmed some biological properties especially antidepressive, antitumoral, and antiviral activities (Awang, 1991). The antiviral
activity of some Hypericum species has been attributed to hypericin and pseudohypericin, but investigations carried out with other Hypericum species have suggested the antiviral property of compounds other than anthraquinones (Taylor et al., 1996). The lack of hypericin and derivatives in the plants studied here, especially in H. connatum, indicates the presence of other substance(s) with activity against lentivirus. Prior studies described anti-retroviral activity of flavonoids (Lednicer and Narayanan, 1993; Mahmood et al., 1993). In the three Hypericum species analyzed, high amounts of this class of compounds were found. Although the plants had the same class of compounds, the flavonoid chromatographic profile was different. For example, both H. caprifoliatum and H. polyanthemum presented hyperoside while H. connatum had quercitrin as the major component. Besides the known compounds, the plants contain other unknown flavonoids. Considering these differences, their role in the antiviral activity of H. connatum methanolic extract cannot be disregarded. In addition, these same substances found in H. connatum ME could inhibit FIV replication through a synergistic mechanism of action with compounds of low polarity, once the aqueous extracts with low tannin concentration (LTCAE), containing high amount of flavonoids, were not active. Evidences of cytotoxic effect of tannins were clearly shown when aqueous extracts were tested in our work, and this effect was probably due to complexation of tannins with proteins by hydrogen bounds, disrupting the cell membrane. This fact was verified when tannins were partly removed from the aqueous extracts. Although tannins have cytotoxic properties, they have demonstrated antiviral activity against retroviruses (Nonaka et al., 1990). In general, it is believed that tannins act by associating with proteins of viral particles and/or host cell surfaces, resulting in a reduction or prevention of viral adsorption (Vlietinck et al., 1998). Taking it into consideration, we also suppose that low amounts of tannins remaining in the methanolic extract
Table 1 Cytotoxicity of the plant extracts Hypericum species
H. H. H. H. H. H. H. H. H.
caprifoliatum caprifoliatum caprifoliatum polyanthemum polyanthemum polyanthemum connatum connatum connatum
Extracts
AE ME LTCAE AE ME LTCAE AE ME LTCAE
Cytotoxicity 150 mg/ml
100 mg/ml
75 mg/ml
50 mg/ml
+ + − + + − + + +
+ + − + + − + + +
+ + − + + − + + −
+ + − + 9 − + 9 −
+: cytotoxicity, −: no cytotoxicity, 9 : about 80% of cell culture confluence.
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Table 2 Antiviral assay: CPE analysis and detection of FIV genome in cell culture supernatant Species (extracts)
H. H. H. H. H.
polyanthemum (ME) polyanthemum (LTCAE) connatum (ME) connatum (LTCAE) caprifoliatum (LTCAE)
Detection of FIV (RT-PCR)
CPE
50 mg/ml
25 mg/ml
15 mg/ml
10 mg/ml
50 mg/ml
25 mg/ml
15 mg/ml
10 mg/ml
− + − + +
+ + − + +
+ + − + +
+ + − − +
CPE e CD CPE e CD CD CD CPE e CD
CPE CPE e CD CD CD CPE e CD
CPE CPE e CD CPE CPE CPE e CD
CPE e CD CPE e CD CPE CPE CPE
CPE, cytophatic effect characterized by sincycia and cell death; CD, cell death; +, presence of FIV genome in supernatant; −, absence of FIV genome in supernatant.
supernatant, the presence of FIV provirus (integrated virus) was confirmed by PCR (data not shown). Thus, it is important to employ other techniques targeting virus protein or enzymatic activity to identify which step of the virus replication should be targeted by the substance(s) present in the plant, which demonstrated activity. Interestingly, among the three plants studied in this work, H. connatum, which is used in traditional medicine, was the only one active against FIV presenting one or more substances that are related to the antiviral activity in the FIV replication. This fact reinforces the value of ethnopharmacological data in the search of bioactive substances. Thus, more detailed studies should be undertaken to isolate and identify antiviral components of specific parts of this plant and most active extracts contributing to find antiviral agents against lentiviruses.
Acknowledgements
Fig. 1. Amounts of FIV (ng of RT-hemi-nested-PCR products) in cell culture supernatant treated with LTCAE (A) and ME (B) extracts from H. polyanthemum, H. caprifoliatum and H. connatum using RT-‘hemi-nested’-PCR technique mcg + mg.
were not able to cause cytotoxicity and could have an antiviral and/or virucidal activity alone or in synergism with other substance found in H. connatum. In our study we used CPE inhibition and a molecular approach to evaluate the antiviral activity against FIV. We believe that the substances found in ME extract of H. connatum could have a mechanism of action directed to virus protease enzyme, preventing virion maturation and release of particles to extra cellular environment. Although FIV genomic RNA was not detected in the
We thank the Brazilian government for the financial support, CBIOT enzimas for Taq DNA polymerase and Se´ rgio Bordignon for collecting and identifying the plant material. Aline Conceic¸ a˜ o Schmitt was a fellow of CAPES, Coordenac¸ a˜ o de Aperfeic¸ oamento de Pessoal de Nı´vel Superior do Ministe´ rio da Educac¸ a˜ o do Brasil. This work was supported by PRONEX em Virologia Veterina´ ria grant, from the Ministe´ rio da Cieˆ ncia e Tecnologia do Brasil.
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Investigation of some Hypericum species native to Southern of Brazil for antiviral activity A.C. Schmitt a,b, A.P. Ravazzolo a,b, G.L. von Poser c,* a
Departamento de Patologia Clı´nica Veterina´ria, Laborato´rio de Virologia Veterina´ria, Uni6ersidade Federal do Rio Grande do Sul, 90540 -000, Porto Alegre, RS, Brazil b Centro de Biotecnologia, Laborato´rio de Virologia Molecular, Uni6ersidade Federal do Rio Grande do Sul, 91501 -970, Porto Alegre, RS, Brazil c Laboratorio de Farmacognosia, Faculdade de Farma´cia, Uni6ersidade Federal do Rio Grande do Sul, Programa de po´s-graduac¸a´o em Cieˆncı`as Farmaceˆuticas, A6. Ipiranga, 2752, 90610 -000 Porto Alegre, RS, Brazil Received 13 February 2001; received in revised form 11 July 2001; accepted 17 July 2001
Abstract Three plant species, Hypericum connatum, Hypericum caprifoliatum, Hypericum polyanthemum (Guttiferae), growing in Southern of Brazil were chemically investigated and tested for their antiviral activity against feline immunodeficiency virus (FIV). The chemical analysis revealed the presence of polyphenolic compounds such as tannins and flavonoids. Hypericin was not detected in these species. The aqueous extract (AE), the aqueous extract with low tannin concentration (LTCAE) and the methanolic extract (ME) were tested for their cytotoxic properties in concentrations of 50 – 150 mg/ml. AE was toxic to CRFK for the three species in all concentrations. LTCAE and ME varied between different concentrations being not toxic or allowing 80% of cell growth. LTCAE and ME (10–50 mg/ml) were analyzed for antiviral activity by inhibition of CPE and measuring FIV genome from cell culture supernatant. LTCAE of all species in this work did not cause any inhibition of FIV. Although no difference was seen in CPE, a lower number of viral particles in the supernatant was observed when FIV infected cells were treated with ME of H. connatum. These results suggest that some plants of the genus Hypericum from Southern Brazil contain compounds with potential antiviral activity against lentiviruses. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Antiviral; Feline immunodeficiency virus; Guttiferae, Hypericum spp; Plant extracts
1. Introduction The control of the human immunodeficiency virus (HIV-1) infection, responsible for the acquired immunodeficiency syndrome (AIDS), has been considered to be a major goal of public health. The therapeutic utility of anti-HIV-1 compounds is comprised of the appearance of severe side effects and the emergence of drug-resistant isolates of HIV-1 (Vandamme et al., 1998; Colgrove and Japour, 1999). Therefore, it is very important to identify new drugs that treat the human immunodeficiency virus (HIV-1) infection. Feline immunodeficiency virus (FIV) is a causative agent of acquired immunodeficiency syndrome (AIDS)like disease in cats. Pathogenesis of FIV infection is * Corresponding author. E-mail address: [email protected] (G.L. von Poser).
thought to be similar to that of HIV infection (Pedersen et al., 1987; Ishida and Tomoda, 1990). Likewise to HIV, FIV is a RNA enveloped virus belonging to the lenti6irus genus of the Retroviridae family, which replicates in lymphocytes, monocyte/macrophage lineage and dendritic cells (Toyosaki et al., 1993; Cadore´ et al., 1997; Hartmann, 1998), and causes the progressive CD4+ cell decrease leading to immune dysfunction (Ackley et al., 1990). Analogous to the HIV infection in man, the clinical course of FIV infection is defined in five stages: acute, asymptomatic carrier, persistent generalized lymphadenopathy, AIDS related complex phase, and AIDS (Ishida and Tomoda, 1990). The FIV is also an attractive model for AIDS research due to its similarity to HIV-1 in many molecular and biochemical properties. This virus has been used to study vaccine strategies, pharmacokinetics and pharmacodynamics of antiviral compounds against HIV-1
0378-8741/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 1 4 - 2
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(Bendinelli et al., 1995; North and La Casse, 1995; Zhu et al., 1996; Lee et al., 1998). Efforts have been made to evaluate the antiviral activity of a wide array of natural products, including plants, in order to isolate and characterize novel compounds which could inhibit virus replication and/or treat viral infection, or even serve as models for new molecules (Hamburger and Hostettmann, 1991; Lednicer and Narayanan, 1993; Elisabetsky et al., 1994; Vlietinck and Vanden Berghe, 1998; Simo˜ es et al., 1999). Members of the Guttiferae family, for example, have been used in traditional medicine to treat wounds, lymphatitis, parotitis, hepatitis, gastrointestinal disorders and tumors, which could be related to viral agents (Andrade et al., 1998; Ishiguro et al., 1998; Kosin et al., 1998; Likhitwitayawuid et al., 1998; Ali et al., 1990). Many compounds have been isolated from plants of this family and have had their antiviral activity studied. The USA National Cancer Institute has done extensive research in this family and some substances as benzophenones and pyranocoumarins have demonstrated an excellent anti-retroviral activity (Mckee et al., 1998; Fuller et al., 1999a,b). Calanolide-A, a coumarin isolated from tropical plants of the genus Calophyllum, has exhibited both in vitro and in vivo activity against HIV-1 and is under clinical investigation (Kashman et al., 1992; Currens et al., 1996; Xu et al., 1998, 1999). Hypericin and pseudohypericin, isolated from plants of the genus Hypericum also received attention due to the antiviral action on lipid enveloped and non-enveloped DNA and RNA viruses. These substances are highly effective in preventing viral-induced manifestations that follow infection with a variety of viruses in vitro and in vivo, and reduce the spread of HIV-1 in vitro (Meruelo et al., 1998; Vlietinck et al., 1998). Considering the presence of antiviral compounds in the Guttiferae family and the long traditional reputation of Hypericum genus as a medicinal plant for the treatment of a variety of conditions, three species of this genus were chosen and collected in the Southern region of Brazil. One of these species, Hypericum connatum, known as ‘orelha-de-gato’ (cat’s ear), is used in traditional medicine as tonic, astringent, and to treat mouth wounds (Correˆ a, 1984). In the search for bioactive compounds from natural sources we have examined the antiviral action of aqueous and methanolic extracts of Hypericum spp. against FIV, as an experimental model for HIV-1.
2. Methodology
2.1. Plant collection All the plants were collected in Rio Grande do Sul State, Southern Brazil. The aerial parts of Hypericum
caprifoliatum Cham. and Schltdl. were collected in the ‘Morro Santana’, Porto Alegre in May 1998. Hypericum polyanthemum Klotzsch ex Reichardt was collected in Cac¸ apava do Sul, in August 1998, and H. connatum Lam. was collected in Capa˜ o do Lea˜ o in January 1999. The voucher specimens have been deposited in the Herbarium of the Federal University of Rio Grande do Sul (ICN/UFRGS) (H. caprifoliatum, Bordignon, 1400; H. connatum, Bordignon and Salazar, 1527; H. polyanthemum, Bordignon et al., 1429).
2.2. Extracts preparation A 10 g sample of dried and powdered plant material was macerated in 100 ml methanol for 24 h. The sample was then filtered through Whatman number 1 filter paper and the marc was washed with another 100 ml of methanol. Both methanolic solutions were combined and evaporated to dryness under reduced pressure. Aqueous extracts (AE) were obtained by macerating dried and powdered plant material in boiled distilled water for 20 min. As methanolic extracts (ME), aqueous extracts were filtrated and evaporated to dryness under reduced pressure. Low tannin concentration aqueous extracts (LTCAE) were obtained by reducing tannins concentration from AE after an addition of 1 ml of 1% protein (gelatin) and centrifugation (3000×g; 5 min) for three times. The extracts were dissolved in Dulbecco’s modified Eagle medium (D-MEM) (Gibco-BRL) to a final concentration of 10 mg of crude plant extract per ml (10 mg/ml). The ME extracts were additionally dissolved in 0.2% dimethyl sulfoxide (DMSO) and sonicated. Then, the extracts were filtrated in a 0.22 mm membrane and stored at −20 °C until tested. DMSO was diluted at 1, 0.5, 0.25, and 0.125% and also analyzed for its cytotoxicity in CRFK cells.
2.3. Cell culture and cytotoxicity assays Crandell feline kidney cells (CRFK/ATCC —CCL94) were propagated in D-MEM medium with 5% fetal bovine serum (FBS) and 200 000 IU/ml penicillin and 200 mg/ml streptomycin. To test for cytotoxicity, CRFKs monolayers (4×104 cells per well) were grown in 96-well microtiter plates, and exposed to serial dilutions of the extracts, ranging from 50 to 150 mg/ml, as described elsewhere (Axarlis et al., 1998). Parallel control groups received the vehicle DMSO (0.2%) diluted in the same way as the extracts. DMSO (1, 0.5, 0.25, and 0.125%) dilutions were tested. The cells were kept at 37 °C for 72 h and after that time were stained by the May–Gru¨ nwald–Giemsa
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(MGG) technique. Cells were examined microscopically for the presence of cytotoxic effects.
2.4. Anti6iral assay A full-length clone 34TF10 of the Petaluma strain was transfected into CRFK cells by liposome method to produce viral stock. The supernatant was monitored for the presence of virus by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (Caldas et al., 2000), and cells were microscopically observed for the presence of cytopathic effect (CPE) characterized by syncytia and cell death. As described by Tozzini et al. (1992), CRFK cultures may present multinucleated cells without virus infection. To avoid this misinterpretation of viral CPE, we develop a method of viral detection in the supernatant of transfected cells (Caldas et al., 2000). Briefly, three primers were designed to detect viral genomic RNA in the supernatant of transfected and infected cells: one reverse primer and two forward primers specific to gag gene. The viral RNA was extracted from the supernatant, submitted to a reverse transcription reaction with the reverse primer and finally to a hemi-nested PCR. The viral stocks were prepared from medium containing extra cellular virus released from transfected CRFK cells. Aliquots were stored frozen at − 80 °C. To screen for antiviral activity, confluent 1-day-old CRFK monolayers were grown in 24-well plates and cultures of cells (4× 104 cells per well) were infected with 3-fold diluted FIV stock. After 2 h of adsorption, the supernatant was removed and the extracts were added in different concentrations (10– 50 mg/ml). Controls consisted of cells only and infected cells without extracts. After an incubation time of 6 days, the medium was collected and stored at − 80 °C and cells were stained by the MGG technique. The syncytia and cell death were observed. The presence of FIV in the supernatant was monitored by measuring virus genome using the RT-PCR and RT-‘hemi-nested’-PCR techniques and are described above (Caldas et al., 2000). All the tests were performed in quadruplicate, and the test for H. connatum ME was repeated three times. To analyze viral RNAs, these RNAs were extracted from supernatant with Trizol (Gibco-BRL) reagent according to manufacturer’s instructions. The RT-PCR and RT-‘hemi-nested’-PCR techniques were used to amplify a central region of the genome containing the gag gene of FIV. To measure FIV genome in supernatant, 6 ml of the PCR reactions were loaded into 2% agarose gel and the gel photograph was analyzed using Scion Image software (Scion Corporation). This analysis allows a semiquantification of viral genomic RNA by comparison of a known amount of DNA (Low Mass Ladder, GibcoBRL) to the DNA fragments obtained by PCR.
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2.5. Phytochemical analyses The chemical analysis (flavonoids, tannins, anthraquinones, alkaloids, saponins, etc) of the three species was performed using methods described by Harborne (1984). Chromatographic analyses were done from methanolic extracts for the presence of hypericin and pseudohypericin and flavonoids according to methods described by Wagner et al. (1984). Thin layer chromatography (TLC) for hypericin and derivatives was performed on silica gel GF254 using as eluent toluene: ethylformiate: formic acid (50:40:10). The chromatogram was sprayed with pyridine and visualized at a wavelength of 365 nm. Hypericin and derivatives appear as light red spots. TLC for flavonoids and phenolic acids (Wagner et al., 1984) was carried out on silica gel GF254 using as eluent ethylacetate: acetic acid: formic acid: water (100:11:11:27). Chromatograms were sprayed with Natural Reagent/PEG 4000 and visualized at a wavelength of 365 nm. The flavonoids (rutine, quercitrin, isoquercitrin, and hyperoside) and the phenolic acids (chlorogenic and caffeic acids) used as reference substances, appear as orange-yellow and blue spots, respectively.
3. Results The cytotoxicity of the extracts of the three plants in CRFK was determined by microscopic examination of cell death and integrity after an incubation time of 72 h (Table 1). The H. caprifoliatum, H. polyanthemum and H. connatum AE extracts were toxic to CRFK in all concentrations. Based on these results, AE of the three species were not used for antiviral test. LTCAE and ME extracts varied between different concentrations being non-toxic or allowing 80% of cell growth. The ME extracts of the three plants were highly toxic to CRFK in dilutions ranging from 75 to 150 mg/ml. At 50 mg/ml, H. polyanthemum and H. connatum ME extracts allowed 80% of cell growth, but at the same dilution H. caprifoliatum ME extract caused cell death. LTCAE and DMSO dilutions were non-toxic to CRFKs in this study. The results of the antiviral activity of the extracts were expressed by CPE identification and virus detection in supernatant (Table 2). On day 6, after infection and exposure to plant extracts, it was observed that LTCAE of the three species and ME of H. polyanthemum did not affect the viral CPE and were not active against FIV (Table 2). Conversely, no inhibition was seen in CPE although ME of H. connatum was highly active against FIV in supernatant, indicating a putative antiviral activity of this extract. It was not possible to detect FIV genome by RT-PCR in supernatant treated
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with ME extract of H. polyanthemum and LTCAE extract of H. connatum, but amounts of FIV could be measured when RT-‘hemi-nested’-PCR was performed (Caldas et al., 2000). The RT-‘hemi-nested’-PCR technique was performed to evaluate the degree of inhibition. The results indicate that there was no difference between FIV measured in FIV infected cells supernatant treated with LTCAE extract of the three species studied here when compared with virus control, which did not receive any extract treatment. Nevertheless, when FIV infected cells were treated with ME extract of H. connatum, the quantity of virus was dramatically lower than the virus control (Fig. 1). The phytochemical analyses revealed substances like tannins and flavonoids, including anthocyanins, but there was no evidence of the presence of saponins, coumarins, or quinones, such as hypericin and derivatives. The chromatographic analysis of the flavonoids showed the presence of quercitrin in the three plants. Isoquercitrin was found in H. polyanthemum and H. connatum; and hyperoside was not found in H. connatum, being the main flavonoid in H. caprifoliatum and H. polyanthemum. Very small amounts of rutin were found in H. caprifoliatum and H. connatum. In addition, chlorogenic acid was found in the latter.
4. Discussion and conclusions The therapeutic properties of plants have been attributed to crude extracts or isolated compounds reflecting, in many times, their role in traditional medicine. Hypericum perforatum, the most studied species of the Guttiferae family, has a good reputation of an useful medicinal herb. Moreover, by studying its compounds, researchers have confirmed some biological properties especially antidepressive, antitumoral, and antiviral activities (Awang, 1991). The antiviral
activity of some Hypericum species has been attributed to hypericin and pseudohypericin, but investigations carried out with other Hypericum species have suggested the antiviral property of compounds other than anthraquinones (Taylor et al., 1996). The lack of hypericin and derivatives in the plants studied here, especially in H. connatum, indicates the presence of other substance(s) with activity against lentivirus. Prior studies described anti-retroviral activity of flavonoids (Lednicer and Narayanan, 1993; Mahmood et al., 1993). In the three Hypericum species analyzed, high amounts of this class of compounds were found. Although the plants had the same class of compounds, the flavonoid chromatographic profile was different. For example, both H. caprifoliatum and H. polyanthemum presented hyperoside while H. connatum had quercitrin as the major component. Besides the known compounds, the plants contain other unknown flavonoids. Considering these differences, their role in the antiviral activity of H. connatum methanolic extract cannot be disregarded. In addition, these same substances found in H. connatum ME could inhibit FIV replication through a synergistic mechanism of action with compounds of low polarity, once the aqueous extracts with low tannin concentration (LTCAE), containing high amount of flavonoids, were not active. Evidences of cytotoxic effect of tannins were clearly shown when aqueous extracts were tested in our work, and this effect was probably due to complexation of tannins with proteins by hydrogen bounds, disrupting the cell membrane. This fact was verified when tannins were partly removed from the aqueous extracts. Although tannins have cytotoxic properties, they have demonstrated antiviral activity against retroviruses (Nonaka et al., 1990). In general, it is believed that tannins act by associating with proteins of viral particles and/or host cell surfaces, resulting in a reduction or prevention of viral adsorption (Vlietinck et al., 1998). Taking it into consideration, we also suppose that low amounts of tannins remaining in the methanolic extract
Table 1 Cytotoxicity of the plant extracts Hypericum species
H. H. H. H. H. H. H. H. H.
caprifoliatum caprifoliatum caprifoliatum polyanthemum polyanthemum polyanthemum connatum connatum connatum
Extracts
AE ME LTCAE AE ME LTCAE AE ME LTCAE
Cytotoxicity 150 mg/ml
100 mg/ml
75 mg/ml
50 mg/ml
+ + − + + − + + +
+ + − + + − + + +
+ + − + + − + + −
+ + − + 9 − + 9 −
+: cytotoxicity, −: no cytotoxicity, 9 : about 80% of cell culture confluence.
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Table 2 Antiviral assay: CPE analysis and detection of FIV genome in cell culture supernatant Species (extracts)
H. H. H. H. H.
polyanthemum (ME) polyanthemum (LTCAE) connatum (ME) connatum (LTCAE) caprifoliatum (LTCAE)
Detection of FIV (RT-PCR)
CPE
50 mg/ml
25 mg/ml
15 mg/ml
10 mg/ml
50 mg/ml
25 mg/ml
15 mg/ml
10 mg/ml
− + − + +
+ + − + +
+ + − + +
+ + − − +
CPE e CD CPE e CD CD CD CPE e CD
CPE CPE e CD CD CD CPE e CD
CPE CPE e CD CPE CPE CPE e CD
CPE e CD CPE e CD CPE CPE CPE
CPE, cytophatic effect characterized by sincycia and cell death; CD, cell death; +, presence of FIV genome in supernatant; −, absence of FIV genome in supernatant.
supernatant, the presence of FIV provirus (integrated virus) was confirmed by PCR (data not shown). Thus, it is important to employ other techniques targeting virus protein or enzymatic activity to identify which step of the virus replication should be targeted by the substance(s) present in the plant, which demonstrated activity. Interestingly, among the three plants studied in this work, H. connatum, which is used in traditional medicine, was the only one active against FIV presenting one or more substances that are related to the antiviral activity in the FIV replication. This fact reinforces the value of ethnopharmacological data in the search of bioactive substances. Thus, more detailed studies should be undertaken to isolate and identify antiviral components of specific parts of this plant and most active extracts contributing to find antiviral agents against lentiviruses.
Acknowledgements
Fig. 1. Amounts of FIV (ng of RT-hemi-nested-PCR products) in cell culture supernatant treated with LTCAE (A) and ME (B) extracts from H. polyanthemum, H. caprifoliatum and H. connatum using RT-‘hemi-nested’-PCR technique mcg + mg.
were not able to cause cytotoxicity and could have an antiviral and/or virucidal activity alone or in synergism with other substance found in H. connatum. In our study we used CPE inhibition and a molecular approach to evaluate the antiviral activity against FIV. We believe that the substances found in ME extract of H. connatum could have a mechanism of action directed to virus protease enzyme, preventing virion maturation and release of particles to extra cellular environment. Although FIV genomic RNA was not detected in the
We thank the Brazilian government for the financial support, CBIOT enzimas for Taq DNA polymerase and Se´ rgio Bordignon for collecting and identifying the plant material. Aline Conceic¸ a˜ o Schmitt was a fellow of CAPES, Coordenac¸ a˜ o de Aperfeic¸ oamento de Pessoal de Nı´vel Superior do Ministe´ rio da Educac¸ a˜ o do Brasil. This work was supported by PRONEX em Virologia Veterina´ ria grant, from the Ministe´ rio da Cieˆ ncia e Tecnologia do Brasil.
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