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In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L
Life Sciences 76 (2005) 2981 – 2993 www.elsevier.com/locate/lifescie
In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L Munevver Sokmena,1, Maria Angelovab,1, Ekaterina Krumovab, Svetlana Pashovab, Stefka Ivanchevac, Atalay Sokmend, Julia Serkedjievab,* a
Department of Biology, Faculty of Science and Literature, Cumhuriyet University, Sivas, Turkey Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. Georgy Bonchev St., 1113 Sofia, Bulgaria c Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria d Department of Chemistry, Faculty of Science and Literature, Cumhuriyet University, Sivas, Turkey
b
Received 27 July 2004; accepted 8 November 2004
Abstract Recent evidence shows that plant polyphenols exhibit antioxidant and radical scavenging properties. By three separate and complementary methods - DPPH assay, h-carotene-linoleic acid assay and NBT-reduction assay it was established that a polyphenol-rich extract from the medicinal plant Geranium sanguineum L. with strong anti-influenza virus activity, possessed antioxidant and radical scavenging capacities. For comparative reasons caffeic acid and the synthetic antioxidant BHT were used. Total soluble phenolic constituents of the MeOH extract measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). Further it was demonstrated that the EtOAc fraction, retaining the majority of the in vivo protective effect exhibited a strong O2scavenging activity while the n-BuOH fraction, containing the majority of the in vitro antiviral activity provoked generation of O2. The O2 scavenging activity of all three preparations correlated with the rate of the protective effect shown in the murine model of experimental influenza virus infection. The present results are in accordance with our intensive studies on the mode of the protective effect of the plant extract which showed positively that the protection may possibly be attributed to the combination of more than one
T Corresponding author. Tel.: +359 2 979 31 85; fax: +359 2 870 01 09. E-mail address: [email protected] (J. Serkedjieva). 1 These authors equally contributed to this work. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.11.020
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biological activities and that the use of antioxidants might be an useful approach in the treatment of influenza infection. D 2005 Elsevier Inc. All rights reserved. Keywords: Polyphenol extracts; Geranium sanguineum; Radical scavenging activity; Antioxidant effect; Influenza virus inhibition
Introduction There is abundant evidence that a great number of aromatic, spicy, medicinal and other plants contain chemical compounds exhibiting antioxidant properties. With this respect a particular interest has been given to plant polyphenols. The natural polyphenols have an ideal structure for capturing of free radicals (Haslam, 1966) and it was found that their antioxidant activity surpasses the effect of known antioxidants, such as the vitamins A and E (Rice-Evans et al., 1977). There are data about the antioxidant effects of plant extracts (Aruoma et al., 1996; Costantino et al., 1992; Kahkonen et al., 1999; Parejo et al., 2003; Pietta et al., 1998; Rakotoarison et al., 1997) and diverse groups of plant polyphenol compounds - flavonoids (Habtemariam, 1997; Rice-Evans et al., 1977; Vitor et al., 2004), tannins (Haslam, 1966; Yokozawa et al., 1998), catechins and proantocyanidins (Plumb et al., 1998), polyphenolic acids (Rice-Evans et al., 1977). Earlier investigations have proved that a semi-standardized polyphenol extract, obtained from the medicinal plant Geranium sanguineum L., designated as polyphenolic complex (PC), exhibited a pronounced anti-influenza virus effect in cell cultures and protected mice from mortality in the experimental influenza virus infection (Serkedjieva and Manolova, 1992). The virus-inhibitory activity of the plant extract was related to the presence of large quantities of polyphenol compounds. Phytochemical analysis of PC showed that it contained tannins (11.02%) flavonoids (0.14%), catechins and proanthocyanidines (2.1 mg/kg) (Ivancheva et al., 1992, 1996). The current study was undertaken to investigate on the in vitro antioxidant and radical scavenging effects of the plant preparation and its active fractions by applying established in vitro assays and to provide evidence for the possible relation between their antiviral and antioxidant capacities.
Materials and Methods Medicinal plant and extraction The medicinal plant Geranium sanguineum L. (Geraniaceae) collected in June-August in the stage of flowering in the Lulin Mountain. A specimen was deposited in the Herbarium of the Institute of Botany, Bulg. Acad. Sci. (SOM-5/91). The MeOH extract from the aerial roots was obtained as described earlier (Ivancheva et al., 1992) and designated as polyphenolic complex (PC). In short - ground air-dried aerial
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roots, were defatted with petroleum ether and treated with MeOH to fully extract the polyphenol components. The extract was filtered and lyophilized (yield 16%). The polyphenol content of the preparation was controlled by thin layer chromatography and by quantitative determination of tannins, flavonoids and catechins (Ivancheva et al., 1992, 1996). The total extract was extracted further with EtOAc and n-BuOH to obtain the corresponding fractions. Chemicals Rimantadine hydrochloride was obtained from Hoffman - La Roche Inc., Nutley, NJ, USA; protein Ahorseradish peroxidase conjugate was from Bio Rad Lab., Ca, USA; Dulbecco’s Eagle medium was from GibcoBRL, Scotland, UK, fetal calf serum (FCS) was from BioWhittaker Europe, Germany; DPPH, h-carotene, BHT, nitro blue tetrazolium (NBT), methionine, riboflavin, NaCN, superoxide dismutase (SOD) from bovine erytrocytes and trypsin were from Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany; caffeic acid was a gift from Prof. V. Bankova, Institute of Organic Chemistry, Bulgarian Academy of Sciences. Total phenolic constituents Total soluble phenolic constituents of the MeOH extract were determined by employing methods, involving Folin-Ciocalteu reagent and gallic acid as standard. 0.1 mL of the extract solution, containing 1000 Ag extract was taken in a volumetric flask, 46 mL distilled water and 1 mL Folin-Ciocalteu reagent were added and the flask was shaken thoroughly. After 3 min, a 3 mL solution of Na2CO3 (2%, w/v) was added and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance was measured at 760 nm. The same procedure was repeated with all standard gallic acid solutions (0–1000 Ag/0.1 mL) and a standard curve was obtained by the equation given below: Absorbance: 0.0012 Gallic acid (Ag) + 0.0033 Antioxidant activity DPPH assay The hydrogen atoms or electrons donation ability of the corresponding extract was measured from the bleaching of purple colored MeOH solution of DPPH. This spectrophotometric assay uses stable radical 1,1-Diphenyl-2-picrylhydrazyl (DPPH) as a reagent (Burits and Bucar, 2000; Cuendet et al., 1997). Fifty Al of various concentrations of the extract in MeOH were added to 5 mL of a 0.004% MeOH solution of DPPH. After a 30 min incubation period at room temperature the absorbance was read against a blank at 517 nm. Inhibition of free radical DPPH in percent was calculated according the formula: Inhibition % ¼ Ablank Asample =Ablank 100; where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated form the graph plotted inhibition percentage against extract concentration. Tests were carried out in triplicate. The synthetic antioxidant buthylated hydroxy toluene (BHT) was included in experiments as a positive control.
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b-carotene-linoleic acid assay In this assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation, causing discoloration of h-carotene (Dapkevicius et al., 1998). A stock solution of h-carotenelinoleic acid mixture was prepared as follows: 0.5 mg h-carotene was dissolved in 1 mL of chloroform (HPLC grade), 25 AL linoleic acid and 200 mg Tween 40 were added. Chloroform was completely evaporated using a vacuum evaporator. Then 100 mL distilled water saturated with oxygen (30 min, 100 mL/min) was added with a vigorous shaking. 2 500 Al of this reaction mixture was distributed to test tubes and 350 Al portions of the extract, prepared at 2 g/L concentrations were added and the emulsion system was incubated up to 48 hours at room temperature. After this incubation period the absorbance of the mixtures was measured at 490 nm. The antioxidant effect of the sample was compared with those of BHT and blank. Absorbance: 0.0012 Gallic acid (Ag) + 0.0033. Superoxide anion scavenging activity The inhibition of NBT-reduction by photochemically generated SO2 (Beauchamp and Fridovich, 1971) was used to determine the superoxide anion scavenging activity of the test samples. The reaction mixture contained 56 AM (NBT), 0.01 M methionine, 1.17 AM riboflavin, 20 AM NaCN and 0.05 M phosphate buffer, pH 7.8. Superoxide was measured by the increasing amount of the absorbance at 560 nm at 30 8C after 6 min incubation from the beginning of illumination. The plant extracts and the reference substance caffeic acid were assayed at varying concentrations with three repetitions (inhibition between 20 and 80%). IC50 (concentrations, required to inhibit NBT reduction by 50%) values were calculated from dose-inhibition curves. The influence of superoxide dismutase (SOD from bovine erythrocytes, 4870 U/mg) on non-enzymatic reduction of NBT was determined by addition of varying amounts of SOD to the above reaction mixture. Antiviral activity Cells, media and viruses Madin-Darby canine kidney (MDCK) cells were passaged in Dulbecco’s Eagle medium, supplemented with 5% FCS and antibiotics (100 IU/mL benzyl penicillin and 100 Ag/mL streptomycin). MDCK were cultivated at 37 8C in the presence of 5% CO2 till the formation of confluent monolayers. In the antiviral experiments 0.5% FCS was added. MDCK cells were kindly provided by Mrs. I. Roeva, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia. The human influenza virus A/Aichi/2/68 (H3N2) (A/Aichi) was adapted to MDCK cells and was cultivated in the presence of 2 Ag/mL trypsin; the virus stock was stored at 80 8C. The virus infectious titre was 106.3 TCID50 (50% tissue culture infectious doses)/mL and the hemagglutination titre was 4096. The virus was from the collection of the Institute of Microbiology, Bulgarian Academy of Sciences. Cellular toxicity The cell-toxic effect was examined following the cytopathic effect of the preparations (CPE) as described in (Serkedjieva and Hay, 1998). The dose, causing visible changes in cell morphology or sheet density in 50% of intact cells was evaluated from graphic plots (50% toxic concentration, TC50).
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Cytopathogenic effect (CPE) reduction assay was as described earlier (Serkedjieva and Hay, 1998). A reduction of CPE N50% was considered to indicate a significant virus-inhibitory effect. The dose, reducing CPE by 50% with respect to virus control was estimated (50% effective concentration, EC50). The selectivity index (SI) was determined by the ratio TC50/EC50. Hemagglutination (HA) assay was as described in (Serkedjieva and Hay, 1998). Enzyme linked immunosorbent assay (ELISA) of hemagglutinin (HA) expression was performed with monoclonal antibody (MAb) to viral HA and protein A-horseradish peroxidase conjugate as described earlier (Serkedjieva and Hay, 1998). The optical densities (OD450) were measured and expressed as % of virus control. Dr. Alan Douglas of the WHO Influenza Centre, Mill Hill, London, UK kindly provided the MAb (HC3N1B26). In all experiments non-drug treated, mock-infected cells were used as a cell control and non-drug treated, virus-infected cells - as a virus control. The selective anti-influenza drug rimantadine hydrochloride (2 Ag/mL) was used as a positive control. For comparative reasons the effect of caffeic acid was tested. Protective effect in vivo The protective effect was examined in white mice, infected nasally (i.n.) with 10 LD50 of A/ Aichi, adapted to mice lungs (A/Aichi-ad), as described in (Serkedjieva and Ivanova, 1997). PC and its fractions were inoculated under light ether anesthesia by i.n. instillation 3 h before infection, in the dose 10 mg/kg, in the volume of 0.05 mL PBS. Mice were observed for death daily for 14 days after viral challenge. The protection was estimated by the reduction of the rate of mortality and prolongation of mean survival time (MST). The index of protection (PI) was determined from the equation (PR-1)/PR 100, where PR (ratio of protection) is Mcontrol/ Mexperiment and M is mortality. Statistical methods Student’s t-test was used for the statistical analyses of the results. All experiments were done in triplicate. Results are given as mean arithmetic values F SD. TTTp b 0.001, TTp b 0.01, Tp b 0.05.
Results Antioxidant activity Total soluble phenolic constituents of the total MeOH extract, measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). The antioxidant potential of the total MeOH extract was determined by three complementary methods. The free radical scavenging activity of PC, evaluated by the DPPH method, is presented in Fig. 1. The preparation was able to reduce the stable free radical DPPH to the yellow-colored 1,1-Diphenyl-2-picrylhydrazyl with an IC50 = 13.86 F 0.84 Ag/mL. The extract exhibited better free radical scavenging activity than the synthetic antioxidant agent BHT (IC50 = 19.81 F 0.05 Ag/mL). The extract exhibited a strong antioxidant capacity also in the h-carotene-linoleic acid test system, achieving 88–89% inhibition that was as strong as that of BHT (Fig. 2).
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Fig. 1. Free radical scavenging effect of the total MeOH extract from Geranium sanguineum L. (DPPH assay).
Further the antioxidant effects of the total MeOH extract and its BuOH and EtOAc fractions were evaluated by suppression of the superoxide anion radicals generated in a photochemical system in the presence of the test samples. The results are shown in Fig. 3. The total extract and EtOAc fraction exhibited a strong SOD-like effect, comparable to that of caffeic acid, used as a positive control. The best SO2 scavenging activity was shown by the EtOAc fraction, followed by the total extract. These preparations inhibited the development of the color, produced during the reaction of SO2 with NBT by 38 and 55% respectively. The values obtained were similar to that of caffeic acid (43% inhibition). Conversely, the BuOH fraction increased the reduction of NBT; presumably additional O2 radicals were generated in its presence. Moreover, the samples of total extract and its EtOAc fraction suppressed SO2 release in a dosedependent manner (Fig. 4). Inhibition of NBT-reduction by superoxide in the presence of the tested preparations increased with the raise of their concentrations. All measurements were compared with control experiments using enhanced levels of SOD or caffeic acid as reference scavengers. The results showed that the total extract and its EtOAc fraction had antioxidative potential, similar to positive controls. On the other hand,
Total extract
BHT
0
20
40
60
80
100
Inhibition, % Fig. 2. Antioxidant activity of the total MeOH extract from Geranium sanguineum L. (h-carotene-linoleic acid assay). The extract was prepared at 2 g/L concentrations.
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caffeic acid EtOAc fr. BuOH fr. Total extr. Control 0.0
0.1
0.2
0.3
0.4
0.5
0.6
E560 Fig. 3. Inhibitory effect of the polyphenol extracts from Geranium sanguineum L. on the reduction of NBT by photochemically generated superoxide anion radicals. Samples contained 3.5 Ag/mL of the corresponding preparations.
increased amount of BuOH fraction in the incubation mixture led to higher NBT-reduction levels up to 3.5 Ag/mL, approaching saturation thereafter. The 50% SO2 scavenging concentrations (IC50) of the total extract and its EtOAc fraction were calculated from the dose-activity curves. The IC50 were found to be 26.0 and 2.95 Ag/mL, respectively. As positive controls, 50% inhibitory concentrations of SOD and caffeic acid were detected as 1.04 and 4.9 Ag/mL, respectively. Further we studied the combined O2 scavenging effect of the plant preparations in the dose 3.5 Ag/ mL, applied together with 5.5 U/mL of commercial SOD (Fig. 5). The combination resulted in 50% and 70% decrease in NBT-reduction respectively, compared to the inhibition caused by SOD alone. The combined application of the BuOH fraction with SOD caused two-fold increase in NBT-reduction value. Antiviral activity In vitro the virus-inhibitory effects of the total extract and its BuOH and EtOAc fractions were tested in parallel on the reproduction of influenza A/Aichi virus in MDCK cells. TC50 for MDCK cells was 82.0, 169.0 and 65.0 Ag/mL respectively (Table 1). The specific influenza virus-inhibitory 0.5 BuOH fr. EtOAc fr. Total extract caffeic acid SOD
E560 nm
0.4 0.3 0.2 0.1 0.0 0
5
10
15
20
25
30
Concentration, µg/ml Fig. 4. Dose-dependence of the superoxide anion scavenging effect of the polyphenol extracts from Geranium sanguineum L.
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0.1
0.2
0.3
0.4
0.5
E560 nm Fig. 5. Inhibitory effect of the polyphenol extracts from Geranium sanguineum L. on the NBT-reduction by photochemically generated superoxide anions in the presence of SOD. Samples contained 5.5 U/mL SOD from bovine erythrocytes and 3.5 Ag/ mL of the corresponding preparation.
activity of the extracts, tested in the CPE-reduction and the HA-assay, showed that PC and the BuOH fraction significantly reduced the virus-induced CPE (Table 1) and the production of hemagglutinin, while the EtOH fraction was not effective in subtoxic concentrations. EC50-s were 2.5 and 9.3 Ag/mL respectively; the selectivity indices were 32.6 and 16.8 respectively. In the dose 20 Ag/mL both preparations practically abolished viral replication. Caffeic acid inhibited viral reproduction with EC50 = 9.8 Ag/mL and SI = 12.2. Similar results were obtained in ELISA with MAb to the viral HA (Fig. 6). The EC50-s of PC, the BuOH fraction and caffeic acid were 6.3, 2.0 and 10.0 Ag/mL respectively; the EC50 of the EtOAc fraction was N of its TC50. Thus by three complementary methods it was confirmed that the BuOH fraction contained the majority of the antiviral effect in vitro. The protective effect of the plant preparations was studied in the murine model of experimental influenza infection with virus A/Aichi. The EtOAc fraction contained the majority of the protective effect in vivo (PI = 67.0%), the protection was close to that of the whole extract (PI = 71.4%); the protection by the BuOH fraction was not significant (Table 1). Table 1 Anti-influenza and superoxide scavenging effect of the preparations from Geranium sanguineum L Plant preparation
Anti-influenza A/Aichi virus effect
O2 scavenging effect
Tannins T (%) Flavonoids T (%) in MDCK cells (SIa, %) in mice (PI b, %) (IC50 c, Ag/mL) Total MeOH extract 11.02 BuOH fraction 2.1 EtOAc fraction 4.41 Caffeic acid a
0.14 0.071 0.176
32.6 16.8 b1 12.4
71.4 28.6 67.4 + **
26.0 Inactive 4.0 5.4
Selectivity index = TC50/EC50, where TC50 is 50% toxic concentration to MDCK cells, EC50 is 50% effective virusinhibitory concentration. b Protective index = (PR-1)/PR 100, where PR (ratio of protection) is Mcontrol/Mexperiment and M is mortality. c Concentration necessary to inhibit the photochemical reduction of NBT by 50%, the combined O2 scavenging effect of the plant preparations in the dose 3.5 Ag/mL, applied together with commercial 5.5 U/mL SOD. * (Ivancheva et al., 1992, 1996). ** (Polikoff et al., 1966).
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OD450, % of control
100 80 60 40 20 0 0
0,4
2
10
50
dose, µg/mL Total extract EtOAc fr
BuOH fr caffeic acid
Fig. 6. Inhibitory effect of the polyphenol extracts from Geranium sanguineum L. on the expression of hemagglutinin on the surface of A/Aichi-infected MDCK cells (ELISA assay).
The results on the antioxidant and antiviral effects of the tested extracts are summarized in Table 1 together with some earlier data on the polyphenol content of the preparations (Ivancheva et al., 1992, 1996).
Discussion The principal objective of the study was to provide data about the in vitro antioxidant and radicalscavenging potential of the polyphenol extract isolated from the medicinal plant Geranium sanguineum L. This evidence appeared necessary for the evaluation of the marked protective effect of PC in the lethal murine experimental influenza A/Aichi infection (Serkedjieva and Manolova, 1992). PC administered intranasally or by aerosol reduced mortality and prolonged the survival time of infected mice. The investigations on the selectivity and specificity of the virus-inhibitory effect in vitro showed that it was fairly modest (Serkedjieva and Hay, 1998) and this was in contrast with its significant protective effect in vivo. Thus the therapeutic effect of PC remained to be explained. We presumed that the protection may possibly be attributed to a combination of more than one biological activities - selective antiviral action, non-selective immunomodulating activity and some non-specific biological and pharmacological interactions, known for natural polyphenols, such as protein-binding, radical-scavenging and antioxidant activities. In fact we demonstrated in model systems that the extract possessed multiple biological and pharmacological activities. In addition to inhibiting the reproduction of influenza viruses type A and B in vitro and in ovo and protecting mice from mortality in the experimental influenza A virus infection, the preparation suppressed the growth of a series of pathogenic bacteria and fungi (Ivancheva et al., 1992), demonstrated a stimulating effect in vitro on the phagocytic activity of murine blood polymorphous nuclear leucocytes (PMNs) and peritoneal macrophages, showed a beneficial effect on the spontaneous NO production by the macrophages (Toshkova et al., 2004), inhibited the proteolytic activity of trypsin (Antonova-Nikolova et al., 2004). It is accepted that acute viral infections are accompanied by profound changes in cell/tissue metabolism, which lead to intensive generation of reactive oxygen species. The latter may ultimately
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cause aggravation in the pathogenesis of the infection. As the influenza virus infection is concerned, it has been found recently that the main cause of mortality from influenza virus-induced pneumonia is the cytotoxicity, which in its turn is determined by the substantially increased levels of SO2 rather than by the viral replication per se in the bronchial epithelial cells (Akaike et al., 1996). Thus the use of antioxidants could be of great value in preventing the onset or the progression of the disease. All these observations induced us to investigate the antioxidant capacity of the polyphenol extracts from Geranium sanguineum. Two complementary test methods were employed for preliminary antioxidative screening of the total MeOH extract. The free radical scavenging activity was measured using the 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH), which is a stable free radical and in the presence of the total extract it was scavenged; the antioxidant activity was defined as the mean of free radical scavenging capacity (Fig. 1). As second test method employed, inhibition of the lipid oxidation was also used to measure antioxidant capacity. Inhibition of the breakdown of lipid hydroperoxides to unwanted volatile products allowed us to determine secondary antioxidants in related mechanisms. In the absence of antioxidants, oxidation products (lipid hydroperoxides, conjugated dienes and volatile byproducts) of linoleic acid simultaneously attack on h-carotene, resulting in bleaching of its characteristic yellow colour in ethanolic solution. In the presence of the total MeOH extract oxidation products were scavenged and bleaching was prevented (Fig. 2). Total soluble phenolic constituents of the total MeOH extract, measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). This observation leads to the conclusion that high soluble phenolics in the extract could be taken into account for the strong antioxidant activity observed in all used assays. Further investigation of the total extract and its fractions was performed in a non-enzymatic system - NBT, methionine and riboflavin. There SO2 were generated photochemically and SOD inhibited the reduction of NBT in concentration-dependent manner. The best S O2 scavenging activity was shown by the EtOAc fraction, followed by the total extract (Fig. 3). These preparations inhibited the development of the color, produced during the reaction of SO2 with NBT by 38 and 55% respectively. The values obtained were similar to that of caffeic acid (43% inhibition). Parejo et al., 2003 found also remarkably high SO2 scavenging activity mainly in the ethyl acetate fraction among different extracts from Bolivian plants. Conversely, the BuOH fraction increased the NBT-reduction; presumably additional O2 radicals were generated in its presence. The contrasting behavior of the EtOAc and BuOH fractions towards SO2 can not be explained simply in terms of their qualitative polyphenol content as the two extracts do not differ significantly in this respect (unpublished results). The varying proportions and possible interactions between the separate ingredients seem to be more significant for the observed opposite effects. Moreover, the samples of total extract and its EtOAc fraction suppressed SO2 release in a dose-dependent manner (Fig. 4). The 50% SO2 scavenging concentrations of the total extract and its EtOAc fraction were 26.0 and 2.95 Ag/mL respectively. These values were comparable to the polyphenol-containing extract from other medicinal plants (Khanom et al., 2000). In all experimental schemes the total MeOH extract and its EtOAc fraction showed similar antioxidative potential to positive controls, e.g. caffeic acid and SOD. On the other hand, enhanced amount of BuOH fraction in the incubation mixture led to higher NBT-reduction suggesting its prooxidant potential. The SO2 scavenging effect of the extracts, obtained from Geranium sanguineum L. was confirmed by measurement of inhibition of NBT-reduction in presence of commercial SOD (Fig. 5). The combination of SOD (5.5 U/mL) with the total extract or its EtOAc fraction (3.5 Ag/mL) resulted in 50% and 70% decrease in NBT- reduction respectively, compared to the inhibition by SOD alone. These results demonstrated synergistic effect between the antioxidant enzyme and the tested plant preparations,
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suggesting SOD-like activity of the extracts from Geranium. On the contrary, addition of the BuOH fraction to SOD-containing incubation mixture caused increase in NBT-reduction value. The results emerged a strong negative correlation between the superoxide scavenging enzyme and the BuOH fraction, confirming its prooxidant activity. A similar effect has been found for the water extract from Hypericum hyssopifolium subsp. elongatum (Cakir et al., 2003). In general, superoxide scavenging as well as superoxide generating activities have been reported for a variety of polyphenolic substances (Costantino et al., 1992; Habtemariam, 1997; Rice-Evans et al., 1977). In Table 1 we summarized the results from the present research together with some data on the polyphenol content of the preparations. The in vivo protective effect of the total MeOH extract and its EtOAc fraction show a distinct correlation with their SO2scavenging potential. The reverse effect, shown by the BuOH fraction (generation of SO2) is connected with the mode of its in vitro antiviral activity. It should be noted that the prooxidant BuOH fraction has lower tannin and flavonoid content although the differences are not great. This is in accordance with the findings that the antioxidant properties of flavonoids were effected mainly via scavenging of superoxide anions (Haslam, 1966; Rice-Evans et al., 1977). Interestingly a comparison between the two classes of compounds with respect to their DPPHscavenging activity showed that tannins have more potential than flavonoids because almost all the tannins demonstrated significant effect within a low concentration range, whereas the activity of flavonoids varied distinctively among the different compounds (Yokozawa et al., 1998). The medicinal plant Geranium sanguineum L. (Geraniaceae) is wide spread in Bulgaria. Aqueous and alcoholic extracts from its root are used in traditional medicine to treat gastrointestinal disorders and various infections and inflammatory conditions. It is frequently used in folk medicine also for the treatment of eruptive skin disease and as a disinfectant bath and poultice to the affected area. This species is reported to contain considerable amounts of polyphenol compounds, namely tannins, flavonoids and phenolic acids as the main constituents. Phytochemical investigation of the total MeOH extract from Geranium sanguineum L. revealed the presence of flavonoids - aglycones and glycosides (quercetin, quercetin 3-0-galactoside, morin, myricetin, kaempferol, rhamnasin, retusin, apigenin), phenolic acids (caffeic, ellagic, quinic, chlorogenic), gallotannins, catechins and maltol (Ivancheva et al., 1992, 1996). These phenols are well-known antioxidants and our results are in agreement with previous reports (Habtemariam, 1997; Haslam, 1966; Plumb et al., 1998; Rice-Evans et al., 1977; Vitor et al., 2004; Yokozawa et al., 1998). On the other hand though the antioxidant and radical scavenging potential of some pure polyphenol compounds is already known, it remains unclear how a complex mixture obtained from plant extracts functions. Moreover, the cooperative effect, which exists between different antioxidant constituents, means the overall action would be greater than the sum of the individual antioxidant activities. As the virus-inhibitory effect of the extract is concerned, we have demonstrated that this effect could not be attributed to one or few separate ingredients (unpublished). The presence of a diversity of biologically active compounds, as well as the possible synergistic action between them seemed to be more significant for the total antiviral effect. There are few reports on the comparative evaluation of the antioxidant and the antiviral activities of plant extracts and plant-derived substances. Two herbal preparations were screened for potential antiHIV activities (Aruoma et al., 1996), Euphorbia thymifolia L. was concluded to possess antioxidant and anti-HSV-2 action (Lin et al., 2002), the anti-HSV-1 and antioxidant effects of some fractions and flavonoids and proanthocyanidins, obtained from Crataegus sinaica were evaluated (Shahat et al., 2002), the in vitro antioxidant and anti-influenza virus activities of the essential oil and various extracts from Origanum acutidens were reported (Sokmen et al., 2004).
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The antioxidant effect of PC and its EtOAc fraction, established in the present investigation (Figs. 1– 5), might explain in part the marked protective effect of PC in the experimental influenza infection (Table 1, Serkedjieva and Manolova, 1992). These results need to be combined with in vivo data for adequate assessment of the antioxidant capacity of PC. Experiments in vivo are in progress to investigate on the interactions between the various biological activities of the plant preparation and to evaluate their role in the protective effect of PC in the murine experimental influenza infection. Some preliminary results show that PC beneficially modulated reactive oxygen species production in alveolar macrophages. The plant extract reduced significantly the level of H2O2 and NO, secreted by alveolar macrophages as a response to influenza virus infection. The effect on O2 production was moderate.
Conclusion The results from the present experiments demonstrated that the MeOH extract from the aerial roots of Geranium sanguineum L. manifested strong antioxidant and radical scavenging activities in model systems. The in vitro antioxidant properties of PC might be contributing to the overall protective effect of the extract in the lethal influenza A/Aichi virus infection.
Acknowledgments This study was supported by a research grant N K-1007 from the National Council for Science, Bulgaria and a collaborative project between the Bulgarian Academy of Sciences and TUBITAK, Turkey.
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Dapkevicius, A., Venskutonis, R., Van Beek, T.A., Linssen, P.H., 1998. Antioxidant activity of extracts obtained by different isolation procedures from some aromatic herbs grown in Lithuania. Journal of Food and Agricultural Sciences 77, 140 – 146. Habtemariam, S., 1997. Flavonoids as inhibitors or enhancers of the cytotoxicity of tumor necrosis factor-alpha in L-929 tumor cells. Journal of Natural Products 60, 775 – 778. Haslam, E., 1966. Natural polyphenols (vegetable tannins) as drugs: possible modes of action. Journal of Natural Products 59, 205 – 215. Ivancheva, S., Manolova, N., Serkedjieva, J., Dimov, V., Ivanovska, N., 1992. Polyphenols from Bulgarian medicinal plants. Basic Life Sciences 59, 717 – 728. Ivancheva, S., Stancheva, B., Serkedjieva, J., 1996. Polyphenol compounds in Geranium sanguineum L. Comptes Rendus of the Bulgarian Academy of Sciences 49, 41 – 43. Kahkonen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S., Heinonen, M., 1999. Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chemistry 47, 3562 – 3954. Khanom, F., Kayahara, H., Tadasa, K., 2000. Superoxide scavenging and prolyl endopeptidase inhibitory activities of Bangladeshi indigenous medicinal plants. Bioscience, Biotechnology and Biochemistry 64, 837 – 840. Lin, C.C., Cheng, H.Y., Yang, C.M., Lin, T.C., 2002. Antioxidant and antiviral activities of Euphorbia thymifolia L. Journal of Biomedical Sciences 9, 656 – 664. Parejo, I., Viladomat, F., Bastida, J., Rosas-Romero, A., Saavedra, G., Murcia, M.A., Jimenez, A.M., Codina, C., 2003. Investigation of Bolivian plant extracts for their radical scavenging activity and antioxidant activity. Life Sciences 73, 1667 – 1681. Pietta, P., Simonetti, P., Mauri, P., 1998. Antioxidant activity of selected medicinal plants. Journal of Agricultural and Food Chemistry 46, 4487 – 4490. Plumb, G.W., De Pascual-Teresa, S., Santos-Buelga, C., Cheynier, V., Williamson, G., 1998. Antioxidant properties of catechins and proanthocyanidins: effect of polymerisation, galloylation and glycosylation. Free Radical Research 29, 351 – 358. Polikoff, R., Lieberman, M., Cochran, K.W., Pascale, A.M., 1966. Effect of caffeic acid on mouse and ferret lung infected with influenza A virus. Antimicrobial Agents and Chemotherapy 5, 561 – 566. Rakotoarison, D.A., Gressier, B., Trotin, F., Brunet, C., Dine, T., Luyckx, M., Vasseur, J., Cazin, M., Cazin, J.C., Pinkas, M., 1997. Antioxidant activities of polyphenolic extracts from flowers, in vitro callus and cell suspension cultures of Crataegus monogyna. Pharmazie 52, 60 – 64. Rice-Evans, C.A., Miller, N.J., Paganga, J., 1977. Antioxidant properties of phenolic compounds. Trends in Plant Sciences 2, 152 – 159. Shahat, A.A., Cos, P., De Bruyne, T., Apers, S., Hammouda, F.M., Ismail, S.I., Azzam, S., Claeys, M., Goovaerts, E., Pieters, L., Vanden Berghe, D., Vlietinck, A.J., 2002. Antiviral and antioxidant activity of flavonoids and proanthocyanidins from Crataegus sinaica. Planta Medica 68, 539 – 541. Serkedjieva, J., Hay, A.J., 1998. In vitro anti-influenza virus activity of a plant preparation from Geranium sanguineum L. Antiviral Research 37, 221 – 230. Serkedjieva, J., Ivanova, E., 1997. Combined protective effect of an immunostimulatory bacterial preparation and rimantadine hydrochloride in experimental influenza A virus infection. Acta virologica 41, 65 – 70. Serkedjieva, J., Manolova, N., 1992. Plant polyphenol complex inhibits the reproduction of influenza and herpes simplex viruses. Basic Life Sciences 59, 705 – 715. Sokmen, M., Serkedjieva, J., Daferera, D., Gulluce, M., Polissiou, M., Tepe, B., Akpulat, A., Sahin, F., Sokmen, A., 2004. In vitro antioxidant, antimicrobial and antiviral activities of the essential oil and various extracts from herbal parts and callus cultures of Origanum acutidens. Journal of Agricultural and Food Chemistry 52, 3309 – 3312. Toshkova, R., Nikolova, N., Ivanova, E., Ivancheva, E., Serkedjieva, J., 2004. In vitro investigation on the effect of a plant polyphenol extract with antiviral activity on the functions of mice phagocyte cells. Pharmazie 59, 150 – 154. Vitor, R.F., Mota-Filipe, H., Teixeira, G., Borges, C., Rodrigues, A.I., Teixeira, A., Paulo, A., 2004. Flavonoids of an extract of Pterospartum tridentatum showing endothelial protection against oxidative injury. Journal of Ethnopharmacology 93, 363 – 370. Yokozawa, T., Chen, C.P., Dong, E., Tanaka, T., Nonaka, G.I., Nishioka, I., 1998. Study on the inhibitory effect of tannins and flavonoids against the 1,1-diphenyl-2 picrylhydrazyl radical. Biochemical Pharmacology 56, 213 – 222.
In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L Munevver Sokmena,1, Maria Angelovab,1, Ekaterina Krumovab, Svetlana Pashovab, Stefka Ivanchevac, Atalay Sokmend, Julia Serkedjievab,* a
Department of Biology, Faculty of Science and Literature, Cumhuriyet University, Sivas, Turkey Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. Georgy Bonchev St., 1113 Sofia, Bulgaria c Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria d Department of Chemistry, Faculty of Science and Literature, Cumhuriyet University, Sivas, Turkey
b
Received 27 July 2004; accepted 8 November 2004
Abstract Recent evidence shows that plant polyphenols exhibit antioxidant and radical scavenging properties. By three separate and complementary methods - DPPH assay, h-carotene-linoleic acid assay and NBT-reduction assay it was established that a polyphenol-rich extract from the medicinal plant Geranium sanguineum L. with strong anti-influenza virus activity, possessed antioxidant and radical scavenging capacities. For comparative reasons caffeic acid and the synthetic antioxidant BHT were used. Total soluble phenolic constituents of the MeOH extract measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). Further it was demonstrated that the EtOAc fraction, retaining the majority of the in vivo protective effect exhibited a strong O2scavenging activity while the n-BuOH fraction, containing the majority of the in vitro antiviral activity provoked generation of O2. The O2 scavenging activity of all three preparations correlated with the rate of the protective effect shown in the murine model of experimental influenza virus infection. The present results are in accordance with our intensive studies on the mode of the protective effect of the plant extract which showed positively that the protection may possibly be attributed to the combination of more than one
T Corresponding author. Tel.: +359 2 979 31 85; fax: +359 2 870 01 09. E-mail address: [email protected] (J. Serkedjieva). 1 These authors equally contributed to this work. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.11.020
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biological activities and that the use of antioxidants might be an useful approach in the treatment of influenza infection. D 2005 Elsevier Inc. All rights reserved. Keywords: Polyphenol extracts; Geranium sanguineum; Radical scavenging activity; Antioxidant effect; Influenza virus inhibition
Introduction There is abundant evidence that a great number of aromatic, spicy, medicinal and other plants contain chemical compounds exhibiting antioxidant properties. With this respect a particular interest has been given to plant polyphenols. The natural polyphenols have an ideal structure for capturing of free radicals (Haslam, 1966) and it was found that their antioxidant activity surpasses the effect of known antioxidants, such as the vitamins A and E (Rice-Evans et al., 1977). There are data about the antioxidant effects of plant extracts (Aruoma et al., 1996; Costantino et al., 1992; Kahkonen et al., 1999; Parejo et al., 2003; Pietta et al., 1998; Rakotoarison et al., 1997) and diverse groups of plant polyphenol compounds - flavonoids (Habtemariam, 1997; Rice-Evans et al., 1977; Vitor et al., 2004), tannins (Haslam, 1966; Yokozawa et al., 1998), catechins and proantocyanidins (Plumb et al., 1998), polyphenolic acids (Rice-Evans et al., 1977). Earlier investigations have proved that a semi-standardized polyphenol extract, obtained from the medicinal plant Geranium sanguineum L., designated as polyphenolic complex (PC), exhibited a pronounced anti-influenza virus effect in cell cultures and protected mice from mortality in the experimental influenza virus infection (Serkedjieva and Manolova, 1992). The virus-inhibitory activity of the plant extract was related to the presence of large quantities of polyphenol compounds. Phytochemical analysis of PC showed that it contained tannins (11.02%) flavonoids (0.14%), catechins and proanthocyanidines (2.1 mg/kg) (Ivancheva et al., 1992, 1996). The current study was undertaken to investigate on the in vitro antioxidant and radical scavenging effects of the plant preparation and its active fractions by applying established in vitro assays and to provide evidence for the possible relation between their antiviral and antioxidant capacities.
Materials and Methods Medicinal plant and extraction The medicinal plant Geranium sanguineum L. (Geraniaceae) collected in June-August in the stage of flowering in the Lulin Mountain. A specimen was deposited in the Herbarium of the Institute of Botany, Bulg. Acad. Sci. (SOM-5/91). The MeOH extract from the aerial roots was obtained as described earlier (Ivancheva et al., 1992) and designated as polyphenolic complex (PC). In short - ground air-dried aerial
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roots, were defatted with petroleum ether and treated with MeOH to fully extract the polyphenol components. The extract was filtered and lyophilized (yield 16%). The polyphenol content of the preparation was controlled by thin layer chromatography and by quantitative determination of tannins, flavonoids and catechins (Ivancheva et al., 1992, 1996). The total extract was extracted further with EtOAc and n-BuOH to obtain the corresponding fractions. Chemicals Rimantadine hydrochloride was obtained from Hoffman - La Roche Inc., Nutley, NJ, USA; protein Ahorseradish peroxidase conjugate was from Bio Rad Lab., Ca, USA; Dulbecco’s Eagle medium was from GibcoBRL, Scotland, UK, fetal calf serum (FCS) was from BioWhittaker Europe, Germany; DPPH, h-carotene, BHT, nitro blue tetrazolium (NBT), methionine, riboflavin, NaCN, superoxide dismutase (SOD) from bovine erytrocytes and trypsin were from Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany; caffeic acid was a gift from Prof. V. Bankova, Institute of Organic Chemistry, Bulgarian Academy of Sciences. Total phenolic constituents Total soluble phenolic constituents of the MeOH extract were determined by employing methods, involving Folin-Ciocalteu reagent and gallic acid as standard. 0.1 mL of the extract solution, containing 1000 Ag extract was taken in a volumetric flask, 46 mL distilled water and 1 mL Folin-Ciocalteu reagent were added and the flask was shaken thoroughly. After 3 min, a 3 mL solution of Na2CO3 (2%, w/v) was added and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance was measured at 760 nm. The same procedure was repeated with all standard gallic acid solutions (0–1000 Ag/0.1 mL) and a standard curve was obtained by the equation given below: Absorbance: 0.0012 Gallic acid (Ag) + 0.0033 Antioxidant activity DPPH assay The hydrogen atoms or electrons donation ability of the corresponding extract was measured from the bleaching of purple colored MeOH solution of DPPH. This spectrophotometric assay uses stable radical 1,1-Diphenyl-2-picrylhydrazyl (DPPH) as a reagent (Burits and Bucar, 2000; Cuendet et al., 1997). Fifty Al of various concentrations of the extract in MeOH were added to 5 mL of a 0.004% MeOH solution of DPPH. After a 30 min incubation period at room temperature the absorbance was read against a blank at 517 nm. Inhibition of free radical DPPH in percent was calculated according the formula: Inhibition % ¼ Ablank Asample =Ablank 100; where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated form the graph plotted inhibition percentage against extract concentration. Tests were carried out in triplicate. The synthetic antioxidant buthylated hydroxy toluene (BHT) was included in experiments as a positive control.
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b-carotene-linoleic acid assay In this assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation, causing discoloration of h-carotene (Dapkevicius et al., 1998). A stock solution of h-carotenelinoleic acid mixture was prepared as follows: 0.5 mg h-carotene was dissolved in 1 mL of chloroform (HPLC grade), 25 AL linoleic acid and 200 mg Tween 40 were added. Chloroform was completely evaporated using a vacuum evaporator. Then 100 mL distilled water saturated with oxygen (30 min, 100 mL/min) was added with a vigorous shaking. 2 500 Al of this reaction mixture was distributed to test tubes and 350 Al portions of the extract, prepared at 2 g/L concentrations were added and the emulsion system was incubated up to 48 hours at room temperature. After this incubation period the absorbance of the mixtures was measured at 490 nm. The antioxidant effect of the sample was compared with those of BHT and blank. Absorbance: 0.0012 Gallic acid (Ag) + 0.0033. Superoxide anion scavenging activity The inhibition of NBT-reduction by photochemically generated SO2 (Beauchamp and Fridovich, 1971) was used to determine the superoxide anion scavenging activity of the test samples. The reaction mixture contained 56 AM (NBT), 0.01 M methionine, 1.17 AM riboflavin, 20 AM NaCN and 0.05 M phosphate buffer, pH 7.8. Superoxide was measured by the increasing amount of the absorbance at 560 nm at 30 8C after 6 min incubation from the beginning of illumination. The plant extracts and the reference substance caffeic acid were assayed at varying concentrations with three repetitions (inhibition between 20 and 80%). IC50 (concentrations, required to inhibit NBT reduction by 50%) values were calculated from dose-inhibition curves. The influence of superoxide dismutase (SOD from bovine erythrocytes, 4870 U/mg) on non-enzymatic reduction of NBT was determined by addition of varying amounts of SOD to the above reaction mixture. Antiviral activity Cells, media and viruses Madin-Darby canine kidney (MDCK) cells were passaged in Dulbecco’s Eagle medium, supplemented with 5% FCS and antibiotics (100 IU/mL benzyl penicillin and 100 Ag/mL streptomycin). MDCK were cultivated at 37 8C in the presence of 5% CO2 till the formation of confluent monolayers. In the antiviral experiments 0.5% FCS was added. MDCK cells were kindly provided by Mrs. I. Roeva, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia. The human influenza virus A/Aichi/2/68 (H3N2) (A/Aichi) was adapted to MDCK cells and was cultivated in the presence of 2 Ag/mL trypsin; the virus stock was stored at 80 8C. The virus infectious titre was 106.3 TCID50 (50% tissue culture infectious doses)/mL and the hemagglutination titre was 4096. The virus was from the collection of the Institute of Microbiology, Bulgarian Academy of Sciences. Cellular toxicity The cell-toxic effect was examined following the cytopathic effect of the preparations (CPE) as described in (Serkedjieva and Hay, 1998). The dose, causing visible changes in cell morphology or sheet density in 50% of intact cells was evaluated from graphic plots (50% toxic concentration, TC50).
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Cytopathogenic effect (CPE) reduction assay was as described earlier (Serkedjieva and Hay, 1998). A reduction of CPE N50% was considered to indicate a significant virus-inhibitory effect. The dose, reducing CPE by 50% with respect to virus control was estimated (50% effective concentration, EC50). The selectivity index (SI) was determined by the ratio TC50/EC50. Hemagglutination (HA) assay was as described in (Serkedjieva and Hay, 1998). Enzyme linked immunosorbent assay (ELISA) of hemagglutinin (HA) expression was performed with monoclonal antibody (MAb) to viral HA and protein A-horseradish peroxidase conjugate as described earlier (Serkedjieva and Hay, 1998). The optical densities (OD450) were measured and expressed as % of virus control. Dr. Alan Douglas of the WHO Influenza Centre, Mill Hill, London, UK kindly provided the MAb (HC3N1B26). In all experiments non-drug treated, mock-infected cells were used as a cell control and non-drug treated, virus-infected cells - as a virus control. The selective anti-influenza drug rimantadine hydrochloride (2 Ag/mL) was used as a positive control. For comparative reasons the effect of caffeic acid was tested. Protective effect in vivo The protective effect was examined in white mice, infected nasally (i.n.) with 10 LD50 of A/ Aichi, adapted to mice lungs (A/Aichi-ad), as described in (Serkedjieva and Ivanova, 1997). PC and its fractions were inoculated under light ether anesthesia by i.n. instillation 3 h before infection, in the dose 10 mg/kg, in the volume of 0.05 mL PBS. Mice were observed for death daily for 14 days after viral challenge. The protection was estimated by the reduction of the rate of mortality and prolongation of mean survival time (MST). The index of protection (PI) was determined from the equation (PR-1)/PR 100, where PR (ratio of protection) is Mcontrol/ Mexperiment and M is mortality. Statistical methods Student’s t-test was used for the statistical analyses of the results. All experiments were done in triplicate. Results are given as mean arithmetic values F SD. TTTp b 0.001, TTp b 0.01, Tp b 0.05.
Results Antioxidant activity Total soluble phenolic constituents of the total MeOH extract, measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). The antioxidant potential of the total MeOH extract was determined by three complementary methods. The free radical scavenging activity of PC, evaluated by the DPPH method, is presented in Fig. 1. The preparation was able to reduce the stable free radical DPPH to the yellow-colored 1,1-Diphenyl-2-picrylhydrazyl with an IC50 = 13.86 F 0.84 Ag/mL. The extract exhibited better free radical scavenging activity than the synthetic antioxidant agent BHT (IC50 = 19.81 F 0.05 Ag/mL). The extract exhibited a strong antioxidant capacity also in the h-carotene-linoleic acid test system, achieving 88–89% inhibition that was as strong as that of BHT (Fig. 2).
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Fig. 1. Free radical scavenging effect of the total MeOH extract from Geranium sanguineum L. (DPPH assay).
Further the antioxidant effects of the total MeOH extract and its BuOH and EtOAc fractions were evaluated by suppression of the superoxide anion radicals generated in a photochemical system in the presence of the test samples. The results are shown in Fig. 3. The total extract and EtOAc fraction exhibited a strong SOD-like effect, comparable to that of caffeic acid, used as a positive control. The best SO2 scavenging activity was shown by the EtOAc fraction, followed by the total extract. These preparations inhibited the development of the color, produced during the reaction of SO2 with NBT by 38 and 55% respectively. The values obtained were similar to that of caffeic acid (43% inhibition). Conversely, the BuOH fraction increased the reduction of NBT; presumably additional O2 radicals were generated in its presence. Moreover, the samples of total extract and its EtOAc fraction suppressed SO2 release in a dosedependent manner (Fig. 4). Inhibition of NBT-reduction by superoxide in the presence of the tested preparations increased with the raise of their concentrations. All measurements were compared with control experiments using enhanced levels of SOD or caffeic acid as reference scavengers. The results showed that the total extract and its EtOAc fraction had antioxidative potential, similar to positive controls. On the other hand,
Total extract
BHT
0
20
40
60
80
100
Inhibition, % Fig. 2. Antioxidant activity of the total MeOH extract from Geranium sanguineum L. (h-carotene-linoleic acid assay). The extract was prepared at 2 g/L concentrations.
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caffeic acid EtOAc fr. BuOH fr. Total extr. Control 0.0
0.1
0.2
0.3
0.4
0.5
0.6
E560 Fig. 3. Inhibitory effect of the polyphenol extracts from Geranium sanguineum L. on the reduction of NBT by photochemically generated superoxide anion radicals. Samples contained 3.5 Ag/mL of the corresponding preparations.
increased amount of BuOH fraction in the incubation mixture led to higher NBT-reduction levels up to 3.5 Ag/mL, approaching saturation thereafter. The 50% SO2 scavenging concentrations (IC50) of the total extract and its EtOAc fraction were calculated from the dose-activity curves. The IC50 were found to be 26.0 and 2.95 Ag/mL, respectively. As positive controls, 50% inhibitory concentrations of SOD and caffeic acid were detected as 1.04 and 4.9 Ag/mL, respectively. Further we studied the combined O2 scavenging effect of the plant preparations in the dose 3.5 Ag/ mL, applied together with 5.5 U/mL of commercial SOD (Fig. 5). The combination resulted in 50% and 70% decrease in NBT-reduction respectively, compared to the inhibition caused by SOD alone. The combined application of the BuOH fraction with SOD caused two-fold increase in NBT-reduction value. Antiviral activity In vitro the virus-inhibitory effects of the total extract and its BuOH and EtOAc fractions were tested in parallel on the reproduction of influenza A/Aichi virus in MDCK cells. TC50 for MDCK cells was 82.0, 169.0 and 65.0 Ag/mL respectively (Table 1). The specific influenza virus-inhibitory 0.5 BuOH fr. EtOAc fr. Total extract caffeic acid SOD
E560 nm
0.4 0.3 0.2 0.1 0.0 0
5
10
15
20
25
30
Concentration, µg/ml Fig. 4. Dose-dependence of the superoxide anion scavenging effect of the polyphenol extracts from Geranium sanguineum L.
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0.1
0.2
0.3
0.4
0.5
E560 nm Fig. 5. Inhibitory effect of the polyphenol extracts from Geranium sanguineum L. on the NBT-reduction by photochemically generated superoxide anions in the presence of SOD. Samples contained 5.5 U/mL SOD from bovine erythrocytes and 3.5 Ag/ mL of the corresponding preparation.
activity of the extracts, tested in the CPE-reduction and the HA-assay, showed that PC and the BuOH fraction significantly reduced the virus-induced CPE (Table 1) and the production of hemagglutinin, while the EtOH fraction was not effective in subtoxic concentrations. EC50-s were 2.5 and 9.3 Ag/mL respectively; the selectivity indices were 32.6 and 16.8 respectively. In the dose 20 Ag/mL both preparations practically abolished viral replication. Caffeic acid inhibited viral reproduction with EC50 = 9.8 Ag/mL and SI = 12.2. Similar results were obtained in ELISA with MAb to the viral HA (Fig. 6). The EC50-s of PC, the BuOH fraction and caffeic acid were 6.3, 2.0 and 10.0 Ag/mL respectively; the EC50 of the EtOAc fraction was N of its TC50. Thus by three complementary methods it was confirmed that the BuOH fraction contained the majority of the antiviral effect in vitro. The protective effect of the plant preparations was studied in the murine model of experimental influenza infection with virus A/Aichi. The EtOAc fraction contained the majority of the protective effect in vivo (PI = 67.0%), the protection was close to that of the whole extract (PI = 71.4%); the protection by the BuOH fraction was not significant (Table 1). Table 1 Anti-influenza and superoxide scavenging effect of the preparations from Geranium sanguineum L Plant preparation
Anti-influenza A/Aichi virus effect
O2 scavenging effect
Tannins T (%) Flavonoids T (%) in MDCK cells (SIa, %) in mice (PI b, %) (IC50 c, Ag/mL) Total MeOH extract 11.02 BuOH fraction 2.1 EtOAc fraction 4.41 Caffeic acid a
0.14 0.071 0.176
32.6 16.8 b1 12.4
71.4 28.6 67.4 + **
26.0 Inactive 4.0 5.4
Selectivity index = TC50/EC50, where TC50 is 50% toxic concentration to MDCK cells, EC50 is 50% effective virusinhibitory concentration. b Protective index = (PR-1)/PR 100, where PR (ratio of protection) is Mcontrol/Mexperiment and M is mortality. c Concentration necessary to inhibit the photochemical reduction of NBT by 50%, the combined O2 scavenging effect of the plant preparations in the dose 3.5 Ag/mL, applied together with commercial 5.5 U/mL SOD. * (Ivancheva et al., 1992, 1996). ** (Polikoff et al., 1966).
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OD450, % of control
100 80 60 40 20 0 0
0,4
2
10
50
dose, µg/mL Total extract EtOAc fr
BuOH fr caffeic acid
Fig. 6. Inhibitory effect of the polyphenol extracts from Geranium sanguineum L. on the expression of hemagglutinin on the surface of A/Aichi-infected MDCK cells (ELISA assay).
The results on the antioxidant and antiviral effects of the tested extracts are summarized in Table 1 together with some earlier data on the polyphenol content of the preparations (Ivancheva et al., 1992, 1996).
Discussion The principal objective of the study was to provide data about the in vitro antioxidant and radicalscavenging potential of the polyphenol extract isolated from the medicinal plant Geranium sanguineum L. This evidence appeared necessary for the evaluation of the marked protective effect of PC in the lethal murine experimental influenza A/Aichi infection (Serkedjieva and Manolova, 1992). PC administered intranasally or by aerosol reduced mortality and prolonged the survival time of infected mice. The investigations on the selectivity and specificity of the virus-inhibitory effect in vitro showed that it was fairly modest (Serkedjieva and Hay, 1998) and this was in contrast with its significant protective effect in vivo. Thus the therapeutic effect of PC remained to be explained. We presumed that the protection may possibly be attributed to a combination of more than one biological activities - selective antiviral action, non-selective immunomodulating activity and some non-specific biological and pharmacological interactions, known for natural polyphenols, such as protein-binding, radical-scavenging and antioxidant activities. In fact we demonstrated in model systems that the extract possessed multiple biological and pharmacological activities. In addition to inhibiting the reproduction of influenza viruses type A and B in vitro and in ovo and protecting mice from mortality in the experimental influenza A virus infection, the preparation suppressed the growth of a series of pathogenic bacteria and fungi (Ivancheva et al., 1992), demonstrated a stimulating effect in vitro on the phagocytic activity of murine blood polymorphous nuclear leucocytes (PMNs) and peritoneal macrophages, showed a beneficial effect on the spontaneous NO production by the macrophages (Toshkova et al., 2004), inhibited the proteolytic activity of trypsin (Antonova-Nikolova et al., 2004). It is accepted that acute viral infections are accompanied by profound changes in cell/tissue metabolism, which lead to intensive generation of reactive oxygen species. The latter may ultimately
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cause aggravation in the pathogenesis of the infection. As the influenza virus infection is concerned, it has been found recently that the main cause of mortality from influenza virus-induced pneumonia is the cytotoxicity, which in its turn is determined by the substantially increased levels of SO2 rather than by the viral replication per se in the bronchial epithelial cells (Akaike et al., 1996). Thus the use of antioxidants could be of great value in preventing the onset or the progression of the disease. All these observations induced us to investigate the antioxidant capacity of the polyphenol extracts from Geranium sanguineum. Two complementary test methods were employed for preliminary antioxidative screening of the total MeOH extract. The free radical scavenging activity was measured using the 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH), which is a stable free radical and in the presence of the total extract it was scavenged; the antioxidant activity was defined as the mean of free radical scavenging capacity (Fig. 1). As second test method employed, inhibition of the lipid oxidation was also used to measure antioxidant capacity. Inhibition of the breakdown of lipid hydroperoxides to unwanted volatile products allowed us to determine secondary antioxidants in related mechanisms. In the absence of antioxidants, oxidation products (lipid hydroperoxides, conjugated dienes and volatile byproducts) of linoleic acid simultaneously attack on h-carotene, resulting in bleaching of its characteristic yellow colour in ethanolic solution. In the presence of the total MeOH extract oxidation products were scavenged and bleaching was prevented (Fig. 2). Total soluble phenolic constituents of the total MeOH extract, measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). This observation leads to the conclusion that high soluble phenolics in the extract could be taken into account for the strong antioxidant activity observed in all used assays. Further investigation of the total extract and its fractions was performed in a non-enzymatic system - NBT, methionine and riboflavin. There SO2 were generated photochemically and SOD inhibited the reduction of NBT in concentration-dependent manner. The best S O2 scavenging activity was shown by the EtOAc fraction, followed by the total extract (Fig. 3). These preparations inhibited the development of the color, produced during the reaction of SO2 with NBT by 38 and 55% respectively. The values obtained were similar to that of caffeic acid (43% inhibition). Parejo et al., 2003 found also remarkably high SO2 scavenging activity mainly in the ethyl acetate fraction among different extracts from Bolivian plants. Conversely, the BuOH fraction increased the NBT-reduction; presumably additional O2 radicals were generated in its presence. The contrasting behavior of the EtOAc and BuOH fractions towards SO2 can not be explained simply in terms of their qualitative polyphenol content as the two extracts do not differ significantly in this respect (unpublished results). The varying proportions and possible interactions between the separate ingredients seem to be more significant for the observed opposite effects. Moreover, the samples of total extract and its EtOAc fraction suppressed SO2 release in a dose-dependent manner (Fig. 4). The 50% SO2 scavenging concentrations of the total extract and its EtOAc fraction were 26.0 and 2.95 Ag/mL respectively. These values were comparable to the polyphenol-containing extract from other medicinal plants (Khanom et al., 2000). In all experimental schemes the total MeOH extract and its EtOAc fraction showed similar antioxidative potential to positive controls, e.g. caffeic acid and SOD. On the other hand, enhanced amount of BuOH fraction in the incubation mixture led to higher NBT-reduction suggesting its prooxidant potential. The SO2 scavenging effect of the extracts, obtained from Geranium sanguineum L. was confirmed by measurement of inhibition of NBT-reduction in presence of commercial SOD (Fig. 5). The combination of SOD (5.5 U/mL) with the total extract or its EtOAc fraction (3.5 Ag/mL) resulted in 50% and 70% decrease in NBT- reduction respectively, compared to the inhibition by SOD alone. These results demonstrated synergistic effect between the antioxidant enzyme and the tested plant preparations,
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suggesting SOD-like activity of the extracts from Geranium. On the contrary, addition of the BuOH fraction to SOD-containing incubation mixture caused increase in NBT-reduction value. The results emerged a strong negative correlation between the superoxide scavenging enzyme and the BuOH fraction, confirming its prooxidant activity. A similar effect has been found for the water extract from Hypericum hyssopifolium subsp. elongatum (Cakir et al., 2003). In general, superoxide scavenging as well as superoxide generating activities have been reported for a variety of polyphenolic substances (Costantino et al., 1992; Habtemariam, 1997; Rice-Evans et al., 1977). In Table 1 we summarized the results from the present research together with some data on the polyphenol content of the preparations. The in vivo protective effect of the total MeOH extract and its EtOAc fraction show a distinct correlation with their SO2scavenging potential. The reverse effect, shown by the BuOH fraction (generation of SO2) is connected with the mode of its in vitro antiviral activity. It should be noted that the prooxidant BuOH fraction has lower tannin and flavonoid content although the differences are not great. This is in accordance with the findings that the antioxidant properties of flavonoids were effected mainly via scavenging of superoxide anions (Haslam, 1966; Rice-Evans et al., 1977). Interestingly a comparison between the two classes of compounds with respect to their DPPHscavenging activity showed that tannins have more potential than flavonoids because almost all the tannins demonstrated significant effect within a low concentration range, whereas the activity of flavonoids varied distinctively among the different compounds (Yokozawa et al., 1998). The medicinal plant Geranium sanguineum L. (Geraniaceae) is wide spread in Bulgaria. Aqueous and alcoholic extracts from its root are used in traditional medicine to treat gastrointestinal disorders and various infections and inflammatory conditions. It is frequently used in folk medicine also for the treatment of eruptive skin disease and as a disinfectant bath and poultice to the affected area. This species is reported to contain considerable amounts of polyphenol compounds, namely tannins, flavonoids and phenolic acids as the main constituents. Phytochemical investigation of the total MeOH extract from Geranium sanguineum L. revealed the presence of flavonoids - aglycones and glycosides (quercetin, quercetin 3-0-galactoside, morin, myricetin, kaempferol, rhamnasin, retusin, apigenin), phenolic acids (caffeic, ellagic, quinic, chlorogenic), gallotannins, catechins and maltol (Ivancheva et al., 1992, 1996). These phenols are well-known antioxidants and our results are in agreement with previous reports (Habtemariam, 1997; Haslam, 1966; Plumb et al., 1998; Rice-Evans et al., 1977; Vitor et al., 2004; Yokozawa et al., 1998). On the other hand though the antioxidant and radical scavenging potential of some pure polyphenol compounds is already known, it remains unclear how a complex mixture obtained from plant extracts functions. Moreover, the cooperative effect, which exists between different antioxidant constituents, means the overall action would be greater than the sum of the individual antioxidant activities. As the virus-inhibitory effect of the extract is concerned, we have demonstrated that this effect could not be attributed to one or few separate ingredients (unpublished). The presence of a diversity of biologically active compounds, as well as the possible synergistic action between them seemed to be more significant for the total antiviral effect. There are few reports on the comparative evaluation of the antioxidant and the antiviral activities of plant extracts and plant-derived substances. Two herbal preparations were screened for potential antiHIV activities (Aruoma et al., 1996), Euphorbia thymifolia L. was concluded to possess antioxidant and anti-HSV-2 action (Lin et al., 2002), the anti-HSV-1 and antioxidant effects of some fractions and flavonoids and proanthocyanidins, obtained from Crataegus sinaica were evaluated (Shahat et al., 2002), the in vitro antioxidant and anti-influenza virus activities of the essential oil and various extracts from Origanum acutidens were reported (Sokmen et al., 2004).
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The antioxidant effect of PC and its EtOAc fraction, established in the present investigation (Figs. 1– 5), might explain in part the marked protective effect of PC in the experimental influenza infection (Table 1, Serkedjieva and Manolova, 1992). These results need to be combined with in vivo data for adequate assessment of the antioxidant capacity of PC. Experiments in vivo are in progress to investigate on the interactions between the various biological activities of the plant preparation and to evaluate their role in the protective effect of PC in the murine experimental influenza infection. Some preliminary results show that PC beneficially modulated reactive oxygen species production in alveolar macrophages. The plant extract reduced significantly the level of H2O2 and NO, secreted by alveolar macrophages as a response to influenza virus infection. The effect on O2 production was moderate.
Conclusion The results from the present experiments demonstrated that the MeOH extract from the aerial roots of Geranium sanguineum L. manifested strong antioxidant and radical scavenging activities in model systems. The in vitro antioxidant properties of PC might be contributing to the overall protective effect of the extract in the lethal influenza A/Aichi virus infection.
Acknowledgments This study was supported by a research grant N K-1007 from the National Council for Science, Bulgaria and a collaborative project between the Bulgarian Academy of Sciences and TUBITAK, Turkey.
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