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Antimicrobial activity of some Hypericum species
Phytomedicine 10: 511–516, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Antimicrobial activity of some Hypericum species R. Dall’Agnol1, A. Ferraz1, A. P. Bernardi1, D. Albring1, C. Nör1, L. Sarmento2, L. Lamb2, M. Hass2, G. von Poser1, and E. E. S. Schapoval1 1 2
Programa de Pós Graduação em Ciências Farmacêuticas, UFRGS. Porto Alegre, RS, Brazil TANAC SA. Montenegro, RS, Brazil
Summary The crude methanolic extracts of six species of Hypericum [H. caprifoliatum Cham. & Schlecht., H. carinatum Griseb., H. connatum Lam., H. ternum A.St. Hil., H. myrianthum Cham. & Schlecht. and H. polyanthemum Klotzsch ex Reichardt] growing in southern Brazil were analyzed for antimicrobial activity against several microorganisms (bacteria and fungi). The most active plant was H. caprifoliatum, which showed activity against Staphylococcus aureus. Only H. polyanthemum and H. ternum extracts were active against Bacillus subtilis. None of the crude methanolic extracts showed activity against S. epidermidis, Escherichia coli or Saccharomyces cerevisiae. Extracts from these species were evaluated chemically and tannin, flavonoid and phenolic acids were the prominent compounds. The plants contained quercitrin, hyperoside (except H. connatum) and, less frequently, isoquercitrin and chlorogenic acid. In contrast to H. perforatum, which has high concentrations of rutin, these species do not produce this flavonoid or it appears as traces. The tannin concentration varied between 5.1 and 16.7% in H. myrianthum and H. ternum, respectively. Key words: Hypericum, Guttiferae, antimicrobial activity, bacteria, fungi, flavonoids, tannins
Introduction Guttiferae is a large family comprised of more than 1000 species. The best-known genus is Hypericum, which encompasses various species used in traditional medicine around the world. Several antifungal (Décosterd et al. 1986), antibiotic (Ishiguro et al. 1986), antiviral (Jacobson et al. 2001) and anticancer (Jayasuriya and McChesney, 1989) compounds have been isolated from these species. The majority of the active compounds isolated are phenolic in nature. These plants have a strong tendency to accumulate phenolic compounds with the phloroglucinol substitution pattern. From the genus Hypericum many phloroglucinol derivatives have also been isolated, of which some are related to the well-known hyperforin, isolated from H. perforatum (Trifunovic et al. 1998) while others present a phloroglucinol unit conjugated with a filicinic acid moiety (Ishiguro et al. 1986; Rocha et al. 1995).
The phloroglucinol derivatives found frequently in the lipophilic fractions of several Hypericum species have demonstrated antifungal and antibacterial activities against microorganisms such as Staphylococcus aureus, Bacillus cereus, B. subtilis and Nocardia gardenen (Ishiguro et al. 1986; Décosterd et al. 1991; Jayasuriya et al. 1991; Rocha et al. 1995; Trifunovic et al. 1998). Their presence could justify the popular use of some Hypericum species as wound healing agents and in the treatment of some microbiological diseases (Ishiguro et al. 1986; Ishiguro et al. 1987; Jayasuria et al. 1991; Yamaki et al. 1994; Rocha et al. 1995). Other substances present in some species of Hypericum have also shown antimicrobial activity against various bacteria and fungi, including the benzopyrans (Jayasuriya et al. 1989; Décosterd et al. 1986), the xanthones (Ishiguro et al. 1999), the flavonoids (Ishiguro et al. 1993), and the tannins, recognized antimicrobial compounds (Scalbert, 1991). 0944-7113/03/10/06–07-511 $ 15.00/0
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The indiscriminate use of antibiotics has led to drug resistance in many bacterial strains, and the development of new antimicrobial compounds for resistant organisms is becoming critically important. Although the most important sources of antibiotics are molds, actinomycetes and bacteria, higher plants also present many classes of secondary metabolites with this property. Thus, efforts have been made to evaluate the antimicrobial activity of a wide array of natural products, including plant metabolites, in order to isolate and characterize novel compounds which could inhibit bacteria and fungi, or even serve as models for new molecules (Martini et al. 1998; Rates, 2001; Srinivasan et al. 2001). In this work we report the antimicrobial activity of methanolic extracts of six species of this genus (H. connatum, H. caprifoliatum, H. carinatum, H. polyanthemum, H. myrianthum and H. ternum evaluated against S. aureus, S. epidermidis, M. luteus, Escherichia coli, B. subtilis, Candida albicans and Saccharomyces cerevisiae) and two antibiotics used in therapy (chloramphenicol and nystatin) by the agar diffusion method. In our continuing research on the phenolic compounds present in Hypericum species, the plants were also investigated for their tannin, flavonoid and phenolic acid contents.
Material and Methods Plant material
The aerial parts of Hypericum caprifoliatum Cham. & Schlecht. were collected in the “Morro Santana”, Porto Alegre, in May, 1998. H. myrianthum Cham. & Schlecht. and H. polyanthemum Klotzsch ex Reichardt were collected in Paraíso do Sul and Caçapava do Sul, in July and August, 1998, respectively. H. connatum Lam. was collected in Capão do Leão in January, 1999. H. carinatum Griseb. was collected in Glorinha, RS, in January, 1999 and H. ternum A.St.Hil. in São Francisco de Paula, RS, in October, 1999. Voucher specimens were deposited in the herbarium of the Universidade Federal do Rio Grande do Sul (ICN): H. caprifoliatum, Bordignon 1400; H. carinatum, Bordignon et al. 1520; H. connatum, Bordignon & Salazar 1527; H. ternum, Bordignon et al. 1715; H. myrianthum, Bordignon 1402; H. polyanthemum, Bordignon et al. 1429. Preparation of plant extracts
The dried and powdered plant material (aerial parts) were extracted with methanol by maceration (3 × 24h), yielding total methanol crude extracts (TMCE) which were evaporated to dryness in vacuo at 45 °C. The dried extracts were reconstituted in methanol to a final concentration of 100 µg/ml and 200 µg/ml.
Bacterial and fungal strains
Tests were performed against the following microorganisms: S. aureus (ATCC 6538P), S. epidermidis (ATCC 12228), M. luteus (ATCC 9341), B. subtilis (ATCC 6633), E. coli (ATCC 25922) and C. albicans (ATCC 10231) purchased from The American Type Culture Collection (ATCC). The strain of S. cerevisiae (ATCC 1600) was obtained from the Universidade Federal de Santa Maria (UFSM). The agars used to maintain the microorganisms were No. 1 Groove-Randal medium (Merck) for bacteria and Sabouraud dextrose (Merck) for fungi. Assay for antimicrobial testing
Antimicrobial activity of the above mentioned samples (TMCE) was assayed separately using an agardiffusion method as described by Schapoval et al. (1988). Cultures of the microorganisms were obtained from stocks maintained at the Laboratório de Controle de Qualidade in the Faculdade de Farmácia (UFRGS). The microorganisms were maintained on agar slants, and subcultures were freshly prepared before use. Bacterial inocula were made in 5 ml of No. 3 medium broth (Merck) and grown for 24 h at 37 °C. The fungi were inoculated in Sabouraud broth (Merck) and grown for 48 h at 25 °C. Freshly prepared No. 1 medium (Merck) and Sabouraud agar (Merck) cooled at 45 °C were poured into 20 × 100mm Petri plates. A 5-ml portion of the No. 1 medium or Sabouraud medium at ca. 48 °C, seeded with the test microorganism (0.5%), was poured directly over the surface of the prepared plate. The plates were then allowed to solidify for 5 min and the stainless steel cylinders (6 per plate) were applied to the surface of the inoculated plates with sterile forceps. Volumes of 200 µl of the crude extracts (at concentrations of 100 and 200 µg/ml) were inoculated in each cylinder. Agar plates were incubated overnight at 35 °C and 37 °C (fungi and bacteria, respectively). At the end of the incubation periods, inhibition zones were recorded as the diameter of the growth-free zones around the cylinders. Each extract and doses were tested in quintuplicate. In all plates, two control cylinders were used: one contained 200 µl of methanol and the other, 200 µl of water. The standard antibacterial agent, chloramphenicol (40 µg/ml, 200 µl), was used as a positive control for bacteria and the standard antifungal agent, nystatin (30 µg/ml, 200 µl) as positive control for fungi. Statistical analysis
The results obtained were analyzed statistically using the Student’s t-test.
Antimicrobial activity of Hypericum species Phytochemical screening
The chemical analysis of the plants was performed using standard phytochemical methods to determine the presence of alkaloids, flavonoids, tannins, saponins, anthraquinones, etc. (Harborne, 1984). Flavonoid content
The dried and powdered plant material was extracted with boiling water and the aqueous extracts were partitioned with N-butanol. The fractions obtained were evaporated to dryness under reduced pressure. After that, the extracts were resuspended in methanol and analyzed by TLC, performed on silica gel GF254 using ethyl acetate:acetic acid:formic acid:water (100:11:11:27) as eluent. The chromatogram was sprayed with Natural Reagent/PEG 4000 and visualized at a wavelength of 365 nm. Flavonoids appeared as light yellow spots (Wagner et al. 1996). For reference, purified rutin, quercitrin, isoquercitrin, hyperoside and phenolic acids (caffeic, chlorogenic and isochlorogenic acids), obtained in the Laboratório de Farmacognosia, Faculdade de Farmácia-UFRGS, were used. Tannin determination
For tannin and non-tannin determination, 50 g of the plant material were extracted with water (2 l) for 24 h at 45 °C (12 h), 75 °C (5 h), 100 °C (1 h); the rest of the time the solution was kept at room temperature. The procedure was performed three times with each plant extract. For determination of the tannin level, the tanning substances were removed with hide powder, based on the ability to complex with proteins. The solution without tannins was evaporated, and dried in a stove (100 °C ) to obtain a constant weight. The difference between the initial and final weight gave the tannin level.
Results and Discussion Microorganisms have been developing resistance to many antibiotics due to the indiscriminate use of antimicrobial drugs, increasing clinical problems in the treatment of infectious diseases. In addition, antibiotics are sometimes associated with adverse effects in the host which include hypersensitivity, depletion of gut and mucosal microorganisms, immunosuppression and allergic reactions. Therefore, there is a need for alternative antimicrobial drugs for the treatment of infectious diseases. One approach is to screen local medicinal plants for possible antimicrobial properties. Medicinal herbs represent a rich source from which novel antibacterial and antifungal chemotherapeutic agents may be obtained (Rates, 2001).
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In this work, crude methanolic extracts of 6 Hypericum species were tested for antimicrobial activity against 7 microorganisms. Among the microorganisms chosen were S. aureus, a pyogenic bacterium known to play a significant role in invasive skin diseases, including superficial and deep follicular lesions, and C. albicans, a fungal microorganism which causes serious systemic infections, especially opportunistic infection in patients infected with HIV (Srinivasan et al. 2001). The results obtained with the native species of Hypericum are presented in Table 1. According to these data, H. caprifoliatum TMCE was the most active against S. aureus, followed by H. myrianthum TMCE. Nevertheless, no significant difference was observed between 100 and 200 µg/ml doses. H. polyanthemum and H. ternum extracts were active against B. subtilis. The former was active in both concentrations used, while the latter showed antimicrobial activity only at the dose of 200 µg/ml. These extracts were also active, at both doses, against M. luteus. In the assays with S. epidermidis, E. coli and S. cerevisiae, no significant difference was observed between the extracts and the negative controls. In the experiments with C. albicans no microorganism growth was observed in the plates containing the samples. Nevertheless, after increasing the incubation time to 36 h, colony growth was observed. These results could indicate that all the plants contain substances which delay Candida growth and could justify the use of some of these plants (i.e. Hypericum connatum) in traditional medicine to treat mouth wounds, including aphthas (Corrêa, 1984; Mentz et al. 1997). Most clinically used antibiotics are active at a concentration of 10 µg/ml. If a pure substance is not active at 100 µg/ml, it will probably not be clinically useful. Plant extracts that are active at 100 µg/ml have a good potency level and recommend the determination of the active components by subsequent purification (Rios et al. 1988). In order to identify the active compounds, a phytochemical analysis of the six species was performed. Polyphenol compounds such as tannins, flavonoids and phenolic acids were the most prominent components of the crude extracts investigated and could contribute to the antimicrobial activity of the Hypericum species extracts. It is known that polyphenols can form heavy soluble complexes with proteins. Polyphenols may bind to bacterial adhesins and by doing so disturb the availability of receptors on the cell surface. The literature demonstrates that antibacterial activity can also be due to tannins, the active compounds of several medicinal plants (Haslam, 1996). Tannins have been shown to form irreversible complexes with proline-rich proteins (Hager-
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Table 1. Antimicrobial activity (Inhibition zone, mean of 5 determinations, measured in mm) of the crude methanolic extracts of Hypericum species. Organism Plant
S. aureus
S. epidermidis
100 µg/ml
100 µg/ml
200 µg/ml
100 µg/ml
200 µg/ml
100 µg/ml
200 µg/ml
100 µg/ml
200 µg/ml
H. caprifoliatum 16.40 19.38 ± ± 0.07 0.12
8.34 ± 0.07
8.46 ± 0.10
8.21 ± 0.12
8.60 ± 0.08
7.30 ± 0.21
7.13 ± 0.15
8.24 ± 0.04
8.06 ± 0.89
6.87 ± 0.04
6.06 ± 0.15
H. carinatum
7.79 ± 0.68
7.85 ± 0.84
7.26 ± 0.10
8.63 ± 0.02
7.84 ± 0.19
7.61 ± 0.16
7.55 ± 0.07
7.79 ± 0.91
7.94 ± 0.23
8.30 ± 0.04
6.94 ± 0.26
6.63 ± 0.19
H. connatum
7.41 ± 0.17
8.13 ± 0.11
8.42 ± 0.06
8.34 ± 0.14
8.03 ± 0.03
8.27 ± 0.12
7.82 ± 0.13
7.82 ± 0.87
8.71 ± 0.21
8.57 ± 0.04
6.07 ± 0.10
7.31 ± 0.25
H. ternum
7.50 ± 0.29
8.23 ± 0.29
8.26 ± 0.16
8.52 ± 0.07
7.97 ± 0.14
10.63 ± 0.54
7.37 ± 0.12
8.15 ± 0.70
8.51 ± 0.06
17.46 ± 0.58
6.17 ± 0.18
7.76 ± 0.44
H. myrianthum
13.14 14.42 ± ± 1.26 1.39
8.33 ± 0.08
8.37 ± 0.17
7.47 ± 0.06
7.85 ± 0.12
7.49 ± 0.24
7.49 ± 0.38
8.07 ± 0.06
7.72 ± 0.04
6.42 ± 0.09
6.91 ± 0.19
7.73 ± 0.11
8.42 ± 0.19
10.45 ± 0.33
16.23 ± 0.22
9.44 ± 2.58
7.51 ± 0.06
10.92 ± 0.34
12.58 ± 0.23
6.65 ± 0.21
6.60 ± 0.09
200 µg/ml
H. polyanthemum 7.99 ± 0.13 Water Methanol
8.01 ± 0.22
M. luteus
E. coli
B. subtilis
S. cerevisiae 100 200 µg/ml µg/ml
7.59 ± .32 7.77 ± 0.33
7.41 ± 0.22 7.97 ± 0.50
7.54 ± 0.41 8.04 ± 0.05
8.80 ± 1.45 7.68 ± 1.04
7.73 ± 0.17 7.97 ± 0.17
7.01 ± 0.21 6.65 ± 0.57
Chloramphenicol 30.61 ± 0.82 40 µg/ml
16.23± 1.67
29.46 ± 0.64
20.51 ± 4.12
14.73 ± 0.54
–
–
–
–
–
10.62 ± 0.35
Nystatin 30 µg/ml
–
± standard deviation
Table 2. Phenolic compounds (tannin and non-tannin) present in some Hypericum species.
Tannins (%) Non-tannins (%)
H. caprifoliatum
H. polyantemum
H. myrianthum
H. connatum
H. carinatum
H. ternum
6.4 13.3
6.7 12.7
5.1 10.3
11.5 11.8
9.1 11.0
16.7 7.3
man and Butler, 1981), which would result in the inhibition of cell wall protein synthesis. This property could explain the mechanisms of action of the plant extracts. Reports of several in vitro assays demonstrate potentially significant interactions with biological systems, such as virus, bacteria, and mollusk, and enzymeinhibiting, antioxidant, and radical-scavenging properties (De Bruyne et al. 1999). Their tendency to interfere with biological systems is, at least in part, due to a characteristic ability to form complexes with macro-
molecules, combined with a polyphenolic nature (Haslam, 1996; De Bruyne et al. 1999). Routine estimations of tannin concentrations in commercial extracts are normally performed using a weight-difference method. An aqueous infusion is prepared and the total solids determined by evaporation of an aliquot. The procedure is then repeated with a further aliquot after removal of the tannins by adsorption onto hide powder and comparison of the two results allows the tannin to non-tannin ratio in the extract to be
Antimicrobial activity of Hypericum species determined (Haslam, 1966). The tannin fraction contains triflavonoids and heptaflavonoids (3 to 7 condensed monomers, formed by flavan-3-ol or flavan3,4-diol units). The non-tannin fraction contains phenolic compounds (monomeric flavonoid compounds and hydroxycinnamic acid derivatives), carbohydrates (glucose, fructose, saccharose and hydrocolloidal gums) and nitrogen compounds (such as amino and imino acids). The tannin content of the six species of Hypericum was determined using the hide method. The results (Table 2) were obtained using the “Procter extraction” which is the method currently used for the analysis of black wattle (Acacia mearnsii) barks. After extraction, the tannin content was quantified by filter analysis. Condensed tannins are found frequently in Hypericum species and can be present in high levels (up to 16%) (Kartnig et al. 1989). In this work, all the plants contained tannins of levels between 5 to 16%. Among these plants, H. ternum and H. connatum were the richest source of these compounds. Even though tannins are antimicrobial agents, other compounds in these plants may be responsible for the activity when the tannin concentration is inversely proportional to the activity: H. ternum, with 15.4% tannins, was the least active plant against S. aureus while the most active, H. caprifoliatum and H. myrianthum, showed 6.1 and 4.9% tannins, respectively (i.e., the tannin concentration is inversely proportional to the activity). Flavonoids are a diverse group of plant secondary metabolites, present almost ubiquitously in higher plants, often at relatively high concentrations. They have a wide range of biological activities that stem largely from their ability to bind to proteins. Some substances act as inhibitors of bacterial type-II topoisomerases. There is one report on the preferential topo IV inhibitory activity of the flavonoids rutin and isoquercitrin (Reece and Maxwell, 1996). In Hypericum species, flavonoids are abundant, representing 11% in the flowers and 7% in the leaves of H. perforatum (Kartnig et al. 1989). The glycosides reported most commonly for the genus are quercitrin, isoquercitrin, hyperoside and rutin, all derived from quercetin (Rocha et al. 1995; American Herbal Pharmacopeia, 1997). The non-tannin fractions of the plants were analyzed for flavonoid glucosides and phenolic acid compounds and the following substances were characterized: H. caprifoliatum: quercitrin, rutin (traces) and hyperoside; H. carinatum: quercitrin, hyperoside and chlorogenic acid; H. connatum: quercitrin, rutin (traces), isoquercitrin (traces) and chlorogenic acid; H. ternum: quercitrin (traces), hyperoside, caffeic acid, chlorogenic acid and isochlorogenic acid; H. myrianthum:
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quercitrin (traces), hyperoside and chlorogenic acid; H. polyanthemum: quercitrin, hyperoside and isoquercitrin. The species presented a certain homogeneity and the most frequent flavonoids were: quercitrin, present in all species, hyperoside (except in H. connatum); and, less frequently, isoquercitrin. As mentioned previously, these substances were found in H. perforatum. A notable characteristic in the species studied is the absence or the presence of only a trace of rutin, a flavonoid present in high amounts in H. perforatum. Nevertheless, it would be interesting to analyze the same plants collected in different places, as some authors have found a correlation between the altitude of the growing site and the flavonoid content (Umek et al. 1999; Tekel’ová et al. 2000). Apparently, tannins and flavonoids can not be considered the only active compounds present in these plants; among the 3 species presenting higher tannin concentrations – H. carinatum, H. ternum and H connatum – the first was not active against any screened microorganism and the others presented activity only against one bacteria and at the higher dose. Moreover, as the flavonoid profile of this plant group was similar, if they were the active compounds, a similar antibacterial activity would be expected, and this was not the case. Although tannins, flavonoids and phenolic acids are the main components of the crude methanolic extracts of these Hypericum plants, it worthwhile to mention that the most apolar fractions of H. caprifoliatum and H. myrianthum contain conjugated phloroglucinol derivatives (Daudt et al. 2000; Ferraz et al. 2002) and the extract of H. polyanthemum contains benzopyrans (Ferraz et al. 2001). These compounds, even in relatively low concentrations, could be responsible for the activity detected. The present investigation represents a preliminary screening on plant antimicrobials and work is progressing for the isolation and identification of the active compounds. Crude extracts of other native Hypericum species will also be evaluated against bacteria and fungi. The most active plants will be fractionated with solvents of increasing polarity and the fractions will be re-assayed. The active fractions will be evaluated using bio-autographed chromatography in order to detect the active compounds. In the sequence of this work, the active compounds will be isolated and their minimum inhibitory concentrations (MIC) will be determined. Acknowledgements
The authors want to thank Sérgio Bordignon for collecting plant material and CNPq, FAPERGS and CAPES for financial support.
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Jayasuriya H, McChesney JD (1989) Antimicrobial and cytotoxic activity of rottlerin-type compounds from Hypericum drummondii. J Nat Prod 52: 325–331 Kartnig T, Gruber A, Sauer H (1989) Comparative phytochemical investigations of Hypericum species. Planta Med 55: 215 Martini N, Eloff JN (1998) The preliminary isolation of several antibacterial compounds from Combretum erythrophyllum (Combretaceae). J Ethnopharmacol 62: 255–263 Mentz LA, Lutzemberger LC, Schenkel EP (1997) Da flora medicinal do Rio Grande do Sul: notas sobre a obra literária de D’Ávila. Cad Farm 13: 25–47 Rates SMK (2001) Plants as source of drugs. Toxicon 39: 603–613 Reece RJ & Maxwell A (1996) DNA gyrase: Structure and function mechanism of DNA topoisomerase II. Nature 379: 225–232 Rios JL, Recio MC, Villar A (1988) Screening methods for natural products with antimicrobial activity: A review of the literature. J Ethnopharmacol 23: 127–149 Rocha L, Marston A, Potterat O, Kaplan MAC, StoeckliEvans H, Hostettmann K (1995) Antibacterial phloroglucinols and flavonoids from Hypericum brasiliense. Phytochemistry 40: 1447–1152 Scalbert A (1991) Antimicrobial properties of tannins. Phytochemistry 30: 3875–3883 Schapoval EE, Alice CB, Zuanazzi JA, Silva GAAB, Henriques AT (1988) Determinação da actividade antimicrobiana dos extrato de Sygygium cuminii. Rev Port Farm 38: 55–57 Srinivasan D, Nathan S, Suresh T, Perumalsamy PL (2001) Antimicrobial activity of certain Indian medicinal plants used in folkloric medicine. J Ethnopharmacol 74: 217–220 Tekel’ová D, Repcák M, Zemková E, Tóth J (2000) Quantitative changes of dianthrones, hyperforin and flavonoids content in the flower ontogenesis of Hypericum perforatum. Planta Med 66: 778–780 Trifunovic S, Vais V, Macura S, Juranic N (1998) Oxidation products of hyperforin from Hypericum perforatum. Phytochemistry 49: 1305–1310 Umek A, Kreft S, Kartnig T, Heydel B (1999) Quantitative phytochemical analyses of six Hypericum species growing in Slovenia. Planta Med 65: 388–390 Wagner H, Bladt S (1996) Plant Drug Analysis. A Thin Layer Chromatography Atlas 2nd ed. Springer Verlag, Berlin Yamaki M, Ishiguro K (1994) Antimicrobial activity of naturally ocurring and synthetic phloroglucinol against Staphylococcus aureus. Phytother Res 8: 112–114
Address G. Lino von Poser, Faculdade de Farmácia – UFRGS, Av. Ipiranga, 2752, 90610-000 Porto Alegre, Brazil Tel.: ++55-(51)3316 5456; Fax: ++55-(51)3316 5437; e-mail: [email protected]
Phytomedicine
Antimicrobial activity of some Hypericum species R. Dall’Agnol1, A. Ferraz1, A. P. Bernardi1, D. Albring1, C. Nör1, L. Sarmento2, L. Lamb2, M. Hass2, G. von Poser1, and E. E. S. Schapoval1 1 2
Programa de Pós Graduação em Ciências Farmacêuticas, UFRGS. Porto Alegre, RS, Brazil TANAC SA. Montenegro, RS, Brazil
Summary The crude methanolic extracts of six species of Hypericum [H. caprifoliatum Cham. & Schlecht., H. carinatum Griseb., H. connatum Lam., H. ternum A.St. Hil., H. myrianthum Cham. & Schlecht. and H. polyanthemum Klotzsch ex Reichardt] growing in southern Brazil were analyzed for antimicrobial activity against several microorganisms (bacteria and fungi). The most active plant was H. caprifoliatum, which showed activity against Staphylococcus aureus. Only H. polyanthemum and H. ternum extracts were active against Bacillus subtilis. None of the crude methanolic extracts showed activity against S. epidermidis, Escherichia coli or Saccharomyces cerevisiae. Extracts from these species were evaluated chemically and tannin, flavonoid and phenolic acids were the prominent compounds. The plants contained quercitrin, hyperoside (except H. connatum) and, less frequently, isoquercitrin and chlorogenic acid. In contrast to H. perforatum, which has high concentrations of rutin, these species do not produce this flavonoid or it appears as traces. The tannin concentration varied between 5.1 and 16.7% in H. myrianthum and H. ternum, respectively. Key words: Hypericum, Guttiferae, antimicrobial activity, bacteria, fungi, flavonoids, tannins
Introduction Guttiferae is a large family comprised of more than 1000 species. The best-known genus is Hypericum, which encompasses various species used in traditional medicine around the world. Several antifungal (Décosterd et al. 1986), antibiotic (Ishiguro et al. 1986), antiviral (Jacobson et al. 2001) and anticancer (Jayasuriya and McChesney, 1989) compounds have been isolated from these species. The majority of the active compounds isolated are phenolic in nature. These plants have a strong tendency to accumulate phenolic compounds with the phloroglucinol substitution pattern. From the genus Hypericum many phloroglucinol derivatives have also been isolated, of which some are related to the well-known hyperforin, isolated from H. perforatum (Trifunovic et al. 1998) while others present a phloroglucinol unit conjugated with a filicinic acid moiety (Ishiguro et al. 1986; Rocha et al. 1995).
The phloroglucinol derivatives found frequently in the lipophilic fractions of several Hypericum species have demonstrated antifungal and antibacterial activities against microorganisms such as Staphylococcus aureus, Bacillus cereus, B. subtilis and Nocardia gardenen (Ishiguro et al. 1986; Décosterd et al. 1991; Jayasuriya et al. 1991; Rocha et al. 1995; Trifunovic et al. 1998). Their presence could justify the popular use of some Hypericum species as wound healing agents and in the treatment of some microbiological diseases (Ishiguro et al. 1986; Ishiguro et al. 1987; Jayasuria et al. 1991; Yamaki et al. 1994; Rocha et al. 1995). Other substances present in some species of Hypericum have also shown antimicrobial activity against various bacteria and fungi, including the benzopyrans (Jayasuriya et al. 1989; Décosterd et al. 1986), the xanthones (Ishiguro et al. 1999), the flavonoids (Ishiguro et al. 1993), and the tannins, recognized antimicrobial compounds (Scalbert, 1991). 0944-7113/03/10/06–07-511 $ 15.00/0
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The indiscriminate use of antibiotics has led to drug resistance in many bacterial strains, and the development of new antimicrobial compounds for resistant organisms is becoming critically important. Although the most important sources of antibiotics are molds, actinomycetes and bacteria, higher plants also present many classes of secondary metabolites with this property. Thus, efforts have been made to evaluate the antimicrobial activity of a wide array of natural products, including plant metabolites, in order to isolate and characterize novel compounds which could inhibit bacteria and fungi, or even serve as models for new molecules (Martini et al. 1998; Rates, 2001; Srinivasan et al. 2001). In this work we report the antimicrobial activity of methanolic extracts of six species of this genus (H. connatum, H. caprifoliatum, H. carinatum, H. polyanthemum, H. myrianthum and H. ternum evaluated against S. aureus, S. epidermidis, M. luteus, Escherichia coli, B. subtilis, Candida albicans and Saccharomyces cerevisiae) and two antibiotics used in therapy (chloramphenicol and nystatin) by the agar diffusion method. In our continuing research on the phenolic compounds present in Hypericum species, the plants were also investigated for their tannin, flavonoid and phenolic acid contents.
Material and Methods Plant material
The aerial parts of Hypericum caprifoliatum Cham. & Schlecht. were collected in the “Morro Santana”, Porto Alegre, in May, 1998. H. myrianthum Cham. & Schlecht. and H. polyanthemum Klotzsch ex Reichardt were collected in Paraíso do Sul and Caçapava do Sul, in July and August, 1998, respectively. H. connatum Lam. was collected in Capão do Leão in January, 1999. H. carinatum Griseb. was collected in Glorinha, RS, in January, 1999 and H. ternum A.St.Hil. in São Francisco de Paula, RS, in October, 1999. Voucher specimens were deposited in the herbarium of the Universidade Federal do Rio Grande do Sul (ICN): H. caprifoliatum, Bordignon 1400; H. carinatum, Bordignon et al. 1520; H. connatum, Bordignon & Salazar 1527; H. ternum, Bordignon et al. 1715; H. myrianthum, Bordignon 1402; H. polyanthemum, Bordignon et al. 1429. Preparation of plant extracts
The dried and powdered plant material (aerial parts) were extracted with methanol by maceration (3 × 24h), yielding total methanol crude extracts (TMCE) which were evaporated to dryness in vacuo at 45 °C. The dried extracts were reconstituted in methanol to a final concentration of 100 µg/ml and 200 µg/ml.
Bacterial and fungal strains
Tests were performed against the following microorganisms: S. aureus (ATCC 6538P), S. epidermidis (ATCC 12228), M. luteus (ATCC 9341), B. subtilis (ATCC 6633), E. coli (ATCC 25922) and C. albicans (ATCC 10231) purchased from The American Type Culture Collection (ATCC). The strain of S. cerevisiae (ATCC 1600) was obtained from the Universidade Federal de Santa Maria (UFSM). The agars used to maintain the microorganisms were No. 1 Groove-Randal medium (Merck) for bacteria and Sabouraud dextrose (Merck) for fungi. Assay for antimicrobial testing
Antimicrobial activity of the above mentioned samples (TMCE) was assayed separately using an agardiffusion method as described by Schapoval et al. (1988). Cultures of the microorganisms were obtained from stocks maintained at the Laboratório de Controle de Qualidade in the Faculdade de Farmácia (UFRGS). The microorganisms were maintained on agar slants, and subcultures were freshly prepared before use. Bacterial inocula were made in 5 ml of No. 3 medium broth (Merck) and grown for 24 h at 37 °C. The fungi were inoculated in Sabouraud broth (Merck) and grown for 48 h at 25 °C. Freshly prepared No. 1 medium (Merck) and Sabouraud agar (Merck) cooled at 45 °C were poured into 20 × 100mm Petri plates. A 5-ml portion of the No. 1 medium or Sabouraud medium at ca. 48 °C, seeded with the test microorganism (0.5%), was poured directly over the surface of the prepared plate. The plates were then allowed to solidify for 5 min and the stainless steel cylinders (6 per plate) were applied to the surface of the inoculated plates with sterile forceps. Volumes of 200 µl of the crude extracts (at concentrations of 100 and 200 µg/ml) were inoculated in each cylinder. Agar plates were incubated overnight at 35 °C and 37 °C (fungi and bacteria, respectively). At the end of the incubation periods, inhibition zones were recorded as the diameter of the growth-free zones around the cylinders. Each extract and doses were tested in quintuplicate. In all plates, two control cylinders were used: one contained 200 µl of methanol and the other, 200 µl of water. The standard antibacterial agent, chloramphenicol (40 µg/ml, 200 µl), was used as a positive control for bacteria and the standard antifungal agent, nystatin (30 µg/ml, 200 µl) as positive control for fungi. Statistical analysis
The results obtained were analyzed statistically using the Student’s t-test.
Antimicrobial activity of Hypericum species Phytochemical screening
The chemical analysis of the plants was performed using standard phytochemical methods to determine the presence of alkaloids, flavonoids, tannins, saponins, anthraquinones, etc. (Harborne, 1984). Flavonoid content
The dried and powdered plant material was extracted with boiling water and the aqueous extracts were partitioned with N-butanol. The fractions obtained were evaporated to dryness under reduced pressure. After that, the extracts were resuspended in methanol and analyzed by TLC, performed on silica gel GF254 using ethyl acetate:acetic acid:formic acid:water (100:11:11:27) as eluent. The chromatogram was sprayed with Natural Reagent/PEG 4000 and visualized at a wavelength of 365 nm. Flavonoids appeared as light yellow spots (Wagner et al. 1996). For reference, purified rutin, quercitrin, isoquercitrin, hyperoside and phenolic acids (caffeic, chlorogenic and isochlorogenic acids), obtained in the Laboratório de Farmacognosia, Faculdade de Farmácia-UFRGS, were used. Tannin determination
For tannin and non-tannin determination, 50 g of the plant material were extracted with water (2 l) for 24 h at 45 °C (12 h), 75 °C (5 h), 100 °C (1 h); the rest of the time the solution was kept at room temperature. The procedure was performed three times with each plant extract. For determination of the tannin level, the tanning substances were removed with hide powder, based on the ability to complex with proteins. The solution without tannins was evaporated, and dried in a stove (100 °C ) to obtain a constant weight. The difference between the initial and final weight gave the tannin level.
Results and Discussion Microorganisms have been developing resistance to many antibiotics due to the indiscriminate use of antimicrobial drugs, increasing clinical problems in the treatment of infectious diseases. In addition, antibiotics are sometimes associated with adverse effects in the host which include hypersensitivity, depletion of gut and mucosal microorganisms, immunosuppression and allergic reactions. Therefore, there is a need for alternative antimicrobial drugs for the treatment of infectious diseases. One approach is to screen local medicinal plants for possible antimicrobial properties. Medicinal herbs represent a rich source from which novel antibacterial and antifungal chemotherapeutic agents may be obtained (Rates, 2001).
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In this work, crude methanolic extracts of 6 Hypericum species were tested for antimicrobial activity against 7 microorganisms. Among the microorganisms chosen were S. aureus, a pyogenic bacterium known to play a significant role in invasive skin diseases, including superficial and deep follicular lesions, and C. albicans, a fungal microorganism which causes serious systemic infections, especially opportunistic infection in patients infected with HIV (Srinivasan et al. 2001). The results obtained with the native species of Hypericum are presented in Table 1. According to these data, H. caprifoliatum TMCE was the most active against S. aureus, followed by H. myrianthum TMCE. Nevertheless, no significant difference was observed between 100 and 200 µg/ml doses. H. polyanthemum and H. ternum extracts were active against B. subtilis. The former was active in both concentrations used, while the latter showed antimicrobial activity only at the dose of 200 µg/ml. These extracts were also active, at both doses, against M. luteus. In the assays with S. epidermidis, E. coli and S. cerevisiae, no significant difference was observed between the extracts and the negative controls. In the experiments with C. albicans no microorganism growth was observed in the plates containing the samples. Nevertheless, after increasing the incubation time to 36 h, colony growth was observed. These results could indicate that all the plants contain substances which delay Candida growth and could justify the use of some of these plants (i.e. Hypericum connatum) in traditional medicine to treat mouth wounds, including aphthas (Corrêa, 1984; Mentz et al. 1997). Most clinically used antibiotics are active at a concentration of 10 µg/ml. If a pure substance is not active at 100 µg/ml, it will probably not be clinically useful. Plant extracts that are active at 100 µg/ml have a good potency level and recommend the determination of the active components by subsequent purification (Rios et al. 1988). In order to identify the active compounds, a phytochemical analysis of the six species was performed. Polyphenol compounds such as tannins, flavonoids and phenolic acids were the most prominent components of the crude extracts investigated and could contribute to the antimicrobial activity of the Hypericum species extracts. It is known that polyphenols can form heavy soluble complexes with proteins. Polyphenols may bind to bacterial adhesins and by doing so disturb the availability of receptors on the cell surface. The literature demonstrates that antibacterial activity can also be due to tannins, the active compounds of several medicinal plants (Haslam, 1996). Tannins have been shown to form irreversible complexes with proline-rich proteins (Hager-
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Table 1. Antimicrobial activity (Inhibition zone, mean of 5 determinations, measured in mm) of the crude methanolic extracts of Hypericum species. Organism Plant
S. aureus
S. epidermidis
100 µg/ml
100 µg/ml
200 µg/ml
100 µg/ml
200 µg/ml
100 µg/ml
200 µg/ml
100 µg/ml
200 µg/ml
H. caprifoliatum 16.40 19.38 ± ± 0.07 0.12
8.34 ± 0.07
8.46 ± 0.10
8.21 ± 0.12
8.60 ± 0.08
7.30 ± 0.21
7.13 ± 0.15
8.24 ± 0.04
8.06 ± 0.89
6.87 ± 0.04
6.06 ± 0.15
H. carinatum
7.79 ± 0.68
7.85 ± 0.84
7.26 ± 0.10
8.63 ± 0.02
7.84 ± 0.19
7.61 ± 0.16
7.55 ± 0.07
7.79 ± 0.91
7.94 ± 0.23
8.30 ± 0.04
6.94 ± 0.26
6.63 ± 0.19
H. connatum
7.41 ± 0.17
8.13 ± 0.11
8.42 ± 0.06
8.34 ± 0.14
8.03 ± 0.03
8.27 ± 0.12
7.82 ± 0.13
7.82 ± 0.87
8.71 ± 0.21
8.57 ± 0.04
6.07 ± 0.10
7.31 ± 0.25
H. ternum
7.50 ± 0.29
8.23 ± 0.29
8.26 ± 0.16
8.52 ± 0.07
7.97 ± 0.14
10.63 ± 0.54
7.37 ± 0.12
8.15 ± 0.70
8.51 ± 0.06
17.46 ± 0.58
6.17 ± 0.18
7.76 ± 0.44
H. myrianthum
13.14 14.42 ± ± 1.26 1.39
8.33 ± 0.08
8.37 ± 0.17
7.47 ± 0.06
7.85 ± 0.12
7.49 ± 0.24
7.49 ± 0.38
8.07 ± 0.06
7.72 ± 0.04
6.42 ± 0.09
6.91 ± 0.19
7.73 ± 0.11
8.42 ± 0.19
10.45 ± 0.33
16.23 ± 0.22
9.44 ± 2.58
7.51 ± 0.06
10.92 ± 0.34
12.58 ± 0.23
6.65 ± 0.21
6.60 ± 0.09
200 µg/ml
H. polyanthemum 7.99 ± 0.13 Water Methanol
8.01 ± 0.22
M. luteus
E. coli
B. subtilis
S. cerevisiae 100 200 µg/ml µg/ml
7.59 ± .32 7.77 ± 0.33
7.41 ± 0.22 7.97 ± 0.50
7.54 ± 0.41 8.04 ± 0.05
8.80 ± 1.45 7.68 ± 1.04
7.73 ± 0.17 7.97 ± 0.17
7.01 ± 0.21 6.65 ± 0.57
Chloramphenicol 30.61 ± 0.82 40 µg/ml
16.23± 1.67
29.46 ± 0.64
20.51 ± 4.12
14.73 ± 0.54
–
–
–
–
–
10.62 ± 0.35
Nystatin 30 µg/ml
–
± standard deviation
Table 2. Phenolic compounds (tannin and non-tannin) present in some Hypericum species.
Tannins (%) Non-tannins (%)
H. caprifoliatum
H. polyantemum
H. myrianthum
H. connatum
H. carinatum
H. ternum
6.4 13.3
6.7 12.7
5.1 10.3
11.5 11.8
9.1 11.0
16.7 7.3
man and Butler, 1981), which would result in the inhibition of cell wall protein synthesis. This property could explain the mechanisms of action of the plant extracts. Reports of several in vitro assays demonstrate potentially significant interactions with biological systems, such as virus, bacteria, and mollusk, and enzymeinhibiting, antioxidant, and radical-scavenging properties (De Bruyne et al. 1999). Their tendency to interfere with biological systems is, at least in part, due to a characteristic ability to form complexes with macro-
molecules, combined with a polyphenolic nature (Haslam, 1996; De Bruyne et al. 1999). Routine estimations of tannin concentrations in commercial extracts are normally performed using a weight-difference method. An aqueous infusion is prepared and the total solids determined by evaporation of an aliquot. The procedure is then repeated with a further aliquot after removal of the tannins by adsorption onto hide powder and comparison of the two results allows the tannin to non-tannin ratio in the extract to be
Antimicrobial activity of Hypericum species determined (Haslam, 1966). The tannin fraction contains triflavonoids and heptaflavonoids (3 to 7 condensed monomers, formed by flavan-3-ol or flavan3,4-diol units). The non-tannin fraction contains phenolic compounds (monomeric flavonoid compounds and hydroxycinnamic acid derivatives), carbohydrates (glucose, fructose, saccharose and hydrocolloidal gums) and nitrogen compounds (such as amino and imino acids). The tannin content of the six species of Hypericum was determined using the hide method. The results (Table 2) were obtained using the “Procter extraction” which is the method currently used for the analysis of black wattle (Acacia mearnsii) barks. After extraction, the tannin content was quantified by filter analysis. Condensed tannins are found frequently in Hypericum species and can be present in high levels (up to 16%) (Kartnig et al. 1989). In this work, all the plants contained tannins of levels between 5 to 16%. Among these plants, H. ternum and H. connatum were the richest source of these compounds. Even though tannins are antimicrobial agents, other compounds in these plants may be responsible for the activity when the tannin concentration is inversely proportional to the activity: H. ternum, with 15.4% tannins, was the least active plant against S. aureus while the most active, H. caprifoliatum and H. myrianthum, showed 6.1 and 4.9% tannins, respectively (i.e., the tannin concentration is inversely proportional to the activity). Flavonoids are a diverse group of plant secondary metabolites, present almost ubiquitously in higher plants, often at relatively high concentrations. They have a wide range of biological activities that stem largely from their ability to bind to proteins. Some substances act as inhibitors of bacterial type-II topoisomerases. There is one report on the preferential topo IV inhibitory activity of the flavonoids rutin and isoquercitrin (Reece and Maxwell, 1996). In Hypericum species, flavonoids are abundant, representing 11% in the flowers and 7% in the leaves of H. perforatum (Kartnig et al. 1989). The glycosides reported most commonly for the genus are quercitrin, isoquercitrin, hyperoside and rutin, all derived from quercetin (Rocha et al. 1995; American Herbal Pharmacopeia, 1997). The non-tannin fractions of the plants were analyzed for flavonoid glucosides and phenolic acid compounds and the following substances were characterized: H. caprifoliatum: quercitrin, rutin (traces) and hyperoside; H. carinatum: quercitrin, hyperoside and chlorogenic acid; H. connatum: quercitrin, rutin (traces), isoquercitrin (traces) and chlorogenic acid; H. ternum: quercitrin (traces), hyperoside, caffeic acid, chlorogenic acid and isochlorogenic acid; H. myrianthum:
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quercitrin (traces), hyperoside and chlorogenic acid; H. polyanthemum: quercitrin, hyperoside and isoquercitrin. The species presented a certain homogeneity and the most frequent flavonoids were: quercitrin, present in all species, hyperoside (except in H. connatum); and, less frequently, isoquercitrin. As mentioned previously, these substances were found in H. perforatum. A notable characteristic in the species studied is the absence or the presence of only a trace of rutin, a flavonoid present in high amounts in H. perforatum. Nevertheless, it would be interesting to analyze the same plants collected in different places, as some authors have found a correlation between the altitude of the growing site and the flavonoid content (Umek et al. 1999; Tekel’ová et al. 2000). Apparently, tannins and flavonoids can not be considered the only active compounds present in these plants; among the 3 species presenting higher tannin concentrations – H. carinatum, H. ternum and H connatum – the first was not active against any screened microorganism and the others presented activity only against one bacteria and at the higher dose. Moreover, as the flavonoid profile of this plant group was similar, if they were the active compounds, a similar antibacterial activity would be expected, and this was not the case. Although tannins, flavonoids and phenolic acids are the main components of the crude methanolic extracts of these Hypericum plants, it worthwhile to mention that the most apolar fractions of H. caprifoliatum and H. myrianthum contain conjugated phloroglucinol derivatives (Daudt et al. 2000; Ferraz et al. 2002) and the extract of H. polyanthemum contains benzopyrans (Ferraz et al. 2001). These compounds, even in relatively low concentrations, could be responsible for the activity detected. The present investigation represents a preliminary screening on plant antimicrobials and work is progressing for the isolation and identification of the active compounds. Crude extracts of other native Hypericum species will also be evaluated against bacteria and fungi. The most active plants will be fractionated with solvents of increasing polarity and the fractions will be re-assayed. The active fractions will be evaluated using bio-autographed chromatography in order to detect the active compounds. In the sequence of this work, the active compounds will be isolated and their minimum inhibitory concentrations (MIC) will be determined. Acknowledgements
The authors want to thank Sérgio Bordignon for collecting plant material and CNPq, FAPERGS and CAPES for financial support.
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Address G. Lino von Poser, Faculdade de Farmácia – UFRGS, Av. Ipiranga, 2752, 90610-000 Porto Alegre, Brazil Tel.: ++55-(51)3316 5456; Fax: ++55-(51)3316 5437; e-mail: [email protected]