Comparative Antioxidant and Antimicrobial Activities of Phenolic Compounds Extracted from Five Hypericum Species

Abstract

Phenolic compounds were extracted from five Hypericum species (H. perforatum, H. oblongifolium, H. monogynum, H. choisianum and H. dyeri Redher) using ethanol. The crude extract (called fraction 1) was then fractionated using re-extraction to water (fraction 2), ethyl acetate (fraction 3), and acetone (fraction 4). The final residue was marked as fraction 5. The content of total phenolics in the fractions ranged from 21 mg of gallic acid equivalents per g (fraction 5 of H. dyeri) to 100 mg of gallic acid equivalents per g (fraction 5 of H. choisianum). Phenolic compounds present in the fractions showed antioxidant and antiradical properties investigated using DPPH radical scavenging activity, molybdate method, and reducing power. The strongest antiradical properties were noted for fraction 3 of H. choisianum (EC^sub 50^=11.2 µg/mL), whereas the weakest was for fraction 5 of H. dyeri (EC^sub 50^=139.2 µg/mL). Fractions 1 and 5 of H. dyeri showed good antibacterial activity against Escherichia coli, while fractions 3-5 of H. perforatum were active against Staphylococcus aureus and Pseudomonas aeruginosa. Fractions 1-4 of H. perforatum were found most active against Helminthosporium maydis as determined by antifungal screening. [PUBLICATION ABSTRACT]

Headnote

Summary

Phenolic compounds were extracted from five Hypericum species (H. perforatum, H. oblongifolium, H. monogynum, H. choisianum and H. dyeri Redher) using ethanol. The crude extract (called fraction 1) was then fractionated using re-extraction to water (fraction 2), ethyl acetate (fraction 3), and acetone (fraction 4). The final residue was marked as fraction 5. The content of total phenolics in the fractions ranged from 21 mg of gallic acid equivalents per g (fraction 5 of H. dyeri) to 100 mg of gallic acid equivalents per g (fraction 5 of H. choisianum). Phenolic compounds present in the fractions showed antioxidant and antiradical properties investigated using DPPH radical scavenging activity, molybdate method, and reducing power. The strongest antiradical properties were noted for fraction 3 of H. choisianum (EC^sub 50^=11.2 µg/mL), whereas the weakest was for fraction 5 of H. dyeri (EC^sub 50^=139.2 µg/mL). Fractions 1 and 5 of H. dyeri showed good antibacterial activity against Escherichia coli, while fractions 3-5 of H. perforatum were active against Staphylococcus aureus and Pseudomonas aeruginosa. Fractions 1-4 of H. perforatum were found most active against Helminthosporium maydis as determined by antifungal screening.

Key words: Hypericum monogynum, H. oblongifolium, H. perforatum, H. choisianum, H. dyeri, phenolic compounds, antioxidant activity, antimicrobial activities

(ProQuest: ... denotes formulae omitted.)

Introduction

Hypericum (Guttiferae, syn. Clusiaceae) is a large genus of herbs or shrubs which grows widely in temperate regions. There are about 400 species of Hypericum genus and they are used as traditional medicinal plants in various parts of the world (1). Most of these species have been used for a long time for the treatment of external wounds and gastric ulcers, and also as sedative, antiseptic, and antispasmodic herbs in folk medicine (1).

In Pakistan the genus Hypericum is represented by nine species. Several studies have been published concerning Hypericum perforatum L., known as St. John's wort. This species has been reported to have antidepressant, anxiolytic, antiviral, wound-healing and antimicrobial activities (1).

Hypericum species have shown good antioxidant activity in vitro (2). Several studies have revealed antimicrobial activity of various extracts of H. perforatum (3,4). The components of Hypericum perforatum chiefly respons- ible for the antidepressant activity, hypericin and hyperforin, have been shown to increase serotonin levels. It has been reported that serotonin possesses a protective effect against oxidative damage in neuronal cells (5). An extract of Hypericum perforatum shoot exhibited an antilipid peroxidation property (6) and superoxide radical scavenging activity (7). Hypericum hyssopifolium exhibited significant DPPH radical scavenging activity (1) and free radical scavenging properties in cell-free and human vascular systems (7). Antioxidant activities of Hypericum perforatum, H. androsaemum, H. triquetrifolium Turra and H. hyssopifolium have also been evaluated (8-10). The antioxidant activities of methanol extracts of nine Hypericum species from the Balkans have been reported by Radulovi} et al. (11). Other species of the genus Hypericum also exhibited antimicrobial properties (12-16).

The aim of the present study is to evaluate comparative antioxidant and antimicrobial potential in vitro of five Hypericum species; i.e. H. monogynum, H. oblongifolium, H. perforatum, H. choisianum and H. dyeri.

Materials and Methods

Chemicals

All chemicals used were of analytical grade. Gallic acid was purchased from Acros (Geel, Belgium), 2,2'-diphenyl- 1-picrylhydrazyl (DPPH) radical and trichloroacetic acid from Sigma-Aldrich (Buchs, Switzerland), sodium phosphate from Panreac (Castellar del Vallès, Barcelona, Spain), and ammonium molybdate from ABSCO Materials (Haverhill, Suffolk, UK). Folin-Ciocalteu phenol reagent, gallic acid, sodium carbonate, ascorbic acid, potassium ferricyanide, ferric chloride, sulphuric acid and other reagents were purchased from Merck (Darmstadt, Germany).

Plant material

Hypericum perforatum L., H. oblongifolium Wall, H. monogynum L., H. choisianum Wall and H. dyeri Redher were collected during the flowering period from different areas of Pakistan. All species were authenticated by Dr Habib Ahmad (Faculty of Science, Hazara University, Mansehra, Pakistan).

Test organisms for bioassays

Microorganisms used in the determination of antimicrobial activities of different plant extracts were as follows: Gram-positive bacteria Staphylococcus aureus; Gram- -negative bacteria Pseudomonas aeruginosa, Salmonella Typhi, Escherichia coli, Proteus vulgaris, Enterobacter aerogenes; and fungal strains Aspergillus niger, Aspergillus flavus, Alternaria solani and Helminthosporium maydis. Pure bacterial and fungal cultures (clinical isolates) were obtained from the Centre of Biotechnology and Microbiology, University of Peshawar, Peshawar, Pakistan. Different bacterial and fugal strains were maintained on nutrient agar (NA; Oxoid, Basingstoke, Hampshire, UK) and Sabouraud dextrose agar (SDA; Oxoid), respectively. Bacterial cultures were prepared by transferring two to three colonies into a tube containing 20 mL of nutrient broth (Oxoid) and grown overnight at 37 °C, while fungal cultures were prepared in SDA. Fresh culture suspensions equivalent to a 0.5 McFarland standard (108 CFU/mL) were used for inoculation.

Preparation of extracts and fractions

The air-dried and powdered aerial parts of five species were extracted with ethanol using a Soxhlet apparatus. The respective extracts were filtered and dried under reduced pressure at a temperature below 50 °C. Various fractions were obtained (Fig. 1) by solvent extraction.

Total phenolic content

The content of total phenolic compounds in each fraction was estimated using the Folin-Ciocalteu phenol reagent (17). The results were reported as mg of gallic acid equivalents (GAE) per g of extract.

Antioxidant activity

The antioxidant activity of the fractions was evaluated by the method of Prieto et al. (18) with slight modifications. Briefly, an aliquot of 0.3 mL of sample solution in methanol was combined in an Eppendorf tube with 2.7 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The effective concentration of the sample was 50 mg/mL in the reaction mixture. For the blank, 0.3 mL of ethanol were mixed with 2.7 mL of the reagent. The tubes were capped and incubated in water bath at 95 °C for 90 min. After the samples had cooled to room temperature, the absorbance of the aqueous solutions was measured at 695 nm against a blank. The results were reported as mmol of ascorbic acid equivalents (AAE) per g of extract.

DPPH radical scavenging activity

DPPH radical scavenging activity of the phenolic fractions of Hyperium species was evaluated according to the method described by Blois (19) with slight modifications. Briefly, a 1-mM solution of DPPH radical solution in methanol was prepared and 1 mL of this solution was mixed with 3 mL of sample solutions in ethanol (containing 20-100 mg of phenolic fraction) and control (without sample). After 30 min, the absorbance was measured at 517 nm.

Radical scavenging activity was calculated with the following equation:

... /1/

Reducing power

Reducing power of phenolics present in the fractions was determined as described by Oyaizu (20). The suspension of the fractions (5-25 mg/mL) in 1 mL of distilled water was mixed with 2.5 mL of 0.2 M phosphate buffer (pH=6.6) and 2.5 mL of 1 % (by mass per volume) potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. Following this, 2.5 mL of 10 % (by mass per volume) trichloroacetic acid were added and the mixture was then centrifuged at 1750'g for 10 min. An aliquot of 2.5 mL of the upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1 % (by mass per volume) FeCl3, and the absorbance of the mixture was read at 700 nm. The results were reported as mg of ascorbic acid equivalents (AAE) per g of extract.

Antibacterial screening

The antibacterial tests were performed using the agar well diffusion assay (21-23). Agar plates were prepared using sterile nutrient agar (Oxoid). Bacterial strains of standardized cultures were evenly spread onto the surface of the agar plates using sterile swab sticks. Five wells (6 mm diameter) were made in each plate with a sterile borer. A volume of 100 mL of ethanolic, aqueous, ethyl acetate, acetone and final extracts (10 mg/mL) of five Hypericum species were added to each well. A volume of 100 mL of absolute alcohol per well was used as a negative control. For a positive control, 100 mL of streptomycin (2 mg/mL) were used. Diffusion of the extracts and controls was allowed at room temperature for 1 h in a laminar flow cabinet. The agar plates were then covered with lids and incubated at 37 °C for 24 h. The plates were observed for the presence of inhibition of bacterial growth, which was indicated by a clear zone around the wells. The size of the zones of inhibition was measured and the antibacterial activity expressed in terms of the average diameter of the inhibition zone in millimeters. The absence of a zone of inhibition was interpreted as the absence of activity.

Antifungal activity assay

For antifungal activity, the tube dilution test was employed (23,24). A volume of 5 mL of medium (SDA) was added to each screw-capped test tube and they were autoclaved at 121 °C for 15 min. To the tubes containing 5 mL of sterile SDA, ethanolic, aqueous, ethyl acetate, acetone and final extracts (400 mg/mL) of five Hypericum species and fuconazole (200 mg/mL) in absolute alcohol were added. The tubes were kept in the upright position overnight to check the sterility. On the next day, the tubes were inoculated with the fungal culture in the upright position, and then incubated for 10 days at 27-30 °C. Each compound or extract was tested against four fungal cultures. The negative control tubes contained 5 mL of SDA, 1.0 mL of absolute alcohol and 0.1 mL of fungal culture. The positive control tube contained 5 mL of SDA, 200 mg/mL of fuconazole in 1 mL of absolute alcohol and fungal cultures. After 10 days, the results were recorded according to the formula:

... /2/

The degree of activity was recorded in five grades according to the percentage inhibition of growth: inactive (0), low (0-30 %), moderate (30-50 %), good (50-70 %) and significant (70 % and above).

Statistical analysis

Statistical analysis was performed with the analysis of variance (ANOVA) followed by Tukey's post hoc test. Statistical calculations were done using Minitab v. 11.0 software (Minitab Inc, State College, PA, USA).

Results and Discussion

Total phenolic content

The content of total phenolics in fractions 1-5 of the five Hypericum species is reported in Fig. 2. The crude extracts (marked as fraction 1) obtained from H. perforatum, H. oblongifoilum, H. monogynum, H. choisianum and H. dyeri were characterized by their contents of total phenolics expressed as GAE: 71.6, 76.8, 72.7, 96.7, and 58.1 mg/g, respectively. Fraction 2, obtained from the crude extracts using water re-extraction, showed a content of total phenolics expressed as GAE ranging from 46.0 (H. monogynum) to 92.7 mg/g (H. choisianum). The following re-extractions using ethyl acetate and acetone resulted in the content of total phenolics expressed as GAE in the range of 46.3 to 73.1 (fraction 3) and 46.0 to 87.8 mg/g (fraction 4). The content of total phenolics in the final residue (fraction 5) ranged from 21.1 (H. dyeri) to 100 mg/g (H. choisianum).

The yield of ethanol extract (content of fraction 1) ranged from 8.0 (H. oblongifolium) to 18.0 % (H. dyeri) (Fig. 2). In the study by Radulovi} et al. (11), the methanol extracts from Hypericum species from the Balkans ranged from 0.8 to 35.3 %. The content of crude aqueous extract of H. perforatum was 28.6 % (4).

Antioxidant activity

The antioxidant activity of the fractions was measured spectrophotometrically using phosphomolybdenum method, which is based on the reduction of Mo(VI) to Mo(V) by the sample analyte and the subsequent formation of green phosphate/Mo(V) compounds with a maximum absorption at 695 nm. The antioxidant capacity of various fractions of the five Hypericum species was compared (Table 1). All fractions showed remarkable activities. The same trend in activity was observed in most cases as in the DPPH radical scavenging activity and reducing power. The highest antioxidant activities as AAE were noted for fraction 2 of H. dyeri (1385 mmol/g), and fractions 1 (1345 mmol/g) and 4 (1287 mmol/g) of H. monogynum. The lowest value (96 mmol/g) was noted in the final residue (fraction 5) of H. dyeri.

Antiradical activity

The DPPH radical is a stable organic free-radical with an absorption band between 515 and 528 nm. It loses this absorption when accepting an electron or a free radical species, which results in a visually noticeable discolouration from purple to yellow. Because the DPPH radical can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentrations, it has been extensively used for screening antiradical activities of plant extracts (8). The DPPH radical scavenging activity of various fractions of five Hypericum species was studied.

All fractions separated from the five Hypericum species showed scavenging activity against the DPPH radical, but their antiradical properties were different (Fig. 3, Table 2). For H. perforatum, the highest activity was noted for fraction 5. For H. monogynum, the activities were shown by all fractions even at the lowest concentration (20 mg/mL). Among the crude extracts of the five plant species, the highest activity (EC50=28.5 mg/mL) was observed in H. monogynum, while the aqueous extract of H. dyeri exhibited the lowest activity (EC50=49.2 mg/mL). The ethyl acetate fraction of all species had lower activity, except that of H. choisianum (EC50=11.2 mg/mL). A very strong activity was shown by the acetone fraction of H. oblongifolium, to a maximum (EC50=19.8 mg/mL). Among the residual fractions, the highest antiradical activity (EC50=15.5 mg/mL) was observed for H. oblongifolium, while the weakest (EC50=139.2 mg/mL) for H. dyeri. Gallic acid in the same assay exhibited EC50=11.2 mg/mL. The significant DPPH radical scavenging activities of the fractions were due to their corresponding phenolic contents; fractions with higher phenolic contents showed remarkable scavenging activities (Table 1). In the studies of Zou et al. (8) and Silva et al. (25), the antiradical activity of the ethanol extract obtained from H. perforatum was characterized by EC50 of 10.6 and 49.0 mg/mL, respectively.

The reducing power serves as a strong indicator of the antioxidant activity and was determined using a modified iron(III) to iron(II) reduction assay. In this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of the extracts or compounds. The presence of reductants in the solution causes the reduction of the Fe3+/ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm (6). Comparative reducing power of various extracts/fractions (crude ethanolic, aqueous, ethyl acetate, acetone, and final residue) of five Hypericum species was studied (Fig. 4, Table 2). All samples showed some degree of reducing power; however, as anticipated, their reducing power was inferior to that of the standard. Like the scavenging activity, the reducing power of the samples increased with the increase of concentration. There were statistically significant differences observed among the same fractions (p<0.05) of different plants except for the final fractions of H. dyeri, where no significant difference (p>0.05) was observed in various fractions of the same plant. The crude extract of H. oblongifolium showed the highest reducing power expressed as AAE (449 mg/g) for all five species. For the aqueous and acetone fractions, the highest activity expressed as AAE (449 and 498 mg/g, respectively) was observed in H. oblongifolium. The ethyl acetate fractions of all species possessed lower activity expressed as AAE except for H. dyeri (402 mg/g). The greatest reducing power expressed as AAE (528 mg/g) was observed in the final fraction of H. choisianum, while the lowest (145 mg/g) was in the final fraction of H. dyeri. The reducing power might be due to either the presence of phenolics or some other reducing agents present in the plant; correlation in phenolic content, reducing power, and DPPH radical scavenging activity was observed in most cases (Table 2). The fraction with higher phenolic content showed remarkable scavenging activity and reducing power.

Antimicrobial activity

As shown in Table 3, the phenolic fractions from the five studied Hypericum species showed some antibacterial activity against all of the tested microorganisms, with the diameters of the zone of inhibition ranging between 10 and 23 mm. Of the studied plants, the most active fractions were fraction 1 and fraction 5 obtained from H. dyeri against Escherichia coli. Fractions 3, 4 and 5 from H. perforatum and fractions 1, 3 and 4 from H. oblongifolium showed positive antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, and so did fractions 1 obtained from H. monogynum and H. choisianum. Fractions 3, 4 and 5 from H. monogynum showed activity against Pseudomonas aeruginosa. All other fractions from all plants exhibited weak activity on the tested strains. All of the bacteria in the study were sensitive to streptomycin with Staphylococcus aureus and Pseudomonas aeruginosa being the most sensitive (inhibition zone values of 31 and 32 mm, respectively).

Antimicrobial activities of Hypericum species have been reported by several authors (11,15,25-28). Radulovi} et al. (11) studied the antibacterial activities of nine Hypericum species which showed significant activity even at a dose of 5 mg/disc and the zone of inhibition ranged from 12 to 42 mm. In our study, the zone of inhibition ranged between 10 and 23 mm, which is almost the same as that previously reported (15,26). Our findings correlate with the observations of previous screenings of medicinal plants for antimicrobial activity, where most of the active plants showed activity against Gram-positive strains only (few are active against Gram-negative bacteria) (15). Moreover, our current results show an antimicrobial activity against the Gram-negative bacterium Escherichia coli and this microorganism has been isolated from infected wounds of humans.

Results reported in Table 4 show that extracts from the five Hypericum species examined showed some antifungal activity against the tested fungal strains, with the zone of inhibition ranging between 10 and 52 %. The crude, aqueous, ethyl acetate, and acetone fractions of H. perforatum were found most active against Helminthosporium maydis, while all fractions of the same plant showed moderate activity against the tested fugal strains. Aqueous, ethyl acetate, acetone, and final residue of H. oblongifolium showed moderate activity against Aspergillus niger, Helminthosporium maydis and Alternaria solani, and weak activity against Aspergillus flavus. Aqueous, ethyl acetate, and acetone extracts of H. monogynum showed moderate activity against Aspergillus niger and Alternaria solani, and weak activity against Helminthosporium maydis and Aspergillus flavus. Moderate activity against Aspergillus niger, Helminthosporium maydis and Aspergillus flavus was also noted for fractions 1 and 5 of the same plant. Activity was also found in acetone fractions of H. choisianum and final residue against Aspergillus niger. The extract obtained from H. dyeri also had an activity up to some extent against Aspergillus niger; water and ethyl acetate fractions showed activity against Helminthosporium maydis, Alternaria solani and Aspergillus flavus. All the fungi tested in the study were sensitive to fuconazole, with Aspergillus niger and Alternaria solani being the most sensitive (inhibition zone values of 76 and 74 %, respectively), which is significant. None of the studied samples showed high activity, and most of them exhibited moderate or weak activity.

With regard to the components responsible for the antimicrobial activity shown, several compounds of distinct nature must be acting as antimicrobial agents in these plants. This is not unexpected: in the majority of Hypericum species studied so far, there are large varieties of active compounds, including naphthodianthrones, flavonoids, xanthones, tannins, essential oils and phloroglucinols (15). Because the antimicrobial activity in other species of this genus has been found to be closely related to the levels of flavonoids and phloroglucinol derivatives (9), it is reasonable to assume that these compounds are responsible for the antimicrobial activities reported here. Results suggest that further work is needed to locate the bioactive compounds from various extracts and that such efforts could result in the discovery of new compounds possessing a wide range of bioactivity.

Conclusions

From the achieved results, it can be concluded that the phenolic fractions from five Hypericum species from Pakistan possess antioxidant and antimicrobial activities. After toxicological studies of some potentially harmful compounds present in the extracts or their fractions, we suggest that these materials be used as natural antioxidants in food, functional food, and nutraceuticals.

Acknowledgements

Higher Education Commission of Pakistan is acknowledged for financial support. We thank Mr Momin Khan, Lecturer, Centre of Biotechnology and Microbiology, University of Peshawar, Pakistan, for his help in antimicrobial studies.

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