Next Article in Journal
New Approach of the Variable Fractional-Order Model of a Robot Arm
Next Article in Special Issue
The Potential of Amaranth as a Basic Raw Material for the Production of Pasta for a Vegan Diet
Previous Article in Journal
Effect of Three-Dimensional (3D) Scanning Factors on Minimizing the Scanning Errors Using a White LED Light 3D Scanner
Previous Article in Special Issue
Anti-Mutagenic and Immunomodulatory Effects of Astragali Radix Extract on a Cyclophosphamide-Induced Immunosuppressed Mouse Model
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Sciences
Volume 13
Issue 5
10.3390/app13053305
applsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Study of the Antibacterial Activity, Total Phenolic and Total Flavonoid Content of Nine Hypericum Species Grown in Greece

by
Eleni Kakouri
1,
Dimitra Daferera
1,
Panayiotis Trigas
2,
Despina Charalambous
3,
Maria Pantelidou
3,
Petros A. Tarantilis
1 and
Charalabos D. Kanakis
1,*
1
Laboratory of Chemistry, Department of Food Science and Human Nutrition, School of Food and Nutritional Sciences, Agricultural University of Athens, IeraOdos 75, 118 55 Athens, Greece
2
Laboratory of Systematic Botany, Department of Crop Science, School of Plant Sciences, Agricultural University of Athens, IeraOdos 75, 118 55 Athens, Greece
3
Department of Pharmacy, School of Health Sciences, Frederick University, 7, Y. Frederickou Str., Pallouriotissa, Nicosia 1036, Cyprus
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(5), 3305; https://doi.org/10.3390/app13053305
Submission received: 8 February 2023 / Revised: 27 February 2023 / Accepted: 3 March 2023 / Published: 5 March 2023
(This article belongs to the Special Issue Bioactive Compounds from Various Sources: Beneficial Effects and Technological Applications II)
Browse Figures
Versions Notes

Abstract

:
Hypericum perforatum is an herb whose use dates back centuries. Extracts of the plant are available as over-the-counter treatment options for depression. The genus consists of approximately 500 species, most of which have not yet been studied. Antimicrobial resistance has reached alarming levels, indicating a post-antibiotic era as many of the available treatment options become less effective. For this reason, nine Hypericum species were studied for their antimicrobial activity and their total phenolic and flavonoid content. Extracts were tested against Gram-negative and Gram-positive bacteria. Extracts inhibited the growth of Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis). The lowest MIC and MBC values were calculated for the extract of H. perforatum against both microorganisms tested, reaching 0.06 mg/mL for S. aureus and 0.13 mg/mL for E. faecalis. Total phenolic content was the highest in the H. perforatum extract (86 ± 12.90 mg GAE/g dry plant material). H. tetrapterum presented the highest flavonoid content, equal to 1.58 ± 0.4 mg RE/g of dry plant material. The Hypericum species studied herein are less common or have not yet been examined compared to H. perforatum; therefore, our study adds new data to the knowledge of the genus Hypericum.

1. Introduction

Antimicrobial resistance towards monotherapy or multiple antibiotic therapy is a spiraling problem because a growing number of infections are becoming harder to treat with current antibiotic treatments, and thereafter serious or even lethal health risks are raised. Multidrug-resistant organisms (MDROs), as reported by official organizations (WHO, ECDC), include, among others, Mycobacterium tuberculosis, Enterococcus faecium, Enterobacter cloacae, Klebsiella pneumoniae, and Staphylococcus aureus [1,2]. Hence, there is a critical need for new antimicrobial compounds to fight drug-resistant microorganisms. Most antibiotics that are used originate from microbes, which means they are basically naturally derived products. Consequently, plant-derived products are a great alternative to the discovery of new biologically active compounds to use against hospital-acquired infections or community-acquired pathogens. Plant-derived products are mixtures of secondary metabolites that consist of many diverse compounds classified based on structural differences. Among them, phenolic compounds are the largest and best studied group. More than 8000 phenolics with documented molecular structures have been described so far [3,4,5]. These secondary metabolites are studied with great interest by researchers due to their multiple biological activities [6,7].Especially, the study of phenolic compounds against antimicrobial resistance is challenging, and several studies confirm theirin vitroefficacy against a vast range of Gram-positive and Gram-negative bacteria [6,8,9,10].
The genus Hypericum belongs to the Hyperiaceae family, which encompasses approximately 500 species divided into 36 sections based mainly on morphological characteristics [11,12]. Historically, the use of the plant as a remedy has its roots in ancient civilizations. Treatment of wounds, burns, and bruises was among the most popular uses of H. perforatum, whereas gastrointestinal diseases, dysmenorrhea problems, acute mastitis, and depression were also handled with the herb [13,14,15,16]. Nowadays, these healing properties of Hypericum perforatum and other species of the genus are attributed to its complex chemical composition. Naphtodianthrones (including hypericin), phloroglucinols (including hyperforin), flavonoids (such as rutin, quercetin, and myricetin glucosides), xanthones (including cis-kielcorin), and phenolic acids (such as coumaroylquinic acid and chlorogenic acid) are the main classes of compounds identified in Hypericum extracts [2,17,18,19].
However, despite the large number of recognized species, the one most studied for its bioactivity remains H. perforatum, and many formulations of dietary supplements designed to ease central nervous system disorders are available [20]. In addition, studies on the antimicrobial activity of H. perforatum and other species of the genus have been published in the past few years [21,22,23]. Most focus was given to hypericin and hyperforin as the compounds responsible for the antimicrobial activity [21,22,23]. Nevertheless, the complex chemical profile of Hypericum species in combination with the already known activity of the secondary metabolites produced by the plants indicates that such biological activity is the result of synergistic action between all or some of the compounds that are present in the species [9].
Despite the frequent use of H. perforatum in modern society, studies on other species of the genus are lacking. Screening of more Hypericum species or species not yet studied is important and can be a strong stimulus for further research on the genus Hypericum and the development of new products to introduce to the pharmaceutical market. Therefore, with the aim of providing new data regarding infrequently studied species of the genus Hypericum, this study evaluates nine Hypericum species, namely H. perforatum and H. tetrapterum (sect. Hypericum), H. perfoliatum, H. rumeliacum subsp. apollinis, H. vesiculosum and H. cycladicum (sect. Drosocarpium), H. fragile (sect. Taeniocarpium), H. olympicum (sect. Olympia), and H. delphicum (sect. Adenosepalum), for their antimicrobial activity, total phenolic content, and total flavonoid content. The present work enhances the knowledge regarding the bioactive compounds of Hypericum species and consists of an additional step to further research the biological activity of more species of the genus.

2. Materials and Methods

2.1. Plant Material

Plant material was collected during the flowering period from different localities in Greece. The specimens were deposited at the herbarium of the Agricultural University of Athens (ACA). Precise information about the geographic locations and voucher numbers of the collected plant material is given in Table 1.

2.2. Preparation of Extracts

Two grams of each sample (leaves, flowers, and stems) were weighted and soaked for 2 min in a hydroalcoholic solution (70% v/v). An ultrasonic bath extraction at a stable temperature (25 ± 1 °C) and frequency (35 kHz) was implemented to isolate secondary metabolites from the plant material. Extraction was repeated three times in the dark. The extract was transferred to a rotary evaporator to remove the organic solvent. The remaining aqueous extract was cooled and lyophilized. The obtained powder was kept at −20 °C until further use.

2.3. Quantification of Total Phenolic Content

The total phenolic content of the extracts was estimated with the FolinCiocalteuPhenol Reagent (Sigma-Aldrich, Hamburg, Germany). Crude extracts were dissolved in methanol and water (70% v/v). The experiment took place in the dark, and the procedure followed the description in our previous study [24]. Extracts were incubated for 11/2 h and their absorbance was read at 765 nm. Quantification of phenolic compounds was performed using a standard curve of gallic acid monohydrate (Riedel-de Haën AG, Seelze, Germany) at concentrations ranging from 100–800 μg/mL. Results were expressed as mg of gallic acid equivalents (GAE) per gram of dry plant material using the equation derived from the plot: y = 0.0011x + 0.0148 (r = 0.999, n =3).

2.4. Quantification of Total Flavonoid Content

The total flavonoid content of the extracts was estimated using the aluminum chloride (AlCl3 anhydrous crystallized ≥ 99.0%, Sigma-Aldrich Germany) colorimetric assay. Crude extracts were dissolved in methanol. Then 200 μL of each extract were added to 1 mL of a 2% AlCl3 methanolic solution, followed by the addition of 3.8 mL of methanol to make a final volume of 5 mL. Solutions were vortexed and incubated for 15 min in the dark. Absorbance was read at 430 nm against a blank solution using methanol. A calibration curve was constructed following the above steps, using rutin dissolved in methanol at concentrations ranging from 200–1700 μg/mL. Results were expressed as mg of rutin equivalents (RE) per gram of dry plant material using the equation derived from the plot: y = 0.0539x + 0.0585 (r = 0.986, n =3).
Rutin was chosen as a standard solution, as indicated by the European Pharmacopoeia, Section II, hypericin herba [25], and according to preliminary experiments described as follows: For the total flavonoids assay, preliminary experiments with quercetin and rutin standard solutions (ExtraSynthese, Genay, France) after complexing with AlCl3 showed a maximum absorbance for quercetin at 458 nm, while the maximum absorbance for rutin was read at 435 nm after reaction with AlCl3 [Figure 1]. Afterwards, the reaction of the flavonoids with the AlCl3 complex was performed on the extracts. The results showed that, in the presence of the extracts, absorbance was near 430 nm. According to the literature data, most studies use a range of 410–430 nm to measure TFC [26]. Therefore, for the reasons mentioned above, rutin was chosen for the quantification of TFC, and the selected wavelength was 430 nm.

2.5. Antibacterial Activity

The antibacterial activity of the extracts was evaluated by determining the minimum inhibition concentration (MIC) values and the minimum bactericidal concentration (MBC) values of the extracts. The MIC was determined using the broth micro-dilution method as described in a previous study [27]. Hypericum extracts (10 mg/mL starting concentration) were dissolved in DMSO (a final concentration of 4%), and 200 μL of each extract was transferred to a 96-well plate in order to achieve a 2-fold serial dilution with 100 μL of Mueller Hinton Broth (MHB). Isolated cultures of E. coli (NCTC 9001, Sigma Aldrich, Hamburg, Germany), S. aureus (NCTC 6571, Sigma Aldrich, Germany), E. faecalis (NCTC775, Sigma Aldrich, Hamburg, Germany), and S. enteritidis (WDCM 00030, Sigma Aldrich, Hamburg, Germany)were prepared in MHB at a concentration of about 1 × 106 cfu/mL. Onehundred microliters (100 µL) of each bacterial inoculum were added to each well of extract or control. Blank samples of each extract (without bacteria) were subjected to a 2-fold serial dilution with MHB (blank control). Controls with bacteria (100 µL) but no extract were used as growth controls. Also, a solvent control and a sterility control were used with MHB, with no bacteria and no extract, respectively. Ampicillin (0.516 mg/mL, Sigma Aldrich, Gillingham, UK) or Gentamycin (0.064 mg/mL, Molekula, Darlington, UK) were included as positive controls. After 18 h of incubation at 37 °C, 30 µL of 0.2 mg/mL p-iodonitrotetrazolium chloride (INT) (Sigma Aldrich, Gillingham, UK) was added to each plate, followed by incubation at 37 °C for 30 min. The absorbance of each plate was measured at 492 nm with a microplate reader (Sunrise, Tecan Trading Ltd., Zürich, Switzerland). The MIC of each extract was defined as the sample concentration that inhibited bacterial growth as compared with that of the blank control.The MBC of the extracts was also determined by subculturing 2 μL aliquots of the MIC assay preparations in 100 μL MHB and incubating for 24 h at 37 °C. The MBC was defined as the lowest concentration of each sample that did not exhibit a color change after the addition of INT, as described above.

2.6. Statistical Analysis

All experiments were performed in triplicates. The results were presented as the mean value ± the estimated SD. Significance differences between sample means were determined by a student’s t test using GraphPad Prism (ver. 8.4.2). The significance level was set to 0.05, and the confidence intervals were at ±95% CI. The data were presented as mean ± standard deviation (SD).

3. Results

3.1. Total Phenolic and Total Flavonoid Content

Results demonstrated that the extract of H. perforatum was the most abundant in phenolic compounds (86 ± 13.34 mg GAE/g dry plant material), while the lowest TPC was determined in the extract of H. vesiculosum (26.92 ± 10.32 mg GAE/g dry plant material). For the total flavonoid content shown in Table 2, the most abundant extract in flavonoids was that of H. tetrapterum (1.58 ± 0.08 mg RE/g dry plant material). Similarly, the lowest TFC was calculated for H. perfoliatum (0.21 ± 0.14 mg RE/g dry plant material). The UV-Vis spectra of some of the Hypericum species studied are presented in Figure 2. Figure 2a presents the UV-Vis spectra of the less studied species of Hypericum, namely H. cycladicum, H. delphicum, and H. fragile, as well as the UV-Vis spectrum of H. perforatum, to allow comparison with the other species. Figure 2b presents the UV-Vis spectra of the same species after complexing with AlCl3. As shown in Figure 2a, the UV-Vis spectra of H. cycladicum and H. fragile present a λmax at 334 nm and 330 nm, respectively. This range of absorption is typical for flavones and flavonols [28]. To distinguish between these two groups, Marby et al. [29] observed that the UV wavelength of the B ring is near 304–350 nm for flavones, while flavonols absorb at a longer wavelength of 328–357 nm. Therefore, according to the literature data, flavonols are compounds that seem to prevail in the extracts of H. cycladicum and H. fragile. Moreover, the hypsochromic shift at band I in the case of H. delphicum is attributed to the presence of sugar moieties, more probably attached at position 3 of the C ring [29].
As demonstrated in Table 2, there seems to be no proportional relationship between TPC and TFC. The mechanism of action of the Folin Ciocalteu assay is the transfer of electrons from phenolic compounds. Nevertheless, non-phenolic compounds such as ascorbic acid, aromatic amines, and sugars can also reduce the Folin Ciocalteu reagent, resulting in an additive effect on the interpreted results [30]. Consequently, a high TPC is not always accompanied by a high TFC, as in the case of H. perforatum. Fewer limitations exist for the AlCl3 colorimetric assay. This method is accepted among researchers for the determination of total flavonoid content because the reagent does not interfere with other subclasses of phenolic compounds. On the contrary, AlCl3 forms stable complexes with flavonoids by binding at position 4 of the cheto-group (C ring) and occupying at the same time hydroxyl groups from positions 5 or 3. The other binding site is at positions 3′ and 4′ [Figure 3]. However, most of the flavonoids are produced as glycosides. A glycosidic moiety or moieties may occupy one or more of the binding sites of AlCl3; therefore, AlCl3 will not bind the positions occupied by the sugar moiety [31].Phenolic compounds are produced mostly in their glycosylated form [32]. Hence, it is possible that the stereochemistry of glycosylated flavonoids presented in the extracts hinders the binding of AlCl3.

3.2. Antimicrobial Activity

According to the results shown in Table 3, none of the extracts exhibited antibacterial activity against Gram-negative bacteria (E. coli and S. enteritidis). On the contrary, all Hypericum extracts demonstrated some degree of inhibition (weak, moderate, or strong) and bactericidal effect against Gram-positive bacteria (S. aureus and E. faecalis). More specifically, H. perforatum demonstrated strong growth inhibition and bactericidal activity against both S. aureus (MIC: 0.06 mg/mL; MBC: 0.51 mg/mL) and E. faecalis (MIC: 0.13 mg/mL; MBC: 0.51 mg/mL). Similarly, H. delphicum also demonstrated a strong growth inhibition against both Gram-positive species. On the other hand, H. cycladicum and H. olympicum exhibited strong bacterial inhibition activity against S. aureus (MIC: 0.31 mg/mL) but moderate inhibition against E. faecalis (MIC: 0.63 mg/mL). All the other Hypericum species demonstrated moderate bacterial inhibition activity (MIC: 0.13 mg/mL), except the H. fragile extract, which exhibited weak inhibition activity against both S. aureus and E. faecalis (1.75 mg/mL and 3.50 mg/mL, respectively).

4. Discussion

Resistance to antibiotics is a spiraling health problem. Current antibiotic therapies gradually become ineffective or are sometimes inadequate to treat infections, an issue that, according to the WHO, prolongs hospitalization and increases costs and mortality rates. In this regard, the discovery of new antibiotics is urgent. Auspicious solutions against antibiotic resistance are naturally derived products [35,36]. Plants have been used to heal numerous infectious diseases since antiquity, and traditional medicine is still widely used to prevent and manage health issues [37,38]. Motivated by traditional medicine and based on scientific knowledge, several medications have been derived from medicinal plants, or naturally derived molecules were the key to the synthesis of new ones [35]. In our study, nine Hypericum species were tested for their antibacterial activity against four microorganisms: E. coli, S. enteritidis, S. aureus, and E. faecalis. All the tested samples showed selective antibacterial activity against Gram-positive bacteria. H. perforatum was the most potent extract against both S. aureus and E. faecalis, while the extract of H. fragile showed the weakest antibacterial activity. Comparing the activities of the species that belong to sect. Drosocarpium, namely H. perfoliatum, H. rumeliacum subsp. apollinis, H. vesiculosum, and H. cycladicum, the latter was the most active to inhibit the growth of S. aureus and E. faecalis (MIC: 0.31 and 0.63 mg/mL, respectively), while the extracts of H. perfoliatum, H. rumeliacum subsp. apollinis, and H. vesiculosum were equally effective. Within the Drosocarpium section, non-statistically significant differences were observed between its TPC and those calculated for H. perfoliatum and H. rumeliacum subsp. apollinis extracts. On the other hand, statistically significant differences were observed between the TFC of the extracts, with H. cycladicum being the extract with the highest TFC. Therefore, the better antibacterial activity of the extract can be mainly attributed to its TFC. Regarding H. rumeliacum subsp. apollinis, the concentration required to inhibit bacterial growth is equal to that of H. perfoliatum and H. vesiculosum. Within these extracts, non-statistically significant differences were observed regarding their TPC, and all the extracts differed significantly for their TFC.
As for the Hypericum section, which includes H. perforatum and H. tetrapterum, the best antibacterial activity for both of the bacteria tested was observed in the H. perforatum extract. The total phenolic content of H. perforatum exceeds that of H. tetrapterum. This study discusses the phytochemical profile of the extracts based on the TPC and TFC. However, according to the literature, Hypericum species also contain other classes of compounds, specifically hyperforin and hypericins. Hyperforin and similar compounds (for example, adhyperforin) are compounds with strong antibacterial activity [39,40]. Therefore, the better antibacterial result observed in the case of H. perforatum indicates that, apart from phenolic compounds, the presence of other classes of compounds in this extract contributes to the overall antibacterial activity.
Between the extracts of H. fragile, H. olympicum, and H. delphicum, the TPC of the latter extract was the highest among the three species, while their TPC and TFC presented statistically significant differences. Naphtodiantrones are also found in Hypericum species of the Adenosepalum and Taeniocarpium sections [41]. Consequently, the strongest antibacterial activity of H. delphicum can be attributed not only to its high phenolic content but also to other compounds, such as phenolic acids with interesting antimicrobial activity [42] and the presence of naphtodianthrones and/or phloroglucinols [41].
In general, regarding the results of the antibacterial activity of the extracts, it should be noted that apart from flavonoids such as luteolin [43,44] and kaempferol [43,44,45,46], other compounds such as chlorogenic acid and neo-chlorogenic acid are also discussed as occurring in the genus Hypericum [44,45,46,47]. In addition to flavonoids, it has been demonstrated that chlorogenic acid is a compound with exceptional antibacterial activity [48,49]. Differences regarding the bactericidal activity of the above extracts indicate that not only quantitative but also qualitative differences may produce synergistic effects that potentiate the activity of the extract and contribute to its overall activity.
A broad spectrum of antimicrobial activity has been demonstrated for various Hypericum species against Gram-positive and Gram-negative microorganisms. H. perforatum is the most studied species, and several solvents, such as ethanol, methanol, and chloroform, have been used by researchers to isolate its secondary metabolites [21,23,50,51]. Similarly, various microorganisms, such as S. subtilis, S. aureus, E. faecalis, and E. coli, have been tested for their susceptibility to Hypericum extracts [50,51]. For example, in the study by Avato et al. [51], six Gram-positive bacteria were treated with different extracts of H. perforatum. It was concluded that the ethanolic and chloroform extracts were the most potent because the MIC values were lower. Nevertheless, all extracts presented antimicrobial activity, and when such activity was compared to that of standard compounds, namely hyperforin and hypericin, it was demonstrated that both standard compounds inhibited the majority of the microorganisms under investigation, while the concentration requested to inhibit growth of the microorganisms was, for most of the bacteria, close to that of the extracts [51]. Similarly, different subspecies of H. perforatum and other Hypericum species that belong to different sections from Central Italy were examined for their antimicrobial activity [50]. Among all the tested extracts, those of the H. perforatum subspecies were the most active, while only the extract of H. perforatum subsp. veronense managed to inhibit the growth of E.coli. Additionally, H. tetrapterum inhibited fewer microorganisms, and the zone of inhibition of bacterial growth was smaller than that formed when microorganisms were treated with the extract of H. perforatum. In the same study, the antimicrobial activity of H. hirsutum (sect. Taeniocarpium) was also evaluated. The H. fragile reported in our study belongs to the same section, thus we compared the antimicrobial activity of this extract with H. hirsutum, for which no data have been published so far. In contrast to H. hirsutum, for which no inhibition against E. faecalis was observed, H. fragile showed a broader spectrum of antimicrobial activity because the extract hindered both S. aureus and E. faecalis. It is possible that different extraction procedures resulted in differences in the phytochemical composition of these extracts, which in turn affected the obtained results [50]. H. delphicum is another species for which data regarding its antimicrobial activity are not available. However, the above-mentioned study [50] and the study by Dall’Agnol et al. [23] examine H. montanum and H. caprifolium, respectively, species that belong to the same section (sect. Adenosepalum) as H. delphicum. The results are partially in accordance with our findings because the extract of H. caprifolium was also active against Gram-negative bacteria, a result not observed for H. delphicum.
H. olympicum was examined by Radulović et al. [43,52] and, according to their results, a broad spectrum of antimicrobial activity was observed against both Gram-positive and Gram-negative bacteria, including S. aureus and E.coli. However, in our study, H. olympicum did not inhibit Gram-negative bacteria. Nevertheless, new information is now available about its antibacterial activity against E. faecalis, a microorganism resistant to many antibiotics [53,54].
There are limited studies that deal with the antibacterial activity of H. perfoliatum and H. rumeliacum subp. apollinis [52,55], while no previous studies were found for H. vesiculosum and H. cycladicum. All these species are classified within sect. Drosocarpium. H. perfoliatum was studied for its antimicrobial activity by Del Monte et al. [55]. The methanolic extract did not exhibit antimicrobial activity; on the contrary, chloroform and chloroform-methanol extracts exerted significant antimicrobial activity against all the microorganisms tested. In our study, the H. perfoliatum extract inhibited only Gram-positive bacteria, and in fact, the MIC and MBC values were equivalent for both microorganisms. Similar results were reported by Radulović et al. [52] regarding H. rumeliacum Boiss. The extract showed strong antimicrobial activity against life-threatening microorganisms, such as K. pneumonia and S. enteritis. In our study, H. rumeliacum subsp. apollinis inhibited only Gram-positive bacteria, and the inhibition of E. faecalis by H. perfoliatum and H. rumeliacum subsp. apollinis was reported for the first time.
All the tested extracts were active against Gram-positive bacteria; however, considering the literature data, generally, Hypericum spp. seem better for combating Gram-positive bacteria. S. aureus and E. faecalis are two microorganisms that resist some kinds of antibiotics, such as β-lactames [56], tetracyclines, and macrolides [57]. Certainly, the biological activity of an extract is dependent on its phytochemical profile and the extraction technique used to isolate secondary metabolites. Consequently, considering the different geographical origins of the Hypericum species used in this study, differences regarding their potency or inability to inhibit microorganisms are expected.
Regarding the total phenolic and total flavonoid content, the most studied Hypericum species are those of the Drosocarpium section, followed by those of the Hypericum section [17,58,59]. For example, Zheleva-Dimitrova et al. [59] studied, among others, some species from the Drosocarpium and Hypericum sections. Species from the Hypericum section, specifically H. perforatum and H. tetrapterum, were reported to have the highest total tannin content (8.67 ± 0.02 g/100 g DW for H. perforatum) and TFC (1.13 ± 0.02 g/100 g DW for H. tetrapterum), respectively. Our results demonstrated a similar bioactivity when referring to the Hypericum section, as H. perforatum contained the highest content of phenolic compounds, while TFC was highest in the H. tetrapterum extract. According to Zheleva-Dimitrovaet al. [59], H. olympicum, a species of the Olympia section, demonstrated a moderate quantity of total tannins (3.28 ± 0.03 g/100 g DW), while the TFC was the lowest (0.20 ± 0.03 g/100 g DW) compared to all the other studied species investigated. In our study, similar results were demonstrated regarding the TPC of H. olympicum. However, the TFC of the extract exceeded that of H. perfoliatum and H. vesiculosum, belonging to the Drosocarpium section. At the same time, for the TFC and the Drosocarpium section, H. cycladicum and H. rumeliacum subsp. apollinis were the most abundant.

5. Conclusions

In this study, nine Hypericum species collected in Greece were evaluated for their total phenolic and total flavonoid content, as well as for their antibacterial activity. Among the studied species, H. perforatum showed the highest TPC, while the TFC was the highest in the case of H. tetrapterum, followed by H. cycladicum and H. rumeliacum subsp. apollinis, extracts that belong to the Drosocarpium section. The obtained results indicated Hypericum species as a good source of secondary metabolites; therefore, their biological activity was evaluated against Gram-positive and Gram-negative bacteria. Extracts demonstrated weak to strong antibacterial activity. Gram-negative bacteria were all resistant to the tested extracts, while Gram-positive bacteria were successfully inhibited. The extracts showed a minor antibacterial potential with respect to the antibiotic gentamycin. However, the need for new antimicrobial agents is continually increasing. In this regard, the study of natural products as potential alternatives to available antibiotic treatments is of critical importance. The findings of this study provide valuable insights into the biological activity of Hypericum species. We demonstrate that, apart from H. perforatum, screening other species of the genus is also likely to contribute to the field of new drug development.

Author Contributions

Conceptualization, E.K., D.D., P.A.T. and P.T.; methodology, E.K., D.D. and D.C.; investigation, E.K. and D.C.; data curation, E.K., D.D. and D.C.; writing—original draft preparation, E.K. and D.C.; writing—review and editing, D.D., D.C., M.P., P.T., C.D.K. and P.A.T.; supervision, C.D.K., P.A.T. and M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data supporting this study are not publicly available. Please contact [email protected], [email protected].

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Levy, S.B.; Marshall, B. Antibacterial resistance worldwide: Causes, challenges and responses. Nat. Med. 2004, 10, S122–S129. [Google Scholar] [CrossRef]
  2. European Centre for Disease Prevention and Control. Antimicrobial Resistance Surveillance in Europe 2011; Annual Report of the European Antimicrobial Resistance Surveillance Network (EARS-Net); ECDC: Stockholm, Sweden, 2012. [Google Scholar]
  3. Pandey, K.B.; Rizvi, S.I. Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Med. Cell. Longev. 2009, 2, 270–278. [Google Scholar] [CrossRef] [Green Version]
  4. Soobrattee, M.A.; Neergheen, V.S.; Luximon-Ramma, A.; Aruoma, O.I.; Bahorun, T. Phenolics as potential antioxidant therapeutic agents: Mechanism and actions. Mutat. Res./Fundam. Mol. Mech. Mutagen. 2005, 579, 200–213. [Google Scholar] [CrossRef]
  5. Zhang, Y.; Cai, P.; Cheng, G.; Zhang, Y. A brief review of phenolic compounds identified from plants: Their extraction, analysis, and biological activity. Nat. Prod. Commun. 2022, 17, 1934578X211069721. [Google Scholar] [CrossRef]
  6. Kumar, S.; Pandey, A.K. Chemistry and biological activities of flavonoids: An overview. Sci. World J. 2013, 29, 162750. [Google Scholar] [CrossRef] [Green Version]
  7. Mutha, R.E.; Tatiya, A.U.; Surana, S.J. Flavonoids as natural phenolic compounds and their role in therapeutics: An overview. Future J. Pharm. Sci. 2021, 7, 25. [Google Scholar] [CrossRef]
  8. Cushnie, T.T.; Lamb, A.J. Antimicrobial activity of flavonoids. Int. J. Antimicrob. Agents 2005, 26, 343–356. [Google Scholar] [CrossRef]
  9. Bouarab-Chibane, L.; Forquet, V.; Lantéri, P.; Clément, Y.; Léonard-Akkari, L.; Oulahal, N.; Degraeve, P.; Bordes, C. Antibacterial properties of polyphenols: Characterization and QSAR (Quantitative structure–activity relationship) models. Front. Microbiol. 2019, 10, 829. [Google Scholar] [CrossRef] [Green Version]
  10. Ecevit, K.; Barros, A.A.; Silva, J.M.; Reis, R.L. Preventing Microbial Infections with Natural Phenolic Compounds. Future Pharmacol. 2022, 2, 460–498. [Google Scholar] [CrossRef]
  11. Robson, N.K.B. Studies in the genus Hypericum L. (Hypericaceae) 5(1). Sections 10. Olympia to 15/16 Crossophyllum. Phytotaxa 2010, 4, 5–126. [Google Scholar] [CrossRef]
  12. Crockett, S.L.; Robson, N.K. Taxonomy and Chemotaxonomy of the Genus Hypericum. Med. Aromat. Plant Sci. Biotechnol. 2011, 5, 1–13. [Google Scholar]
  13. Linden, M.; Wurzendorf, K.; Ploch, M.; Schaefer, M. Self medication with St. John’s wort in depressive disorders: An observational study in community pharmacies. J. Affect. Disord. 2008, 107, 205–210. [Google Scholar] [CrossRef]
  14. Lewis, S.; Willis, K.; Kokanovic, R.; Pirotta, M. ‘I’m managing myself’: How and why people use St John’s wort as a strategy to manage their mental health risk. Health Risk Soc. 2015, 17, 439–457. [Google Scholar] [CrossRef]
  15. Solati, K.; Karimi, M.; Rafieian-Kopaei, M.; Abbasi, N.; Abbaszadeh, S.; Bahmani, M. Phytotherapy for wound healing: The most important herbal plants in wound healing based on Iranian ethnobotanical documents. Mini. Rev. Med. Chem. 2021, 21, 500–519. [Google Scholar] [CrossRef]
  16. Gunther, R. Dioscorides, ‘De Materia Medica’, The Greek Herbal of Dioscordes; Hafner Publishing Co.: New York, NY, USA, 1968. [Google Scholar]
  17. Öztürk, N.; Tunçel, M.; Potoğlu-Erkara, İ. Phenolic compounds and antioxidant activities of some Hypericum species: A comparative study with H. perforatum. Pharm. Biol. 2009, 47, 120–127. [Google Scholar] [CrossRef]
  18. Babotă, M.; Frumuzachi, O.; Mocan, A.; Tămaș, M.; Dias, M.I.; Pinela, J.; Stojković, D.; Soković, M.; Bădărău, A.S.; Crișan, G.; et al. Unravelling Phytochemical and Bioactive Potential of Three Hypericum Species from Romanian Spontaneous Flora: H. alpigenum, H. perforatum and H. rochelii. Plants 2022, 11, 2773. [Google Scholar] [CrossRef]
  19. Tanaka, N.; Kashiwada, Y. Characteristic metabolites of Hypericum plants: Their chemical structures and biological activities. J. Nat. Med. 2021, 75, 423–433. [Google Scholar] [CrossRef]
  20. Oliveira, A.I.; Pinho, C.; Sarmento, B.; Dias, A.C. Neuroprotective Activity of Hypericum perforatum and Its Major Components. Front. Plant Sci. 2016, 7, 1004. [Google Scholar] [CrossRef] [Green Version]
  21. Saddiqe, Z.; Naeem, I.; Maimoona, A. A review of the antibacterial activity of Hypericum perforatum L. J. Ethnopharmacol. 2010, 131, 511–521. [Google Scholar] [CrossRef]
  22. Ilieva, Y.; Marinov, T.; Trayanov, I.; Kaleva, M.; Zaharieva, M.M.; Yocheva, L.; Kokanova-Nedialkova, Z.; Najdenski, H.; Nedialkov, P. Outstanding Antibacterial Activity of Hypericum rochelii-Comparison of the Antimicrobial Effects of Extracts and Fractions from Four Hypericum Species Growing in Bulgaria with a Focus on Prenylated Phloroglucinols. Life 2023, 13, 274. [Google Scholar] [CrossRef]
  23. Dall’Agnol, R.; Ferraz, A.; Bernardi, A.P.; Albring, D.; Nör, C.; Sarmento, L.; Lamb, L.; Hass, M.; von Poser, G.; Schapoval, E.E. Antimicrobial activity of some Hypericum species. Phytomedicine 2003, 10, 511–516. [Google Scholar] [CrossRef]
  24. Kakouri, E.; Kanakis, C.; Trigas, P.; Tarantilis, P.A. Characterization of the chemical composition of Drimianumidica plant parts using high-resolution mass spectrometry: Study of their total phenolic content and antioxidant activity. Anal. Bioanal. Chem. 2019, 411, 3135–3150. [Google Scholar] [CrossRef] [PubMed]
  25. European Pharmacopoeia 7th Edition (7.5). Ref. 07/2008:1874. Available online: https://file.wuxuwang.com/yaopinbz/EP6/EP6.2_01__114.pdf (accessed on 30 January 2023).
  26. Shraim, A.M.; Ahmed, T.A.; Rahman, M.M.; Hijji, Y.M. Determination of total flavonoid content by aluminum chloride assay: A critical evaluation. LWT 2021, 150, 111932. [Google Scholar] [CrossRef]
  27. Charalambous, D.; Eliades, N.-G.H.; Christoforou, M.; Kakouri, E.; Kanakis, C.; Tarantilis, P.A.; Pantelidou, M. Chemical Characterization, Antioxidant and Antimicrobial Properties of Different Types of Tissue of Cedrusbrevifolia Henry Extracts. Molecules 2022, 27, 2717. [Google Scholar] [CrossRef] [PubMed]
  28. Santos-Buelga, C.; García-Viguera, C.; Tomás-Barberán, F.A. On-line identification of flavonoids by HPLC coupled to diode array detection. In Methods in Polyphenol Analysis, 1st ed.; Santos-Buelga, C., Williamson, G., Eds.; The Royal Society of Chemistry: Cambridge, UK, 2003; pp. 92–127. [Google Scholar]
  29. Mabry, T.J.; Markham, K.R.; Thomas, M.B.; Mabry, T.J.; Markham, K.R.; Thomas, M.B. The ultraviolet spectra of flavones and flavonols. In The Systematic Identification of Flavonoids, 1st ed.; Springer: Berlin/Heidelberg, Germany, 1970; pp. 41–164. [Google Scholar]
  30. Everette, J.D.; Bryant, Q.M.; Green, A.M.; Abbey, Y.A.; Wangila, G.W.; Walker, R.B.J. Thorough Study of Reactivity of Various Compound Classes toward the Folin-Ciocalteu Reagent. Agric. Food Chem. 2010, 58, 8139–8144. [Google Scholar] [CrossRef] [Green Version]
  31. Mammen, D.; Daniel, M. A critical evaluation on the reliability of two aluminum chloride chelation methods for quantification of flavonoids. Food Chem. 2012, 135, 1365–1368. [Google Scholar] [CrossRef]
  32. Hollman, P.C. Absorption, bioavailability, and metabolism of flavonoids. Pharm. Biol. 2004, 42 (Suppl. S1), 74–83. [Google Scholar] [CrossRef]
  33. Aligiannis, N.; Kalpoutzakis, E.; Mitaku, S.; Chinou, I.B. Composition and antimicrobial activity of the essential oils of two Origanum species. J. Agric. Food Chem. 2001, 49, 4168–4170. [Google Scholar] [CrossRef]
  34. Fabry, W.; Okemo, P.O.; Ansorg, R. Antibacterial activity of East African medicinal plants. J. Ethnopharmacol. 1998, 60, 79–84. [Google Scholar] [CrossRef] [Green Version]
  35. Atanasov, A.G.; Zotchev, S.B.; Dirsch, V.M.; Supuran, C.T. Natural products in drug discovery: Advances and opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216. [Google Scholar] [CrossRef]
  36. Shamsudin, N.F.; Ahmed, Q.U.; Mahmood, S.; Ali Shah, S.A.; Khatib, A.; Mukhtar, S.; Alsharif, M.A.; Parveen, H.; Zakaria, Z.A. Antibacterial Effects of Flavonoids and Their Structure-Activity Relationship Study: A Comparative Interpretation. Molecules 2022, 27, 1149. [Google Scholar] [CrossRef] [PubMed]
  37. Yuan, H.; Ma, Q.; Ye, L.; Piao, G. The Traditional Medicine and Modern Medicine from Natural Products. Molecules 2016, 21, 559. [Google Scholar] [CrossRef] [Green Version]
  38. Li, F.S.; Weng, J.K. Demystifying traditional herbal medicine with modern approach. Nat. Plants 2017, 3, 17109. [Google Scholar] [CrossRef] [PubMed]
  39. Alahmad, A.; Alghoraibi, I.; Zein, R.; Kraft, S.; Dräger, G.; Walter, J.G.; Scheper, T. Identification of Major Constituents of Hypericum perforatum L. Extracts in Syria by Development of a Rapid, Simple, and Reproducible HPLC-ESI-Q-TOF MS Analysis and Their Antioxidant Activities. ACS Omega 2022, 7, 13475–13493. [Google Scholar] [CrossRef]
  40. Bridi, H.; de Carvalho Meirelles, G.; von Poser, G.L. Structural diversity and biological activities of phloroglucinol derivatives from Hypericum species. Phytochemistry 2018, 155, 203–232. [Google Scholar] [CrossRef] [PubMed]
  41. de Carvalho Meirelles, G.; Bridi, H.; Rates, S.M.K.; von Poser, G.L. Southern brazilian Hypericum species, promising sources of bioactive metabolites. Stud. Nat. Prod. Chem. 2018, 59, 491–507. [Google Scholar]
  42. Liu, J.; Du, C.; Beaman, H.T.; Monroe, M.B.B. Characterization of phenolic acid antimicrobial and antioxidant structure–property relationships. Pharmaceutics 2020, 12, 419. [Google Scholar] [CrossRef]
  43. Saddiqe, Z.; Naeem, I.; Hellio, C.; Patel, A.V.; Abbas, G. Phytochemical profile, antioxidant and antibacterial activity of four Hypericum species from the UK. S. Afr. J. Bot. 2020, 133, 45–53. [Google Scholar] [CrossRef]
  44. Barnes, J.; Anderson, L.A.; Phillipson, J.D. St John’s wort (Hypericum perforatum L.): A review of its chemistry, pharmacology and clinical properties. J. Pharm. Pharmacol. 2001, 53, 583–600. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Ion, V.; Ielciu, I.; Cârje, A.G.; Muntean, D.L.; Crişan, G.; Păltinean, R. Hypericum spp.—An Overview of the Extraction Methods and Analysis of Compounds. Separations 2022, 9, 17. [Google Scholar] [CrossRef]
  46. Rusalepp, L.; Raal, A.; Puessa, T.; Maeeorg, U. Comparison of chemical composition of Hypericum perforatum and H. maculatum in Estonia. Biochem. Syst. Ecol. 2017, 73, 41–46. [Google Scholar] [CrossRef]
  47. Daskalaki, A.; Grafakou, M.E.; Barda, C.; Kypriotakis, Z.; Heilmann, J.; Skaltsa, H. Secondary metabolites from Hypericum trichocaulon Boiss. & Heldr., growing wild in the island of crete. Biochem. Syst. Ecol. 2021, 97, 104294. [Google Scholar]
  48. Lou, Z.; Wang, H.; Zhu, S.; Ma, C.; Wang, Z. Antibacterial activity and mechanism of action of chlorogenic acid. J. Food Sci. 2011, 76, M398–M403. [Google Scholar] [CrossRef] [PubMed]
  49. Wu, Y.; Liang, S.; Zhang, M.; Wang, Z.; Wang, Z.; Ren, X. The effect of chlorogenic acid on Bacillus subtilis based on metabolomics. Molecules 2020, 25, 4038. [Google Scholar] [CrossRef]
  50. Cecchini, C.; Cresci, A.; Coman, M.M.; Ricciutelli, M.; Sagratini, G.; Vittori, S.; Lucarini, D.; Maggi, F. Antimicrobial activity of seven hypericum entities from central Italy. Planta Med. 2007, 73, 564–566. [Google Scholar] [CrossRef] [Green Version]
  51. Avato, P.; Raffo, F.; Guglielmi, G.; Vitali, C.; Rosato, A. Extracts from St John’s Wort and their antimicrobial activity. Phytother. Res. 2004, 18, 230–232. [Google Scholar] [CrossRef] [PubMed]
  52. Radulović, N.; Stankov-Jovanović, V.; Stojanović, G.; Šmelcerović, A.; Spiteller, M.; Asakawa, Y. Screening of in vitro antimicrobial and antioxidant activity of nine Hypericum species from the Balkans. Food Chem. 2007, 103, 15–21. [Google Scholar] [CrossRef]
  53. Gagetti, P.; Bonofiglio, L.; García Gabarrot, G.; Kaufman, S.; Mollerach, M.; Vigliarolo, L.; Specht, M.; Toresani, I.; Lopardo, H.A. Resistencia a los β-lactámicosenenterococos. Rev. Argent. Microbiol. 2019, 51, 179–183. [Google Scholar] [PubMed]
  54. Feßler, A.T.; Scholtzek, A.D.; Schug, A.R.; Kohn, B.; Weingart, C.; Hanke, D.; Schink, A.-K.; Bethe, A.; Lübke-Becker, A.; Schwarz, S. Antimicrobial and Biocide Resistance among Canine and Feline Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii Isolates from Diagnostic Submissions. Antibiotics 2022, 11, 152. [Google Scholar] [CrossRef] [PubMed]
  55. Del Monte, D.; De Martino, L.; Marandino, A.; Fratianni, F.; Nazzaro, F.; De Feo, V. Phenolic content, antimicrobial and antioxidant activities of Hypericum perfoliatum L. Ind. Crops Prod. 2015, 74, 342–347. [Google Scholar] [CrossRef]
  56. Guo, Y.; Song, G.; Sun, M.; Wang, J.; Wang, Y. Prevalence and Therapies of Antibiotic-Resistance in Staphylococcus aureus. Front. Cell. Infect. Microbiol. 2020, 10, 107. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Foster, T.J. Antibiotic resistance in Staphylococcus aureus. Current status and future prospects. FEMS Microbiol. Rev. 2017, 41, 430–449. [Google Scholar] [CrossRef]
  58. Kızıl, G.; Kızıl, M.; Yavuz, M.; Emen, S.; Hakimoğlu, F. Antioxidant Activities of Ethanol Extracts of Hypericum triquetrifolium. and Hypericum scabroides. Pharm. Biol. 2008, 46, 231–242. [Google Scholar] [CrossRef] [Green Version]
  59. Zheleva-Dimitrova, D.; Nedialkov, P.; Kitanov, G. Radical scavenging and antioxidant activities of methanolic extracts from Hypericum species growing in Bulgaria. Pharmacogn. Mag. 2010, 6, 74. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Applsci 13 03305 g001 550
Figure 1. UV-Vis spectrum of quercetin and rutin standard solutions after complexing with AlCl3 reagent.
Figure 1. UV-Vis spectrum of quercetin and rutin standard solutions after complexing with AlCl3 reagent.
Applsci 13 03305 g001
Applsci 13 03305 g002 550
Figure 2. (a) UV-Vis spectra of H. cycladicum, H. delphicum, H. fragile, and H. perforatum extracts. (b) UV-Vis spectra of H. cycladicum, H. delphicum, H. fragile, and H. perforatum extracts after complexing with AlCl3 reagent.
Figure 2. (a) UV-Vis spectra of H. cycladicum, H. delphicum, H. fragile, and H. perforatum extracts. (b) UV-Vis spectra of H. cycladicum, H. delphicum, H. fragile, and H. perforatum extracts after complexing with AlCl3 reagent.
Applsci 13 03305 g002
Applsci 13 03305 g003 550
Figure 3. General structure of flavonoids.
Figure 3. General structure of flavonoids.
Applsci 13 03305 g003
Table
Table 1. Collection data of the Hypericum species examined.
Table 1. Collection data of the Hypericum species examined.
TaxonSectionCollection SiteCollection DateLatitudeLongitudeElevation (m)HabitatVoucher Number
H. cycladicumDrosocarpiumAndros Island12 May201937°54′24°52′30Phrygana12285
H. delphicumAdenosepalumEvvia Island14 June201938°53′23°52′1000Forest clearings12281
H. fragileTaeniocarpiumEvvia Island15 June 201938°32′24°01′420Cliffs12283
H. olympicumOlympiaEvvia Island14 June 201938°36′23°51′890Rocky slopes12288
H. perfoliatumDrosocarpiumMt. Parnon1 June 201937°15′22°39′1050Forest12282
H. perforatumHypericumAndros Island8 June 201937°50′24°53′560Rocky slopes12280
H. rumeliacum subsp. apollinisDrosocarpiumMt. Parnassos27 May 201938°33′22°34′1760Rocky slopes12286
H. tetrapterumHypericumEvvia Island14 June 201938°36′23°51′910Wet places12287
H. vesiculosumDrosocarpiumMt. Chelmos5 June 202238°05′22°10′910Woodland12284
Table
Table 2. Total phenolic and total flavonoid content of the Hypericum species examined.
Table 2. Total phenolic and total flavonoid content of the Hypericum species examined.
SpeciesTPC ± SDTFC ± SD
mg GAE/g dry plant materialmg RE/g dry plant material
H. perfoliatum59.31 ± 16.47 a,*0.21 ± 0.14 A
H. rumeliacum subsp. apollinis54.87 ± 2.73 a1.18 ± 0.22 B
H. vesiculosum26.92 ± 10.32 b0.30 ± 0.12 C
H. cycladicum54.59 ± 8.55 a1.34 ± 0.05 D
H. perforatum86 ± 13.34 c0.76 ± 0.11 E
H. tetrapterum51.26 ± 21.36 a1.58 ± 0.08 F
H. fragile39.72 ± 6.05 d0.54 ± 0.12 G
H. olympicum32.63 ± 17.42 b0.64 ± 0.09 H
H. delphicum54.09 ± 4.29 a0.6 ± 0.17 I
* Results are presented as mean ±standard deviation (SD) (n = 3) (p < 0.05). a–d and A–I values with the same letter are non-significantly different (p < 0.05).
Table
Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Hypericum species against E. coli, S. enteritidis, S. aureus, and E. faecalis bacteria.
Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Hypericum species against E. coli, S. enteritidis, S. aureus, and E. faecalis bacteria.
Hypericum Species ExtractsE. coliS. enteritidisS. aureus *E. faecalis **
MIC 2
(mg/mL) ± SD		MBC 3
(mg/mL) ± SD		MIC 2
(mg/mL) ± SD		MBC 3
(mg/mL) ± SD		MIC 2
(mg/mL) ± SD		MBC 3
(mg/mL) ± SD		MIC 2
(mg/mL) ± SD		MBC 3
(mg/mL) ± SD
H. perfoliatum----1.25 ± 0.02 a1.25 ± 0.02 A1.25 ± 0.02 a1.25 ± 0.02 A
H. rumeliacum subsp. apollinis----1.25 ± 0.02 a1.25 ± 0.02 A1.25 ± 0.02 a5.00 ± 0.04 B
H. vesiculosum----1.25 ± 0.02 a2.50 ± 0.03 B1.25 ± 0.04 a5.00 ± 0.05 B
H. cycladicum----0.31 ± 0.04 b1.25 ± 0.04 A0.6 ± 0.05 b1.25 ± 0.02 A
H. perforatum----0.06 ± 0.01 c0.51 ± 0.06 C0.13 ± 0.02 c0.51 ± 0.05 C
H. tetrapterum----1.25 ± 0.03 a1.25 ± 0.03 A1.25 ± 0.05 a2.50 ± 0.24 D
H. fragile----1.75 ± 0.05 d1.75 ± 0.05 D3.50 ± 0.06 d5.00 ± 0.06 B
H. olympicum----0.31 ± 0.05 b1.25 ± 0.04 A0.63 ± 0.04 b2.50 ± 0.05 D
H. delphicum----0.16 ± 0.02 e0.63 ± 0.05 E0.31 ± 0.03 e0.63 ± 0.03 E
Amp 10.03 ± 0.0020.03 ± 0.0020.02 ± 0.0010.02 ± 0.001NANANANA
Gen 1NANANANA0.02 ± 00.010.02 ± 0.0010.02 ± 0.0030.02 ± 0.003
1 Ampicillin and gentamycin were used as antibacterial control samples against Gram-negative and Gram-positive bacteria, respectively; 2 The smaller the MIC value, the less extract is required for bacterial growth inhibition. Substances with MIC values of <0.6 mg/mL are regarded as strong inhibitors, 0.6–1.6 mg/mL moderate, 1.6–8.0 mg/mL weak, and >8.0 mg/mL low [33,34]; 3 MBC indicates the lowest concentration of the extract that is bactericidal. The smaller the MBC value, the less extract is required to kill the bacteria. -: not active; Amp: Ampicillin; Gen: Gentamycin; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; ΝA: not available. * a–e; A–E values with the same letter are not significantly different (p < 0.05). ** a–e; A–E values with the same letter are not significantly different (p < 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kakouri, E.; Daferera, D.; Trigas, P.; Charalambous, D.; Pantelidou, M.; Tarantilis, P.A.; Kanakis, C.D. Comparative Study of the Antibacterial Activity, Total Phenolic and Total Flavonoid Content of Nine Hypericum Species Grown in Greece. Appl. Sci. 2023, 13, 3305. https://doi.org/10.3390/app13053305

AMA Style

Kakouri E, Daferera D, Trigas P, Charalambous D, Pantelidou M, Tarantilis PA, Kanakis CD. Comparative Study of the Antibacterial Activity, Total Phenolic and Total Flavonoid Content of Nine Hypericum Species Grown in Greece. Applied Sciences. 2023; 13(5):3305. https://doi.org/10.3390/app13053305

Chicago/Turabian Style

Kakouri, Eleni, Dimitra Daferera, Panayiotis Trigas, Despina Charalambous, Maria Pantelidou, Petros A. Tarantilis, and Charalabos D. Kanakis. 2023. "Comparative Study of the Antibacterial Activity, Total Phenolic and Total Flavonoid Content of Nine Hypericum Species Grown in Greece" Applied Sciences 13, no. 5: 3305. https://doi.org/10.3390/app13053305

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop