Chemical Composition and Biological Activities of St John’s Wort (Hypericum perforatum L.) Essential Oil from Bulgaria

Abstract

Since ancient times, essential oils obtained from various aromatic plants have been utilized as bioactive ingredients in medicines, foods and cosmetics. The present study aimed to investigate the chemical composition and biological activities of St John’s Wort (Hypericum perforatum L.) essential oil (SJW EO) from Bulgaria, which is known to possess various biological properties. Gas chromatography and mass spectrometry (GC–MS) analysis, determination of antioxidant activity (by the ABTS method), an antimicrobial activity test and an in vitro anti-inflammatory activity test were performed. The main classes of compounds identified by GC–MS analysis were monoterpenes (43.55%), followed by sesquiterpenes (36.81%) and alkanes (16.92%). The predominant chemical components of SJW EO were α-pinene (27.52%), followed by β-pinene (10.08%), β-caryophyllene (6.77%), germacrene D (6.37%) and caryophyllene oxide (4.48%). The highest antibacterial activity was observed against the Gram-negative bacteria Klebsiella pneumoniae ATCC 13883 (inhibition zone of 12.0 mm) and Pseudomonas aeruginosa ATCC 9027 (inhibition zone of 11.0 mm). SJW EO exhibited significant in vitro anti-inflammatory activity, as the results demonstrated that its anti-inflammatory effect was stronger than those of the conventional anti-inflammatory drugs Prednisolon Cortico and acetylsalicylic acid (Aspirin), which were used as controls (all in concentration of 1 mg/mL). The obtained results demonstrated that Bulgarian SJW EO can be used as an active ingredient in the composition of new products for the pharmaceutical and cosmetic industries.

1. Introduction

Essential oils (EOs) are aromatic and volatile liquids produced from different parts of the plants—flowers, roots, stems, barks, leaves, seeds, peels or fruits—or from whole plants [1] and stored in their secretory cells, cavities, canals, epidermic cells or glandular trichomes [2]. EOs represent multicomponent, water-insoluble mixtures of polar and nonpolar natural compounds including terpenes (monoterpenes and sesquiterpenes), aromatic compounds (aldehydes, alcohols, phenols, methoxy derivatives) and terpenoids (isoprenoids) [3]. The rich chemical compositions of EOs determine their broad spectrum of biological activities, such as antimicrobial, antiviral, antioxidant, antiparasitic, insecticidal and many others, which find pharmaceutical, cosmetic, fragrance, agricultural and food industry applications [4].

Hypericum perforatum L., widely known as St John’s Wort (SJW), is a perennial herbaceous plant that belongs to the Hypericaceae family. It is largely distributed in Europe, Asia, North America and Northern Africa. SJW prefers sunny locations with well-drained, limey soil, mostly at roadsides, slopes, wood borders and stone quarries, and reaches a height of 50–100 cm. SJW flowers are bright yellow, star-shaped, clustered in a trio and possessing five petals. SJW leaves have specific tiny, transparent oil glands resembling perforations [5].

The genus Hypericum unifies approximately 480 species worldwide, of which 61 are spread throughout Europe. Twenty-two species of this genus are found in Bulgaria, five of which are endemic to the country. H. perforatum L. is among the most popular and commonly used herbs in Bulgarian folk medicine. Its extracts possess strong antioxidant, anti-inflammatory, neuroprotective and gastroprotective properties [6]. SJW is a popular herbal remedy recommended by Traditional Chinese Medicine for the treatment depressive disorders [7]. It was found that the antidepressant and anxiolytic properties of SJW have been attributed to one of its major phytocomponents, known as hyperforin, which plays the role of a reuptake inhibitor for neurotransmitters such as dopamine, norepinephrine, serotonin and glutamate [8,9]. According to some authors, several flavonoids, such as quercetin, hyperoside, quercitrin, isoquercitrin, miquelianin and others, also contribute to the antidepressant activity of SJW [10]. SJW preparations in the form of oil macerates or tinctures have been used for the topical treatment of skin disorders, such as superficial wounds, abrasions, sunburns, dermatitis and eczema, due to their anti-inflammatory and wound healing effects [11,12].

EO obtained from SJW is colourless or light yellow, possessing a distinctive odour and liquid consistency at room temperature. While SJW EO is generally found in large amounts in the roots, leaves and flowers of the plant, it is presented in small quantities in other plant organs, such as bark and stems [6]. The most commonly detected chemical components in SJW EO are monoterpenes, sesquiterpenes and their oxygenated derivatives, but their phytochemical profile is also comprised of naphthodianthrones (hypericin and pseudohypericin), hyperforin, proanthocyanins, flavonoids, biflavonoids, xanthones, phenylpropanes, phenolic acids and volatile compounds [13]. The phytochemical profile of the EO of Hypericum spp. vary widely, influenced by the characteristics of geographical region, some environmental factors and phenological cycle, as well as the type of plant organ in which the EO is produced [14].

Although Hypericum spp. extracts have been extensively studied in terms of their biological properties, the number of studies conducted with their EOs is still limited. It was established that SJW EO exhibits diverse biological activities and health benefits, such as antiseptic, antifungal, antioxidant, antimicrobial, antiviral, antiparasitic, anti-angiogenetic, insecticidal [6,14], antimalarial, cytotoxic, neuroprotective, hepatoprotective, tyrosinase inhibitory, immunomodulatory and wound-healing effects, proven on different in vitro and in vivo experimental models [15].

Despite the large number of scientific publications concerning the phytochemical composition of SJW EO from different countries worldwide, the knowledge of EO obtained from Hypericum perforatum L. grown in Bulgaria and its biological properties is still very limited. Therefore, the present study aimed to investigate the composition of EO from one of the most abundant Hypericum species in Bulgaria, namely Hypericum perforatum L., as well as to determine some of its biological activities (antioxidant, antimicrobial and anti-inflammatory).

2. Materials and Methods

2.1. Plant Material

The essential oil from H. perforatum was purchased from the company Kateko Ltd., Plovdiv, Bulgaria. The information on the habitat of the plants used as raw material, as well as the production technology, was provided by the manufacturer.

The flowers of St. John’s Wort (H. perforatum) were collected during the flowering phase (late June) in 2022 from the Uzana area, with geographic coordinates 42°45′58″ N and 25°14′18″ E, at an altitude of 1300 m. The plant material was transported and processed at the distillery on the same day in “The Old Rose Distillery” in Strelcha, Bulgaria. Voucher specimens of the species used were deposited in the Herbarium of the Agricultural University, Plovdiv, Bulgaria (SOA).

2.2. Extraction Method of Essential Oil

The genus Hypericum is low in essential oil, yielding very low levels below 1%. To increase the yield of essential oil, the inflorescences of the plant were used, and harvesting was done at the flowering stage.

The method used for extraction of the SJW EO was steam distillation. Distillation pots manufactured by the engineering team at Pharmachim, Bulgaria were used (Figure 1). Fresh flowers were loaded into the distillation pots and periodically pressed down to increase the density of the plant material. This pressing made it more difficult for steam to pass through, which improved oil extraction. Superheated steam at 160 °C and 4–5 atm pressure was used. The steam carried the essential oil from the plant, passed through the “pipe” of the lid, and entered the condenser for cooling. The extraction of the essential oil was conducted via distillation over a period of 2 h, resulting in a yield of about 1–1.5% per kg of fresh material. The obtained SJW EO was a clear, yellow liquid with viscous consistency.

In future research, we will attempt to apply methods such as hydrodistillation and supercritical extraction, as recommended by the European Pharmacopoeia, in order to increase the yield and optimize extraction efficiency.

2.3. Test Microorganisms

Sixteen microorganisms, including five Gram-positive bacteria (Bacillus subtilis ATCC 6633, Bacillus cereus NCTC 11145, Staphylococcus aureus ATCC 25923, Listeria monocytogenes NBIMCC 8632, Enterococcus faecalis ATCC 19433), five Gram-negative bacteria (Salmonella enteritidis ATCC 13076, Klebsiella pneumoniae ATCC 13883, Escherichia coli ATCC 25922, Proteus vulgaris ATCC 6380, Pseudomonas aeruginosa ATCC 9027), two yeasts (Candida albicans NBIMCC 74, Saccharomyces cerevisiae ATCC 9763) and four fungi (Aspergillus niger ATCC 1015, Aspergillus flavus, Penicillium chrysogenum, Fusarium moniliforme ATCC 38932) from the collection of the Department of Microbiology at the University of Food Technologies, Plovdiv, Bulgaria, were selected for the antimicrobial activity test.

2.4. Culture Media

2.4.1. Luria–Bertani Agar Medium with Glucose (LBG Agar)

LBG agar was used for cultivation of the test bacteria. A quantity of 50 g of LBG-solid substance mixture (containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, 10 g glucose and 15 g agar) was dissolved in 1 L of deionized water, pH 7.5 ± 0.2.

2.4.2. Malt Extract Agar (MEA)

MEA was used for cultivation of the test yeasts and fungi. A quantity of 50 g of the MEA-solid substance mixture (containing 30 g malt extract, 5 g mycological peptone and 15 g agar) was dissolved in 1 L of deionized water, pH 5.4 ± 0.2.

The culture media were prepared according to the manufacturer’s instructions (Scharlab SL, Barcelona, Spain) and autoclaved at 121 °C for 20 min before use.

2.5. Gas Chromatography and Mass Spectrometry (GC–MS) Analysis

A system consisting of a gas chromatograph Agilent Technology Hewlett Packard 7890A, coupled with a mass detector Agilent Technology 5975C inert XL EI/CI MSD (Agilent, Santa Clara, CA, USA) at 70 eV was used to analyse the composition of SJW EO. The quantity of 20 µL of SJW EO was dissolved in 380 µL of hexane. Separation of the metabolites was performed on an HP-5MS column (30 m × 0.32 mm × 0.25 μm) at the following temperature program: from 40 °C to 300 °C, with a step of 5 °C/min, and holding at 300 °C for 10 min. The carrier gas (helium) was maintained at a constant flow rate of 1 mL/min. The mass spectrometer detector scan range was m/z = 40–400. The injector temperature was set at 250 °C with a split injection ratio of 100:1 and an injection volume of 1 μL [16].

2.6. Identification of Metabolites

The obtained mass spectra were analysed using 2.64 AMDIS (Automated Mass Spectral Deconvolution and Identification System, National Institute of Standardization and Technology, NIST, Gaithersburg, MD, USA). The separated polar and nonpolar compounds were identified by comparison of their GC–MS spectra and Kovach retention indices (RIs) with referent compounds in the NIST 08 database (NIST Mass Spectral Database, PC-Version 5.0, 2008). The RIs of the compounds were recorded with a standard n-hydrocarbon calibration mixture (C10–C40, Fluka) using the 2.64 AMDIS software [16].

2.7. Antioxidant Activity

ABTS Radical Scavenging Ability

ABTS radical scavenging ability was determined according to the method of Ivanov et al. [17]. The ABTS radical was prepared by mixing aliquot parts of 7.0 mM 2,2′azinobis(3)-ethylbenzthiazoline-6-sulfonic acid—ABTS (Sigma, Kawasaki, Japan) in distilled water and 2.45 mM potassium persulfate (Merck, Darmstadt, Germany) in distilled water. The reaction was performed for 16 h at ambient temperature in darkness. The generated ABTS radical was stable for several days. Before analysis, 2.0 mL of the generated ABTS•+ solution was diluted with methanol at a proportion of 1:30 (v/v); thus, the obtained final absorbance of the working solution was about 1.0 ÷ 1.1 at 734 nm.

To conduct the assay, 2.85 mL of the ABTS•+ solution was mixed with 0.15 mL of SJW EO. After incubation at 37 °C for 15 min in darkness, the absorbance was measured at 734 nm against methanol. The antioxidant activity was expressed as mM (TE)/g EO by using a calibration curve built in a range of 0.05–0.5 mM Trolox (Fluka) dissolved in methanol (Merck, Darmstadt, Germany).

2.8. Antimicrobial Activity Assay

The antimicrobial activity of St. John’s Wort EO was determined by the agar well diffusion method according to Tumbarski et al. [18]. The bacteria B. subtilis and B. cereus were cultured on LBG agar at 30 °C for 24 h, while S. aureus, L. monocytogenes, E. faecalis, S. enteritidis, K. pneumoniae, E. coli, P. vulgaris and P. aeruginosa were cultured on LBG agar at 37 °C for 24 h. The yeast C. albicans was cultured on MEA at 37 °C, while S. cerevisiae was cultured on MEA at 30 °C for 24 h. The test fungi A. niger, A. flavus, P. chrysogenum and F. moniliforme were grown on MEA at 30 °C for 7 days or until sporulation.

The bacterial/yeast inocula were prepared by homogenization of a small amount of biomass in 5 mL of sterile 0.5% NaCl. The fungal inocula were prepared by the addition of 5 mL of sterile 0.5% NaCl directly into the cultivation tubes. After stirring by vortex V-1 plus (Biosan, Riga, Latvia), the fungal inocula were filtered and replaced in other tubes before use. The number of viable cells and fungal spores was determined using a bacterial counting chamber Thoma (Poly-Optik, Bad Blankenburg, Germany). Their final concentrations were adjusted to 10⁸ cfu/mL for bacterial/yeast cells and 10⁵ cfu/mL for fungal spores and then inoculated in LBG/MEA preliminarily melted and tempered at 45–48 °C. Next, the inoculated media were transferred, at a quantity of 18 mL, in sterile Petri plates (d = 90 mm) (Gosselin™, Borre, France) and allowed to harden. Then, six wells (d = 6 mm) per plate were cut and triplicates of 60 μL of the SJW EO (10 mg/mL) were pipetted into the agar wells. The Petri plates were incubated in identical conditions.

The antimicrobial activity was determined by measuring the diameter of the inhibition zones (IZ) around the wells on the 24th and 48th hours of incubation. Test microorganisms with IZ of 18 mm or more were considered sensitive; moderately sensitive were those in which the IZ were from 12 to 18 mm; resistant were those in which the IZ were up to 12 mm or completely missing.

2.9. In Vitro Anti-Inflammatory Activity Assay

The in vitro anti-inflammatory activity was assessed by percentage of inhibition of albumin denaturation. The anti-denaturation assay was performed as previously described by Milusheva et al. [19]. The reaction mixture contained 0.5 mL of a 5% aqueous solution of human serum albumin (Albunorm 20, Octapharma, Brussels, Belgium) and 0.2 mL of the SJW EO, which was pre-diluted in DMSO at a concentration of 1 mg/mL. The sample and the controls were incubated at 37 °C for 15 min. Each tube was filled with 2.5 mL of phosphate-buffered saline—PBS (pH 6.3), heated to 80 °C for 30 min, and then cooled for 5 min. The turbidity of the samples was measured at 660 nm by a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). A mixture of 2.5 mL of PBS and 0.2 mL of DMSO was used for a blank, while the positive contained 0.5 mL of albumin and 2.5 mL of PBS. The percentage of inhibition of protein denaturation (% IPD) was calculated according to the following Equation (1):

$$\%\mathrm{I}\mathrm{P}\mathrm{D}={\displaystyle \frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

(1)

The control represents 100% of protein denaturation. The results were expressed also as a half of the maximal inhibitory concentration, or IC₅₀. Commercially available steroid (Prednisolon Cortico) and non-steroid (acetylsalicylic acid or Aspirin) anti-inflammatory drugs at the same concentration (1 mg/mL) were used as controls. Their anti-inflammatory effect was determined using the same method as for the EO sample.

2.10. Statistical Analysis

Data from triplicate experiments were processed with MS Office Excel 2010 software using statistical functions to determine the standard deviation (±SD) and maximum estimation error at significance levels of p < 0.05.

3. Results

3.1. GC–MS Analysis of the Chemical Composition of SJW EO

The identified volatile compounds in the essential oil of H. perforatum were classified in four main groups, with the relevant subgroups, as follows: alkanes, alkylbenzene, monoterpenes (monoterpene hydrocarbons and oxidized monoterpenes), and sesquiterpenes (sesquiterpene hydrocarbons and oxidized sesquiterpenes) (

Table 1. Groups of chemical compounds identified in Bulgarian St John’s Wort (H. perforatum L.) essential oil by GC–MS analysis.

Groups of Chemical Compounds	% of Total Ion Current
Alkanes	16.92 ± 0.13
Alkylbenzene	2.05 ± 0.33
Monoterpenes	43.55 ± 0.35
Monoterpene hydrocarbons	41.04 ± 0.51
Oxygenated monoterpenes	2.51 ± 0.12
Sesquiterpenes	36.81 ± 0.37
Sesquiterpene hydrocarbons	27.29 ± 0.24
Oxygenated sesquiterpenes	9.52 ± 0.38
Total identified	99.33 ± 0.90

). Monoterpene hydrocarbons were the predominant group in the SJW EO (41.04%), followed by sesquiterpene hydrocarbons (27.29%), alkanes (16.92%), oxygenated sesquiterpenes (9.52%), oxygenated monoterpenes (2.51%) and alkylbenzene (2.05%). A total of 42 chemical compounds were identified in SJW EO, which are presented in

Table 2. Phytochemical profile of Bulgarian St John’s Wort (H. perforatum L.) essential oil by GC–MS analysis.

Peak	RT	RI	Name	% of TIC
Alkanes
1	8.68	900	n-Nonane	5.85 ± 0.12
5	11.03	966	3-Methylnonane	5.63 ± 0.11
11	14.01	1077	2-Methyldecane	3.42 ± 0.24
12	15.11	1101	n-Undecane	1.80 ± 0.13
18	19.94	1299	2-Methyldodecane	0.49 ± 0.04
Alkylbenzene
8	12.69	1022	p-Cymene	2.05 ± 0.33
Monoterpenes
Monoterpene hydrocarbons
2	9.46	923	α-Thujene	0.90 ± 0.09
3	9.78	930	α-Pinene	27.52 ± 1.05
4	10.23	948	Camphene	0.39 ± 0.07
6	11.19	977	β-Pinene	10.08 ± 1.43
7	11.55	989	β-Myrcene	0.46 ± 0.08
9	12.84	1026	Limonene	0.89 ± 0.78
10	13.40	1045	β-Ocimene	0.80 ± 0.09
Oxygenated monoterpenes
13	16.36	1136	L-Pinocarveol	0.41 ± 0.13
14	16.51	1142	(E)-Verbenol	0.46 ± 0.10
15	18.00	1187	α-Terpineol	0.69 ± 0.23
16	18.33	1203	Verbenone	0.52 ± 0.11
17	18.65	1238	(E)-Chrysanthenyl acetate	0.43 ± 0.06
Sesquiterpenes
Sesquiterpene hydrocarbons
19	23.01	1376	α-Copaene	0.60 ± 0.06
20	23.23	1390	β-Elemene	0.44 ± 0.11
21	24.07	1412	β-Funebrene	0.80 ± 0.14
22	24.21	1417	β-Caryophyllene	6.77 ± 1.23
23	24.31	1423	β-Cedrene	0.43 ± 0.07
24	24.42	1434	β-Copaene	0.55 ±0.05
25	24.65	1439	Aromandendrene	0.73 ± 0.08
26	24.95	1444	(Z)-β-Farnesene	0.47 ± 0.10
27	25.07	1452	α-Caryophyllene	0.31 ± 0.05
28	25.58	1478	γ-Muurolene	2.76 ± 0.75
29	25.76	1486	Germacrene D	6.37 ± 0.87
30	25.98	1492	Viridiflorene	0.95 ± 0.07
31	26.07	1499	Bicyclogermacrene	0.39 ± 0.07
32	26.18	1505	α-Muurolene	0.89 ± 0.23
33	26.50	1516	γ-Cadinene	1.71 ± 0.11
34	26.62	1524	δ-Cadinene	1.74 ± 0.21
35	26.69	1533	(E)-Cadina-1,4-diene	0.77 ± 0.12
36	26.88	1541	α-Cadinene	0.61 ± 0.14
Oxygenated sesquiterpenes
37	27.62	1560	(E)-Nerolidol	0.55 ± 0.14
38	28.10	1579	Spathulenol	2.54 ± 0.78
39	28.16	1585	Caryophyllene oxide	4.48 ± 1.12
40	28.45	1592	Viridiflorol	0.60 ± 0.05
41	29.40	1640	epi-α-Cadinol	0.57 ± 0.06
42	29.85	1655	α-Cadinol	0.78 ± 0.15

RT—retention time; RI—retention index; TIC—total ion current.

in the order of their elution from the column.

The GC–MS analysis of the phytochemical profile of SJW EO showed that α-pinene (27.52%) and β-pinene (10.08%) were the predominant chemical compounds in the group of monoterpene hydrocarbons. The major sesquiterpenes presented in SJW EO were β-caryophyllene (6.77%) and germacrene D (6.37%), followed by caryophyllene oxide (4.48%), γ-muurolene (2.76%) and spathulenol (2.54%).

The chemical composition of the essential oil and volatile components of different vegetative parts (flowers and stems) of St. John’s Wort populations growing in different regions of the world were previously investigated. It has been found that in EOs of wild-growing SJW populations from Tunisia, Kosovo, Portugal and Greece, the predominant compounds were the monoterpene hydrocarbons, in particular α-pinene, which was in agreement with our results. Hosni et al. [20] identified 32 compounds in the EO of Hypericum perfoliatum from Tunisia, with α-pinene (13.1%), allo-aromadendrene (11.4%), germacrene-D (10.6%), n-octane (7.3%), α-selinene (6.5%) and β-selinene (5.5%) as major constituents. Hajdari et al. [21] detected 67 compounds in EOs from the aerial parts of H. perforatum in Kosovo and stated that the main constituents were α-pinene (3.7–36.5%), caryophyllene oxide (3.3–17.7%), 2-methyl-octane (1.1–15.5%), β-caryophyllene (1.2–12.4%) and n-tetradecanol (3.6–10.4%). Using GC–MS analysis, Nogueira et al. [22] studied the chemical composition of EOs of four Portuguese Hypericum species—Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum. The authors stated that monoterpene hydrocarbons group constituted the main fraction in all EOs (43–69%, 53–85%, 28–45% and 48–65% for H. perfoliatum, H. humifusum, H. linarifolium and H. pulchrum, respectively), followed by the sesquiterpene hydrocarbons group (2–13%, 6–18%, 21–27% and 16–18%, respectively), as well as non-terpenic compounds (20–29%, 3–16%, 2–14% and 5–11%, respectively). The GC–MS analyses of the phytochemical composition of EOs of H. perforatum L., Hypericum tetrapterum Fries and Hypericum olympicum L., growing in Greece, showed that α-pinene (21.0%) and 2-methyl-octane (12.6%) were the most abundant constituents of H. perforatum, whereas α-copaene (11.3%) and α-longipinene (9.7%) were the major components of the EO obtained from H. tetrapterum. The EO of H. olympicum was characterized by the highest concentration of germacrene D (16.0%) and (E)-caryophyllene (7.4%) [23].

Bardhi et al. [24] investigated 11 SJW populations in Southern Albania and concluded that, depending on the content of major components, two types of SJW EOs can be distinguished: the pinene type, which includes EOs rich in α-pinene, and the caryophyllene type, which includes EOs rich in trans-(E)-caryophyllene and caryophyllene oxide. Many research teams reported that the EO of some SJW populations contained β-caryophyllene or its oxide (11–35%) and germacrene D (17–39%) as the predominant components. Baser et al. [25] analysed EOs from the aerial parts of two Hypericum species from Uzbekistan and determined that the main components of the EO of Hypericum scabrum L. were α-pinene (11.2%), spathulenol (7.2%), p-cymene (6.1%), acetophenone (4.8%) and carvacrol (4.7%), while the EO of H. perforatum L. contained as the main components β-caryophyllene (11.7%), caryophellene oxide (6.3%), spathulenol (6.0%) and α-pinene (5.0%). The analyses of SJW EOs from Serbia revealed that sesquiterpenes exhibited the highest concentration (44.4%), as the caryophyllene oxide (15.3%) was the major component [26]. Different chemotypes of the EO of 10 wild-growing populations of H. perforatum L. in Eastern Lithuania have been isolated and studied by Mockutė et al. [27]. The samples were collected at full flowering and analysed by GC–MS. The authors identified 46 constituents in the investigated oils. The EOs were classified into three groups as follows: β-caryophyllene (4 samples, 10.5–19.1%), caryophyllene oxide (4 samples, 13.3–35.8%) and germacrene D (2 samples, 16.1–31.5%).

Variations in the chemotypes and chemical compositions of EOs among the SJW species growing in Bulgaria were also observed. Semerdjieva et al. [28] investigated EOs obtained from the flowers and leaves of seven Bulgarian Hypericum species—H. perforatum, Hypericum cerastoides, Hypericum rumeliacum, Hypericum montbretii, Hypericum maculatum, Hypericum hirsutum and Hypericum calycinum. The identified EO constituents belonged to four chemical classes—alkanes, monoterpenes, sesquiterpenes and fatty acids. The main class of compounds detected in H. maculatum and H. perforatum were sesquiterpenes. H. montbretii EO contained monoterpenes (38.09%) and sesquiterpenes (37.09%) as major groups, while H. hirsutum EO contained predominately alkanes (67.19%). Interestingly, H. hirsutum EO contained cedrol (5.04%), identified for the first time in Hypericum species. Fatty acids were the main compounds in H. cerastoides, while monoterpenes were the most abundant class in H. rumeliacum and H. calycinum EOs. α-Pinene and germacrene D were the major EO constituents of all analysed Hypericum species except for H. hirsutum and H. cerastoides. The analysis of EO from H. perforatum harvested in 2020 from the region of Dulovo, Dobrich district, Bulgaria, showed that oxygenated monoterpenes were 5.4 times higher, but monoterpene hydrocarbons were 11.5 times lower in comparison with the sample we studied. The oxygenated sesquiterpenes were 1.4 times lower, and sesquiterpene hydrocarbons had 1.7 times lower values compared with the results for our SJW EO [6].

The phytochemical composition of SJW EO varies within a wide range depending on the geographic region, season, climatic characteristics, soil and period of harvesting, as well as the plant species and organs from which it is obtained. Based on the differences in chemical profile, the research teams divide the Hypericum species into two main chemotypes: the chemotype with predominant monoterpenes in its chemical composition, and the chemotype with predominant sesquiterpenes as the main components [14,29].

3.2. Antioxidant Activity

The results presented in

Table 3. Antioxidant activity of Bulgarian St John’s Wort (H. perforatum L.) essential oil by ABTS method.

EO Concentration, mg/mL	Antioxidant Activity
	Inhibition of ABTS Radical, %	mM TE/g EO
100.00	92.52 ± 0.12	6.23 ± 0.01
50.00	81.68 ± 0.43	10.99 ± 0.06
25.00	62.97 ± 0.43	16.93 ± 0.12
12.50	42.30 ± 0.48	22.70 ± 0.80
6.25	25.98 ± 0.68	27.79 ± 0.73
3.13	13.40 ± 0.37	28.44 ± 0.80
1.56	12.45 ± 0.12	52.75 ± 0.53

demonstrate that the degree of antioxidant activity determined by the ABTS method was dependent on the SJW EO concentration. It was found that the value of IC₅₀ by the ABTS method was 18.07 ± 0.22 mg/mL (Figure 2). The low IC₅₀ value indicates an active ability of the EO to act as a radical scavenger. It is assumed that the antioxidant activity of EOs could be attributed to their hydrogen-donating ability [30].

Presently, the literature is lacking thorough research on the antioxidant capacity of Hypericum spp. essential oils. Vuko et al. [31] investigated the chemical composition and antioxidant activity of H. perforatum L. ssp. veronense EO from the region of Split, Croatia, and reported values for the percentage of inhibition of 44.03 ± 0.74% and IC₅₀ = 23.07 ± 0.49 mg/mL, determined by the DPPH method. Similar values for the percentage of inhibition by the DPPH method (30.46 ± 2.88%) were reported by Akhbari et al. [32] for H. perforatum L. EO from the Karkas Mountains of Kashan, central Iran. Pirbalouti et al. [30] studied the antioxidant activity of H. perforatum L. EO from the Bakhtiari Zagros Mountains, Iran, and reported that the value for IC₅₀ = 311.7 ± 21.6 μg/mL, determined by the DPPH assay. Sharopov et al. [33] investigated the biological properties of the EOs of 18 plant species originating from Tajikistan (Central Asia) and stated that H. perforatum exhibited values of IC₅₀ = 3.71 mg/mL (determined by DPPH assay), IC₅₀ = 0.48 mg/mL (ABTS assay) and 98.25 µM Fe (II)/mg sample (FRAP assay).

3.3. Antimicrobial Activity

As seen from the results presented in

Table 4. Antimicrobial activity of Bulgarian St John’s Wort (H. perforatum L.) essential oil.

Test Microorganisms	Inhibition Zones (IZ), mm
	St. John’s Wort EO (10 mg/mL)	Control (MeOH)
Bacillus subtilis ATCC 6633	9 ± 0.00	-
Bacillus cereus NCTC 11145	9 ± 0.00	-
Staphylococcus aureus ATCC 25923	8 ± 0.00	-
Listeria monocytogenes NBIMCC 8632	9 ± 0.00	-
Enterococcus faecalis ATCC 29212	8 ± 0.00	-
Salmonella enteritidis ATCC 13076	9 ± 0.00	-
Klebsiella pneumoniae ATCC 13883	12 ± 0.71	-
Escherichia coli ATCC 25922	9 ± 0.00	-
Proteus vulgaris ATCC 6380	-	-
Pseudomonas aeruginosa ATCC 9027	11 ± 0.00	-
Candida albicans NBIMCC 74	8 ± 0.00	-
Saccharomyces cerevisiae ATCC 9763	10 ± 0.00	-
Aspergillus niger ATCC 1015	-	-
Aspergillus flavus *	-	-
Penicillium chrysogenum *	10 ± 0.00	-
Fusarium moniliforme ATCC 38932	8 ± 0.00	-

* plant isolates.

, the St. John’s Wort EO at the concentration of 10 mg/mL exhibited the highest inhibitory activity against the Gram-negative bacteria K. pneumoniae ATCC 13883 (IZ = 12.0 mm) and P. aeruginosa ATCC 9027 (IZ = 11.0 mm). The antimicrobial activity against the Gram-positive bacteria B. subtilis ATCC 6633, B. cereus NCTC 11145, S. aureus ATCC 25923, L. monocytogenes NBIMCC 8632 and E. faecalis ATCC 19433, the Gram-negative bacteria S. enteritidis ATCC 13076 and E. coli ATCC 25922, the yeasts C. albicans NBIMCC 74 and S. cerevisiae ATCC 9763 and the fungi P. chrysogenum and F. moniliforme ATCC 38932 was low. The St. John’s Wort EO failed to inhibit the Gram-negative bacterium P. vulgaris ATCC 6380, as well as the fungi A. niger ATCC 1015 and A. flavus. The methanol (MeOH) used as a solvent of the EO and a control did not show antimicrobial activity against the test microorganisms used in the study.

Jianu et al. [34] screened for antimicrobial activity in EOs obtained from six medicinal plants from Romania and stated that H. perforatum L. EO inhibited the growth of the tested food-borne pathogens in a dose-dependent manner. The SJW EO at a dose of 10 μg/disc showed inhibitory activity against S. aureus ATCC 25923 (IZ = 10.24 mm), Salmonella typhimurium ATCC 14028 (IZ = 9.89 mm), E. coli ATCC 25922 (IZ = 9.87 mm), K. pneumoniae ATCC 13882 (IZ = 11.8 mm), E. faecalis ATCC 29212 (IZ = 9.79 mm) and C. albicans ATCC 10231 (IZ = 22.12 mm) but did not inhibit P. aeruginosa ATCC 27853. Pirbalouti et al. [30] reported the efficacy of SJW EO against B. cereus, L. monocytogenes, P. aeruginosa and S. typhimurium, with values of minimum inhibitory concentration (MIC) varying between 250 and 500 μg/mL. Using the agar diffusion method, Kalaba et al. [35] evaluated the antimicrobial activity of H. perforatum EO and reference antimicrobial drugs (antibiotics) against the growth of S. aureus, S. typhimurium and P. aeruginosa. The tested EO (applied singly or diluted with ethanol of olive oil) exhibited an inhibitory effect on S. aureus and P. aeruginosa but failed to inhibit S. typhimurium. The results from antimicrobial screening of six Hypericum species from Serbia revealed that SJW EO was active against B. cereus, Micrococcus luteus, Sarcina lutea, S. aureus (MIC = 12.5 μg/mL), Agrobacterium tumefaciens, E. coli, Pseudomonas tolaasii, S. enteritidis (MIC = 25 μg/mL), Proteus mirabilis, P. aeruginosa and C. albicans (MIC = 50 μg/mL) [36].

In addition to the inhibitory effect on some food-borne pathogens, it was also found that SJW EO was an effective antimicrobial agent against standard strains and clinical isolates of periodontal pathogens, such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. The authors determined that the MIC values were between 0.312 and 0.625 μg/mL [37]. Rančić et al. [38] observed low antifungal activity of SJW EO against five fungal strains—A. niger ATCC 6275, A. flavus ATCC 9170, Cladosporium cladosporioides ATCC 13276, Penicillium funiculosum ATCC 10509 and Trichoderma viride IAM 5061. The authors determined an MIC value of 15 μg/mL against all tested strains and assumed that the most probable reason for the weak antifungal effect of the studied SJW EO was the low concentration of p-cymene (4.8%)—an aromatic monoterpene with antifungal activity. This was in agreement with the results obtained for the antifungal activity of our SJW EO, in which the concentration of p-cymene was also low (2.05%).

3.4. In Vitro Anti-Inflammatory Activity

The denaturation of proteins is an indicator for the presence of an inflammatory process. During this process, the secondary and tertiary structures of proteins are destroyed and a disturbance in their biological functions is observed. The ability of a certain substance (natural or synthetic) to reduce inflammation is associated with an anti-inflammatory effect [39]. Some anti-inflammatory drugs have been shown to inhibit the thermally induced protein denaturation in a dose-dependent manner [40].

The in vitro anti-inflammatory activity was evaluated as inhibition of albumin denaturation, assessing the degree of resistance to thermal denaturation of the human albumin molecule in the presence of SJW EO. The results are presented as the percentage of inhibition of albumin denaturation (Figure 3A), as well as a half-maximal inhibitory concentration or IC₅₀ (Figure 3B).

The SJW EO at concentration of 1 mg/mL exhibited the highest anti-inflammatory effect, expressed as inhibition of the thermally induced albumin denaturation (44.9 ± 0.08%). This value was higher than those of the conventional anti-inflammatory drugs used as controls (22.57 ± 0.12% for acetylsalicylic acid or Aspirin and 43.62 ± 0.04% for Prednisolon Cortico) at the same concentration. SJW showed the lowest IC₅₀ value (1.11 ± 0.01 mg/mL), compared to those of the controls (2.22 ± 0.02 mg/mL for acetylsalicylic acid and 1.15 ± 0.01 mg/mL for Prednisolon Cortico).

The non-steroidal and steroidal anti-inflammatory drugs (NSAIDs and SAIDs) also effectively inhibit inflammation, which, from a medical point of view, is a normal healing process; however, these drugs cause many adverse effects, especially with high dosages and prolonged intake. NSAIDs can cause gastric ulcers, stomach irritation and lower gastrointestinal disorders [41,42], while SAIDs increase the risk of hyperglycaemia, predisposition to infections, peptic ulcer disease, glaucoma, cataracts, psychosis, depression, adrenal insufficiency, diabetes and osteoporosis [43]. Consequently, the results in the present study reveal the great anti-inflammatory potential of SJW EO that can find application in the development of new medical formulations as alternatives to conventional anti-inflammatory drugs.

4. Conclusions

In recent years, essential oils have gained great popularity and wide application as bioactive ingredients in the food, cosmetic and pharmaceutical industries. The obtained results for the studied essential oil of St John’s Wort (Hypericum perforatum L.) revealed that it belongs to the monoterpenic type of SJW EOs and possesses high antioxidant activity (IC₅₀ by the ABTS method was 18.07 mg/mL) and moderate antimicrobial activity against Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027, Saccharomyces cerevisiae ATCC 9763 and Penicillium chrysogenum. Moreover, for the first time, an in vitro anti-inflammatory activity assay by inhibition of protein denaturation was applied to determine the anti-inflammatory effect of H. perforatum L. essential oil. The SJW EO exhibited significant in vitro anti-inflammatory potential (IC₅₀ = 1.11 mg/mL), which was higher compared to that of the conventional anti-inflammatory drugs Prednisolon Cortico and acetylsalicylic acid (Aspirin). This emphasizes the high biological value of Bulgarian SJW EO, which can find future practical application in the development of novel natural pharmaceutical and cosmetic products with anti-inflammatory properties.