Antimicrobial Potential of Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze Essential Oils for Food Applications: A Review of Their Chemical Compositions and Antimicrobial Efficacy

Abstract

The rising demand for natural and safe products has increased interest in essential oils (EOs) as alternatives to synthetic preservatives. EOs could be encapsulated in active packaging or incorporated in nano-emulsion systems and help extend food shelf life by inhibiting the growth of pathogens. H. officinalis and Agastache foeniculum (Lamiaceae) are widely used in food and beverages. This review aims to explores their potential food applications, focusing on their antimicrobial activities, chemical compositions, and toxicity. H. officinalis EO mainly consists of oxygenated monoterpenes (27.32–92.25%), with 1,8-cineole, isopinocamphone, and pinocamphone as key compounds. It also contains monoterpene hydrocarbons (3.84–67.24%), including β-pinene, β-phellandrene, and β-ocimene. A. foeniculum EO is rich in phenylpropanoids (22.39–84.67%), primarily estragole (3.2–94.89%) and methyl eugenol, along with oxygenated monoterpenes (0.08–54.51%), mainly menthone (31.58–34.3%). H. officinalis EO exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella Typhimurium, Pseudomonas aeruginosa, and various fungi, including Penicillium, Cladosporium, Candida, and Aspergillus species. A. foeniculum EO seemed to be effective against fungi and Gram-positive bacteria but showed lower activity against Gram-negative bacteria. H. officinalis EO showed no mutagenic or genotoxic effects in the available studies, while the toxicity of A. foeniculum EO remains unstudied. H. officinalis EO exhibited potential preservative properties when added to ground meat or used as coating for cheese and shrimp. The results of this study provide critical insights into the possibilities of integrating these EOs into food preservation strategies and their potential contributions to enhancing food safety and sustainability.

1. Introduction

The growing consumer preference for “natural” and “safe” products has sparked a growing interest in essential oils (EOs) as a promising natural alternative to synthetic preservatives [1]. Microorganisms and their toxins play significant roles in food spoilage, biodegradation, and food safety, making them key factors in food insecurity within the food chain. EOs and their phytoconstituents are known for their wide range of biological activities, including antibacterial, insecticidal, antiviral, and antifungal properties [2]. Their unique aromas, flavors, and antimicrobial qualities make them particularly valuable in food preservation [3]. As the food industry shifts toward becoming more natural, EOs offer an effective solution for extending the shelf life of perishable foods by inhibiting the growth of foodborne pathogens [4]. Despite the proven potential of EOs and their constituents in vitro, their application as food preservatives remains limited due to the high concentrations required for effective antimicrobial activity [5]. Also, their application in food preservation remains restricted due to their strong aromas and potential toxicity [6].

EOs, also referred to as volatile oils, are concentrated liquid extracts containing volatile aromatic compounds derived from the flowers, buds, leaves, and bark of the plant, with steam distillation being the most commonly used method of extraction. They are natural antimicrobial agents used to protect against plant infections, and they hold significant potential for combating foodborne pathogens and spoilage organisms. The effectiveness of a specific EO is largely determined by its concentration and chemical composition. These oils are typically complex mixtures containing 20 to 80 different chemical compounds, which could be categorized into two main groups: terpenoids and non-terpenoid hydrocarbons. Terpenoids are derived from the combination of isoprene units, forming monoterpenes (two units), sesquiterpenes (three units), or diterpenes (four units). Phenylpropanoids, which fall under non-terpenoid hydrocarbons, represent another structural class of phenolic compounds found in EOs. While terpenes and their oxygenated derivatives (terpenoids) are typically the most prevalent and abundant components in EOs, certain plant species contain high concentrations of phenylpropanoids that contribute to the unique fragrance and flavor of the plant [1,2].

Numerous studies have demonstrated a strong correlation between the chemical compositions of EOs and their antimicrobial properties. These oils and their constituents exhibit broad-spectrum antimicrobial activity against a range of foodborne pathogens, including Gram-positive, Gram-negative, and spoilage microorganisms. Their antimicrobial effectiveness largely depends on the primary constituents and their interactions with minor components. Their antimicrobial properties are primarily attributed to terpenes, terpenoids, and phenols [1].

The antimicrobial properties of EOs result from a complex mixture of compounds with diverse chemical structures, leading to multiple mechanisms of action. Their high lipophilicity plays a crucial role, enabling penetration of cell and mitochondrial membranes, disrupting their structure and function, and increasing permeability [7].

EOs exhibit antibacterial effectiveness (Figure 1) that can be either bacteriostatic (inhibiting bacterial growth and allowing microbial cells to recover reproductive capabilities) or bactericidal (destroying bacterial cells). Various methods can be employed to assess these antibacterial properties, including agar dilution, agar/disc diffusion, broth micro/macro dilution, direct bioautography, antimicrobial gradient method (Etest), time-kill test, adenosine triphosphate (ATP) bioluminescence assays, and flow cytofluorometric analysis [2].

The Lamiaceae family has a long-standing tradition of use in flavoring, food preservation, and medicine owing to its curative and preventive properties. These plants have gained popularity as functional foods due to the bioactive components in their EOs, which are known for their distinctive tastes and fragrances and biological effects, such as antimicrobial, antioxidant, and anticancer activities [8]. This family includes more than 7000 aromatic species, such as lavender, mint, thyme, basil, oregano, rosemary, sage, hyssop, and anise hyssop, among others. [9]. The EOs from the last two species are less used in the food sector compared with the others. However, their bioactive proprieties (especially antimicrobial effects) and flavors could make them suitable for this industry.

H. officinalisus L. spp. and Agastache L. spp. are two representative genera from Lamiaceae family, Nepetoideae subfamily [9]. Morphologically, the Nepetoideae subfamily is characterized by herbaceous, shrubby, or occasionally arboreal plants. These plants are typically aromatic due to the variety of terpenoids they contain, along with the presence of the rosmarinic acid. Many species within this subfamily, found in various genera, are recognized for their medicinal and culinary uses as condiments [10]. In food industry, the total aerial parts of H. officinalisus L. spp. are used to spice several beverages and foods, such as green salads, chicken soup, fruit soup, fruit salads, liqueurs, lamb stew, poultry, fish, and meat [11], while A. foeniculum (Pursh) Kuntze is used mostly as an insecticide [10].

H. officinalisus is a genus comprising herbaceous or semiwoody plants, encompassing approximately 10 to 15 species. H. officinalis L. is particularly prevalent in Mediterranean countries, including Central Asia and Northwest India [12]. H. officinalis L. contains numerous bioactive compounds, such as EOs, polyphenols, acids (e.g., rosmarinic acid and caffeic acid, among others), flavonoids, polysaccharides, tannins, pigments, and resins [13]. Due to its mildly bitter taste and minty aroma, it has been used for centuries to create flavors and fragrances in foods, particularly in sauces, seasonings, bitters, and liqueurs [8]. As a food ingredient, H. officinalis is valued for its distinctive flavor and it is commonly used in sauce formulations. Despite its slightly bitter taste, it is often incorporated as a minty flavoring and condiment. Additionally, H. officinalis extracts have been shown to inhibit lipid oxidation and prevent the degradation of heme pigments during cooking and storage. This suggests their potential as a valuable additive in meat processing to reduce lipid oxidation and discoloration [14]. The ethanolic extract of H. officinalis obtained from the plant residue that was subjected to industrial steam distillation exhibited antifungal proprieties against Penicillium verrucosum during the ripening process of cheese. This property can be due to the cyanidin 3-rutinoside content, a phenolic compound found at a concentration of more than 94% in the extract [15]. H. officinalis EO proves to be a valuable ingredient in the formulation of Benedictine and Chartreuse liqueurs [11].

The genus Agastache comprises 22 species of perennial ornamental and medicinal plants [16]. Agastache species are found mostly in North America and East Asia [17]. Agastache foeniculum (Pursh) Kuntze, also known as Lophanthus anisatus (Nutt.) Benth., is a perennial herbaceous and honey-bearing plant widely valued for its EO production [18]. It has a diverse phytochemical composition, containing EOs and non-volatile compounds such as flavones, flavone glycosides, phenolic compounds (e.g., rosmarinic acid and caffeic acid), lignans, and terpenoids like triterpenoids, diterpenes, and sterols. Its leaves and purple flowers are widely utilized for ornamental purposes and as flavoring agents for sweets and other foods [16]. The leaves of A. foeniculum are commonly used for infusions, food flavoring, and in various low-alcohol beverages. For its unique aroma and flavor, blending anise seed and mint, A. foeniculum leaves and flowers are traditionally consumed raw or cooked to flavor salads, bread, and dishes such as pea and lamb preparations [18]. EOs of A. foeniculum are predominantly composed of methyl chavicol (estragole), which imparts a distinct anise-like flavor, making it a popular ingredient in the production of perfumes, liqueurs, various foods, and beer. However, variations in other aromatic compounds, such as menthone or pulegone, can modify the scent profile, occasionally imparting a minty fragrance [19].

The purpose of this comprehensive review is to explore the possible food utilization of the two EOs (H. officinalis L. and Agastache foeniculum (Pursh) Kuntze EOs) from the antimicrobial activity, chemical composition and toxicity points of view. As far as we know, there are no reviews on the food utilization of these two EOs, and so this study proposes to cover this gap in the scientific literature.

2. Methodology of the Literature Review

The data for Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze EOs were extracted from scientific databases and analyzed in order to present comprehensive insights into their volatile compositions, antibacterial and antifungal activities, and toxicity. Google Scholar, Web of Science, Scopus, and PubMed databases were used to search peer-reviewed research articles and book chapters written in English on Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze. The period between 2009 and 2024 was set in order to cover enough information about the EOs, which leads to the most accurate conclusions. The keywords used in the search were essential oils, H. officinalis EO, Agastache foeniculum EO, antimicrobial activity, antifungal activity, bioactive compounds, chemical composition, toxicity, food, food preservatives, and AEMS test, alone or in various combinations. The inclusion criteria were articles and book chapters that analyzed the EOs obtained by hydrodistillation and steam distillation, articles that analyzed the chemical composition by GC-MS, articles that tested the antimicrobial activity against microorganisms relevant for food applications by the microdilution method and inhibition zone, articles that used the AMES test or Comet assay, and articles that tested EOs’ antimicrobial activities after food incorporation. The exclusion criteria were articles that analyzed the chemical composition and antimicrobial activity of the EOs obtained under plant-independent growing conditions (such as weather conditions, soil composition, etc.) and articles that did not clearly present the methods or results. According these criteria, 33 articles and book chapters were included in the current literature review.

3. Chemical Composition of Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze EOs

Table 1 and Table 2 summarize the main insights related to the chemical compositions of the two plants. Hydrodistillation and steam distillation of the aerial parts of H. officinalis L. and Agastache foeniculm (Pursh) Kuntze produced light-yellow EOs, with a yield between 0.1 and 2% [20,21] for H. officinalis and 0.37 and 2.78% [19,22] for A. foeniculum. The difference in the yield of EOs could be attributed to harvest country, even to various regions of the same country. The differences may also depend on the type of plant used, as well as the harvesting period. Miladinovic et al., 2024 [21], state that the fluctuation in EO yield could vary throughout the vegetation cycle, being largely influenced by genetic factors, developmental stages, and harvest timing.

Similarly, the composition of the EO is influenced by various factors, including climatic conditions, the origin of the plant material, subspecies or variety, the plant’s developmental stage, age, cultivation methods, growing location and soil type, harvest timing, the specific plant parts collected and phenotype [7], extraction techniques, and storage conditions [1]. The differences in oil composition determine its organoleptic and physiological properties and, therefore, its potential for application in different industries [23].

Table 1. Presentation of the data for the H. officinalis EO.

Method of Extraction	Part of the Plant Used for Extraction	Stage of the Plant at Harvest	Mass of the Plant Material Used for Extraction	Yield	Origin of the Plant	Reference
Steam distillation	NM	NM	NM	NM	Bulgaria	[24] 1
Steam distillation	Leaves	NM	100 g	NM	Spain	[25]
Steam distillation	NM	Flowering	NM	0.21% (v/w)	Romania	[26]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	100 g	NM	Serbia	[8]
Hydrodistillation using a Deryng apparatus	Aerial parts	Flowering	40 g	EO1: 0.7% EO2: 0.5%	Poland	[7] 2
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	200 g	0.6% (w/w)	Serbia	[14] 3
NM	Aerial parts	NM	NM	NM	Italy	[27]
NM	Aerial parts	NM	10 kg	NM	Egypt	[28]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts (stems, leaves, and flowers)	Flowering	25 g	EO1: 0.70% EO2: 0.28% EO3: 0.40%	Italy	[29] 4
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	50 g	EO1: 0.42–0.56 (v/w) EO2: 0.24–0.56 (v/w) EO3: 0.60–0.90 (v/w) EO4: 1.70–2.00 (v/w) EO5: 0.80–1.06 (v/w)	Kosovo	[20] 5
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	50 g	NM	Iran	[30]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	1.1% (w/w)	Iran	[31]
Hydrodistillation with a Clevenger-type apparatus	NM	NM	160 g	NM	Iran	[32]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	EO1: 0.40% (v/w) EO2: 0.54% (v/w) EO3: 0.65% (v/w) EO4: 0.79% (v/w) EO5: 0.48% (v/w)	Montenegro	[23] 6
Hydrodistillation with a Clevenger-type apparatus	Inflorescence parts	Flowering	200 g	EO1: 0.40 ± 0.09% EO2: 0.45 ± 0.11%	Iran	[33] 7
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	1.1% (w/w)	Italy	[34] 8
Hydrodistillation with an Aura Distillateur installation	Aerial parts (shoots and inflorescences)	Flowering	10 kg	0.27% (v/w)	Romania	[35] 9
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Vegetative	100 g	0.17% (v/v)	Serbia	[36]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Vegetative (June) Flowering (September) Flowering (November)	150 g	J: EO1: 1.3% (w/w) EO2: 1.2% (w/w) EO3: 1.4% (w/w) S: EO1: 0.5% (w/w) EO2: 0.4% (w/w) EO3: 0.4% (w/w) N: EO1: 0.1% (w/w) EO2: 0.1% (w/w) EO3: 0.1% (w/w)	Serbia	[21] 10
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	10 g	2015: EO1: 1.20% (v/w) EO2: 1.30% (v/w) EO3: 1.00% (v/w) 2016: EO1: 0.83% (v/w) EO2: 0.79% (v/w) EO3: 0.83% (v/w)	Poland	[37] 11
Hydrodistillation with a Clevenger-type apparatus	Leaves and flowers	Flowering	500 g	NM	Turkey	[38]
Steam distillation	NM	NM	NM	NM	Balkan	[39] 12

NM—not mentioned. ¹—commercial EO. ²—EO1, extracted from a white flower plant; EO2, extracted from a pink flower plant. ³—H. officinalis subsp. pilifer (Pant.) Murb (also named H. officinalis L. subsp. aristatus (Godr.) Nyman). ⁴—The EOs were obtained from plants from 3 different regions of the same country. ⁵—The EOs were obtained from plants from 5 different regions of the same country. ⁶—The EOs were obtained from plants from 5 different regions of the same country (H. officinalis subsp. aristatus (Godr.) Nyman). ⁷—EO1, extracted from a white flower plant; EO2, extracted from a purple flower plant (H. officinalis L. subsp. angustifolius (Bieb.). ⁸—H. officinalis L. subsp. aristatus (Godr.) Nyman. ⁹—H. officinalis L. variety “Catalin”. ¹⁰—Three EOs extracted from plants harvested at in 3 different months: June—J, September—S, November—N (H. officinalis subsp. aristatus (Godr.) Nyman). ¹¹—EOs obtained in 2015 and 2016; EO1, extracted from a blue flower plant; EO2, extracted from a white flower plant; EO3, extracted from a pink flower plant. ¹²—commercial EO.

Table 1. Presentation of the data for the H. officinalis EO.

Method of Extraction	Part of the Plant Used for Extraction	Stage of the Plant at Harvest	Mass of the Plant Material Used for Extraction	Yield	Origin of the Plant	Reference
Steam distillation	NM	NM	NM	NM	Bulgaria	[24] 1
Steam distillation	Leaves	NM	100 g	NM	Spain	[25]
Steam distillation	NM	Flowering	NM	0.21% (v/w)	Romania	[26]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	100 g	NM	Serbia	[8]
Hydrodistillation using a Deryng apparatus	Aerial parts	Flowering	40 g	EO1: 0.7% EO2: 0.5%	Poland	[7] 2
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	200 g	0.6% (w/w)	Serbia	[14] 3
NM	Aerial parts	NM	NM	NM	Italy	[27]
NM	Aerial parts	NM	10 kg	NM	Egypt	[28]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts (stems, leaves, and flowers)	Flowering	25 g	EO1: 0.70% EO2: 0.28% EO3: 0.40%	Italy	[29] 4
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	50 g	EO1: 0.42–0.56 (v/w) EO2: 0.24–0.56 (v/w) EO3: 0.60–0.90 (v/w) EO4: 1.70–2.00 (v/w) EO5: 0.80–1.06 (v/w)	Kosovo	[20] 5
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	NM	50 g	NM	Iran	[30]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	1.1% (w/w)	Iran	[31]
Hydrodistillation with a Clevenger-type apparatus	NM	NM	160 g	NM	Iran	[32]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	EO1: 0.40% (v/w) EO2: 0.54% (v/w) EO3: 0.65% (v/w) EO4: 0.79% (v/w) EO5: 0.48% (v/w)	Montenegro	[23] 6
Hydrodistillation with a Clevenger-type apparatus	Inflorescence parts	Flowering	200 g	EO1: 0.40 ± 0.09% EO2: 0.45 ± 0.11%	Iran	[33] 7
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	1.1% (w/w)	Italy	[34] 8
Hydrodistillation with an Aura Distillateur installation	Aerial parts (shoots and inflorescences)	Flowering	10 kg	0.27% (v/w)	Romania	[35] 9
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Vegetative	100 g	0.17% (v/v)	Serbia	[36]
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Vegetative (June) Flowering (September) Flowering (November)	150 g	J: EO1: 1.3% (w/w) EO2: 1.2% (w/w) EO3: 1.4% (w/w) S: EO1: 0.5% (w/w) EO2: 0.4% (w/w) EO3: 0.4% (w/w) N: EO1: 0.1% (w/w) EO2: 0.1% (w/w) EO3: 0.1% (w/w)	Serbia	[21] 10
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	10 g	2015: EO1: 1.20% (v/w) EO2: 1.30% (v/w) EO3: 1.00% (v/w) 2016: EO1: 0.83% (v/w) EO2: 0.79% (v/w) EO3: 0.83% (v/w)	Poland	[37] 11
Hydrodistillation with a Clevenger-type apparatus	Leaves and flowers	Flowering	500 g	NM	Turkey	[38]
Steam distillation	NM	NM	NM	NM	Balkan	[39] 12

NM—not mentioned. ¹—commercial EO. ²—EO1, extracted from a white flower plant; EO2, extracted from a pink flower plant. ³—H. officinalis subsp. pilifer (Pant.) Murb (also named H. officinalis L. subsp. aristatus (Godr.) Nyman). ⁴—The EOs were obtained from plants from 3 different regions of the same country. ⁵—The EOs were obtained from plants from 5 different regions of the same country. ⁶—The EOs were obtained from plants from 5 different regions of the same country (H. officinalis subsp. aristatus (Godr.) Nyman). ⁷—EO1, extracted from a white flower plant; EO2, extracted from a purple flower plant (H. officinalis L. subsp. angustifolius (Bieb.). ⁸—H. officinalis L. subsp. aristatus (Godr.) Nyman. ⁹—H. officinalis L. variety “Catalin”. ¹⁰—Three EOs extracted from plants harvested at in 3 different months: June—J, September—S, November—N (H. officinalis subsp. aristatus (Godr.) Nyman). ¹¹—EOs obtained in 2015 and 2016; EO1, extracted from a blue flower plant; EO2, extracted from a white flower plant; EO3, extracted from a pink flower plant. ¹²—commercial EO.

Table 2. Presentation of the data for Agastache foeniculum (Pursh) Kuntze EO.

Method of Extraction	Part of the Plant Used	Stage of the Plant at Harvest	Mass of the Plant Material Used for Extraction	Yield	Origin of the Plant	Reference
Steam distillation using a copper distillation apparatus	Aerial parts (flower and leaves)	NM	500 g	0.37%	Bulgaria	[19]
Hydrodistillation with a Clevenger-type apparatus	Stem, leaves, and flowers	Flowering	50 g	1.86 ± 0.64% (v/w)	Romania	[16] 1
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	25 g	NM	Iran	[40]
Hydrodistilled using a Likens–Nickerson apparatus with continuous extraction with dichloromethane	Aerial parts	Flowering	EO1: 24.78 g EO2: 28.22 g EO3: 13.77 g	EO1: 1.48% EO2: 2.08% EO3: 2.30%	Alabama	[41] 2
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	0.83%	Finland	[42]
Hydrodistillation using a Neo-Clevenger type apparatus	Aerial parts	Flowering	100 g	EO1: 1.74 ± 0.10 mL/100 g EO2: 1.76 ± 0.11 mL/100 g	Romania	[17] 3
Hydrodistillation	EO1: Aerial parts EO2: Leaves EO3: Flowers	NM	2000 g	EO1: 0.62 ± 0.020 g/100 g EO2: 0.75 ± 0.008 g/100 g EO3: 1.22 ± 0.011 g/100 g	Romania	[43] 4
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	40 g	EO1: 1.32% (w/w) EO2: 2.78% (w/w)		[22] 5
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	50 g		Irana	[44]

NM—not mentioned. ¹—Variety “Aromat de Buzău. ²—Three samples of EOs that differ in the mass of the plants subjected to extraction. ³—Two EOs: Agastache foeniculum (Pursh) Kuntze and A. foeniculum variety “Aromat de Buzău”. ⁴—The percentage of aerial parts subjected to extraction: leaves 27.3%; flowers 36.4%; strain 36.3%. ⁵—EO1, extracted from diploid plants; EO2, extracted from tetraploid plants; the aerial parts were composed of thin stems, flowers, and leaves in equal parts.

Table 2. Presentation of the data for Agastache foeniculum (Pursh) Kuntze EO.

Method of Extraction	Part of the Plant Used	Stage of the Plant at Harvest	Mass of the Plant Material Used for Extraction	Yield	Origin of the Plant	Reference
Steam distillation using a copper distillation apparatus	Aerial parts (flower and leaves)	NM	500 g	0.37%	Bulgaria	[19]
Hydrodistillation with a Clevenger-type apparatus	Stem, leaves, and flowers	Flowering	50 g	1.86 ± 0.64% (v/w)	Romania	[16] 1
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	25 g	NM	Iran	[40]
Hydrodistilled using a Likens–Nickerson apparatus with continuous extraction with dichloromethane	Aerial parts	Flowering	EO1: 24.78 g EO2: 28.22 g EO3: 13.77 g	EO1: 1.48% EO2: 2.08% EO3: 2.30%	Alabama	[41] 2
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	NM	0.83%	Finland	[42]
Hydrodistillation using a Neo-Clevenger type apparatus	Aerial parts	Flowering	100 g	EO1: 1.74 ± 0.10 mL/100 g EO2: 1.76 ± 0.11 mL/100 g	Romania	[17] 3
Hydrodistillation	EO1: Aerial parts EO2: Leaves EO3: Flowers	NM	2000 g	EO1: 0.62 ± 0.020 g/100 g EO2: 0.75 ± 0.008 g/100 g EO3: 1.22 ± 0.011 g/100 g	Romania	[43] 4
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	40 g	EO1: 1.32% (w/w) EO2: 2.78% (w/w)		[22] 5
Hydrodistillation with a Clevenger-type apparatus	Aerial parts	Flowering	50 g		Irana	[44]

NM—not mentioned. ¹—Variety “Aromat de Buzău. ²—Three samples of EOs that differ in the mass of the plants subjected to extraction. ³—Two EOs: Agastache foeniculum (Pursh) Kuntze and A. foeniculum variety “Aromat de Buzău”. ⁴—The percentage of aerial parts subjected to extraction: leaves 27.3%; flowers 36.4%; strain 36.3%. ⁵—EO1, extracted from diploid plants; EO2, extracted from tetraploid plants; the aerial parts were composed of thin stems, flowers, and leaves in equal parts.

3.1. Chemical Composition of Hyssopus officinalis L. EO

The effective quality control of H. officinalis EO is based on the identification of plant samples at the intraspecific level and subjected to a precise chemical analysis. The H. officinalis EO international standard (ISO 9841:2013) [35] provides 13 compounds as representative and characteristic, where pinocamphone, isopinocamphone, and pinene are the most significant.

The volatile compounds of H. officinalis L. EO are listed in detail in

Table 3. Volatile compounds of H. officinalis EO.

Total Number of Identified Compounds	Total Identified Compounds (%)	Main Classes of Compounds (%)	Main Compounds (%)	Reference
59	99.4	Oxygenated monoterpenes: 67.8 Monoterpene hydrocarbons: 20.9 Sesquiterpene hydrocarbons: 6.4 Oxygenated sesquiterpenes: 2.4 Other: 0.1	cis-Pinocamphone: 41.1; trans-Pinocamphone: 20.5; β-Pinene: 12; Myrcene: 1.5	[8]
EO1: 44 EO2: 49	99.6 99.0	Oxygenated monoterpenes: EO1: 70.6, EO2: 59.3 Monoterpene hydrocarbons: EO1: 23.2, EO2: 17.4 Sesquiterpene hydrocarbons: EO1: 4.9, EO2: 10.2 Oxygenated sesquiterpenes: EO1: 0.6, EO2:10.9 Other compounds: EO1:0.3, EO1:1.2	β-Pinene: EO1: 12.4, EO2: 9.8; 1,8-Cineole: EO1: 7.6, EO2: 1.9; Pinocamphone: EO1: 51.0, EO2: 28.8; Isopinocamphone: EO1: 1.9, EO2: 21.9; Myrcene: EO1: 3.9, EO2: 1.7	[7]
EO1: 66 EO2: 55	97.2 98	Oxygenated monoterpenes: 61.69 Monoterpene hydrocarbons: 20.77 Sesquiterpene hydrocarbons: 15.19 Oxygenated sesquiterpenes: 1.39 Other compounds: 0.97	β-Pinene: EO1: 13.38, EO2: 13.54; Myrcene: EO1: 1.90, EO2: 1.94; cis-Pinocamphone: EO1: 48.98, EO2: 50.77; trans-Pinocamphone: EO1: 5.78, EO2: 5.94; β-Phellandrene: EO1: 4.44, EO2: 5.17; Myrtenol: EO1: 1.62, EO2: 1.39; Germacrene D: EO1: 1.92, EO2: 1.87; Bicyclogermacrene: EO1: 1.53, EO2: 2.08; Sabinene: EO1: 1.54, EO2: 1.70; Limonene: EO1: 1.48, EO2: 1.50	[24] 1
31	98.73	Oxygenated monoterpenes: 59.78 Monoterpene hydrocarbons: 33.51 Sesquiterpene hydrocarbons: 4.54 Oxygenated sesquiterpenes: 0.87 Other compounds: 0.03	1,8-Cineole: 36.43; β-Pinene: 19.55; iso-Pinocamphone: 15.32; Pinocamphone: 6.39; Myrcene: 1.95; Sabinene: 2.90; Germacrene D: 1.65	[14]
57	96.81	Oxygenated monoterpenes: 54.08 Monoterpene hydrocarbons: 28.39 Sesquiterpene hydrocarbons: 13.64 Oxygenated sesquiterpenes: 0.35 Other compounds: 0.35	β-Pinene: 18.20; iso-Pinocamphone: 29.10; trans-Pinocamphone: 11.12; Lavandulol: 4.43; Myrcene: 1.84; β-Phellandrene: 1.85; Sabinene: 1.43; Limonene: 1.27; Myrtenol: 1.26	[27]
36	99.83	Oxygenated compounds: 58.77 Nonoxygenated compounds: 41.03 Monoterpene compounds: 88.76 Sesquiterpene compounds: 11.07	Isopinocamphone: 34; Pinocamphone: 21.27; β-Pinene: 13.19; β-Phellandrene: 13.10; Sabinene: 1.84; trans-Caryophyllene: 1.77; Germacrene-D: 1.28; bicyclo-Germacrene: 3.47	[28]
EOEO1: 13 EOEO2: 33 EOEO3: 12	99.3 96.1 98.9	NM	1,8-Cineole: EO1: 15.5, EO2: 4.4, EO3: 39.7; Limonene: EO1: 5.8, EO2: 0.2, EO3: 7.6; trans-Pinocamphone: EO2: 11.0; iso-Pinocamphone; EO2: 43.2; Methyleugenol: EO2: 15.8, EO3: 41.5; (-)-Limonen-10-yl-acetate: EO1: 67.9; β-Pinene: EO1: 1.7, EO2: 3.1, EO3: 4.3; trans-Sabinol: EO2: 1.9; Spathulenol: EO1: 0.4, EO2: 2.1, EO3: 0.9; Caryophyllene oxide: EO2: 2.7, EO3:1.5; Limonen-10-ol: EO1: 2.8; Methyl ether: EO1: 1.9;	[29]
EO1: 57 EO2: 52 EO3: 44 EO4: 65 EO5: 66	NM	NM	β-Pinene: EO1:3.3, EO2: 3.41, EO3:23.31, EO4: 13.66, EO5: 11.23; Limonene, EO1: 3.36, EO3: 14.47, EO4: 7.75, EO5: 3.92; Eucalyptol (cineol): EO1: 45.27, EO2: 7.15, EO3: 0.95, EO4: 12.29, EO5: 16.67; iso-Pinocamphone: EO1: 0.33, EO2: 14.67, EO3: 1.01, EO4: 0.61, EO5: 1.46; Pinocamphone: EO2: 57.73, EO3: 33.81, EO4: 37.86, EO5:30.44; E-Caryophyllene: EO1: 2.15, EO3: 13.73, EO4: 9.04, EO5: 4.98; Myrtenol: EO1: 1.55, EO2:2.36, EO3: 1.46; Sabinene: EO1: 1.33, EO3: 1.22, EO4: 1.10, EO5: 1.54; Z-β-Ocimene: EO3: 1.93, EO4: 4.43, EO5: 9.04; Borneol: EO3: 1.17, EO4: 3.81, EO5: 4.11; α-Pinene: EO1: 1.82, EO5: 1.12; Myrcene: EO1: 1.39, EO3: 1.03, EO5: 1.02; E-Anethenol: EO2: 4.83; Bicyclogermacrene: EO1: 2.16, EO4: 1.13;	[20]
24	98.12	NM	β-Pinene: 10.39; iso-Pinocamphone: 55.49; Pinocamphone: 1.75; Elemol: 3.74; D-Germacrene: 4.55; Myrtenol: 1.67; Myrtenyl acetate: 2.26; β-Bourbonene: 2.27; Caryophyllene: 3.30; Alloaromadendrene: 1.76; γ-Elemene: 2.70	[26]
18	93.9	NM	Sabinene: 5.2; iso-Pinocamphone: 44.7; Pinocamphone: 14.1; Elemol: 5.6; Germacrene D-11-ol: 5.7; β-Phellandrene: 2.4; Myrtenol: 2.8; Germacrene D: 1.6; Spathulenol: 2.8; Caryophyllene oxide: 1.6; β-Caryophyllene: 1.4; cis-α-Bergamotene: 1.4	[30]
43	93.7	NM	Isopinocamphone: 22.1; Isopinocamphopinone: 39.3; 2-Hydroxypinocamphone: 5.4; β-pinene: 2.9; β-Bourbonene: 1.7; Myrtenal: 2.0; Cymene: 1.2; Spathulenol: 2.8; Elemol: 1.7; cis-Pinonsaeure: 1.8; Caryophyllene oxide: 1.2	[31]
33	99.75	NM	Pinocamphone: 11.81; Isopinocamphone: 35.45; β-Pinene: 10.12; Elemol: 5.11; Germacrene D: 3.68; Bicyclogermacrene: 4.04; β-Phellandrene: 5.65; β-Myrcene: 1.21; Linalool: 1.14; β-Bourbonone: 1.73; E-Caryophyllen: 3.25; γ-Eudesmol: 1.47	[32]
NM	NM	NM	cis-Pinocamphone: 47.83; trans-Pinocamphone: 14.65; β-Pinene: 15.21; Myrtenol: 2.26; Sabinene: 1.53; Spathulenol: 1.52; β-Phellandrene: 1.47; Limonene: 1.33; β-Bourbonene: 1.06	[45]
EO1: 16 EO2: 13 EO3: 13 EO4: 12 EO5: 14	EO1: 86.84 EO2: 97.59 EO3: 99.21 EO4: 99.43 EO5: 98.47	Oxygenated monoterpenes: EO1:57.94, EO2: 41.7, EO3: 27.35, EO4: 58.14, EO5: 57.9 Monoterpene hydrocarbons: EO1:28.9, EO2: 36.65, EO3: 43.53, EO4: 37.77, EO5: 26.2 Sesquiterpene hydrocarbons: EO5: 0.67 Phenylpropanoids: EO2: 19.24 EO3: 28.33, EO4: 3.52, EO5: 13.70	1,8-Cineole: EO1: 42.07, EO2: 9.77, EO3: 1.42, EO4: 38.19, EO5: 56.08; cis-Pinocamphone: EO1: 5.61, EO2: 22.75, EO3: 14.72, EO4: 14.54; β-Pinene: EO1: 9.13, EO2: 16.33, EO3: 15.79, EO4: 9.69, EO5: 5.48; Limonene: EO1: 7.99, EO2: 16.11, EO3: 23.81, EO4: 21.77, EO5: 15.43; trans-Pinocamphone: EO1:1.84, EO2: 3.34, EO3: 8.34, EO4: 4.72; Methyl eugenol: EO2: 19.24, EO3: 28.33, EO4: 3.52, EO5: 13.70; Sabinene: EO1: 1.24; Z-β-Ocimene: EO1: 2.94, EO2: 2.06, EO3: 1.88, EO4: 3.11, EO5: 3.06; Myrtenal: EO1: 3.71, EO2: 1.02	[23] 2
EO1: 26 EO2: 22	WF: 97.75 PF: 97.38	Oxygenated monoterpenes: WF: 74.22, PF: 63.82 Monoterpene hydrocarbons: WF: 17.93, PF: 28.96 Sesquiterpene hydrocarbons: WF: 4.59, PF: 4.60 Oxygenated sesquiterpenes: WF: 1.01, PF: 0.0	cis-Pinocamphone: EO1: 30.11, EO2: 55.14; Camphor: EO1: 31.85; β-Pinene: EO1: 12.26, EO2: 17.06; Trans-pinocamphone: EO1: 6.09, EO2: 3.37; Sabinene: EO1: 1.61, EO2: 3.17; δ-3-Carene: EO1: 1.34, EO2: 2.57; Myrtenol: EO1: 2.62, EO2: 3.50; Germacrene D: EO1: 1.61, EO2: 1.69; Bicyclogermacrene: EO1: 1.33, EO2: 1.47; α-Pinene: WF: 0.EO1, EO2: 1.14; Myrcene: EO2: 1.60	[33]
44	99.6	Oxygenated monoterpenes: 51.8 Monoterpene hydrocarbons: 22.7 Sesquiterpene hydrocarbons: 10.4 Oxygenated sesquiterpenes: 4.9 Phenylpropanoids: 9.9%	Linalool: 47.7; (Z)-β-Ocimene: 6.2; (E)-β-Ocimene: 5.4; Germacrene D: 5.8; Methyl eugenol: 9.9; β-Pinene: 1.7; Myrcene: 1.5; Limonene: 4.6; Palustrol: 2.4; Ledol: 2.2	[34]
28	96.15	Oxygenated monoterpenes: 55.33 Monoterpene hydrocarbons: 24.51 Sesquiterpene hydrocarbons: 17.62 Oxygenated sesquiterpenes: 2.19	cis-Pinocamphone: 36.63; trans-Pinocamphone: 11.72; β-Pinene: 10.46; Germacrene D: 7.27; Terpiene: 7.19; β-Elemene: 6.20; α-Thujene: 2.05; Myrcene: 1.78; Carrenol: 1.86; Pinenol: 1.74, β-Caryophyllen: 2.63; Longifolene: 2.02	[35]
43	98.6	NM	1,8-Cineole: 49.09; (Z)-β-Ocimene: 2.47; Isopinocamphone: 22.69; Limonene: 1.46; Sabinene: 1.33; α-Pinene: 1.13; β-Pinene: 11.26; trans-Pinocamphone: 1.24	[36]
NM	NM	NM	Campholenone: 38.39; trans-3-Pinanone: 25.05; β-Pinene: 11.07; Myrtenyl methyl ether: 2.87; Caryophyllene: 2.16; β-Phellandrene: 1.71; (-)-β-Bourbonene: 1.66; (-)-Myrtenol: 1.63; Terpinene-4-ol: 1.22; Linalool: 1.14; D-Limonene: 1.10; Germacrene D: 1.08	[39]
38	93.47	-	β-Pinene + Mircene: 16.36; 1,8-Cineole: 48.23; Pinocamphone: 3.42; Isopinocamphone: 4.38; Limonene: 6.02; 1-Octen-3-ol: 3.32; Pinocarvone: 3.16; α-Pinene: 2.67; β-e-Ocimene: 1.97; α-Terpineol: 1.87	[25]
J: EO1: 25 EO2: 26 EO3: 23 S: EO1: 41 EO2: 51 EO3: 51 N: EO1: 53 EO2: 54 EO3: 52	J: EO1: 97.86 EO2: 99.06 EO3: 99.67 S: EO1: 99.73 EO2: 99.01 EO3: 97.87 N: EO1: 98.05 EO2: 99.06 EO3: 98.82	Oxygenated monoterpenes J: EO1: 61.43, EO2: 48.9, EO3: 27.32 S: EO1: 68.89, EO2: 78.74, EO3: 76.9 N: EO1: 86.47, EO2: 91.5, EO3: 92.25 Monoterpene hydrocarbons J: EO1: 31.12, EO2: 45.12, EO3: 67.24 S: EO1: 28.98, EO2: 14.51, EO3: 14.98 N: EO1: 3.89, EO2: 3.84, EO3: 3.65 Sesquiterpene hydrocarbons J: EO1: 5.31, EO2: 5.04, EO3: 5.11 S: EO1: 1.62, EO2: 2.09, EO3: 4.09 N: EO3: 3.14, EO2: 1.19, EO3: 0.98 Oxygenated sesquiterpenes J: 0 S: EO1: 0.16, EO2: 0.44, EO3: 1.58 N: EO1: 3.05, EO2: 1.9, EO3: 1.29 Others J: 0 S: EO1: 0.08, EO2: 3.23, EO3: 0.32 N: EO1: 1.5, EO2: 0.63, EO3: 0.65	β-Pinene: J: EO1: 13.62, EO2: 19.44, EO3: 45.43; S: EO1: 12.75, EO2: 6.1 EO3: 5.79, N: EO1: 2.72, EO2: 2.51, EO3: 2.22 Eucalyptol: J: EO1: 16.46, EO2: 33.35, EO3: 25.88 S: EO1: 40.34, EO2: 63.91, EO3: 47.99 N: EO1: 17.15, EO2: 53.42, EO3: 53.85 Z-β-Ocimene: J: EO1: 9.89, EO2: 14.32, EO3: 10.75 S: EO1: 11.3, EO2: 5.18, EO3: 6.59 N: EO1: 0.35, EO2: 0.12, EO3: 0.15 cis-Pinocamphone: J: EO1: 41.69, EO2: 13.19 S: EO1: 18.41, EO2: 9.64, EO3: 21.53 N: EO1: 52.25, EO2: 25.99, EO3: 26.84 Sabinene: J: EO1: 1.63, EO2: 2.35, EO3: 2.08 trans-Pinocarveol: N: EO1: 2.02, EO2: 1.6, EO3: 1.42 trans-Pinocamphone: S: EO1: 7.14 N: EO1: 5.92, EO2: 2.6, EO3: 2.68 Methyl eugenol: S: EO2: 3.04 E-Caryophyllene: J: EO1: 1.1, EO2: 1.04, EO3: 2.17 Germacrene D: J: EO1: 3.27, EO2: 2.33, EO3: 1.27 Caryophyllene oxide: S: EO3: 1.4 N: EO1: 2.68, EO2: 1.69, EO3: 1.15 α-Pinene: J: EO2: 1.96, EO3: 2.76 S: EO1: 1.48	[21]
EO1: 2015: 57 2016: 65 EO2: 2015: 52 2016: 62 EO3: 2015: 57 2016: 60	EO1: 2015: 99.80 2016: 99.43 EO2: 2015: 99.24 2016: 99.40 EO3: 2015: 99.76 2016: 98.81	Oxygenated monoterpenes: EO1: 2015: 50.73, 2016: 38.48 EO2: 2015: 53.03, 2016: 45.29 EO3: 2015: 54.04, 2016: 48.11 Monoterpene hydrocarbons: EO1: 2015: 13.77, 2016: 10.15 EO2: 2015: 13.78, 2016: 12.68 EO2: 2015: 12.34, 2016: 10.46 Sesquiterpene hydrocarbons: EO1: 2015: 20.79, 2016: 28.31 EO2: 2015: 19.22, 2016: 24.45 EO3: 2015: 19.76, 2016: 23.12 Oxygenated sesquiterpenes: EO1: 2015: 14.24, 2016: 21.08 EO2: 2015: 12.90, 2016: 15.91 EO3: 2015: 13.48, 2016: 16.00 Other compounds: EO1: 2015: 0.27, 2016: 1.41 EO2: 2015: 0.31, 2016: 1.07 EO3: 2015: 0.14, 2016: 1.12	Isopinocamphone: EO1: 2015: 28.02, 2016: 20.05 EO2: 2015: 43.02, 2016: 33.33 EO3: 2015: 31.85, 2016: 21.26 Pinocamphone: EO1: 2015: 15.83, 2016: 12.17 EO2: 2015: 1.68, 2016: 3.45 EO3: 2015: 15.25, 2016: 19.62 β-Pinene: EO1: 2015: 7.13, 2016: 4.53 EO2: 2015: 6.95, 2016: 5.83 EO3: 2015: 7.49, 2016: 5.82 β-Phellandrene: EO1: 2015: 3.47, 2016: 2.99 EO2: 2015: 4.23, 2016: 4.37 EO3: 2015: 1.66, 2016: 1.46 δ-Elemene: EO1: 2015: 2.38, 2016: 2.73 EO2: 2015: 1.80, 2016: 2.36 EO3: 2015: 2.15, 2016: 2.34 β-Caryophyllene: EO1: 2015: 2.73, 2016: 3.36 EO2: 2015: 2.81, 2016: 4.13 EO3: 2015: 3.23, 2016: 3.87 Alloaromadendrene: EO1: 2015: 2.12, 2016: 3.28 EO2: 2015: 2.93, 2016: 2.93 EO3: 2015: 2.38, 2016: 2.47 Germacrene D: EO3: 2015: 4.34, 2016: 5.76 EO3: 2015: 3.85, 2016: 5.32 EO4: 2015: 4.11, 2016: 5.20 Bicyclogermacrene: EO1: 2015: 3.01, 2016: 4.15 EO2: 2015: 2.40, 2016: 2.91 EO3: 2015: 2.30, 2016: 2.43 Elemol: EO1: 2015: 8.74, 2016: 11.37 EO2: 2015: 8.04, 2016: 11.02 EO3: 2015: 8.77, 2016: 9.56 Spathulenol: EO1: 2015: 1.78, 2016: 1.72 EO2: 2015: 2.00, 2016: 0.90 EO3: 2015: 1.68, 2016: 1.14	[37]
46	NM	NM	β-Myrcene: 10.16; β-Phellandrene: 49.79; Linalool: 10.04; α-Elemol: 3.62; Germacrene D: 2.79; Linalyl acetate: 2.46; Bicyclogermacrene: 2.08; Alloaromadendrene: 2.07	[39]

NM—not mentioned. ¹—EO1, SE-54 column for the separation and identification of the compounds; EO2, CW20M column for the separation and identification of the compounds. ²—commercial EOs, no details mentioned.

. The H. officinalis EO constituents are categorized as oxygenated monoterpenes (27.32–92.25%) [21], monoterpene hydrocarbons (3.84–67.24%) [21], sesquiterpene hydrocarbons (0.64–28.31%) [23,37], and oxygenated sesquiterpenes (0–21.08%) [37]. Figure 2 presents the distribution of these classes.

The preponderance of the oxygenated monoterpenes in the oil composition is reflected in its major constituents: 1,8-cineole, isopinocamphone, and pinocamphone. This class is completed by linalool, myrtenol, and borneol, among others. The prominent monoterpene hydrocarbons were β-pinene, β-phellandrene, and β-ocimene, but α-pinene, sabinene, myrcene, and limonene can also be found. The main sesquiterpene hydrocarbons found in H. officinalis EO are β-caryophyllene, germacrene D, bicyclogermacrene, and alloaromadendrene. Spathulenol, caryophyllene oxide, and elemol are the most abundant oxygenated sesquiterpenes. Some authors reported compounds from the phenylpropanoid class, such as methyl eugenol. The chemical structures of the main compounds are presented in Figure 3.

The amounts of main compounds can vary, as can be seen in Table 3. The highest content of isopinocamphone was observed in the oils obtain from pink blooming plants of H. officinalis, compared with white and blue flower EOs [37]. Baj et al., 2018 [7] found that the main compounds of H. officinalis EO vary between plants with pink (pinocamphone 28.8%, isopinocamphone 21.9%) and white (pinocamphone 51.0%, isopinocamphone 1.9%) flowers. Miladinovic et al. (2024) [21] reported that H. officinalis should be harvested in November, as the concentrations of key EO compounds, such as eucalyptol and cis-pinocamphone (which significantly influence its antimicrobial effectiveness) are higher, but, from an economic point of view, the best time to harvest H. officinalis should be in June, during the vegetative stage.

3.2. Chemical Composition of Agastache foeniculum (Pursh) Kuntze EO

The volatile compounds of A. foeniculm (Pursh) Kuntze EO are listed in

Table 4. Volatile compounds of Agastache foeniculum (Pursh) Kuntze EO.

Total Number of Identified Compounds	Total Identified Compounds (%)	Main Classes of Compounds (%)	Main Compounds (%)	Reference
7	99.99	NM	Estragole: 94.89; Limonene: 2.91	[16]
12	99.26	NM	Estragole: 94; 1,8-Cineole: 3.33	[44]
7	95.4	NM	Estragole: 83.; Limonene: 3.4; Spathulenol: 3.1; Caryophyllene oxide: 3.1	[40]
EO1: 31 EO2: 45 EO3: 44	100	Monoterpene hydrocarbons: EO1: 2.4, EO2: 4.9, EO3: 2.9 Oxygenated monoterpenoids: EO1: 1.0, EO2: 0.1, EO3: 0.1 Sesquiterpene hydrocarbons: EO1: 1.8, EO2: 2.5, EO3: 2.8 Oxygenated sesquiterpenoids: EO1: 0.2, EO2: 0.2, EO3: 0.3 Benzenoid aromatics: EO1: 94.1, EO2: 91.3, EO3: 92.9 Others: EO1: 0.5, EO2: 1.0, EO3: 0.9	Estragole: EO1: 93.2, EO2: 88.4, EO3: 91.5; Limonene: EO1: 1.5, EO2: 4.9, EO3: 2.9; β-Caryophyllene: EO1: 1.2, EO2: 1.6, EO3: 1.9	[41]
13	100	Oxygenated aliphatics: 1.98 Monoterpene hydrocarbons: 10.23 Oxygenated monoterpenes: 0.08 Sesquiterpene hydrocarbons: 3.04 Phenylpropanoids: 84.67	Methyl chavicol: 82.03 ± 0.80; Limonene: 9.90 ± 0.09; β-Caryophyllene: 2.35 ± 0.02; 1-Octen-3-ol, acetate: 1.84 ± 0.01	[19]
17	NM	NM	Methyl chavicol: 3.2; Menthone: 34.3; Isomenthone: 14.4; Pulegone: 9.1; Methyl eugenol: 3.1	[42]
27	100	Phenylpropanoids: 69.79 Sesquiterpene hydrocarbons: 19.19 Monoterpene hydrocarbons: 6.29 Oxygenated monoterpenes: 0.46 Oxygenated sesquiterpenes: 2.42 Other: 1.85	Estragole: 63.27; Caryophyllene: 10.44; Limonene: 6.29; Methyl isoeugenol: 4.34;	[17]
34	99.63	Phenylpropanoids: 22.39 Sesquiterpene hydrocarbons: 9.23 Mnoterpene hydrocarbons: 8.69 Oxygenated monoterpenes: 54.51 Oxygenated sesquiterpenes: 3.96 Others: 0.85	Estragole: 21.80; Menthone: 31.58; Pulegone: 21.44; Caryophyllene: 5.03
EO1: 10 EO2: 8 EO3: 8	NM	NM	Estragole: EO1: 66.48, EO2: 30.16, EO3: 88.09; Limonene: EO1: 5.24, EO3: 8.01; Chavicol: EO1: 1.04, EO2: 14.22; Methyl eugenol: EO1: 9.77; Eugenol: EO2: 13.96; Benzaldehyde: EO1: 5.20, EO2: 2.44; Pentanol: EO1: 4.45, EO2: 3.13; Benzyl alcohol: EO2: 2.44; Phenyl ethyl alcohol: EO2: 20.19; Ethyl lactate: EO2: 4.53	[43]
43	EO1: 96.32% EO2: 98.63%	-	Methyl chavicol: EO1: 78.75 ± 3.18, EO2: 81.02 ± 2.00; Limonene: EO1: 2.71 ± 0.12, EO2: 3.00 ± 0.12; 1,8-Cineole: EO1: 3.23 ± 0.03, EO2: 3.04 ± 0.07; Globulol: EO1: 1.43 ± 0.04, EO2: 1.67 ± 0.11	[22]

NM—not mentioned.

. The EO constituents are categorized as phenylpropanoids, (22.39–84.67%) [17,19], monoterpene hydrocarbons (2.4–10.23%) [19,41], oxygenated monoterpenes (0.08–54.51%) [17,19], sesquiterpene hydrocarbons (1.8–19.19%) [17,41], and oxygenated sesquiterpenes (0.2–3.96%) [17,41]. Figure 4 presents the distribution of these main classes of EO. Also, some researchers reported benzenoid aromatics (approximately 90%) or oxygenated aliphatics.

The high amount of phenylpropanoids can be explained through the concentrations of estragole or methyl chavicol (3.2–94.89%) [16,42], methyl eugenol, eugenol, and methyl isoeugenol. Some authors reported a high amount of oxygenated monoterpene menthone (31.58–34.3%) [17,42]. Limonene is the most representative compound (1.5–9.9%) [19,41] from the monoterpene hydrocarbons class; meanwhile, β-caryophyllene is the most common compound from the sesquiterpene hydrocarbon group. Caryophyllene oxide and spathulenol are the most representative of the oxygenated sesquiterpene class. The chemical structures of the main compounds are presented in Figure 5.

According to Lawson et al., 2021 [41], five distinct chemotypes of A. foeniculum were identified based on EO chemical profiles: methyl chavicol (estragole), spathulenol/bornyl acetate, δ-cadinene/β-cadinol, limonene and isomenthone. They also tested three samples of H. officinalis EO obtained from plants grown under similar conditions (full sun, clayey–loamy sand) and reported no significant differences in the composition of the compounds. Nechita et al., 2024 [17] showed a difference between two species of A. foeniculum in which one had the main compound estragole and the other menthone and pulogone. Stefan et al., 2022 [43] demonstrated a significant difference between H. officinalis EO obtained from different parts of the plant. EO obtained from flowers had the higher amount of estragole, at 88.09%, followed by limonene (8.01%). Meanwhile, EO obtained from leaves had a more balanced composition; the main compounds being estragole, chavicol, eugenol, and phenyl ethyl alcohol. Additionally, significant amounts of methyl eugenol were reported besides estragole in the EO extracted from the entire aerial parts. The major considerations related to the various chemotypes must be identified and standardized to ensure their efficacy and safety, thereby guaranteeing their safe use by consumers [29].

4. Antimicrobial Activities of Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze EOs

4.1. Antimicrobial Activity of H. officinalis EO

The EO extracted from H. officinalis exhibited a stronger antimicrobial activity than its components (α-pinene, β-pinene, trans-pinocamphone, cis-pinocamphone, and β-phellandrene) [24].

4.1.1. Antifungal Activity

The EO from H. officinalis demonstrated antifungal activity against P. verrucosum attributed to its main compounds: pinocamphone (22.1%) and isopinocamphone (25.5%). When used as a natural cheese coating, it effectively inhibited the growth of contaminant flora during cheese ripening. The correlation between the chemical composition and antifungal efficacy highlighted 16 significant compounds, with structural groups comprising α- and β-phellandrene emerging as the most influential in the antifungal model [46]. DeMartino et al., 2009 [27] demonstrated the inhibition of fungal growth of strains of agro-food interest: Penicillium simplicissimum, Aureobasidium pullulans, Penicillium citrinum, and Penicillium aurantiogriseum. Meanwhile, Tancinova et al., 2023 [39], demonstrated that H. officinalis EO had a potential antifungal effect against Cladosporium sp. obtained from moldy fruits.

Hristova et al., 2015, [24] reported the antifungal activity of H. officinalis EO against clinical isolates and reference strains from the genus Candida. C. albicans exhibited the highest sensitivity to the EO, followed by C. krusei, C. parapsilosis, and C. tropicalis, with C. glabrata having the lowest sensitivity. Other research groups also demonstrated antifungal activity against Candida albicans [7,26,47]. Mahboubi et al., 2011, [31] demonstrated the inhibitory effect of EO on A. niger and C. albicans. A higher concentration (8 µL/mL and 2 µL/mL, respectively) of oil was required to inhibit spore germination compared to the amount needed to suppress hyphal growth (0.5 µL/mL and 1 µL/mL, respectively). Dazmic et al., 2013 [14] reported the antifungal activity of EO against ten micromycetes. Aspergillus niger was the most resistant fungus, followed by Penicillium funiculosum. Cladosporium species, Aspergillus versicolor, and Trichoderma viride proved to be the most sensitive ones. Karbin et al., 2009 [30] demonstrated the effective inhibition of mold growth, demonstrating strong anti-Aspergillus flavus activity by suppressing its proliferation. Harcarova et al., 2021 [48] showed an inhibitory effect of H. officinalis EO on Fusarium graminearum, but mycelial growth inhibition was the least effective across all tested concentrations. Stan et al., 2022 [35] also demonstrated the antifungal activity of H. officinalis EO against a Fusarium strain, F. oxysporum. The data are comprehensively detailed in

Table 5. Minimum inhibitory concentration and minimum bactericidal/fungicidal concentrations of H. officinalis EO.

Microorganism		MIC			MBC/MFC			Reference
		Bacterial concentration: 106 cfu/mL						[8]
Bacillus cereus ATCC 11778		14.20 μL/mL			28.40 μL/mL
Escherichia coli ATCC 8739		227.25 μL/mL			227.25 μL/mL
Salmonella Enteritidis ATCC 13076		227.25 μL/mL			227.25 μL/mL
Staphylococcus aureus ATCC 25923		227.25 μL/mL			227.25 μL/mL
Enterococcus faecalis ATCC 29212		454.50 μL/mL			454.50 μL/mL
Pseudomonas aeruginosa ATCC 27853		454.50 μL/mL			454.50 μL/mL
Staphylococcus epidermidis ATCC 12228		227.25 μL/mL			227.25 μL/mL
Proteus hauseri ATCC 13315		454.50 μL/mL			454.50 μL/mL
		Bacterial concentration: 106 cfu/mL						[7]
Staphylococcus aureus ATCC 25923		EO1: 10 mg/mL EO2: 5 mg/mL			EO1: 20 mg/mL EO2: 10 mg/mL
Bacillus subtilis ATCC 6633		EO1:5 mg/mL EO2: 0.625 mg/mL			EO1: 5 mg/mL EO2: 2.5 mg/mL
Streptococcus pyogenes ATCC 19615		EO1: 0.625 mg/mL EO2: 0.312 mg/mL			EO1:1.25 mg/mL EO2: 0.625 mg/mL
Escherichia coli ATCC 25922		EO1: 5 mg/mL EO2: 5 mg/mL			EO1: 10 mg/mL EO2: 5 mg/mL
Proteus mirabilis ATCC 12453		EO1: 5 mg/mL EO2: 5 mg/mL			EO1: 10 mg/mL EO2: 10 mg/mL
Klebsiella pneumoniae ATCC 13883		EO1: 5 mg/mL EO2: 5 mg/mL			EO1: 10 mg/mL EO2: 10 mg/mL
Pseudomonas aeruginosa ATCC 9027		EO1: 5 mg/mL EO2: 5 mg/mL			EO1: 10 mg/mL EO2: 10 mg/mL
Staphylococcus epidermidis ATCC 12228		EO1: 5 mg/mL EO2: 2.5 mg/mL			EO1: 20 mg/mL EO2: 5 mg/mL
Micrococcus luteus ATCC 10240		EO1: 2.5 mg/mL EO2: 2.5 mg/mL			EO1: 5 mg/mL EO2: 5 mg/mL
Streptococcus pneumoniae ATCC 49619		EO1: 0.625 mg/mL EO2: 0.312 mg/mL			EO1: 1.25 mg/mL EO2: 0.625 mg/mL
Streptococcus mutans ATCC 25175		EO1: 1.25 mg/mL EO2: 0.625 mg/mL			EO1: 1.25 mg/mL EO2: 1.25 mg/mL
Streptococcus pyogenes ATCC 19615		EO1: 0.625 mg/mL EO2: 0.312 mg/mL			EO1: 1.25 mg/mL EO2: 0.625 mg/mL
		Fungal concentration: 5 × 104 cfu/mL
Candida albicans ATCC 102231		EO1: 0.625 mg/mL EO2: 0.625 mg/mL			WF: 25 mg/mL PF: 2.5 mg/mL
Candida parapsilosis ATCC 102231		EO1: 1.25 mg/mL EO2: 0.625 mg/mL			WF: 5 mg/mL PF: 1.25 mg/mL
		Fungal concentration: 0.5 × 103–2.5 × 103 cfu/mL						[24]
28 strains of Candida albicans: 1 × ATCC 10231 and 27 × clinical isolates		128–256 μL/mL			256–512 μL/mL
8 strains of Candida glabrata: 1 × ATCC 90030 and 7 × clinical isolates		512–1024 μL/mL			1024–2048 μL/mL
6 strains of Candida tropicalis: 1 × NBIMCC 23 and 5 × clinical isolates		512–1024 μL/mL			512–1024 μL/mL
6 stains of Candida parapsilosis: 1 × ATCC 22019 and 5 × clinical isolates		256–512 μL/mL			512–1024 μL/mL
Candida krusei: 4 × clinical isolates		128–256 μL/mL			256–512 μL/mL
		Fungal concentration: 1.0 × 106 spore/mL						[14]
Aspergillus niger ATCC 6275		52.20 mg/mL			104.40 mg/mL
Aspergillus ochraceus ATCC 12066		26.10 mg/mL			52.20 mg/mL
Aspergillus versicolor ATCC 11730		10.44 mg/mL			26.10 mg/mL
Cladosporium cladosporioides ATCC 13276		10.44 mg/mL			26.10 mg/mL
Cladosporium fulvum TK 5318		26.10 mg/mL			26.10 mg/mL
Penicillium funiculosum ATCC 10509		52.20 mg/mL			52.20 mg/mL
Penicillium ochrochloron ATCC 9112		26.10 mg/mL			52.20 mg/mL
Trichoderma viride AM 5061		10.44 mg/mL			26.10 mg/mL
Proteus vulgaris ATTC 13315		20 μg/mL			-			[28]
Escherichia coli ATCC 35218		80 μg/mL
Staphylococcus aureus ATCC 25923		80 μg/mL
Staphylococcus aureus resistant		30 μg/mL
		Fungal concentration: 106 cfu/mL						[48]
Fusarium graminearum CCM F-683		0.4 mg/mL			-
Fusarium graminearum CCM 8244		0.4 mg/mL			-
		Bacterial concentration: 1 × 107–1 × 108 cfu/mL						[31]
Staphylococcus aureus ATCC 25923		0.5 μL/mL			0.5 μL/mL
Staphylococcus saprophyticus ATCC 13518		1 μL/mL			1 μL/mL
Bacillus cereus ATCC 1247		1 μL/mL			1 μL/mL
Escherichia coli ATCC 8739		4 μL/mL			4 μL/mL
Pseudomonas aeruginosa ATCC 9027		4 μL/mL			4 μL/mL
		Fungal concentration: 1 ×107–1×108 cfu/mL
Candida albicans ATCC 10231		1 μL/mL			2 μL/mL
Aspergillus niger ATCC 16404		0.5 μL/mL			8 μL/mL
Bacillus cereus ATCC 10876		4.1 mg/mL			-			[34]
Bacillus subtilis ATCC 6633		4.1 mg/mL
Staphylococcus aureus ATCC 29213, ATCC 6538, ATCC 25923		4.1 mg/mL
Staphylococcus aureus ATCC 43300		Resistant
Enterococcus faecalis ATCC 29212		4.1 mg/mL
Acinetobacter baumanni ATCC 19606		4.1 mg/mL
Pseudomonas aeruginosa ATCC 27853		Resistant
Escherichia coli ATCC 25922		4.1 mg/mL
Klebsiella pneumoniae ATCC 13883		4.1 mg/mL
Staphylococcus epidermidis ATCC 12228		4.1 mg/mL
Clinical isolates: Staphylococcus aureus (IG22)		4.1 mg/mL
Staphylococcus aureus (IG23)		2.0 mg/mL
Staphylococcus aureus (IG24)		Resistant
Staphylococcus aureus (IG5)		Resistant
Staphylococcus epidermidis (IG1)		Resistant
Staphylococcus epidermidis (IG6)		4.1 mg/mL
Serratia marcescens (IG)		4.1 mg/mL
Acinetobacter baumannii (BS1) MDR		4.1 mg/mL
Acinetobacter baumannii (BS2) MDR		4.1 mg/mL
Acinetobacter baumannii (BS3) MDR		4.1 mg/mL
Klebsiella pneumoniae (BS1) MDR		Resistant
Klebsiella pneumoniae (BS2) MDR		4.1 mg/mL
		Bacterial concentration: 106 cfu/mL						[36]
MDR clinical isolates:
Wound swab isolates:
Staphylococcus aureus		22.15 mg/mL			44.3 mg/mL
Streptococcus pyogenes		88.60 mg/mL			88.60 mg/mL
Enterococcus faecalis		22.15 mg/mL			22.15 mg/mL
Escherichia coli		44.3 mg/mL			44.3 mg/mL
Pseudomonas aeruginosa		44.3 mg/mL			88.60 mg/mL
Acinetobacter sp.		22.15 mg/mL			22.15 mg/mL
Proteus Mirabilis		44.3 mg/mL			88.6 mg/mL
Nasal swab isolates:
Klebsiella sp.		88.6 mg/mL			88.6 mg/mL
Streptococcus pneumoniae		22.15 mg/mL			22.15 mg/mL
Staphylococcus aureus		22.15 mg/mL			22.15 mg/mL
Throat swab isolates:
Streptococcus pyogenes		22.15 mg/mL			22.15 mg/mL
Escherichia coli		44.3 mg/mL			44.3 mg/mL
Sputum isolates:
Pseudomonas aeruginosa 1		11.08 mg/mL			11.08 mg/mL
Pseudomonas aeruginosa 2		44.30 mg/mL			88.60 mg/mL
Klebsiella sp.		11.08 mg/mL			22.15 mg/mL
Aspirate isolates:
Escherichia coli		44.30 mg/mL			44.3 mg/mL
		Fungal concentration: 106 spores/mL						[39]
6 fruits from which Cladosporium cladosporioides was isolated:		7 days of incubation			14 days of incubation
KMi-1034		500 µL/L			>500 µL/L
KMi-1035		500 µL/L			>500 µL/L
KMi-1036		500 µL/L			500 µL/L
KMi-1037		250 µL/L			500 µL/L
KMi-1038		500 µL/L			>500 µL/L
		Bacterial concentration: 107–108 cfu/mL						[21]
		J	S	N	J	S	N
Staphylococcus aureus ATCC 25923	EO1	38.5 mg/mL	64.0 mg/mL	8.8 mg/mL	154.0 mg/mL	64.0 mg/mL	70.5 mg/mL
	EO2	39.4 mg/mL	65.5 mg/mL	72.5 mg/mL	157.5 mg/mL	65.5 mg/mL	72.5 mg/mL
	EO3	160.0 mg/mL	66.0 mg/mL	37.8 mg/mL	160.0 mg/mL	66.0 mg/mL	75.5 mg/mL
Bacillus cereus ATCC 11778	EO1	9.6 mg/mL	64.0 mg/mL	70.5 mg/mL	9.6 mg/mL	64.0 mg/mL	70.5 mg/mL
	EO2	39.4 mg/mL	65.5 mg/mL	72.5 mg/mL	39.4 mg/mL	65.5 mg/mL	72.5 mg/mL
	EO3	40.0 mg/mL	66.0 mg/mL	37.8 mg/mL	40.0 mg/mL	66.0 mg/mL	75.5 mg/mL
Enterococcus faecalis ATCC 19433	EO1	38.5 mg/mL	8.0 mg/mL	8.8 mg/mL	77.0 mg/mL	64.0 mg/mL	70.5 mg/mL
	EO2	78.8 mg/mL	8.2 mg/mL	9.1 mg/mL	78.8 mg/mL	65.5 mg/mL	72.5 mg/mL
	EO3	160.0 mg/mL	16.5 mg/mL	9.4 mg/mL	160.0 mg/mL	66.0 mg/mL	75.5 mg/mL
Salmonella enteritidis ATCC 13076	EO1	9.6 mg/mL	16.0 mg/mL	8.8 mg/mL	9.6 mg/mL	64.0 mg/mL	70.5 mg/mL
	EO2	19.7 mg/mL	32.8 mg/mL	9.1 mg/mL	39.4 mg/mL	65.5 mg/mL	72.5 mg/mL
	EO3	80.0 mg/mL	66.0 mg/mL	18.9 mg/mL	80.0 mg/mL	66.0 mg/mL	75.5 mg/mL
Escherichia coli ATCC 25922	EO1	77.0 mg/mL	32.0 mg/mL	35.3 mg/mL	77.0 mg/mL	64.0 mg/mL	70.5 mg/mL
	EO2	78.8 mg/mL	16.4 mg/mL	18.1 mg/mL	78.8 mg/mL	65.5 mg/mL	72.5 mg/mL
	EO3	160.0 mg/mL	16.5 mg/mL	18.9 mg/mL	160.0 mg/mL	66.0 mg/mL	75.5 mg/mL
Enterobacter aerogenes ATCC 13048	EO1	38.5 mg/mL	64.0 mg/mL	70.5 mg/mL	38.5 mg/mL	64.0 mg/mL	70.5 mg/mL
	EO2	39.4 mg/mL	65.5 mg/mL	72.5 mg/mL	78.8 mg/mL	65.5 mg/mL	72.5 mg/mL
	EO3	40.0 mg/mL	66.0 mg/mL	75.5 mg/mL	80.0 mg/mL	66.0 mg/mL	75.5 mg/mL
Pseudomonas aeruginosa ATCC 9027	EO1	38.5 mg/mL	16.0 mg/mL	8.8 mg/mL	38.5 mg/mL	16.0 mg/mL	8.8 mg/mL
	EO2	157.5 mg/mL	32.8 mg/mL	9.1 mg/mL	157.5 mg/mL	32.8 mg/mL	9.1 mg/mL
	EO3	160.0 mg/mL	16.5 mg/mL	9.4 mg/mL	160.0 mg/mL	16.5 mg/mL	18.9 mg/mL
		Fungal concentration: 107–108 cfus/mL
		J	S	N	J	S	N
Candida albicans ATCC 24433	EO1	2.4 mg/mL	8.0 mg/mL	4.4 mg/mL	2.4 mg/mL	16.0 mg/mL	17.6 mg/mL
	EO2	19.7 mg/mL	8.2 mg/mL	4.5 mg/mL	19.7 mg/mL	32.8 mg/mL	18.1 mg/mL
	EO3	10.0 mg/mL	8.3 mg/mL	4.7 mg/mL	160.0 mg/mL	33.0 mg/mL	9.4 mg/mL
		Bacterial concentration: 2 × 108 ufc/mL						[47] 1
		EO1	EO2	EO3	EO4	EO5
Staphylococcus aureus ATCC 6538		>500 µg/mL	400 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL	-
Bacillus subtilis ATCC 6633		>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL
Escherichia coli ATCC 8739		>500 µg/mL	400 µg/mL	>500 µg/mL	400 µg/mL	500 µg/mL
Klebsiella pneumoniae NCIMB 9111		>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL
Salmonella Typhimurium ATCC 14028		>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL
Pseudomonas aeruginosa ATCC 9027		>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL	>500 µg/mL
		Fungal concentration: 2 × 106 ufc/mL
		EO1	EO2	EO3	EO4	EO5	-
Candida albicans ATCC 10231		500 µg/mL	500 µg/mL	500 µg/mL	500 µg/mL	500 µg/mL

¹—The description and the chemical compounds are described in Micovic et al., 2021 [23], and presented in the tables above.

and

Table 6. Inhibition zone of H. officinalis EO.

Microorganism		Inhibition Zone of Microorganism Growth						Reference
		Bacterial concentration: 1 × 108 ufc/mL						[27]
		µg of EO/5 mm disc 1
		93 µg		185 µg		463 µg
Bacillus cereus DSM 4313		Not active		Not active		0.63(±0.06) cm
Bacillus cereus DSM 4384		Not active		Not active		0.60(±0.05) cm
Escherichia coli DSM 8579		Not active		Not active		0.77(±0.12) cm
Enterococcus faecalis DSM 2352		Not active		Not active		0.60(±0.04) cm
Staphylococcus aureus DSM 25923		Not active		Not active		0.60(±0.00) cm
		Fungal concentration: 1 × 106 ufc/mL
		µg of EO/5 mm
		93 µg		185 µg		463 µg
Penicillium simplicissimum DSM 1097		0.60(±0.00) cm		0.77(±0.6) cm		0.27(±0.06) cm
Aureobasidium pullulans DSM 62074		0.43(±0.06) cm		0.53(±0.06) cm		0.80(±0.00) cm
Penicillium citrinum DSM 1997		0.50(±0.00) cm		0.60(±0.00) cm		0.73(±0.06) cm
Penicillium aurantiogriseum DSM 2429		0.27(±0.06) cm		0.30(±0.00) cm		1.03(±0.06) cm
Penicillium expansum DSM 1994		Not active		Not active		Not active
Debaryomyces hansenii DSM 70238		Not active		Not active		Not active
Proteus vulgaris ATTC 13315		5 ± 0.2 mm						[28]
Escherichia coli ATCC 35218		11 ± 0.3 mm
Staphylococcus aureus ATCC 25923		8 ± 0.24 mm
Staphylococcus aureus resistant		4 ± 0.23 mm
		5 mm mycelial disc (10 days old)						[48]
		Media with EOs (concentration)
		100 μg/mL		500 μg/mL		1000 μg/mL
Fusarium graminearum CCM F-683	Day 10	49.00 ± 2.00 mm		42.00 ± 2.65 mm		37.67 ± 1.15 mm
Fusarium graminearum CCM 8244		50.00 ± 0.00 mm		40.67 ± 1.15 mm		39.33 ± 0.58 mm
		Bacterial concentration: 106 ufc/mL						[26] 2
		6 mm discs containing
		5 μL	15 µL		10 µL		20 µL
Staphylococcus aureus ATCC 25923		6.93 ± 0.26 mm	12.62 ± 0.69 mm		13.91 ± 0.28 mm		16.16 ± 0.33 mm
Salmonella Typhimurium ATCC 14028		6.69 ± 0.31 mm	9.03 ± 0.22 mm		14.66 ± 0.69 mm		18.16 ± 0.29 mm
Pseudomonas aeruginosa ATCC 25923		7.06 ± 0.17 mm	8.32 ± 0.32 mm		9.21 ± 0.34 mm		10.81 ± 0.17 mm
Escherichia coli ATCC 25922		6.76 ± 0.35 mm	9.09 ± 0.3 mm		14.84 ± 0.35 mm		18.13 ± 0.29 mm
Klebsiella pneumoniae ATCC 13882		16.58 ± 0.28 mm	17.6 ± 0.46 mm		18.59 ± 0.48 mm		19.51 ± 0.45 mm
Enterococcus faecalis ATCC 29212		10.4 ± 0.44 mm	12.73 ± 0.3 mm		13.64 ± 0.36 mm		15.02 ± 0.37 mm
		Fungal concentration: 106 ufc/mL
		6 mm discs containing the following EO volumes
		5 μL	15 µL		10 µL		20 µL
Candida albicans ATCC 10231		15.56 ± 0.3 mm	19.89 ± 0.4 mm		25.82 ± 0.32 mm		29.81 ± 0.33 mm
		5 mm mycelial disc (7 days old)						[30]
		Media with EOs (concentrations)
		0.125%	0.25%		0.375%		0.5%
Aspergillus flavus isolated from rotted and injured fruits	Day 2	260.28 ± 0.5 mm2	173.52 ± 0.5 mm2		173.52 ± 0.5 mm2		129.53 ± 0.5 mm2
	Day 4	1711.06 ± 0.5 mm2	2418.65 ± 0.5 mm2		1976.76 ± 0.5 mm2		1474.75 ± 0.5 mm2
	Day 6	5204.55 ± 0.5 mm2	3002.36 ± 0.5 mm2		2534.25 ± 0.5 mm2		1516.52 ± 0.5 mm2
		Bacterial concentration: 1 × 107–1 × 108 cfu/mL						[31]
		6 mm disc containing the following EO volumes
		1 µL			2 µL
Staphylococcus aureus ATCC 25923		10			20
Staphylococcus saprophyticus ATCC 13518		7			16
Bacillus cereus ATCC 1247		9			19
Escherichia coli ATCC 8739		-			8
Pseudomonas aeruginosa ATCC 9027		-			-
		Fungal concentration: 1 × 107–1 × 108 cfu/mL
		6 mm disc containing the following EO volumes
		1 µL			2 µL
Candida albicans ATCC 10231		-			-
Aspergillus niger ATCC 16404		-			10 µL
		Mycelial plugs, 8 mm diameter 5 mm discs containing 10 µL of EO						[35]
		EO concentration
		25%	50%		75%		100%
Fusarium oxysporum ZUM 2407	Day 10	insignificant compared with the control	43.6%		62.4%		82.4%

¹ The data do not include the diameter of the discs. ² The data include the disc diameter.

.

4.1.2. Antibacterial Activity

Baj et al., 2018 [7] demonstrated moderate activity of H. officinalis EO against Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus mutans, and weak activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, and Micrococcus luteus. Micovic et al., 2023 [47] demonstrated a slightly better activity against E. coli and S. aureus. DeMartino et al., 2009 [27] tested H. officinalis EO against several Lactobacillus strains (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus sakei, and Lactobacillus rhamnosus) and found no antibacterial activity, suggesting its suitability for food applications. Also, they showed slight antibacterial activity against Bacillus cereus, E. coli, Enterococcus faecalis, and S. aureus. Dezmic et al., 2013 [14] showed moderate antibacterial activity of H. officinalis EO against E. coli and S. aureus and weak antibacterial effect against Proteus vulgaris and S. aureus resistant strains. Mahboubi et al., 2011 [31] demonstrated that the oil showed considerable activity against S. aureus, B. cereus, and Staphylococcus saprophyticus. Gram-negative bacteria were less susceptible than Gram-positive bacteria. Additionally, the oil displayed bactericidal effects against the tested bacteria. Rosato et al., 2018, [34] demonstrated antibacterial activity against B. cereus, B. subtilis, S. epidermidis, Acinetobacter baumanni, E. coli, and most of the S. aureus strains, standard strains, and clinical isolates. Stankovic et al., 2016 [36] showed strong inhibitory and bactericidal effects against the strain of P. aeruginosa from sputum, while the weakest effect was against the strains S. pyogenes and Klebsiella sp. from wound swabs. Jianu et al., 2016 [26] showed that the H. officinalis EO is effective against S. aureus, S. typhimurium, E. coli, and P. aeruginosa. The data are comprehensively detailed in Table 5 and Table 6.

4.2. Antimicrobial Activity of Agastache foeniculum (Pursh) Kuntze

To the best of our knowledge, data regarding the antimicrobial activity of A. foeniculum EO is significantly scarcer compared with the data on H. officinalis EO.

4.2.1. Antifungal Activity

Ownagh et al., 2010 [49] showed the fungistatic effect of A. foeniculum EO on A. flavus, F. solani, and A. fumigatus. Also, they demonstrated a fungicidal effect on the first two strains. Mollova et al., 2024 [19] demonstrated antifungal activity against two strains of yeasts: C. albicans and S. cerevisiae. Meanwhile, Hashemi et al., 2017 [40] showed antifungal effects on A. flavus and A. niger. The data are comprehensively detailed in

Table 7. Minimum inhibitory concentration and minimum bactericidal/fungicidal concentrations of Agastache foeniculum EO.

Microorganism	MIC		MBC/MFC		Reference
	Bacterial concentration: 1.5 × 106 cfu/mL				[40]
Staphylococcus aureus ATCC 6538	125 µg/mL		125 µg/mL
Escherichia coli ATCC 43894	500 µg/mL		1000 µg/mL
Bacillus cereus BC 6830	125 µg/mL		250 µg/mL
Bacillus subtilis ATCC 6633,	62.5 µg/mL		125 µg/mL
Salmonella Enteritidis ATCC 13076	250 µg/mL		250 µg/mL
Salmonella Typhimurium ATCC 13311	250 µg/mL		500 µg/mL
Listeria monocytogenes ATCC 19118	150 µg/mL		250 µg/mL
	Fungal concentration: 106 spore/mL
Food-borne fungal strains:	20 µL EO		5 µL EO
Aspergillus flavus	200 ppm		400 ppm
Aspergillus niger	400 ppm		800 ppm
	Bacterial concentration: 1.5 × 108 cfu/mL				[17]
	ABF	AF	ABF	AF
Escherichia coli ATCC 25922	18.72 ± 6.86 μL/mL	18.72 ± 6.86 μL/mL	22.68 ± 0.00 μL/mL	18.72 ± 6.86 μL/mL
Salmonella Enteritidis ATCC 13076	30.99 ±14.39 μL/mL	22.68 ± 0.00 μL/mL	47.62 ± 0.00 μL/mL	22.68 ± 0.00 μL/mL
Staphylococcus aureus ATCC 6538P	22.68 ± 0.00 μL/mL	10.80 ± 0.00 μL/mL	47.62 ± 0.00 μL/mL	18.72 ± 6.86 μL/mL
Listeria monocytogenes ATCC 19114	22.68 ± 0.00 μL/mL	22.68 ± 0.00 μL/mL	22.68 ± 0.00 μL/mL	22.68 ± 0.00 μL/mL
	Fungal concentration: 107 spores/mL				[49] 1
Aspergillus fumigates (PTCC 5009)	1000 μL/mL		-
Aspergillus flavus (PTCC 5006)	500 μL/mL		2000 μL/mL
Fusarium solani (PTCC 5284)	500 μL/mL		2000 μL/mL

¹ The EO was obtained in Iran.

and

Table 8. Inhibition zone of Agastache foeniculum EO.

Microorganisms	Inhibition Zone of Microorganism Growth				Reference
	0.1 mL of the bacterial suspension/plate; concentration 1.5 × 106 cfu/mL 20 µL of EO/6 mm disc				[40]
	EO concentration 10–15 mg/mL
Staphylococcus aureus ATCC 6538	16.33 ± 0.88 mm
Escherichia coli ATCC 43894	9 ± 0.58 mm
Bacillus cereus BC 6830,	14.67 ± 0.33 mm
Bacillus subtilis ATCC 6633,	19.33 ± 1.85 mm
Salmonella Enteritidis ATCC 13076	12.67 ± 1.2 mm
Salmonella Typhimurium ATCC 13311	11 ± 1.15 mm
Listeria monocytogenes ATCC 19118	15.67 ± 1.2 mm
	100 µL of the fungal suspension/plate; concentration: 106 spores/mL 10 µL of EO dissolved in methanol
	EO concentration
	1 mg/mL	2.5 mg/mL	5 mg/mL	10 mg/mL
Foodborne fungal strains: Aspergillus flavus	13.2 ± 1 mm	18.67 ± 0.88 mm	21.33 ± 0.67 mm	25.33 ± 1.2 mm
Aspergillus niger	12 ± 0.78 mm	15.3 ± 0.3 mm	19.67 ± 1.2 mm	22.67 ± 0.67 mm
	Fungal concentration: 108 spores/mL 10 µL of EO/6 mm disc				[49]
	EO concentration
	250 μL/mL	500 μL/mL	1000 μL/mL	2000 μL/mL
Aspergillus fumigates (PTCC 5009)	-	7 mm	16 mm	21 mm
Aspergillus flavus (PTCC 5006)	7 mm	12 mm	18 mm	24 mm
Fusarium solani (PTCC 5284)	7 mm	11 mm	19 mm	28 mm
	200 µL of the bacterial suspension/plate; concentration: 1.5 × 108 CFU/mL				[19]
	50 µL of EO; concentration: 0.1 mL/mL
Staphylococcus aureus ATCC 6538	25.7 ± 0.5 mm
Bacillus cereus ATCC 10876	12.3 ± 0.2 mm
Candida albicans ATCC 10231	16.5 ± 0.5 mm
Saccharomyces cerevisiae ATCC 9763	16.3 ± 0.4 mm
	Bacterial concentration: 1.5 × 108 CFU/mL EO concentration: 10 µL on discs with a diameter of 6 mm 25 µL on discs with a diameter of 9 mm 100 µL in glass cylinders with a diameter of 6 mm				[43]
Staphylococcus aureus hospital flora	Flower EO: 18 mm Leaf EO: 9 mm Whole plant EO: 10.5 mm

.

4.2.2. Antibacterial Activity

Stefan et al., 2022 [43] concluded that the EO extracted from the flowers of A. foeniculum was the most effective against S. aureus, followed by the EOs extracted from whole aerial parts and from leaves. Nechita et al., 2024 [17] tested the EO against Escherichia coli, Salmonella Enteritidis, S. aureus, and Listeria monocytogenes and demonstrated its efficiency. Mollova et al., 2024 [19] showed that the EO was active against the Gram-positive bacteria S. aureus and B. cereus. Significant antifungal activity was also demonstrated by Hashemi et al., 2017 [40] against Gram-positive bacteria (B. subtilis, S. aureus, L. monocytogenes, and B. cereus). The Gram-negative bacteria were more resistant (S. enteritidis, S. typhimurium, and E. coli). The data are comprehensively detailed in Table 7 and Table 8.

5. Toxicity of Some Compounds of Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze EOs

Various international organizations, including the World Health Organization (WHO), FDA (Food and Drug Administration), Food and Agriculture Organization of the United Nations (FAO), FAO/WHO Codex Alimentarius Commission (CAC), and the European Commission (EC), among others, are firmly committed to regulating safety [50]. The FDA has classified 160 EOs as Generally Recognized as Safe (GRAS) for use in food preparation, cosmetics, and pharmaceuticals [51].

A community list of authorized substances is currently being developed, considering the European Food Safety Authority (EFSA) evaluation of each compound, and they are included in Regulation (EC) 1334/2008 of the European Parliament and of the Council (EC, 2025) on flavorings and certain food ingredients with flavoring properties for use in and on foods [52].

Table 9. Foods in which the presence of the EO compounds are restricted.

Name of the Compound	Foods in Which the Compounds Are Restricted	Maximum Level mg/kg	Reference
Estragole	Dairy products	50	[52]
	Processed fruits and vegetables, nuts, and seeds	50
	Fish products	50
	Non-alcoholic beverages	10
Methyl eugenol	Dairy products	25
	Meat preparations and meat products	15
	Fish preparations and fish products	10
	Soups and sauces	60
	Ready-to-eat savories	20
	Non-alcoholic beverages	1
Pulegone	Mint/peppermint-containing confectionery, except micro breath-freshening confectionery	250
	Micro breath-freshening confectionery	2000
	Chewing gum	350
	Mint/peppermint-containing non-alcoholic beverages	20
	Mint/peppermint-containing alcoholic beverages	100

presents some of the compounds found in A. foeniculum EO that have restrictive use. Also, the Regulation provides the EFSA’s opinion on a large number of compounds found in A. foeniculum and H. officinalis EOs that can be use as food flavorings, such as limonene, α-phellandrene, β-caryophyllene, β-ocimene, linalool, menthol, myrtenol, elemol, and 1,8-cineole, among others [52].

There are few studies presenting the in vitro toxicity of H. officinalis EO. DeMartio et al., 2009 [27] and Guerini et al., 2021 [29] tested the EO from H. officinalis by the AMES test and demonstrated that it did not present any mutagenic effect. Micovic et al., 2021 [23] tested the genotoxic and antigenotoxic potential of EO using the Comet assay, and it did not exhibit a genotoxic effect. Regarding the antigenotoxic effect, EO displayed weaker yet statistically significant activity. These results could make H. officinalis EO suitable for food utilization, but further research is needed in order to establish the optimal dosage for food utilization.

To the best of our knowledge, there are no studies assessing the toxicity of the EO of A. foeniculum. There are, however, some studies regarding the insecticidal effects. The primary active compounds responsible for it are monoterpenes [51]. It was demonstrated that the EO of A. foeniculum had strong toxicity against Tribolium castaneum (the red flour beetle) larvae [53], against adult T. castaneum and Rhyzopertha dominica (the lesser grain borer) that affect grains during storage [44], and against adult Callosobruchus maculatus F. (cowpea seed beetle), an important pest of several plants [54].

6. Food-Related Applications of Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze EOs

In many food products, due to the hydrophobic nature of EO components, the bioactive potential is hindered by interactions with the food matrix, including fat, starch, and proteins [5]. Pure EOs are highly sensitive to environmental stress, making their long-term use in food and beverages challenging. Their water insolubility and volatility are key limitations, while additional factors such as light, heat, humidity, pH, and oxygen further impact their stability. These environmental stressors can lead to reactions between EO compounds and food matrix components, potentially reducing their antioxidant and antimicrobial effectiveness [55]. Besides these factors, their antimicrobial efficacy is also influenced by the microbial load of the food product. As a result, extrapolating in vitro findings to real food systems is challenging, often leading to reduced effectiveness in practical applications [5].

To preserve their biological activity, enhance their effectiveness, and minimize their impacts on food organoleptic properties, EOs could be encapsulated in a delivery system compatible with food applications. Encapsulation not only stabilizes EOs but also increases their contact area with food, ensuring better dispersion in regions where microorganisms thrive and proliferate [56].

One approach is incorporating EOs into active packaging instead of directly adding them to food products. They can be encapsulated in edible and biodegradable polymer coatings or sachets, allowing for controlled release onto the food surface or into the package headspace, which also facilitates their application in foods by improving their distribution in areas where microorganisms thrive, thus enhancing their antimicrobial effectiveness. Sachets releasing volatile EOs into the package headspace provide a simple yet effective preservation method for products such as fruits, meat, and fish [5,56]. Also, biodegradable films can enhance food quality. In meat preservation, they help reduce moisture loss and lipid oxidation [6].

Another method of incorporating EOs into food is by forming nano-emulsion systems. Nano-emulsions play a crucial role in food safety by enabling the controlled release of key compounds, ensuring prolonged antimicrobial effectiveness, and extending the shelf life of food and beverages. Additionally, they could enhance organoleptic properties and improve bioavailability by protecting the bioactive components of EOs during consumption [57]. The use of nano-emulsions in the food industry addresses the degradation of EOs caused by environmental exposure. Nano-emulsions act as a protective barrier for foods against bacteria, fungi, and viruses through a mechanism that involves the selective binding of their transparent or semi-transparent particles to the cell walls of prokaryotic cells, leading to destabilization. This passive absorption process does not affect eukaryotic cells, as their complex membrane structure provides resistance to disruption [55].

As far as we know, the incorporation of A. foeniculum EO in food has not yet been studied. Regarding H. officinalis EO, there are few studies available. Michalczyk et al., 2012 [45] tested the effect of the addition of H. officinalis EO to ground meat. They concluded that the most important benefit was on the sensory changes and Enterobacteriaceae growth. However, maintaining the samples at lower temperature for 3 days was more favorable. Nikolic et al., 2024 [58] tested the coating effect of a mixture of H. officinalis EO and Salvia officinalis EO on a cheese model against staphylococcal isolates. Mehraie et al., 2023, [32] demonstrated the effect of H. officinalis EO on shrimp preservation. They were coated by immersion in a chitosan nano-emulsion containing the EO. After drying the coating and storage for 12 days at 4 °C, the samples were tested for total psychrophilic and mesophilic counts. The conclusion was that the EO nano-emulsion had a better effect on the microbial evaluations, as well as on the chemical and sensory ones.

In general, the effectiveness of both added and naturally occurring antimicrobials may be diminished by certain food components. To successfully apply EOs in food systems, preliminary studies using representative food model media should be conducted to assess potential interactions between EOs and food components that could affect their antimicrobial efficacy. Also, combining EOs can enhance efficacy against both spoilage and pathogenic target organisms. Whole plant extracts often exhibit greater antimicrobial activity compared to mixtures of major components alone. Additionally, minor components in plant EOs may play a critical role in their activity, offering potentiating influences or synergistic effects [59].

7. Future Perspectives of the Food Utilization of Hyssopus officinalis L. and Agastache foeniculum (Pursh) Kuntze EOs

The research studies do not suggest any limitations on the use of EOs in food products, and their use as preservatives is even encouraged. However, conducting comprehensive safety evaluations and adhering to food regulations are crucial to ensure the safety, efficacy, and suitability of EOs. Further research may also be required to assess their impacts on food flavor, stability, and sensory attributes [19]. The acceptability of food products depends on the amount of EOs added, as their strong flavors may lead to palatability issues. Consequently, the use of EOs as preservatives in food has been limited, as achieving effective antimicrobial activity often requires high concentrations that could impact the sensory characteristics of the final product [5]. When used responsibly in food formulations, EOs can enhance shelf-life and food safety by utilizing their natural preservative qualities. Ongoing research in food science may broaden their applications, helping to overcome challenges in food preservation and catering to consumer demand for clean-label, minimally processed products [19].

Looking ahead, the chemical compositions of the studied plants present significant potential for applications in cosmetics and food products, particularly due to their strong antimicrobial properties. Nevertheless, more research is necessary to optimize their effectiveness for these applications. Furthermore, investigating synergistic combinations with other natural compounds could enhance their beneficial effects. Ongoing explorations of their properties and uses will drive innovative advancements in both industries, offering consumers safer and more sustainable options [19].

Many EOs are classified as Generally Recognized as Safe (GRAS) by the United States FDA. However, regulatory restrictions exist regarding their acceptable daily intake, necessitating comprehensive daily intake surveys to assess safety. In Europe, a community list of authorized substances is being developed based on the EFSA’s evaluations. The level of toxicological data required for inclusion in this list depends on the anticipated migration into food. Therefore, careful consideration is necessary when determining suitable concentrations of active substances for use in food packaging to ensure both safety and efficacy [60].

8. Conclusions

The data presented in this review indicate that A. foeniculum and H. officinalis EOs exhibit antimicrobial activity, particularly against Gram-positive bacteria, and demonstrate antifungal efficacy against a wide range of strains. Oxygenated monoterpenes and monoterpene hydrocarbons, represented by isopinocamphone, pinocamphone, and β-pinene, are the main classes found in H. officinalis EO. Estragole and methyl eugenol (phenylpropanoids), and oxygenated monoterpene menthone are the main compounds found in A. foeniculum EO. These chemical compounds are responsible for the bioactive proprieties of these EOs. These findings highlight their potential as natural food preservatives, offering a promising alternative to synthetic additives. The antifungal activity of H. officinalis EO during the cheese ripening process and having no effect against Lactobacillus strains, but inhibiting the growth of S. aureus, makes it suitable for utilization in cheese factories. Additionally, its antifungal effects on mold strains on fruits make it suitable for preventing fruit spoilage. Moreover, the bitter and minty aroma of H. officinalis EO, along with the anise–minty flavor of A. foeniculum EO, can enhance the taste and overall acceptability of food products that incorporate these EOs. A key gap identified in this literature review is the lack of studies specifically investigating the application of these findings into the food industry. While various research efforts have explored the potential of bioactive compounds and antimicrobial properties, their direct implementation in food systems remains underexplored. Future studies should focus on evaluating these EOs in food matrices by assessing their performance in food preservation, sensory impacts, and regulatory compliance.