Composition of the Essential Oil of Coristospermum cuneifolium and Antimicrobial Activity Evaluation

Abstract

For the first time, the chemical composition and antimicrobial evaluation of Coristospermum cuneifolium (previously named Ligusticum lucidum subsp. cuneifolium) essential oil obtained from the aerial parts are reported in this work. Approximately 85% of the total constituents were identified by GC-MS analysis, evidencing the presence of 12 chemical components which belong to several classes of natural compounds. Most of them are reported for the first time in the Ligusticum genus (s.l.) and in the Apiaceae family. Their presence was able to provide a rationale for essential oil use in the field similar to those obtained from other species of the Ligusticum genus (s.l.). Moreover, the huge presence of aromatizing and flavoring components, accounting for 44.4% of the essential oil composition, might make C. cuneifolium a useful natural source of aromatic components for the food and cosmetic fields. In addition to this, a deep comparison of the essential oil of this species with that of other entities within the Ligusticum genus (s.l.) was performed and discussed on a chemotaxonomic basis.

The essential oil was tested for its antimicrobial activity at both high and low inoculum (~5×10⁵ and ~ 5×10³ cfu/mL, respectively) against several bacterial and fungal strains, including methicillin-susceptible Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Candida albicans (ATCC 14053), methicillin-resistant S. aureus (clinical strain), carbapenem-susceptible Klebsiella pneumoniae (clinical strain), carbapenem-resistant K. pneumoniae (clinical strain), and carbapenem-resistant Acinetobacter baumannii (clinical strain).

A high potency against C. albicans was shown, with an absence of growth at the concentration of 3.01 mg/mL; similarly, for methicillin-susceptible S. aureus and methicillin-resistant S. aureus, a reduction of 1.73 and 2 log10 cfu/mL at the concentration of 3.01 mg/mL was observed. With regard to gram-negative microorganisms, only slight potency against A. baumannii was shown, whereas no activity was found against E. coli and K. pneumoniae.

Key words

Coristospermum cuneifolium - Apiaceae - essential oil composition - ethnopharmacology - comparison within the genus - antimicrobial activity

Introduction

Coristospermum cuneifolium (Guss.) Bertol. [synonym of Ligusticum lucidum Mill. subsp. cuneifolium (Guss.) Tammaro] is a perennial herbaceous plant belonging to the Apiaceae family. From the systematic point of view, this plant was originally classified as Ligusticum, a genus which is no longer recognized as valid since it resulted in not being a monophyletic genus from both the morphological and genetic profile [1]. For this reason, it was decided to use the name Coristospermum, and the subspecies L. lucidum subsp. cuneifolium has been upgraded to the species rank and classified as C. cuneifolium [2].

The name of the genus derives from the greek terms κορυστός (korystos) and σπέρμα (spérma), which together mean “full of seeds”, while the name of the species derives from Latin and refers to the typical wedge-shaped leaves.

From the morphological point of view, this species is characterized by an erect stem that is fully branched, striated, and fluted. The leaves are long in the lower part of the plant, and linear and lanceolate in the upper portion. The inflorescence is formed by a composed umbel and is constituted by small white flowers that bloom between June and July. Lastly, the fruits are oblong ([Fig. 1]) [3].

Fig. 1 C. cuneifolium (Guss.) Bertol.

This species is endemic in the Italian territory, especially in central regions of Italy such as Latium, Abruzzo, and Molise [4]. There are only a couple of articles in the literature on entities belonging to the Coristospermum genus because of its relative recent recognition as an autonomous genus, and these mainly report on the genetic diversity of isolated populations [5] and karyological aspects [6]. There is, instead, only a single work reporting on the activities and chemical composition of L. lucidum subsp. cuneifolium (botanical denomination no longer valid, but referred to as C. cuneifolium) solvent extract showing several pharmacological properties, among which the anti-inflammatory and antioxidant ones are the most well known and important [7]. By consequence, many of the Ligusticum species are used in the folk medicine of several countries, especially China and America. In particular, Ligusticum striatum DC. is used in China to treat pain and to cure several hematological disorders such as thrombosis and ischemia due to its high cardiovascular, neuroprotective, and anti-fibrotic properties [8] [9] [10]. Indeed, Ligusticum porteri J. M. Coult. & Rose is still used by native Americans to cure the flu, colds, and toothaches [11] but, to date, there is no record of the use of C. cuneifolium in local ethnomedical traditions.

From a literature survey on essential oil (EO) from plants of the Ligusticum s.l. genus, many have seen that numerous reports indicated several pharmacological activities, i. e., analgesic, cicatrizing, antipyretic, antioxidant, insecticidal, and anticonvulsant [12] [13] [14]. Several publications report on the Ligusticum s.l. genus EO composition and its associated pharmacological properties [15] [16] [17], while no information was found regarding C. cuneifolium EO (CCEO) and associated pharmacological activity.

With the aim to verify the presence of certain chemical components that might justify the use of C. cuneifolium in the ethnopharmacological field [similarly as for other Ligusticum (s.l.) species] and to complete our previous study on the C. cuneifolium non-volatile phytochemical pattern [18], herein the CCEO isolation is reported along with its chemical composition analysis and antimicrobial activity evaluation against several bacterial and fungal strains.

Finally, a detailed comparison of the CCEO chemical composition and other EOs obtained from species of the Ligusticum (s.l.) genus is reported.

Results and Discussion

The CCEO GC-MS analysis led to the identification of 12 compounds. This corresponds to 84.2% of the total area of the gas chromatogram ([Table 1]).

Table 1 Essential oil composition of C. cuneifolium and comparison among C. cuneifolium and other species of the Ligusticum s.l. genus.

Compounds	C. cuneifolium	Retention index	Area %	L. striatum [11]	L. mutellina [15]	L. porteri [16]	L. sinense [37]	L. jeholense [37]	L.grayi
β-Farnesene (1)	0.8%	1682	0.8	0.18%	1.6%	0.3%	–	–	–
β-Caryophyllene (2)	5.2%	1629	5.2	–	3.1%	–	–	–	–
3-Methylbutanoic acid (3)	3.5%	1686	3.5	–	–	–	–	–	–
β-Copaene (4)	0.5%	1512	0.5	–	0.1%	–	–	–	–
3-Methyl-2-butenoic acid (5)	10.6%	1821	10.6	–	–	–	–	–	–
2,5-Bornanedione (6)	2.5%	2006	2.5	–	–	–	–	–	–
Caryophyllene oxide (7)	9.8%	2035	9.8	–	1.9%	–	–	–	–
2,4,6-Trimethyl-benzaldehyde (8)	14.8%	2065	14.8	–	–	–	–	–	–
Spathulenol (9)	7.1%	2152	7.1	0.26%	5.1%	–	3.3%	0.9%	–
Mintlactone (10)	24.3%	2316	24.3	–	–	–	–	–	–
2,4,6-Trimethylbenzyl alcohol (11)	3%	2340	3.0	–	–	–	–	–	–
Phytol (12)	2%	2620	2.0	1.14%	–	–	–	–	–
Total identified			84.1	–	–	–	–	–	–

Compounds 1, 2, 4, 7, and 9 are sesquiterpenes, compounds 3 and 5 are organic acids, compound 6 is a diketone, compound 8 is a trisubstituted benzaldehyde, compound 10 is a bicyclic monoterpene lactone, compound 11 is a substituted benzyl alcohol and, lastly, compound 12 is an acyclic diterpene alcohol. The structures of the main components are reported in [Fig. 2].

Fig. 2 Structures of the three major constituents of the essential oil of C. cuneifolium.

β-farnesene (1), β-caryophyllene (2), β-copaene (4), caryophyllene oxide (7), spathulenol (9) mintlactone (menthalactone) (10), and phytol (12) represent new constituents of the EO for the C. cuneifolium species, 2,4,6-trimethyl-benzaldehyde (8) is a new constituent of the EO for the Ligusticum s.l. genus, 3-methyl-butanoic acid (3), 3-methyl-2-butenoic acid (5), 2,5-bornanedione (6), and 2,4,6-trimethylbenzyl alcohol (11) are, instead, new constituents of the EO also for the Apiaceae family.

β-Farnesene (1) was already evidenced in Ligusticum s.l. [15] [19] [20] and, in general, represents one of the major constituents of the EO of plants belonging to the Apiaceae family [21] [22] [23]. This compound was reported to have insecticidal properties [24]. β-Caryophyllene (2) was already reported in the Ligusticum s.l. genus [19] and has anti-inflammatory, gastroprotective, and anesthetic properties [25] [26] [27].

3-Methyl-butanoic acid (3; common name isovaleric acid) and 3-methyl-2-butenoic acid (5) (also known as 3-methyl-crotonic acid) were found only in traces in the EOs of plants belonging to the Berberidaceae, Lamiaceae, and Asteraceae families [28] [29] and were recognized for the first time during the present study as constituents of the EO obtained from an Apiaceae species. It is interesting to note that these compounds resulted instead as being present in quite a high amount in CCEO, accounting for 3.5 and 10.6%, respectively. The former has a very pungent smell and seems to have anticonvulsant properties [30], while for the latter, no pharmacological properties are reported in the literature but, in medicine, its presence is linked with a disease known as organic aciduria [31].

On the other side, β-copaene (4) was already evidenced, even if in small traces, in Mutellina purpurea (Poir.) Reduron, Charpin & Pimenov [19] [considered as syn. of Ligusticum mutellina (L.) Crantz] and in other entities of the family, i. e., Ferula glauca L. [32]. It was reported to exhibit antioxidant effects on human lymphocyte cultures [33].

The oxygenated monoterpene 2,5-bornanedione (6) resulted in being a compound more typical of the Lamiaceae family [34]. This compound, which is an oxidation product of camphor, showed many aromatizing properties [34].

Caryophyllene oxide (7) was previously identified only in L. mutellina [19]. From the pharmacological point of view, this compound presents strong anti-inflammatory and cytotoxic properties [35] [36].

2,4,6-Trimethyl-benzaldehyde (8), also known as mesitaldehyde, was already found only in entities belonging to the Apiaceae family, even in small concentrations. In particular, its presence was reported in Eryngium corniculatum Lam. [37], Eryngium foetidum L. [38], Trachydium roylei Lindl. [39], and Prangos ferulacea (L.) Lindl. [40]. It also has aromatizing properties and was present as one of the main components in the CCEO with 14.8% of the total composition.

Indeed, 2,4,6-trimethylbenzyl alcohol (11), also known as mesityl-methanol, was never recognized as an EO component from species of the Apiaceae family, and it accounted for 3.0% of the total composition in CCEO. Also, this compound is a good flavoring agent. Its presence is chemically related to compound 8, which represents its oxidized form.

Spathulenol (9) represents one of the major constituents of the EOs of a plant belonging to the Ligusticum s.l. genus [15] and to the Apiaceae family, which was also identified in T. roylei Lindl., Kundmannia anatolica Hub. Mor., and Seseli rigidum Waldst. Kit. [39] [41] [42]. This compound was reported to exert strong antibacterial properties [43].

Mintlactone (menthalactone; 10) was originally identified as a minor component of Mentha piperita [44] [45] [46] and Mentha aquatica [47] EOs and, more recently, its presence was also evidenced in aged red wines [48]. It is a very interesting odorant compound related to the p-menthane bicyclic γ-lactone (3,6-dimethyl-4,5,6,7-tetrahydro-benzo[b]-furan-2(H)-one) derivatives that are widely used as flavoring agents in foods and cosmetics [49] [50] [51] [52]. For this reason, in the literature there are a great number of works reporting on organic synthetic approaches [53] [54] [55] [56] for the preparation of mintlactone and its isomers. The presence of mintlactone (10) in CCEO (as well as for Ligusticum s.l.) is herein reported for the first time, and it is worth to note that it accounted for 24.1% of the total composition, therefore, C. cuneifolium may be regarded as an abundant natural source of this compound.

Lastly, phytol (12) was already evidenced in the Ligusticum s.l. genus [57] as well as in the Apiaceae family [58] and showed anti-inflammatory, antimicrobial, and diuretic properties [59].

[Table 1] shows a comparison between the CCEO chemical composition and those cited in the literature for other species of the Ligusticum s.l. genus, focusing only on overlapping compounds.

As can be seen, seven compounds were exclusively found in C. cuneifolium: 3-methyl-butanoic acid (3), 3-methyl-2-butenoic acid (5), 2,5-bornanedione (6), 2,4,6-trimethyl-benzaldehyde (8), mintlactone (menthalactone) (10), and 2,4,6-trimethylbenzyl alcohol (11).

Spathulenol (9) was, instead, the most common compound and, in fact, it was found in all of the compared species besides L. porteri and L. grayi Coult. & N. E. Rose, both coming from the USA. Anyway, in the studied species (C. cuneifolium), the total percentage of this compound was much more abundant compared to any other Ligusticum s.l. species. This might be one peculiarity of the plant growing in Majella National Park and might be used as distinctive marker of this area.

The other distinctive trait is the presence of mintlactone (10) as a major component of the EO, which, to the best of our knowledge, has not been previously recognized in any Ligusticum s.l. EOs. This additional peculiarity, as well as the total absence of ligustilide and related butyl-phthalides which, on the contrary, are often recognized as the principal components of the EOs from Ligusticum s.l. species, contributed to the very unique chemotype observed in C. cuneifolium.

From a chemosystematic point of view, the total absence of butyl-phthalidic derivatives and the presence of peculiar components not yet demonstrated in Ligusticum s.l. genus is further evidence to enhance the recent upgrade to the species rank of C. cuneifolium, also on the phytochemical basis.

The EO composition from the species L. mutellina, harvested in Poland, is more similar to that of C. cuneifolium, likely indicating a similar environmental and climatic influence of growth area. β-Farnesene (1) is the only component present in almost every species listed in [Table 1] with similar relative abundance in all of them. The EO composition of L. grayi [60] is the only one not presenting any constituent in common with that of C. cuneifolium.

The activities of reference compounds and CCEO are reported in [Table 2], [3], respectively. As expected, both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) were susceptible to vancomycin and rifampin, whereas a high level of meropenem resistance was observed in clinical strains of Klebsiella pneumoniae and Acinetobacter baumannii.

Table 2 Activity of antimicrobials commonly used in clinical practice against different bacterial strains at high (A) and low (B) inocolum.

Strain	MIC/MBC (mg/L)
	A				B
	VAN	MEM	RIF	FLU	VAN	RIF	MEM	FLU
MSSA (ATCC 29213)	0.50/1.00	–	0.03/0.07	–	0.50/0.50	0.03/0.03	–	–
MRSA*	1.00/1.00	–	0.07/0.07	–	0.50/0.50	0.07/0.07	–	–
Candida albicans (ATCC 14053)	–	–	-	0.25/1.00	–	–	–	0.25/0.50
CS Klebsiella pneumoniae*	–	0.25/0.25	–	–	–	–	0.03/0.03	–
CR Klebsiella pneumoniae*	–	16/32	–	–	–	–	16/32	–
Escherichia coli (ATCC 25922)	–	0.1/0.25	–	–	–	–	0.03/0.03	–
CR Acinetobacter baumannii*	–	256/256	–	–	–	–	32/32	–

* Clinical strain. MSSA: methicillin-susceptible S. aureus; MRSA: methicillin-resistant S. aureus; CS: carbapenem susceptible; CR: carbapenem resistant. VAN: vancomycin; RIF: rifampin; MEM: meropenem; FLU: fluconazole

Table 3 Activity of C. cuneifolium against different bacterial strains at high (A) and low (B) inocolum.

Strains	A			B
	Δlog10 cfu/mL 3.01 mg/mL*	Δlog10 cfu/mL 0.30 mg/mL*	MBC (mg/mL)°	Δlog10 cfu/mL 3.01 mg/mL§	Δlog10 cfu/mL 0.30 mg/mL§	MBC (mg/mL)°
MSSA (ATCC 29213)	1.73	1.52	>3.01	1.69	0.11	>3.01
MRSA**	2	NA	>3.01	3.69	1.15	3.01
Candida albicans (ATCC 14053)	5.30	NA	3.01	3.69	0.52	3.01
CS Klebsiella pneumoniae**	NA	NA	–	NT	NT	NT
CR Klebsiella pneumoniae**	NA	NA	–	NT	NT	NT
Escherichia coli (ATCC 25922)	NA	NA	–	NT	NT	NT
CR Acineto bacterbaumannii**	1.75	NA	>3.01	NT	NT	NT

^* Difference between the starting inoculum (5.30 log10 cfu/mL) and the number of residual viable colonies after 24 h incubation; ^**clinical strain; ^§difference between the starting inoculum (3.69 log10 cfu/mL) and the number of residual viable colonies after 24 h incubation; °minimal bactericidal concentration. MSSA: methicillin-susceptible S. aureus; MRSA: methicillin-resistant S. aureus; CS: carbapenem susceptible; CR: carbapenem resistant. NA: not active; NT: not tested

CCEO showed high potency against Candida albicans both at high and low inocolum, with an absence of growth at a concentration of 3.01 mg/mL. Likely, compared with the starting inoculum (5.30 log10 cfu/mL), we observed a reduction of 1.73 and 2 log10 cfu/mL for MSSA and MRSA at the concentration of 3.01 mg/mL, respectively; at a lower inoculum (3.69 log10 cfu/mL), the potency of CCEO was bactericidal only against MRSA, with an absence of growth after 24 h incubation. At high inoculum, no activity was observed against Escherichia coli and K. pneumoniae (both carbapenem susceptible and resistant strains); interestingly, CCEO demonstrated a slight potency against a clinical strain of carbapenem-resistant A. baumannii, with a reduction of 1.75 log10 cfu/mL compared with the initial inoculum.

The antimicrobial activity of C. cuneifolium might be very interesting when considering its possible use as a part of the treatment of infections caused by MSSA, MRSA, and, in particular, against C. albicans, both at high and low inocolum.

The activity against carbapenem-resistant A. baumannii deserves further investigation in order to evaluate the role of CCEO in the setting of multidrug-resistant A. baumannii infections, which represent a global threat due to the limited available therapeutic options and the high mortality [61].

From an analytical point of view, CCEO resulted in being constituted by 12 compounds, namely, β-farnesene (1), β-caryophyllene (2), 3-methyl-butanoic acid (3), β-copaene (4), 3-methyl-2-butenoic acid (5), 2,5-bornanedione (6), caryophyllene oxide (7), 2,4,6-trimethyl-benzaldehyde (8), spathulenol (9), mintlactone (10), 2,4,6-trimethylbenzyl alcohol (11), and phytol (12). Seven of them are new constituents of CCEO, one is a new constituent of the EO for the Ligusticum s.l. genus and five are new constituents of the EO oil for the Apiaceae family. The presence of all these peculiarities in the CCEO composition, together with the absence of ligustilide and related compounds that are widely distributed among the Ligusticum genus, may give additional evidence, from a chemosystematic standpoint, to support the upgrade of C. cuneifolium to an autonomous species.

The presence of these components also provide a rationale for the use of CCEO in the ethnopharmacological field, similar to that reported for the EOs from other species of the Ligusticum (s.l.) genus, due to their outstanding medical properties. In addition, the huge presence of components widely known for their aromatizing and flavoring properties (i. e., 6, 8, 10, and 11, which accounted for 44.4% of the total composition) might make the studied species a useful natural source of aromatic components for the food and cosmetic fields.

When making a comparison, it emerged that seven compounds were exclusively present in C. cuneifolium and that the EO composition showed several similarities with that reported for L. mutellina. This fact would lead us to believe that these species have very similar environmental conditions. Indeed, spathulenol (9) was also present in the majority of the Ligusticum species. It is interesting to note that 9 might be regarded as a sort of a marker compound because it has been found in CCEO at a much higher concentration, but we cannot exclude that it represents a peculiarity of the plants growing in Majella National Park without further studies on different populations.

Last but not least, the observed antimicrobial properties of CCEO provide useful information for its possible role in the therapy of infections caused by MSSA, MRSA, and, in particular, C. albicans, whereas the activity against a carbapenem-resistant strain of A. baumannii deserves further investigation.

Materials and Methods

Plant material

A sample of the C. cuneifolium plant aerial parts (500 g) was harvested during July 2015 in the territory of the Majella National Park in Abruzzo and, more precisely, in the town of Santa Eufemia a Majella (Pescara province; geographical coordinates: 42°10'04'' N, 14°05'97'' E) at 1500 m a.s.l. of altitude.

The botanical identification was performed by the botanists of the park (Dr. Mirella Di Cecco and Dr. Giampiero Ciaschetti) by means of available literature [3] [4]. The plant materials were dried at room temperature with the use of a desiccator immediately after the harvest. A specimen is stored in the Sapienza University of Rome Chemistry Department for further reference under voucher number CC08072015B.

Chemicals

The following reagents and material were utilized: distilled water for the steam distillation process; diethyl ether for the liquid/liquid extraction of the EO from the oil/water biphasic distillate; methanol as the solvent used for the GC-MS analysis; anhydrous sodium sulfate to dry the organic extracts. All the solvents having RPE analytical purity grade, if not differently specified, were purchased from Sigma-Aldrich. Antimicrobial agents were provided as purified powders by the manufacturer (Sigma-Aldrich). Stock solutions at different concentrations were prepared in sterile and pyrogen-free 0.9% saline or water according to manufacturer’s instructions. The following antimicrobials were used as references: vancomycin (VAN, Sigma-Aldrich, purity >99%) and rifampin (RIF, Sigma-Aldrich, purity ≥97%) for gram-positive microorganisms, meropenem (MEM, Sigma-Aldrich, purity ≥98%) for gram-negative bacteria, and fluconazole (FLU, Sigma Aldrich, purity ≥98%) for fungi.

Biologicals

For the determination of C. cuneifolium antimicrobial activity, the following microorganisms were used: MSSA (ATCC 29213), E. coli (ATCC 25922), C. albicans (ATCC 14053), MRSA (clinical strain), carbapenem-susceptible K. pneumoniae (clinical strain), carbapenem-resistant K. pneumoniae (clinical strain), and carbapenem-resistant A. baumannii (clinical strain). Until further analyses, bacteria were stored on a cryovial bead preservation system (Microbank, Pro-Lab Diagnostics) at −80°C.

Inoculum was prepared by spreading one cryovial bead on a blood agar plate and then incubation overnight at 37°C. One colony was resuspended in 5 ml tryptic soy broth (TSB) and incubated at 37°C without shaking. Overnight cultures were then adjusted to a turbidity of 0.5 McFarland, corresponding to ≈1 s× 10⁸ cfu/mL.

Instruments

A 20 L steel apparatus was used for steam distillation according to the method described in the next paragraph. For separation and identification of the constituents of the EO, a GC-MS/GC-FID TurboMass Clarus 500 from Perkin Elmer Instruments was used, characterized by a Stabilwax fused silica gel capillary column (Restek; 60 m long, 0.25 mm I.D., 0.25 μm film thickness). Helium was used as the carrier gas (1.0 mL/min) and the oven temperature was kept at 60°C for 5 min and raised up to 220°C with a speed of 5°C/min and kept constant at 220°C for 30 min. MS spectrometry was performed at 70 eV with a mass range from 30 to 350 m/z.

The main components of the EO were identified by comparison of their MS spectra with those present in the NIST and Wiley libraries. A second confirmation was achieved by calculating the GC retention indices (RI). The relative abundances of the separated compounds were achieved utilizing the same instrumentation with a FID detector.

Isolation of coristospermum cuneifolium essential oil

Similarly as previously reported [62] [63], dried plant material (200.0 g) was subjected to steam distillation. After 8 h, the accumulated oil/water double phase (800 mL) was extracted 3 times with 100 mL of diethyl ether. The unified organic layers were dried over anhydrous sodium sulfate (Na₂SO₄), filtered, and deprived of the solvent in vacuum to furnish oils (28.6 mg of CCEO, yield 0.0143%). The prepared oils were stored in tightly closed dark vials until further analysis.

Coristospermum cuneifolium essential oil antimicrobial activity evaluation

The CCEO sample was dissolved in a 25% DMSO water solution, with the highest tested concentration of 3.01 mg/mL. The used concentration of DMSO did not interfere with bacterial and fungal viability (data not shown).

Antimicrobial activity determination

Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined for each reference compound ([Table 2]) at high and low bacterial inoculum (~5×10⁵ and ~5×10³ cfu/mL, respectively). For CCEO, only the MBCs (at high and low inoculum) were evaluated, as the starting solution was opalescent. The decision to test both high and low inoculum was based on the likely bacterial amount in case of systemic and local infections, respectively ([Table 3]). Cation-adjusted Mueller Hinton (CAMHB) and Sabouraud broths were used for bacteria and fungi, respectively.

Briefly, two fold serial dilutions of CCEO and each antimicrobial agent were prepared in 2 mL Mueller Hinton broth (MHB) in borosilicate glass tubes and incubated for 18 h at 37°C. For antimicrobials agents, the MIC was defined as the lowest concentration of antibiotic that completely inhibited visible growth whereas MBC, for both references and CCEO, was defined as ≥99.9% (i. e., ≥3-log10 CFU/mL) reduction of the initial bacterial count at each time point [64]. Furthermore, CCEO antibacterial activity was assessed as the difference between the starting inoculum and the number of residual viable colonies after 24 h of incubation (expressed as Δlog10 cfu/mL) [65].

Conflicts of Interest

The authors declare no conflict of interest.

• References

• 1 Jiménez-Mejías P, Vargas P. Taxonomy of the tribe Apieae (Apiaceae) revisited as revealed by molecular phylogenies and morphological characters. Phytotaxa 2015; 212: 57-79
• 2 Banfi E, Galasso G, Soldano A. Notes on systematics and taxonomy for the Italian vascular flora. Atti Soc It Sci Nat Museo Civ Stor Nat Milano 2005; 146: 219-244
• 3 Pignatti S. Flora d’Italia. Vol. 2 Bologna, Italy: Edagricole; 1982: 227
• 4 Conti F, Abbate G, Alessandrini A, Blasi C. An annotated checklist of the Italian vascular flora. Roma: Palombi Editori; 2005: 428
• 5 López-Pujol J, Martinell MC, Massó S, Blanché C, Sáez L. The 'paradigm of extremes': Extremely low genetic diversity in an extremely narrow endemic species, Coristospermum huteri (Umbelliferae). Plant Syst Evol 2013; 299: 439-446
• 6 Castro M, Rosselló JA. Karyological observations on plant taxa endemic to the Balearic Islands. Bot J Linn Soc 2007; 153: 463-476
• 7 Menghini L, Epifano F, Genovese S, Marcotullio MC, Sosa S, Tubaro A. Antiinflammatory activity of coumarins from Ligusticum lucidum Mill. subsp. cuneifolium (Guss.) Tammaro (Apiaceae). Phytother Res 2010; 24: 1697-1699
• 8 Chen DR. Clinical and experimental study of Ligusticum wallichii and aspirin in the treatment of transient ischemic attack. Chin J Integr Trad West Med 1992; 12: 672-674
• 9 Ran X, Ma L, Peng C, Zhang H, Qin LP. Ligusticum chuanxiong Hort: A review of chemistry and pharmacology. Pharm Biol 2011; 49: 1180-1189
• 10 Sinclair S. Chinese herbs: A clinical review of Astragalus, Ligusticum, and Schizandrae. Altern Med Rev 1998; 3: 338-344
• 11 Jackson A. The cytotoxic effect of Oshá on human breast cancer cells, normal human blood cells, and embryonic mouse cells. McNair Schol J 2004; 14: 60-71
• 12 Zhang J, Zhou Z, Chen R, Xie F, Cheng G, Yu D, Zhou T. Study on chemistry and pharmacology of genus Ligusticum. Chin Pharmacol J 2002; 37: 654-657
• 13 Passreiter CM, Akhtar Y, Isman MB. Insecticidal activity of the essential oil of Ligusticum mutellina roots. Z Naturforsch C 2005; 60: 411-414
• 14 Jeong JB, Ju SY, Park JH, Lee JR, Yun KW, Kwon ST, Lim JH, Chung GY, Jeong HJ. Antioxidant activity in essential oils of Cnidium officinale makino and Ligusticum chuanxiong hort and their inhibitory effects on DNA damage and apoptosis induced by ultraviolet B in mammalian cell. Cancer Epidemiol 2009; 33: 41-46
• 15 Sim Y, Shin S. Combinatorial anti-trichophyton effects of Ligusticum chuanxiong essential oil components with antibiotics. Arch Pharm Res 2008; 31: 497-502
• 16 Cégiéla-Carlioz P, Bessière JM, David B, Mariotte AM, Gibbons S, Dijoux-Franca MG. Modulation of multi-drug resistance (MDR) in Staphylococcus aureus by Osha (Ligusticum porteri L., Apiaceae) essential oil compounds. Flavour Fragr J 2005; 20: 671-675
• 17 Wang J, Xu L, Yang L, Liu Z, Zhou L. Composition, antibacterial and antioxidant activities of essential oils from Ligusticum sinense and L. jeholense (Umbelliferae) from China. Rec Nat Prod 2011; 5: 314-318
• 18 Venditti A, Frezza C, Gatto Agostinelli V, Di Cecco M, Ciaschetti G, Serafini M, Bianco A. Study on the molecular composition of an indigenous Italian species: Coristospermum cuneifolium (Guss.) Bertol. Int J Indig Med Plants 2016; 48: 1930-1938
• 19 Sieniawska E, Losb R, Baja T, Malmb A, Glowniak K. Antimicrobial efficacy of Mutellina purpurea essential oil and α-pinene against Staphylococcus epidermidis grown in planktonic and biofilm cultures. Ind Crops Prod 2013; 51: 152-157
• 20 Collin G, Gagnon H, St-Gelais A, Turcotte M. Composition of the essential oil and the hydrosol of the roots of Ligusticum porteri. Am J Essent Oils Nat Prod 2014; 1: 4-10
• 21 Labed A, Labed I, Safaei-Ghomi J, Touzani R, Kabouche A, Kabouche Z. GC-MS analysis of Oenanthe virgata Poiret (Apiaceae) from Algeria. J Essent Oil Bear Pl 2011; 14: 481-483
• 22 Liu ZL, Chu SS. Jiang GH.Insecticidal activity and composition of essential oil of Ostericum sieboldii (Apiaceae) against Sitophilus zeamais and Tribolium castaneum. Rec Nat Prod 2010; 5: 74-81
• 23 Borg-Karlson K, Valterová I, Nilsson LA. Volatile compounds from flowers of six species in the family Apiaceae: Bouquets for different pollinators?. Phytochem 1993; 35: 111-119
• 24 Gibson RW, Pickett JA. Wild potato repels aphids by release of aphid alarm pheromone. Nature 1983; 302: 608-609
• 25 Martin S, Padilla E, Ocete MA, Galvez J, Jiménez J, Zarzuelo A. Anti-inflammatory activity of the essential oil of Bupleurum fruticescens. Planta Med 1993; 59: 533-536
• 26 Tambe Y, Tsujiuchi H, Honda G, Ikeshiro Y, Tanaka S. Gastric cytoprotection of the non-steroidal anti-inflammatory sesquiterpene, β-caryophyllene. Planta Med 1996; 62: 469-470
• 27 Ghelardini C, Galeotti N, Di Cesare Mannelli L. Mazzanti Bartolini A. Local anaesthetic activity of β-caryophyllene. Il Farmaco 2001; 56: 387-389
• 28 Bajpai VK, Shukla S, Kang SC. Chemical composition and antifungal activity of essential oil and various extract of Silene armeria L. Biores Technol 2008; 99: 8903-8908
• 29 Ding J, Xuejian Y, Yu W, Ding Z, Chen Z. Aromatic components of the essential oils of four Chinese medicinal plants (Asarum petelotii, Elsholtzia souliei, Eupatorium adenophorum, Micromeria biflora) in Yunnan. Z Naturforsch C 1994; 49: 703-706
• 30 Eadie MJ. Could valerian have been the first anticonvulsant?. Epilepsia 2004; 45: 1338-1343
• 31 Duran M, Wadman SK, Jakobs C, Schutgens RBH, Veder HA. Organic acid excretion in a patient with 3-hydroxy-3-methylglutaryl-Coa lyase deficiency: Facts and artefacts. Clin Chim Acta 1978; 90: 187-193
• 32 Maggi F, Cecchini C, Cresci A, Coman MM, Tirillini B, Sagratini G, Papa F. Chemical composition and antimicrobial activity of the essential oil from Ferula glauca L. (F. communis L. subsp. glauca) growing in Marche (central Italy). Fitoterapia 2008; 80: 68-72
• 33 Türkez H, Çelik K, Toğar B. Effects of copaene, a tricyclic sesquiterpene, on human lymphocytes cells in vitro. Cytotechnology 2014; 66: 597-603
• 34 Guillén MD, Manzanos MJ. Characteristics of smoke flavourings obtained from mixtures of oak (Quercus sp.) wood and aromatic plants (Thymus vulgaris L and Salvia lavandulifolia Vahl). Flavour Fragr J 2005; 20: 676-685
• 35 Tung YT, Chua MT, Wang SY, Chang ST. Anti-inflammation activities of essential oil and its constituents from indigenous cinnamon (Cinnamomum osmophloeum) twigs. Biores Technol 2007; 99: 3908-3913
• 36 Jun NJ, Mosaddik A, Moon JY, Jang KC, Lee DS, Ahn KS, Cho SK. Cytotoxic activity of β-Caryophyllene oxide isolated from Jeju Guava (Psidium cattleianum Sabine) leaf. Rec Nat Prod 2011; 5: 242-246
• 37 Palá-Paúl J, Brophy JJ, Pérez-Alonso MJ, Usano J, Soria SC. Essential oil composition of the different parts of Eryngium corniculatum Lam. (Apiaceae) from Spain. J Chromatogr 2007; 1175: 289-293
• 38 Chowdhury JU, Nandi NC, Yusuf M. Chemical constituents of essential oil of the leaves of Eryngium foetidum from Bangladesh. Bangladesh J Sci Ind Res 2007; 42: 347-352
• 39 Wang YT, Zhu L, Zeng D, Long W, Zhu SM. Chemical composition and anti-inflammatory activities of essential oil from Trachydium roylei. J Food Drug Anal 2016; 24: 602-609
• 40 Massumi MA, Fazeli MR, Alavi SHR, Ajani Y. Chemical constituents and antibacterial activity of essential oil of Prangos ferulacea (L.) Lindl. fruits. Iranian J Pharma Sci 2007; 3: 171-176
• 41 Şanli A, Karadoğan T. Geographical impact on essential oil composition of endemic Kundmannia Anatolica Hub. Mor. (Apiaceae). Afr J Trad Complement Altern Med 2017; 14: 131-137
• 42 Marčetić MD, Lakušić BS, Lakušić DV, Kovačevic N. Variability of the root essential oils of Seseli rigidum Waldst. Kit. (Apiaceae) from different populations in Serbia. Chem Biodivers 2013; 10: 1653-1666
• 43 Bougatsos C, Ngassapa O, Runyoro DKB, Chinou IB. Chemical composition and in vitro antimicrobial activity of the essential oils of two Helichrysum species from Tanzania. Z Naturforsch C 2004; 59: 368-372
• 44 Takahashi K, Someya T, Muraki S, Yoshida T. A new keto-alcohol, (−)-mintlactone, (+)-isomintlactone and minor components in peppermint oil. Agric Biol Chem 1980; 44: 1535-1543
• 45 Akhila A, Srivastava R, Rani K, Thakur RS. Biosynthesis of (-)-mintlactone and (+)-isomintlactone in Mentha piperita. Phytochem 1991; 30: 485-489
• 46 Frérot E, Bagnoud A, Vuilleumier C. Menthofurolactone: A new p-menthane lactone in Mentha piperita L.: Analysis, synthesis and olfactory properties. Flav Fragr J 2002; 17: 218-226
• 47 Sutour S, Tomi F, Bradesi P, Casanova J. Chemical composition of the essential oil from Corsican Mentha aquatic–combined analysis by GC(RI), GC-MS and 13C NMR spectroscopy. Nat Prod Comm 2011; 6: 1479-1482
• 48 Picard M, de Revel G, Marchand S. First identification of three p-menthane lactones and their potential precursor, menthofuran, in red wines. Food Chem 2017; 217: 294-302
• 49 Guth H. Identification of character impact odorants of different white wine varieties. J Agric Food Chem 1997; 45: 3022-3026
• 50 Näf R, Velluz A. Phenols and lactones in Italo-Mitcham peppermint oil Mentha × piperita L. Flav Fragr J 1998; 13: 203-208
• 51 Gaudin JM. Synthesis and organoleptic properties of p-menthane lactones. Tetrahedron 2000; 56: 4769-4776
• 52 Serra S, Fuganti C, Brenna E. Biocatalytic preparation of natural flavours and fragrances. Trends Biotechnol 2005; 23: 193-198
• 53 Carda M, Marco JA. Total synthesis of the monoterpenes (-)-mintlactone and (+)-isomintlactone. Tetrahedron 1992; 48: 9789-9800
• 54 Tanyeli C, Çalişkan Z, Demir AS. A facile synthesis of (±)-mintlactone. Synth Comm 1997; 27: 3471-3476
• 55 Ferraz HMC, Grazini MVA, Ribeiro CMR, Brocksom U, Brocksom TJ. A short route to (-)-mintlactone by thallium(III)-mediated cyclization of (-)-isopulegol. J Org Chem 2000; 65: 2606-2607
• 56 Gao P, Xu P, Zhai H. Expeditious construction of (+)-mintlactone via intramolecular hetero-Pauson-Khand reaction. J Org Chem 2009; 74: 2592-2593
• 57 Chu SS, Guo HJ, Zhi LL. Insecticidal components from the essential oil of Chinese medicinal herb, Ligusticum chuanxiong Hort. J Chem 2011; 8: 300-304
• 58 Nurzyńska-Wierdak R. Essential oil composition of the coriander (Coriandrum sativum L.) herb depending on the development stage. Acta Agrobot 2012; 66: 53-60
• 59 Sermakkani M, Thangapandian V. GC-MS analysis of Cassia italica leaf methanol extract. Asian J Pharma Clin Res 2012; 5: 90-94
• 60 Cool LG, Vermillion KE, Takeoka GR, Wonga RY. Irregular sesquiterpenoids from Ligusticum grayi roots. Phytochem 2010; 71: 1545-1557
• 61 Kaye KS, Pogue JM. Infections caused by resistant gram-negative bacteria: epidemiology and management. Pharmacotherapy 2015; 35: 949-962
• 62 Garzoli S, Pirolli A, Vavala E, Di Sotto A, Sartorelli G, Božović M, Angiolella L, Mazzanti G, Pepi F, Ragno R. Multidisciplinary approach to determine the optimal time and period for extracting the essential oil from Mentha suaveolens Ehrh. Molecules 2015; 20: 9640-9655
• 63 Božović M, Garzoli S, Pepi F, Baldisserotto A, Andreotti E, Romagnoli C, Mai A, Manfredini S, Ragno R. Essential oil extraction, chemical analysis and anti-candida activity of Calamintha nepeta (L.) Savi subsp. glandulosa (Req.) Ball – new approaches. Molecules 2017; 22: 203-214
• 64 Clinical and Laboratory Standards Institute . Performance standards for antimicrobial susceptibility testing: Fifteenth informational supplement M100-S15. Wayne, Pennsylvania: CLSI; 2005
• 65 Oliva A, Scorzolini L, Cipolla A, Mascellino MT, Cancelli F, Castaldi D, D'Abramo A, D'Agostino C, Russo G, Ciardi MR, Mastroianni CM, Vullo V. In vitro evaluation of different antimicrobial combinations against carbapenemase-producing Klebsiella pneumoniae: The activity of the double-carbapenem regimen is related to meropenem MIC value. J Antimicrob Chemother, advance online publication 2017; DOI: 10.1093/jac/dkx084.