Chemical Composition and Inhibitory Activity Against Helicobacter pylori of the Essential Oil of Apium nodiflorum (Apiaceae)

Abstract

The chemical composition of the essential oil obtained from Apium nodiflorum (L.) Lag. (Family Apiaceae), a plant used in the ethnomedical traditions of the Abruzzo region (Central Italy) as a culinary herb, as a diuretic, and to cure stomachache, was analyzed by gas chromatography/mass spectrometry, and 14 components were identified. Limonene (27.72 %), p-cymene (23.06%), myristicine (18.51%), and β-pinene (6.62%) were the main components. The antimicrobial activity of the essential oil was assayed in vitro against Helicobacter pylori (strain DSMZ 4867), resulting in a minimum inhibitory concentration value of 12.5 μg/mL.

Introduction

A pium nodiflorum (L.) Lag. (syn. Helosciadium nodiflorum Koch.) (Family Apiaceae), commonly known as “fool's watercress” but also known with several local common names such “sedanina d'acqua,” “gorgalestro,” “erba canella,” or “cannizzelle,” is a glaborous perennial herb growing up to 30 cm in height that is typically found in shallow streams, lakes, ponds, and marshes. It grows in all of the Mediterranean region. A. nodiflorum is used in the ethnomedical traditions of people from the Abruzzo region (Central Italy) as a culinary herb, mixed with fried garlic and Capsicum. Extracts from hypogenous parts are used as diuretics, and a decoction of leaves is used against stomachache.¹ A culinary use, for salad or boiled and preserved in olive oil, is reported also in other Italian regions and Mediterranean countries.² In Guarrera, traditional uses of the plant include in digestive disorders, in dysfunctions of the gastrointestinal or respiratory tract, as a depurative, for the treatment of cough and inflammation, and for postpartum disorders, in veterinary medicine.³ A. nodiflorum has been regarded also as an antipolluting plant being capable of removing several heavy metals from aqueous solutions.⁴ However, few data about the chemical composition and pharmacological activity of phytochemicals from A. nodiflorum have been reported to date in the literature. More data are available from ethnobotanical research, where some traditional uses, not experimentally verified, are reported.

Based on local ethnomedical traditions indicating A. nodiflorum as a food helpful in curing stomachache, in the present article we report the chemical composition and in vitro inhibitory activity against Helicobacter pylori of the essential oil produced by the aerial parts of A. nodiflorum collected in the Abruzzo region of Italy. The activity was investigated against H. pylori because the organism is well known as the main causal factor in the etiogenesis of many pathologies involving the gastric tract. It is noteworthy that all these data are reported herein for the first time.

Materials and Methods

Plant material

Aerial parts of A. nodiflorum were collected along the Tirino River around Bussi sul Tirino (in the province of Pescara, Abruzzo, Italy) in September 2006, and the sample was authenticated by one of the authors (L.M., Dipartimento di Scienze del Farmaco, Università “G. D'Annunzio” Chieti-Pescara, Chieti Scalo, Italy). Voucher specimens (number AN-2006) of this plant have been deposited at the herbarium of the “Giardino dei Semplici” of the University “G. D'Annunzio” of Chieti-Pescara.

Extraction of the essential oil

Freshly collected aerial parts of A. nodiflorum were subjected for 3 hours to steam/water distillation using a Clevenger apparatus to produce the essential oil in a yield of 0.08% (vol/wt). Oil was dried over anhydrous sodium sulfate and, after filtration, stored at 4°C until tested and analyzed.

Gas chromatography (GC)-mass spectrometry (MS) analysis

Oil was analyzed by GC and GC-MS. Analyses were performed on a Hewlett-Packard (Palo Alto, CA, USA) gas chromatograph (model 5890) equipped with a flame ionization detector and coupled to an electronic integrator. The chromatograph was fitted with a methyl silicone column (12.5 m × 0.25 mm, 0.25 μm film thickness). The carrier gas was helium (0.9 mL/minute); injector and detector temperatures were 280°C and 250°C, respectively. The oven temperature was programmed from 50°C to 270°C at a rate of 4°C/minute. Quantitative data were obtained by electronic integration of flame ionization detector area data without the use of response factor correction. GC-MS analyses were performed using a Hewlett-Packard model 6890 chromatograph combined with HP Chemstation software, equipped with a model 5973 mass selective detector, and the mass spectrometer was operated at 70 eV, a scanning speed of 1 s over the 40–300 atomic mass units range, and an ion source temperature of 180°C. Compounds were identified by comparison of GC retention indices relative to retention times of a series of n-alkanes (C₇–C₂₅) with those reported in the literature⁵ and by comparison of mass spectra from the NIST 98 Mass Spectral Database. GC analyses using a polar column were carried out on a Varian (Cernusco s/N [MI], Italy) model 3400 GC system equipped with a fused silica CP-Sil 88 column (50 mm × 0.22 mm, 0.2 μm film thickness) and with a nonpolar Rtx-5MS column. The carrier gas was helium (0.9 mL/minute). The oven temperature was programmed from 50°C to 270°C at a rate of 4°C/minute. Quantitative data were obtained by electronic integration of flame ionization detector area data without the use of response factor correction. Component identification was carried out by comparison of calculated retention indices with those reported in the literature.⁵

Antimicrobial activity

Strains of H. pylori (DSMZ 4867, originated from human gastric samples) were cultured on Columbia agar (Difco, Detroit, MI, USA) with 4% horse blood (Difco). Before tests, H. pylori plates were prepared by subculturing onto Mueller-Hinton agar supplemented with 5% defibrinated horse blood and incubated for 48 hours microaerobically. An evaluation of the activity of the essential oil with reference drugs was made by comparison of bacterial growth degree in each plate of H. pylori. The essential oil was dissolved in dimethyl sulfoxide. By serial double dilutions of antimicrobial agents, the oil was diluted in melted Mueller-Hinton agar supplemented with 5% defibrinated horse blood to give concentrations ranging from 200 to 0.25 μg/mL. They were inoculated with 5 μL of bacterial suspension (10⁷ colony-forming units/mL) and incubated at 37°C for 3 days under microaerobic conditions. An antimicrobial agent-free plate and plates with corresponding dilutions of dimethyl sulfoxide were used as negative controls to ensure bacterial viability and no contaminants in inocula. The minimum inhibitory concentration (MIC) tests were performed by the agar dilution method according to guidelines provided by the NCCLS.⁶ The MIC was defined as the lowest concentration capable of inhibiting bacterial colony formation.

Results and Discussion

In the essential oil of the aerial parts of A. nodiflorum, 14 compounds were identified by GC-MS analysis, mainly monoterpenes (Table 1).

Table 1.

Chemical Composition of the Essential Oil of A. nodiflorum

			Retention index (minutes)
Compound number	Name	Mean ± SE (%)	RI₁	RI₂
1	α-Pinene	1.21 ± 0.02	942	1,038
2	β-Pinene	6.62 ± 0.03	978	1,120
3	β-Myrcene	0.74 ± 0.05	986	1,166
4	Limonene	27.72 ± 0.06	1,005	1,187
5	cis-β-Ocimene	3.70 ± 0.05	1,013	1,228
6	p-Cymene	23.06 ± 0.01	1,016	1,273
7	trans-β-Ocimene	3.13 ± 0.05	1,038	1,250
8	Terpinolene	3.48 ± 0.03	1,062	1,289
9	Citronellal	0.74 ± 0.04	1,147	1,465
10	p-Cymenene	1.78 ± 0.01	1,277	1,468
11	Thymol	2.95 ± 0.03	1,279	1,640
12	Germacrene D	4.16 ± 0.02	1,468	1,712
13	δ-Cadinene	2.20 ± 0.03	1,506	1,784
14	Myristicine	18.51 ± 0.01	1,808	1,585

Values are averages of three determinations. RI₁, retention index calculated on the nonpolar Rtx-5MS column; RI₂, retention index calculated on the polar CP-Sil 88 column.

Limonene (27.72%), p-cymene (23.06%), myristicine (18.51%), and β-pinene (6.62%) represented the most abundant compounds. Whereas in Apium graveolens four relevant compounds were detected, namely, limonene, β-caryophyllene, 3-butyl-4,5-dihydrophthalide, and γ-terpinene, at 18.3%, 13.5%, 32.1%, and 4.3%, respectively, the pattern of main mono- and sesquiterpenes recorded in A. nodiflorum was substantially different, with limonene, p-cymene, and myristicine being the most abundant compounds.⁷ The chemical composition of essential oil can show huge differences, quantitatively rather than qualitatively, not only between different species, but also within a single species comparing different varieties.⁸ As noted previously, because A. nodiflorum is used for alimentary purposes, we decided to test the activity of the essential oil in vitro against H. pylori. This bacterium is now well known to be the main causal factor in the etiogenesis of chronic active or type B gastritis, peptic and duodenal ulcers, gastric carcinoma, and mucosa-associated lymphoid tumors. Pharmacological treatment of H. pylori infections includes administration of a proton pump inhibitor and a combination of two or more antibiotics, among which the most active ones are amoxicillin, clarithromycin, metronidazole, and tetracyclines. However, bacterial resistance to these antibiotics is a growing problem: for example, metronidazole is no longer effective in 10–50% of H. pylori-positive subjects, and also amoxicillin and clarithromycin resistance has increased in the last few years. For these reasons, the search for alternative therapeutic remedies for H. pylori infections is a field of current interest.⁹ The essential oil of A. nodiflorum was assayed as an inhibitory agent of growth of H. pylori in vitro using metronidazole, amoxicillin, tetracycline, and clarithromycin as reference drugs (Table 2).

Table 2.

In Vitro Inhibitory Activity Against Growth of H. pylori of the Essential Oil of A. nodiflorum

Sample	MIC (μg/mL)
Essential oil	12.50
Metronidazole	>200
Amoxicillin	0.75
Tetracycline	5.00
Clarithromycin	1.25

Values were obtained from three determinations.

As shown in Table 2, the essential oil of leaves of A. nodiflorum showed an appreciable activity against H. pylori with an MIC value of 12.5 μg/mL, although only slightly comparable to values obtained from three of the four more commonly used antibiotics in current therapy for infections caused by this bacterium. Data obtained by treating our H. pylori strain with metronidazole are not significant for a comparison to those obtained from the essential oil as this strain is clearly resistant to this antibiotic. However, data of Table 2 confirm that resistance of H. pylori strains of different origins to antibiotics has become nowadays quite a common phenomenon and that mixtures, like essential oils of secondary metabolites, extracted from alimentary plants, could be used as a valuable alternative to bypass this disadvantage.

In conclusion, data reported in our study showed that the essential oil obtained from leaves of A. nodiflorum exerted an appreciable inhibitory activity against H. pylori in vitro. It is noteworthy that this effect, together with the qualitative and quantitative analysis of its components, are reported herein for the first time and could account for the ethnomedical use of A. nodiflorum as a food able to cure stomachache and related diseases. Results obtained in this study prompted us to examine further the profile of secondary metabolites of A. nodiflorum, to perform tests in vivo using a suitable animal model, and to perform in vitro and in vivo tests aimed at evaluating the activity of the essential oil and other secondary metabolites of A. nodiflorum against strains of H. pylori isolated from clinical patients, and these investigations are underway in our laboratories.

Footnotes

Acknowledgment

The authors wish to acknowledge the financial support from Regione Abruzzo (L.R. 35/97) Project “Tutela della Biodiversita.”

Author Disclosure Statement

We thereby declare that the present manuscript does not create any conflict of interest with any commercial associations.

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