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Chemical composition and biological assays of essential oils of Calamintha nepeta (L.) Savi subsp. nepeta (Lamiaceae) a

a

a

a

B. Marongiu , A. Piras , S. Porcedda , D. Falconieri , A. b

c

c

Maxia , M.J. Gonçalves , C. Cavaleiro & L. Salgueiro

c

a

Dipartimento di Scienze Chimiche , Università degli Studi di Cagliari, Cittadella Universitaria di Monserrato , SS 554, Km 4.500, 09042 Cagliari, Italy b

CoSMeSe and Dipartimento di Scienze Botaniche , Università degli Studi di Cagliari , Viale Sant’Ignazio, I-09123, Cagliari, Italy c

Laboratorio de Farmacognosia, Facultade de Farmacia/CEF, Universidade de Coimbra , 3000, Coimbra, Portugal Published online: 27 Oct 2010.

To cite this article: B. Marongiu , A. Piras , S. Porcedda , D. Falconieri , A. Maxia , M.J. Gonçalves , C. Cavaleiro & L. Salgueiro (2010) Chemical composition and biological assays of essential oils of Calamintha nepeta (L.) Savi subsp. nepeta (Lamiaceae), Natural Product Research: Formerly Natural Product Letters, 24:18, 1734-1742, DOI: 10.1080/14786410903108944 To link to this article: http://dx.doi.org/10.1080/14786410903108944

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Natural Product Research Vol. 24, No. 18, 10 November 2010, 1734–1742

Chemical composition and biological assays of essential oils of Calamintha nepeta (L.) Savi subsp. nepeta (Lamiaceae)


B. Marongiua*, A. Pirasa, S. Porceddaa, D. Falconieria, A. Maxiab, M.J. Gonçalvesc, C. Cavaleiroc and L. Salgueiroc a Dipartimento di Scienze Chimiche, Universita` degli Studi di Cagliari, Cittadella Universitaria di Monserrato, SS 554, Km 4.500, 09042 Cagliari, Italy; bCoSMeSe and Dipartimento di Scienze Botaniche, Universita` degli Studi di Cagliari, Viale Sant’Ignazio, I-09123 Cagliari, Italy; cLaboratorio de Farmacognosia, Facultade de Farmacia/CEF, Universidade de Coimbra, 3000 Coimbra, Portugal

(Received 7 May 2009; final version received 10 June 2009) Aerial parts of wild Calamintha nepeta (L.) Savi subsp. nepeta growing spontaneously on the Mediterranean coast (Sardinia Island, Italy) and on the Atlantic coast (Portugal) were used as a matrix for the supercritical extraction of volatile oil with CO2. The collected extracts were analysed by GC-FID and GC-MS methods and their compositions were compared with that of the essential oil isolated by hydrodistillation, but the differences were not relevant. A strong chemical variability was observed in the essential oils depending on the origin of the samples. The results showed the presence of two chemotypes of C. nepeta. In all Italian samples, pulegone, piperitenone oxide and piperitenone were the main components (64.4– 39.9%; 2.5–19.1%; 6.4–7.7%); conversely, the oil extracted from Portuguese C. nepeta is predominantly composed of isomenthone (35.8– 51.3%), 1,8-cineole (21.1–21.4%) and trans-isopulegone (7.8–6.0%). The minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) were used to evaluate the antifungal activity of the oils against Candida albicans, Candida tropicalis, Candida krusei, Candida guillermondii, Candida parapsilosis, Cryptococcus neoformans, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, Epidermophyton floccosum, Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus. The Italian oil, rich in pulegone, exhibited significant antifungal activity against Aspergillus and dermatophyte strains, with MIC values of 0.32–1.25 mL mL1. Keywords: Calamintha nepeta; essential oil; supercritical carbon dioxide; antifungal activity

1. Introduction Calamintha nepeta (L.) Savi (Lamiaceae) is a perennial aromatic plant widespread in the Mediterranean region. This odorous herb is used as a mint-like spice in food

*Corresponding author. Email: [email protected]

ISSN 1478–6419 print/ISSN 1029–2349 online 2010 Taylor & Francis DOI: 10.1080/14786410903108944 http://www.informaworld.com


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preparations and in the composition of some recipes during religious feasts. It is known in folk medicine as a stimulant, antiseptic and antispasmodic. The essential oils of C. nepeta have been the subject of several studies (Ristorcelli, Tomi, & Casanova, 1966) and show great diversity in their chemical compositions. It was found that the essential oil of the plant possessed fungistatic and fungicidal properties against the Microsporum canis and Microsporum gypseum mycetes responsible for human cutaneous mycoses spread by domestic animals. Recently, it was demonstrated that pulegone is the constituent responsible for the antimicrobial activity (Flamini, Cioni, Puleio, Morelli, & Panizzi, 1999). In this article, we continue our studies on the extraction and characterisation of extracts by means of the supercritical CO2 and hydrodistillation (HD) of plants growing spontaneously in Sardinia and Portugal, as well as their antifungal activity. Supercritical fluid extraction (SFE) is an interesting technique for the extraction of compounds from vegetable material. The main advantage of SFE is the that solvent power and selectivity can be controlled, to some extent, by changing the pressure and temperature of the fluid. No studies have been found in the literature concerning the composition of the oil obtained from the supercritical CO2 extraction of aerial parts of Calamintha nepeta. Furthermore, to our knowledge, despite some work published on the antibacterial activity of C. nepeta oil, this is the first report of its antifungal activity against dermatophyte strains.

2. Results and discussion The volatile compounds in C. nepeta were analysed by GC-FID and GC-MS. The results are summarised in Table 1 according to their elution order on an SPB-1 column. A total of 41 compounds were identified which accounted for 82.3– 96.2% of the total composition. All the essential oils obtained by HD and SFE from the Italian C. nepeta were characterised by high contents of pulegone (64.4–39.9%), piperitenone (6.4–7.7%) and piperitenone oxide (PO) (2.5–19.1%). Other important compounds were limonene (4.8–2.8%), menthone (2.8–2.5%) and isomenthone (1.9–2.0%). The chemical composition of Portuguese C. nepeta is quite different from the Italian variety. Isomenthone (35.8–51.3%), 1,8-cineole (21.1–21.4%) and trans-isopulegone (7.8–6.0%) were the main components. Some other compounds include: neo-isoisopulegol (4.1–1.4%), neo-iso-menthol (3.1–3.9%), pulegone (2.7–2.4%) and -pinene (2.3–1.5%). Our results and the literature data clearly indicate the presence of a remarkable chemical polymorphism. Data reported earlier (Baldovini, Ristorcelli, Tomi, & Casanova, 2000) reveal that in Corsica (France), an important chemical variability has been observed for C. nepeta: the composition was observed to be independent of the geographical origin of the sample. Although all the essential oils are characterised by the predominance of monoterpenes bearing the p-menthane skeleton, three chemotypes can be distinguished. The first chemotype consists of menthone as the major component, associated with a wide range of pulegone, limonene and POs. The second chemotype was characterised by high content of piperitone oxide II and

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Table 1. Retention index (RI) and chromatographic area percentages of compounds found in C. nepeta essential oil.


RI (SPB-1)

RI (SuperW-10)

1

2

4

0.7 0.1 0.3. 0.7 2.2 0.1 0.2 0.3 4.8 0.2 – – 0.1

– – – – 1.9 – 0.3 0.4 2.8 – – – –

932 945 966 972 980 983 1014 1023 1023 1027 1037 1049 1053

1126 1117 1385 1161 1272 1213 1205 1232 1248 1248 1461

1076 1084

1286 1540

0.1 0.1

– 0.1

– –

– –

1084 1132 1144

1539 1461 1557

0.2 0.8 0.3

0.16 0.8 –

0.7 2.8 –

0.4 2.5 –

1141 1149 1149 1151 1151 1156 1161 1161 1168 1172 1172 1216 1225

1488 1578 1567 1594 1662 1631 1594 1600 1620 1690 1657 1642 1720

1.9 0.4 0.5 – – – 0.4 – – 0.3 – 64.4 0.4

2.0 0.3 0.5 – – 0.3 0.2 – – – – 39.9 2.2

1225 1228

1716 1701

0.1 –

– –

0.5 0.2

1.0 –

1254 1308 1330 1413 1471 1562

1907 1943 1594 1701 1967

– – – 0.4 0.3 –

– – – 0.2 – 0.6

0.3 6.4 2.5 0.2 0.1 0.2

– 7.7 19.1 – – 0.8

89.1 96.2 2.9 3.4

91.6 3.0

82.3 3.8

Total identified Percent yield

1.1 0.6 0.1 0.1 1.4 1.1 2.3 1.5 0.2 0.2 1.2 0.5 0.1 – 21.1 21.4 1.6 1.1 0.5 – 0.1 – 0.1 – 0.1 0.4

3

35.8 51.3 1.4 0.6 7.8 6.0 0.7 – 0.8 1.1 – – – – 4.1 1.4 3.1 3.9 0.6 0.9 0.2 – 2.7 2.4 – –

Compound -Pinene Camphene Sabinene -Pinene 3-Octanol Myrcene p-Cymene 1,8-Cineole Limonene Z- -Ocimene E- -Ocimene

-Terpinene trans-Sabinene hydrate Terpinolene cis-Sabinene hydrate Linalool Menthone neo-Isopulegol þ iso-Isopulegol Isomenthone cis-Isopulegone trans-Isopulegone neo-Menthol -Terpineol Menthol Terpinene-4-ol neo-iso-Isopulegol neo-iso-Menthol -Terpineol iso-Menthol Pulegone cis-Piperitone oxide Piperitone trans-Piperitone oxide Isopulegyl acetate Piperitenone Piperitenone oxide E-Caryophyllene Germacrene D Caryophyllene oxide

Identificationa MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,

IR, IR, IR, IR, IR, IR, IR, IR, IR, IR IR IR, IR

Inj Inj Inj Inj Inj Inj Inj Inj Inj Inj

MS, IR, Inj MS, IR MS, IR, Inj MS, IR, Inj MS, IR MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,

IR IR IR IR IR IR IR, Inj IR IR IR, Inj IR IR, Inj IR

MS, IR MS, IR MS, MS, MS, MS, MS, MS,

IR IR IR IR, Inj IR IR, Inj

Notes: Compounds are listed in order to their elution on the SPB-1 column: 1 – HD Portugal; 2 – SFE Portugal; 3 – HD Italy; 4 – SFE Italy. aIdentification has been realised by comparing mass spectra (MS), retention indices (IR) and injection of authentic compound (Inj).



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piperitone oxide. The third chemotype was characterised by a strong predominance of pulegone over the menthone. Flamini et al. (1999) reported the chemical composition of the essential oil of C. nepeta collected in Tuscany (Italy). The main constituent was pulegone (about 50%); menthone (9.4%), limonene (7.0%), menthol (4.6%), PO (4.6%) and piperitone oxide (3.9%) were also present. Similar results have been obtained by Panizzi, Flamini, Cioni, and Morelli (1993): pulegone was the main constituent of the essential oil from C. nepeta gathered in Tuscany. Sarer and Pancali (1998) investigated the essential oil of C. nepeta (L.) Savi subsp. glandulosa collected in Turkey; the major components of the oil were pulegone (40.5%) and menthone (23.6%). The components piperitone and piperitone oxide in the oil were 9.3%. Souleles, Argyriadou, and Philianos (1987) reported pulegone as the main compound found in the oil of C. nepeta from Greece, and the presence of menthone, isomenthone and piperitone shows that the biogenesis of the therpenes followed the pulegone pathway. In conclusion, our results reinforce previous data on the variability of the essential oils, depending on the origin of the samples. In the essential oil of C. nepeta that grows wild in Sardinia, pulegone is predominantly associated with PO and piperitenone. From these data, it can be seen that the chemical composition of the Sardinian oil is very similar to the oils obtained from other C. nepeta growing in Corsica. The chemical composition of the Portuguese oil is peculiar and rather different from those of other origins; in fact, it would appear that it is a new oil type, characterised by high amounts of isomenthone and 1,8-cineole and by low amounts of pulegone. Considering the extraction techniques, the composition of the Portuguese volatile concentrate obtained by SFE is similar to the composition of essential oil (HD), differing only in the higher percentage of isomenthone (51.3 vs. 35.8%, HD); the SFE extract from Sardinian C. nepeta is different from the essential oil obtained by HD, which is richer in PO (19.1 vs. 2.5%, HD) and poorer in pulegone (39.9 vs. 64.4%, HD). Indeed, PO being a moderately polar compound, its solubility in water is not negligible (Sousa, Magalhaes, Lima, Oliveira, & Leal-Cardoso, 1997). So PO is preferentially dissolved in water and therefore not fully recovered in the oil. As a counterbalance, the HD oil contains higher percentages of pulegone and therefore these phenomena determine the production of an oil whose components are not in their original proportion, and have, as a consequence, an altered aroma with respect to the starting material. Evaluation of MIC and MLC showed that the Italian oil is more active than the Portuguese oil, with MIC values ranging from 0.32 to 1.2 mL mL1 (Table 2). For Candida and dermatophyte strains, MIC values are similar to MLC values, indicating a fungicidial activity of the oil. The highest antifungal activity of the Sardinian oil can be associated with the contribution of pulegone. Other oils rich in pulegone, like Mentha pulegium and Mentha cervina, also present significant antifungal activity (Gonçalves, Vicente, Cavaleiro, & Salgueiro, 2007). The Italian oil, with a high amount of pulegone (64.4%), could be used for therapeutical purposes, particularly in the treatment of dermatophytosis and aspergillosis.

2.5 2.5 2.5 2.5 2.5 1.25 5 2.5 2.5–5 5 2.5 5–10 5 10

Candida albicans ATCC 10231 Candida tropicalis ATCC 13803 Candida krusei H9 Candida guillermondii MAT23 Candida parapsilosis ATCC 90018 Cryptococcus neoformans CECT 1078 Trichophyton mentagrophytes FF7 Microsporum canis FF1 Trichophyton rubrum CECT 2794 Microsporum gypseum CECT 2905 Epidermophyton floccosum FF9 Aspergillus niger ATCC 16404 Aspergillus fumigatus ATCC 46645 Aspergillus flavus F44

2.5 2.5 2.5 2.5 5 1.25 5 2.5 2.5–5 5 2.5 420 10 20

MLCa 1.25 1.25 1.25 1.25 1.25 0.32–0.64 0.64 0.64 0.64 1.25 0.64 0.32 0.64 1.25

MICa

Italy 3

1.25 1.25 1.25 1.25 2.5 1.25 1.25 0.64 1.25 1.25 0.64 5 2.5 2.5

MLCa 1 4 64 8 51 16 16–32 128 16 128 16 N.T N.T N.T

MIC 4128 4128 64–128 8 51 128 32–64 128 64 4128 16 N.T N.T N.T

MLC

Fluconazole

MLCb N.T N.T N.T N.T N.T N.T N.T N.T N.T N.T N.T 4 4 8

MICb N.Tc N.T N.T N.T N.T N.T N.T N.T N.T N.T N.T 1–2 2 2

Amphotericin B

Notes: Results were obtained from three independent experiments performed in duplicate. aMIC and MLC were determined by a macrodilution method and expressed in mL mL1 (V/V). bMIC and MLC were determined by a macrodilution method and expressed in mg mL1 (W/V). cNot tested.

MICa

Strains

Portugal 1

Table 2. Antifungal activity (MIC and MLC) of C. nepeta oils for yeasts, dermatophyte and Aspergillus strains.


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3. Experimental 3.1. Materials Aerial parts of C. nepeta (L.) Savi subsp. nepeta were collected at the flowering stage of the plant from two different sites: Vilarinho de Baixo (Coimbra, Portugal) and Baunei (Sardinia, Italy). The plant was identified by Dr Andrea Maxia, Dipartimento di Scienze Botaniche, Universita` di Cagliari, and a voucher specimen was deposited in the Herbarium of Cagliari, Sardinia, with the number 1182. Aerial parts of C. nepeta were air-dried in a hot air-drier at 40 C with forced ventilation for two days. Before utilisation, the vegetable matter was ground with a Malavasi mill (Bologna, Italy), taking care to avoid overheating. The aerial parts were subjected to two different extraction methods: HD (samples 1 and 3) and SFE (samples 2 and 4). CO2 (purity 99%) was supplied by Air Liquid Italia (Cagliari, Italy). All solvents used, of the highest purity available, were purchased from Sigma– Aldrich (Milan, Italy). Camphene, sabinene, -pinene, limonene, -terpinene, linalool, menthone, terpinen-4-ol, -terpineol, (E)-caryophyllene, caryophyllene oxide and amphotericin B were obtained from Sigma–Aldrich (Milan, Italy). -Pinene, 3-octanol, myrcene, p-cymene, 1,8-cineole terpinolene and pulegone were obtained from Extrasynthese (Genay, France). Fluconazole was purchased from Pfizer (New York, USA).

3.2. SFE apparatus Supercritical CO2 extractions were performed in a laboratory apparatus, equipped with a 320 cm3 extraction vessel and two separator vessels of 300 and 200 cm3, respectively, connected in series. Experiments were carried out at different conditions in the extraction section. In the first separator, the temperature was set at 10 C and the pressure at the same value as the extraction section. The second separator was set at 15 bar and 10 C. Extraction was carried out in a semi-batch mode: batch charging of vegetable matter and continuous flow solvent. About 180 g of material were charged in each run.

3.3. Hydrodistillation Hydrodistillation was performed for 3 h in a circulatory Clevenger-type apparatus up to the exhaustion of the oil contained in the matrix, according to the procedure described in the European Pharmacopoeia (Council of Europe, 1997).

3.4. GC and GC/MS analysis Analysis of the volatile oil was carried out by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS). Analytical GC was carried out in a Hewlett Packard 6890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph with the HP GC ChemStation Rev. A.05.04 data handling system, equipped with a single injector and two flame ionisation detectors (FIDs). A Graphpak divider (Agilent Technologies, Part Number 5021-7148) was used for simultaneous sampling


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to two Supelco (Supelco Inc., Bellefont, PA, USA) fused silica capillary columns with different stationary phases: SPB-1 (polydimethylsiloxane 30 m 0.20 mm i.d., film thickness 0.20 mm), and SupelcoWax 10 (polyethyleneglycol 30 m 0.20 mm i.d., film thickness 0.20 mm). The oven temperature was settled at 70 C, raising at 3 C min1 to 220 C and then held for 15 min at 220 C; injector temperature: 250 C; carrier gas: helium, adjusted to a linear velocity of 30 cm s1; splitting ratio 1 : 40; detector temperature: 250 C. GC-MS analyses were carried out in a Hewlett Packard 6890 gas chromatograph fitted with a HP1 fused silica column (polydimethylsiloxane 30 m 0.25 mm i.d., film thickness 0.25 mm), interfaced with a Hewlett Packard mass selective detector 5973 (Agilent Technologies) operated by HP Enhanced ChemStation software, Version A.03.00. GC parameters are set as given above; interface temperature: 250 C; MS source temperature: 230 C; MS quadrupole temperature: 150 C; ionisation energy: 70 eV; ionisation current: 60 mA; mass range: 35–350 m; scan rate: 4.5 scans s1. The identity of the components was assigned by the comparison of mass spectra and retention indices for two different chromatographic stationary phases calculated by linear interpolation to the retention of a series of n-alkanes. Experimental data were compared with corresponding data of reference oils and commercially available standards banked at a home-made library or from literature data (Adams, 2004; Joulain & Konig, 1998). Percentages of individual components were calculated based on GC peak areas without FID response factor correction.

3.5. Antifungal strains Antifungal activity of two oils obtained from the plants collected in Portugal (sample 1) and in Italy (sample 3) were evaluated against yeasts, Aspergillus and dermatophyte strains: two clinical Candida strains were isolated from recurrent cases of vulvovaginal candidosis (C. krusei H9, C. guillermondii MAT23), three types of strains from the American Type Culture Collection (Candida albicans ATCC 10231, C. tropicalis ATCC 13803, C. parapsilosis ATCC 90018) and one type of strain from the Coleccioń Espanõla de Cultivos Tipo (Cryptococcus neoformans CECT 1078); three dermatophyte clinical strains were isolated from nails and skin (Epidermophyton floccosum FF9, Trichophyton mentagrophytes FF7, Microsporum canis FF1) and two types of strains from the Coleccioń Espanõla de Cultivos Tipo (Trichophyton rubrum CECT 2794, M. gypseum CECT 2908); and one Aspergillus clinical strain was isolated from bronchial secretions (A. flavus F44) and two types of strains from the American Type Culture Collection (Aspergillus niger ATCC 16404, A. fumigatus ATCC 46645). The fungal isolates were identified by standard microbiology methods and stored on Sabouraud broth with glycerol at 70 C. Prior to antifungal susceptibility testing, each isolate was inoculated on Sabouraud agar to ensure optimal growth characteristics and purity.

3.6. Antifungal activity A macrodilution broth method was used to determine the minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC), according to



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NCCLS references M27-A2 (National Committee for Clinical Laboratory Standards, 2002a) and M38-A (National Committee for Clinical Laboratory Standards, 2002b) for yeasts and filamentous fungi, respectively. The serial doubling dilution of each oil was prepared in dimethyl sulphoxide (DMSO), with concentrations ranging from 0.16 to 20 mL mL1. The final concentration of DMSO never exceeded 2%. Recent cultures of each strain were used to prepare the cell suspension, adjusted to 1–2 103 cells mL1 for yeasts and 1– 2 104 cells mL1 for filamentous fungi. The concentration of cells was confirmed by viable count on Sabouraud agar. The test tubes were incubated aerobically at 35 C for 48 h/72 h (Candida spp. and Aspergillus spp./Cryptococcus neoformans) and at 30 C for seven days (dermatophytes), and MICs were determined. To evaluate MLC, aliquots (20 mL) of broth were taken from each negative tube after MIC reading, and cultured in Sabouraud dextrose agar plates. Plates were then incubated for 48 h at 35 C (Candida spp. and Aspergillus spp.), 72 h for Cryptococcus neoformans and seven days at 30 C (dermatophytes). In addition, two reference antifungal compounds, amphotericin B (Fluka) and fluconazole (Pfizer), were used to control the sensitivity of the tested microorganisms. All tests were performed in RPMI medium. For each strain tested, the growing conditions and the sterility of the medium were checked in two control tubes. The innocuity of the DMSO was also checked at the highest tested concentration. All experiments were performed in triplicate and repeated if the results differed.

Acknowledgements This work was supported by funds from the Progetto PON 1.4L-2006-16 ‘Naturalmente Sardegna’. We are grateful to Ca´tia Ineˆs Santos (Faculdade de Farmaćia, Coimbra) for her collaboration in the biological experiments.

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National Committee for Clinical Laboratory Standards. (2002b). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved Standard M38-A, Wayne, PA, USA. Panizzi, L., Flamini, G., Cioni, P.L., & Morelli, I. (1993). Composition and antimicrobial properties of essential oil of four Mediterranean Lamiaceae. Journal of Ethopharmacology, 39, 167–170. Ristorcelli, D., Tomi, F., & Casanova, J. (1996). Essential oils of Calamintha nepeta subsp. nepeta and subsp. glandulosa from Corsica (France). The Journal of Essential Oil Research, 8, 363–366. Sarer, E., & Pancali, S.S. (1998). Composition of the essential oil from Calamintha nepeta (L.) Savi ssp. glandulosa (Req.) P.W. Ball. Flavour and Fragrance Journal, 13, 31–32. Souleles, C., Argyriadou, N., & Philianos, S. (1987). Constituents of the essential oil of Calamintha nepeta. Journal of Natural Products, 50, 510–522. Sousa, P.J.C., Magalhaes, P.J.C., Lima, C.C., Oliveira, V.S., & Leal-Cardoso, J.H. (1997). Effects of the piperitenone oxide on the intestinal smooth muscle of the guinea pig. Brazilian Journal of Medical and Biological Research, 30, 787–791.