Introduction

Throughout the world, the life expectancy of the human species continues to increase; the desire to satisfy its needs and those of future generations allows it to surely strive to discover the extraordinary beneficial virtues of aromatic and medicinal plants. Nowadays, the majority of the world population is turning to traditional medicine and pharmacopoeia and thus to the use of plants and plant extracts to deal with health problems. Indeed, aromatic and medicinal plants are very valuable source for the production of new, precious and miraculous chemical molecules. These molecules are often likened to active ingredients with a specific properties that give them a unique character. Today, no one ignores the importance of the plants and the discovery of natural products.

Mentha pulegium L. is a species of the family Lamiaceae and of the genus Mentha (Quezel et Santa 1962; Guignard et Dupont 2004). Pennyroyal (Mentha pulegium L.) is one of the most important aromatic and medicinal plants most used in traditional medicine and is marketed in Morocco as an essential oil, and its production fluctuates very dramatically from year to year (Direction de la protection de végétaux-Rabat 1999). According to several researches conducted, the flowering aerial parts of Pennyroyal are frequently used for their antimicrobial properties to treat colds, sore throats, coughs, hoarseness, bronchitis, lung infections and chills of all kinds; cholera, food poisoning, and tuberculosis. It also plays as an excellent digestive material (Zargari 1990; Bellakhdar 1997; Chraibi et al. 2018; Lahsissene et Kahouadji 2010). It is recognized as stimulating and exciting chemical for the nervous system (Aid et al. 2003). The dried leaves of this species are rolled into cigarettes and smoked to relieve asthma (Salhi et al. 2010). Furthermore, it is also used as an anti-flatulent, carminative, expectorant, diuretic, antitussive and menstruation agent (Newall 1996). Several publications describe the chemical composition of the essential oil of the aerial part of pennyroyal in which the authors report that the essential oil of this plant is characterized by the majority presence of ketones, although it is rich in oxygenated monoterpenes. Indeed, the described compositions are dominated either by the pulegone; menthone; piperitone; piperitenone; or sometimes by neo-menthol or menthol (El Arch et al. 2003; Silva et al. 2015; Ouakouaket al. 2015; Zantar et al. 2015; Mohammadhosseini et al. 2021; Mollaeia et al. 2021; Teixeira et al. 2012; Sarikurkcu et al. 2012). Within the framework of the valorization of aromatic and medicinal plants of Morocco, the present study aims to identify the chemical composition of the essential oil of pennyroyal and to isolate its majority compounds.

Material and methods

Plant material

The plant material consists of the flowering tops of Mentha pulegium L. harvested from M'Rirt (Middle-Atlas, Morocco) in the flowering month of July 2017. The aerial part of the plant has been dried away from the light and humidity at room temperature. This plant under study was identified at the Botany and Plant Ecology Laboratory of the Scientific Institute of Rabat (Morocco).

Extraction of the essential oil

The extraction of the essential oil was done by hydrodistillation in a Clevenger type device. This extraction was repeated three times in order to confirm the yield obtained by the used mode. The essential oil collected at the end of the distillation, measured in mL per 100 g of the dry plant, was introduced into a hermetically sealed dark glass bottle to preserve it from heat and light (Afnor 1996), then kept in the refrigerator at a temperature of 4 °C.

Analysis and identification of the chemical composition of the EO by GC and GC/MS

The chromatographic analysis of the EO from the aerial part of Mentha pulegium L. was performed on a gas phase chromatography of the Thermo Electron type (Trace GC Ultra) coupled to a mass spectrometer of the Thermo Electron Trace MS system type (Thermo Electron: Trace GC Ultra; Polaris Q MS), the fragmentation is performed by an electron impact with an intensity of 70 eV. The chromatography is equipped with a DB-5 type column (5% phenyl-methyl-siloxane) (30 m × 0.25 mm × 0.25 μm film thickness), a flame ionization detector (FID) powered by an H2/Air gas mixture. The temperature of the column is programmed at the rate of a rise of 4 °C/min from 50 to 200 °C during 5 min. The injection mode is split (leakage ratio: 1/70, flow rate ml/min) and the used vector gas is the nitrogen with a flow rate of 1 ml/min. The identification of the chemical composition of the EO of Mentha pulegium L. was performed based on the comparison of their kovats indices (KI) and Adams with those of the reference products known in the literature (Kovats 1965; Adams 2007). It was supplemented by a comparison of indices and mass spectra with different references (Adams 2007). Kovats indices compare the retention time of any product with that of a linear alkane of the same carbon number. They are determined by injecting a mixture of alkanes (standard C7-C40) under the same operating conditions.

Fractionation of the essential oil of pennyroyal by chromatography in liquid phase at low atmospheric pressure

In this present study, we were interested in fractionating the components of the essential oil extracted from M. pulegium consisting of its main compounds: pulegone (71.97%) and piperitenone (26.04%). After the glass column was filled and stacked with a silica-eluent mixture, 3 g of the essential oil was weighed, diluted in ether and then gently added to the silica column by agitating it against the walls. The essential oil was then gently incorporated into the silica column until it reaches the upper limit of the column. Then, the eluent slided over the walls of the latter in order to cover the silica grains. A reservoir filled with the eluting solvent was finally placed above the column in order to feed (alimenter) it gradually during the experiment. The polarity gradient of the solvent in the reservoir gradually increased from a hexane-ether solvent (95/5) to a final hexane-ether solvent (90/10). The eluted products were collected progressively in numbered glass tubes. The plates of the chromatography on thin layer (TLC) plates with a migration solvent formed from hexane-ether (90/10) were used at each step for monitoring and controlling the purifications. At the end of the experiment, the column was washed with an increasingly high polarity solvent until the hexane-ether composition is reached (50/50). Due to the discoveries revealed by TLC plates, the identical compounds were grouped together to form the preponderant fractions of the essential oil containing the eluent. After the elimination of the eluting solvent by rotary evaporator, the obtained various fractions were weighed and stored away from light at temperature below 4 °C. This step permitted to collect two oxygenated fractions F1 and F2. The step of the fractionation of the EO is shown in Fig. 1. The molecular structures of the two fractions will be identified and confirmed later using different chromatographic and spectroscopic methods.

Fig. 1
figure 1

Scheme of fractionation of the essential oil of M. pulegium

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Spectroscopic analyzes by nuclear magnetic resonance (1H and 13C)

Due to the analysis of the fractions obtained by GC/MS, we have used the methods of proton nuclear magnetic resonance (1H NMR) and carbon 13 (13C NMR) to confirm the identities of the compounds and lead to their exact structural identifications. The fractions F1 and F2 of the EO of M. pulegium obtained by LPC, have checked their purity by GC and GC/MS, were then analyzed by proton and carbon 13 Nuclear Magnetic Resonance (1H NMR and 13C NMR) in order to confirm their structural identities. The 1H NMR and 13C NMR spectra were recorded on a nuclear magnetic resonance spectrometer of the «AVANCE 300 (MHz) type from BRUKER». The chemical shifts δ are given relative to the reference (TMS) universally accepted by 1H NMR and 13C NMR. The used solvent is the deuterated chloroform (CDCl3).

Results and discussion

Yield and chemical composition of the essential oil of Mentha pulegium

The average yield of the EO extracted from M. pulegium is around 5.2 ± 0.25. The identification of the volatile constituents of the EO of M. pulegium was performed by gas chromatography and gas chromatography coupled with mass spectrometry (GC/MS). The relative chromatogram for this analysis is shown in Fig. 2.

Fig. 2
figure 2

Chromatogram of the essential oil of M. pulegium L.

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Twelve chemical substituents have been identified in the EO of M. pulegium (Table 1). The results presented in Table 1 indicate that the essential oil of M. pulegium is dominated by monoterpenes (99.66%), with a preponderance of oxygenated compounds (99.39%) and marked by high percentages of pulegone (71.97%) and piperitenone (26.04%). The hydrocarbon monoterpenes (0.27%) and sesquiterpenes are in the minority in this essence (0.12%).

Table 1 Chemical composition of the EO of M. pulegium
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Liquid chromatographic analysis of the fractionated essential oil of M. pulegium

The fractionation of 3 g of the essential oil of M. pulegium by the liquid phase chromatography method (LPC) on silica gel made possible to obtain two fractions F1 and F2, representing 54.33% (1.63 g) and 32.67% (0.98 g), respectively of the total essential oil. The TLC analysis made it possible to check the purity of the two fractions of the crude EO, and to compare the migration of the sample of the commercial pulegone to that fractionated by LPC. We have found that the separation of the two fractions is perfect and that the degree of the purity of our sample (pulegone) is better than that of the commercial pulegone.

The more precise study of the chemical composition of the two fractions required other spectroscopic and analytical analyses in order to rigorously give the structure of each compound. These are the analyses: GC, GC/SM, RMN 1H, RMN 13C and DEPT.

Fraction analysis F1 and F2 of the EO by GC and GC/SM

According to the fractionation of the essential oil by chromatography on an open column of silica, both fractions were analyzed by GC and GC/MS. The chromatographic spectra (GC) of EO and its fractions are shown in Fig. 3.

Fig. 3
figure 3

Chromatograms of the EO of M. pulegium and its fractions (F1, F2)

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Analysis of the fraction F1

The analysis of the fraction F1 made it possible to identify a total of 55 compounds (Table 2), by comparing the retention indices in GC and the mass spectra in GC/MS with those of the reference products known in the literature. These compounds are distributed as follows:

  • Four compounds previously identified in the crude essential oil are: α-pinene, β-pinene, 1,8-cineole and the pulegone.

  • 51 newly identified compounds are: 3-methyl-2-buten-1-ol, 5-methylene-2-norbornene, santolinatriene, 2E, 4E hexadienol, α-thujene, sabinene, p-cymene, limonene, linalool, myrcenol, α-campholenal, thymol, methyl ether, menthofuran, borneol, cyclopent-2-enone, myrtenal, trans-4-caranone, p-cymen-9-ol, 2-methoxy thiophenol, trans-cyclocitral, carvotanacetone, piperitone, cis-piperitone epoxide, trans-piperitone epoxide, carvenone, Perilla aldehyde, p-menth-1-en-7-al, 2-ethyl-endo-fenchol, 3-oxo-p-menth-1-en-7-al, transdimethoxy citral, mentho thiophene, dihydroisojasmatone, piperitenone, 4aα,7α, 7aβ-nepetalactone, piperitenone oxide, trans-mentholactone, carvacrol acetate, trans-p-mentha-8-thiol-3-one, dihydrojasmone, 8-epi-dictaminol, 2E, 6Z nonadienal diethylacetal, 4a-α,7-β, 7a-α-nepetalactone, Cis-jasmone, methyl perillate, (2E,3Z)-2-ethylidene-6-methyl-3,5-heptadienal, (Z)-isoeugenol, 2 (3H)-furanone, dihydro-5,5-dimethyl-4-(3-oxobutyl), α-thujaplicin, cis-caryophellene, tans-caryophyllene, and 4,8-β-epoxy caryophellene.

  • Seven compounds of the parent essential oil are absent from this fraction: trans-p-menth-2-en-1-ol, cis-chrysanthenol, α–terpineol, trans-pulegol, thymol, α-guaiene and 11- α H-himachal -4-en-1- β-ol.

Table 2 Chemical compounds identified in the fraction F1 of the EO of M. pulegium
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Thus, the main constituent of the fraction F1 is the pulegone which presented the highest percentage (77.92%). This compound was also predominant in the essential oil of M. pulegium (71.97%). The other six compounds with a percentage greater than 1% are: Trans-cyclocitral (2.15%); Cis-epoxide of piperitenone (1.24%); p-menth-1-en-7-al (5.28%); dihydro-isojasmatone (1.20%), piperitenone oxide (2.73%) and menthalactone (1.60%). For other 48 compounds (remaining) of the fraction F1, they are given with a percentages lower than 1%.

Analysis of the fraction F2

The chromatographic analysis of fraction F2 revealed 51 chemical compounds (Table 3). In this fraction, three chemical compounds are initially identified in the essential oil «mother»: pulegone, trans-p-menth-2-en-ol and piperitenone. This latter constitutes a majority compound of this fraction with a very high percentage of 84.72%, and it represents a percentage of 26.04% in the parent EO. Other 48 constituents are newly identified compounds. All of these 48 compounds have an abundance less than 1%, except for three compounds which have a percentage greater than 1%. It is artemisia ketone (1.09%); p-menth-3-en-8-ol (1.58%) and perilla aldehyde (2.31%).

Table 3 Chemical compounds identified in the fraction F2 of the EO of M. pulegium
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Discussion of the results of the two fractions F1 and F2

The seven chemical compounds: 2E, 4E-hexadienol, linalool, borneol, pulegone, piperitenone, piperitenone oxide and carvacrol acetate were present in both the fractions F1 and in F2 with different proportions. These results show that fractionation of the essential oil by liquid chromatography on silica gel is an effective method that effeciently separates the majority of compounds and reveals new compounds of the essential oil. However, it is necessary to optimize the parameters to obtain even better results. These parameters are, among others, the length and width of the column, the elution flow rate, the progressive variations of the level of polarity of the eluent, the size of the silica beads and the elution time. According to the chromatographic results obtained above, we noted that the majority compound (pulegone) of the fraction F1 contains 77.92% of its total chemical composition; while it represents only 71.97% of all constituents of the EO of M. pulegium. Concerning the fraction F2, of which the piperitenone contains 84.72% of the totality of its chemical composition, it only represents 26.04% of the totality of the constituents of the same EO. Then, we can conclude that the fractionation of the EO by chromatography on a silica column made it possible to highlight a number of compounds greater than the number highlighted in the starting essential oil. This number is explained by their presence in very low percentages. The fractionation also permits separating and concentrating the major compounds of the essential oil. The EO of M. pulegium initially studied contained 12 identified compounds, while the fraction F1 contains 55 compounds and the fraction F2 contains 51 identified compounds. In addition, the separation on a silica column allowed us to achieve the objective which was to separate, identify and confirm the structure of pulegone and piperitenone which are the main compounds of the essential oil of M. pulegium, responsible for its important biological properties and its very appreciable therapeutic virtues. The fractionation of the EO permits highlighting 92 chemical compounds instead of 12 compounds identified in the crude EO. The disappearance of these compounds in the EO is probably linked to the presence of the two major compounds (pulegone and piperitenone) in the crude EO with very high percentages (98.01%) or their appearance is due to degradation of the chemical composition of the crude EO during the fractionation.

1H and 13C NMR analyzes of the two fractions F1 and F2

1H NMR analyzes of the fraction F1

The NMR spectrum of the proton in Fig. 4 revealed the presence of two singlets and a doublet, with little different chemical shifts, attributable to the protons of three methyl groups of the pulegone. For the other protons of the three CH2 groups, they are distinguished by several unresolved massifs. The results are given in Table 4.

Fig. 4
figure 4

1H NMR spectrum of pulegone

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Table 4 Characteristics of 1H NMR spectrum of pulegone
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1H NMR is an essential spectroscopic method for identifying and distinguishing the types of protons in an organic structure. Indeed, the comparison of the 1H NMR spectrum of our sample (F1) with that of the commercial sample (Pulegone) showed that they are practically identical.

Analysis by 13C NMR and 13C DEPT-NMR of the fraction F1

To identify the carbons of the studied molecule, we have performed two analyses giving rise to two spectra: that one of the 13C NMR and the other of the 13C NMR-DEPT (Fig. 5).

Fig. 5
figure 5

13C NMR and 13C NMR-DEPT spectrum of the pulegone

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The analysis of the first spectrum made it possible to compare the number and the position of the signals with the number and the nature of the carbons in the molecule. Concerning the counting of the second spectrum, it contributed to assigning the different signals to the types of primary, secondary, tertiary or quaternary carbons. We were able to confirm the presence of ten signals relating to the ten carbons present in the structure of the pulegone. We remember that the analysis of a product by NMR-DEPT spectroscopy of 13C (135) provides relevant information to facilitate the identification of organic structure. NMR-DEPT of 13C indicates the appearance of the signals associated with the CH3 and CH groups upwards. While the signals due to the CH2 groups point downwards, and the atoms of quaternary 13C do not appear. The simple method to approach the interpretation of 13C-NMR-DEPT spectra is to start by looking for the signals that are present in the 13C-NMR spectrum and absent in the 13C-NMR-DEPT. The signals that are absent in the latter are attributed to quaternary carbons. Indeed, three signals absent in the DEPT correspond to the three quaternary carbons of the molecule (C1, C6 and C8). For the four signals which point upwards, they correspond to the three primary carbons of the CH3 group (C7, C9 and C10) and to the single tertiary carbon of the group CH (C3). For the other three signals that point down, they correspond to the secondary carbons C2, C4 and C5. The results of the analysis of the NMR spectrum of 13C are shown in Fig. 6 and Table 5. In order to attribute to each carbon its adequate chemical shift, we relied on the data of electronic effects (electronic charges and densities, polarity of atoms) and on the bibliographic reference of David (David et al. 2016). Figure 7 summarizes the characteristics of the 13C NMR spectrum of the pulegone from our sample and those found by David et al. in 2016.

Fig. 6
figure 6

NMR-DEPT spectrum of 13C and 13C NMR of pulegone

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Table 5 Characteristics of the 13C NMR spectrum of the pulegone
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Fig. 7
figure 7

Identification of the pulegone of the essential oil of M. pulegium by 13C NMR

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1H and 13C NMR analysis of the fraction F2

The analysis of the 1H NMR and 13C NMR spectra (Fig. 8) of the fraction F2 made it possible to verify the presence of 14 protons and 10 carbons in the structure of the piperitenone. The results are shown in Tables 6 and 7.

Fig. 8
figure 8

1H NMR, 13C NMR and 13C NMR-DEPT spectrum of piperitenone

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Table 6 Characteristics of the 1H NMR spectrum of piperitenone
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Table 7 Characteristics of the 13C NMR spectrum of the pulegone
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The four signals absent in the DEPT correspond to the four quaternary carbons of the molecule (C1, C3, C6 and C8) (Fig. 7). Concerning the four signals which point upwards, they correspond to the three primary carbons of the CH3 group (C7, C9 and C10) and to the single tertiary carbon CH (C2). For the two signals that point down, they correspond to the secondary carbons C4 and C5. The attribution of the chemical shifts to the different carbons of piperitenone (Fig. 9) is completed using the electronic effects (electronic charges and densities, polarity of the atoms) which distribute the charges inside the molecule. Figure 10 summarizes the characteristics of the 13C NMR spectrum of piperitenone of our sample. The chemical shifts attributed to the various carbons are recorded in Table 7.

Fig. 9
figure 9

NMR-DEPT spectrum of 13C and 13C NMR of piperitenone

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Fig. 10
figure 10

Identification of piperitenone from the EO of M. pulegium by 13C NMR

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Conclusion

In this present work, we have studied the chemical composition of the essential oil of Mentha pulegium L. from M'Rirt (Middle Atlas, Morocco). Interesting results have been found; in particular, pulegone and piperitenone are the main compounds of this oil sample with a rate of 71.97% and 26.04%, respectively. In addition, the isolation of these two main compounds was done for the purpose of using their therapeutic properties.