Phytochemical study and insecticidal activity of Mentha pulegium L . oils from Morocco against Sitophilus Oryzae

There is a growing interest of industry to replace synthetic chemicals by natural products with bioactive properties from plant origin. The aim of this study was to validate the therapeutic properties of Mentha pulegium L. by conducting a phytochemical study, to determine the chemical composition of its essential oils (EO) and evaluate its insecticidal activity against stored cereals pests. Leaves and flowers of M. pulegium L. were collected from three regions of the Moroccan Middle Atlas. Phytochemical tests on pennyroyal aerial parts revealed the presence of gallic tannins, flavonoids, alkaloids, sterols and triterpenes and saponins. The chemical composition of essential oils was analyzed by gas chromatography coupled with mass spectrometry. The main components were pulegone and piperitenone. The EO from Khénifra is dominated by pulegone (81.46%), and those from Azrou and M'rirt are rich both in pulegone (68.86 and 71.97%) and piperitenone (24.79% and 26.04%) respectively. Pennyroyal oil has showed an important fumigant effect against Sitophilus oryzae (L.) adults. This effect is influenced by the tested doses and exposure periods. The potential of this plant to be used to control stored product insects was discussed.


Introduction
Mentha pulegium L. is a herbaceous perennial that belongs to the Lamiaceae family, commonly known as pennyroyal/European pennyroyal, native to North Africa, Europe, Asia Minor and the Middle East 1 .It grows wildly in humid areas of the plains and mountains.In Morocco, M. pulegium L. is known as the Arabic name "Fliyou".It is among the top five national mints and most widely used and commercialized 2 .
Aerial parts of this plant contain a wide diversity of secondary metabolites such as: tannins, resins, pectins, bitter principles and essential oils 1,3 .Fresh or dried leaves and flowering tops are commonly used for their healing and culinary properties.The whole plant and its essential oil have a strong and characteristic smell 3 .
It was even used to promote menstruation, cure headaches, treat bronchitis, relieve bites from scorpions and snakes and help against vomiting and kidney disease 1,3 .It also served as a repellent against fleas and other insects [2][3][4] .It was effective in relieving acne and other skin conditions.This plant has been used as a spice and flavoring in various foods, especially desserts 1,4 , also as perfume in cosmetics [3][4] and corrosion inhibitor for steel in the chemical industries 5 .
Pennyroyal is not yet described by the Pharmacopoeia 15 .Even if its properties were traditionally well-known, this species has never been investigated pharmacologically 15 .However, its essential oil is highly suspected to be hepatotoxic.It was responsible for fatal accidents.Its liver toxicity is mainly related to pulegone and its metabolites that are responsible for tissue necrosis 15,22 .The consumption of several milliliters of pennyroyal oil is highly probable to cause toxic effects.Small amounts (10 ml) can produce a moderate to severe poisoning 23 , large quantities (25ml) can cause a fatal liver necrosis 22 .The most important hazard associated with this oil, is the potential for accidental consumption by a child 22 (LD 50 = 3 ml).The right dosage and good administration of the plant could avoid such accidents 22,24 .Note that the exact mechanism of toxicity of pennyroyal oil and pulegone is not yet revealed 15 .
In Middle Atlas, a mountainous region of Morocco, pennyroyal is considered among plants frequently used by people in traditional medicine.In this context, the aim of this work is to identify the own secondary metabolites of pennyroyal that collected from three regions of Middle Atlas, to study the variability of the chemical composition of its essential oils and to evaluate its insecticidal potential against pests of stored cereals.

Essential oils extraction
Flowers and leaves (100g) were air-dried at room temperature and hydro-distilled using a Clevenger-type apparatus for 3 hours.The essential oils were dried with anhydrous sodium sulphate and stored in a refrigerator at 4°C until use.For calculations of essential oil yields, three replicates were performed for each plant material.

Chromatographic analysis
The chromatographic analyses were performed using a gas chromatograph Hewlett Packard (HP 6890 series) type equipped with a HP-5 capillary column (30m x 0.25 mm x 0.25 microns film thickness) , a FID detector set at 250 ° C and fed with a gas mixture H2/air.The mode of injection is split; the carrier gas used is nitrogen with a flow rate of 1.7 ml / min.The column temperature is programmed at a rate of 4 mounted ° C / min from 50 to 200 °C for 5 min.The unit is controlled by a computer system type "HP ChemStation" managing the operation of the device and to monitor chromatographic analyzes.GC-MS was carried out by a chromatograph Hewlett Packard (HP 6890) coupled to a mass spectrometer (HP 5973 series).Fragmentation is performed by electron impact at 70 eV.The used column was a capillary-type HP 5SM (30 mx 0.25 mm x 0.25 mm).The column temperature is programmed at a rate of 4 mounted °C / min from 50 to 200°C for 5 min.The carrier gas is helium with a flow rate set at 1.7 ml / min.The injection mode is split type.
For compound identification, the Kovàts Index 25 of each compound was calculated in relation to the retention time of a series of linear alkanes (C7-C40).The calculated index was then compared to those of Adams 26 reference.The mass spectra of compounds were also matched with those stored in the NIST library / EPA / NIH MASS SPECTRAL LIBRARY; Version 2.0, build libraries Jul, 1, 2002.

Phytochemical tests
The phytochemical study needed the preparation of plant material.Leaves and flowers of M. pulegium were dried in the open air, milled in an electric grinder and used to prepare extracts, infusions and decoctions.
Selective extractions of homogenates were made specifically on each family of compounds studied.The extracts have been obtained by extraction with solvents.The solvents used are petroleum ether, methanol, ethanol, chloroform and distilled water.
The phytochemical screening was also based on several reagents.Research of alkaloids was performed by Dragendorff reagent.Characterization of catechin tannins was carried out by isoamyl alcohol and hydrochloric acid and gallic tannins by Stiasny reagent, sodium acetate and ferric chloride.To detect sterols and triterpenes, we used acetic anhydride and concentrated sulphuric acid.Diluted alcohol hydrochloric acid, magnesium chips and isoamyl alcohol were used to seek the flavonoids.Chloroform, dilute ammonia and hydrochloric acid have to look for quinonic substances.
Characterization tests of different chemical groups were performed as described by Bruneton 15 , Harborne 27 and N'Guessan 28 .

Pests
Adults of Sitophilus oryzae (L.), rice weevils belonging to the Curculionidea family were brought from the grain market.They were raised thereafter at the expense of wheat grains in glass jars at a temperature of 24 ± 1°C and a relative humidity of 76 ± 5% in the dark in order to obtain a homogeneous population.

Biotests
Fumigation tests were carried out in plastic boxes of 1 liter volume.Thus, in each box was placed a Petri dish by airy chiffon gauze containing 10 adults of S. oryzae and filter paper soaked with M. pulegium essential oil.The concentrations of essential oil in the air were: 0.75; 0.25; 0.5; 1 and 2 µl /l air.For each concentration, five replicates were conducted.

Data analysis
To detect the toxic fumigant effect of tested essential oil, an analysis of variance performed using the function Arsin (square root (percentage mortality)) in the software Microsoft Excel 2007.The calculation of the survival probabilities and comparison of the effect of each concentration tested were taken respectively by the test of Kaplan-Mayer 29 and the log-rank test 30 .The lethal concentrations 50% (LC 50 ) and 99% (LC 99 ) fumigated insects were determined by the Probits method according to Finey 31 .
The variation of yields and chemical composition of essential oils depends on several factors: the method used, the used plant parts, the products and reagents used in the extraction, the environment, the plant genotype, geographical origin, the harvest period of the plant, the degree of drying, the drying conditions, temperature and drying time and the presence of parasites, viruses and weeds [32][33] .
Pennyroyal can be considered as an important source of pulegone.This ketone presents approximately 3/4 of the overall chemical composition of the studied oils.It is usually used in the manufacture of industrial and cosmetic products as it can be transformed into menthol by semisynthetis which is highly demanded for flavouring food, in cosmetics and pharmaceutical industry 34 .
The chemical composition of the studied oils is similar to that reported by several studies already carried out in Morocco.The EO of M. pulegium from Morocco is characterized by its high rate of pulegone.The EO from Asilah (North east) studied by Hmiri 18 contains a very attractive pulegone rate (80.28%).The content of pulegone in pennyroyal from Meknes is about 65% 14 .In Taouirt region (North-East) 69.8% 35 ; in Rabat region (Ain Aouda), it is about 73.33% 11 and in Southern Morocco, it reached 85.4% 17 .Similarly, pennyroyal oils from different Algerian sites are characterized by the predominance of pulegone with different proportions 43.3 to 87.3% 36 .Similar results were obtained from Uruguay 7 and Tunisia 12 , the main compounds are respectively pulegone (73.4% and 61.11%) and isomenthone (12.9% and 17.02%).In Egypt, the pennyroyal oil is rich in pulegone (43.5%) and piperitone (12.2%) 9 .While the species from Iran 10 and Skoura (Morocco) 13 contain two particular chemotypes: piperitone (38 and 35.56%) and piperitenone (33 and 21.12%) while the pulegone rate does not exceed 2.3 and 6.42% respectively.Similarly, those from Portugal 6 and Yugoslavia 37 are characterized by different chemical composition whose major compounds are menthone (35.9 and 30.9%) and pulegone (23.2 and 14.1%) respectively.

Phytochemical screening
The results of phytochemical screening are assembled in Table 2. Different groups occurring in leaves and flowers of pennyroyal were identified.
According to the results of the characterization tests, the aerial parts of pennyroyal from three regions contain gallic tannins, saponins, flavonoids, sterols and triterpenes, alkaloids, mucilages and reducing compounds.However, it is devoid of anthraquinones, oses and holosides, cyanogenic glycosides and catechin tannins.The effective presence of some of them in the plant does not exclude its therapeutic properties 38 .

Table 2: Results of phytochemical screening of pennyroyal aerial parts by colored reactions.
Alkaloids have different pharmacological activities such as strengthening the heart activity, excitation of the central nervous system and nerves symptomatic, stimulating blood circulation 38 .The presence of alkaloids may also justify the use of the plant in the treatment of certain diseases 28 .The tannins show the properties of vitamin D, they could be used to strengthen blood vessels and contribute to the accumulation of vitamin C in the body 38 .The saponins have a healing effect and sterols and triterpenes have bactericidal properties 28 .
These properties were linked to the identified classes of phytoconstituents in pennyroyal.Therefore, this plant exhibits important therapeutic effects.These results justified the wide use of this plant in traditional medicine by people in Middle Atlas.

Insecticidal activity of M. pulegium essential oil against adults of S. oryzae (L.)
In this research, we evaluated the insecticidal activity of pennyroyal oil from Azrou against weevil of S. oryzae.Concentrations tested have showed a significant fumigant activity.The degree of this activity depends on the applied concentration (F concentrations = 18.38 > F (0.05, 5-192) = 2.2141) and the exposure duration (F time = 12.83> F (0.05, 7-192) = 2.0096).The survival rate of insects decreases as the concentration and exposure period increased (Figure 1).
The interaction between weevils and essential oil lasted eight days.The mortality within the weevil was observed on the first day and even with the lowest concentration.At 2μl of essential oil/l air, insects have been totally decimated the fourth day.As at low concentrations, the mortality of insects reached gradually the entire population on the eighth day after the beginning of fumigation.
The survival time of 50% (LT 50 ) and 99% (LT 99 ) of adults who were exposed to different concentrations of essential oil vary respectively from 24 hours to 4 days and about 4 to 8 days depending on the concentration, whereas in the control group, adults live an average of 29 and 57 days respectively (Table 3).Furthermore, the LT 50 and LT 99 are negatively correlated with the tested concentrations.

Chemical group Observations
Gallic  The toxicity parameters of the tested essential oil are summarized in Table 4.The calculated lethal concentrations LC 50 and LC 99 vary according to the time.They decrease gradually as the fumigation time increases.Extreme lethal concentrations LC 50 and LC 99 vary from 2.65 to 0.044μl /l air and 143.9 to 0.518μl /l air respectively.
Results similar to ours have been reported by Benayad 11 which a concentration of 3 μl of M. pulegium oil (73.33% of pulegone) caused mortality of all weevils of S .oryzaeafter one day of treatment.Similarly, these insects were also decimated at a dose of 1.7 10 -2 μl/ cm 3 of M. suaveolens oil (85.5% of pulegone) after 7 days of treatment and at a dose of 3.5 10 -2 μl/ cm 3 of oil after 24 hours 44 .
The fumigant effect of this oil against adults of S. oryzae could also be explained by the high content of pulegone.The toxicity of this ketone against S. oryzae was also observed by Lee 30 and Hannin 45 .
Furthermore, the insecticidal activity of the essential oil is not limited only to its major constituents; it could also be due to some minor constituents or a synergistic effect of several constituents [46][47] .Table 4: Toxicity parameters of pennyroyal oil against S. oryzae (L.) adults.
The toxic effect of M. pulegium essential oils against S. oryzae may be attributed to the inhibition of neurotransmitters such as acetylcholinesterase 41,48 and octopamine 49 .
Essential oils as natural insecticides are therefore an alternative to synthetic insecticides because of their physicochemical properties that make them very volatile and biodegradable, which presents no risk of residues on treated products or on the germination of processed grains.Moreover, the fact that essential oils act on octopaminergic sites insects 46 , they are less toxic to mammals.

Conclusion
In the present research, we performed a phytochemical study of M. pulegium L., determined the chemical composition of essential oils and assessed its insecticidal activity.The results allowed concluding that the yields and chemical composition of essential oils vary according to the plant origin.Essential oils originating from Azrou, M'rirt and Khénifra are characterized by diverse chemical profiles.Thus, species of Azrou and M'rirt are dominated mainly by pulegone (68.86% and 71.97%) and piperitenone (24.97% and 26.04%) while that of Khénifra is very rich in pulegone (81.46%).
Moroccan pennyroyal may therefore be an important source of pulegone.This active component is highly required for the manufacture of cosmetics and industrial products.
Different secondary metabolites were identified: flavonoids, gallic tannins, sterols and triterpenes, alkaloids and saponins.Therefore, pennyroyal can be seen as a potential source of useful drugs.Further studies are going on this plant in order to isolate, identify and characterize the structure of the bioactive compounds.
Pennyroyal oil has showed an important fumigant effect against S. oryzae weevils.This effect could be attributed to the chemical composition and particularly to the abundance of pulegone and piperitenone without ignoring the synergistic role of minor compounds.
Furthermore, the use of essential oil M. pulegium in fumigation to control populations of S. oryzae is possible.Fumigation can handle large masses of seeds without moving.In this way, the pennyroyal oil has a huge potential as alternative to synthetic pesticides in cereals stored and crop protection.Further studies should evaluate the safety and toxicity of M. pulegium oil to human consumption before their use for medicinal and food purposes.

Table 1 :
Chemical composition of pennyroyal oils from Middle-Atlas.

Table 1 (
Continued): Chemical composition of pennyroyal oils from Middle-Atlas.

Figure 1 .
Survival rate of S. oryzae (L.) adults fumigated by pennyroyal oil (Concentrations affected with the same letter do not differ statistically between them (log-rank test)).

Table 3 :
LT 50 and LT 99 of S. oryzae adults fumigated with M. pulegium essential oil.