Chemical Composition and Evaluation of the Antibacterial and Antifungal Activities of Pre-Rif Teucrium polium Essential Oil

Oussama Chauiyakh^1*, Aziz Et-Tahir¹, Badr Satrani², Mohamed Ghanmi², Sara Cherrad³, Kamal Kettani¹,

Abdelaziz Chaouch³, Malika Tiskar³

¹Mohammed V University in Rabat, Higher School of Technology of Salé, Material,

Energetic and Acoustics Team, Salé, Morocco.

²Forest Research Center, Rabat, Morocco.

³Ibn Tofail University of Kenitra, Faculty of Sciences, Laboratory of Agrophysiology, Biotechnology, Environment and Quality.

*Corresponding Author E-mail: oussama.chauiyakh@gmail.com

ABSTRACT:

As part of the valorization of Aromatic and Medicinal Plants (AMP) in Morocco, we studied the chemical composition and the antibacterial and antifungal activities of Teucrium polium Essential Oil harvested in a region of Pre-Rif, Taza in Morocco (BniKrama). After extraction of the essential oil (EO) by hydro-distillation process, the study of the chemical composition of this EO was carried out by gas chromatography (GPC) and GC/MS (gas chromatography- mass spectrometry), and the evaluation of antibacterial and antifungal activity was carried out against eleven microbial strains by determining the minimum concentration inhibitor. The yield after extraction by hydro-distillation is 0.71%. After (CPG) of the essence of this plant, sixty-eight components have been identified, of which α-Pinene is the major component (21.96%). It is followed by Limonene (18.77%), and β-Pinene (8.46%). The essential oil of T. polium (EOTP) has shown strong antibacterial and antifungal activity. This bioactivity is mainly due to the richness of this essence in terpenes known by their effectiveness against microbial agents. This essential oil is very rich in chemical molecules. Antimicrobial tests show that this EOTP has a broad spectrum on molds, fungi and bacteria.

KEYWORDS: Antibacterial activity; Antifungal Activity; Aromatic and Medicinal Plant (AMP); Gas Chromatography (GC); Essential oil (EO); Teucrium Polium.

INTRODUCTION:

Today, microbial resistance to antibiotics has become one of the major global health problems. In fact, the world health organization published in its 2017 report, the list of the most resistant bacteria for which it is necessary to look for new antibiotics hence the need for other natural antimicrobial agents to fight against this resistance^1,2,3. She adds that almost 80% of the world's population depends on traditional medicine and herbal medicine⁴, especially in developing countries⁵ and also increasing in developed countries⁶, they use aromatic and medicinal plants for their antimicrobial activities due to their essential oils⁷, they contain the compounds of therapeutic values^8,9 and the quality and efficacy of these drugs^10,11. All herbs have some potential medicinal value¹², more than 20,000 to 25,000 plants are used in the pharmacopoeia and more than 50% of the pharmaceutical products available on the market are of natural origin¹³. Morocco is rich in aromatic and medicinal plants (4, 500 plant species, 940 genera and 135 families)¹⁴. Of the 7000 existing species and subspecies 537 are endemic and 1625 rare or threatened¹⁵. This wealth is mainly due to the geographic location and the Mediterranean bioclimatic atmospheres of Morocco.

Teucrium polium is a plant native to the southwest of Asia, Europe and North Africa (abundantly found in the Irano-Turanian sector and mainly Mediterranean and western). It grows in arid meadows, low altitude rockeries, hills and arid deserts¹⁶. In Morocco this plant is called 'Jaada' or 'Khayata' or 'Barhemane' according to the region, and it has been used for over 2000 years as a diuretic, inotropic, chronotropic, tonic, antipyretic, cholagogue and anorexics¹⁷. Studies have shown several therapeutic properties of this plant. It is used against inflammations, hypertension, fever, diabetes, rheumatism, parasitic diseases such as amoebicides¹⁸, anti-inflammatory and anti-cancer¹⁹, and also as an antibacterial²⁰. This work will focus firstly on the characterization of the chemical composition essential oil of Teucrium polium, and secondly on the study of antibacterial and antifungal activity of this species against four pathogenic bacteria, four fungi and three molds.

MATERIALS AND METHODS:

Plant material:

The Teucrium polium plant was harvested in July after flowering, in the region of ‘BniKrama’, ‘Tainast’ town, Province of Taza in Morocco. It was dried for two weeks in the shade at room temperature. The characterization of the plant specimen was identified by a botanist in the Laboratory of applied chemistry and quality control, IbnTofail University, Kenitra, Morocco, the following taxonomy of this plant is confirmed by the same botanist (Kingdom: Plantae; Phylum: Spermatophyta; Subphylum: Angiospermae; Class: Dicotyledonae; Order: Lamiales; Family: Lamiaceae; Genus: Teucrium ; Species: Teucrium polium).

Extraction of essential oil:

The extraction of EO was carried out by the hydro-distillation process using a Clevenger type apparatus²¹. The operation consists in introducing a mass of 200g of the aerial part of the dried plant, in a balloon containing 3 Liters of distilled water. The extraction operation is carried out in three hours from the start of boiling. Finally, the oil obtained is stored in well-sealed smoked bottles at a temperature of 5°C. The extraction yield is expressed as a percentage, it is calculated by the following relation:

                        V_HE

Rdt (%) = ––––––––– × 100

                       m_ms

With: Rdt (%): Yield of essential oils (ml/g)

V_HE  : Volume collected of essential oil

m_ms  : Mass of dry matter

Chromatographic analysis:

The chromatographic analyzes were performed on a gas chromatograph with electronic pressure regulation of the Hewlett Packard type (HP 6890 series). It is equipped with a capillary column HP-5 (30mx 0.25mm) with a film thickness of 0.25μm, an FID detector adjusted to 250°C and supplied with a mixture of H2/Air gas and a split-splitless injector set at 250°C. The injection mode is split (leakage ratio: 1/50, flow rate: 66ml.min-1). The gas used is nitrogen with a flow rate of 1.7ml.min-1. The column temperature is programmed from 50 to 200°C for 5 min at a rate of rise of 4°C. min-1. The device is controlled by a computer system such as "HP ChemStation" and allows to follow the progress of chromatographic analyzes. The volume injected is 1 μl. Identification of components was performed based on their Kováts indices (IK)²² and on gas chromatography (Hewlett-Packard: HP 6890 series) coupled with mass spectrometry (HP 5973 series). The fragmentation is carried out by electronic impact at 70eV. The column used is a capillary column HP-5MS (30m x 0.25mm), the film thickness is 0.25μm. The column temperature is programmed from 50 to 200°C for 10 min at a rate of rise of 4°C.min-1. The carrier gas was helium with a flow rate set at 1.5ml.min-1. The mass range scanned m/z: 50-650 and the interface temperature is 280°C. The injection mode is split (leakage ratio: 1/70 flow 112 ml.min-1). The device is connected to a computer system managing a mass spectrum library NIST 98.

Microbial strains studied:

The microorganisms used to determine the minimum inhibitory concentration (MIC) of T. polium essential oil are:

Bacteria:Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus.

Fungi :Coniophora puteana, Coriolus versicolor, Gloeophyllum trabeum and Poria placenta.

Mold  :Aspergillus niger, Penicillium digitatum and Penicillium expansum.

The bacterial strains, in the form of ATCC batches (American Type Culture Collection), are cultivated on TSB medium (Tryptic Soy Broth). Molds and fungi belong to the collection of the mycotheque of the mycology laboratory of the Forest Research Center (Morocco), they are cultivated on the Malt-Agar medium. They were chosen for their high frequency of contaminating foodstuffs and for their pathogenicity for humans.

Microbiological procedure:

To ensure the consistency of our solution containing the essential oil T. polium (EOTP), we mixed it with the 0.2% Agar solution^23,24. The 1/10 dilution of the EOTP in the Agar solution was the first step, then quantities of this solution are added to test tubes containing TSB agar for bacteria and Malt-Agar for fungi and molds. Finally, these tubes are sterilized for 20 min at 121°C, cooled to 45°C and poured into Petri dishes to obtain the following final concentrations : 1/100, 1/250, 1/500, 1/1000, 1/2000, 1/3000 and 1/5000. Controls containing the culture medium plus the 0.2% Agar-Agar solution were also prepared.

RESULTS AND DISCUSSIONS:

Extraction efficiency:

The high yield of hydro-distillation extraction of EO from the aerial parts of the plant reached 0.71%, unlike other authors who had less^25,26,27 and who obtained respectively 0.7%, 0.21% and 0.66% of EO yield. On the other hand^28,29 had very high yields compared to our case respectively 1.66% and 0.9%. By comparing our performance with that of other studies on other species of the same genus, we notice that we obtained one of the highest values compared to those of^30,31 who obtained 0.6% and 0.58% respectively.

It can be deduced that the yield of essential oil can vary for species of the same genus, and also for the same species. This variation depends on several parameters, such as harvest time and extraction method, temperature, relative humidity, total duration of sunshine, wind regime, origin (geographic location) and part of the plant treated^32,33,34.

Chemical composition:

Figure 1 shows the chromatogram of Teucrium polium essential oil by Gas Chromatography. Sixty-eight compounds are identified representing a total of 99, 62 % of this species (Table 1). It is noted that there are in the first class two majority compounds α-Pinene (21.96%) and Limonene (18.77%) which alone represent 40.73%. They are followed by the second class which includes β-Pinene (8.46%), Sabinene (5.22%), (E) -iso-ɤ-Bisabolene (3.69%) and Myrcene (3.57%), and finally the third class of minority compounds (% <2%).

Figure 1 : GC chromatogram of Teucrium polium essential oil

The chemical composition of our essential oil is confirmed by the results obtained by³⁰, of which α-Pinene represents a majority compound (28, 8%) followed by β -Pinene (7, 2%). On the other hand, other studies have found that the majority compound is D-Germacrene (18.92%)²⁸, followed by β-E-Ocimene (12, 71%) and β-Pinene (9, 03%). In the case of²⁶, D-Germacrene is the majority compound whose content is 25.81%, followed by Bicyclogerma-crene (13%) and β- Pinene (11.69%), and²⁷ with a content of 23.6% in D-Germacrene, followed by 16.5% of p-Calyophyllene and 9.8% of α -Pinene. In the case of different species. The study carried out in Morocco by²⁹ on two species of Teucrium showed:

For the first T. polium subsp. Aurum, the majority element is Caryophyllene (19.13%) followed by c-Muurolene (13.02%), and s-cadinol (11.01%), with a low percentage in Monoterpenes 15, 75% (Monoterpenes Hydrocarbon: 11, 93%, Oxygenated Monoterpenes: 3, 82%) and very high in Sesquiterpenes 76, 41% (Sesquiterpenes Hydrocarbon: 64, 11%, Oxygenated Sesquiterpenes: 12, 3%) compared to our case.

For the second T. polium subsp. Polium the majority element is 3-Carene (16.49%), followed by c-Muurolene (14.03%), and α-Pinene (9.94%), with a low percentage in Oygenated Terpenes 11, 96% (Oxygenated Sesquiterpenes: 9, 66%, Oxygenated Monoterpenes: 2, 3%) and very high in Terpenes Hydroarbon 92, 53% (Sesquiterpenes Hydrocarbon: 54, 3%, Monoterpenes Hydrocarbon 38, 23%) compared to our case. it is noted that the sum of the percentages of Terpenes in the chemical composition of this species presents an error since this sum is 104.49%.

We can deduce that the chemical composition and the content of bioactive molecules of the same species can vary from one region to another and from one period to another due to several factors such as temperature, climate, and the harvest period.

Table 1: Chemical composition of the T. polium EO

N	IK	compound	%	N	IK	compound	%
1	922	α-Thujène	2, 7	35	1243	Ketoneperilla	0, 05
2	932	α-Pinène	21, 96	36	1265	Perillaldehyde	0, 13
3	939	5-methyl-3-heptanone	0, 5	37	1275	Iso-3-acetate thujanol	0, 74
4	944	Camphène	0, 19	38	1285	2-ethyl-endo-Fenchol	0, 89
5	949	Ethyl 3 methyl pentanoate	0, 23	39	1315	δ –Acetateterpinyl	0, 16
6	969	Sabinène	5, 22	40	1319	Neo-acetateVerbanol	0, 07
7	973	β-Pinène	8, 46	41	1334	δ –Elemene	0, 18
8	983	Myrcène	3, 57	42	1350	Αlpha-longipinene	0, 3
9	997	n-Octanol	0, 12	43	1368	Oxidepiperitenone	0, 1
10	1003	α-Phellandrene	1, 18	44	1375	α Copanene	0, 13
11	1009	Δ-3-Carene	0, 36	45	1380	2-epi-α Funebrene	0, 21
12	1017	α-terpinene	1, 57	46	1414	β Funebrene	1, 11
13	1025	Limonène	18, 77	47	1428	2, 5-dimethoxy-ρ-Cymene	1, 03
14	1037	Ethyl (hex-2E)	0, 07	48	1434	ɤ- Elemene	0, 05
15	1041	β –(E) –ocimene	0, 15	49	1448	Cis-Muurola-3, 5-diene	0, 32
16	1054	ɤ -Térpinène	0, 96	50	1455	Sesquisabinene	0, 17
17	1059	(2E) –Octen-1-Ol	0, 11	51	1463	9-epi-(E -Caryophyllene	0, 7
18	1079	Camphenilone	0, 75	52	1473	α-Terpinylisobutanoate	0, 6
19	1086	Terpinolene	0, 63	53	1480	ɤ-Curcumene	0, 3
20	1107	6-Camphenol	0, 35	54	1487	Cis-Eudesma-6, 11-diène	0, 46
21	1110	Trans Thujone	0, 35	55	1492	Trans-Muurola-4(14), 5-diène	0, 8
22	1115	Endo- Fenchol	0, 28	56	1509	Germacrène A	1, 48
23	1130	1-Terpiniol	1, 52	57	1528	(E)-iso-ɤ-Bisabolene	3, 69
24	1138	Cis-Verbenol	1, 95	58	1538	α-Cadinene	0, 71
25	1149	3-Neo thujanol	0, 23	59	1563	8-acetoxy-Carvotanacetone	0, 48
26	1158	Trans-β-Terpeniol	0, 85	60	1568	Longipinanol	0, 41
27	1170	Cis-oxyde linalool	1, 57	61	1580	Oxidecaryophyllene	0, 22
28	1175	Terpinen-4-Ol	0, 19	62	1589	β-Cis-Elemenone	0, 12
29	1182	Neo-verbanol	0, 27	63	1602	Trans-β-Elemenone	0, 28
30	1189	Alpha-Terpineol	1, 07	64	1631	ɤ-Eudesmol	1, 01
31	1203	Verbenone	0, 66	65	1646	Cubenol	0, 92
32	1209	Trans-Piperitol	0, 7	66	1659	DihydroEudesmol	2, 39
33	1220	Cis-acetate hydrate sabinene	0, 21	67	1743	8α-11-Elemodiol	0, 07
34	1234	(E)-Ocimenone	1, 45	68	1775	14-hydroxy-α-Muurolene	0, 08
Monoterpenes Hydrocarbon: 19, 11 % Oxygenated Monoterpenes: 33, 82 % Sesquiterpenes Hydrocarbon: 20, 58 % Oxygenated Sesquiterpenes: 11, 76 % Others: 14, 70% TOTAL : 99, 97%

Antimicrobial activity:

Table 2 shows the results of the antibacterial and antifungal activity of the essential oil of T. polium. We have found that bacteria are more resistant to EOTP studied, compared to molds and fungi which are the most sensitive. The in vitro antimicrobial effect of essential oil tested versus bacteria, mold and wood rot fungi was revealed according to the presence or absence of the proliferation of the tested species. The four bacterial strains studied were inhibited at a concentration of 1/250 which shows the strong effect of EOTP on bacteria. On the other hand, the fungi showed a different sensitivity behavior towards EO:1/2000 which is sufficient to inhibit the growth of P. placenta, followed by C. puteana with a minimum inhibition concentration (MIC) 1/1000 and finally G.trabeum and C.versicolor (1/500). Molds also showed a different sensitivity behavior towards EO. The most sensitive strain is P. expansum with an MIC of 1/500 followed by P. digitatum and A. niger (1/250).

A study by²⁶ has also shown that this EO has good activity against E.coli and S. aureus with an MIC of 3 µL/ml and 4µl/ml respectively. That carried out by²⁸ revealed an activity of the EO of T.polium against S. aureus which was inhibited at an MIC of (1/8) 125µl/ml with an inhibition diameter of 14.5mm, and E. coli inhibited at an MIC of (1/2) 500μl/ml with an inhibition diameter of 11.5 mm.

Concerning the Teucrium polium from Morocco and according to the study by²⁹, the EO of two species studied (T.polium subsp. Polium and T.polium subsp. Aurum) have antibacterial activity. The MIC varies respectively according to the two species between 2.81 mg/ml and 5.62mg/ml for E. coli and an MIC = 0.17mg /ml for S.aureus. These results obtained confirm the results of our study, which demonstrated good activity against these two bacterial strains.

Table 2 : Results of Essential Oil Teucrium polium antimicrobial activity

Concentration	C	1/100	1/250	1/500	1/1000	1/2000	1/3000	1/5000
Bacteria
E. coli	+	-	-	+	+	+	+	+
B. subtilus	+	-	-	+	+	+	+	+
M. luteus	+	-	-	+	+	+	+	+
S. aureus	+	-	-	+	+	+	+	+
Molds
A. niger	+	-	-	+	+	+	+	+
P. expansum	+	-	-	-	+	+	+	+
P. digitatum	+	-	-	+	+	+	+	+
wood rot fungi
G. trabeum	+	-	-	-	+	+	+	+
P. placenta	+	-	-	-	-	-	+	+
C. puteana	+	-	-	-	-	+	+	+
C. versicolor	+	-	-	-	+	+	+	+

By comparing our results with others studies we notice that our essential oil is stronger with MICs always lower than the other results. We can deduce that this essential oil has a broad spectrum of inhibition for fungal and bacterial strains which show, however, different sensitivity behaviors. These antimicrobial activities can be enhanced by extracting and purifying the bioactive molecules as well as determining their dosage for appropriate administration³⁵.

CONCLUSION:

As part of the promotion of MAP in Morocco, we studied the essential oil of Teucrium polium known by these therapeutic effects. Since ancient times it has been used as an antimicrobial, diuretic, inotropic, chronotropic, tonic, antipyretic, cholagogue and anorexics. Indeed, after the hydro-distillation of the plant and the obtaining of its EO we moved on to the characterization of the chemical composition and the evaluation of the antimicrobial activities of this EO.

The EO yield is 0.71%, this value is one of the highest values obtained in other study. Characterization of the chemical composition by GPC showed chemical variability in components, we identified Sixty-eight compounds representing a total of 99, 62 %, with a predominance of α-Pinene (21.96%), followed by Limonene (18.77%) and β-Pinene (8.46%).

After studying the chemical composition of T. polium essential oil, we determined, in vitro, its antimicrobial properties on four bacteria (E. coli, B. subtilus, S. aureu, and M. luteus), three molds (A. niger, P. digitatum and P. expansum) and four wood rot fungi (G. trabeum, P. placenta, C. puteana and C.versicolor), these microbial strains were chosen for their high frequencies to contaminate food and for their pathogenicity. The results obtained show that the essential oil tested has an antimicrobial action, which differs depending on the microorganism tested.

It is noted that bacteria are the most resistant to the EO studied with a MIC of 1/250, followed by molds with a MIC that reaches 1/500 for P.expensum, on the other hand the fungi, which are the most sensitive, with an MIC that reaches up to 1/2000 for P.placenta.

 Finally, it was concluded that one of the highest yields of essential oil was obtained compared to the other studies, with an important richness in chemical molecules. Antimicrobial tests show that this essential oil has a broad spectrum on molds, fungi and bacteria. For this reason, it is noted that this essential oil is an effective antimicrobial and bio pesticide agent against the microorganisms.

ACKNOWLEDGEMENTS:

Thanks to the researchers at the National Centre for Forestry Research for their advice and guidance.

CONFLICT OF INTEREST:

The authors do not have any conflict of interest.

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Received on 25.12.2020                Modified on 13.07.2021

Accepted on 12.10.2021               © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4):1755-1760.

DOI: 10.52711/0974-360X.2022.00294