Eucalyptus sideroxylon A. Cunn.:

A Natural Eucalyptol (1,8-cineole) source in Morocco

 

Loubna Koursaoui^1,2, Badr Satrani*¹, Mohamed Ghanmi¹, Sara Cherrad^1,2, Imane Jaouadi^1,2, Said Hajib¹, El Mahjoub Aouane², Abdelaziz Chaouch²

¹Laboratories of Microbiology and Chemistry of Aromatic and Medicinal Plants,

Forest Research Center, BP 763, Agdal, Rabat, Morocco.

²Biotechnology, Environment and Quality Laboratory, Faculty of Science,

Ibn-Tofail University, BP 133, Kenitra, 14000, Morocco.

 

ABSTRACT:

The main objective of this work is to determine the effect of the harvest period on the yield and chemical quality of the essential oils of the Eucalyptus sideroxylon A. Cunn. leaves of the Mamora forest, Dayet Zerzour Bnifdel region, Rabat. Essential oil yields are remarkably high above 2.3%, with an ultimate rate of 5.48% for the month of April. The chemical quality of these essential oils is characterized by the presence of two major monoterpenes, 1,8-cineole (eucalyptol) and α-terpineol, which are proportionally inverted in terms of quantity. During the wet months between September and March, the cineole predominates with levels ranging from 72.67% to 86.11% and the other dry months are characterized by an increase of α-terpineol rate from 12.05% to 25.61%. This inverted chemical variability reveals a change in the orientation of cineole and α-terpineol biosynthesis under the control of climatic factors. This work allows us to discern favorable periods for the harvest of Eucalyptus sideroxylon leaves in terms of yield and quality of essential oils.

 

 

 

INTRODUCTION:

Morocco's geographical situation, between the Mediterranean to the north, the Atlantic Ocean to the west, the Sahara to the south and south-east, and its particular orographic nature give it a very remarkable climatic, ecological, faunistic and floristic diversity. According to the first national forest inventory (NFI), carried out between 1990-2005, the Moroccan forest domain covers an area of approximately 9 million hectares, representing a cover rate of 12.7% of the national territory¹. The natural tree forest formations cover 5.814.000 ha, with 63% of leafy trees (green oak, argan, cork oak) and 20% of resinous (cedar, thuya, pines, junipers, cypress and fir).

 

The rest of the area, 17%, is populated by low-lying formations (Matorrals) resulting from forest degradation. Artificial or reforestation plantations cover nearly 490.518 hectares, 5.4% of the forest area, represented mainly by Eucalyptus and Acacia as introduced species¹.

 

For several decades, at the beginning of the 20th century, Morocco has been introducing and acclimatizing fast-growing Australian Eucalyptus species in the Sidi Yahia region of Gharb, capable of adapting to the country's ecological conditions to improve the productivity of reforestation and overcome the wood deficit. The first plantations were carried out by the farmer H. Ménager in an arboretum, for an economic interest to supply the pulpwood and the paper industry².

 

In the north-western region of Morocco, Eucalyptus plantations are spread over an area of 112,855 ha. The major species are represented by the hybrid clonal Eucalyptus (E. camaldulensis x E. grandis), E. camaldulensis (44%), E. grandis, E. saligna, E. gomphocéphala (31%), E. globulus and E. cladocalyx².

The forest of Mamora is dominated by cork oak (133 853 ha) which represents 50% of the total forest stands, 65 000 ha. The rest of the area is occupied by well-reproducible Eucalyptus species (wood and cellulose) such as E. camaldulensis and E. gomphocephala to replace the lack of profitability of cork oak³. The other species such as E. grandis and E. sideroxylon come in third place as they cover only 4% of the total forest stands of the Mamora⁴.

 

The genus Eucalyptus is an Australian tree plant belonging to the Myrtaceae family, represented by 800 species of which 300 species are very rich in secondary metabolites, that make up the essential oils extracted from their evergreen leaves⁵, considered as protective molecules against herbivores and microbial agents^6–9 thanks to the bioactivity of essential oils proven in the laboratory^10–13. These oils were once used in traditional Chinese medicine as an analgic, anti-inflammatory¹⁴ and antipyretic symptoms of respiratory infections¹⁵. Indeed, several medical laboratory research worldwide have revealed the antiviral, antibacterial, anticancer and antifungal bioactivity of these essential oils, particularly related to the presence of the 1,8-cineole known as eucalyptol^5,16–22.

 

However, the majority of research has shown that E. camaldulensis and E. globulus leaves and their hybrids are the main source of 1,8-cineole-rich essential oil, as an active ingredient sought in pharmaceutical and cosmetological industry, with a rate of more than 70%¹⁷. Eucalyptol, or 1,8-cineole, is a colourless compound, appreciated for its refreshing and spicy scent similar to camphor. This constituent has several pharmaceutical properties and is therefore used for human medical treatment. It is also indispensable in pharmaceuticals owing to its antifungal, anti-infective, bactericidal, antiviral and expectorant properties. The broadest field of application for 1,8-cineole is the treatment of serious lung diseases such as asthma, where the compound has a mucolytic, bronchodilator and anti-inflammatory effect. Similar effects contribute to the treatment of acute sinusitis, where 1,8-cineole is the pharmacologically active agent in some over-the-counter drugs²³.

 

After a thorough investigation in the bibliography much work has targeted the study of the species of E. camaldulensis and E. globulus and their hybrids, however, no work has been devoted in Morocco to the chemical quality and biological activities of E. sideroxylon essential oils.

 

E. sideroxylon is an arborescent species whose height reaches 15 to 20m of conical summit type. The trunk of this tree has a black, persistent, thick and deeply furrowed bark impregnated with kino ironbark type, giving rise to a more or less dark red wood. The mature leaves are alternating, petiolate, lanceolate, narrow, linear in green and shiny on the faces. This species is characterized by an axillary umbel inflorescence of 3 to 7 flowers and pedicelled fruits ovoid, globular, thin and flat or oblique disc, deeply enclosed valves. Flowering occurs in May and February²⁴.

 

The introduction of E. sideroxylon in Morocco, particularly in the Oued Cherrat and Sidi Yahia du Gharb region, has revealed a good acclimatization to Mediterranean ecological conditions which are similar to those of South West Australia. It requires a biotope characterized firstly by temperatures ranging from an average of the maximum of 32°C to an average of the minimum of 3 to 5°C, then by winter and summer rains with an average annual total of 375 to 625mm and a dry period lasting between 6 and 8 months. Regarding the nature of the soil on which it grows, it is a poor, superficial, sandy soil rich in gravel and ferruginous concretions²⁴. The implantation of this species and in general the Eucalyptus, which are introduced mainly in the north-western region of Morocco, is justified by the production of paper pulp⁴.

 

Preliminary work has been undertaken on the chemical composition of essential oils of this species has shown that it is rich in 1,8-cineole with a rate higher than 70% required by industry. With this in mind, we have been interested in the one-year monthly monitoring of the yeld and chemical quality of E. sideroxylon essential oils with a view to its use for better exploitation in the pharmaceutical and cosmetological industry. In particular, it is a pure-lined forest species more genetically adapted to its biotope. Unlike the hybrid species of Eucalyptus studied by Farah and al¹⁷ whose chemical profile is dominated by 1,8-cineole with a rate greater than 70%, however, they showed excessive vulnerability to attacks by pathogenic fungi compared to their parent species planted in the same climatic and edaphic conditions.

 

MATERIAL AND METHODS:

Study site:

The Mamora site is the largest cork oak forest in Morocco and probably in the world, it is located in the Northwest of Morocco, on the edge of the Atlantic, between latitude 34°15'52' North and longitude 6°39'27' West. It is part of a rectangle 60 km long, from West to East, and 30km wide, from North to South, bounded to the South by the Bou Regreg valley and the Central Plateau foothills and to the North by the Gharb plain²⁵.

 

Plant material:

Eucalyptus sideroxylon leaves were collected monthly (1 to 2kg approximately) in the same plot located in the Dayet Zerzour Bnifdel region (34°19’62’ North; 6°18’70’ West) of the Mamora forest, for a period of one year from September 2016 until August 2017, according to the french standardization association (AFNOR) standard²⁶.

 

Extraction of essential oil:

The extraction of Essential Oils (EO) was performed by hydrodistillation in a Clevenger-type apparatus²⁷. The method applied is that described in the European Pharmacopoeia in 2008²⁸ and according to the 2008 recommendations of the French Health Products Safety Agency.

 

We performed three distillations by boiling 200g of vegetal material cut with pruning shears and introduced into a 2 liter flask containing 1 liter of water. The extraction time is of the order of 3 hours on average. This extraction time was determined by preliminary tests that showed that the yield did not change after this time. The (EO) obtained is dehydrated with anhydrous sodium sulphate and then stored at a low temperature (below 4 °C) and in the dark before use. Then it was diluted in methanol (1/20 v/v) prior to analysis by GC and GC/MS according to AFNOR standard²⁶.

 

Determination of moisture content:

The determination of the moisture content is carried out by weighing 25g of each sample and placed in the oven at 60°C for 48 to 60 hours. After cooling, the average weight loss is calculated and the moisture content is determined by the following equation (1):

 

%mc = (W_w – W_d)/W_w ×100                                         (1)

 

With: %mc: moisture content, W_w: wet weight and W_d: dry weight

 

Essential oil yield calculation:

The EO yield is expressed in ml of the distillate per 100 g dry matter according to the following equation (2): Yld (%) = [V/W_d x 100] ± [∆V/ W_d x 100] (2)

With: Yld%: Yield of EO (ml/g);V: Volume EO collected (ml); ΔV: Reading on Error; W_d: dry plant weight

 

Analysis of essential oil:

Chromatographic analysis of the E. sideroxylon Essential oils samples was performed on a gas chromato-graph with electronic pressure control, type Hewlett Packard (HP series 6890) equipped with a capillary column HP-5(5% diphenyl, 95% dimethylpolysiloxane) (30m x 0.25mm) with a film thickness of 0.25μm, with an FID detector set at 250°C and fed by a gas mixture and a H2/Air split-splitless injector set at 250°C. The volume injected is 1μl. The injection mode was split (split ratio: 1/50 flow: 66ml/min). The gas used is nitrogen with a flow rate of 1.7ml/min. The column temperature is pro-grammed to increase from 50 to 200°C at a rate of 4 °C/min and held for 5 minutes at the final temperature. The detection limit is less than 1ppm. The device is controlled by a computer system type "HP Chem Station", managing the operation of the device and monitoring the changes in chromato-graphic analysis.

 

Identification of components was performed based on their Kovats indices (KI)^29,30, and on gas chromatography coupled with mass spectrometry electron im-pact (GC-SMIE). The latter is performed on a gas chromatograph, type Hewlett- Packard (HP series 6890) coupled with a mass spectrometer (HP 5973 series). Fragmentation is per-formed by electron impact at 70 eV. The column used was a HP-5MS capillary column (30m x 0.25mm); the film thickness is 0.25μm. The column temperature is programmed from 50 to 200 °C at 4°C/min. The carrier gas is helium with a flow rate set at 1.5ml/min. The injection method is the split mode (split ratio: 1/70). The device is connected to a computer system that manages a library of mass spectra NIST 98. Indeed, the index system is based on the concept of relative retention. It compares the retention of any product to that of a linear alkane. This system is applicable in gas chromatography to any compound on any column. By definition, it assigns an index of 800 in the linear alkane C8 (n-octane), 1000 to C10 linear alkane (n-decane), and this, whatever the stationary phase, the length of the column, the temperature or flow rate. KI are determined by injecting a mixture of C9 to C24 alkanes in the same operating conditions³⁰.

 

RESULTS AND DISCUSSION:

Essential oils yield:

Average yields in EO were expressed in ml compared to 100g of dry plant matter. Results for the yields of the twelve EO obtained in a year are grouped in table1.

 

Table 1: Monthly monitoring of E. sideroxylon essential oil yield

Months	Jan	Feb	Mar	Apr	May	Jun	Jul	Aug	Sep	Oct	Nov	Dec
Yields (%)	2.38	2.43	3.04	5.48	3.85	3.99	3.57	3.42	3.04	3.64	3.30	3.23

 

These results show that this species is very rich in EO with a rate that exceeds 3% for all months of the year except the two months of January and February when the yield reaches 2.38% and 2.43% respectively. The best rate of EO is obtained during the spring season, in the month of April with a percentage of 5.48%, while the winter season provided a low rate of yield in January (2.38%). A comparison between the Summer and Autumn seasons shows that the yield of essential oil is higher in the Summer season, between 3.42 and 3.99% compared to 3.04 and 3.64% in Autumn. These results suggest that the EO yield of the species E. sideroxylon increases mainly during the spring and winter seasons, which coincide with the flowering period between May and February²⁴.

 

Moreover, the yield obtained by Zrira and al³¹for the species E. sideroxylon is relatively low, in the order of 2.08% and 1.36% respectively from two origins (Takerkoust and Jbillet) in the Marrakech region during the April harvest. Similarly, work underway in the Oued Cherrat arboretum on the species E. sideroxylon showed a very low yield of 0.55%³². Another study conducted in Pakistan on E. sideroxylon EO showed almost the same low yield of 0.51%¹⁵. These latest results are similar to those of Algeria's E. sideroxylon collected in two regions, which also revealed low EO yields of 0.77% in the Bainem forest, west of Algiers³³ and another rate of 0.7% in the Canstantine-Bkira³⁴. On the one hand, the Dellacassa et al³⁵ research team found a very low yield of 0.17% in E. sideroxylon EO from Uruguay. While the yield of E. sideroxylon EO obtained in Argentina has increased relatively compared to previous studies, at a rate of 1.65% for the March harvest³⁶.

 

Yields in all of this work remain lower than the results found in our study of Mamora's E. sideroxylon (2.38 to 5.48%). Furthermore, only one study is consistent with our results is that of Sebei and al²⁰, whose species E. sideroxylon from Tunisia provided a 3% profitability.

 

This observed variation in the yield of E. sideroxylon EO in different countries is probably due to climatic and geographical conditions, age of exploitation and collection, vegetative cycle of the organ, extraction mode and soil nature³⁷.

 

Chemical composition of the essential oil

The results of the one-year monthly monitoring of the chemical composition of E. sideroxylon essential oils are grouped in (Table 2). It emerges from this table, a qualitative and quantitative difference of the compounds identified, the number of constituents varies between 14 and 48 compounds with a rate that varies between 68.25 and 99.96% of the total constituents.

 

The molecules that make up the EO of the species studied belong to six chemical families: terpenes (mono and sesquiterpenes), ketones, alcohols, esters, aldehydes and oxides (Table 3). There is an abundance of the compounds in the oxides, alcohols and terpenes family according to the following decreasing classification: Oxides > Alcohol > Terpenes, usually for all months of the year.

 

It is noted that during the same period between April and August, there is an increase in the rates of terpenes (α-thujene, δ-elemene, β-humulene and caryophyllene oxide) and alcohols (terpin-4-ol, α-terpineol, spathulenol, globulol, widdrol and α-eudesmol) with percentages ranging from (18.5% - 46.39%) and (30.22 % - 33.34%) respectively. As for the oxides, the period between September and March is the exponential phase of the production of 1,8-cineole, which is the only representative of this family with a very high rate that oscillates between 72.67% and 84.73%

 

Indeed, the chemical profiles of E. sideroxylon EO, studied monthly for one year, were generally dominated by 1,8-cineole as a major compound during September until April, reaching high levels of 28,48% to 84,73% (Figure 1). This active principle is accompanied by other minor compounds with low levels: α-terpineol (1.07 % - 2.58%), α-thujene (2.67% - 6.02%), trans-dihydro-β-terpineol (0.47% - 2.02%), δ-elemen (0.97% - 1.81%), globulol (0.22% - 1.93%) and widdrol (0.07% - 1.44 %). For samples collected between April and July, they were distinguished by the presence of α-terpineol as the majority compound with rates ranging from 12.05% to 25.61%, with the exception of August, which is marked by the joint abundance of trans-muurola 4(14)5-diene and α-terpineol at values close to 17.60% and 12.05% respectively. During the latter period, α-terpineol is followed by minority compounds at lower percentages: globulol (2.91% - 12.09%), δ-elemène (7.07% - 9.37%), widdrol (0.31% - 5%), caryophyllane 4,8-α-epoxy (1.55 % - 4.78%), terpinyl α-isobutanoate (0.74% - 4.69%), terpin-4-ol (1.01% - 4.23%), carvone (0.24% - 3.50%) and α-eudesmol (2.61% - 3.22%).

 

Table 2: Monthly monitoring of the chemical composition of Eucalyptus sideroxylon essential oil

N°	KI	Components (%)	Jan	Feb	Mar	Apr	May	Jun	Jul	Aug	Sep	Oct	Nov	Dec
1	926	α-thujene	3.83	2.99	2.67	-	-	-	-	-	3.40	6.02	6.01	5.03
2	941	α-fenchone	0.12	0.12	-	-	-	-	-	-	-	0.10	0.12	0.12
3	969	Sabinene	0.10	0.11	0.18	-	-	-	-	-	-	0.13	0.19	0.14
4	979	β-pinene	0.27	0.19	0.47	-	-	0.04	-	-	0.25	0.18	0.30	0.22
5	994	Linalool trans- hydroxy oxide	0.52	0.62	0.20	-	-	-	-	-	-	0.29	0.46	0.22
6	1026	1.8-Cineole	76.44	80.68	84.73	28.48	10.32	6.36	5.42	7.19	86.11	73.10	72.67	79.74
7	1048	(E)-β-ocimene	0.64	0.13	0.56	-	-	-	-	-	0.19	0.14	0.30	0.24
8	1063	Sabinene cis- hydrate	0.05	-	-	1.02	-	-	-	-	-	-	-	-
9	1077	Camphenilone	0.19	0.13	0.14	-	0.12	0.09	-	-	-	0.14	0.16	0.15
10	1089	Dehydro-linalool	0.43	-	0.30	0.26	0.27	0.45	-	-	0.23	0.42	0.41	-
11	1105	2,6-Dimehyl phenol	0.14	0.14	-	-	0.16	0.16	-	-	-	0.12	0.09	0.08
12	1120	α-campholenal	-	-	-	0.92	-	-	-	-	-	-	tr	-
13	1133	Trans-dihydro-β-terpineol	1.74	2.02	0.77	0.90	0.33	0.61	-	-	0.47	1.57	0.95	0.56
14	1143	Trans-dihydro-α-terpineol	0.07	-	-	0.60	0.28	0.56	-	0.33	-	0.07	0.12	0.10
15	1155	Pinene β-oxide	0.26	0.25	0.12	1.81	-	-	-	-	-	0.21	0.20	-
16	1160	cis-dihydro-β-terpineol	-	-	-	-	1.49	1.43	0.90	0.81	-	-	0.29	0.39
17	1169	Terpin-4-ol	0.70	0.39	0.45	2.57	4.23	3.36	1.01	1.20	0.48	0.45	0.68	0.62
18	1180	p-cymen-8-ol	1.12	0.83	-	-	-	-	-	-	-	-	-	-
19	1182	α-Terpineol	1.44	1.07	2.36	18.06	25.61	21.11	14.72	12.05	1.43	2.34	3.14	2.58
20	1189	cis-Dihydro-carvone	0.14	-	-	-	-	-	-	-	-	-	0.11	-
21	1202	Verbenone	0.09	-	-	0.26	0.11	0.26	-	-	-	-	-	-
22	1209	Linalool formate	0.23	0.19	-	0.43	0.19	0.27	-	0.33	-	0.15	0.13	0.13
23	1219	Citronellol	1.39	1.12	0.50	1.13	1.41	1.53	1.35	1.93	0.31	0.89	0.93	0.65
24	1229	cis-Pulegol	-	-	-	-	0.40	0.20	-	-	0.17	-	-	-
25	1234	Pulegone	0.10	-	-	-	-	-	-	-	-	-	-	-
26	1240	Carvone	tr	-	0.24	1.55	3.50	3.23	2.41	1.01	-	-	-	-
27	1255	Linalool acetate	-	-	-	0.63	0.61	0.30	-	-	-	-	-	-
28	1261	Carvone cis-oxide	-	-	-	0.52	0.22	-	-	-	-	-	-	-
29	1275	Linalool dehydro acetate	-	-	-	0.58	0.09	0.14	-	-	-	-	-	-
30	1281	α-Terpinen-7-al	tr	-	-	-	0.25	0.14	-	-	-	-	-	-
31	1286	γ-Terpinen-7-al	0.13	0.06	0.06	0.76	-	0.07	-	-	-	-	tr	-
32	1304	Terpinen- 4-ol acetate	-	-	-	-	0.96	0.12	-	-	-	-	-	-
33	1307	Verbanol iso-acetate	-	-	-	0.49	-	-	-	-	-	-	-	-
34	1320	Verbanol neo-acetate	-	-	-	0.27	-	tr	-	-	-	-	-	-
35	1330	Piperonal	0.12	0.18	0.11	0.35	0.78	1.17	0.94	1.69	-	0.19	0.12	0.17
36	1337	δ-Elemene	1.21	1.81	1.25	7.78	6.15	-	7.07	9.37	1.00	0.97	0.98	1.63
37	1361	Citronellol hydroxy	-	-	0.19	-	0.13	0.23	-	-	-	-	-	-
38	1369	Piperitenone oxide	-	-	0.68	-	0.16	0.16	-	-	-	-	-	-
39	1382	Isobornyl propanoate	-	-	-	-	0.07	0.16	-	0.60	-	0.08	-	-
40	1404	Sesquithujene	-	-	-	-	0.20	0.35	-	0.55	-	0.09	0.06	-
41	1418	Caryophyllane 4,8-α-epoxy	0.30	0.24	0.09	1.74	1.55	1.54	1.70	4.78	0.15	0.90	0.49	0.18
42	1438	β-Humulene	0.26	0.14	0.13	2.58	1.75	2.72	1.73	5.32	0.19	0.75	0.47	0.39
43	1450	α-Humulene	-	-	-	-	0.43	0.39	-	0.91	-	0.13	0.07	-
44	1457	Sesquisabinene	-	0.08	-	0.87	0.65	1.34	-	1.97	-	0.32	0.22	0.16
45	1460	(E)-β-Farnesene	0.12	-	-	-	-	-	-	-	-	-	-	-
46	1475	terpinyl α-isobutanoate	0.23	0.15	-	0.74	1.99	2.14	1.61	4.69	-	0.32	0.32	-
47	1488	Germacrene D	-	-	-	1.07	-	-	-	-	-	-	-	-
48	1492	Trans-muurola-4(14),5-diene	0.45	0.41	0.52	-	6.64	9.94	8.99	17.60	-	1.86	0.90	0.35
49	1500	α-Muurolene	-	-	-	-	0.07	0.25	-	-	-	-	-	-
50	1511	(Z)-g-Bisabolene	-	-	-	-	0.89	0.18	-	0.43	-	-	-	-
51	1544	cis-Hydrate sesquisabinene	-	-	0.08	0.76	1.14	0.51	-	-	-	-	-	-
52	1549	Elemol	-	-	-	0.53	-	-	-	-	-	-	-	-
53	1560	Germacrene B	0.16	-	0.08	1.08	1.78	1.30	1.24	1.33	-	0.21	0.17	0.13
54	1566	Z-Acetate isoeugénol	-	-	-	0.86	-	-	-	0.43	-	-	-	-
55	1570	Caryophyllenyl alcohol	0.23	0.21	0.12	-	1.17	1.14	2.69	1.56	-	0.35	0.36	0.16
56	1575	Spathulenol	-	0.48	0.14	2.05	0.81	1.92	2.66	2.80	-	0.38	0.29	0.15
57	1579	Caryophyllene oxide	0.39	0.88	0.57	8.25	-	-	-	8.91	-	-	-	1.18
58	1587	Globulol	1.31	0.75	-	2.91	10.99	8.37	12.09	5.78	0.22	1.93	1.54	0.63
59	1596	Widdrol	0.88	0.07	0.38	0.64	0.31	3.73	5.00	0.49	0.19	1.39	1.44	-
60	1603	Trans-β-elemenone	0.40	-	-	-	3.81	0.97	-	-	-	0.41	0.47	0.19
61	1606	Epoxide II humulene	-	-	-	-	-	1.24	-	-	-	-	-	-
62	1619	Z-8-Hydroxy-linalool	-	-	0.13	0.39	-	-	3.40	1.80	-	-	-	-
63	1624	Pentanoate citronellyl	0.28	0.25	-	0.57	-	0.76	-	0.27	-	0.46	-	0.27
64	1637	γ-Eudesmol	0.11	0.12	-	0.75	1.49	1.19	1.28	1.07	-	0.27	0.11	0.13
65	1653	α-Eudesmol	0.25	0.23	0.21	-	3.11	3.22	2.61	3.19	-	0.27	0.23	-
66	1666	14-Hydroxy-(Z)-caryophyllene	-	-	-	-	0.24	0.25	-	-	-	-	-	-
67	1699	Eudesm-7(11)-en-4-ol	-	-	-	-	0.39	0.22	-	-	-	-	-	-
68	1716	(2Z-6E)-Farnesal	-	-	-	-	0.13	0.14	-	-	-	-	-	-
69	1734	Curcumenol	-	-	-	-	0.13	0.17	0.49	-	-	-	-	-
70	1757	β-(Z)-Curcumen-12-ol	-	-	-	-	0.30	0.38	1.03	-	-	-	-	-
Number of components			37	32	26	33	46	48	20	28	14	35	37	29
Total (%)			96.84	96.48	96.99	83.34	92.14	84.78	68.25	99.96	94.6	96.21	94.06	95.93

KI: Kovats  indices; tr: Trace

 

Table 3 :Monthly quantitative evolution of chemical families composing Mamora's Eucalyptus sideroxylon essential oil

% of chemical families	Jan	Feb	Mar	Apr	May	Jun	Jul	Aug	Sep	Oct	Nov	Dec
Terpenes	7.29	6.99	6.63	25.22	23.75	18.51	19.03	46.39	5.03	11.01	9.87	9.12
Oxides	76.44	80.68	84.73	28.48	10.32	6.36	5.42	7.19	86.11	73.10	72.67	79.74
Esters	0.51	0.40	-	1.31	2.06	3.06	1.61	1.56	-	0.86	0.32	0.27
Ketones	0.92	0.25	0.38	2.33	7.92	4.71	0	1.01	0	0.65	0.86	0.46
Aldehydes	1.07	1.1	0.46	2.85	2.71	3.06	2.64	6.47	0.15	1.38	1.07	0.57
Alcohols	10.56	6.38	5.26	30.22	52.89	48.79	37.14	33.34	3.08	9.08	9.32	5.77

 

Fig1: Evolution of the 1,8-cineole content of Eucalyptus sideroxylon essential oils according to the months of the year

 

These results allow us to see a diametrically opposite evolution over the year between the two components, 1,8-cineole and α-terpineol. In fact, there was a simultaneous rise in the rate of 1,8-cineole and a decrease in α-terpineol during the period (September - April) and the opposite for the other slice of the year (May - August). Therefore these results can be considered as indicators to differentiate the collections and also to guide the exploitation of E. sideroxylon during the period when the plant is richer in active principle in the Mamora forest.

 

In general, this metabolic relationship between the plant's 1,8-cineole and α-terpineol content could be attributed to the process of biosynthesis of these two constituents. Indeed, the orientation of biosynthesis is controlled by the alternation between the flowering and stress periods of the plant. During flowering, the two pathways of α-terpineol and 1,8-cineole biosynthesis are simultaneously favored with the preponderance of the latter between the months of September and March. While in April until August, which coincides with the plant’s period of biotic and abiotic stress, biosynthesis is more α-terpineol-oriented (Fig. 2). Similarly, recent studies have indicated that the biosynthesis of 1,8-cineole is reported by α-terpineol through the α-terpinyl cation³⁸, but the molecular details of the 1,8-cineole reaction mechanism remain elusive.

 

Fig 2: Percentage of 1,8 cineole and α-terpineol biosynthesis by month of the year

 

In general, the chemical composition of E. sideroxylon EO from Mamora (Morocco) is characterized by 1,8-cineole as the main chemical compound. On the other hand, previous studies in different national and international localities have also shown that E. sideroxylon EO are rich in this monoterpene with varying percentages. Indeed, E. sideroxylon EO, which are marked by high rates of 1,8-cineole, are collected in Morocco in the Marrakech region (Jbilet and Takarkoust) (76.9%; 80.9%)³¹, Algeria (77.1%)³³, Tunisia (80.75%)²⁰ and Argentina (91.27%)³⁹. As for other E. sideroxylon EO reported with a 1,8-cineole rate of less than 70%, they were found in the Oued Cherrat Arboretum in Morocco, where the 1,8-cineole only reaches 38,7%³². Similarly, in Tunisia⁴⁰ and Uruguay³⁵, the study of the chemical composition of these oils attests that 1,8-cineole thus acquires relatively low rates of 37.07% and 36.7%, respectively. South Korea’s E. sideroxylon essential oil is moderately rich in 1,8-cineole with a content of 54.8%⁴¹.

 

All this shows that the chemical composition of E. sideroxylon EO is highly variable and depends on several factors, mainly the period and origin of the harvest.

 

CONCLUSION:

This study determined monthly variations over a year in chemical composition and yield in EO extracted from the leaves of the species E.sideroxylon from the Mamora forest (Morocco). Quantitatively, the yields expressed by the studied plant are important, exceeding 2.3% with a maximum rate of 5.48 % recorded in April. Chromatographic analyzes showed that the chemical map of these oils is dominated by 1,8-cineole for an eight-month period of the year from September to April, including the autumn, winter and spring seasons. During this phase of the year, the levels of this active principle range from 72.67% to 86.73%. The other months that correspond to the dry season are marked by a decrease in 1,8-cineole levels by promoting an increase in the α-terpineol content, which varies between 12.05% and 25.6%. From the above, we have just realized that the pathways of cineole and terpineol biosynthesis are dependent on the dry and wet months of the year.

 

This work could contribute to discerning the favourable periods for harvesting E. sideroxylon leaves from the Mamora forest in terms of essential oils yield and quality particularly rich in the active principle "1,8-cineole" much demanded on the global market for better use in industrial sectors.

 

REFERENCES:

1.      Haut Commissariat aux Eaux et Forêts et à la Lutte Contre la désertification. Programme de conservation et de development des Écosystèmes Forestiers 2005-2014. 2014:1-14.

2.      Jean-Noel, M. et Azami AI. Le Développement Des Plantations Des Eucalyptus Clonales Au Maroc.; 2011.

3.      Lepoutre B. LA Mamora.; 1967.

4.      Aafi, A., EL Kadmiri, A., Benabid, A and Rochdi M. Richesse et diversité floristique de la suberaie de la Maamora (Maroc). Divers Floristique Mamor. 2005:127-138.

5.      Elaissi, A., Salah, K H., Mabrouk, S., Larbi, K M., Chemli, R., Harzallah-Skhiri F. Antibacterial activity and chemical composition of 20 Eucalyptus species’ essential oils. Food Chem. 2011;129(4):1427-1434.

6.      Joshi, R.K.; Joshi, B.C.; Sati MK. Chemical and chemotaxonomic aspects of some aromatic and medicinal plants species from Utrrakhand: a review. Asian J Pharm Technol. 2014;4(3):157-162.

7.      Krishnaveni, M.; Santhoshkumar J. Secondary metabolite, antioxidant, phyto nutrient assay of essential oil from dry Coriandrum sativum seed black variety. Res J Pharm Technol. 2016;9(7):853-856. doi:10.5958/0974-360X.2016.00161.X

8.      Prabhjot, K.; Anupam T. Medicinal values of secondary metabolites of Withania somnifera. Res J Pharm Technol. 2018;11(7):3167-3170. doi:10.5958/0974-360X.2018.00582.6

9.      Gurunani, S.G.; Yeole, M.P.; Gholse, Y.N.; Chaple DR. Hairy root culture: An experimental system for secondary metabolite production. Res J Pharm Technol. 2015;8(6):728-730. doi:10.5958/0974-360X.2015.00115.8

10.   Subasree, S.; Geetha R V. Essential oils in prevention of dental caries - An in-vitro study. Res J Pharm Technol. 2015;8(7):909-911. doi:10.5958/0974-360X.2015.00148.1

11.   Insira Sarbeen J. Preliminary phytochemical analysis of peppermint oil and tulsi oil. Res J Pharm Technol. 2015;8(7):929-931. doi:10.5958/0974-360X.2015.00154.7

12.   Vaishali, M.; Geetha R V. Popular medicinal plants used for dental diseases in India. Res J Pharm Technol. 2014;7(7):805-809.

13.   Vaishali, M.; Geetha R V. Antibacterial activity of orange peel oil on Streptococcus mutans and enterococcus-an in-vitro study. Res J Pharm Technol. 2018;11(2):513-514. doi:10.5958/0974-360X.2018.00094.X

14.   Tiwari, P.K.; Sahu P. Eucalyptus tereticornis: Phytochemical constituents and medicinal properties. Res J Pharm Technol. 2018;11(6):2677-2680.

15.   Shahwar, D., Raza, M A., Bukhari S and, Bukhari G. Ferric reducing antioxidant power of essential oils extracted from Eucalyptus and Curcuma species. Asian Pac J Trop Biomed. 2012;2(3): S1633-S1636.

16.   Ashour H. Antibacterial, antifungal, and anticancer activities of volatile oils and extracts from stems, leaves, and flowers of Eucalyptus sideroxylon and Eucalyptus torquata. 2008:399-403.

17.   Farah, A., Fechtal M, Chaouch A. Effet de l’hybridation interspécifique sur la teneur et la composition chimique des huiles essentielles d’eucalyptus cultivés au Maroc. Biotechnol Agron Soc Environ. 2002; 6(3): 163-169.

18.   Ghedira, K. Goetz, P., Le Jeune R. Eucalyptus globulus Labill. Phytothérapie. 2008; 6(3): 197-200.

19.   Joshi, R.K.; Joshi, B.C.; Sati MK. Artemisia capillaris: medicinal uses and future source for commercial uses from Western Himalaya of Uttarakhand. Asian J Res Pharm Sci. 2013; 3(3): 137-140. http://asianpharmaonline.org/AJPS/6_AJPS_3_3_2013.pdf.

20.   Sebei K, Sakouhi F, Herchi W, Khouja ML, Boukhchina S. Chemical composition and antibacterial activities of seven Eucalyptus species essential oils leaves. Biol Res. 2015;48. doi:10.1186/0717-6287-48-7

21.   Hmiri, S., Rahouti, M., Habib, Z., Satrani, B. Ghanmi, M and EL Ajjouri M. Évaluation Du Potentiel Antifongique Des Huiles Essentielles De Mentha Pulegium Et d’eucalyptus Camaldulensis Dans La Lutte Biologique Contre Les Champignons Responsables De La Détérioration Des Pommes En Conservation. Bull la Soc R des Sci liège. 2011: 824-836.

22.   Cimanga K, Apers S, De Bruyne T, et al. Chemical composition and antifungal activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J Essent Oil Res. 2002;14(5): 382-387. doi:10.1080/10412905.2002.9699894

23.   Kirsch F, Buettner A. Characterization of the metabolites of 1,8-cineole transferred into human milk: Concentrations and ratio of enantiomers. Metabolites. 2013; 3(1): 47-71.

24.   Fechtal,M and El Kadmiri A. Atlas Des Eucalyptus Du Maroc. [s.n.]; 1994.

25.   Aafi. A. Etude de la diversité floristique de l’écosystème de chêne-liège de la forêt de la Maâmora. Rabat. 2007.

26.   Association française de normalisation . Huiles Essentielles. Tome 2, Monographies Relatives Aux Huiles Essentielles. AFNOR; 2000.

27.   Clevenger JF. Apparatus for the Determination of Volatile Oil*. J Am Pharm Assoc. 1928;17(4):345-349.

28.   France E, Nf A. Pharmacopée européenne huiles essentielles. 2008:6-13.

29.   Adams R. Identification of essential oil components by gas chromatography/mass spectrometry. 2007; 456: 811.

30.   Kovats E. Gas chromatographic characterization of organic substances in the retention index system. Adv Chromatogr. 1965;1: 229-247.

31.   Zrlra, S., El Khirani, F., Benjilali B. Huiles essentielles de six espèces xérophyles d’ Eucalyptus: effet du milieu sur les rendements et la composition-chimique. Actes inst Agron Vet (Maroc). 1994; 14:5-9.

32.   Zrira, S and Benjilali B. Essential Oils of Twenty-seven Eucalyptus Species Grown in Morocco. J Essent Oil Res. 1992; 4(3): 259-264.

33.   Foudil-Cherif, Y., Meklati, B.Y., Verzera, A., Mondello, L., Dugo G. Chemical examination of essential oils from the leaves of nine eucalyptus species growing in Algeria. J Essent Oil Res. 2000; 12(2):186-191.

34.   Benayache, S., Benayache, F., Benyahia, S., Chalchat, J C., Garry R. Leaf Oils of some Eucalyptus Species Growing in Algeria. J Essent Oil Res. 2001; 13(3): 210-213.

35.   Dellacassa, E.Menéndez, P., Moyna P, Soler E. Chemical composition of Eucalyptus essential oils grown in Uruguay. Flavour Fragr J. 1990; 5(2):91-95.

36.   Lucia, Al., Licastro, S., Zerba, E., Masuh H. Yield, chemical composition, and bioactivity of essential oils from 12 species of Eucalyptus on Aedes aegypti larvae. Entomol Exp Appl. 2008; 129(1): 107-114.

37.   Khia, A., Ghanmi, M., Satrani, B., Aafi, A. Aberchane, M., Quaboul, B. Chaouch, A., Amusant, N., Charrouf Z. Effet de la provenance sur la qualité chimique et microbiologique des huiles essentielles de Rosmarinus officinalis L. du Maroc. Phytotherapie. 2014; 12(6): 341-347.

38.   Degenhardt J, Köllner TG, Gershenzon J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry. 2009; 70(15-16): 1621-1637.

39.   Toloza, AC., Alejandro, L., Zerba, E., Masuh, H., Picollo M. Eucalyptus essential oil toxicity against permethrin-resistant Pediculus humanus capitis (Phthiraptera: Pediculidae). Parasitol Res. 2010; 106(2): 409-414.

40.   Yangui, I., Zouaoui Boutiti, M, Mohamed Boussaid CM. Essential oils of Myrtaceae species growing wild in Tunisia: Chemical variability and antifungal activity against Biscogniauxia Mediterranea, the causative agent of charcoal canker. Islem. Int J Lab Hematol. 2017.

41.   Hyun, S R., Byung HL., Chung Gyoo BC. Acaricidal and repellent effects of myrtacean essential oils and their major constituents against Tetranychus urticae (Tetranychidae). J Asia Pac Entomol. 2013; 16(3): 245-249.

 

 

Received on 06.08.2020           Modified on 12.12.2020

Accepted on 18.03.2021         © RJPT All right reserved