The technological influence of the different methods of extraction on the initial concentration of thymol in the essential oil of thyme (Thymus satureioides)

The present study deals with the influence of the technological factor in relation to the technique used for the extraction of essential oil of thyme, its chemical composition, and in particular, the concentration of thymol as the principal chemo-typical active substance of this variety of thyme from the Moroccan High Atlas. The working protocol includes the extraction of the essential oil by three different methods (First method: Academic hydro-distillation; Second method: Training with water vapor using artisanal alembic; and Third method: Traditional hydro-distillation using artisanal alembic) and the comparative study of the individual thymol areas in the three spectrograms corresponding to the three essential oil samples and calculation of the differences. It emerges that the first method produces an essential oil very rich in thymol, followed by second method which produces an essential oil moderately concentrated in thymol, while the latter method produces an essential oil with very low concentration of thymol. It has been deduced that an important influence of the technological factor in relation to the type of the technique used for the extraction of the essential oil of thyme was exerted on the initial chemical composition, in particular, on the concentration of thymol.


INTRODUCTION
Among the critical points highlighted during the elaboration of the general strategic policy (GSP) of the national system of management (NSM) of the S: Safety/E: Efficiency/Q: Quality/E: Ecology ("SEQE") relating to medicinal plants (MP) and health products based on medicinal plants (HP.b.MP) abbreviated to "The P.S.G of N.S.M-S.E.Q.E relating to M.P/H.P.b.M.P.", especially in its axis dedicated to production itself noted the question "the performance of different methods and techniques of extraction of essential oil (EO)".
Of course, this question becomes even more critical when it highlights, in the light of the day, the performance of the technology (technique, method and equipment) used in the extraction of EO and its influence on quality.Of the latter, knowing that this is a very sensitive point that falls within the framework of the professional secrecy of the producers in the field and who exercise their trade in artisanal, academic or industrial (modern) ways.
It was in this context that the present study was conducted to test some techniques (methods) of extractions of EO: one academic (academic hydrodistillation) and two sub-techniques falling under a traditional method (training with water vapor using artisanal alembic and traditional hydro-distillation using artisanal alembic).
The main raw material used is a medicinal plant "thyme = Thymus satureioides", chosen to represent its category at the national level, both in terms of its endemism, nationwide (Morocco) production and its phytopharmacological interest.

Plant
After completing all the administrative requirements and procedures and following the Good Harvesting Practice Guidelines, sufficient quantity of the raw material "flowering aerial parts of thyme" at the level of the zone (Bin Elouidane in the province of Azilal/Beni Mellal, Morocco) was gathered during a two-day period, June 10 and 11, 2016, with the help of local collectors.

Identification the specimen
A preliminary identification of the plant was made on the spot, even before harvesting in accordance with the specific botanical monograph of T. satureioides resulting from the manual of determination of the vascular plants of Morocco, Practical flora of Morocco (Fennane et al., 2007).
After the harvest, a representative sample of the plant was deposited at the Scientific Institute of Rabat, Department of Herbal Botany and Ecology, for official botanical identification.

Extraction of essential oil of T. satureioides
After about 5 days of drying the raw material, the extraction of the essential oil of the saturated thyme was carried out by three methods, during three successive days, at the rate of one extraction per method and per day.
Three techniques (methods) of extraction of the essential oil include: one academic and two sub-techniques of a traditional method: The academic method (method 1) Academic, called this way because it is practiced in the context of academic research within the university (Laboratory of the Faculty of Medicine and Pharmacy of Rabat).Essential oil extraction method of thyme (aerial flowered parts) by academic hydrodistillation, ecological variant.
This was done using hydro-distillation apparatus, "Clevenger" type (Figure 1) and its principle consists of immersing the plant raw material, which in this case consists of aerial parts of thyme, in a bath of water boiled.Once released outside the cells, under internal pressure and chemical action of water, the essential oil of thyme is transported by stream of water vapor, cooled and condensed, further, through a condensation system and special refrigeration and subsequently discharged into a receiving container.During settling, the difference in the density between the water and the aromatic compounds causes the formation of an aqueous phase which composed essentially of hydrolate and an organic phase which composed essentially of essential oil.The latter and in the present case (ecological procedure) is separated, physically, from the first aqueous phase, using a simple separating funnel or other equivalent physical separation device, apart from any other chemical substance or solvent of separation.This gives the present procedure the title ecological.

The traditional methods
Extraction of the essential oil using artisanal alembic which has two sub-techniques.

The traditional sub-technique of training with water vapor (method 2)
Essential oil extraction method of thyme (aerial flowered parts) by training with water vapor using artisanal alembic (Figure 2), ecological variant.
This technique is different from that of the hydro-distillation, because the contact "steam/plant" is done "ex situ" in the case of the present technique (by training with the steam) and which is explained by the fact that the plant is not put in direct contact with water (in situ) as the case of hydro-distillation, but it is placed, at a level apart, in a perforated tank or special grid and the contact can only be done with water vapor (and not directly with water) and can only take place after evaporation of water in the boiler located at the lower level of the still.In fact, water is boiled in the lower tank also called "boiler" to generate water vapor that is passed through the plant to recover the oil and placed at the level of the perforated tank also called central tank-grid.Steam, by the effect of its temperature and its osmotic tension exerted on the plant, will destroy the cellulosic structure of the plant cells which will release their essential oils.Under the effect of heat, the oil evaporate and as two gases are always miscible, the water and oil vapors will mix and the water vapor carries with it the essential oils of the plant.Once vaporized, the volatile compounds of the essential oil are transported by steam flow, cooled and condensed, further, through a special condensing and refrigeration system (through the cooling water tank) and subsequently discharged into a receiving container.The decantation and separation steps are the same as in method 1.

The traditional sub-technique of hydro-distillation (method 3)
Method of extraction of essential oil of thyme (aerial flowered parts) by traditional hydrodistillation using artisanal alembic (Figure 2), ecological variant.
In essence, this technique is identical to that of academic hydrodistillation (method 1) with the only difference compared to the latter; it is done in a traditional way using traditional alembic (Figure 2).It is therefore and above all a difference of form between the two techniques.Indeed, the raw material is brought into direct contact with water (in situ) in the lower tank also called "boiler".The perforated central tank, also called a tank-grid or a "couscoussière", will remain vacant throughout the extraction process unless it has played the role of intermediation and passage of the volatile mixture between the lower tank (or boiler) and the upper tank cooling.The  mixture of water vapor and the essential oil obtained by heating is generated.This time directly in the boiler and by passing through the central tank, it is cooled at the level of the upper tank to recover the level of the harvesting container.The decantation and separation steps are the same as in the two previous methods.

Raw materials
The raw vegetable material is the flowering aerial parts of thyme (50 g for method 1, 1000 g for method 2, and 1250 g for method 3) in the semi-fresh state (only 5 days of drying in the open air and at room temperature).The parallel raw material, source of water vapor, is the tap water (1000 ml for method 1 and 8000 ml for methods 2 and 3).

The conditioning
The quantity of essential oil of T. satureoides (EO/TS), thus recovered, was put in vial(s), as it was, using a sterile syringe.No specific method of preservation (such as the sub vacuum or under nitrogen) was considered in this case study.

Storage
The three essential oil samples were kept separately, at a temperature of 4°C in the dark to preserve their original composition.

Initial chemical characterization of the essential oil extract HE/TS obtained
A first analysis (at t = 0) of the chemical composition of the essential oil (EO/TS) was carried out at the National Center for Scientific and Technical Research "CNRST"/Division of Technical Support Units for Research Scientific "UATRS", Rabat, Morocco, under the following conditions: (1) GPC/MS Analysis conditions: UATRS Standard Conditions (Essential Oil) (2) Apparatus: Gas chromatograph (TRACE GC ULTRA) coupled with a mass spectrometer (Polaris Q MS with ion trap).

Data processing
The data obtained after the analysis, were processed on computer, in the form of digital data thus allowing the development of a "spectrogram" which is a graphical presentation of the spectral map (peaks) of the various components of the analyzed product.
Special software, integrated with the GC-MS system, manages the digital data from the detector, by making an automatic comparative study with reference scientific data contained in computerized libraries belonging to the laboratory.

Comparism of the individual thymol areas at the three spectrograms corresponding to the three essential oil samples and calculating the percentage difference
All the parameters of the analysis (GC/MS) of all the samples at t = Elkacimi et al. 1183 0 months were fixed and identical, in particular, those linked to the technique of analysis, to the used equipment, to the manipulation of the analyst, to the analytical environment (temperature, humidity, pressure, etc.), and the automatic interpretation of the software.A very simple calculation approach was used by direct comparison of the individual areas corresponding to the thymol fractions, in the three different samples of the thyme essential oil.The calculation was done as follows: AT 1: Thymol area in spectrogram 1 corresponding to sample 1 of the essential oil extracted by method 1. AT 2: Thymol area in spectrogram 2 corresponding to sample 2 of the essential oil extracted by method 2. AT 3: Thymol area in spectrogram 3 corresponding to sample 3 of the essential oil extracted by method 3.
The largest of the three areas (AT X) corresponds to 100% of the substance.The percentages (Y) and (Z) of the two remaining areas (AT Y) and (AT Z) relative to (AT X) were calculated, using the equation (rule of three): Subsequently, the difference "ET A" (in percentage) between AT X and AT Y was calculated by: ET A = 100% -Y The difference "ET B" (in percentage) between AT X and AT Z:

Harvesting stage
At the harvest level, about 20 kg of the raw material was collected which composed of the aerial parts of the fresh plant of the saturated thyme, gathered from the mountainous area of Bin Elouidane (Morocco), in accordance with the good practices of harvests recommended by World Health Organization (WHO).An image of the plant sample is as shown in Figure 3.

Identification stage
The working sample was officially identified by the Scientific Institute of Rabat with the reference: "T.saturioides Cosson in Bull.Soc.Bot. France 58: 436. 1911 AS AA MA MA Mam -2-3 and registered at the herbarium of the same institute under the index number: RAB98065.

Extraction step
Approximately 1.5 ml of EO/TS essential oil was extracted from almost 50 g of the raw material in the laboratory of the faculty and in accordance with the aforementioned academic procedure.19 ml of the essential oil was extracted by method 2 from 1000 g of thyme, while using method 3 (artisanal), only 13 ml of essential oil was extracted from 1250 g of the plant.

Initial analysis step: Initial chemical characterization (at t = 0) of the essential oil EO/TS
The results of initial qualitative analysis (at t = 0) of the various samples of the essential oil, performed using gas chromatography coupled with mass spectrometry (GC/MS), are summarized in the following spectrograms: (1) Spectrogram 1 (Figure 4): Corresponds to the chemical characterization of sample 1 of the EO/TS (extracted by method 1); (2) Spectrogram 2 (Figure 5): Corresponds to the chemical characterization of sample 2 of the EO/TS (extracted by method 2); (3) Spectrogram 3 (Figure 6): Corresponds to the chemical characterization of sample 3 of the EO/TS (extracted by method 3).

Calculation of individual percentages and deviations
The calculated difference results between thymol areas (A T 2 and A T 3 versus A T 1 ) are listed in Table 3.These results are in accordance with the following basic principles of calculation: Given the importance of its area relative to the other two, A T 1 is considered to be 100% of the substance.The percentages (Y) and (Z) of the two remaining areas (A T 2 ) and (A T 3 ) are calculated on the basis of (A T 1 ), through the equation (rule of three): For Y The differences are calculated subsequently as follows: (1) The difference "E T A " (in percentage) between A T 1 and A T 2 :    (2) The difference "E T B " (in percentage) between A T 1 and A T 3 :

Deviation ET A Deviation ET B
Between AT 1 and AT 2 Between AT 1 and AT 3 47.81% 76.75% extraction of thyme, a significant decrease of about 47.81% was noticed in the thymol concentration in sample 2 as compared to that of the same substance in sample 1 and even greater decrease was noticed in the thymol concentration approaching 76.75% in sample 3 as compared to the same reference in sample 1.
This means that essential oil extraction methods: method 2 (artisanal extraction method by steam distillation) and method 3 (artisanal extraction method by hydrodistillation), produce essential oils of thyme, less concentrated in thymol (approximately 47% less for method 2 and about 75% less for method 3), as compared to method 1 (method of academic extraction by hydrodistillation).It is deduced that an important influence of the technological factor in relation to the type of technique and the method used for the extraction of the essential oil of thyme, was exerted on the initial chemical composition of the oil and in particular, on the concentration of thymol.

Conclusion
In view of the foregoing, it can be concluded that the technological factor in relation to the type of technique and method used for the extraction of thyme essential oil has a direct and important influence on the qualitative and quantitative aspect of the production of thyme essential oil, and therefore, its characteristics and consequently its properties, both physico-chemical and phyto-pharmacological.
The technological factor consists of a set of sub-factors and/or interactive cofactors that are related, as the case may be, to the different elements of the extraction method (or procedure), in particular and mainly the following elements: (1) The equipment used for extraction: Its nature (main component materials), for example copper, aluminium, stainless steel, galvanized iron, glass, etc.The technology of its manufacture (technological quality of its different parts as well as their arrangement and their sequence etc.) and technique of its use; (2) The technique itself, the extraction procedure: Is the choice of such a technique favorable or compatible with the medicinal plant; (3) The environment: Is the environment of the procedure favorable or not to the extraction of the plant, for example, the climatic conditions such as: temperature, pressure, humidity, etc; The ability and competence of the personnel responsible for carrying out the extraction procedure Faced with such a problem, both scientific, technical and practical, it is hoped the present study should open the way (at least at the national level) to other similar or even Elkacimi et al. 1187 more advanced and/or more specialized studies, to better elucidate this enigmatic set that constitutes the technological factor and its influence on the qualitative and quantitative process of the extraction.The results of this type of study will serve the cause of the national production of essential oils, in general, since they will form a basis of main guiding elements for better selection of suitable extraction equipment, to better target the exact procedure and optimal exercise environment and better performance and for a rational and intelligent production.

Figure 3 .
Figure 3. Photo taken on the premises of the raw vegetable material.Source: Elkacimi (2016).

Table 1 .
Initial spectrogram (graphic presentation of the spectral map (peaks)) of the different components of the Thymus satureioides essential oil extracted by method 3, at t=0.Retention times: RT = 18.46 correspond to "Thymol".Source: Elkacimi (2016).Representation of injection conditions.

Table 2 .
Representation of separation conditions.

Table 3 .
Representation of the differences (%) of the thymol areas, successively AT 2 and AT 3 compared to AT 1.