الفهرس 
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Abstract
Essential oils are volatile natural complex products; each is made up of many, sometimes hundreds, of distinct molecules (terpene alcohols, aldehydes, ketones and esters (>90%) and/or phenylpropane derivatives) which come together to form the oil’s characteristic aroma and therapeutic properties. At present, approximately 3000 essential oils are known, 300 of which are commercially important especially in the industries of pharmaceutical products, food preservation, cosmetics and perfumery. Medically, they exhibit a broad range of useful biological activities as antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic and local anesthetic, in addition to the popular use in aromatherapy.
The research accomplished in this thesis includes six plant species; three species belonging to family Apiaceae (Umbelliferae): Carum carvi (Caraway), Apium graveolens (Celery), Anethum sowa (Dill), and three species belonging to family Lamiaceae (Labiatae): Mentha × piperita (Peppermint), Rosmarinus officinalis (Rosemary), and Thymus vulgaris (Thyme). The oil content and composition of these species were studied and relevant comparisons were made between different methods of extraction in order to ensure the best choice of technology. The present study also determines the relationship between the composition of essential oils and their biological activities; antibacterial and antioxidant activities.
The present study comprises three main parts:
1. Extraction of essential oils.
2. Analysis of essential oils.
3. Biological activities of essential oils.
1. Extraction of Essential Oils
Among the different extraction methods described for the extraction of essential oils from aroma-active plants, both hydrodistillation (water distillation and steam distillation) and super critical fluid extraction (SFE) were used for the selected plants.
1.1. Hydrodistillation
Laboratory scale water distillation was done on both entire and ground (fine) plants materials to study the effect of grinding on the quantity and quality of essential oil. While in pilot scale steam distillation (10kg), only entire plants materials were selected to compare between water distillation and steam distillation considering; yield and time of distillation process, as well as composition of different fractions obtained by both methods. In steam distillation, the recovery of all volatile organic constituents depends on their partitioning between the water and oil phases of the distillate. Therefore, water-soluble (polar) components are usually lost in the water phase, which is considered the major disadvantage of this method, due to the decrease in the yield and loss of valuable aroma-active components. Studies have been done to qualify and quantify these water-soluble compounds in distillation wastewater. In order to optimize the recovery of essential oils, the distillate was cohobated by further water distillation.
Grinding of plant materials before its water distillation leads to considerable loss in their oil contents and changes in oil compositions, which is variable and directly related to the composition of oil in each plant. The percentages of loss in oils of plants under investigation ranged from 2.90% to 65.28%. Members of family Apiaceae showed highest losses in oil contents (37.5% - 65.28%) which may be due to rupture of the oil ducts (schizogenous oleoresin canals known as vittae) and escape of some oil components due to the slight increase in fruits temperature during crushing. Also, these plants contained mainly the monoterpene hydrocarbon limonene which is a highly volatile compound; the % loss in celery oil was 65.28% (limonene was 66.23% in entire fruit while 40.54% in ground one), dill was 47.83% (30.93% to 6.43%), and caraway was 37.5% (31.77% to 10.64%). While plants of family Lamiaceae showed lower losses in oil contents (2.90% - 23.10%) as the oil is stored in glandular hairs and cells that not easily crushed. Peppermint showed lowest losses only 2.9 – 6.25% as it contained mainly oxygenated compounds with low volatilities, while the loss in rosemary was 9.7% as 1, 8 cineole content dropped from 15.94% to 11.98%, finally thyme which contained 25.2% p-cymene and became 10.0% after grinding showed 23.10% loss in oil content.
The efficiency of steam distillation was less than water distillation; it ranged from 41.3% to 84.38%. Steam distillation was more efficient in plants of family Lamiaceae (59.7% in rosemary, 75.0% in thyme, and 80.43 – 84.38% in peppermint) than plants of family Apiaceae (41.3% in dill, 51.32% in celery, and 59.24% in caraway). After cohobation (water distillation of distillate), the recovered oil obtained increased the efficiency of steam distillation and it ranged from 54.72% to 96.88%, which is dependant on the amount of water soluble compounds in each plant oil. Concerning plants of family Apiaceae: in caraway which contains a high amount of oxygenated monoterpene, carvone (65.9%), about 46.10% of the steam distilled oil was recovered, in dill about 36.85% was recovered as it contains also carvone (50.8%), while in celery the recovery was only 6.63% as the oil does not contain highly water soluble compounds. While plants of family Lamiaceae including peppermint (menthol about 38.0%) and thyme (thymol 35.96%) recovered about 14.5% of the steam distilled oil, and in rosemary the recovery was 35.01% as it contained both camphor (15.8%) and verbenone (5.14%).
Fruits of family Apiaceae needed more time for distillation than leaves of family Lamiaceae. Steam distillation of Apiaceae fruits consumed more time than water distillation; caraway required 13.0 hrs versus 5.5, celery required 11.7 hrs versus 10.5 hrs, and dill required 9.3 hrs versus 7.0 hrs. In contrast to leaves of family Lamiaceae; peppermint required only 1.3 hrs versus 3.0 hrs, rosemary required 2.2 hrs versus 4.0 hrs, and thyme required 3.6 hrs versus 8.0 hrs. It was also observed that about 80.0% of the oil is obtained from fruits at about half of the total time of distillation, while from leaves it was only quarter of the time.
1.2. Supercritical Fluid Extraction
Supercritical fluid extraction (SFE) which involves the use of carbon dioxide in the supercritical state (the pressure and temperature must be above the critical point of CO2 i.e. 31oC and 74bars) as the ’solvent’ which is pumped through the plant material to dissolve and carries the essential oil away, then with release of pressure, carbon dioxide escapes in its gaseous form, leaving the essential oil behind. In SFE experiments, the samples were extracted at 121 bar (1759 p.s.i.), 40C, and density was 0.72 g/ml at a flow rate 2.0 ml/min. Extraction time was 40 min; 5 min of static extraction (to equilibrate the contents of the extractor cell), followed by 35 min of dynamic extraction.
It was observed that celery SFE showed higher yield than WD (5.04% versus 0.92%), followed by dill (2.62% versus 1.2%), then rosemary (2.5% versus 0.56%), caraway (1.72% versus 1.5%), thyme (1.25% versus 1.5%), and finally peppermint (1.1% versus 1.0%).
2. Analysis of Essential Oils
The characteristics of oils and extracts produced in Egypt were found to be unique and specific to this region. This assumption was tested to quantify and qualify properties of essential oils to give scientific information to the essential oil industry in Egypt as well as to authenticate and specify the preferred chemical profile of the essential oils. This information may not have to meet international specifications, which were established in different countries using reference material originating in different climates, from limited plant varieties and using different horticultural practices.
By comparing the essential oil obtained by water distillation and the product obtained by SFE, it was found that water distillation oil contained higher percentages of terpene hydrocarbons. In contrast, the SFE oil contained a higher percentage of oxygenated compounds. However, sometimes SFE revealed to be not suitable to obtain essential oils; producing concrete like extracts, due to co-extraction of cuticular waxes and fatty acids.
Hydrodistilled caraway volatile oil fractions contained six compounds mainly; carvone decreasing from 87.84% to 14.80% and limonene increasing from 11.37% to 84.57%. The recovered oil was rich in the oxygenated compound carvone (87.36%). Caraway SFE extract contained mainly carvone (72.0%), trans-dihydrocarvone (10.0%), and trans-carveol (9.0%), limonene was nearly absent. While in water distilled oil of ground caraway fruit, also carvone was the main compound but with a higher percentage (85.07%) and limonene content was 10.64%. trans-carveol was completely absent.
Hydrodistilled celery volatile oil contained twelve compounds mainly; limonene decreasing from 85.28% to 28.03%, ethyl mandelate increasing from 4.18% to 29.91%, p-ethylacetophenone from 3.23% to 20.59%, and -selinene from 1.25% to 8.85%. Recovered oil contained mainly limonene (69.9%) and -selinene (14.5%). SFE extract of ground celery fruits contained mainly ethyl mandelate (74.78%), ethylacetophenone (10.92%), and -selinene (6.47%), while monoterpene hydrocarbons especially limonene were nearly absent. In water distilled oil of ground celery fruit, limonene was the main constituent and was 40.54%, also -selinene was present at higher percent (14.98%). Both ethyl mandelate and ethylacetophenone were present at lower percentages; 22.96% and 8.01%, respectively.
Hydrodistilled dill volatile oil contained eight compounds mainly; carvone decreasing from 67.4% to 4.54%, limonene increasing from 12.5% to 43.98%, and a phenylpropanoid called dillapiole increasing from 4.61% to 50.13%. The recovered oil con-tained high % of carvone (62.0%), however, limonene was only 12.7%, and dillapiole was only 8.7%. SFE extract of ground dill fruits contained mainly dillapiole with high percentage (68.43%), low percentage of carvone (21.15%), and limonene was nearly absent. While in water distilled oil of ground dill fruit, both dillapiole and carvone nearly have the same percentages (42.22% and 41.77%, respectively), and limonene was 6.43%.
Hydrodistilled peppermint volatile oils contained about twenty five compounds mainly; menthol a monocyclic monoterpene alcohol ranging from 16.83% to 48.78%, menthone a monocyclic monoterpene ketone decreasing from 31.77% to 2.97%, 1, 8-cineole decreasing from 8.23% to 0.0%, menthyl acetate a monoterpene ester increasing from 4.07% to 18.38%, and bi-cyclic sesquiterpenes hydrocarbons;-caryophellene from 1.31% to 18.85%, and germacrene-D from 0.86% to 11.39%. The recovered oil contained high % of menthol (50.40 - 51.69%), also contained 18.37% menthone, 8.93% menthyl acetate and 6.44% 1,8 cineole. Peppermint SFE extract contained mainly menthol (50.91%), menthone (17.94%), and nearly all monoterpene hydrocarbons were absent. While in water distilled oil of ground peppermint leaves, the main constituents were also menthol (36.63%), menthone (22.07%), and menthyl acetate (7.79%). Some differences were observed in hydrodistilled peppermint (3) volatile oils; menthone was very low decreasing from 11.12% to 2.38%, isomenthone was larger than menthone (18.68 - 9.94%), menthyl acetate was very high increasing from 14.25% to 42.07%, and 1, 8-cineole (1.95 – 0.0%), -caryophellene and germacrene-D were very low.
Hydrodistilled rosemary volatile oil contained about twenty five compounds mainly; -pinene a monocyclic monoterpene hydrocarbon increasing from 10.14% to 35.68% (the reverse in SD; decreasing from 26.75% to 5.17%), 1, 8-cineole a thermolabile monoterpene ether decreasing from 26.39% to 0.0%, endo-borneol a monoterpene alcohol decreasing from 8.61% to 3.99% (the reverse in SD; increasing from 4.79% to 15.56%), and monoterpene ketones; L-camphor decreasing from 22.35% to 0.83% (the reverse in SD; increasing from 14.26% to 20.23%), and verbenone increasing from 3.73% to 13.31%.The recovered oil con¬tained a high percentages of oxygenated monoterpenes; L-camphor (22.83%), endo-borneol (17.01%), verbenone (28.85%), -pinene was not present completely. Rosemary SFE extract contained mainly verbenone (17.02%), camphor (10.10%), endo-borneol (7.9%), bornyl acetate (6.3%) and nearly all monoterpene hydrocarbons were absent. While in water distilled oil of ground rosemary leaves, the main constituents were camphor (16.48%), -pinene (14.65%), 1, 8 cineol (11.98%), and verbenone (11.0%).
Hydrodistilled thyme volatile oil contained mainly twenty eight compounds mainly; alcoholic aromatic monoterpenes thymol increasing from 19.28% to 71.0% and carvacrol from 0.0% to 7.33%, p-cymene an aromatic monoterpene hydrocarbon decreasing from 39.4% to 2.56%, and -terpinene a monocyclic monoterpene hydrocarbon from 18.99% to 2.87%. The recovered oil con¬tained high percentages of oxygenated compounds, particularly thymol (76.31%) and carvacrol (5.24%), while monoterpenes hydrocarbons p-cymene and -terpinene were very low (only 6.71% and 2.19%). SFE extract of ground thyme leaves main constituents were thymol (69.76%) and carvacrol (7.92%), and nearly all monoterpene hydrocarbons were absent. While in water distilled oil of ground thyme leaves, the main constituents were thymol (59.4%), carvacrol (4.74%), and p-cymene (10.0%).
3. Biological Activities of Essential Oils
Generally, the major components are found to reflect the biological features of the essential oils, the amplitude of their effects being just dependent on their concentration when they were tested alone or comprised in essential oils. However, it is possible that the activity of the main components is modulated by other minor molecules since interactions have shown to lead to additive, synergistic or antagonistic effects. In that sense, for biological purposes, it is more informative to study entire oil rather than some of its components because synergism appears to be more meaningful.
3.1. Antibacterial Activities
The antimicrobial activity of essential oils is assigned to a number of small terpenoid and phenolic compounds (thymol, carvone, limonene, 1, 8 cineole, pinene and their precursors), which also in pure form have exhibited antibacterial or antifungal activity. The differences in chemical composition and relative proportions of the individual constituents in the essential oils of the plant affect the activities. Essential oils could possibly be rendered more active if the relative concentrations of antimicrobial components were adjusted to levels that consistently provide the required strength and spectrum of inhibition. This could be accomplished by distillation of the crude oil to yield preparations with a constant and reproducible composition, or by mixing of individual fractions to achieve a desired level of activity.
The antibacterial activity and minimum inhibitory concentration (MIC) of the different obtained volatile oils fractions were screened using the ”Agar-well diffusion method”. Since the goal of the study is to evaluate the impact or the reflection of different oil extraction methods &/or conditions on the biological activity of the obtained oil, it was preferred to determine the antimicrobial susceptibility as well as MIC using bacteria against which most of the oils show consistent and promising antimicrobial activity. So according to our results in the antimicrobial susceptibility testing, a robust antibacterial activity was observed against Staphylococcus aureus ATCC 6538.
Recovered thyme oil was the most active oil fraction (MIC was 0.3125%v/v) followed by recovered peppermint oil (MIC was 2.5%v/v) and thyme oil rich in p-cymene (MIC was 2.584%v/v), then celery SD2 fraction (MIC was 4.5%v/v), recovered dill oil (MIC was 4.838%v/v), recovered caraway oil (MIC was 5.13%v/v), and finally recovered rosemary oil which was least active (MIC was 8.57%v/v).
It was observed that carvone may be responsible for antibacterial activities in both caraway and dill. Recovered oils containing highest level of carvone (87.36% and 62.2%, respectively) were the most active among other oil fractions. Celery oil’s main active constituent may be limonene; and the first hydrodistilled oil fractions with highest content of limonene (86.71%) were more active than the last fractions. Menthol was the most active constituent in peppermint; as the recovered oil containing the highest level of menthol (50.4%) showed highest activities. In rosemary oil, two components showed activity, verbenone and 1, 8 cineole. Verbenone was more active than 1, 8 cineole at levels above 12.0% (recovered oil containing 28.85% verbenone), followed by 1, 8 cineole (descending activities were observed when its level decreases from 26.39% to 5.97%). In thyme oil, synergism was observed between thymol and p-cymene; thymol was the most active constituent, but p-cymene potentiated it. In fractions with highest p-cymene levels and lowest thymol levels, larger inhibition zones were observed (first hydrodistilled oil fraction, p-cymene was 38.03% and thymol was 19.28%) but was less potent as MIC was 2.584%v/v, while last hydrodistilled oil fraction and also recovered oil containing highest level of thymol (76.31%) and lowest level of p-cymene (6.71%) gave smallest IZ but was more potent as MIC was 0.3125%v/v.
3.2. Anti-Oxidant Activities
Antioxidant activity of volatile oils was assayed spectrophotometricaly with diphenylpicrylhydrazyl (DPPH) radical. Quercetin was used as a positive control (IC50 = 3.42 ± 0.21 µg/ml). Essential oils which gave lower IC50 indicate their higher potency than those which have higher IC50.
Thyme oil showed lowest IC50 (0.161 l/ ml) indicating that it is the most powerful antioxidant (contained strong antioxidant phenolic compound thymol 52.8% and p-cymene 18.75%), followed by rosemary with IC50 3.560 l/ ml (contained camphor 23.11%, endo-borneol 11.68%, 1,8 cineole 10.17%, and verbenone 9.73%), then dill with IC50 4.931 l/ ml (contained carvone 54.4%, limonene 19.1%, and dillapiole 13.1%), peppermint with IC50 9.768 l/ ml (contained menthol 43.35% and menthone 18.94%), celery with IC50 10.158 l/ ml (contained limonene 57.8% and ethyl mandelate 18.7%), and the least active as antioxidant was caraway with IC50 19.567 l/ ml (contained carvone 74.8% and limonene 24.8%).