Retention indexes of important compounds from 10 randomly selected essential oils.
Essential oil, sent by nature, is a complex mixture of volatile secondary metabolites. Its composition varies along with many parameters that can lead to misunderstanding of its wonderful pharmacological property. In fact, from post-harvest treatment to the compound’s identification through extraction approaches, the original chemotype of essential oils can be misdescribed. The pharmacological potentials of these oils are well known in the traditional system since time immemorial. Nowadays, some chemotypes of these oils had shown the effect against WHO’s top 10 killer diseases. But the misuses of these essential oils are in part due to the lack of robust and easy analysis strategy that can allow the quality of the essential oils.
- essential oils
- Kovats indexes
- 10 leading death diseases
Essential oils are a complex mixture of plant volatile compounds. Those compounds are essentially composed of terpenoids and phenolic compounds. The biosynthesis of these flavoring volatile compounds is done in dedicated cell types present in almost all parts of the plant, from the leaves or flower to the roots depending on the plant’s genus. These cells are glandular trichome, adduct cavities and osmophores were the compounds are biosynthesized and accumulated . According to the scent of these compounds, the plant that can produce those compounds that when extracted are called essential oils are then called aromatic plants. Aromatic plants are not specific to a given taxonomic group, but they are present widely across the plant kingdom. It must be mentioned that the composition of the essential oils is different from plant taxonomic group to another [2, 3, 4, 5]. It should be noted as well that, in the plant, compounds that will later form the essential oils are considered as secondary metabolites with a volatile characteristic. The variation in chemical composition of the essential oil can change from plant to plant even in the same species. These changes in the chemical profile are associated with many factors such as abiotic and biotic factors, postharvest treatment, extraction methods, and conservation conditions.
Abiotic factors group all the nonliving factors that affect the plant’s secondary metabolite production. This includes the soil hydrology, pH and salinity, and the climate in general but more interestingly the microclimate in which plant is growing [6, 7, 8, 9, 10].
Biotic factors group the living organisms that can affect the plant’s metabolite productions. These factors include, in one part, soil organism and microorganism. In fact, secondary metabolites present in essential oils are produced to help the plant to fight against plant invaders, interact with the symbiotic organism, and attract insects for pollination, among others. In another part, biotic factors are inherent in the plant itself.
Postharvest treatment include all the procedure that occurs between the plant collection and essential oil extraction. There are numerous reports that highlight the fact that drying plant material before extraction increases drastically the yield of extraction [5, 11, 12]. The counterpart of this method is the fact that after plant collection, there is a biochemical reaction that occurs between secondary metabolites. The output of this biochemical interactions is the changes in the chemistry of the obtained essential oil in comparison with that originally present in plant. At that point, the balance is between biological activity and the yield [5, 11, 12].
The extraction method can also lead to significant modification of the chemical profile of the essential oil. There are a huge number of approaches that can help to obtain an extract from the aromatic plant, including distillation methods, expression, solvent extraction, enfleurage, and microwave-assisted extraction. Apart from hydrodistillation, microwave assisted, and expression (specifically the citrus pericarp expression), all the other methods lead to a product that is not recognized as essential oil sensu stricto. In fact, with all the other methods, the extract obtained usually contains nonvolatile compounds that are extractable by the process and must be further removed. Also, the hydrodistillation method can lead to the chemical transformation of the compounds such as ester and other compounds that are highly hydrolyzable due to the long stay of the essential oil in water in the extraction system [13, 14, 15, 16, 17].
Regarding the conservation method that group all the process between the extraction and chemical analysis, the nature of the oil can lead to some modifications. In fact, most of the compounds that are present in essential oils are unsaturated (contain double bound) and most of them are sensitive to light (photosensitive) and to oxygen (oxidable).
Essential oils are used for their wonderful biological properties. The biological properties include the effect on humans, animals, plants, insects, and microorganisms. In humans, every single part of the human life can be affected by essential oils. In fact, essential oil is employed in nutrition as a food preservative or flavoring, in cosmetics as an odorant part, and in pharmacology as an active ingredient. The essential oil’s pharmacological properties comprise their effect on transmissible and non-transmissible diseases.
This chapter aims at going through the mechanism of essential secondary metabolite biosynthesis, essential oil extraction, essential oil chemical profile, and its pharmacological potential against the top list of human killer diseases as presented by WHO.
2. Chemical composition
Terpenoid and phenylpropanoid derivatives are the main components found in essential oils. In most plants, their essential oils contain terpenoids at around 80%. But the presence of phenylpropanoid derivatives affords the essential oils significant flavor, odor, and piquant. These two groups of compounds are derived from two different pathways from different primary metabolites .
2.1.1 Biosynthesis of terpenoids
The name terpene was first attributed to the compounds with molecular formula C10H16 by Kekule, a German chemist, due to its abundance in turpentine oil. The derivatives C10H16O and C10H18O were named generically camphor and related to terpenes. Wallach, Kekule’s assistant, characterized pinenes, limonene, dipentene, phellandrene, fenchone, terpinolene, and sylvestrene, which at that period were recognized as artifacts for turpentine oil . But nowadays, they are considered as compounds of some essential oils.
Terpenoids are a heterogeneous group of terpenes (compounds with double bonds) and their oxygenated derivative. Sensu stricto, an essential oil terpene, is a group of compounds derived from isoprene. Isoprene in its part is an organic scaffold with 5 carbon units with 1 double bond. Terpenoids are also called isoprenoids.
To start at the beginning, there are two pathways that lead to the synthesis of isoprene. These two pathways occur in the different parts of the upstate specialized cell. In the cytosol, the so-called mevalonic acid pathway is used, while in the plastid, the Rohmer or 1-deoxy-d-xylulose-5-phosphate is used. As this part is the most important part to understand the differences between essential oils’ chemistry and thereby their pharmacological properties, let us walk you through some chemistry (Figure 1).
According to plant species and foremost to the plant enzymatic ability, there is interconversion between certain compounds. The most observed case is that of conversion of thymol into carvacrol or vice versa. This depends on what plants need and environmental conditions [22, 23]. This leads in some case to a huge difference between chemotype of the same plants collected in different areas or different period of time during the same day or during different maturity stages. This has been reported for
2.1.2 Biosynthesis of phenylpropanoids
The synthesis of this class of compounds in aromatic plant leads to a wide variety of compounds, but in this chapter, we will focus on the pathway that leads to volatile compounds.
In comparison to isoprenoids, volatile phenylpropanoid compound synthesis occurs less often. But this is not the case for almost all plants; in fact for clove oil, eugenol is the major compounds .
The enzymatic arsenal involved in the synthesis of volatile compounds in the plant is not well known. Therefore, there are many approaches to explain the synthesis of those groups of compounds. But from a metabolite point of view, the starting point of these volatile phenols is the phenyl-alanine that is transformed into cinnamic acid by the phenylalanine amino lyase. The cinnamic acid in turn is transformed into para-COUMARIC acid. This latter compound depending on the enzymatic ability of the plants can undergo two different ways of synthesis (Figure 2).
2.2 Essential oil extraction
Essential oil extraction is one of the critical points that can affect the chemical profile of the essential oil. Sensu stricto, essential oils are a volatile odorant complex mixture obtained by distillation. Many techniques have been developed to obtain essential oil such as microwave-assisted extraction, expression, enfleurage, and solvent extraction. Most of these extraction methods lead most of the time to artefactual products as well as transformed products. To better understand, the next paragraphs will present the most used methods and their principal limit in the way of modification of the original chemical profile of the essential oil.
2.2.1 Distillation methods
Distillation methods are a group of methods using steam as compound vector or transporter. In fact, in distillation method, the plant material may be immersed or not in water, and after heating to water boiling point, the impression created in the reactor by steam as well as the high temperature will create the vaporization of these volatile compounds from their stockade cell to the environment of the reactor. The gas is pouched throughout a cooler. The condensation of the water and volatilized compounds from their vapor to water phase form a mixture that can be separated according to their density. There are two varieties of distillation methods: the hydrodistillation and the so-called steam distillation methods [38, 39, 40].
Hydrodistillation is an essential oil extraction method in which the material is immersed in water, but in the steam distillation, the plant material is not in direct contact with water but will go through, for example, steam flow before entering the cooler.
The principal limits of this method regarding the impact on the chemical profile of the essential oil are as follow:
The thermosensitive compounds will undergo a transformation or simply degradation. In fact, the high temperature can catalyze some chemical reactions that the normal cell will not, and this will lead to the chemical entities that were not present in the plants.
Lasting contact with water increases hydrolysis. Esterified compounds are highly sensitive to water as they will be broken down into compounds that do not exist in the essential oil storage cells.
When the glass Clevenger is used, the essential oil is exposed to light and can, therefore, undergo photo-oxidation and the chemical profile will reveal a high number of oxygenated derivatives that do not really exist in the plant.
Many reports have highlighted these observations and one of them is that of Kamii et al. . In fact when analyzing the chemical profile of
2.2.2 Microwave-assisted essential oil extraction
Microwave-assisted essential oil extraction is a variant of the distillation method where the heating source has been changed from the normal electric heating cap by the microwave. The plus here is the hypothetic increasing in extraction yield: hypothetic because the increase in yield is not as spectacular as tough [41, 42, 43]. It is true that it is better to crush the plant material, but in comparison to the classic distillation method, the essential oil yield is systematically the same. The principle of this method is based on the change of the polarity of water by the waves and of course the heating that will play the same role as in classic distillation method. This method has in addition the limit of the normal distillation method, the fact that the microwave can lead to chemical stereo switching from one isomer to another.
This method is specific for citrus pericarp. In fact, to avoid the thermal destruction, cold pressing of the citrus fruit rinds as the essential oil is store at that part of the plant has led to a good quality oil. The limit of this technique is the fact that it is not applicable to other parts of the plant [38, 39, 40].
Enfleurage is an old technique of essential aroma extraction. It is based on the solubilization of the essential oil’s component on a greasy wax. When using cold extraction, it can take more than a month, but this can be reduced to a few hours by heating. The aromatized wax is then called concrete. This later undergoes solubilization in a polar solvent and then partitioned with absolute ethanol. The product obtained after this process is at most a part of the essential oil as present in plant, and it contains many other terpenes that can be solubilized in fats used; that is, while at the end, the product is not called essential oil but absolute.
2.2.5 Solvent extraction
This is the oldest method for obtaining crude extract from plants. The principle is based on the solubilization of the compounds in the cells by the solvent. This method also has two variants as it can be run in room temperature or in high temperature. But no matter whether the extraction was performed in room temperature or not, the solvent will be separated by Rotavapor regarding the volatility of the solvent. This method does not really lead to obtaining the essential oil as all the nonvolatile compounds are also extracted by this approach. Therefore, the chemical profile will not be that of the volatile fraction of the plants but for the compound that is soluble in the solvent used in the process [13, 14, 15, 16, 17].
2.2.6 Supercritical fluid extraction
This method is the most modern and sophisticated. It uses gases at their supercritical stage. The gas at supercritical stage is liquid due to the high pressure applied to it. Many solvents can be used as the method brings the solvent at its temperature and pressure above its thermodynamic critical point, but the most used is CO2 for the reason that it needs less pressure to be liquified, it is less reactive than other, it is noninflammable, it is nontoxic and available at low cost with high purity, and most importantly it can be removed from the plant material using just the press release. This method is based on the fact that gas at the supercritical state can enter throughout the plant material like a gas and dissolve component like a liquid. After the extraction procedure, the essential oils compounds are mixed with the supercritical fluid (in liquid form). The separation is performed by reducing temperature and increasing the pressure up to room conditions [13, 14, 15, 16, 17]. The principal limit of this method is the complexity of the system.
2.3 Method for chemical analysis
There is a myriad of technics and methods for essential oil chemical profiling. All the methods used in organic chemistry can be used here. Due to their volatility nature, the compounds that constitute essential oil are preferably analyzed by gas chromatography (GC). Gas chromatography alone does not provide enough data for good chemical proofing. Therefore, many other analytical tools have been used such as mass spectrometry (MS), infrared spectroscopy (IR), and nuclear magnetic resonance (NMR). As well, many technics have been used to make the GC a better tool for chemical profiling, and these include chiral selective GC and multidimensional GC. Advances in liquid chromatography have highlighted the usefulness of high-pressure liquid chromatography (HPLC) as a tool for essential oil analysis. Many variants are available to date as multidimensional HPLC, HPLC-MS, and HPLC-GC. The following section will walk you through gas chromatography and gas chromatography coupled with mass spectrometry for their popularity and the Fourier transform infrared spectroscopy for its simplicity and for being environment-friendly and long-term cost-effectiveness.
2.3.1 Gas chromatography and variant in essential oil analysis
Gas chromatography is an old essential oil chemical profiling method. As all the chromatographic methods, it is based on the single compound separation from a complex matrix between two phases regarding their affinity between the phases, their shape, and mass. The mobile phase is the one which moves by capillarity or pressure transporting compounds faster or slower toward the fixed phase. In GC, the mobile phase is a gas and the fixed phase is a solid at room temperature, but at a high temperature, this phase will be slightly melted but will remain fixed on the column.
For analysis, the essential oil is mixed with a solvent (mostly hexane or pentane) and introduced into the injector room set at 200°C with the help of a syringe. The mixture is carried by the mobile phase into the column. Here the temperature drops down to about 50°C. All the compounds settle down at different distance in the column. This column itself is placed in an oven set with gradient increasing temperature (1–5°C/min). Each compound has it vaporization point and therefore when the oven temperature will reach this point, it will be volatilized thereby take off from the stationary (fixed) phase and carry and spit off out of the column.
Usually, the column is connected to a detector, which in the case of essential oil analysis is the flame ionization detector (FID). In this case, when the compound comes out of the column, it is burned and the increasing temperature in the flame, proportional to the compound scaffold, is detected and transformed into a peak. The compound identification is done by using the retention time and/or the Kovats index. The following formula as presented in Adams is used to calculate the Kovats index.
The linearization of this formula presented in Adams is as follows:
After calculation of index, the value is compared with that of the online libraries or the Adams library .
When the GC is connected to MS, the compound is accelerated and ionized into the different compartment of its scaffold and detected as bands.
Table 1 summarizes few retention or Kovats indexes reported in the literature for randomly selected plant and randomly selected papers. The diversity of chemotype obtained per essential oil is interesting. But the question about the accuracy of this profiling method has come up. Moreover, the Internet-based data bank proposes a huge range of retention indexes for the same compounds. For illustration mater, let us take the eucalyptol also called cineol-1,8. In the NIST database in the slightly polar column, here dimethyl-silicone with 5% phenyl group (DIMS 5P), this compound have a retention indexes ranging from 1021 to 1044 . This range represents 23 units in the retention indexes. Taking into consideration the Kovats or Van Den Dool and Kartz formula, this range (8 min 57 s to 9 min 44 s) represents a gap of 47 s. For the same column, in terms of polarity, in Adams Database, during this period, 24 single compounds can be identified . More complexly, there are overlappings in the range proposed by the NIST as reported by Babushok  as it can be observed in Table 1; for the same DIMS5P column, α pinene (921–944) is overlapping with camphene (936–959) and eucalyptol (1021–1044) is overlapping with ocimene (1028–1047) and camphor (1127–1155). These observations are the real pitfalls of these identification approaches and can lead to misuses of the valuable data from those data banks. In fact, these overlappings lead to compound identification oriented to the chemotype available in the literature rather than the real chemotype that is analyzed. And the previous paragraph had highlighted that the chemical profile thereby the pharmacological profile can change because of a myriad of factors. All these lead to questioning the robustness of this actual gold essential oil chemical profiling strategy not in the view of the provided database or analytic tools but in the view of data interpretation by the users.
|Allyl methyl disulfide||891–915||887–928||1241–1322||916||918||—||908||915|
|Allyl methyl trisulfide||1100–1132||1123–1165||1587–1605||1138||1116||1145||1126||1131|
2.3.2 Infrared spectroscopy and chemometric analysis
The idea besides the spectroscopy analysis is the fact that, in the electromagnetic radiation, there is a range of a tiny part of visible light that exists as a wave. This light moves in a straight line if the part of this light is not reflected, refracted, or absorbed by the matter. In biology and chemistry, this technique is used to produce an infrared spectrum in the case of infrared spectroscopy by passing an infrared radiation through a given sample knowing that the atom constituting the sample will lead to absorption of a part of the light energy. When a part of the molecule present in the sample absorbs this energy, it will become unstable and will react by twisting, stretching, bending, rocking, wagging, or scissoring depending on the bond linking these atoms with the rest of the molecule. The energy absorbed, the different reactions of the atom lead to the appearance of a specific peak on the spectrum [46, 47, 48, 49].
The spectrum can be at least qualitatively interpreted without prior or additional chemometric algorithm as it provides a high level of specific information on the molecular aspect of the essential oil . In fact, the spectrum regions are known and correspond to established characteristic group absorbances  and therefore the assignment can lead to specific chemical identification (Figure 3). But this is when the sample is a single compound. When the mixture is analyzed, the peak cannot be simply attributed to the compounds as it is most of the time the result of the overlapping small vibration of different closely related groups from a different molecule in the mixture.
For the quality purpose, the fingerprint region is most important. In fact, the fingerprint region is a unique vibrational signature that a given essential oil can have. It is as specific as the fingerprint is specific to a human. But to be more efficient in the analysis of the area, chemometric tool should be used such as derivation. This approach is a simple application of the derivate formula to the equation of the fingerprint region or a given region. This technique provides with key benefits. In fact, it enhances the resolution for the first derivative and the second derivative gives negative peaks of each band in the examined region  an example from Figure 3 is given inFigure 4A. It is also possible to apply other data processing such as baseline correction, smoothing, or curve fitting .
Using this approach, an increasing number of authors have succeeded in discriminating the essential oil of various plants based on their chemotype [46, 51, 52, 53, 54, 55, 56]. Those authors have suggested that for a known essential oil, the fingerprint of the oil should be used as a tool for rapid essential oil quality control.
3. Pharmacological property of essential oils
This part of the chapter is huge and covers various aspects of human and animal care. It will be easier to find a disease that no essential oil as a whole or its component can exert any effect. Essential oils are used to manage even diseases that are not scientifically measurable these days. These include protecting against misfortune and bad spirit and protecting the soul after death. This aspect of the therapeutic use of the essential oil will not be described here. The following paragraphs will be devoted to the review of essential oil use against top 10 human killers. Regarding the classification of 10 global causes of death around the world as published by WHO , ischemic heart disease, stroke, chronic obstructive pulmonary disease, lung cancer, diabetes, lower respiratory infection, diarrheal disease, tuberculosis, HIV/AIDS, and road injuries were respectively the most important human killers in 2016. Unfortunately, this chapter could not review the potential of essential oils against road accident.
3.1 Essential oil against ischemic heart disease and stroke
During the last 18 years, ischemic heart disease had won the gold medal in killing a human. In the second position with silver medal comes the stroke. In 2016, these affections were incriminated in 15.2 million human death . This is clear, known, and localizable but a silent terrorist against which all efforts should be focused on. These two affections share the same origin but different target. In fact, they are both cardiovascular diseases. Cardiovascular disease is known as a disease that affects the heart and blood vessels of the circulatory system . In this case, ischemic heart disease and stroke are specific to blood vessels. Ischemic heart disease is a class of cardiovascular disease that appears on the vessels that supply the heart cells with blood in terms of nourishing rather than pumping for other organs . Stroke on its part can be defined as neurological shortfall caused by crucial injury of the central nervous system. The reason behind is the cerebral infarction or intracerebral hemorrhage. Ischemic stroke accounts for 80%  of all types of stroke this chapter will focus on these later on.
Death happens when cells of these two vital organs are not supplied with nutrients and oxygen and are not free from their metabolic wastes. The main cause is the obstruction of the vessels by a clot (thrombus) from venal or upper artery atherosclerosis in coronary (in the heart) or arterioles (in the brain) and/or atherosclerosis on these two vessels [58, 59]. It is therefore clear that targeting specific clot dissolution or arteriosclerosis can help to manage ischemic heart and stroke disease.
In that regards, a massive number of essential oils have been studied for their effect on cardiovascular diseases. These reports were previously reviewed by Grenier et al.  for the vasodilatation effect. The major compounds from these essential oils were eucalyptol (
3.2 Chronic obstructive pulmonary disease and lower respiratory infection other than tuberculosis
Chronic obstructive pulmonary disease and lower respiratory infection other than tuberculosis are respectively the third and the fourth leading causes of death worldwide. These diseases caused cumulatively 6 million deaths in 2016. The lower respiratory infection disease owns the gold medal in communicable disease. It is important to notice that, in the developing world, the lower respiratory infectious disease is the first leading cause of death .
The chronic obstructive pulmonary disease (COPD) can be defined as a progressive respiratory condition clinically characterized by dyspnea, cough, and sputum production. This is the manifestation of alveolar enlargement and destruction as well as inflammation of lung parenchyma airways .
Lower respiratory infection disease is mainly caused by microorganisms including bacteria (other than Mycobacterium), fungi and viruses. A myriad of reports has proved the potential of essential oils as a possible source or as a drug against pathogens causing respiratory infection. Essential oils from
3.3 Lung cancer
Lung cancer is the fifth leading cause of death worldwide, and in 2016, WHO estimated that 1.7 million persons were dying from lung cancer. It is established that there is an urgent need for anticancer drugs due to the high selectivity that anticancer drug should possess. There are an important number of publications in the literature on the anticancer effect of the essential oils. More specifically, a certain number of essential oils have been claimed to possess anticancer effect against lung cell line; all these experiments are as per now
Diabetes is the sixth leading cause of death worldwide. In 2016, this affection caused 1.6 million deaths with an increase of 0.6 million compared to 2000. As the number of affected persons is increasing, this means that the management method is not that efficient, and alternatives are needed. Essential oils have been analyzed for their antidiabetic effect.
3.5 Diarrheal disease
Diarrhea is a disease caused by infection of the gastrointestinal tract by bacteria, fungi, virus, and ameba. The manifestation includes the urination of more than 3 liquid stools per day. The epidemiology of diarrheal disease has decreased from 200 with 2.4 million death cases to 1.4 million cases in 2016 . This reduction is mostly made by hygiene management. Essential oils have been analyzed for their beneficial effect in the therapeutic management of diarrheal infection [77, 78, 79, 80]. Some components as linalool, eucalyptol, terpineol, geraniol, thymol, carvacrol, spathulenol, caryophyllene, elemene, viridiflorol, bisabolol, eugenol, t-anetol, cinnamaldehyde, and allyl isothiocyanate were used to optimize the effect of the available drugs against some of the pathogens with successful results .
Tuberculosis is the infection of the lung by
HIV/AIDS is the acquired immunodeficiency syndrome, caused by human immunodeficiency virus. The disease is the ninth more important terrorist worldwide, killing in 2016 1 million persons . The mortality due to this virus has decreased along the years due to the generations of antiretrovirals as well as the protective measures adopted by the populations. There are only a few publications available on the effect of the essential oils on this virus. The reason can be the fact that this virus is very dangerous, and the testing equipment must be sophisticated to allow this test. However,
Essential oils are the oil of nature, sent by nature, the spirit of nature, and the soul of the forest. Their scent depends on their composition that in turn depends on the enzymatic package of the plant species. This enzymatic package can be influenced by external factors that can be biotic, depending on a living organism, or abiotic depending on molecules or climate and geography. The pharmacological potential of this spirit of the forest is a fact. But the scientific proof of that fact is subject to misunderstanding or misinterpretation. That may be why it is difficult to have essential oil recommended as an official drug for a disease. In fact, there are many variations that could affect the chemical composition not in the plant but during essential oil processes from plant collection time to the analytic method and the data interpretation. As well, Aromatic plants containing these scent of the nature are mostly present in tropical part of the word, and most of those tropical countries are developing countries and as such they do not have robust technology equipment to test and prove the whole efficiency of that essential oils, spirit of nature. To solve these small but crucial issues, scientists should come along with a robust analytical tool (FTIR, for example) and standard postharvest and extraction protocols for standardization of all essential oils not only in few local pharmacopeias but on a world level.
Conflict of interest
The authors declare no conflict of interest.