Chemical Characterization and Chemotaxonomic Significance of Essential Oil Constituents of Matricaria aurea Grown in Two Different Agro-Climatic Conditions

A comprehensive study on chemical characterization of essential oil (EO) constituents of a rarely explored plant species (Matricaria aurea) of the Asteraceae family grown in Saudi Arabia and Jordan was carried out. Analyses were conducted employing gas chromatographic approaches such as GC-MS, GC-FID, and Co-GC, as well as RT, LRI determination, and database and literature comparisons, on two diverse stationary phase columns, which led to the identification of a total of 135 constituents from both EOs. Oxygenated sesquiterpenes were found to be the most predominant chemical class of Saudi M. aurea EOs, in which α-bisabolol (27.8%), γ-gurjunenepoxide (21.7%), (E, E)-α-farnesene (16.3%), and cis-spiroether (7.5%) were present as major components. In contrast, the most dominant chemical class of Jordanian M. aurea oil was found to be sesquiterpene hydrocarbons, where (E, E)-α-farnesene (50.2%), γ-gurjunenepoxide (8.5%), (E)-β-farnesene (8.1%), and (Z, E)-α-farnesene (4.4%) were detected as chief constituents. It is interesting to mention here that Saudi and Jordanian M. aurea EOs showed quite interesting chemical compositions and were found to have different chemotypes when compared to previously reported M. aurea EO compositions.


Introduction
Currently, the increasing threats of contagious diseases and epidemics have forced scientists to explore different natural resources, such as medicinal plants and other marine organisms, for the development of novel pharmaceutics based on traditional knowledge [1][2][3].As of now, a variety of plant materials have been significantly explored and applied as precursors in different pharmaceutical, fragrance, and cosmetic industries to derive novel drugs, perfumes, and other applied ingredients for different medicinal applications [4,5].Over the years, despite significant success in using synthetic substances in the development of novel pharmaceutics, natural products, and particularly plant-derived materials, are still regarded as reliable sources of medicines and other applied materials, and are thus very popular in different industries [6][7][8].Indeed, due to the recent advancements in the techniques of synthesis and identification of phytochemicals, an enormous number of phytoconstituents have been extracted from plants and tested for their medicinal potential by utilizing modern methodologies and traditional knowledge [9,10].Among these medicinal plants, Matricaria aurea, which is an effective herb of the genus Matricaria, is an indigenous drug of the Kingdom of Saudi Arabia, and is largely known for its therapeutic potential and considered a promising source of antimicrobials and antioxidant agents.This plant exhibits various resemblances with M. chamomilla (chamomile), particularly in terms of the composition of its phytoconstituents of essential oils, such as flavones and flavonoids [11].
Plants 2023, 12, 3553 2 of 14 Moreover, the traditional applications of M. aurea are also similar to those of the main species (chamomile), and thus it is extensively applied and globally considered to be one of the ancient medicinal plants [12].
This crucial medicinal (M.aurea) plant is a member of the Asteraceae family, and is typically found in Saudi Arabia and in several other parts of the world.It has been widely applied in folk medicines for several diseases including cough, spasmodic, asthma, flatulence, common cold, and influenza [13].In addition to these, this plant is also known to exhibit analgesic and anti-inflammatory activities [14].So far, different types of phytoconstituents, including coumarins and quercetins, caffeic acid, apigenin-7-O-glucoside, umbelliferone, and naringenin, have been extracted from different species of Matricaria.Still, the detailed exploration of the phytochemical profile of M. aurea has been rarely performed, except in a few studies, which have superficially highlighted types of phytomolecules present in the species.For example, in our previous study, we prepared different extracts of M. aurea, which were explored for their anticorrosive properties against mild steel (MS) in corrosive media (1.0 M HCl).Among the different studied extracts, including water, n-hexane, and methanol, the methanolic extract demonstrated superior anticorrosive property, and was chosen for further detailed analysis to determine the active phytomolecules responsible for the anticorrosive action.This detailed analysis led to the discovery of a novel green corrosion inhibitor named apigetrin [15].Subsequently, in another study, Ahmad et al. studied the antibacterial efficacy of the ethanolic extract of M. aurea, which was tested against a variety of clinical isolates [12].Similarly, the same group further explored the remarkable medicinal properties of M. aurea by testing the anticancer properties of the plant against human breast adenocarcinoma (MCF-7) and other cell lines [16].Apart from these, and a few other studies, the aforementioned plant has not been explored appropriately according to its medicinal potential; in particular, the plant species of M. aurea from Saudi Arabia has been rarely explored.
It is worth mentioning that species of the same plant growing in different regions of the world generally exhibit vast chemical diversity, which is typically attributed to the presence of varying chemotypes.For example, the comparative analysis of the phytomolecules of the essential oils of the leaves and stems of Achillea fragrantissima of Saudi Arabia has revealed the presence of different types of major constituents when compared to the essential oils of the same plant grown in other regions of the world, such as Egypt, Jordon, and Yemen [17].Generally, this type of chemical diversity can be attributed to different factors, such as genetic variations and ecological and environmental factors [18].In addition, different atmospheric conditions, such as radiation levels, climatic conditions, temperature, and photoperiod, may also exert significant effects on the quantity and quality of the phytoconstituents [19,20].Indeed, these types of chemical diversities (specific variety of the chemicals produced by the plants) have been effectively utilized for the classification of the plants.This is referred to as chemotaxonomic classification [21], which is a modern strategy used to classify the plants.For example, A. fragrantissima grown in different regions of the same country (Egypt) has exhibited varying chemotypes; a plant of the Sinai region showed cis-thujone as a major component, while plants grown in Saint Catherine and Sharkia possessed α-thujone and santolina alcohol as their lead component, respectively [17,[22][23][24].In addition, A. fragrantissima obtained from different regions of Jordan, namely Mafraq and Amman, have exhibited the presence of artemisia ketone and α-thujone as major components, respectively [25,26].Similarly, Plectranthus cylindraceus grown in different parts of the world has exhibited various chemotypes; for instance, P. cylindraceus from Oman showed carvacrol as a major component, while the same plants grown in Yemen, Ethiopia, and Saudi Arabia possessed thymol, camphor, and patchouli alcohol as the most dominant compounds, respectively [27][28][29][30].Therefore, chemotaxonomic evaluation of M. aurea grown in different regions of the world may reveal interesting information, and, to the best of our knowledge, chemical characterization of M. aurea grown in different agro-climatic conditions has not been done yet.Moreover, as discussed earlier, M. aurea has wide applications in traditional medicine, but despite its vast medicinal potential, it has been relatively less explored.Thus, herein, the essential oils of the plant species M. aurea, which were grown in Saudi Arabia and Jordan, were subjected to extraction and analyzed in detail using GC approaches such as GC-MS, GC-FID, and Co-GC, and RT and LRI determination techniques.

Results and Discussions
In order to analyze and compare the phytochemical profiles of both the essential oils of M. aurea from Saudi Arabia and Jordan, the essential oils of the plants were isolated using a conventional hydro-distillation process, which was performed for three hours in a Clevenger-type apparatus [31].At the end of the extraction process, light-yellowcolored oils from both the plant materials were generated at the yields of 0.03% and 0.05%, respectively, which were measured as per the fresh weight of the plant materials.The chemical characterization of the extracted essential oils was carried out by applying GC-MS (gas chromatography-mass spectrometry) and GC-FID (gas chromatography-flame ionization detector) techniques on two different stationary phase (nonpolar and polar) columns.The GC analysis indicated the presence of 135 phytochemical constituents in the essential oils of both the plants from Saudi Arabia and Jordan.Out of these 135 identified constituents, only 56 phytomolecules were found to be present in both the essential oils, while 62 compounds were specific to the plant from Jordan and only 17 constituents were only associated with the essential oil of Saudi M. aurea.Notably, most of the specific phytochemical constituents of the Jordanian species were present in very minute quantities, i.e., 0.1 to 0.3%.The respective quantities of all the determined phytochemicals from both oils are presented in the form of a table (Table 1) based on their order of elution on a HP-5MS column.* Components are recorded as per their order of elution from an HP-5MS column; compounds higher than 1.0% are highlighted in boldface; LRI Lit = linear retention index from the literature [32][33][34][35]; LRI Exp a = linear retention index computed with reference to the n-alkanes mixture (C8-C31) on an HP-5MS column; LRI Exp p = linear retention index computed with reference to the n-alkanes mixture (C8-C31) on a DB-Wax column; SMA = Saudi M. aurea oil; JMA = Jordanian M. aurea oil; b = Mean percentage calculated from a flame ionization detector (FID).
As per the information given in Table 1, sesquiterpene hydrocarbons were present in the largest amount in Jordanian species, whereas the Saudi plant sample was mostly dominated by oxygenated sesquiterpenes.
For instance, the essential oil of Jordanian plant consists of 66.4% sesquiterpene hydrocarbons, while the Saudi species demonstrated the occurrence of 58.2% oxygenated sesquiterpenes.Notably, the sesquiterpene hydrocarbons and their oxygenated derivatives were dominant in both the essential oils; however, their amounts were different, i.e., the sesquiterpene hydrocarbons were present in 24.0% of the total contents in Saudi plant, whereas only 18.5% of oxygenated sesquiterpenes were present in the Jordanian species.On the second position, the Saudi species consisted of 7.7% polyacetylenic, while a similar Plants 2023, 12, 3553 6 of 14 group of compounds was found in the amount of 4.0% in the Jordanian species.It is worth mentioning that, in both the species, oxygenated aliphatic hydrocarbons were detected in a distant third position, and were present in almost the same quantity, i.e., 4.0% and 3.9% oxygenated aliphatic hydrocarbons in the Saudi and Jordanian plants, respectively.After these three major kinds of phytoconstituents, which were present in relatively large quantities, oxygenated monoterpenes (2.3% and 1.7%), monoterpene hydrocarbons (0.3% and 0.4%), aliphatic hydrocarbons (0.7% and 0.9%), diterpenoids (1.3% and 1.6%), and aromatics (1.0% and 1.3%) were also present in notable quantities in the Saudi and Jordanian M. aurea, respectively.In addition to these groups of compounds, other components were individually detected in miniscule amounts, but together they were significant, amounting to between 11 and 15% in both species.The total percentage of the identified compounds was found to be 98.3% and 98.7% in Saudi and Jordanian species, respectively.
There were only a few compounds that heavily dominated the list of major constituents of the Saudi species, and, out of the list of 73 compounds, only 11 compounds were present in more than 1% of the total phytochemical constituents (see Table 1).The major compounds in the Saudi species were α-bisabolol (27.8%), γ-gurjunenepoxide (21.7%), (E, E)-α-farnesene (16.3%), cis-spiroether (7.5%), (E)-β-farnesene (2.7%), diepicedrene-1-oxide (2.0%), artemesia ketone (1.8%), palmitic acid (1.6%), (Z, E)-α-farnesene (1.4%), (Z)-β-farnesene (1.3%), phytol (1.2%), and germacrene D (1.0%).The remaining 62 compounds were only present in <1% amounts of the total constituents of Saudi M. aurea.Notably, among the total 118 phytoconstituents present in the Jordanian M. aurea, only 7 compounds were present in relatively large quantities (>1% of the total compounds), while the remaining 111 compounds were present in minute quantities.Almost all the major compounds were same in both species, except α-bisabolol, which was completely absent in the Jordanian species (Figure 1).The chemical structures of lead compounds identified from both Saudi and Jordanian essential oils of M. aurea are given in Figure 2. Indeed, the major difference between the two species is the presence of α-bisabolol, which can be exploited for the chemotaxonomic identification of the Saudi M. aurea [36].The heatmap and dendrograms (Figure 3) were created using a total of twenty-four different types of phytomolecules found in each oil sample in variable quantities.For this analysis, only the phytomolecules with a quantity of 0.5% or more were included.The data obtained from these phytomolecules (>0.5%) revealed that the Jordanian and Saudi samples clearly form distinct clusters, which further confirmed our initial analysis as detailed earlier, i.e., the samples obtained from Jordanian and Saudi EOs exhibit distinct essential oil profiles.Saudi samples are marked by the higher content of oxygenated sesquiterpenes, including α-bisabolol, γ-gurjunenepoxide, gossonorol, and dehydrosesquicineole (Table 2), whereas Jordanian samples have a distinctly higher content of sesquiterpene hydrocarbons, Plants 2023, 12, 3553 7 of 14 such as (E, E)-α-farnesene, (E)-β-farnesene, and (Z, E)-α-farnesene.The biplot for PC1 and PC2 further confirmed that the essential oil profiles associated with Jordanian EOs are quite distinct from those of Saudi EOs (Figure 3B).The PCA analysis and the dendrogram therefore confirm that the Saudi samples are quite different than the Jordanian samples.A rigorous analysis based on a higher number of samples is required in future.α-Bisabolol belongs to the class of unsaturated monocyclic sesquiterpene alcohols, and is widely considered as one of the "most-used herbal constituents" globally [37].So far, a broad range of biological and therapeutic properties of α-bisabolol have been reported, including anti-oxidative and anti-cancer properties, for the treatment of inflammatory and metabolic disorders and neurodegenerative diseases [38].Four different stereoisomers of α-bisabolol possibly exist in nature, i.e., (-)-α-bisabolol (known as levomenol), (-)-epi-αbisabolol, (+)-α-bisabolol, and (+)-epi-α-bisabolol [39].Bisabolol is a low-density (0.93 in 20 • C) and pale-yellowish liquid, which can be easily oxidized to produce two bisabolol oxides (bisabolol-oxide A and B) [40].Notably, this compound is not present in all the species of M. aurea growing in different regions of the world; indeed, this substance is specific to the plants such as M. aurea and other plants of genus Matricaria which are found under harsh climatic conditions, such as very humid and hot summers with annual precipitation (ranging from 235 to 455 mm), e.g., the Persian Gulf with mild winters and the hot regions of Saudi Arabia, as specifically found in this study [41].For instance, this compound is not present in the Jordanian M. aurea as revealed in the present study; in addition, it is also not found in the Tunisian and Indian species [42,43].However, other than M. aurea, α-bisabolol can also be found in other plant species including M. recutita, Salvia runcinata, Silene stenophylla, Vanillosmopsis pohlii, Vernonia arborea, Myoporum crassifolium, and Eremanthus erythropappus.Indeed M. chamomilla is considered one of the major sources of this compound and consists of up to 50.0% α-bisabolol [44].When the overall components of the Saudi and Jordanian EOs were compared to the same species of plants from other regions, it was clearly revealed that the major components were completely different from each other (cf.Table 2).

No.
Country City Major Components (%) Reference

Saudi Arabia Alkharj
Bisabolol oxide A (64.8), n-nonadecane (6.7), 2R,3R, ALL-E)-2,3-Epoxy-2,6,10,14-tetramethyl-16-(phenylthio) hexadeca-6,10,14-triene (5.8), trans-β-farnesene (3.0), 1-fluorododecane (2.1), β-bisabolene (1.9). [45] 3 In the present study, we have, for the first time, discovered the presence of α-bisabolol in the M. aurea of Saudi Arabia, which is typically known to be present within the genus Matricaria of the family Asteraceae, but is only found in plants that grow under specific climatic conditions [42].Particularly, to the best of our knowledge, to date α-bisabolol has not been found in the M. aurea plants of other regions, including Jordan, Tunisia, and India [42,43].Therefore, it can be effectively used as a valuable marker to support the taxonomic classification of M. aurea species.Furthermore, several other derivatives of bisabolol were identified in the studied plants, such as β-bisabolol, epi-α-bisabolol, α-bisabolol oxide A, and α-bisabolol oxide B, which can be of vital importance as chemotaxonomic markers of the genus Matricaria.Notably, none of the derivatives of bisabolol were found in the Jordanian species.Therefore, compounds 101, 105, 106, 107, and 113 from Table 1, which are reported for the first time in the M. aurea plant of Saudi Arabia, can be used as further chemical markers to distinguish Saudi M. aurea from other Matricaria species growing in other regions of the world [42,43,45].Since the M. aurea plant is widely applied in Saudi Arabia for various medicinal purposes, the biological/toxicological profile of the phytoconstituents of M. aurea may offer useful information.In particular, the isolation of α-bisabolol and its in vitro and in silico studies, which we plan to perform in our future research, may offer valuable information.

Plant Material
Whole aerial parts of M. aurea grown wildly in two different agro-climatic conditions, namely Riyadh, Saudi Arabia and Amman, Jordan, were procured in the month of March.Fresh plant materials were taxonomically identified at the herbarium division of King Saud University (Riyadh, Saudi Arabia) and then processed further for the isolation of essential oils.

Extraction of M. aurea Essential Oils
Firstly, the fresh whole aerial parts of the procured M. aurea from Saudi Arabia and Jordan were chopped into small pieces and subjected to hydro-distillation in a conventional Clevenger apparatus for three hours as described previously [31].After hydro-distillation of Saudi and Jordanian M. aurea plant materials, light-yellow-colored oils with yields of 0.03 and 0.05% on a fresh weight basis, respectively, were obtained.These essential oils obtained from the aerial parts of the M. aurea were dried over anhydrous Na 2 SO 4 and stored at 4 • C until they were analyzed.

GC and GC-MS Analysis of M. aurea Essential Oils
In order to identify the chemical constituents of the Saudi and Jordanian M. aurea essential oils, volatile oils were dissolved in diethyl ether and subjected to GC-FID and GC-MS analyses.The GC analysis was carried out employing two different stationary phase columns, i.e., a nonpolar (HP-5MS) and a polar (DB-Wax) column using the same method as described previously [30].The detailed methodology is given in Supplementary Materials (Scheme S1).The identified constituents from the Saudi and Jordanian M. aurea essential oils and their relative percentages are given in Table 1 and the identified constituents are listed according to their elution order on the HP-5MS column.

Calculation of Linear Retention Indices (LRIs)
LRI values of chemical constituents of Saudi and Jordanian M. aurea essential oils were determined employing previously reported procedures [30], and LRI values of each component are listed in Table 1.The detailed methodology is provided in Supplementary Materials (Scheme S2).

Identification of Volatile Chemical Components
Identification of the chemical constituents of Saudi and Jordanian M. aurea essential oils was achieved through analysis of both oils on two different stationary phase columns, namely the HP-5MS and DB-Wax columns, as described previously [30].The detailed methodology for the identification of chemical constituents of Saudi and Jordanian M. aurea essential oils is provided in detail in Supplementary Materials (Scheme S3) [32][33][34][35].GC chromatograms of the analysis of both essential oils on an HP-5MS column are given in Figure 4.

Statistical Analysis
Heatmap and Principal Component Analysis (PCA) analyses were performed to evaluate the difference in the chemical constituents of the EOs of Saudi and Jordanian M. aurea.For the purpose of statistical analysis, each sample of EO was injected three times into a GC to obtain the standard deviation of the contents of the oil components.The data sets of the Saudi EO were named SMA-1, SMA-2, and SMA-3, while the Jordanian oil was referred to as JMA-1, JMA-2, and JMA-3.The data obtained were further used for heatmap and PCA analyses.An overall clustering of the six samples based on twenty-four different phytomolecules showing a content of more than or equal to 0.5% was carried out by calculating dendrograms, heat maps, and PCA using the web-based tool Clustvis [46].To visualize the relationship between the Saudi and Jordanian EOs, Clustvis-based R tools such as ggplot2, pheat, and pcaplot were used.

Conclusions
In this study, we explored the phytoconstituents of the essential oils of M. aurea obtained from different countries, i.e., Saudi Arabia and Jordan.The detailed chemical characterization of the volatile compounds of collected M. aurea plants was performed and the results of both the plants were extensively compared.The essential oils of M. aurea from Saudi Arabia exhibited a significant difference in their chemical compositions when compared to its counterpart collected from Jordan.Here, the presence of α-bisabolol was revealed as a major component (~27%) of Saudi M. aurea, which has so far not been found in the same plant from other regions, including Jordan, India, and Tunisia.By comparison, the Jordanian M. aurea consisted of γ-gurjunenepoxide (~22%) as its major constituent, which is also present in the Saudi plant, but in a relatively small quantity (~9%).Furthermore, the studied plants also contain (E, E)-α-farnesene (16.3%), cis-spiroether (7.5%), (E)-β-farnesene (2.7%), diepicedrene-1-oxide (2.0%), artemesia ketone (1.8%), palmitic acid (1.6%), (Z, E)-α-farnesene (1.4%), (Z)-β-farnesene (1.3%), phytol (1.2%), and germacrene D (1.0%).Therefore, α-bisabolol can be used as a valuable marker to support the taxonomic classification of M. aurea species.Furthermore, this study also reaffirms the same plant having different origins can have different phytochemical profiles which can be effectively used for the purpose of chemotaxonomic classifications.This was also confirmed by dendrograms and PCA analysis.Furthermore, it is important to mention that, in the present study, detailed chemical investigation of M. aurea revealed the presence of various antimicrobial agents.In particular, the presence of α-bisabolol as a major component in the Saudi M. aurea oil may provide great opportunity for the isolation of bioactive compounds, which could be used as potential candidates in the chemotherapy of infectious diseases.

Figure 1 .
Figure 1.Comparison of major components of Saudi and Jordanian M. aurea essential oils.

Figure 2 .
Figure 2. Chemical structure of the most dominant compounds from M. aurea essential oils.

Figure 3 .
Figure 3. (A) Dendrogram heatmaps showing comparative quantities of various essential oil components detected in Saudi (SMA) and Jordanian (JMA) M. aurea samples.Twenty-four components with a content of more than or equal to 0.5% of the total oil composition are used for analysis.(B) PCA of essential oil composition of Saudi and Jordanian samples, showing a distinct clustering of the two samples.

Table 1 .
Percentage composition of essential oils from aerial parts of M. aurea grown in Saudi Arabia and Jordon.

Table 2 .
Major components of M. aurea from different parts of the world.