Comparative study on the essential oils of Artemisia judaica and A. herba-alba from Saudi Arabia

1 Introduction

Curative plant species are nature’s endowment to individuals to shape a healthy life, and play a vital role to preserve our health that are implicit to be significantly nontoxic and proven elixir in the cure of different diseases. Due to its potentially medical and/or economical components, it has been considered significantly important to several phytochemical and pharmacological researchers in the past several decades. The major family of the flowering plants, Asteraceae consists of 13 tribes, 1000 genera and 23,000 species. The genus Artemisia comprises of about 500 species, is widely distributed in the northern moderate region (0–50 cm precipitation area) of the world (Marco and Barbera, 1990). Artemisia medicinal plant species are extensively used in folk medicine reducing phlegm, relieving cough, stopping pain, inducing sweat, dieresis, invigorating blood circulation, anti-hypertension, anti-helminthic, anti-toxic, and anti-allergy, anti-malarial, anti-bacterial, anti-fungal, anti-oxidant activity and anti-feedent (Kordali et al., 2005; Kelsey and Shafizadeh, 1979; Negahban et al., 2007). Bioactive phytoconstituents such as mono- and sesquiter penoids, flavonoids, coumarins, isoprenylcoumaric acid derivatives, caffeoylquinic acids, sterols and acetylenes constitute major classes of this genus (Kundan and Anupam, 2011; Tan et al., 1998).

Artemisia judaica L. is a perennial fragrant small shrub with pubescent leaves, bushy herbs, strongly aromatic, with timbered bases and burly spreading branches, covered by woolly hairs, leaves grayish, dissected, short, crowded, heads are rounded, crowded and made of tubular floret, which grow widely in the Kingdom of Saudi Arabia (Friedjung, 2010; Abu-Darwish et al., 2016). This medicinal plant has been traditionally used to treat gastro-intestinal disorders, poor eye sight, cardiovascular disease, skin disorders, and weak immune systems as well as to decrease the risk of atherosclerosis, cancer and arthritis (Janaćković et al., 2015; Abd-Elhady, 2012). Precedents showed it to be a great source of flavonoids including apigenin, cirsimaritin, and various novel compounds (Liu et al., 2003, 2004), the oxygen containing monoterpenes (54.2%) such as peritone, camphor and ethyl cinnamate (Abu-Darwish et al., 2016). Moreover, it included piperitone from Libyan plant (Janaćković et al., 2015), piperitone from Algeria that makes up about half of the oil (Stoyka, 2002; Dob and Chelghoum, 2006), terpinen-4-ol, bornyl acetate, chrysanthenone and cis-chrysanthenyl acetate (Soković et al., 2002) and a mixture of esters, ketone and aldehydes (Saleh, 1985; Mahmoud, 1985). Several biological assays have been depicted to A. judaica essential oils e.g. anti-helmintic, anti-inflammatory, analgesic and anti-pyretic effects (Batanouny et al., 1999), anti-helicobacter (El-Sayed, 2013), anti-oxidant (El-Massry et al., 2002), anti-microbial (Janaćković et al., 2015, Al-Gaby and Allam, 2000, Mahasneh, 1986), the increase in the isotonic concentration of isolated muscles of aorta, duodenum, ureter and coronary artery (Abdalla, 1979) and anti-angiogenic activity (Zihlif et al., 2011).

A. herba-alba Asso, a greenish-silver perennial herb grows 20–40 cm in height; it is a chamaephyte (i.e. new growth through the buds each year and are borne close to the ground) and is distributed in North Africa (Libya), Middle East (Saudi Arabia) and most of Europe (Jafri and El-Gadi, 1983). Traditionally it has been used as a medicine for healing external wounds (Baiky and Danin, 1981), gastric disturbances such as diarrhea, abdominal cramps and a decoction against fever, menstrual and nervous problems (Alzweiri et al., 2011). The phytochemical screening of essential oils (with golden yellow color and strong aroma) of this plant revealed the presence of herbal bin, cis-chryanthenyl acetate, flavonoids (hispidoline and cirsilineol), phenolic compounds, monoterpenes and sesquiterpenes (Moufid and Eddouks, 2012), oxygen-containing monoterpenes (77.3%) such as camphor, α-thujone, chrysanthenone (Belhattab et al., 2014), nonpolar irregular monoterpene alcohols: artemisia alcohol, santolina alcohol, yomogi alcohol and lyratol (Segal et al., 1980). Furthermore, the variety in oil composition from plants grown in different countries or different areas in the same country indicated the many oil-dependent chemotypes assigned to the plant (Abou El-Hamd et al., 2010; Lawrence, 1989). For example, artemisia ketone was reported as the principle constituent of an Egyptian chemotype (El-Sayed and Seida, 1990), while 1,8-cineole, camphor and chrysanthenone were a part of a French chemo-type (Hurabielle et al., 1981). Similarly, regular monoterpenes such as α- and β-thujones, sabinyl acetate, germacrene D, α-eudesmol and caryophyllene acetate were predominant in Jorden (Hudaib and Aburjai, 2006). Whereas in Libya, chrysanthenone, cis-chrysanthenylacetate, cis-thujone, filifolone, trans-thujone, trans-pinocarveol, and trans-sabinyl acetate were major constituents (Janaćković et al., 2015). Likewise, α-thujone and β-thujone from Spain (Salido et al., 2001), trans-sabinyl acetate, germacrene D, α-eudesmol and caryophyllene acetate from Jorden (Hudaib and Aburjai, 2006), 1,8-cineole, α- and β-thujone, terpinen-4-ol, borneol from Judean desert (Fleisher et al., 2002), monoterpene hydrocarbons, sesquiterpenes, p-menthane and pinane from Spain (Feuerstein et al., 1988), cineole, thujones, chrysanthenone, camphor, borneol, chrysanthenyl acetate, sabinyl acetate, davana ethers and davanone from Tunis (Haouari and Ferchichi, 2009; Neffati et al., 2008), camphor, α/β-thujones from Tunis (Mighri et al., 2009), 1,8-cineole and chrysanthenyl derivatives, camphene, borneol, davana ether, davanone from Algeria (Vernin and Parkanyi, 2001; Dahmani-Hamzani and Baaliouamer, 2005). Pharmacological activities of A. herba-alba have been reported as anti-diabetic (Al-Khazarji et al., 1993), anti-microbial (Zouari et al., 2010), anti-malarial, anti-oxidant and anti-radial (Kardi et al., 2011), anti-mutagenic (Neffati et al., 2008), anti-spasmodic and mild anti-biotic activities (Ross et al., 1980) and anti-Leishmania activities (Aloui et al., 2016).

Since both of these important medicinal plants belong to the same family, Asteraceae but a scientific approach about the aspects of the therapeutic uses of the essential oil of A. judaica and A. herba-alba from Saudi Arabia, as well as their chemical composition, remains scarce and incomplete. Hence, as part of our research interest on aromatic plants of Saudi Arabia (Khan et al., 2014, 2016a,b), in this paper, we aimed to determine the chemical composition of the essential oil of A. judaica and A. herba-alba from Saudi Arabia, and to assess their antimicrobial activity.

2 Experimental

2.6

2.6 Identification of volatile components and chemical constituents

GC–FID chromatogram of the essential oils of A. judaica (Fig. 1) and A. herba-alba (Fig. 2) have shown the identified peaks of major components on HP-5MS column. The identification of components was done by matching their mass spectra with the library entries (WILEY 9th edition, NIST-08 MS library version 2.0f as well as the Adams and Flavor libraries) of a mass spectra database as well as by comparing their mass spectra and linear retention indices (LRI) with published data obtained using both polar and nonpolar columns (Adams, 2007; Babushok et al., 2011; NIST, 2015; El-Sayed, 2015; Acree and Arn, 2012; Davis, 1990) and the co-injection of authentic standards available in our laboratory.

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Fig. 1 GC-FID chromatogram of A. judaica.

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Fig. 2 GC-FID chromatogram of A. herba-alba.

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3 Results and discussion

Perusal of literature on A. judaica and A. herba-alba illustrate that these plants have been previously studied for its phytochemical constituents. They differed in their reported aroma, physicochemical properties and in the composition of their main constituents. Of the Saudi Arabian plants, so far a detailed chemical characterization of essentialoils of A. judaica and A. herba-alba were studied here for the first time through (GC–MS and GC–FID) using both polar and non-polar columns which resulted in the identification of a total of 116 and 81 chemical constituents from the plants oil respectively (Table 2). Out of 110 compounds (96.7%) identified for A. judaica, the prominent volatile components were characterized as: β-eudesmol (13.1%), hexadecanoic acid (5.7%), spathulenol (3.7%), eudesma-4 (15), 7-dien-1-β-ol (3.5%), carvacrol (3.5%), thymol (3.5%), trans-sabinylacetate (3.3%), 14-hyroxy-α-muurolene (2.7%), cis-thujone (2.5%), α-cadinol (2.3%), pentadecanal (1.9%), cis-methyljasmonate (1.7%), germacrene-D (1.7%), β-sesquiphellandrene (1.7%), cis-jasmone (1.5%), 2-methylbutyl-3-phenylpropanoate (1.5%), aristolone (1.5%), cis-carvyl acetate (1.3%), iso-amylphenylacetate (1.3%), (Z)-lanceol (1.3%), α-cedrene-epoxide (1.1%), tetradecanal (1.1%), α-bisabolol (1.1%) and hexahydrofarnesyl acetone (1.1%). The ratio of the class of components present in essential oils of A. judaica varies greatly, from the oxygenated sesquiterpenes (45.1%) to oxygenated monoterpenes (25.4%), oxygenated aliphatic hydrocarbons (10.4%), sesquiterpenes hydrocarbons (10.0%) and esters (5.8%). Major constituents can form up to 60.4%, while the remaining components can be present in only trace amounts. We report the presence of valerenol, α-bisabolol, β-selinene, cis-carvyl acetate, hexahydrofarnesyl acetone, α-cedrene epoxide, iso-amyl phenylacetate, 2-methylbutyl-3-phenylpropanoate, hexadecanoic acid and aristolone for the first time from the oil of this plant. An interesting finding was the detections of 65 additional volatile components not previously mentioned in the literature of this plant. Additionally, the identified volatile components were also found to be exceptionally high in percentage composition such as carvacrol (3.5%), α-cadinol (2.3%) and β-eudesmol (13.1%) as compared to previously identified in 0.3% and 0.2%, 1.2% concentrations respectively (Abd-Elhady, 2012). Likewise spathulenol (3.7%) was found in high concentration as compared to 1.4% (Abu-Darwish et al., 2016) and 1.7% (Janaćković et al., 2015) and for α-thujone (2.5%) was higher than that reported in other Artemisia species (Negahban et al., 2007).

While crushed flowers, leaves and stems of A. herba-alba were hydrodistilled and 81 compounds (99.6%) were identified. The investigated essential oils of Saudi Arabian A. herba-alba was characterized by an exceptionally high percentage (almost half of the total oil composition) for piperitone (44.6%) followed by (E)-ethylcinnamte (14.7%), (Z)-ethylcinnamate (4.6%), thymol (3.4%), myrtenyl acetate (3.3%), spathulenol (3.3%), isophorone (1.9%), filifolide-A (1.5%), artemisia ketone (1.1%), piperitenone (1.1%). The ratio of the class of components present in essential oils of A. herba-alba varies greatly, from the oxygenated monoterpenes (65.2%) to esters (19.8%), oxygenated sesquiterpenes (6.6%) and others (3.6%). While hydrocarbons (monoterpene, sesquiterpenes and oxygenated aliphatic) were present in small amount. The major constituents can form up to 78% of the total essential oilsand many constituents have not been detected earlier in any A. herba-alba essential oils analyzed up to now. Although piperitone (44.6%) and (E)-ethylcinnamate (14.7%) were isolated in high percentage composition as compared to the previous report (Janaćković et al., 2015).

The comparison of chemical compositions of essential oils of A. judaica and A. herba-alba growing in Saudi Arabia between the present data and an updated survey of the existing literature reinforces the major variability of both plant essential oils (Table 1). It is also evident that the studied plant species has found to be high in compositions for spathulenol, thymol as compared to the same species in trace amount originated from Algeria (Belhattab et al., 2014) and Libya (Janaćković et al., 2015). Moreover, previously traced compound piperitone, was detected as a leading compound in the current study. It is significant to mention here that the chemical composition of A. judaica and A. herba-alba essential oils studied until now from different regions of the world has shown prodigious variations (see Table 1). However, it has been noticed that spathulenol was the only compound that was found either as a major or in appreciable amount in all the A. judaica and A. herba-alba essential oils studied so far. Thus, spathulenol could be used as a chemical marker for the Artemisia essential oils. The difference in ratio of some molecules in essential oils can be affected by different factors such as time of plant collection, humidity, climate and other environmental conditions.

Comparative findings on both plant species described different class of compounds where the monoterpenes and esters etc. were the major class of A. herba-alba, while sesquiterpenes and hydrocarbons were the main class in A. judaica (Fig. 3). It is pertinent to mention that among all the major identified compounds, besides α-thujone, trans-sabinyl acetate, cis-jasmone, isoamylphenyl acetate, β-sesquiphellandrene, α-cedrene epoxide, tetradecanal, α-cadinol, α-bisabolol, eudesma-4 (15)-7-dien-1-β-ol, pentadecanal, (Z)-lanceol, 14-oxy-α-muurolene, aristolone, (Z,Z)-hexahydrofarnesyl acetone and hexadecanoic acid (from A. judaica) and isophorone, filifolide-A, piperitenone from (A. herb-alba), only spathulenol and thymol were found to have similar percentage value in both plant species. Furthermore carvacrol, cis-carvyl acetate, germacrene-D, 2-methylbutyl-3-phenylpropanoate, cis-methyljasmonate, and β-eudesmol from A. judaica have high values as compared to A. herba-alba and similarly artemisia ketone, piperitone, myrtenyl acetate, (Z)-ethylcinnamate and (E)-ethylcinnamate were found to be the dominant essential oil components of A. herba-alba. While camphene, yomogi alcohol, 1,8-cineole, trans-pinocarveol, iso-borneol, terpinen-4-ol, cryptone, trans-carveol, p-cuminaldehyde, (E)-anethole, cis-patchenol, ethyldihydrocinnamate, trans-β-damascenone, cis-threo-davanafuran, β-selinene, α-muurolene, caryophellene oxide, α-muurolol and γ-dodecalactone were identified in both plants as trace components (see Table 2). This divergence in the profile of volatile constituents of A. judaica and A. herba-alba should be possibly attributed either due to geographical and ecological factors or an indication of the existence of different chemotypes of these plants.

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Fig. 3 Class of compounds in A. herba alba and A. judaica (A graphical representation).

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4 Anti-microbial study of A. herba-alba and A. judaica essential oils

It is pertinent to mention here that the plants secondary metabolites (PSMs), identified previously from A. judaica have been demonstrated to have various important biological activities and industrial applications. Precedents revealed that different compounds from both plants have different activities. For example from A. judaica, β-eudesmol is known for anti-tumor activity and anti-angiogenic activity (Long et al., 2008), hexadecanoic acid for anti-microbial and anti-oxidant (Abubakar and Majinda, 2016; Patra et al., 2015), while spathulenol (an oxygenated sesquiterpene) is identified for its immunomodulatory and MDR reversal activities (Ziaei et al., 2011). It is also used as a vital ingredient in perfumery, food, pharmaceutical, detergent and cosmetic industries (Leendert et al., 1988). Whereas, major compounds from A. herba-alba for example piperitone (a natural monoterpene ketone) is known for insecticidal activity and anti-fungal activity (Grudniewska et al., 2015) while (E)-ethylcinnamate showed anti-feedant activity (Singh, 2011). Hence, precedents on biological screening for both species have demonstrated efficacy against fungi but did not show activity against gram-positive and gram-negative bacteria except in one case, where A. judaica was able to inhibit against E. coli (Al-Gaby and Allam, 2000). Anti-microbial mechanism of action of different PSM-mixture is highly dependent on a number of factors such as the features of target cells (bacterial/fungal cell, Gram-positive/Gram-negative bacteria), and also the environment where the anti-microbial action should be revealed. Environmental conditions, hydrophilicity (i.e. solubility in water), concentration, temperature and pH are very important for the final effect of PSM-mixture (Denyer and Stewart, 1998).

In the present study, for the possible synergistic/antagonistic effects between PSMs and with controlled standard antibiotics, PSM-mixtures (e.g. essential oils) of both plant species (A. judaica and A. herba-alba) has been specifically considered for the anti-microbial activity as shown in Table 3, Fig. 4.

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Fig. 4 Antimicrobial study of A. herba-alba and A. judaica essential oil.

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Where the hydrodistilled oils of both plant exhibited moderate anti-fungal activities against some human pathogens such as A. fumigates, S. racemosum, G. candidum, C. albicans, S. pneumoniae, B. subtilis and E. coli. Comparison with the controlled drugs amphoterocin B, ampicillin and gentamycin is clearly revealed that both A. judaica and A. herba-alba oils showed high activities against all microorganisms. However, A. judaica was found to be much prominent as compared to A. herba-alba against all microbes except for Gram- –ve bacteria (P. aeruginosa) that was completely resistant against both plants. Generally, Gram- +ve bacteria is highly sensitive as compared to Gram- –ve bacteria. This sensitivity can be attributed to the cell membrane constituents, where the Gram- +ve bacteria surrounds an ineffective permeability barrier (an outer peptidoglycan layer) (Baba and Malik, 2014), whereas Gram- −ve bacteria holds lipopolysaccharides in their outer membrane, and therefore, the Gram- −ve strains are more resistance than Gram- +ve strains (Alzoreky and Nakahara, 2003). The anti-microbial action of PSMs points to the fact that their primary target site is the cytoplasmic membrane where some anti-fungal agents interact with ergosterol (the main sterol of the fungal membrane) involved in maintaining the fluidity, integrity of the membrane and regulation of enzymes necessary for the growth and division of fungal cells (Ahmad et al., 2011). For example, thymol, an aromatic p-menthane type monoterpene phenol and one of the common plant’s active compound provides interaction with both outer and inner cytoplasmic cell membranes, by integrating at the polar head group region of the lipid bi-layer. This alternates the cell membrane and leads to its increased permeability/disintegration (Kohanski et al., 2010). Similarly carvacrol, a compound isomeric to thymol, interacts with the cytoplasmic membrane by inserting the acyl chains of phospholipids (Di Pasqua et al., 2006). The efficacy of this compound is significantly enhanced by its hydroxyl group which functions as a transmembrane carrier of monovalent cations, leading to the disturbance of the membrane potential (Ben Arfa et al., 2006). Also, it was proved that carvacrol (a structure responsible for higher resistance of Gram- −ve bacteria) also affects the outer membrane by measuring the release of lipopolysaccharides, (La Storia et al., 2011).

Assays involving in vitro MIC methodology are commonly used and provides data on the lowest concentration of an anti-microbial that inhibits the visible growth of a microorganism after an overnight incubation. The value of MIC is an accepted standard for measuring the sensitivity of microorganisms (e.g. bacteria in their planktonic phase) to inhibitors (Kalemba and Kunicka, 2003). The effectiveness of the least amount of volatile oils separated from A. judaica and A. herba-alba was also tested to determine the Minimum Inhibitory Concentration (MIC) of the microbes (Table 4, Fig. 5).

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Fig. 5 MIC of A. herba-alba and A. judaicaessential oil.

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The comparison results of the essential oils from both plants with the reference controlantibiotics (amphoterocin B, ampicillin and gentamicin) showed that oils from A. herba-alba inhibited satisfactory counter effect for MIC against all the phytopathogens as compared to A. judaica. Although the values of MIC in both plants were found to be comparably higher than the standard antibiotics.

5 Conclusion

A. judaica and A. herba-alba essential oils have shown significant variations in their chemical compositions in relation to their place of collection. In the present study, comparison of essential oils of both Artemisia species growing in Saudi Arabia have shown a distinct composition among all major constituents except for thymol and spathulenol. To the best of our knowledge, α-bisabolol, cis-carvyl acetate, hexahydrofarnesyl acetone, α-cedrene epoxide, iso-amylphenylacetate, 2-methylbutyl-3-phenylpropanoate, hexadecanoic acid and aristolone were isolated first time from the oil of A. judaica. β-eudesmol, a natural sesquiterpenoid alcohol effective in neuromuscular blocking of succinylcholine (SuCh) in diabetic muscles, was also detected as one of the major components in the present study. Similarly, piperitone and (E)-ethylcinnamate isolated from the essential oils of A. herba-alba were detected with high percentage composition as compared to the previous reports. Considering the importance of major components of the essential oil of both Artemisia plants (abundantly available in Saudi Arabia). These two Artemisia species could be used as a cheap and renewable source for industrial isolation of commercially useful phytomolecules. Moreover, the hydrodistilled oils of both plants were found to possess significant antimicrobial activities.