Comparative Studies of Chemical Compostion, Antimicrobial and Antioxidant Activity of Essential Oil of Some Species from Genus Artemisia

Background and Objective: Some species from genus Artemisia were used in ancient medicine since Pharaonic civilization, most species of this genus are grown in arid or desert zones in Egypt. The present study aimed to evaluate and compare the chemical constituents, antimicrobial and antioxidant potential of the essential oils of three species of Artemisia (Artemisia sieberi, Artemisia judaica and Artemisia monosperma) grown in Egypt. Materials and Methods: Chemical constituent of the essential oils of these species were analysed by GC-MS, antibacterial activities were carried out using disc-diffusion test and antioxidant properties were investigated with Iron, Fe (III) to Fe (II) reduction and DPPH radical scavenging capacity. Results: The results of chemical analysis revealed similarity between the three species in 20 compounds, the major compounds identified in essential oils were verbenol, (7.5 % and 11.51%) in A. sieberi and A. monosperma, β-caryophyllene oxide (1.25 % – 0.78%) and Methyl jasmonate (0.9 % and 1.21 %) in A. sieberi and A. judaica, respectively. While the major compound in A. judaica was Lilac alcohol C (9.6%) and camphor (4.5%). The antibacterial investigation exhibited significant and broad-spectrum antibacterial efficacy of A. monosperma and A. judaica against different strains of Gram-positive and Gram-negative bacteria. Whereas, A. sieberi showed higher antibacterial efficacy against the gram-positive bacteria but weak or no effect against the Gram-negative bacteria. The results of the antioxidant investigation showed that A. sieberi present the higher reduction capacity with an IC50 of 0.17 ± 0.03g/L, followed by A. judaica with an IC50 of 0.58 ± 0.04g/L and A. monosperma with an IC50 of 6.35 ± 0.41g/L. However, the reducing capacity of ascorbic acid and quercetin were 0.091 ± 0.002g/L and 0.026 ± 0.002g/L respectively. Conclusion: The present study revealed that the essential oils of A. sieberi, A. judaica and A. monosperma possesses significant antioxidant and antibacterial activity, which attributed to the plenty of varied chemical compounds in these medicinal plants.


Introduction
Medicinal plants and natural products have attracted the scientific interest all over the globe, medicinal plants are essential components of primary health care for up to 80% of the Earth's inhabitants, they are also intervened in 11% of up to 252 drugs of modern pharmaceutical industries 1 .
Essential oils are important compounds used in traditional medicine extensively in industries. From 3000 known essential oils only 10% of them are commercially important in pharmaceutical, food, cosmetic, perfumery industries 2,3 .
Generally, little knowledge is known about desert plants, which exists in harsh environmental condition. Artemisia is genus of small herbs and shrubs, belong to Asteraceae, comprises over 500 species, which are mostly found in Asia, Europe and North America. Number of these genus member of Anthemideae tribe which is famous with medicinal and ethnopharmacological properties is prescribed in the cosmetic, flavor and fragrance industries 4 . Interestingly, members from this genus are used in ancient Egyptian civilization as found in some medical papyri dating back to 1850 B.C. 5 A. judaica L. is a perennial fragrant shrub which grows widely in the deserts and it is very common in Sinai Peninsula in Egypt, it is used as an anthelmintic drug in most North African and Middle-Eastern countries where it is known by the Arabic name of "shih" 6 . A. sieberi is traditionally used for the treatment of different ailments. Some studies analysed the chemical constituents of A. sieberi growing in Iran and France using gas chromatography-GC-mass spectrometry (GC-MS), and showed antimicrobial activity 7 . A. monosperma is growing in different regions of Egypt, it grows in desert and semi-desert climate, and have many medicinal properties 8 .
There are many factors influencing on the variations in the essential oils contents of plant species belonging to the same plant family, the climatic variation that occur over the course of a year is one of the major impacts, which is important to identify the most appropriate time of the year for optimal extractions in terms of yield and/or compound concentration. When different climatic factors in seasonal climates with two well-determined seasons is modified, these variations could act on the plants and alter their metabolism 9,10 .
The chemical composition of essential oils from the Artemisia genus has been extensively studied in several species from around the world, however, little studies is known about them in Egypt, many studies have shown that Artemisia species display significant intraspecific variations in the terpene constituents of their essential oils. In some cases, the variation in the volatile components of these plants may occur during plant growth at different altitudes. For the cultivated species, the quality and yield of essential oils from Artemisia species is influenced by the harvesting season, fertilizer and pH of soil, the choice and stage of drying conditions, the geographic location, the plant part, the plant genotype, or the extraction method 11 .
In literature, an exhaustive survey have been conducted on different species from genus Artemisia, which showed that they have a vast range of biological activities including antimalarial, cytotoxic, antihepatotoxic, antibacterial, antifungal and antioxidant activity. Some very important drug leads have been discovered from this genus, notably artemisinin, the well known antimalarial drug isolated from the Chinese herb A. annua. Terpenoids, flavonoids, coumarins, caffeoylquinic acids, sterols and acetylenes constitute major classes of phytochemical constituents of that genus 12 .
The aim of this study was to determine the potential variations occurring in the essential oils yield and chemical composition of three plants from genus Artemisia, grown in agro-climatic condition of Egypt in addition to their antioxidant and antimicrobial properties.

Plant Material
Flowering aerial parts of three species from genus Artemisia, A. sieberi, A. monosperma and A. judaica ( Figure 1)

Extraction and Analysis of Essential Oils
Two ml of extract prepared in a soxhelt with petrlum ether: ether (1:1) as decribed above were hydrodistllied for 4hours, the distillate was extracted by n-hexane, seprated the organic layer in separating fynle and concentrated under reduced pressure to 1 ml and dried over anhydrous sodium sulphate. The compounds were analyzed using a Thermo GC-Trace ultra system (Thermo Co. USA), they were separated on 30m X 0.25 mm X 0.25 µm Elite-5MS column (Thermo Scientific GC Column). The column temperature was increased from 40 o C to 220 o C at a rate of 4 o C/ min; injector temperature, 250 o C; injection volume, 1 µl; helium carrier gas flow rate 20 ml/min; transfer temperature, 280 o C. MS parameters were as follows: EI mode, with ionization voltage 70 ev, ion source temperature. The constituents were identified by matching their mass spectra in the Wiley 275.L library and by comparison of their retention indices with literature values 13 . Retention indices were determined using retention times of n-alkanes that have been injected to the same instrument and under the same chromatographic conditions. Relative percentage amounts were calculated from the total area under the peaks by the software of the apparatus 14 .

Fe (III) to Fe (II) Reduction Capacity
The reduction capacity of Fe (III) to Fe (II) was done as described previously (Abd Alla et al., 2016) 15 . Briefly, 1 ml of each concentration was mixed with 2.5 ml of phosphate buffer (0.2 mol/l, pH 7.0) and 2.5 ml of potassium hexacyanoferrate K 3 Fe(CN) 6 solution and they were incubated for 30 min at 50°C. Then, 2.5 ml of trichloroacetic acid (10%) was added to this mixture to stop the reaction. Afterwards, 2.5 ml of this mixture was homogenized with FeCl 3 (0.5 ml, 0.1%) and distilled water (2.5 ml). The absorbance was measured at 700 nm and the concentration of the samples at which the absorbance of 0.5 (EC 50 ) was determined. Quercetin and Ascorbic acid were used as positive control for comparison.

DPPH Radical Scavenging Capacity
In this test, 0.5 ml of each concentration was homogenized with 0.5 ml of DPPH methanolic solution (0.04 g/L). Then, this solution was mixed vigorously and putting in darkness for 30 min at a temperature of 25 °C. Afterwards, The absorbance of the mixtures was measured at 517 nm 15 , and the percentage inhibition was calculated as: A blank 100 Quercetin and ascorbic acid was used as positive control and the concentration providing 50% inhibition (IC 50 ) was calculated from the graph of inhibition percentage plotted.

Antibacterial Testing
Ten bacterial strains (Gram-posttive and Gram-negative) including referenced bacterial strains and clinically isolated strains were used to evaluate the antibacterial properties of the flowering aerial parts of A. sieberi 15 . Briefly, using sterile loop one colony from each of the over-night subcultures containing the above motioned microorganisms was dipped in to a tube containing sterile normal saline and adjusted to be equivalent to 0.5 McFarland standard (Approximately 1-2 × 10 8 CFU/ml). This adjusted microorganism was swabbed over a previously prepared sterile Mueller-Hinton agar. The dry volatile oils were reconstituted in Ether at concentration 500 mg/ml, and without delay 20 µl from that concentration was dropped over 6 mm blank discs (Whatman No.1) and left to dry under aseptic conditions. The dried discs were loaded over the swabbed sterile Mueller-Hinton plates, Erythromycin (15 μg/disc) was used as positive control, blank disc loaded with Ether only and dried with the tested discs was used as negative control. Mean zone of inhibition was calculated after overnight incubation at 35 o C.

Statistical Analysis
For quantitative data, One-way ANOVA analysis was used, P ≤ 0.05 considered significant. SPSS version 11 was used in statistical analysis of quantitative data and for graph drawing.

Fatty Acids
Fatty Acids Analysis of the three investigated species from genus Artemisia revealed the presence of varied bioactive fatty acids as shown in (Table 2), Linolic acids are present in the three species where Arachidonic acid, Erucic acid and Oleic acid are found in two species and not detected in other one. Whereas, Linolenic acid was found only in A. sieberi.

Antioxidant Activity
In the present study, the FRAP method and DPPH scavenging capacity was used to determine the antioxidant capacity of the tested Artemisia essential oils by reducing the ferric ion (Fe 3+ ) to the ferrous ion (Fe 2+ ), and reduction of DPPH. The results of this study showed that A. sieberi present the higher antioxidant activity, followed by A. judaica and A. monosperma (Table 3). However, the standards substances used in this study (Ascorbic acid and Quercetin) present an antioxidant activity of 0.033±0.001g/l and 0.017±0.001 using DPPH reduction and 0.091±0.002g/l and 0.026±0.002g/l using FRAP method, respectively (Table 3).

Antibacterial Activity
The results of the antibacterial evaluation are presented in      showed no antibacterial effect on the Gram-negative bacteria (6 mm), only weak effect recorded against Acinitobacter baumannii (8 mm). However, it showed higher antibacterial activity against the Gram-positives only. Surprisingly, A. judaica exhibited mean zone of inhibition ranged between 31.5±0.5 to 14.5±0.5 mm against the Gram-positive bacteria only, which lead to assuming that this plant has a great antibacterial and narrow spectrum effect against the Gram-positive bacteria in particular.

Discussion
The strong fragrance of leaves and flower of genus Artemisia is associated to high concentration of sesquterpenes, and other constituent in their essential oils which was determined in the current study. In our study the yield of Essential Oils (EO) are varied according to species, where A. judaica have higher amount of EO yields than A. monosperma and A. sieberi. Similar results were obtained by van Wyk, and Wink 6 . The EO yields of A. monosperma detected in the present study were higher than thet detected by El Zalabani et al. 16 in plant grow in Libya, where the yield of the essential oils was 0.16 % v/w., this higher yield of essential oils related to the tolerance level of these plants under drought conditions, plants that grow in such harsh conditions are adapted by means of accumulating some chemical compounds. When plant exposed to mild or severe drought condition, plant exhibit arrange of specific responses, to reduce the water loss or optimize water uptake. Amoung of these responses, closure of stomata, and decrease the rate of photosynthesis 17,18 . Upon exposure to osmotic stress due to high temperature and drought, plant accumulate number of osmolytes for maintenance a turgor presuer in the cell.
From the investigation on the basis of chemotaxonomy, there is similarity in major compounds of the essential oils from those three species. It was concluded that most of the essential oils are rich in oxygenated monoterpenes and monoterpene hydrocarbons compounds. However, sesequiterpene is found as a major group of compounds in those three species.
Whereas, our result clearly indicate that the composition of the essential oils varies significantly due to drastic condition and drought stress in which the plant grow and survive, some compounds were detected in other chemical forms like, thujone which found as cis 4-methoxy-thujane, verbenol and Lilac alcohol C were accumulate in higher amount to adapt the drought stress, this confirmed by Putievsky et al., 22 who established that Pelargonium graveolens plants which grow under moisture stress stress yelided essential oil richer in citronellol. He was also reported, as acrop plant accumulate amino acid proline when subjected to stress, citronellol accumulation in the test crop during summer could be similar mechanism for adaptation to stresses 22 .
Regarding the antioxidant testing, the results showed that the ability of these essential oils to reduce the iron and DPPH was less than that of ascorbic acid and querectin because they are pure products and are known as antioxidant substances 23 . Furthermore, the results showed a difference in the antioxidant capacity between the tested Artemisia species, this can be explained by the differences in chemical compositions and the harvested origin. These results are supported by a study carried out for different species of the genus Artemsia and showed a difference in the antioxidant capacity 24 . In this study, A. judaica has a similar antioxidant capacity to that reported in Ethiopia with an IC 50 of 0.289 mg/ml 25 . Moreover, the earlier studies have reported the antioxidant capacity of essential oil of other Artemisia species 26,29 .
Our study is in conformity with the study of Guetat. 30 who reported that A. monosperma and A. sieberi as well as another two species A. scoparia, A. judaica showed high antibacterial activity. However, it disagrees with their findings with the Gram-negatives. Our results suggests that A. sieberi has higher antibacterial activity against the Gram positives and weak or no activity with the gram negative bacteria, this claim was confirmed by Mahboubi and Farzin 31 , who cited that the Grampositive bacteria and fungi were more sensitive towards oil of A. sieberi than Gram negative ones.

Conclusion
Genus Artemisia, family asteraceae, tribe anthemideae, famous with important medicinial plants, which are currently the area under discussion of phytochemical consideration due to their chemical and biological diversity. Three species of Artemisia show more than thirty compounds identified from essential oils, 20 compounds are similar and some compounds are different, the major compounds are Lilac alcohol C, verbenol and camphor, three species of Artemisia showed high antimicrobial and antioxidant activity, the antibacterial and antioxidant activity are attributed to the richness of phytochemical components of essential oil of these plants. There is a variety of compostion of essential oils between three species, finally recommended by study the mechanism of action of essential oil in antimicrobial and antioxidant.

Acknowledgment
The authors were grateful to king Abd-Alaziz University, Faculty of Metrology, Environment and Arid Land Agriculture for supporting in analysis of GC-MS, and also we are grateful to Desert Research Center, Centre Lap for approval and support.

Competing Interest
The authors have declared that no competing interest exists.

Significance Statement
The current study found that the essential oils of three medicinal plant species from the genus Artemisia, which are growing in the arid zone in Egypt, namely A. sieberi, A. judaica and A. monosperma, are rich in chemical compounds of potential bioactive properties, confirming the use of some species from genus Artemisia in the traditional medicine since the ancient Pharaonic civilization. The study also highlights some similarities in chemical constituents between species of this genus as well as some significant antibacterial and antioxidant properties.