Antileishmanial and Antibacterial Activity of Essential Oils of Medicinal Plant Achillea santolina L

Corresponding Author: Dr. Abdulkarim Dakah Faculty of Pharmacy, International University for Sciences and Technology, Damascus, Syria Email: abdu83alkarim@yahoo.com Abstract: Achillea santolina is medicinal plant widely used in folk medicine for gastrointestinal disorders, anti-inflammatory and anti-diuretic. The World Health Organization (WHO) reports that about 350 million people are considered to be at risk of contracting leishmaniasis especially among people living in the developing countries. This research will be the first time that evaluate antileishmanial activity of essential oils of A. santolina before and after flowering. Wild plants of Achillea santolina were collected from the Kalamoon Mountains in Syria before and after flowering. Essential oil of wild plants was identified by GC/MS analysis. The activity of essential oil was evaluated against five bacterial strains by well diffusion method and Promastigote lysis percentage was determined by an MTT assay. Chemical composition of essential oils of A. santolina was variable. According to our results the samples from Assal Al-ward and Deir Atiyah after flowering gave light yellow oils and the main constituents were Borneol 17.34% and Camphor 27.69% respectively. While the sample before flowering from Assal Al-ward gave blue oil due to presence Azulene 0.69% which absence in oils after flowering and the main component was 2-Cyclohexen-1-ol, 1methyl-4-(1-methylethyl) 13.05%. The essential oil of A. santolina at concentration 30% after flowering from Deir Atyiah showed the highest inhibitory effect on Klebsiella pneumonia with diameter 21 mm. Also essential oil of A. santolina before flowering gave the highest antileishmanial activity with an IC50 value (56.17 μg/mL). The antileishmanial activity of oils from Achillea santolina before flowering was better than after flowering, in contrast the antibacterial activity of oils before flowering was less than after flowering. Depending on the results, the oils from plants before flowering It could be constitute a potential treatment in the future.


Introduction
Increased interest in products of medicinal plants has been observed within recent years, due to the cosmetics, natural antimicrobial agents, pharmaceutical and foodpreservation systems. Leishmaniasis is a major public health problem especially in the developing countries. Leishmaniasis is recognized as one of the most neglected tropical diseases (Barrett and Croft, 2012). Visceral leishmaniasis is a disease caused by the Leishmania infantum in Europe, northern Africa and Latin America and Leishmania donovani complex in East Africa and the Indian subcontinent (Lukes et al., 2007).
Anthroponotic cutaneous leishmaniasis is caused by Leishmania tropica and transmitted between humans by the Phlebotomus sergenti sand fly. Incidence in cutaneous leishmaniasis in Syria was 23,000 cases in year (Hayani et al., 2015). In early 2013, an alarming increase to 41,000 cutaneous leishmaniasis cases was reported (Haddad et al., 2015;Hayani et al., 2015). The World Health Organization (WHO) reports that about 350 million people are considered to be at risk of contracting leishmaniasis especially among people living in the developing countries (WHO, 2010). The antileishmania and antimicrobial properties of plants have been investigated by a number of researchers around the world, this is due to compounds synthesized in the secondary metabolism of the plant like the essential oils. There are continued studies for searching on treatments, especially search for new and better compounds that were produced of plant origin, because they are easily obtained at low cost. According to the World Health Organization about 80% of the people around the world turn to traditional medicine for their health care (Avijit et al., 2007).
Latin name of Achillea comes from the name of Achilles, who used Achillea herb to healed the wounds at the time of the Trojan War, It was immortalized in the Iliad by Homer (Nowak et al., 2010), in Arabic it is known as Qaysoom. Species name santolina means Sacred. Achillea is one of the youngest genera of the Asteraceae family, which is present throughout the world (Farajpour et al., 2012). More than 100 species have been recognized in this genus (Goli et al., 2008). Achillea santolina L. grows as a common herbal plant (perennial) in the north western Mediterranean region, this plant usually grows in barley and fallow fields, olive and figure plantations and at the edges of the small canals and roads (Darier and Tammam, 2012). These plants are native to Europe and western Asia but are also found in Australia, New Zealand and North America (Rechinger, 1963) and in some parts of Iran (Czygan, 2004). A. santolina L., is perennial herb, 10-30 cm long, with aromatic smell, the leaves are narrow, linear, pinnatisect, heads are small, yellow, 5-8 cm in diameter, arranged in small, flat inflorescence (Al-Eisawi, 1998). Achillea santolina L. Greyish woolly herb, stems branched, erect or ascending (Boulos, 2002). Flowering occurs during the spring season extending from March to May. Achillea has been used in medicine for its antihemorrhagic, healing and analgesic properties (Ody, 1993;Fleming, 2000). A. santolina is medicinal plant, widely used in folk medicine for gastrointestinal disorders (Bimbiraitė et al., 2008), its flower heads are used to relieve toothache, rheumatic pains and for treatment of colic and diabetes mellitus, also used as an anthelmintic and stomachic drug (El-Darier et al., 2005). A. santolina has some traditional uses: Antiinflammatory, antidiuretic and antimicrobial effects (Al-Hindawi et al., 1989;Twaij et al., 1985;Cowan, 1999). This plant contains several polyphenols, a family of compounds with a great antidiabetic potential and uses in Iraq, Egypt and Pakistan as antidiabetic, anti-inflammatory and to relieve pain. Also as vermifugal and carminative and also for stomach pain and hypertension (Alwwadi, 2013). A. santolina is dysentery and insect repellant (Khalil et al., 2009). Eddouks and his colleagues had shown that A. santolina has some antimicrobial Activity and chemical analysis reported that A. santolina contains flavones, particularly flavonoids and polyphenols that have some beneficial antidiabetic effects (Eddouks et al., 2003). Khalil et al. (2009) studied antimicrobial activity of Achillea santolina exerted against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans and showed that this medicinal plant can be used as natural antimicrobial agents in pharmaceutical and food preservation systems (Khalil et al., 2009). Ardestani and Yazdanparast evaluated the effect of Achillea santolina extracts on lipid peroxidation, protein oxidation and antioxidant defense system such as Super Oxide Dismutase (SOD), Catalase (CAT) and reduced Glutathione (GSH) in the liver of Streptozotocin (STZ)-induced diabetic rats. Also they reported that the hydroalcoholic extracts of Achillea santolina reduced the blood glucose levels in rats (Ardestani and Yazdanparast, 2006). The essential oil of A. santolina contained 54 volatile components, the major components were 1, 8-cineole, fragranol, fragranyl acetate and terpin-4-ol (Si et al., 2006;El-Shazly et al., 2004). Motavalizadehkakhky et al. (2013) and his colleagues isolated and analysised the essential oils of Achillea pachycephala Rech.f. and Achillea santolina L. that obtained by steam distillation from the flowers, leaves and stems. The principle components In essential oils and extracts of A. santolina were 1, 8-cineole, camphor, terpinene-4-ol, fragranol, fragranyl acetate, α-terpinyl acetate, caryophyllene oxide, α-muurolol and some alkanes, alkanoic acids and esters. Oils and extracts showed higher activities against the tested gram negative bacterial strains (Motavalizadehkakhky et al., 2013). No previous studies were found about antileishmania of A. santolina, so this research will be the first time that evaluate antileishmanial, so the aim of this study was to analysis essential oils of A. santolina before and after flowering and evaluation antibacterial and antileishmanial activity.

Materials and Extraction of Essential Oil
Wild plants of Achillea santolina were collected from the Kalamoon Mountains in Syria, from two sites. Before and after flowering from Assal Al-ward and after flowering from Deir Atiyah. Voucher specimens of the plants were deposited in the Department of Plant Biology, Faculty of sciences, Damascus University. The collected air parts were washed with clean water to remove soil, dried in room temperature and powdered using grindery. For production of essential oil, 100 g of air dried plant material was hydrodistilled for 3 h using a Clevenger apparatus. The oil was dried over anhydrous Na 2 SO 4 and then was kept in a sealed vial at 4°C.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The GC-MS analysis was performed in Atomic Energy Commission in Syria (AECS) using a Variane 3400 equipped with a HB-5MS column (30×0.25 mm internal diameter, film thickness 0.25 µm and with helium as a carrier gas) with analytic conditions: the injector and detector temperatures were held at 260°C.
The oven temperature program at first was 60°C for 4 minutes and then it was increased to 200°C and it remained at 200°C for 8 minutes. The final temperature was 260°C for 7.5 min. The SFE samples (1 µL) were injected using split mode with a split ratio of 1:40 and carrier gas was helium with split flow of 1ml/min. The ionization energy was 69.922eV with a scan time of 1 second and mass range of 35-450 amu.

Evaluation of Antibacterial Activity
The examined bacteria were obtained from Atomic Energy Commission in Syria (AECS). The bacteria include strains Gram positive: Staphylococcus aureus and Gram negative: Salmonella typhi, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa. The density of each test bacterium suspension was set equalizes to 0.5 McFarland stander, which is 10 8 CFU/ml. Antibacterial activities were evaluated using well diffusion method (wells with diameter = 6mm) on Mueller-Hinton Agar (MHA). Wells were filled with 50 µl with different concentration of essential oils in DMSO (10, 20, 30%) and incubated at 37°C for 24 h. After the incubation period, the diameter of the growth inhibition zones was measured. The DMSO was used as negative standards. All tests were performed in triplicate.

Viability Assay
Promastigote lysis percentage was determined by an MTT [3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay as described (Ebrahimisadr et al., 2013), the bioassays were performed in triplicate. In 96-well microtitre plates, 3×10 6 promastigotes of Leishmania tropica per well were cultured in RPMI 1640 medium in the presence of essential oils in concentration of 25, 50, 75, 100 and 125 µg/mL and allowed to multiply for 24 h in the medium. After these times 20 µL of MTT (5 mg/mL) was added to each well and incubated in 18°C for 4 h then centrifuged in 1000g for 10 min, the supernatant was discarded and 100 µL of DMSO was added to each wells and re-suspended. The OD was read by ELISA reader in 540 nm: Viability % = (Absorbance of Sample/ Absorbance of Control) × 100 Results were expressed as the minimum concentration necessary for inhibition of 50% of parasite growth (IC 50 ). The IC 50 values were calculated using Excel program.

Statistical Analysis
The data were statistically analyzed by one-way ANOVA (SPSS), followed by Tukey's multiple comparison test.

Antibacterial Activity
The results showed that concentration 30% has more activity than lower concentrations 20 and 10% in each tested bacteria Table 2. In this concentration 30% the essential oil of A. santolina after flowering from Deir Atyiah showed the highest inhibitory effect on Klebsiella pneumonia with diameter 21 mm and the essential oil of A. santolina before flowering from Assal Al-ward showed the lowest effect on Escherichia coli and Salmonella typhi with diameter 12 mm. In general essential oils from Deir Atyiah after flowering was more affective at all concentrations. Pseudomonas aeruginosa was not sensitive to all tested essential oil.

Anti-Leishmanial Activity of Essential Oils
Growth inhibition of Leishmania tropica promastigotes was evaluated and the percentage of viability in the presence of various concentrations of essential oils of Achillea santolina in comparison with control for 24 h are showed in Table 3 and Fig. 1. Our findings showed that essential oils of Achillea santolina inhibited promastigote growth with percentage of viability ranged from 33.3% to 97%. With a concentration increase of essential oils the optical density significantly decreased and inhibitory percentage increased. The best activity of essential oils was of A. santolina from Assal Al-ward before and after flowering with viability 33.3 and 33.5% respectively at concentration 125 µg/mL. Essential oil of A. santolina before flowering gave the highest antileishmanial activity with an IC 50 value (56.17 µg/mL) and was more active compared with essential oils of A. santolina after flowering from Assal Al-ward and Deir Atiyah with IC 50 value (60.84 and 87.15 µg/mL), respectively.

Components of Essential Oils
The aim of the present study was to determine the chemical composition of the essential oils of A. santolinafrom different locations before and after flowering from Kalamoon Mountains in Syria. Chemical composition of essential oils of A. santolina was variable. According to our data, Borneol 17.34%, Camphor 27.69% and 2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl) 13.05% were the main constituents of A. santolina from Assal Al-ward and Deir Atiyah after flowering and Assal Al-ward before flowering, respectively. Which may be due to the differences in environmental conditions and date of collection of plants before and after flowering.
Previous studies that have determined the chemical composition of A.santolina essential oils from Iran showedalso identified high levels of Fragranyl acetate 28.4%, 34% and 37% from flowers, leaves and steam, respectively (Motavalizadehkakhky et al., 2013). Our results didn't show differences with percentage of eucalyptol before flowering (9.6%) and after flowering (9.66%) from Assal Al-ward, but there is clear increasing with percentage of camphor from <1% to (17.34%), these findings confirmed that essential oil composition of plant can be different in quality and quantities in different period of growth of plant, that is disagree with another study (Al-Jaber et al., 2016) where A. santolina was collected at different growth stages, eucalyptol was the main constituent at the pre-flowering (18.05%) and increase compare with flowering (20.51%) and camphor decrease from (9.51%) before flowering to (7.66%) after flowering. There are many similarities between the oils although the amounts of some corresponding compounds are different. Our results agree with (Dastjerdi and Mazoji, 2015) suggested that these differences may be related to the different geographical origins of the samples. In general, the differences in oil composition may be due to , plant genetic type, different environmental factors, seasonality, physiological age and developmental stage.

Antibacterial Activity
Antibacterial properties due to many components like Eucalyptol (1,8-cineole) and 3-Cyclohexen-1-ol, 4methyl-1-(1-methylethyl) which were found with high percentage in all tested essential oils. Eucalyptol is one of the most important compounds as antibacterial activity (Leung and Foster, 2003). 3-Cyclohexen-1-ol, 4methyl-1-(1-methylethyl) which is derivative of limonene (Olivera et al., 2001) and Limonene has been implicated to be an effective antimicrobial agent (Magwa et al., 2006). Our results showed that Klebsiella pneumonia was the most sensitive bacteria to the plant oils from Deir Atiyah (21 mm), It is revealed that the Klebsiella pneumonia was sensitive to the essential oil of A. santolina from Iran with inhibition zone from 21.5 to 23 mm (Motavalizadehkakhky et al., 2013). The activity of oils from Deir Atiyah may be due to presence high amount of Camphor 27.69% that has the most efficient antibacterial property (Prudent et al., 1993;Aligiannis et al., 2000). All of oils didn't show any effect against P. aeruginosa, that is disagree with result of Khalil and his colleagues where reported that extracts from Achillea santolina inhibited P. aeruginosa, with the high effect (Khalil et al., 2009) that is because they used ethanol extracts.

Anti-Leishmanial Activity of Essential Oils
Our results showed that the differential antileishmanial activity of these oils against the promastigotes is related to the differential composition of such oils depending on sources of plants and stage of growth. No previous study or reports about effect of A. santolina as antileishmanial, so we suggested the active components in oils especially A. santolina from Assal Al-ward before flowering can interact with mitochondrial membranes leading to its death by apoptosis. Santos and his colleagues tested essential oil activity of Achillea millefolium against Leishmania amazonensis and IC 50 was 7.8 µg/ml, they reported that Azulene has antileishmania activity (Santos et al., 2010), their result supports our findings because the highest activity was in oil that contain Azulene before flowering. Caryophyllene also was found in A. santolina oil before flowering and has antileishmanial activity recording to Soares and his colleagues, their studies pointed out Caryophyllene in Copaifera spp. oil was an effective antileishmanial compound against L. amazonensis (Soares et al., 2013). Also Chouhan and his colleagues reported that Hexane and Ethanolic extracts of Piper nigrum which contain Caryophyllene inhibited the growth of Leishmania donovani promastigotes with IC 5031.6 and 37.8 µg/ml, respectively (Chouhan et al., 2014). Another study showed the antileishmanial activity of Caryophyllene was at least two times more potent than limonene against L. amazonensis with IC 50 value 96 µM (Moura et al., 2012). Another compounds like Eucalyptol and Borneal did not have antileishmanial activity according to Machado and his colleagues, they did not showed any biological activity when tested extracted essential oil from Thymus capitellatus against Leishmania infantum, Leishmania tropica and Leishmania major promastigotes at the tested concentrations (Machado et al., 2014).

Conclusion
Essential oils of Achillea santolina before and after flowering showed clear different with color, chemical components and activity. The antileishmanial activity of oils from plants before flowering was better than after flowering, in contrast the antibacterial activity of oils before flowering was less than after flowering. Also the antileishmanial activity of oils was more than antibacterial activity. Depending on the results, the oils from plants before flowering It could be constitute a potential treatment in the future.