GC-MS Analysis and Antimicrobial Activity of the Essential Oil from the Stem of the Jordanian Toothbrush Tree Salvadora persica.

Abstract

Chemical composition of the essential oil of the stem of the toothbrush tree Salvadora persica. L. grown in Jordan was determined by gas chromatography-mass spectrometry (GC-MS). The oil obtained by hydrodistillation (yield: 0.6% w/w) was determined as a mixture of monoterpene hydrocarbons (11%), oxygenated monoterpenes (54%), and sesquiterpene hydrocarbons (21%). The major components identified were 1,8-cineole (eucalyptol) (46%), α-caryophellene (13.4%), β-pinene (6.3%), and 9-epi.-(E.)-caryophellene. The antimicrobial activity of the volatile oils and aqueous and alcohol extracts of the plant has been also evaluated. Among all tested fractions, the volatile oils exhibited potent activity against both sensitive and resistant strains of Pseudomonas aeruginosa. (Schroeter and Migula) and Staphylococcos aureus. (Rosenbach). Moreover, the oil revealed significant inhibition against Candida albicans. (C. P. Robin) and Trichosporon cutaneum. (Beurm, Govgerot and Vaucher).

Keywords:

• Antimicrobial
• arak
• capillary GC-MS
• essential oil
• miswak
• Salvadoraceae
• Salvadora persica. L
• volatile oil

Introduction

Miswak or arak (chewing sticks) is commonly used in Jordan and in many other areas in the Arab and Islamic world for oral hygiene and medical, religious, and social purposes (Hattab, 1997). The use of the wood stick for brushing the teeth is still an important tool for oral hygiene in these countries. Recently, the World Health Organization (WHO) has recommended and encouraged the use of these sticks as an effective tool for oral hygiene. Arak tree is used as an diuretic, antigastric, to treat hook worn, venereal diseases, for teeth cleaning, amenorrhoea, in rheumatism, cough, and asthma, to lower cholesterol plasma levels, reestablishment of the components of gastric mucosa, and as a laxative (El-tawil, 1983; Wolinsky & Sote, 1984; Rotimi & Mosadomi, 1987; Galletti & Chiavari, 1993). Studies on the antibacterial effect of miswak on oral bacteria demonstrated that the crude extract is effective against Bacteroides gingivalis. (Coykendall) and other black-pigmented Bacteroides (Almas & Al, 1997) as well as Streptococcus mutans., (Clarke) salivaris (Andrews and Horder), mitis (Andrews and Horder), and Staphylococcus aureus. (Rosenbach); with inhibition of 67 to 96% at a 10% concentration of the extract (Hattab, 1997).

Despite the variety of literature on miswak, little has been done to investigate the chemical components of the different plant parts (Faroogi & Srivatava, 1968; Ezmirly et al., 1978; Ezmirly & Seif-Elnasr, 1981; Kamil & Ahmad, 2000). The antibacterial activity of the essential oil and different extracts obtained from the leaves and stems of arak tree was tested against some oral aerobic bacteria using the disk diffusion test (Al-Lafi & Ababneh, 1995; Alali & Al-Lafi, 2002). It was found that the essential oils of the leaves have a considerable effect on several different oral aerobic bacteria with comparable results to known antibiotics (Alali & Al-Lafi, 2002). The major essential oil components identified in the leaves were benzyl nitrile, eugenol, thymol, isothymol, eucalyptol, isoterpinolene, and β-caryophyllene (Alali & Al-Lafi, 2002).

In the current study, stems from Salvadora persica., L. the most common source for miswak, were analyzed for their content and composition of volatile oils. Also, the antimicrobial activity of the volatile oils and aqueous and alcoholic extracts of the plant's stems against susceptible and resistant microbes has been evaluated.

Materials and Methods

Plant material

Samples of the native arak (Salvadora persica. L.) was collected in March 1999 from Ghour Al-Safi (southern part of the Dead Sea in the Jordan Rift Valley), 400 m below sea level, during the flowering period and the vegetative phase. The taxonomic identity of the plant was confirmed by comparing a collected voucher specimen with those of known identity in the herbarium of the Department of Biological Science, Faculty of Science, University of Jordan (Amman, Jordan) and with the assistance of Dr. Kamal Khairallah, Faculty of Agriculture, University of Jordan. A voucher specimen (no. S.p. 13–99) has been deposited in Dr. T. Aburjai's research laboratory at the Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan.

Oil distillation

Oils from air-dried, finely ground stems were obtained by hydrodistillation using a Clevenger-type apparatus. Distillation was performed using 50 g of each dried plant material in 2.5 l distilled water for 4 h. The oil obtained was dried over anhydrous sodium sulfate and stored in a dark glass bottle at 4°C until analysis. The yield of oil (w/w) was 0.6%.

GC/MS analysis

Varian Chrompack CP-3800 GC/MS/MS-2000 (VWR International GmbH, Darmstadt, Germany) equipped with split-splitless injector and DB-5.625 GC column (30 m × 0.25 mm i.d., 0.25 µm film thickness) was used. The injector temperature was set at 220°C for 5 min with a split ratio of 1:10. A 1 µl volume of 1000 ppm oil solution (GC grade n.-hexane, Scharlau, Chemie, Barcelona, Spain) was injected. A linear temperature program was adapted to separate the different oil components as follows: initially, the column maintained at 50°C for 2 min, ramped at a rate of 10°C/min to 150°C at which it was held isothermal for 5 min; a second ramp (20°C/min) was then applied to 220°C and held isothermal for 10 min. The total run time was 30.5 min. The temperatures of the transfer line and ion source were maintained at 230 and 180°C, respectively. The mass detector was set to scan ions between 40 and 400 m./z. using full-scan fixed mode electron impact (EI: 70 eV). The volatile oil compounds were identified by matching their recorded spectra with the data bank mass spectra (Saturn and NIST library databases) provided by the instrument software and by comparing their retention indices values with those in the literature, measured on columns with identical polarity (Adams, 1995). The databases were compiled using more than 80,000 electron impact (EI) mass spectra. Only matching spectra of large degree of certainty using reverse-fit modes were accepted. Concentration (% content) of the oil components were calculated by integrating their peak areas, in the total ion current (TIC) chromatograms, assuming a unity response by all components.

Preparation of plant extracts and microbial cultures

Alcohol and aqueous extracts were prepared by macerating 10 g dried and finely ground stems in 1 l absolute ethanol and distilled water overnight, respectively. Alcohol and aqueous dried residues were obtained by rota-evaporation and freeze-drying, respectively. Alcohol and water extracts and volatile oils of Salvadora persica. were sterilized by filtration, and a 20-µg sample of each was loaded on a filter paper disk (Whatman, Maidstone, UK). The impregnated disks were then tested for their antimicrobial activity. Bacteria including Pseudomonas aeruginosa. (Schroeter and Migula) ATCC 27853, Escherichia coli. (Castellani and Chalmers) ATCC 25922, Staphylococcus aureus. ATCC 25923, and Bacillus subtilis. (Cohn) ATTC 6633 were grown in nutrient broth (Oxoid Limited, Hampshire, UK) overnight at 37°C and maintained on nutrient agar (Oxoid) plates at 4°C. Candida albicans. ATCC 10231 and Trichosporon cutaneum. were grown in malt broth (Oxoid) at 25°C for 24 h and maintained on nutrient agar (Oxoid) plates at 4°C. T. cutaneum., resistant strains of P. aeruginosa. and S. aureus. were clinically isolated from hospitalized patients from the Jordan University Hospital (Amman, Jordan) and were confirmed by biochemical tests.

Antimicrobial activity assay

The crude plant extracts and the volatile oils were tested for their antimicrobial activity using the diffusion technique on solid media (Janssen et al., 1987). Sterile 5-mm-diameter filter paper disks were impregnated with 20 µg of either alcoholic or water plant extracts and placed on nutrient agar seeded with the microorganisms (10⁶ cfu/ml). The plates were incubated for 24 h at 37°C for bacteria and 48 h at 25°C for fungi. Control disks were soaked with the same solvent and treated as the sample disks. The experiments were carried out as duplicate three times and corrected for the control disks. Additionally, erythromycin, nystatin (The Arab Pharmaceutical Manufacturing Co. Ltd., Salt-Jordan), tetracycline (Dar al Dawa, Naur-Jordan), and chloramphenicol (Pliva, Zagreb, Croatia) were tested as positive standards at a concentration of 20 µg/disk. The diameters of the inhibition zones are presented in Table 1.

Table 1. Zones of growth inhibition (mm) of water and alcoholic extracts and volatile oils of Salvadora persica. L.

	Microorganism^a.
Sample	E. coli.	S. aureus.	S. aureus. (R)^b.	B. subtilis.	P. aeruginosa. (R)^b.	P. aeruginosa.	C. albicans.	T. cutaneum.
Water extract	5 ± 0.9	5 ± 0.7	−	−	+/−	−	9 ± 1.1	+/−
Alcoholic extract	11 ± 1.1	2 ± 1.0	−	+/−	+/−	4.1 ± 0.5	11 ± 0.5	−
Volatile oil	13 ± 0.5	12 ± 0.1	3 ± 0.1	3 ± 0.1	2.9 ± 0.1	3.8 ± 0.6	16 ± 0.3	12.4
Erythromycin	30 ± 5.1	22 ± 3.9	7 ± 1.1	22 ± 2.4	10 ± 0.7	16 ± 1.4	−	−
Chloramphenicol	−	10 ± 2.1	2 ± 0.1	8 ± 1.0	7 ± 0.4	17 ± 1.3	−	−
Nystatin	−	−	−	−	−	−	22 ± 3.2	27 ± 3.4

−, No inhibition;+/−, slight inhibition (2 mm).

^a.Values for zone of growth inhibition are presented as means (three determinations in duplicate) ± SE.

^b.Resistant strain.

Results and Discussion

The composition of the essential oil of the stem of the toothbrush tree (Salvadora persica. L.) grown in Jordan was determined by GC-MS as shown in Table 2. In our study, we were able to identify and quantify 16 oil components, the majority of which are monoterpene hydrocarbons (11%), oxygenated monoterpenes (54%), and sesquiterpene hydrocarbons (21%). The major components identified were 1,8-cineole (eucalyptol) (46%) as the principal oil constituent, α-caryophellene (13.4%), β-pinene (6.3%), and 9-epi.-(E.)-caryophellene (6.3%) as according to Table 2. The rest of the oil corresponded to either minor components or unidentified compounds. It is noteworthy that no aromatic monoterpenes were detected in the stem while these components, mainly thymol, isothymol, and eugenol, corresponded to the major volatile oils constituents identified in the leaves of the same plant (Alali & Al-Lafi, 2003). The composition of the essential oil of the stem may contribute to its popular use as a teeth-cleaning tool. The antiseptic properties of cineole, the major essential oil identified in the stem (46%), was established unambiguously in vitro. on many microorganisms (Bruneton, 1995). To the best of our knowledge, this is the first report on the volatile oils present in the stem of the arak tree growing in Jordan and in the region. This study may stimulate further investigation on the chemical and biological properties of these sticks. Also, it may encourage a wider use of these sticks as an alternative tool for oral hygiene especially in poor countries and for those who adapt a natural way of living.

Table 2. The volatile oil components identified in Salvadora persica. L. stems by GC-MS analysis.

RI^a.	Compound	% Content
939	α-Pinene	0.50
980	β-Pinene	6.30
1026	p.-Cymene	1.32
1033	1,8-Cineole	45.90
1088	Terpinolene	2.85
1098	Linalool	1.15
1102	β-Thujone	1.84
1143	Camphor	1.03
1173	cis.-Pinocamphone	0.50
1177	Terpin-4-ol	1.65
1189	α-Terpineol	4.58
1257	Linalyl acetate	1.28
1404	α-Caryophyllene	13.37
1418	β-Caryophyllene	0.72
1439	Aromadendrene	0.63
1581	Caryophyllene oxide	6.34
	Unidentified traces	5.00
	Monoterpene hydrocarbon.	10.97
	Oxygenated monoterpene.	57.93
	Sesquiterpene hydrocarbon.	21.06
	Total	95.00

^a.RI retention index calculated on DB-5 capillary column material.

The results of the antimicrobial activity of aqueous and alcoholic extracts and the essential oil of S. persica. are given in Table 1. These data revealed that the volatile oil of S. persica. exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Moreover, it is worth noting that the oil also exhibits significant activity against resistant strains of P. aeruginosa. and S. aureus.. On the other hand, this oil revealed significant inhibition against C. albicans and T. cutaneum.. Water extract showed weak activity against all tested microorganisms except for C. albicans.. Finally, the alcohol extract of the plant showed good activity against gram-negative bacteria and C. albicans..