CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF ESSENTIAL OILS FROM VARIOUS PARTS OF GLADIOLUS CANDIDUS , RANUNCULUS MULTIFIDUS ,

. The essential oil compositions of Gladiolus candidus , Ranunculus multifidus , Artemisia abyssinica and Crinum abyscinicum were analyzed with gas chromatography-mass spectrometry (GC-MS). The principal components in the leaves, stems and rhizomes of G. candidus were eudesmol, 1-naphthalenepropanol and oleic acid, respectively. α -Terpineol, alloocimene and p -menth-2-en-1-ol from leaves, bulbs and roots were respectively the major constituents of C. abyscinicum . The aerial part of R. multifidus furnished p -mentha-2,8-dien-1-ol. Linalool and terpinenol were identified as the major constituents of A. abyssinica . The essential oils were evaluated for their antibacterial activity. Essential oil from leaves of G. candidus displayed zone of inhibition (IZ) of 15.1±0.3 and 16.7±0.9 mm against E. coli and S. aureus , respectively. Leaves essential oil of C. abyscinicum exhibited IZ of 17.9±1.1 and 15.6±1.1 mm against E. coli and P. aeruginosa , respectively, whereas essential oil from aerial part of R. multifidus displayed IZ of 18.8±0.8 and 19.4±0.6 mm against S. aureus and S. pyogens , respectively. At the same concentration, ceftriaxone showed IZ of 15.1±0.1, 16.2±0.8, 14.3±0.9 and 16.1±2.5mm against E. coli , P. aeruginosa , S. aureus , and S. pyogens , respectively. The findings presented herein support the ethnobotanical uses of these plants against bacteria.


INTRODUCTION
Infectious diseases are responsible for an immense global burden that affects public health [1]. The diseases are reported to be the cause of deaths for over 9 million people in the world [1] with the major causes reported to be lower respiratory tract infections, diarrheal diseases, malaria and tuberculosis [2]. There are also infectious diseases that have newly appeared or have existed but are found resistant to the existing drugs [2]. Resistance developed by pathogenic microorganisms to the existing drugs is also a serious concern worldwide [3]. This makes infectious diseases harder to treat and consequently causes severe illness and death [3]. Medicinal plants that have been used traditionally for the treatment of infectious diseases remain an important source of pharmaceuticals. Hence, these plants might serve as a source of lead drugs [3]. In Ethiopia, many plant species including Gladiolus candidus, Crinum abyscinicum, Ranunculus multifidus and Artemisia abyssinica have been used as traditional medicine for the treatment of various human and animals against various infectious diseases Gladiolus candidus (Iridaceae), locally named as Dallo in Afan Oromo and Milas Golgul in Amharic, is a perennial herb that grows in woodland and dry grassland between 1450 and 2250 m in various parts of Ethiopia such as Arsi, Sidamo, Bale and Harerge floristic regions. The species is also occurring in other African countries such as Djibouti, Somalia, Kenya and

Extraction of essential oil
For the extraction of essential oils from the plant materials by hydro-distillation under optimal operating conditions, a quantity of 50 g of each plant part was added to 400 mL of distilled water in a 1 L flask using Clevenger type apparatus for 3 h. The essential oils were extracted from the distillate using chloroform, dried over anhydrous sodium sulfate, filtered and kept in a glass vial in the fridge at a temperature of 4 o C till analysis.

Yield of essential oils
The yields of essential oil of the G. candidus, R. multifidus, A. abyssinica and C. abyscinicum were expressed in g relative to 100 g of dry plant material and it was calculated according to the following equation: Yield (%) = Amount of extracted oil (g) Amount of dry plant material x 100%

Chromatographic analysis of the essential oils
The essential oils obtained from various parts of the plant materials were analyzed by GC-MS at Adama Science and Technology University using an Agilent 6870. GC with Agilent 5977 mass selective detector [MSD operated in the EI mode (70 eV). Scan range 40-400 amu and scan rate 3.99 scan/s and an Agilent Chem. Station data system. The GC column was HP-5 Fused silica capillary with a (5% phenyl-poly methyl siloxane stationary phase film thickness of 0.25 µm a length of 30 m and internal diameter of 0.25 mM). The carrier gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0 mL/min. The inlet temperature was 20 o C and interface temperature was 280 o C. The GC oven temperature was used as follows 60 o C initial temperature hold for 10 min. increased at 3 o C/min to180 o C hold for 6 min. increased 5 o C/min to 220 o C. A 1% w/v solution of the sample in hexane was prepared and 1 µL was injected using a splitter of 1:20.

Identification of compounds
The retention indices (RIs) for all of the essential oils were determined by co-injection of the sample with a mixture of the homologous series of C8-C25 n-alkanes. Identification of components was based on comparison of their mass spectra (MS) with those of NIST MS search 2.0 and Wiley 275 libraries and with those described by Adams [18].

Antibacterial activity
Clinical bacterial strains with American standards including Escherichila coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Staphylococcus pyogens (ATCC 19615) were used to evaluate the antibacterial activity of the essential oils. The standard bacterial cultures were obtained from Oromia Regional Laboratory and Quality Control, Adama, Ethiopia. McFarland number 0.5 standard was prepared by mixing 9.95 mL 1% H2SO4 in distilled water and 0.05 mL 1% BaCl2 in distilled water in order to estimate bacterial density [19]. The prepared sample was stored in an air tight bottle and used for comparison of bacterial suspension.
The antibacterial susceptibility test was evaluated by the agar disk diffusion method [19]. Essential oil extract of each plant species was prepared in DMSO to give 30, 15, 7.5, and 3.75 μg/mL. Bacteria cell suspensions were adjusted to 0.5 McFarland turbidity standards to prepare 1 × 10 8 bacterial/mL inoculum. Each bacterial suspension was inoculated on Mueller-Hinton agar plates, and the plates were allowed to dry for 5 min. The sterile filter paper disks were soaked in 30, 15, 7.5, and 3.75 μg/mL of each essential oil. The extract-soaked filter paper disks were then placed on the inoculated Mueller-Hinton agar plates and the analysis were conducted in triplicate. Ceftriaxone 30 µg/mL disk was used as the positive control and DMSO was used as the negative control. The plates were incubated for 18 h at 35 ± 2 °C. After incubation, the zones of inhibition were recorded as the diameter of the growth free zones measured in mm using antibiotic zone reader.

Essential oil yield
Oil yields expressed in relation to dry weight of plant materials. It is apparent through observing the extraction yields, the essential oil yields differ significantly according to their different organs and the species (Table 1). Table 1. Botanical names and essential oil yields (%w/w) of selected plants.
The essential oil composition from air dried leaves, stems and rhizomes of G. candidus were 0.29, 0.28, 0.44% w/w, respectively. The results showed that the yield obtained from the rhizome of G. candidus is greater than from the leaves and stems of the same plant. The essential oil yield of the rhizome of G. atroviolaceus, sister species of G. candidus, afford an average yield of 0.033 % [20]. This was smaller compared with the essential oil obtained from rhizome of G. candidus (0.44%). The essential oil composition from dried leaves, bulbs and roots of C. abyscinicum were 0.32, 0.32 and 0.08% w/w, respectively, which were isolated at the maximum growth stage of the plant. The study also exhibit that the essential oil yield obtained from the aerial part of R. multifidus was lesser than the yield displayed by various parts of G. candidus, A. abyssinica and C. abyscinicum. It was also found out that the essential oil yields from different parts of A. abyssinica such as the leaves, stems and roots were found to be 4.63, 0.47 and 3.23% w/w, respectively. The results clearly showed that the leaves of A. abyssinica were rich in essential oil. Previous work has shown different parts of the same plant species had differences in their essential yields [21]. Same parts of plants growing in different geographical regions were also reported to have different essential oil yields. It is noteworthy that the essential oil yield obtained herein from the leaves of A. abyssinica was found to be greater in comparison to the yield obtained by Asfaw et al. for the same plant [22].

Species name
Essential oil (% w/w) Leaves Stems were identified from the stem of G. candidus essential oils, accounting for about 94.55% of the essential oil compositions. Essential oils extract from stem of G. candidus were rich in linalool (6.73%) (1), 1-naphthalenepropanol (20) and ethyl linoleate (31.20%) (27). GC-MS analysis of the essential oil from rhizome of G. candidus revealed a total of 21 compounds with the major constituents of found to be oleic acid (20.3 %) (26), abietadiene (2.94 %) and heneicosane (5.19 %) (7). It was evident from Table 1 that, linalool, viridiflorol and 1-naphthalenepropanol were the only compounds detected in the leaves, stems and rhizome essential oils of G. candidus. The principal component of essential oil from the leaves of G. candidus was eudesmol (6) (30.06%) ( Table 2). This compound is reported to have various pharmacological activities such as anti-tumor, antibacterial and anti-angiogenic [23]. The antitumor property is believed to be by inhibiting angiogenesis by suppressing CREB activation of the growth factor signaling pathway [23]. Therefore, the presence of this compound as a major constituent in the leaves could be the case for the use of this plant as antibacterial and antitumor agent. The other principal component in the essential oil of G. candidus is linalool (1) (2.27%) which has been reported to exhibit immense biological activities such as antimicrobial, anti-inflammatory, anticancer, anti-oxidant properties [24]. The essential oil of the stem of G. candidus furnished viridiflorol (4) (2.09%) as one of the principal constituents. Viridiflorol is a sesquiterpenoid previously reported have antibacterial activity against Mycobacterium tuberculosis [25]. Hexadecanoic acid (15) possesses some biological activities such as antioxidant, hypocholesterolemia, nematicide and pesticide [21]. Ethyl linoleate (6) is used in many cosmetics for its antibacterial and anti-inflammatory properties [25] and is reported to accelerate healing of wounds and clinically proven to be an effective antiacne agent [26]. Ethyl linoleate was also reported as inhibitor of melanogenesis by inhibiting tyrosinase promoter activity [27]. The presence of eudesmol, linalool, viridiflorol and ethyl linoleate suggest the use of the essential oil of this plant as a potential candidate against disease causing microorganisms and cancer.

Essential oil composition of C. abyscinicum
The essential oils of the leaves, bulbs and roots of C. abyscinicum were analyzed using GC-MS and the findings are depicted in Table 3. The essential oil obtained from C. abyscinicum was colorless with a flowery odor. A total of 31 compounds were identified from essential oil extract of the leaves of C. abyscinicum using GC-MS. Essential oil from the leaves of C. abyscinicum were rich in ethyl hexadecanoate (5.75%), pentadecanoic acid (4.71%), 1-hexadecanol (3.86%), and α-terpineol (3.57%). On the other hand, a total of 15 compounds were identified from bulb C. abyscinicum essential oils, accounting for about 92.65% of the essential oil composition. T h e ma j or c on st i t u en t s o f t h e es s e n tia l oi l of t h e b u l b wer e found t o p-menth-2-en-1ol, cis-(4.02%), p-mentha-2,8-dien-1-ol (4.64%), alloocimene (55.49%) and 1-naphthalenepropanol (7.32%).
A total of 18 compounds were identified in the essential oil of the root of C. abyscinicum. Essential oils fr om roots of C. abyscinicum were rich in p-menth-2-en-1-ol, cis-(3.72%), alloocimene (4.08%), and methyllinoleate (4.72%). cis-p-menth-2-en-1-ol (3.72%), reported herein as one of principal components from the bulbs of C. abyscinicum, have some established biological activities including antifungal, insecticidal, larvicidal, anti-inflammatory and antibacterial [28]. Ocimene, the most common monoterpenes found in nature, had anticonvulsant, antifungal, antitumor, and pest resistance properties [29]. Pentadecanoic acid, 14-methyl ester belonging to fatty acid has antibacterial and antifungal activity [30]. In addition, α-terpineol attracts a great interest as it has a wide array of biological applications including an antioxidant, anticancer, anticonvulsant, antiulcer, antihypertensive, antibacterial and anti-nociceptive compound [31]. Therefore, the presence of these compounds in various parts of C. abyscinicum demonstrates the potential use of this plant against many life-threatening diseases.

Essential oil composition of A. abyssinica
The GC-MS analysis of essential oils from the leaves, stems and roots of A. abyssinica has led the identification of 70 compounds, accounting for about 65.94 % of the essential oil compositions. 34 -L = leaves; S= stems; R= roots; RI = retention index and results expressed as % area. Those compounds with %area beyond 0.1% were included in this report.
Linalool reported herein as the principal component was reported to have several biological activities such as antibacterial and antiplasmodial effect [22]. Limonene, which is the main ingredient of lemon essential oil, was found to have antimicrobial activities against L. monocytogenes in minced beef meat [36]. Nerolidol known to have some established biological activities including antioxidant, antifungal, anticancer, and antimicrobial activities [37]. Davanone also shows good antibacterial and anticytotoxicity against selected microorganisms. Terpinen-4-ol significantly enhances the effect of several chemotherapeutic and biological agents. The possible mechanism of the activity of terpinen-4-ol involves induction of cell death rendering this compound a potential anti-cancer drug alone and in combination in the treatment of numerous malignancies [38].

Antibacterial activity of essential oil extract
The in vitro antibacterial activities of the essential oils of the various parts of G. candidus, R. multifidus, A. abyssinica and C. abyscinicum were evaluated by the disc diffusion method using Mueller-Hinton agar against four bacterial strains including E. coli, P. aeruginosa, S. aureus and S. pyogenes with the results given in Table 6.
The essential oils from the leaves, stems and roots of A. abyssinica were assessed for their antibacterial activity. Among these, the essential oil displayed by the roots was significant compared with the activity shown by the leaves and stems. The essential oil from root of A. abyssinica displayed inhibition zone of about 13.5 ± 0.3 mm against E. coli at a concentration of 30 µg/mL ( Table 6). The result is significant compared with ceftriaxone (15.1 ± 0.1 mm at 30 µg/mL). The essential oil of the root also displayed antibacterial activity with inhibition zone of 11.1± 0.8 and 11.5 ± 0.8 mm against P. aeruginosa and S. pyogenes, respectively. The activity demonstrated by the essential oil of the roots of A. abyssinica could probably be due to the presence nerolidol and davanone as the principal components. In fact, the activity of these compounds against bacteria has been reported previously [37].
The essential oil from leaves and roots of C. abyssinicum were shown to have antibacterial activity with inhibition zone of 17.9 ± 1.1 and 11.4 ± 0.7 mm at 30 µg/mL, respectively against E. coli. The activity displayed by the essential oil of the leaves has an inhibition zone of 17.9 ± 1.1 and 15.6 ± 1.1 mm at 30 µg/mL against E. coli and P. aeruginosa, respectively. The results are comparable to the activity shown by ceftriaxone with inhibition zone of about 15.1 ± 0.1 and 16.2 ± 0.8 mm, respectively, at the same concentration. α-Terpineol was reported to have antibacterial activity in addition to its use as anticancer, antiulcer and antihypertensive properties [31]. Indeed, the presence of α-terpineol as one of the principal components of the essential oils might be responsible for the antibacterial activity of the essential oil of the leaves of C. abyscinicum.
The essential oils of the leaves, stems, and rhizomes of G. candidus were evaluated for their antibacterial activity ( Table 6). The essential oil of the leaves displayed activity with inhibition zone of 15.1 ± 0.3, 13.9 ± 0.9, 16.7 ± 0.9 and 10.8 ± 1.1 mm at 30 µg/mL against E. coli, P. aeruginosa, S. aureus and S. pyogenes, respectively. Ceftriaxone displayed zone of inhibition of 15.1 ± 0.1, 16.2 ± 0.8, 14.3 ± 0.9 and 16.1 ± 2.5 against E. coli, P. aeruginosa, S. aureus, and S. pyogens, respectively, at 30 µg/mL. The essential oil was shown to have broad spectrum antibacterial activity. Furthermore, the activity displayed by the essential oil of the leaves is superior to the essential oil from the stem and rhizomes but comparable with the positive control. The major constituent of the leaves essential oil was found to be eudesmol (6). This compound was reported to have antibacterial activity in addition to its use against tumor and angiogenic [23]. Hence, the antibacterial activity displayed by the leaves essential oil might to be due to eudesmol (6). The essential oil from the aerial part of R. multifidus displayed inhibition zone of 18.8 ± 0.8 and 19.4 ± 0.6 mm at 30 µg/mL against S. aureus and S. pyogenes, respectively. This indicates that the essential oil of R. multifidus is active against Gram positive bacteria. Furthermore, the inhibition zone displayed by the essential oil of R. multifidus against S. aureus and S. pyogenes comparable to the activity displayed by ceftriaxone (14.3 ± 0.9 and 16.1 ± 2.5 mm against S. aureus and S. pyogens, respectively, at 30 µg/mL).
One of the main postulated modes of action of essential oil has an ability to disrupt bacterial membranes intracellular materials, such as genetic materials, proteins, and potassium ions, in the event of membrane disruption [39]. The possible mechanism for its activity involves induction of cell death rendering this compound a potential anti-cancer drug and in combination in the treatment of numerous malignancies [38]. The essential oil showed strong antibacterial activity against S. pyogenes, while E. coli, and appeared to be resistant [40]. The borneol, α-terpineol, and terpinen-4-ol showed strong antibacterial activity against all tested bacteria [32].

CONCLUSION
The antibacterial activity of the essential oils from aerial parts of R. multifidus against S. aureus and S. pyogens was found to be significant compared with ceftriaxone. This could probably due to the presence of p-mentha-2,8-dien-1-ol as a major constituents in the essential oil of R. multifidus. The antibacterial activity displayed by the essential oil of the leaves of C. abyscinicum was turned out to be higher than ceftriaxone, a standard drug used as positive control. This might be accounted to the presence α-terpineol in the essential oil of the leaves of C. abyscinicum, which is cited in many reports as antibacterial agent. It was also found that the IZ displayed by the essential oils of leaves of G. candidus is better than ceftriaxone. Remarkable activity displayed by these essential oils could be due to the presence of certain bioactive compounds such as Dlimonene, linalool, eudesmol, alloocimene, α-terpineol, viridiflorol, nerolidol and davanone in the plant species essential oil. The aforementioned findings suggest that the essential oils can be used as natural antibacterial remedies. Furthermore, the results presented herein support the traditional uses of these plants against bacteria.