Phytochemical Composition and Antimicrobial Activities Of

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Phytochemical composition and antimicrobial activities of

the essential oil from Plectranthus tenuiflorus growing in Saudi Arabia

Tagreed Alsufyani1, Asif fatani2, Faten Khorshedm3, Soad Shaker3 and

Hassan A. H. Albar4

1 Department of Chemistry, TaifUniversity,PO Box 1506-Taif, Saudi Arabia

2King Abdulaziz University Hospital, Clinical Microbiology Lab, PO Box 80215-Jeddah21589, Saudi Arabia

3 King Fahad medical research center, King Abdulaziz University, PO Box 9028-Jeddah 21413, Saudi Arabia

4Department of Chemistry, KingAbdulazizUniversity, PO Box80203, Jeddah21589, Saudi Arabia

Abstract

The present study describes the phytochemical profile and antimicrobial activity of Plectranthus tenuiflorus essential oil, collectedin Taif from Saudi Arabia. This sample of essential oil was obtained from the Leaves of the plant by hydrodistillationand analyzed by GC–MS. From the 31 compounds representing 92.79% of the oil, Thymol(62.53%), appears as the main component. The oil also contained smaller Percentages (1-10%) of 1-octen-3-ol, m-cymene, -terpinene, 1-terpinen-4-ol, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol, -bergamotene, 4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl-cycloheptane, and 2,3,4,4a,5,6-hexahydro-1,4a-dimethyl-7-(1-methylethyl)-naphthalene.Furthermore,antimicrobial activity of the oil was evaluated using agar-well diffusion method. The antimicrobial test resultsshowed that the oil had a great potential antimicrobial activity against all 5 bacteria and 1 fungal pathogenic strains. C. albicans yeastis more sensitive to the investigated oil, with aninhibitory area diameter 44.7 mm,then S. aureus bacteria (28 mm) and lastly K. pneumoniae (25.7 mm). It was also observed that inhibitory effect to growth of C. albicans and P. aeruginosa under the essential oil and thymol are symmetrical. Results presented here may suggest that the essential oil of P. tenuifloruspossesses antimicrobialproperties, and is therefore a potential source of antimicrobial ingredients for the pharmaceutical industry.

Keywords: Plectranthus tenuiflorus; Essential oil; Thymol; Antimicrobial activity;GC-MS.

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1. Introduction

The genus Plectranthus (Lamiaceae) consists of some 350 species, distributed from Africa through to Asia and Australia. Several species of them are used as a folk medicine for skin irritations, antiseptics, vermicide and nausea (Narukawa et al., 2001). There are some reports(Sandra et al., 2001; Abdel-Mogibet al., 2002) about the occurrence of unique diterpenoids in several Plectranthus species and moderate antibacterial activity.

Plectranthus tenuiflorus, plant well-known in Saudi Arabia as Sharah, is an aromatic herb widely used in the Saudi folk medicine as a non-specific treatment for earache and inflammation of middle ear in western region (Albar et al., 2006), whereas it is prescribed in Asirregion for a remedy sore throat (Rahman et al., 2004). Essential oil obtained from the leaves of this plant was shown to have antimicrobial activity which∆3-Carene (52.8%) was found the major component (Al-Yahya et al., 1985), while (Mwangi et al., 1993) found the essential oil of P. tenuiflorus (Vatke) Agnew from Kenya contained -terpinene (10.2%), p-cymene (10.9%) and carvacrol (14.3%) as the major components. The last paper(Smith et al., 1996) has reported that the principle component of the oil produced from plant harvested in Saudi Arabiawas found to be Thymol (85.3%).

The aim of this study was to analyze by GC-MS the volatiles of oils responsible for the significant odor impression and provide information for a use in medicinal or applications.

2. Materials and methods

2.1. Plant material

Leaves of P. tenuifloruswere collected fromAl-sirValley near Taifcity in Saudi Arabiain April 2003. Identity of the plant was confirmed by Dr. Farag Aghamdi, Faculty of Science Herbarium, KingAbdulaziz University.

2.2. Extraction of the essential oil

Fresh leaves of the plant were used for the analysis of essential oil composition. A portion (400 g) of the leaves of P. tenuifloruswas submitted for 7 hrs to water-distillation, using a Clevenger-type apparatus. The obtained essential oil was dried over anhydrous magnesium sulphate and 2 l as a solution in diethyl ether was used for GC–MS measurements, the oil was kept in small well closed amber coloured glass container at low temperature.

2.3. GC–MS analysis conditions

The analyses of the volatile compounds were carried out on a Shimadzu Qp 5050 GC–MS system (GC 5890 Series II; MSD 5971A). Fused-Silica 5% phenyl silicon, 95% methyl silicon DB-5 Open Tubular (30 m x 0.25 mm, i.d.; 0.25 m film thickness) was directly coupled to the mass spectrometer. The carrier gas was helium (1.5 ml/min) and the program used was 5 min isothermal at 50 C, followed by 50–150 C at a rate of 10C/min, then held at 150 C for 10 min; the injection port temperature was 260 C. Ionization of the sample components was performed in the E.I. mode (70 eV). source temperature: 230 C; mass range: 40 - 400a.m.u.Components were identified by comparison oftheir mass spectra with those in the NIST98 and PEST GC–MSlibrary.

2.4. Antimicrobial activity

2.4.1. Microbial strains

The antimicrobial activity of P. tenuiflorusesentialoil was evaluated using a panel which included laboratorycontrol strains obtained from the AmericanType Culture Collection (Hospital of king Abdulaziz University):Gram-positive bacteria Staphylococcus aureus (ATCC 29213),Streptococcus pyogenes (ATCC 19615), Gram-negative bacteria: Escherichia coli (ATCC 35218), Klebsiella pneumoniae(ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), and fungal microorganismsCandidaalbicans (ATCC 14053).

2.4.2. Test for antimicrobial activity

In the agar-well diffusion method was used, each microorganisms was suspended in sterile saline and diluted at 0.5 McFarland(1.5x108CFU/mL for bacteria and spore/ml for fungal strains). They were '' flood-incubated'' onto the surface of MHA for bacteria and DSA for fungi.The wells (4 mm in diameter) were cut from the agar and 25 l of essential oilwas delivered into them. After incubation for 24 hrs at 37 C, all plates were examined for any zones of growth inhibition, and the diameters of these zones were measured in millimeters. All the tests were performed intriplicate.

To compare the antimicrobial activities of essential oil and pure sample of thymol [Aldrich], all samples were weighed and dissolved in dimethylsulphoxide (DMSO), 10 mg/mL, the well (8 mm in diameter) were cut from the incubated agar (MHA for P. aeruginosa and DSA for C. albicans) and 0.25 mL of sample solution were delivered into them, these plates were incubated at 37 C for 24 hrs. The diameters of the inhibition zones were measured in millimeters. the tests were repeated thrice.

Positive and negative growth controls were included in a test, A microbial susceptibility control test was performed with Penicillin (10 unit) for Staphylococcus aureusand Streptococcus pyogenes, Gentamicin(10μg) forP. aeruginosa, K. pneumoniae and E. coliandNystatin (25 μL) forC. albicans.

2.4.3. Statistical analysis

The data obtained were statistically analysed by the variance analysis (one-way ANOVA) and the means separated according to LSD test with a significance level (p) of 0.05.

3. Results and discussion

3.1. Chemical composition of the essential oil

Leaves of the plant were subjected tohydrodistillation using a Clevenger apparatus and whitish-coloured essential oil was obtained. The yields of oil was0.23% w/w. The percentage compositions are listed in Table 1. GC–MS analysis resulted in the identification of 32 compounds representing 92.79% of the oil. Monoterpene and their oxygenated derivates dominated the chemical composition. The sesquiterpenes are present in smaller quantities. The major compound was Phenolic monoterpene thymol (62.53%). The essential oil also contained smaller percentages of 1-octen-3-ol (1.57%), m-cymene (1.11%), -terpinene (5.61%), 1-terpinen-4-ol (1.94%), 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol (5.13%),-bergamotene (3.26%),4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl-cycloheptane(2.2%),and2,3,4,4a,5,6-hexahydro-1,4a-dimethyl-7-(1-methylethyl)-naphthalene (2.04%).

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Table 1

Phytochemical composition of P. tenuiflorus essential oil (%).

aPhytochemicals are listed in order of their elution on DB-5 column.

bRetention Time (as minutes).

c non-identified compound.

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Literature review showed that the main phytochemical constituents of the genus Plectranthus are diterpenoids, phenolics and essential oils (Abdel-Mogib, 2002, Grayeret al., 2003).It was mentioned that the reports on the essential oils of several Plectranthus species have shown major differences with regard to the main components; β–Caryophyllene, Epiperitenone oxide and Carvacrol were the common major compounds found(Ascensãoet al., 1998), butthe last studywas a firstreference made to the occurrence of 6,7-dehydroroyleanone in the Plectranthusessential oils despite this component together with other related diterpenes has been found in solvent extracts from several Plectranthus species (Ascensãoet al., 1998). The essential oil of fresh and dried leaves of P. glandulosus were characterized by a high percentage of oxygenated monoterpenes (58.6% and 84.6% respectively) represented by cis-piperiton oxide (3.0% and 35.1%), trans-piperitone oxide (0.5% and 12.6%), fenchone (30.8% and 21.6%) and piperitenone oxide (10.9% and 6.0%), the main monoterpene hydrocarbons were terpinolene (25.2% and 7.7%), limonene (3.2% and 1.7%) and myrecene (2.2% and 1.6%),the sesquiterpene derivatives were found in a very low percentage (2.5%) presented mainly by germacrane D (1.4% and 1.0) (Ngassoum et al., 2001).Compared to other samples of essential oils extracted fromP. tenuiflorus collected from different geographical origins, ∆3-Carene (52.8%)was found the major component of essential oil obtained from plant grown in Abha (south highlands of Saudi Arabia) (Al-Yahya et al., 1985), -terpinene (10.2%), p-cymene (10.9%) and carvacrol (14.3%) were the major components in essential oil from plant cultivated in Kenya (Mwangi et al., 1993), while plant grown in Taif (west highlands of Saudi Arabia), (Smith et al., 1996) has reported that the principle component of the oil was found to be Thymol (85.3%) which symmetrized our result.

3.2. antimicrobial activity of the essential oil

The in vitro antimicrobial activity of P. tenuiflorus essential oil against the microorganisms employed and its activity potentials were qualitatively and quantitatively assessed by the presence or absence of inhibition zones and zone diameters values. According to the results given in Tables 2, the essential oil of theinvestigated plant had great in vitro potential of antimicrobialactivities against all 5 bacteriaand a yeast species tested. In this study, the antimicrobialactivities of essential oil having one concentrationof 25l/well are compared withstandard antibiotics such as gentamicin, penicillinand nystatin used as positive controls. Results fromthe antimicrobial agar-well diffusion assay are summarizedin Table 2. The data obtained from the agar-well diffusionmethod indicated that the essential oil displayed a variabledegree of antimicrobial activity (p 0.05) on different testedstrains. The data indicatedthat yeastC. albicans was the most sensitivestrain tested to the oil of P. tenuiflorus with thestrongest inhibition zones (44.7 mm). TheStaphylococcus group was, in general, found to be more sensitiveamong gram-positive bacteria with S. aureus being themost sensitive (28 mm), then Streptococcus pyogenes, with inhibitionzones of 17 mm. Among these, Gram-negative strains also displayed variable degree of susceptibility against investigated oil. Maximum activity was observed against K. pneumoniae (25.7 mm), followed by E. coli (16 mm). Gram-negative bacteria, P. aeruginosa exhibited weak inhibition zones (13.5 mm), but the response of this microbe for oil was still allergic, since it is known to have high level of intrinsic resistance to virtually all known antimicrobials and antibiotics due to a combination of a very restrictive outer membrane barrier, highly resistant even to synthetic drugs (Skočibušić et al., 2006).

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Table 2

Antimicrobial activity of the P. tenuiflorus essential oil by the agar-well diffusion method1

Data in the column followed by different letters are significantly different at p 0.05 according to LSD test.

(<12 mm), inactive; (12-20 mm), moderately active; (21- 45 mm), highly active; nt, not tested.

1 Diameter of inhibition zone (mm) including well diameter of 4 mm.

2Gentamicin resistance ≤12 mm; Penicillin resistance 28 mm; Nystatin resistance 7 mm.

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Plant essential oil is a potentially useful source of antimicrobial compounds. By inhibiting the growth of all human pathogenic bacteria and the yeast tested, P. tenuiflorusessential oil exerted a broad antimicrobial spectrum. Numerous studiesdemonstrated that the essential oils and extracts of other Plectranthus species are among the most potent extracts with regard to antimicrobial properties (Batista et al., 1994; 1995; 1996;Ascensãoet al., 1998; Rabe & Staden1998; Esther & Johannes, 2003; Grayer et al., 2003; Rijo et al., 2002; Gaspar et al., 2003; 2004; Gaspar-Marques et al.2006; Lukhoba et al., 2006). (Al-Yahya et al., 1985) determined Minimum Inhibitory Concentration (MIC) of P. tenuiflorus essential oilby broth dilution for S. aureus, E. coli, Proteus vulgaris, Bacillus subtilis,P. aeruginosaand C. albicans and the result showed that the oil was found active against all the tried microorganismsparticularly against S. aureus, B. subtilis andC. albicans.Some researchers (Moujir et al., 1993; Dellar et al., 1996) reported that there is a relationship between the chemical structures of the most abundant compounds in the tested extracts and the antimicrobial activity. The essential oils rich in phenolic compounds are widely reported to possess high levels of antimicrobial activity (Dorman & Deans, 2000; Bagamboula et al., 2004; Skaltsa et al., 2003; Knobloch et al., 1989; Hanbali et al., 2005; Skočibušić et al., 2006), which has been confirmed and extended in the present study. The antimicrobial nature of the essential oil studied is apparently related to its phenolic components, such as thymol, oxygenated derivatives (thymol acetate) and its precursorsm-cymene and -terpinene (Table 1). The bacteriostatic and fungistatic properties of the oil are suspected to be associated with the thymol content, which has been tested previously and was found to have a significant antibiotic activity (Cosentino et al., 1999; Dorman & Deans, 2000), this result was proved inpresent study by comparing the antimicrobial activity of essential oil and thymol(p0.05) against C. albicans and P. aeruginosa(Table 3). Thymol has been reported to have significant bactericidal effects towards S. aureus and E. coli (Trombetta et al., 2005).

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Table 3

Antimicrobial activities of the P. tenuiflorus essential oil and its main compound (thymol) by the agar-well diffusion method1.

Data in the row followed by different letters are significantly different at p 0.05 according to LSD test.

(<16 mm), inactive; (16 - 30 mm), moderately active; (31- 50 mm), highly active; nt, not tested.

1 Diameter of inhibition zone (mm) including well diameter of 8 mm.

2 The solvent which used for preparing sample solution (10 mg/mL).

3Gentamicin resistance ≤12 mm; Nystatin resistance 7 mm.

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In conclusion, the results obtained from our screeningconfirm the therapeutic potency of the P. tenuiflorus essential oil analysedand thus provide a rationale for their use in traditionalmedicine. These results also form a good basis for further pharmacologicaland toxicity studies and for conservation purposes. Work iscurrently being conducted in a bid to test the efficiency of essential oil in enhancing wound healing processdue to its highactivityof inhibition pathogenic microbes were selected based on it's direct or opportunistic contribution to wound contamination.

Acknowledgement

The result presented here are basically originated from Ms.Tagreed Alsufyani's M.Sc. thesis. The authors thank Mr. Jabor Albealy of the Department of Organic Chemistry for his help in gas chromatography and mass spectroscopy analysis of essential oil. Thanks also to king AbdulazizUniversityHospitalfor providing microbial strain andexcellent guidance and constructive criticisms throughout this investigation. We wish to thank Mr. Abdulraheam Alsufyani for hiskind hospitality during the collection of plant material from his farm in Alsir valley.

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