Chemical compositions and antimicrobial activity of twig essential oils from three Xylopia ( Annonaceae ) species

The chemical composition of twig essential oils of Xylopia malayana, Xylopia elliptica and Xylopia fusca were analyzed using GC and GCMS. Both X. malayana (12.9%) and X. fusca (11.8%) twig oils contained spathulenol as the major constituent while X. elliptica twig oil was found to be rich in terpinen-4-ol (11.9%). The antimicrobial activities of the essential oils were evaluated against six gram positive bacteria, two gram negative bacteria and yeast by using the broth microdilution method. The twig oil of X. elliptica at a concentration ranging from 156 to 625 μg/ml was found to be active against all bacterial and yeast strains tested. Both X. malayana and X. fusca twig oils demonstrated weak activity toward all microorganisms tested with the MIC value of 5000 μg/ml. Gram negative bacteria’s, Pseudomonas aeruginosa and Escherichia coli seemed to be resistant to the essential oils tested. α-Pinene showed moderate to strong inhibitory effect against all the microorganism strains tested with the MIC value ranging from 325 to 2500 μg/ml, as compared to the other standard compounds which showed weak inhibitory even at the highest concentrations used. This finding demonstrated that the twig oil of X. elliptica possesses antimicrobial activity which may be useful and potential ingredient in the production of health care products.


INTRODUCTION
The family Annonaceae comprises about 120 genera and more than 2000 species (Leboeuf et al., 1982) and is distributed mainly in South America, Paleotropical Africa and Southeast Asia.The plants of the family Annonaceae can be divided into three categories which are trees, shrubs, woody climbers and commonly found in the lowland forest (Kochummen, 1997).The shape of the plant's leaf is simple and alternate.The bark is smooth, brown or dark green in colors and fragrant.The flowers are solitary or clusters and possessing a strong aromatic smell while the fruits are normally oblong in shape, stalked or sessile.Many members of Annonaceae family are used by local native in traditional medicine for the treatment of skin diseases, fever and stomachaches (Wiart, 2006).Phytochemical studies on the Annonaceae plants resulted in the isolation of several compounds for examples isoquinoline alkaline alkaloids (Mukhtar et al., 2000) and acetogenins (Alali et al., 1999) which have been found to possess antimicrobial, insecticidal, antimalarial and antioxidant properties (Iwu et al., 1999).The genus Xylopia comprises about 170 species and they are widely distributed throughout Southeast Asia, Australia and Melanesia (Hyland and Whiffin, 1993).In Malaysia, eight species namely Xylopia malayana, Xylopia caudata, Xylopia ferrugineae, Xylopia magna, Xylopia fusca, Xylopia elliptica, Xylopia stenopetala and Xylopia subdehiscens have been documented by Kochummen (1972).The plants are usually shrubs or small trees in nature and commonly found in lowland, peat swap and mountain forests forest.The secondary metabolites from Xylopia species have been known for their great usage in folk medicine, food flavoring and medical industries (Leboeuf et al., 1982).Besides, the essential oils of some Xylopia sp. are also used in traditional medicine as a remedy in the treatment of stomach-ache and intestines.Xylopia species are sources of volatile components, diterpenes and alkaloid, which have been reported to possess antimicrobial, antiinflammantory and antimalarial properties.Volatile constituents and bioactivity studies are available in the literature on Xylopia aethiopica (Issakou et al., 2014;Sylvain et al, 2014;Vyry et al, 2014), Xylopia longifolia (Fourier et al, 1993), Xylopia parviflora (Woguem et al, 2014;Samuel et al., 2015), Xylopia aromatica (Lago et al, 2003) and Xylopia langsdorfiana (Correia et al., 2015).
X. malayana or locally known as Jangkang is a small tree up to 20 m high and occurs in the northern part of East Malaysia.The morphology of leaves are ellipticoblong whilst flowers are normally small and aromatic.In Peninsular Malaysia, the leaves of X. malayana are traditionally be used for treatment after childbirth (Kamarudin, 1988).The chemical constituent and antimicrobial activity of essential oils from the leaf of X. malayana have been previously reported by Nor Azah et al. (1996).X. elliptica or lilan is a medium sized tree which could grow up to 8 to 10 m tall and is one of the endemic plants.The leaves are membranous and blade elliptic while the flowers are axillary with oblong fruits.X. fusca is widely distributed in Southeast Asia.It is a medium to large trees with 125 m in height and commonly found in peat swamp forest.X. fusca is known as jangkang paya.In traditional medicine practice, the plant used as an abortifacient and as an antidote for insect-bites.The wood of X. fusca is used in making a pineapple box whilst the barks of this plant are also used for walls of huts (Whitmore, 1972).The chemical composition of the leaf essential oils from X. malayana, X. fusca and X. elliptica have been examined by Siti Humeirah et al. (2010) but no information is available on the twig oil of these plants.Therefore, in this study, the chemical compositions of essential oils of twig part from X. malayana, X. elliptica and X. fusca as well as their antimicrobial properties were explored.

Plant
The twig parts of X. malayana (FRI 54729), X. elliptica (FRI 54736) and X. fusca (FRI 54733) were randomly collected from Pasoh Forest Reserve, Negeri Sembilan, Malaysia on November 2007.All the plant materials were identified by Mr. Kamarudin Salleh from Forest Research Institute Malaysia (FRIM) and the voucher specimens were deposited at the FRIM herbarium

Extraction of essential oils
The plant material were cut into small pieces, weighed (200 to 400 g) and hydrodistilled separately in clavenger type apparatus for 6 h.The oily layer obtained from each plant material were separated, dried over anhydrous sodium sulfate and stored at 4°C until used.The yield (%) of essential oil was also determined by Azeotropic methods (British Pharmacopoeia, 2004).

GC and GCMS analysis
The chemical constituents of essential oils were analysed by GC and GCMS.GC analysis was carried out using Shimadzu GC2010 (FID) chromatography with a CBP-5 column (25 mm × 0.25 µm × 0.25 mm film thickness).GCMS analysis was performed using a Agilent GCMS 7890A/5975C series MS with a HP-5MS column (30 mm × 0.25 µm × 0.25 mm film thickness).The components of essential oils were identified by comparison of their retention indices with literature values (Adams, 2001) and confirmed by comparison of their mass spectral data with those from the Wiley (1990), HPCH 2205.L and NIST05a.L libraries.The Kovats indices (Kovats, 1965) of each component were determined relative to the retention time of a series of n alkanes (C8-C20).The relative proportions of the chemical compounds were expressed as percentages obtained by peak-area normalization, all relative response factors being taken as one.

Antimicrobial assay
The twig oils of X. malayana, X. elliptica and X. fusca were screened for their antimicrobial activity against nine strains of microorganisms including six gram positive bacteria Staphylococcus aureus ATCC 25923 (MSSA); S. aureus ATCC 33591 (MRSA); S. aureus ATCC 70069 (VISA); Vancomycin Resistant S. aureus (VRSA156), Vancomycin Intermediate S. aureus (VISA24) and S. epidermidis ATCC 12228.The gram negative bacteria including Pseudomonas aeruginosa ATCC 25668, Escherichia coli ATCC 10536 and yeast Candida albicans ATCC 10231 using the Minimum Inhibitory Concentration (MIC) method as described previously by Saiful et al. (2008).The gram positive and negative bacteria and yeast were purchased from American Type Culture Collection (ATCC) while VRSA156 and VISA24 were lab passage derived mutants from clinical MRSA isolate.

Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) values of twig oils were determined using the double-broth microdilution method.Briefly, the well A was loaded with essential oils stock solution (100 mg/ml) and topped up with respectively broth.The mixture in well A was mixed thoroughly and transferring to well B to H which are already contained with respectively broth.The same procedure (well A) was repeated for well B to H to create a serial doubling dilution of the oils with concentration ranging from 10 to 0.078 mg/ml.Finally, a suspension containing 10 8 colony-forming units (CFU)/ml which are equivalent to McFarland standard tube no.0.5 used (Vandepitte et al., 1991) were inoculated into the mixture to produce a final volume of 100 μl.Oxacillin and cyclohexamide were used as positive control whiles DMSO-broth mixture (5%) was used as a negative control.The experiments were performed in triplicates and repeated twice.The lowest concentration which produces no visible growth or remains clear after observed through the macroscopic evaluation was taken as the MIC.The activity was categorized as strong (MIC ≤ 1000 µg/ml), moderate (999 µg/ml < MIC < 4900 µg/ml) and weak (MIC ≥ 5000 µg/ml) (Ibrahim et al., 2009).
Several studies on the chemical composition of the essential oils of other Xylopia species have shown that monoterpenes and sesquiterpenes are the major constituents of essential oils.Spathulenol, which was detected in the twig oils of the X. elliptica X. fusca and X. malayana was identified as the major component in the X. aromatica (Founier et al., 1994) and Xylopia emarginata (Lago et al, 2003) essential oils.Both monoterpenoid, p-cymene and α-pinene were detected as major constituents in the essential oil of Xylopia sericea (Lemos et al., 1992).Other chemical constituents for example terpinen-4-ol, β-pinene, δ-cadinene, cymene and α-copaene were also found in other part of Xylopia species Nor Azah et al. (1996); Lemos et al. (1992).However, the essential oils obtained from X. fusca contained certain compounds which are not observed in the other oils namely; isopulegyl acetate, gymnomitrol, 14-hydroxy--cadinene and 14-hydroxy-αmuurolene which could be considered unique to the species.The presence of some terpene type compounds in appreciable amounts supported the use of these compounds as chemical marker in the species identification purposes.Phytochemical analysis of the oils indicated that the chemical constituents represent a valuable chemotaxonomic characterization for supporting the identification of the genus Xylopia (Siti Humeirah et al., 2010).

Antimicrobial activity
The antimicrobial assays of the twig oils of X. malayana, X. fusca and X. elliptica, together with eight reference standards which are α-pinene, geranyl acetate, linalool, limonene, geraniol, terpinen-4-ol, 1,8-cineole and βpinene were carried out by broth microdilution method against nine different strains of microorganisms.According to the results in Table 2, the twig oil from X. elliptica have been shown to possess the strongest  antibacterial and antifungal activity against all gram positive bacteria and yeast strains tested with the MIC values ranging from 156 μg/ml to 625 µg/ml.Gram negative bacteria's, P. aeruginosa and E.coli seemed to be resistant to the essential oils tested.Both X. fusca and X. malayana twig oils however showed less inhibitory effect on the growth of bacteria and yeast strains even at the highest doses (5000 µg/ml) used.The results indicated the gram positive bacteria are more sensitive to plant oil than gram negative bacteria due to their restrictive outer membrane barrier which are generally less susceptible and destabilization.The inhibitory effects of X. elliptica twig oil on bacterial strains tested could be related to the present of α-pinene (Magwa et al., 2006), terpinen-4-ol (Barel et al., 1991) and ρ-cymene which have been reported their high level of antimicrobial activity.In addition, it is also possible that the other minor or trace compounds might be involved in some types of antimicrobial synergism with other active components of essential oil.
Various studies have reported antimicrobial activities for essential oils from Xylopia species.Lemos et al. (1992) reported the essential oil of X. sericea which contains 1,8-cineole as the major constituents showed effective antifungal activity against Candida strains.The essential oils of Xylopia longifolia from France were also found to possess antibacterial and antifungal activity (Fourier et al., 1993).The antimicrobial activities of essential oil from Xylopia parvifloral and Xylopia aethiopica from Africa have been reported by Bakarnga et al. (2014) and Woguem et al. (2014).Beside essential oil, the antimicrobial properties of selected standard compounds were also conducted in the present study.Based on the results summarized in Table 2, the antibacterial and antifungal activity of the five reference standards indicated that α-pinene had the moderate to strong inhibitory effect against all bacterial and yeast growth at the concentration of 325 to 2500 μg/ml.Xylopia epidermidis, E. coli and C. albicans were most susceptible to the compound investigated and hence support the earlier finding on α-pinene's significant antimicrobial potential (Dorman and Deans, 2000).The reference standards of β-pinene and 1,8-cineole however inhibited weak (MIC values of 5000 μg/ml) antibacterial and antifungal activity against all microorganisms tested.Terpinen-4-ol, the most abundant component in the twig oil of X. elliptica, has been shown to have a moderate to strong antibacterial activity against P. aeruginosa and Ecoli strains with the MIC values of 2500 and 625 µg/ml.Terpinen-4-ol however showed weak inhibitory effect against MRSA strains tested.The results however was not in agreement with a previous study reported by Barel et al. (1991) which found the bacteriostatic effects of tested compound, terpinen-4-ol against several microorganisms.Linalool was found to be less effective to all microorganisms tested with the MIC values ranging from 1250 to 5000 μg/ml.

Conclusion
The characteristics of the essential oils from three Xylopia species are represent a valuable chemotaxonomic tool for the identification of the genus Xylopia.Considering the relatively high yield of monoterpenoids and bioactive component in the oils, Xylopia could be a potential new source of fragrance ingredient for food, pharmaceutical and cosmetic industries.
Percentages were calculated based on results obtained from gas chromatography on CBP-5 column; RI = retention indices; MS= mass fragmentation; CO= co-chromatography with authentic sample.

Table 2 .
Minimum Inhibitory Concentration (µg/ml) of twig oils from three Xylopia species and standard compounds.