Characterization and Antimicrobial Activity of Volatile Constituents from Fresh Fruits of Alchornea cordifolia and Canthium subcordatum

Bacterial resistance has been increasingly reported worldwide and is one of the major causes of failure in the treatment of infectious diseases. Natural-based products, including plant secondary metabolites (phytochemicals), can be exploited to ameliorate the problem of microbial resistance. The fruit essential oils of Alchornea cordifolia and Canthium subcordatum were obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). The essential oils were subjected to in vitro antibacterial, antifungal and cytotoxic activity screening. Thirty-eight compounds comprising 97.7% of A. cordifolia oil and forty-six constituents representing 98.2% of C. subcordatum oil were identified. The major components in A. cordifolia oil were methyl salicylate (25.3%), citronellol (21.4%), α-phellandrene (7.4%), terpinolene (5.7%) and 1,8-cineole (5.5%). Benzaldehyde (28.0%), β-caryophyllene (15.5%), (E,E)-α-farnesene (5.3%) and methyl salicylate (4.5%) were the quantitatively significant constituents in C. subcordatum fruit essential oil. A. cordifolia essential oil demonstrated potent in vitro antibacterial activity against Staphylococcus aureus (MIC = 78 μg/mL) and marginal antifungal activity against Aspergillus niger (MIC = 156 μg/mL). C. subcordatum showed antibacterial activity against Bacillus cereus and S. aureus (MIC = 156 μg/mL) and notable antifungal activity against A. niger (MIC = 39 μg/mL). However, no appreciable cytotoxic effects on human breast carcinoma cells (Hs 578T) and human prostate carcinoma cells (PC-3) were observed for either essential oil. The antimicrobial activities of A. cordifolia and C. subcordatum fruit essential oils are a function of their distinct chemical profiles; their volatiles and biological activities are reported for the first time.


Introduction
Antimicrobial resistance is one of the most serious public health threats that results mostly from the selective pressure exerted by antibiotic use and abuse [1,2]. During the last few decades, rapid evolution and spread of resistance among clinically important bacterial species have been observed. Due to this increasing resistance, many antimicrobial agents are losing their efficacy [3][4][5]. Consequently, the therapeutic options for the treatment of infections have become limited or even unavailable. According to the World Health Organization (WHO), infectious diseases are the second cause of death around the world [6]. Therefore, it is necessary to search and develop new alternative compounds to ameliorate the problem of microbial resistance.
Alchornea cordifolia (Schumach. and Thonn.) Müll. Arg. (Euphorbiaceae) is a shrub found along the coastal regions of West Africa. It has multipurpose utilization as fodder, food and medicine. The leaves, roots and stem bark extracts are used extensively in traditional medicine in the preparation of drugs for urinary, respiratory and gastro intestinal disorders [7]. A slurry from the fruits is administered for asthma and cough. The leaves are used internally for the management of gastrointestinal, respiratory and urinary tract infections and externally for wounds. A decoction of the leaves is used as eye lotion [8]. The leaves and stem bark, when powdered, are used in the treatment of ringworms and other skin infections [9]. Hitherto, the leaf part of A. cordifolia has been a subject of scientific studies: anti-microbial, antioxidant and anticancer activities, etc. [10][11][12][13]. The gas chromatographic-mass spectral (GC-MS) characterization of the volatile oil from fresh leaves of A. cordifolia has been reported [14]. The aqueous extract of A. cordifolia has demonstrated antibacterial activity against 21 bacterial strains tested and showed the highest levels of antibacterial activity with MICs against methicillin-resistant Staphylococcus aureus (MRSA) in the range of 1.6-3.1 mg/mL and MBCs in the range of 6.3-12.5 mg/mL among 24 other plant species studied [15]. Phyto-constituents, such as steroids, phenolic compounds, flavonoids, flavones, tannins, xanthones and alkaloids, have been isolated from A. cordifolia leaf [16][17][18][19].
Canthium subcordatum DC. (formerly Psydrax subcordata DC., Rubiaceae) is a tree that grows in central and western Africa and reaches a height of more than 10 m [20]. Its roots, leaves and stem bark are used for medicinal purposes. Alcoholic extracts of the stem bark have potential antidiabetic properties [21] and the roots are used to treat malaria fever, inflammation and cardiovascular disease [22]. Recently, five new iridoid dimers were isolated from the fruits of C. subcordatum [23]. A number of iridoids, which include (6S,9R)-roseoside [24] and shanzhisin methyl ester gentiobioside [25] have been isolated from the stem-bark of C. subcordatum and their structures deduced. GC-MS analysis and anticancer activity of ethanol leaf extract of C. parviflorum have also been reported [26,27]. As part of an ongoing search for biologically active essential oils from the rain forest biodiversity of Nigeria, we report the antibacterial and antifungal activities of volatile constituents from the aromatic fresh fruits of A. cordifolia and C. subcordatum.

Plant Material
The mature fresh fruits of A. cordifolia and C. subcordatum were collected in the month of July 2004, from the campus of the University of Ibadan, Nigeria. Plant samples were authenticated by F. Usang of the Herbarium Headquarters, Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria, where voucher specimens (FHI 107409 and FHI 107410, respectively) were deposited.
The fruit essential oils were obtained by hydrodistillation (4 h) of the pulverized air-dried plant samples (500 g) in an all glass Clevenger-type apparatus following the British Pharmacopoeia specifications [28]. The fruit oils were dried over sodium sulfate and kept in refrigeration (4˝C) after estimation of percentage yield.

Gas Chromatographic-Mass Spectral Analysis
The essential oils were subjected to GC-MS analysis on an Agilent system consisting of a model 6890 gas chromatograph, a model 5973 mass selective detector (MSD) (Agilent Technologies, Santa Clara, CA, USA), and an Agilent ChemStation data system (http://www.agilent.com/ en-us/products/software-informatics/massspec-workstations/gc-msd-chemstation-software). The GC column was an HP-5ms fused silica capillary with a (5% phenyl)-methyl polysiloxane stationary phase (30 mˆ0.25 µm film thickness). The carrier gas was helium with a column head pressure of 7.07 psi and flow rate of 1.0 mL/min. Inlet temperature was 200˝C and MSD detector temperature was 280˝C. The GC oven temperature program was used as follows: 40˝C initial temperature, held for 10 min; increased at 3˝C/min to 200˝C; increased 2˝C/min to 220˝C. The sample was dissolved in CH 2 Cl 2 , and 1 µL was injected using a splitless injection technique.
Identification of individual constituents of the essential oils was achieved based on their retention indices (determined with a reference to a homologous series of normal alkanes) and by comparison of their mass spectral fragmentation patterns (National Institute of Standards and Technology, NIST, database/ChemStation data system) and with the literature [29].

Antibacterial Screening
A. cordifolia and C. subcordatum essential oils were screened for antibacterial activity against Plates were incubated at 37˝C for 24 h; the minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive antibiotic control; DMSO was used as a negative control (50 µL plus 50 µL CAMHB, serially diluted as above).

Antifungal Screening
Antifungal activity was determined, as described above for bacteria (i.e., serial dilution, concentrations of 2500, 1250, 625, 313, 156, 78, 39, and 19.5 µg/mL) using Candida albicans (ATCC No. 10231) in a yeast-nitrogen base growth medium with approximately 7.5ˆ10 7 CFU/mL. Amphotericin B was used as the positive control. An additional test for antifungal activity against Aspergillus niger (ATCC No. 16888) was determined as above using yeast mold (YM) broth inoculated with A. niger hyphal culture diluted to a McFarland turbidity of 1.0. Amphotericin B was the positive control.

Cell Culture
Human Hs578T breast ductal carcinoma cells (ATCC No. HTB-129) [31] were grown in a 3% CO 2 environment at 37˝C in Dulbecco's modified Eagle medium (DMEM) with 4500 mg glucose per liter of medium, supplemented with 10% fetal bovine serum, 10 µg bovine insulin, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium, and buffered with 44 mM NaHCO 3 , pH 7.35.
Human PC-3 prostatic carcinoma cells (ATCC No. CRL-1435) [32] were grown in a 3% CO 2 environment at 37˝C in RPMI-1640 medium with l-glutamine, supplemented with 10% fetal bovine serum, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium and buffered with 15 mM Hepes and 23.6 mM NaHCO 3 , pH 7.30.

Cytotoxicity Screening
Hs 578T cells were plated into 96-well cell culture plates at 1.0ˆ10 5 cells per well and PC-3 cells at 1.9ˆ10 4 cells per well. The volume in each well was 100 µL for both cell types. After 48 h, supernatant fluid was removed by suction and replaced with 100 µL growth medium containing either 2.5 or 1.0 µL of dimethylsulfoxide (DMSO) solution of oils (1% w/w in DMSO), giving a final concentration of 250 or 100 µg/mL, respectively, for each oil. Hs 578T cells were tested with final concentrations at 250 µg/mL and PC-3 at final concentration of 100 µg/mL. Solutions were added to wells in four replicates. Medium controls and DMSO controls (25 or 10 µL DMSO/mL) were used. Tingenone (250 or 100 µg/mL) was used as a positive control [33]. After the addition of the sample, plates were incubated for 48 h at 37˝C; medium was then removed by suction, and 100 µL of fresh medium was added to each well. In order to establish percent kill rates, the Cell Titer 96 ® AQ ueous Non-Radioactive Cell Proliferation assay was performed [34]. After colorimetric readings were recorded (using a Molecular Devices SpectraMAX Plus microplate reader, 490 nm, Molecular Devices, LLC, Sunnyvale, CA, USA), average absorbances, standard deviations and percent kill ratios (%kill oil /% kill DMSO ) were calculated.

Results and Discussion
The relative concentrations of the volatile components in A. cordifolia and C. subcordatum fruits, according to their elution order on HP-5ms capillary column are presented in Tables 1 and 2 respectively. The aromatic fruit essential oils were obtained in 0.17% (w/w) yield. Thirty-eight compounds comprising 97.7% of A. cordifolia oil and forty-six constituents representing 98.2% of C. subcordatum oil were identified. The fruit essential oil of A. cordifolia consisted of oxygenated monoterpenoids and aromatic esters (52.9% and 26.5%), monoterpene and sesquiterpene hydrocarbons (17.2% and 14.6%) and low amounts of aliphatic alcohol and aldehyde (3.7%). The major components identified in this sample include methyl salicylate (25.3%), citronellol (21.4%), α-phellandrene (7.4%), terpinolene (5.7%) and 1,8-cineole (5.5%). Other minor constituents detected in considerable quantities were p-cymene (3.6%), α-humulene (3.0%), β-caryophyllene (2.8%) (E)-β-damascenone (2.0%) and 1-octen-3-ol (2.0%). Two uncommon essential oil constituents were identified as (Z)-rose oxide and geosmin in A. cordifolia essential oil. (Z)-Rose-oxide (isobutenyl-4-methyl tetrahydropyran) is a perfumery ingredient and a constituent (inter alia) of Pelargonium essential oils and secretions of Aromia moschata [35]. This cyclic monoterpene ether, found in 0.5% concentration in rose oil, is said to be responsible for the highly volatile floral-green top note [36]. Geosmin (1,10-dimethyl-9-decalol), detected in 1.0% concentration, is reported as a microbial volatile organic compound. Geosmin is described as a powerful aromatic compound with an earthy smell and is implicated as one of the consequences of rot on grapes. It has very low odor threshold and strong odors. Microorganisms such as fungi and bacteria (Actinomycetes, Streptomyces riseus and Streptomyces odourifer) are reported to be present on some fruits, grapes for example, and are known for their ability to produce geosmin during metabolism [37]. According to Dionigi et al. [38], geosmin is derived from a sesquiterpene precursor such as farnesyl pyrophosphate. The major constituents of the fresh leaf essential oil of A. cordifolia reported by Okoye et al. [14] were eugenol (41.7%), cadinol (2.46%), linalool (30.6%), caryophyllene (1.04%) and (E)-α-bergamotene (4.54%). Twenty-five constituents consisting 90.3% of the composition of the leaf essential oil were identified in 0.13% w/w yield. Benzaldehyde (28.0%), β-caryophyllene (15.5%), (E,E)-α-farnesene (5.3%) and methyl salicylate (4.5%) were the quantitatively significant constituents in C. subcordatum fruit essential oil. C. subcordatum fruit oil is comprised of 41.3% hydrocarbons and 56.9% oxygen-containing compounds. The five classes of organic compounds identified and reported are twenty-four hydrocarbons (41.3%), twelve alcohols (18.6%), three aldehydes (28.7%), four aromatic esters (5.8%), one ketone and one ether (3.8%). The fruit volatile oil is dominated by sesquiterpenoid compounds (50.8%), followed by aromatic compounds (33.8%), monoterpenoid compounds (7.3%), simple aldehydes and alcohols (6.3%). The monoterpenoid profile consisted of two monoterpene hydrocarbons (2.8%) and six oxygenated monoterpenes (4.5%). Twenty-two sesquiterpene hydrocarbons and seven oxygenated sesquiterpenes made up the sesquiterpenoid profile of the oil. Two unusual sesquiterpenoids, α-calacorene and longiborneol were identified in C. subcordatum fruit oil. The sesquiterpene alcohol, longiborneol is a constituent of Juniperus, Pinus, Cupressus, Dacrydium species and Cedrus deodara. It is reported to be a plant growth regulator (inhibits cress root growth and promotes wheat germination) [35]. A literature search has revealed no previous work on the analyses of volatile components of this plant or other Canthium species. The high concentration of benzaldehyde in essential oil samples has been reported by other workers. Lei et al. [39] showed that essential oils from fresh flowers of Cerasus subhirtella and C. serrulata contain 31.2% and 42.1% benzaldehyde, respectively as major constituents. Benzaldehyde (96.96%) was also indicated as a major component in the leaf essential oil of Prunus myrtifolia [40].  The antibacterial and antifungal activities of the fruit volatile oils of A. cordifolia and C. subcordatum showed promising antimicrobial activity (Table 3). Alchornea cordifolia oil demonstrated good antibacterial activity against the Gram-positive organism S. aureus (MIC = 78 µg/mL) and moderate antifungal activity against A. niger (MIC = 156 µg/mL); C. subcordatum oil exhibited moderate antibacterial activity against B. cereus and S. aureus (MIC = 156 µg/mL) and good antifungal activity (MIC = 39 µg/mL) against A. niger. It has been documented that Gram-positive bacteria are more sensitive to chemical compounds than Gram-negative bacteria due to differences in the structures of their cell walls; Gram-negative bacteria are less susceptible to hydrophobic small molecules such as essential oil components due to hydrophilic lipopolysaccharides in their outer membrane [41]. This is obvious by the sensitivity of the Gram-positive bacteria to the tested essential oils in the assay; however with differences in the degree of inhibition. The antimicrobial activities observed in the studied fruit essential oils can be attributed to the major constituents or a synergy between the major and some minor compounds. Consistent with the results of A. cordifolia essential oil, Laportea aestuans essential oil, rich in methyl salicylate (54.50%), has been shown to exhibit antimicrobial activity against S. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans at 200 mg/mL compared to the standard drug; however, it was more active against the fungi Rhizopus stolon and A. niger at 25 mg/mL [42]; Pelargonium graveolens essential oil, dominant in citronellol (26.7%), inter alia, and citronellol, also demonstrated strong inhibitory activity against S. aureus and E. coli [43,44]. Similarly, benzaldehyde (96.96% and 90.6%, respectively, in P. myrtifolia and apricot, Prunus armeniaca, seed essential oils) exhibited antimicrobial activity against S. aureus, S. epidermidis, B. subtilis, P. aeruginosa, E. coli and C. albicans [39,40]. Additionally, Stachys cretica essential oil (β-caryophyllene, 51.0%) and β-caryophyllene are reported to exhibit strong antimicrobial activity, particularly against P. aeruginosa and B. subtilis [45]. Both A. cordifolia and C. subcordatum fruit essential oils were screened for in vitro cytotoxic activity against Hs 578T human breast adenocarcinoma and PC-3 human prostatic carcinoma cells. Neither oil showed activity, however, with 0% kill at the concentrations tested.

Conclusions
The antimicrobial activities of A. cordifolia and C. subcordatum essential oils are a function of their distinct chemical profiles. The fruit essential oil of A. cordifolia, rich in methyl salicylate and citronellol, showed antibacterial activity against S. aureus and antifungal activity against A. niger. The antimicrobial activity can be attributed to these two major components, which have shown antimicrobial activities [46][47][48]. C. subcordatum fruit oil was active against B. cereus, S. aureus, and A. niger, but the activity is not likely due to the major component benzaldehyde, which is generally not antimicrobial [49], but rather a synergism between minor essential oil components. The promising antimicrobial activities of A. cordifolia and C. subcordatum essential oils are consistent with traditional uses of these medicinal plants.