Chemical Composition and Antimicrobial Activity of Geniosporum rotundifolium Briq and Haumaniastrum villosum (Bene) AJ Paton (Lamiaceae) Essential Oils from Tanzania

Purpose: To determine the chemical composition and antimicrobial potential of essential oils from two aromatic plants of Tanzania, Geniosporum rotundifolium Briq. and Haumaniastrum villosum (Benè) A.J. Paton (Lamiaceae). Method: Essential oils from the aerial parts of the plants were extracted by hydro-distillation for 3 h using a Clevenger type of apparatus. The constituents were analyzed by gas chromatography – mass spectrometry (GC/MS).The minimum inhibitory concentrations of the essential oils were determined for eight bacterial strains and three pathogenic fungi using agar dilution method. Results: The constituents of G. rotundifolium oil were mainly oxygenated derivatives of mono- and sesquiterpenes; spathulenol (12.46 %), α -terpineol (4.65 %) and germacrene-D (3.71 %) were the most abundant. Those of H. villosum oil were predominantly sesquiterpenes (72.61 %) with caryophyllene oxide (19.01 %), humulene ep oxide II (11.95 %), β - bourbonene (5.7 %), α - humulene (5.63 %) and β caryophyllene (5.39 %) being more abundant. The oil of G. rotundifolium exhibited weak to moderate activity against the bacterial species but showed no activity against the test fungi. However, H. villosum oil showed very promising activity against all the test microorganisms (MIC 0.08 – 10.34 mg/mL). Conclusion: The major components of G. rotundifolium essential oil were oxygenated derivatives of mono- and sesquiterpenes whereas those of H. villosum were sesquiterpenes. All tested microorganisms were susceptible to H. villosum oil.


Geniosporum rotundifolium
Briq. and Haumaniastrum villosum (Benè) A.J. Paton (Lamiaceae) are known as "Nkulilo" in the Nyakyusa dialect of Rungwe District, Mbeya Region, Southwestern Tanzania. Geniosporum rotundifolium (syn. G. paludosum Bak) [1], is a stout, erect, perennial herb which grows in damp grassland at high altitude [2]. It is confined to several African countries including Tanzania [3]. Its leaves, stems and essential oils are given in combination with leaves of other plants for a number of medical uses. In Burundi it is used as an enema, cough remedy, laxative and antiabortion while in Uganda it is used against fungal and bacterial infections [4]. A previous study on G. rotundifolium growing in Cameroon indicated that the essential oil from this plant possessed significant antifungal activities against Fusarium moniliforme and Rhizopus stolonifera. Furthermore, its chemical composition was determined with sesquiterpene hydrocarbons constituting more than 90 % of the oil [5].
Haumaniastrum villosum is an annual or shortlived perennial herb confined to the African continent and Madagascar, in the sub-humid climate [6]. There is scanty information on the medicinal uses and biological activities of H. villosum and to our knowledge there is no information on its phytochemical studies. Its synonym H. galeopsifolium, has been reported to be used traditionally in Burundi, alone or in combination for a number of health problems including urogenital infections [1]. It has also been reported to be used in controlling crop pests in the Democratic Republic of Congo [7].
In the current study, chemical compositions and antimicrobial activities of the essential oils of Geniosporum rotundifolium and Haumaniastrum villosum from Tanzania are reported for the first time.

Isolation of essential oil
All materials were air-dried in the shade, prior to hydro-distillation of essential oils for 3 h in a Clevenger-type apparatus. The essential oils collected over water were separated, dried over anhydrous sodium sulfate and stored at 4-6 o C until chemical analysis and antimicrobial screening.

Gas chromatography
Gas chromatography (GC) analysis was carried out on a Perkin-Elmer 8500 gas chromatograph with a flame ionization detector (FID), fitted with a Supelcowax-10 fused silica capillary column (30 m x 0.32 mm, 0.25 µm film-thickness). The column temperature was programmed from 75 to 200 o C at a rate of 2.5 o C/min. The injector and detector temperatures were programmed at 230 o C and 300 o C, respectively. Helium was used as the carrier gas, at a flow rate of 1 mL/min.

Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC-MS) analysis was carried out using a Hewlett Packard 5973-6890 GC-MS system operating on EI mode (equipped with a HP 5MS 30 m x 0.25 mm x 0.25 µm film thickness capillary column). Helium (2 mL/min) was used as the carrier gas. The temperature of the column was programmed from 60to 280 o C, at a rate of 3 °C/min. Split ratio, 1:10.

Evaluation of antimicrobial activity
Antimicrobial activity of the essential oils against bacteria and fungi was determined using the agar dilution technique. Technical data have been described previously [10]. Briefly, stock solutions of the tested samples were prepared at 10 mg/mL in dichloromethane. Serial dilutions of the stock solutions in broth medium (100 μL of Müller-Hinton broth or on Sabouraud broth for the fungi) were prepared in a microtiter plate (96 wells). Then 1 μL of the microbial suspension (the inoculum, in sterile distilled water) was added to each well. For each strain, the growth conditions and the sterility of the medium were checked and the plates were incubated as referred above. Standard antibiotics, netilmicin and amoxicillin (at concentrations 4-88 μg/ml), were used as positive controls. For each experiment, the pure solvent, dichloromethane, was also applied as negative control. The experiments were repeated three times and the results were expressed as average values. Minimum inhibitory concentrations (MICs) were determined for all the samples and the standard pure compounds, under the same conditions, for comparison purposes. The MICs were taken as the lowest concentrations preventing visible growth.

RESULTS
The oils obtained from both plant species were pale yellow liquids with slight aromatic smell. The yield was 0.06 % v/w for G. rotundifolium and 0.12 % v/w for H. villossum. A total of 59 components, comprising 91.15 % of the oil got separated in the GC of G. rotundifolium, of which 54 constituents were identified (Table 1(a), 1(b) and 1(c). A 44.89 % of the oil was composed of oxygenated derivatives, while mono and sesquiterpene hydrocarbons constituted 36.67 % of the oil. The major compounds identified were spathulenol (12.46 %), α-terpineol (4.65 %) and germacrene-D (3.71 %). In a previous study on plants growing in Cameroon, it was found that sesquiterpene hydrocarbons constituted 90.1 % of the oil with germacrene D, β-caryophyllene and β-gurjunene being the major components [5]. The difference in the composition could be attributed to differences in the geographical location, climate, season and age at which the plants were collected.
In the essential oil of Haumaniastrum villosum, a total of 44 components were identified, representing 85.6 % of the oil ( The oils as well as pure reference compounds were tested for antimicrobial activity against eight bacterial species and three species of Candida. The antimicrobial activity as minimum growth inhibitory concentrations of the essential oils, some pure components and the reference antimicrobial agents, are shown in

DISCUSSION
The major compounds identified for the essential oil of G. rotundifolium were different from those identified previously for plants growing in Cameroon in which sesquiterpene hydrocarbons constituted 90.1 % of the oil with germacrene D, β-caryophyllene and β-gurjunene being the major components [5]. The difference in the composition could be attributed to differences in the geographical location, climate, season and age at which the plants were collected.
It would be worth reporting that H. villosum oil was strongly active against S. mutans, S. viridis, Candida albicans, C. tropicalis and C. glabrata (with MIC's 0.14-0.94 mg/mL), which were resistant to oils from G. rotundifolium and other plants growing in Tanzania, as reported previously [10][11][12]. In addition, the essential from G. rotundifolium was devoid of antifungal activity against the tested Candida species unlike the essential oil growing in Cameroon which was previously reported to have shown significant antifungal activity against Fusarium moniliforme and Rhizopus stolonifera [15].   The observed antimicrobial activity in the studied essential oils could be attributed to their major components. In the case of G. rotundifolium, the activity could be mainly, due to the oxygenated sesquiterpene spathulenol, which showed two to three times more activity than the oil, while the activity of H. villosum oil compared well with that of β-caryophyllene oxide. The antimicrobial activity of these oils could also be attributed to the major and minor constituents of the oils, constituents with the known antimicrobial activity such as spathulenol [11], linalool [13] and camphor [14], and their synergistic effects.

CONCLUSION
The composition and antimicrobial potential of two aromatic plants of Tanzania, Geniosporum rotundifolium and Haumaniastrum villosum have been determined for the first time. H. villosum shows good antimicrobial activity and hence should be further evaluated for possible use in preparations of pharmaceuticals for the management of disease conditions caused by these microorganisms, especially the oral and skin infections caused by Candida species.