Comparative study of the essential oil composition and antimicrobial activity of Leonotis leonurus and L. ocymifolia in the Eastern Cape, South Africa

Species of Leonotis are among the frequently-used herbal remedies to treat various bronchial illness and epilepsy. The essential oils of the leaf and flower of L. leonurus and L. ocymifolia growing in the Eastern Cape of South Africa were analysed by GC-MS. Major constituents of the L. leonurus oils were limonene (7.2–15.6%), (Z)- ββ -ocimene (7.5–10.8%), γγ -terpinene (4.0–4.7%), ββ -caryophyllene (15.2–19.6%), αα -humulene (4.6–6.5%) and germacrene D (18.9–20.0%), while the essential oils of L. ocymifolia had (Z)- ββ -ocimene (13.0–15.2%), nonanal (5.5%) ββ -caryophyllene (21.4– 30.8%), αα -humulene (9.1–11.6%), germacrene D (21.5– 21.7%) and T-muurolol (4.6%) as the prominent compounds. The oils exhibited a broad spectrum antibacterial activity against Gram-positive ( Bacillus subtilis , Bacillus cereus , Micrococcus kiristinae , Staphylococcus aureus , Staphylococcus

Species of Leonotis are widely distributed in Africa where they have a long history of use for medicinal purposes.In South Africa, many members of the genus are found in the southwest, spreading to the Eastern Cape (Watt andBreyer-Brandwijk 1962, Batten 1988).In the Eastern Cape Province, some of the species are used for the treatment of coughs, chest troubles, bronchial asthma, stomach aches, skin diseases, haemorrhoids and epilepsy (Watt and Breyer-Brandwijk 1962, Batten and Bokelmann 1966, Gledhill 1969, Batten 1988).
Leonotis leonurus (L.) R. Br. and Leonotis ocymifolia (Burm.f.) Iwarsson are the two most commonly-used species in the Province.L. ocymifolia grows mostly along the coastal region and is particularly used for the treatment of nerve weakness, while L. leonurus grows in moist areas, along hill-slopes and is commonly used for the treatment of liver inflammation (Watt and Breyer-Brandwijk 1962, Batten and Bokelmann 1966, Gledhill 1969, Van Wyk et al. 1997, Pienaar 1984, Batten 1988, Batten et al. 2001).
Previous essential oil analyses of Leonotis species from Nigeria, South India and Rwanda have been reported.The Nigerian and Rwanda types were dominated by sesquiterpenoids while South India had phenols and monoterpenoids as the major components, indicating a chemotypic difference (Thoppil and Jose 1995, Muhayimana et al. 1998, Oyedeji et al. 1999).L. leonurus flower oil from Portugal had α-pinene, limonene, βcaryophyllene, α-humulene and caryophyllene oxide as the major components (Pedro et al. 1991).
These two important medicinal plants growing in South Africa are highly aromatic, yet, to the best of our knowledge, there is no information on the type and constituents of their volatiles.In this paper, we present a comparative analysis of the oils found in L. leonurus and L. ocymifolia in South Africa with their antimicrobial activity.
Fresh plants of L. leonurus and L. ocymifolia were collected from the wild around the University of Fort Hare campus, and voucher specimens were deposited at the University Herbarium.
The fresh plant materials were carefully separated into flowers and leaves.About 400g of the leaves and 200g of the flowers of each plant were hydrodistilled separately in an all-glass Clevenger-type apparatus in accordance with the British Pharmacopoeia (BP) (1980) method, for 3h.
GC-MS analyses of the oils were performed on a Hewlett Packard Gas Chromatograph HP 5973 interfaced with a VG analytical 70-280s double-focussing mass spectrometer.Electron ionisation was at 70eV with an ion-source temperature at 240°C.HB-5 column was used (30m x 0.25mm id) similar to DB 5, film thickness was 0.25µm, while helium was used as the carrier gas.The oven temperature was 70-240°C at 5°C min -1 .Oil (0.2µl) was injected into the GCMS.n-Alkanes were run under the same conditions for the retention indices determination.
Identification of the oil constituents was accomplished by comparison of their mass spectra and retention indices with those of standard samples and literature (Adams 1989, Joulain andKoenig 1998).
A collection of eight laboratory bacteria which included five Gram-positive and three Gram-negative strains was obtained from the Microbiology Department, Rhodes University.These are Bacillus subtilis, Bacillus cereus, Micrococcus kiristinae, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Shigella sonnei.The MIC values of the essential oil were determined with a microplate dilution method against the bacteria using 96-well microtitre plates.Essential oil in 40µl of hexane was dissolved in 40µl of acetone.Each test organism was prepared by diluting 24hold broth culture with sterile nutrient broth.The culture was then diluted 100-fold to give approximately 10 6 bacteria ml -1 .The microtitre plates were prepared using serial dilution (Afolayan and Meyer 1997, Amvam Zollo et al. 1998, Eloff 1998) and incubated for 24-48h at 37°C.As an indicator of bacterial growth, 40µl of 0.2mg ml -1 p-iodonitrotetrazolium (INT) solution was added to each well and incubated at 37°C for 30min.The colourless tetrazolium salt was reduced to a red-coloured product by biological activity of the organisms, thereby making the inhibition of bacterial growth visible as clear wells.MIC values were recorded as the lowest concentration resulting in complete inhibition of bacterial growth.Each treatment was replicated three times.Streptomycin, chloramphenicol, solvents and sample-free solutions were used as positive and negative controls.
Again, compositional difference was observed in the two species.In the leaf and flower oils of L. leonurus, limonene occurred at higher concentrations (15.6% and 7.2%, respectively), while it was present in a trace amount in the 3.2 3.7 1.5 6.7 t = trace amount <0.01 a = retention index on an HB-5 capillary column at 5°C min -1 from 70-240°C * = correct isomer not identified flowers and 1.93% in the leaves of L. ocymifolia essential oils.Also, β-caryophyllene and germacrene D were found at higher concentrations in the essential oils of L. ocymifolia, making the plant a good source of β-caryophyllene, αhumulene, germacrene D and (Z)-β-ocimene while limonene could be readily obtained from L. leonurus.P-cymene was considerably lower (1.2-1.5%) in the two flower oils.This compound was not detected in the leaf oil of L. ocymifolia but was present in an appreciable amount (3.5%) in the leaf oil of L. leonurus.Furthermore, the concentration of Tmuurolol was fairly high in the flower oil of L. ocymifolia when compared to the remaining three oils.Nonanal was substantially present in the essential oil of L. ocymifolia flowers but not detected in the leaf oil and the flowers of L. leonurus.γ-Terpinene and terpinolene were not detected in the oils of L. ocymifolia.Generally, L. ocymifolia had a higher oil yield (0.06%) when compared to L. leonurus.Information on the oil constituents found in the species of Leonotis may be significant in the chemotaxonomy of the group.This type of information is also important in our understanding of the medicinal use of the group.
The antibacterial test also reveals both oils having a broad spectrum antimicrobial activity against all the tested organisms with MIC ranging from 0.039-1.25mgml -1 (Table 2).The oil was however more active against the Grampositive bacteria than the Gram-negative ones.It is interesting to note that while streptomycin was only effective against B. cereus and B. subtilis bacteria at 2.5mg ml -1 , the essential oil of this plant inhibited the growth of these organisms at concentrations of 0.078-1.250mgml -1 .

Table 1 :
Percentage composition of the leaf and flower oils of L.