South African Journal of Botany 2004, 70(4): 611–617 Copyright © NISC Pty Ltd Printed in South Africa — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299 Essential oil composition and in vitro antimicrobial and anti-inflammatory activity of South African Vitex species E Nyiligira1, AM Viljoen1*, KHC BaÕer2, T Äzek2 and SF van Vuuren1 1 Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa 2 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470 EskiÕehir, Turkey * Corresponding author, e-mail: [email protected] Received 1 March 2004, accepted in revised form 7 May 2004 The essential oil composition of Vitex pooara, V. rehman- inflammatory activity of the essential oils was evaluat- nii, V. obovata ssp. obovata, V. obovata ssp. wilmsii and ed using a 5-lipoxygenase assay and all essential oils V. zeyheri was determined using gas chromatography effectively inhibited 5-lipoxygenase, a key enzyme in and mass spectrometry. The in vitro antimicrobial activ- the inflammatory cascade with V. pooara producing the ity of the essential oils was assessed on Staphylococcus most promising activity (IC50 value of 25ppm). Using the aureus, Bacillus cereus and Escherichia coli and the essential oil data matrix, chemotaxonomic evidence is minimum inhibitory concentration values recorded. All presented which supports the infrageneric placement essential oils were moderately active with V. zeyheri of V. pooara in subgenus Vitex while the other four being the most active (8, 4 and 16mg ml–1 for S. aureus, above mentioned taxa are placed in subgenus B. cereus and E. coli respectively). The in vitro anti- Holmskiodiopsis. Introduction The genus Vitex (Verbenaceae) has a worldwide tropical to nervous system dysfunction (Gupta et al. 1999), allergy subtropical distribution with a few species found in temper- (Shin et al. 2000) and snakebite (Alam and Gomes 2003). ate zones. It comprises approximately 380 taxa, 87 of which The mature fresh leaves of V. negundo, which have been occur in Africa and are divided into the subgenera Vitex and used in traditional Indian and Ayurveda medicines for their Holmskiodiopsis. The major diagnostic characters of the anti-inflammatory, analgesic and anti-itching properties genus Vitex include their form (trees or shrubs); leaves (Dharmasiri et al. 2003, Dìaz et al. 2003) revealed the anti- (opposite, palmately compound with three to five pinnae, inflammatory activity in the early phase of inflammation in margins entire or seldom dentate), presence or absence of the carrageenan model. It was reported that this activity a petiole; inflorescence terminal, racemose or axillary; and might be attributed to prostaglandin synthesis inhibition and flowers bisexual, slightly zygomorphic or zygomorphic antihistamine activities shown by mature fresh leaves of that (Bredenkamp and Botha 1996). Seven species are indige- species (Dharmasiri et al. 2003). Hossain et al. (2001) nous to South Africa: V. harveyana, V. patula, V. pooara, V. reported that an external application of the leaves of V. trifo- ferruginea with two subspecies amboniensis and amanien- lia is considered useful in rheumatic pains, sprains, and sis, V. rehmannii, V. zeyheri, V. obovata with subspecies inflammations in traditional medicine, and found that the obovata and wilmsii. The genus Vitex is important in tradi- petroleum ether and ethanol extracts of V. trifolia leaves tional medicine and has been utilised for many years as an were moderately active against most Gram-positive and integral part of herbal medicines. Reference to literature indi- Gram-negative bacteria, with the petroleum ether fraction cates that Vitex has been a popular subject in phytochemi- being generally more active. cal and ethnobotanical research. These studies have most- Different phytochemical studies conducted on Vitex ly been conducted on plant extracts of species in Europe species revealed the presence of chemical compounds and Asia illustrating its efficacy and safety to treat current known to be pharmacologically active, such as diterpenoids, fatal diseases such as cancer (Hernández et al. 1999), bac- sesquiterpenoids, triterpenoids, lignans, iridoids, sterols and terial infections (Samy et al. 1998, Taiwo et al. 1999), pre- flavonoids (Rimpler 1972, Chawla et al. 1992, Okuyama et menstrual syndrome (Brown 1994, Berger et al. 2000, Carr al. 1998, You et al. 1998, Hernandez et al. 1999, Ono et al. 2000, Schellenberg 2001a, 2001b), inflammatory/immunore- 1999, Mizuki and Nohana 2000, Kawazoe et al. 2001, Ono lated diseases or lymphomas (Chawla et al. 1992, You et al. et al. 2002, Dìaz et al. 2003). 1998, Gupta et al. 1999, Hernández et al. 1999), central Guided by the above-mentioned medicinal uses of Vitex 612 Nyiligira, Viljoen, BaÕer, Äzek and Van Vuuren as well as the recorded ethnobotanical data for some indige- DMSO and Tween 20 to produce a starting concentration of nous species, we embarked on a study of the anti-infective 100ppm. The inhibitory activity of the respective essential and anti-inflammatory properties of Vitex and to specifically oils on the enzyme was assessed by recording the establish the biological activity of the essential oil. To the absorbance at 234nm, of the conjugated diene formed from best of our knowledge, this is the first study on the phyto- linoleic acid used as enzyme substrate. Different dilutions chemistry and biological activity of South African Vitex were made for essential oils exhibiting an anti-inflammatory species. activity. Nordihydroguaiaretic acid (NDGA) was used as a positive control. Percentage enzyme activity was plotted Material and Methods against concentration of test compound and the IC50 value was determined using Enzfitter® version 1.05 software. Fresh plant material was collected from natural populations. Their taxonomy was confirmed by botanists at the National Results and Discussion Botanical Institute (NBI), Pretoria and the voucher speci- mens have been maintained in the Department of Pharmacy The chemical compounds identified in the five taxa are list- and Pharmacology, University of the Witwatersrand. ed in Table 1, while the results of their antimicrobial and anti- Duplicate copies have been deposited in National Botanical inflammatory activities are presented in Table 2. Institute, Pretoria (PRE). Apart from caryophyllene oxide present in substantial Fresh aerial parts were subjected to hydrodistillation in a amounts in the essential oils of all species (except V. zey- Clevenger-type apparatus for three hours. The essential oils heri), some common components appear in all species in were refrigerated in sealed amber vials prior to analysis. trace amounts, such as p-cymene, α-pinene, (Z)-3-hexen-1- ol, linalool, and trans-p-menth-2-en-1-ol. There are signifi- Gas Chromatography (GC) and Mass Spectrometry (MS) cant qualitative and quantitative differences among the five analysis taxa. However, as expected, the essential oils of V. obovata ssp. obovata and V. obovata ssp. wilmsii showed remark- Analysis of samples by GC-MS was performed using HP able similarities. The essential oils of these two subspecies 1800A GCD system operating under the following condi- showed high yields of 1,8-cineole (13.5% and 8.7%), α- tions; column: HP-Innowax (60m x 0.25mm i.d., 0.25µm film copaene (8.5% and 14.2%), caryophyllene oxide (6.9% and thickness), temperatures: injection port 250°C, column 60°C 5.6%) and γ-muurolene (5.7% and 6.1%), for V. obovata ssp. for 10min, 4°C min–1 to 220°C, 220°C for 10min, 1°C min–1 obovata and V. obovata ssp. wilmsii respectively. Terpinen- to 240°C (total = 80min). Library search was carried out 4-ol (5.4%), the anti-microbial compound in Tea Tree using BaÕer Library of Essential Oil Constituents. (Melaleuca alternifolia) oil was found in the essential oil of V. obovata ssp. obovata as one of the main components, while Statistical analysis it is absent in the essential oil of V. obovata ssp. wilmsii. Limonene (19.8%), β-selinene (14.7%), cryptone (10.3%), Cluster analysis was carried out with the NTSYS-PC pack- humulene epoxide II (9.6%) and caryophyllene oxide (7.6%) age version 2.0 (Rohlf 1997). The quantitative essential oil predominate in the essential oil of V. pooara. The essential dataset (Table 1) was analysed using standard clustering oil of V. zeyheri has 1,8-cineole (18.6%), globulol (6.6%), algorithms. linalool (6.4%) and trans-pinocarveol (4.9%) as the major compounds. Spathulenol (10%), caryophyllene oxide Minimum inhibitory concentration (MIC) assay (9.8%), elema-1,11-dien-15-ol (7.2%) and caryophylladienol II (6.5%) are the major compounds in the essential oil of V. The MIC assay (Eloff 1998) was carried out on two Gram- rehmannii. positive (Staphylococcus aureus ATCC 6538, Bacillus Previous studies on the essential oil of two non-indige- cereus ATCC 11778) and one Gram-negative (Escherichia nous Vitex species, V. agnus-castus (Galelleti et al. 1996, coli ATCC 11775) bacteria. Essential oils (51.2mg) were Males and Blazevic 1998, Zoghbi et al. 1999) and V. rotun- dissolved in acetone (400µl) to make a stock-solution of difolia (Kondo et al. 1986), also reported 1,8-cineole, α- 128mg ml–1. Sterile water (100µl) was added to all wells. pinene and limonene in abundance. Serial dilutions of essential oils were made in the micro-titre The results of the in vitro antimicrobial activity assay plates with 32mg ml–1 as a starting concentration, mixed with (Table 2) indicated that all essential oils exhibited poor activ- 100µl of inoculum and incubated overnight at 37°C. After ity ranging from 4mg ml–1 to 32mg ml–1. They were moder- incubation, 40µl of ρ-iodonitrotetrazolium (INT) 0.04% in ately active against the Gram-positive pathogens (S. aureus sterile water were added to each well and the MIC was and B. cereus), with the essential oil of V.