Essential Oil Composition and in Vitro Antimicrobial and Anti-Inflammatory Activity of South African Vitex Species

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. zeyheri being the determined by visualisation of a red colour with INT, indicat- most active against B. cereus. Only the essential oil of V. ing microbial growth. zeyheri exhibited activity towards E. coli. The essential oil of V. pooara displayed lower activity comparatively to other 5-Lipoxygenase assay tested essential oils. This paper is narrowly focused on the volatile (essential oil) fraction. The solvent extracts show The anti-inflammatory activity of different essential oils was more superior antimicrobial activity (Nyiligira et al. in prep) carried out in vitro, using the 5-lipoxygenase assay (Baylac and it is most probably that some additive effects may be at and Racine 2003). The essential oils were dissolved in play. South African Journal of Botany 2004, 70: 611–617 613

Table 1: Essential oil composition of five South African Vitex species

Component RRI V. po V. re V. ob V. ow V. ze Essential oil yield (%) 0.23% 0.19% 0.21% 0.12% 0.04% β-cyclogeraniolene 947 0.02 Decane 1 000 0.02 0.02 α-pinene 1 032 1.69 0.02 2.22 0.02 2.53 Camphene 1 076 0.05 0.31 0.23 Undecane 1 100 0.01 β-pinene 1 118 0.16 1.57 3.01 0.23 Thuja-2,4(10)-diene 1 120 0.03 Sabinene 1 132 0.45 δ-3-carene 1 159 0.08 0.03 Myrcene 1 174 0.59 0.06 β-terpinene 1 177 0.04 0.07 0.02 Pseudo limonene 1 183 3.66 α-terpinene 1 188 0.05 Heptanal 1 194 0.02 0.03 0.40 Limonene 1 203 19.35 0.46 1.56 0.26 1,8-cineole 1 213 0.28 11.69 7.18 15.29 β-phellandrene 1 218 1.25 p-cymene 1 280 2.26 0.05 2.66 2.32 3.29 Terpinolene 1 290 0.02 0.04 (Z)-3-hexenyl acetate 1 327 0.12 0.09 0.24 0.08 (E)-2-heptenal 1 335 0.03 1-hexanol 1 360 0.04 0.03 0.01 0.20 (Z)-3-hexen-1-ol 1 391 0.44 0.83 0.26 0.11 0.96 3-octanol 1 393 0.01 Nonanal 1 400 0.03 Perillen 1 429 0.09 trans-linalool oxide (furanoid) 1 450 0.09 0.03 0.18 0.58 α,p-dimethyl styrene (p-cymenene) 1 452 0.05 1-octen-3-ol 1 452 0.09 0.17 0.54 0.34 cis-1,2-limonene epoxide 1 458 0.18 1-heptanol 1 463 0.06 α-cubebene 1 466 0.06 trans-1,2-limonene epoxide 1 468 0.38 (Z)-3-hexenyl butyrate 1 471 0.22 0.18 cis-linalool oxide (furanoid) 1 478 0.08 0.03 0.42 α-ylangene 1 493 0.02 0.26 0.38 0.57 α-copaene 1 497 7.39 11.79 0.30 α-campholene aldehyde 1 499 0.03 α-bourbonene 1 528 0.28 β-bourbonene 1 535 0.44 0.14 0.30 3.34 β-cubebene 1 549 0.83 Linalool 1 553 1.21 2.24 2.73 0.36 5.24 1-octanol 1 562 0.02 0.02 8,9-limonene epoxide-I 1 565 0.02 trans-p-menth-2-en-1-ol 1 571 0.28 0.02 0.30 0.05 0.08 Pinocarvone 1 586 0.05 0.32 1.52 1.39 β-ylangene 1 589 0.42 0.88 Fenchyl alcohol 1 591 0.91 0.99 β-copaene 1 597 0.59 0.66 β-elemene 1 600 0.28 Terpinen-4-ol 1 611 0.28 4.63 0.30 β-caryophyllene 1 612 3.82 3.64 Hotrienol 1 616 3.39 6,9-guaiadiene 1 617 1.56 trans-dihydrocarvone 1 624 0.08 Aromadendrene 1 628 1.37 3.05 cis-p-menth-2-en-1-ol 1 638 0.40 trans-p-menth-2,8-dien-1-ol 1 639 0.54 3.64 Myrtenal 1 648 0.06 0.19 0.30 0.14 Umbellulone 1 657 0.11 γ-gurjunene 1 659 0.37 Alloaromadendrene 1 661 0.50 1.60 614 Nyiligira, Viljoen, BaÕer, Äzek and Van Vuuren

Table 1 cont.

Component RRI V. po V. re V. ob V. ow V. ze Essential oil yield (%) 0.23% 0.19% 0.21% 0.12% 0.04% rans-pinocarveol 1 670 1.17 2.47 4.05 cis-p-menth-2,8-dien-1-ol 1 678 0.32 δ-terpineol 1 682 0.12 0.60 0.32 2.63 α-humulene 1 687 0.38 0.21 0.27 Cryptone 1 690 10.05 p-mentha-1,8-dien-4-ol (= Limonen-4-ol) 1 700 0.05 0.03 0.11 γ-muurolene 1 704 0.42 3.19 4.90 5.08 α-terpineol 1 706 3.43 Germacrene D 1 726 0.35 Valencene 1 740 0.12 0.05 α-muurolene 1 740 2.55 1.36 1.69 β-selinene 1 742 14.41 1.47 Carvone 1 751 1.49 0.12 0.06 Bicyclogermacrene 1 755 0.06 cis-piperitol 1 758 0.16 0.19 δ-cadinene 1 773 0.67 0.43 0.25 γ-cadinene 1 776 0.03 1.58 1.71 2.28 Drimenene 1 783 0.51 0.18 p-methylacetophenone 1 797 0.18 Cuminaldehyde 1 802 3.33 Myrtenol 1 804 0.13 0.23 0.76 0.44 3,7-guaiadiene 1 810 0.02 0.11 (E,E)-2,4-decadienal 1 827 0.01 0.04 (E)-β-damascenone 1 838 0.25 0.12 0.29 trans-carveol 1 845 0.98 0.05 0.16 0.09 cis-calamenene 1 853 1.52 1.62 2.06 1.30 p-cymen-8-ol 1 864 0.50 cis-carveol 1 882 0.26 Myrtanol 1 889 0.04 cis-p-mentha-1(7),8-dien-2-ol 1 896 0.07 0.01 0.09 1,11-oxidocalamenene 1 898 1.39 Epicubebol 1 900 0.05 1,10-oxidocalamenene 1 918 Silphinene 1 922 0.06 α-calacorene 1 941 0.11 1.07 1,5-epoxy-salvial-4(14)-ene 1 945 0.33 trans-jasmone 1 948 1.89 Palustrol 1 953 0.42 0.38 γ-calacorene 1 984 0.76 Isocaryophyllene oxide 2 001 0.62 Caryophyllene oxide 2 008 7.45 7.93 5.94 4.61 2.36 1-allyl-2,4-dimethoxybenzene 2 012 0.36 Perilla alcohol 2 019 0.16 epi-globulol 2 033 0.79 Salvial-4(14)-en-1-one 2 037 0.16 0.75 0.86 Humulene epoxide I 2 045 0.18 Gleenol 2 051 0.26 0.17 Ledol 2 057 0.88 0.25 0.90 0.34 Humulene epoxide II 2 071 9.45 1.26 0.91 Cubenol 2 080 0.25 0.31 Humulene epoxide III 2 081 0.41 1-epi-cubenol 2 088 0.79 0.87 1.03 0.45 β-oplopenone 2 092 0.51 Globulol 2 098 3.76 1.18 5.46 Viridiflorol 2 104 0.34 1.58 0.38 1.52 Cumin alcohol 2 113 1.40 Rosifoliol 2 144 2.34 1.89 1.52 Spathulenol 2 144 8.06 2.19 1.91 (Z)-3-hexen-1-yl benzoate 2 148 4.86 Neointermedeol 2 153 0.34 Elema-1,11-dien-15-ol 2 161 5.80 (E)-2-hexenyl benzoate 2 170 0.66 South African Journal of Botany 2004, 70: 611–617 615

Table 1 cont.

Component RRI V. po V. re V. ob V. ow V. ze Essential oil yield (%) 0.23% 0.19% 0.21% 0.12% 0.04% Nor-copaonone 2 179 0.79 T-cadinol 2 187 1.64 1.56 1.39 0.84 T-muurolol 2 209 1.11 1.29 1.50 0.71 Carvacrol 2 214 0.66 Torreyol 2 219 1.15 0.79 0.68 Isospathulenol 2 228 0.65 α-cadinol 2 255 2.85 3.27 3.57 4-hydroxycryptone 2 264 1.67 Selin-11-en-4α-ol 2 273 0.81 0.58 Caryophylla-2 (12),6-dien-5β-ol (= Caryophylladienol I) 2 316 1.09 0.46 0.37 Caryophylla-2 (12),6(13)-dien-5α-ol (Caryophylladienol II) 2 324 5.22 3.79 3.27 2.82 Calamenene oxide 2 347 0.94 Caryophylla-2 (12),6- dien-5α-ol (Caryophyllenol I) 2 347 0.29 1.59 0.16 1.11 1.23 9-aristolen-1α-ol 2 353 0.34 Eudesma-4(15),7-dien-1β-ol 2 369 0.21 0.48 0.54 Eremophilone 2 371 1.27 Germacra-4(15),5,10(14)-trien-1α-ol 2 375 2.20 Hexadecanol 2 384 0.13 Caryophyllenol II 2 392 1.77 0.92 1.17 3.62 Drim-8-en-7-ol 2 430 1.04 0.61 0.31 1-octadecanol 2 607 2.88 0.07 0.23 Diterpene alcohol 2 607 0.06 0.11 Phytol 2 622 0.30 2.41 0.06 0.38 0.11 Total 97.87 80.65 86.50 82.83 82.27

V. po = V. pooara, V. re = V. rehmannii, V. ob = V. obovata ssp. obovata, V. ow = V. obovata ssp. wilmsii, V. ze = V. zeyheri

Table 2: MIC values for the antimicrobial activity and IC50 values of the 5-lipoxygenase inhibitory activity for the essential oils of five South African Vitex species

–1 Species Antimicrobial activity MIC values (mg ml ) Anti-inflammatory activity IC50 (ppm) S. aureus B. cereus E. coli V. pooara 32 16 r 25.0 V. rehmannii 16 8 r 40.5 V. obovata ssp. obovata 8 8 r 42.0 V. obovata ssp. wilmsii 8 8 r 48.0 V. zeyheri 8 4 16 64.0 Control 3.10–4* 6.10–4* 3.10–3* 5** r = resistant * = Ciprofloxacin ** = Nordihydroguaiaretic acid (NDGA)

The antibacterial activity of the essential oils may be attrib- The results of the in vitro anti-inflammatory activity assay uted to the content of 1,8-cineole as the activity of the essen- revealed that all essential oils were active with V. pooara tial oil increases proportionally to their respective yield of displaying the most promising activity with an IC50 value of 1,8-cineole which is known to possess some antimicrobial 25ppm. The essential oil of V. zeyheri showed the least activity (Ünlü et al. 2002, Aligiannis et al. 2001, Viljoen et al. activity (64ppm) while V. rehmannii, V. obovata ssp. obo- 2003). The highest antimicrobial activity of the essential oil vata and V. obovata ssp. wilmsii exhibited moderate activ- of V. zeyheri may be due to high levels of alcohols, espe- ity. cially 1,8-cineole and linalool, which also have been shown The anti-inflammatory activity of these essential oils may to have antimicrobial activity (Carson and Riley 1995, be due to the terpenes and sesquiterpenes which have been Knobloch et al. 1988, Pattnaik et al. 1997). The greater shown to be good inhibitors of 5-lipoxygenase (Baylac and activity of these essential oils towards Gram-positive bacte- Racine 2003). The authors reported that d-limonene and ria may be attributed to the resistance of the membrane of some esters strongly inhibited 5-lipoxygenase. The greater Gram-negative pathogens as reported by Martin (1995), and activity of the essential oil of V. pooara may be attributed to Mangena and Muhima (1999). higher levels of limonene which is the major component of 616 Nyiligira, Viljoen, BaÕer, Äzek and Van Vuuren

V. pooara References

Alam MI, Gomes A (2003) Snake venom neutralisation by Indian V. rehmannii medicinal plants (Vitex negundo and Emblica officinalis) root extracts. Journal of Ethnopharmacology 86: 75–80 Aligiannis N, Kalpoutzakis E, Chinou IB, Mitakou S, Gikas E, V. obovata Tsarbopoulos A (2001) Composition and antimicrobial activity of subsp. obovata the essential oils of five taxa of Sideritis from Greece. Journal of Agriculture and Food Chemistry 46: 811–815 V. obovata Baylac S, Racine P (2003) Inhibition of 5-Lipoxygenase by essential subsp. wilmsii oils and other natural fragrant extracts. The International Journal of Aromatherapy 13: 138–142 Berger D, Schaffner W, Schrader E, Meier B, Brattström A (2000) V. zeyheri Efficacy of Vitex Agnus-castus L. extract Ze 440 in patients with Pre-Menstrual Syndrome (PMS). Archives of Gynaecology and Obstetrics 264: 150–153 33.29 27.99 22.69 17.40 12.10 Bredenkamp CL, Botha DJ (1996) FSA Contributions 7: SIMILARITY COEFFICIENT Verbenaceae: Vitex. Bothalia 26: 141–151 Brown DJ (1994) Vitex agnus-castus clinical monograph. Quarterly Review of Natural Medicine, Summer, pp. 111–121 Figure 1: Dendrogram constructed on the quantitative essential oil Carr M (2000) Selections from current literature treatments for pre- data matrix (Table 1) menstrual dysphoric disorder. Family Practice 18: 644–646 Carson CF, Riley TV (1995) Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. Journal of Applied Bacteriology 78: 264–269 this essential oil (19.8%), and two esters, (Z)-3-hexenyl ben- Chawla AS, Sharma AK, Handa SS (1992) Chemical investigation zoate and (E)-2-hexenyl benzoate. and anti-inflammatory activity of Vitex negundo seeds. Journal of To examine the chemotaxonomic relationships between Natural Products 55: 163–167 the five taxa, cluster analysis was used to generate the den- Dharmasiri MG, Jayakody JRAC, Galhena G, Liyanage SSP, drogram. Figure 1 shows the two main distinctive clusters. Ratnasooriya WD (2003) Anti-inflammatory and analgesic activi- Vitex rehmannii, V. obovata ssp. obovata, V. obovata ssp. ties of mature fresh leaves of Vitex negundo. Journal of wilmsii, and V. zeyheri are united into a single coherent clus- Ethnopharmacology 87: 199–206 Dìaz F, Chávez D, Lee D, Mi Q, Chai HB, Tan GT, Kardono LBS, ter while V. pooara is graphically presented as an outlier. As Riswan S, Fairchild CR, Wild R, Farnsworth NR, Cordell GA, is evident from both the statistical analysis (Figure 1) and Pezzuto JM, Kinghorn AD (2003) Cytotoxic flavone analogues of biological activities (Table 2), V. pooara is anomalous. This Vitexicarpin, a constituent of the leaves of Vitex negundo. Journal confirms the results of the previous anatomical and taxo- of Natural Products 66: 865–867 nomical study on South African Vitex species which revealed Eloff JN (1998) A sensitive and quick microplate method to deter- that V. pooara belongs to the subgenus Vitex while V. mine the minimal inhibitory concentration of plant extracts for bac- rehmannii, V. obovata ssp. obovata, V. obovata ssp. wilmsii teria. Planta Medica 64: 711–713 and V. zeyheri belong to the subgenus Holmskiodiopsis Galelletti GC, Russo MT, Bocchini P (1996) Essential oil of leaves (Bredenkamp and Botha 1996). and berries of Vitex agnus-castus L. from Cabria, southern Italy. Inhalation therapy is common practice in African tradition- Rapid Communications in Mass Spectrometry 10: 1345–1350 Gupta M, Mazumder UK, Bhawal SR (1999) CNS activity of Vitex al healing; hence our study is narrowly focused on the negundo Linn. in mice. Indian Journal of Experimental Biology 37: essential oil (volatile) fraction. Preliminary results on the 143–146 non-volatile constituents (Nyiligira et al. in prep) have Hernandez MM, Heraso C, Villarreal ML, Vargas-Arispuro I, Aranda revealed several compounds, most notably diterpenoids, to E (1999) Biological activities of crude plant extracts from Vitex have very good antimicrobial properties. It is most probable trifolia L. (Verbenaceae) Journal of Ethnopharmacology 67: that the essential oils and the non-volatile compounds act in 37–44 a synergistic/additive manner to produce an enhanced ther- Hossain MM, Paul N, Sohrab MH, Rahman E, Rashid MA (2001) apeutic effect. The results presented here provide some in Antibacterial activity of Vitex trifolia. Fitoterapia 72: 695–697 vitro scientific evidence for the traditional uses of Vitex Kawazoe K, Yutani A, Tamemoto K, Yuasa S, Shibata H, Higuti T, species in treating skin diseases, inflammation and rheuma- Takaishi Y (2001) Phenylnaphtalene compounds from the subter- ranean part of Vitex rotundifolia and their antibacterial activity tism. against Methicillin-resistant Staphylococcus aureus. Journal of Natural Products 64: 588–591 Acknowledgements — The authors are thankful to the National Knobloch K, Pauli A, Iberl B, Wies N, Weigand H (1988) Mode of Research Foundation (Indigenous Knowledge Systems) and the action of essential oil components on whole cells of bacteria and Medical Faculty Research Endowment Fund, University of the fungi in plate tests. In: Schreier P (ed) Bioflavor ‘87. Walter de Witwatersrand for financial support. Robertet (Grasse, France) is Gruyter Verlag, Berlin, New York, pp 287–299 acknowledged for assistance in setting up the 5-lipoxygenase Kondo Y, Sugiyama K, Nozoe S (1986) Studies on the constituents assay, and we are indebted to Dr CL Bredenkamp and Ms PM of Vitex rotundifolia L. fil. Chemical and Pharmaceutical Bulletin Burgoyne (NBI) for the identification and assistance with the collec- 34: 4829–4832 tion of the plant material. Males M, Blazevic N (1998) Composition of the essential oil of Vitex agnus-castus L. f. rosea fruits. Pharmazie 53: 728–729 South African Journal of Botany 2004, 70: 611–617 617

Mangena T, Muhima NYO (1999) Comparative evaluation of the Schellenberg R (2001b) Is an extract of the fruit of Vitex agnus-cas- antimicrobial activities of essential oils of Artemisia afra, Pteronia tus (chaste tree or chasteberry) effective for prevention of pre- incana and Rosmarinus officinalis on the selected bacteria and menstrual syndrome (PMS)? British Medical Journal 322: yeast strains. Letters in Applied Microbiology 28: 291–296 134–137 Martin GJ (1995) Ethnobotany. E People and Plants Conservation Shin TY, Kim SH, Lim JP, Such ES, Jeong HJ, Kim BD, Park ES, Manual. Chapman and Hall, London, UK, pp 67–93 Hwang WJ, Rye DG, BaÕek SH, An NH, Kim HM (2000) Effects of Mizuki K, Nohana T (2000) Diterpenoids from the fruits of Vitex tri- Vitex rotundifolia on immediate-type allergic reaction. Journal of folia. Phytochemistry 55: 873–877 Ethnopharmacology 72: 443–450 Okuyama E, Fujimori S, Yamazaki M, Deyama T (1998) Taiwo O, Xu HX, Lee SF (1999) Antimicrobial activities of extracts Pharmacologically active components of Viticis fructus (Vitex from Nigeria chewing sticks. Phytotherapy Research 13: 675–679 rotundifolia). II. The components having analgesic effects. Unlü M, Daferera D, Dönmez E, Polissiou M, Tepe B, Sökmen A Chemical and Pharmaceutical Bulletin 46: 655–662 (2002) Compositions and in vitro antimicrobial activities of the Ono M, Yanaka T, Yamamoto M, Ito Y, Nohara T (2002) New diter- essential oils of Achillea setacea and Achillea teretifolia penes and norditerpenes from the fruits of Vitex rotundifolia. (Compositae). Journal of Ethnopharmacology 83: 117–121 Journal of Natural Products 65: 537–541 Viljoen A, Van Vuuren SF, Klepser ME, Demirci B, BaÕer KHC, Van Ono M, Yamamoto M, Masuoka C, Ito Y, Yamashita M, Nohara T Wyk B-E (2003) Osmitopsis asteriscoides (Asteraceae) — the (1999) Diterpenes from the fruits of Vitex rotundifolia. Journal of antimicrobial activity and essential oil composition of a Cape- Natural Products 62: 1532–1537 Dutch remedy. Journal of Ethnopharmacology 88: 137–143 Pattnaik S, Subramanyam VR, Bapaji M, Kole CR (1997) You KM, Son KH, Chang HW, Kang SS, Kim HP (1998) Vitexicarpin, Antibacterial and antifungal activity of aromatic constituents of a flavonoid from the fruits of Vitex rotundifolia, inhibits mouse lym- essential oils. Microbios 89: 39–46 phocyte proliferation and growth of cell lines in vitro. Planta Rimpler H (1972) Phytoecdysones and Iridoids from Vitex Medica 64: 546–550 megapotamica. Pharmazeutische Gesellschaff 305: 746–751 Zoghbi MGB, Andrade EHA, Maia JGS (1999) The essential oil of Rohlf FJ (1997) NTSYSpc-2.0. Department of Ecology and Vitex agnus-castus L. growing in the Amazon region. Flavour and Evolution, State University of New York, USA Fragrance Journal 14: 211–213 Samy RP, Ignacimuthu S, Sen A (1998) Screening of 34 Indian medicinal plants for antibacterial properties. Journal of Ethnopharmacology 62: 173–182 Schellenberg R (2001a) Treatment of the premenstrual syndrome with V. agnus-castus fruit extract: prospective, randomised, placebo controlled study. British Medical Journal 322: 134–137

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