Assessing the antifungal and aflatoxin B1 inhibitory efficacy of nanoencapsulated antifungal formulation based on combination of Ocimum spp. essential oils.

The aim of the study was to explore the antifungal and aflatoxin B1 inhibitory efficacy of nanoencapsulated antifungal formulation. Mixture design response surface methodology (RSM) was utilized to design the antifungal formulation (SBC 4:1:1) based on the combination of chemically characterized Ocimum sanctum (S), O. basilicum (B), and O. canum (C) against Aspergillus flavus. The SBC was incorporated inside the chitosan nanomatrix (Ne-SBC) using an ultrasonic probe (40 kHz) and interactions were confirmed by SEM, FTIR and XRD analysis. The results showed that the Ne-SBC possessed enhanced antifungal and aflatoxin B1 inhibitory effect over the free form of SBC. The biochemical and in silico results indicate that the antifungal and aflatoxin B1 inhibitory effect was related to perturbance in the plasma membrane function (ergosterol biosynthesis and membrane cation) mitochondrial membrane potential, C-sources utilization, antioxidant defense system, and the targeted gene products Erg 28, cytochrome c oxidase subunit Va, and Nor-1. In-situ observation revealed that Ne-SBC effectively protects the Avena sativa seeds from A. flavus and AFB1 contamination and preserves its sensory profile. The findings suggest that the fabrication of SBC inside the chitosan nano-matrix has promising use in the food industries as an antifungal agent.

Anesthetic activity of plant essential oils on Cyprinus carpio (koi carp).

The aims of this study were to investigate the anesthetic and cytotoxic effects of essential oils (EOs) of Ocimum basilicum (OBO), O. canum (OCO), and O. sanctum (OSO) on Cyprinus carpio (koi carp). For anesthetic effect, induction time to surgical anesthesia and recovery time were determined. For cytotoxicity effect, viability of fish peripheral blood nuclear cells (PBMCs) was investigated. Results indicated that increasing oil concentration caused significant (p < 0.01) decrease of induction time. OSO at 100, 200, and 300 mg/L gave the induction time of 169.5 ± 10.2, 62.8 ± 2.3, 45.3 ± 2.2 sec, respectively, significantly shorter than OCO, and OBO. The recovery time of anesthetized fish was dose dependent (p <0.01). Among them, OCO showed the longest recovery time of 313.0 ± 8.1, 420.7 ± 12.6, 616.6 ± 12.1 sec for concentrations of 100, 200, and 300 mg/L, respectively, followed by OSO and OBO, respectively. Within 10 min contact time of the EOs and fish PBMCs, the fish PBMC viability was higher than 80%. Increase contact time and EO concentration caused an increase in cytotoxicity to fish PBMC. OBO showed less toxic than OSO and OCO. Based on the desired induction and recovery times for anesthetizing koi carp, OBO, OCO, and OSO at 300, 200, and 100 mg/L, respectively were suggested to be the most suitable. It was concluded that OBO, OCO, and OSO can be used as natural anesthetics for fish.

Atividade antifúngica e possível mecanismo de ação do óleo essencial de folhas de Ocimum gratissimum (Linn. ) sobre espécies de Candida / Antifungal activity and potential action mechanisms of essential oil of Ocimum gratissimum (Linn. ) leaves against Candida species

RESUMO A espécie Ocimum gratissimum (Linn.), popularmente conhecida como alfavaca-cravo, é muito utilizada na medicina popular. A planta apresenta inúmeros compostos, sendo o eugenol o constituinte majoritário do seu óleo essencial e o provável responsável pela sua atividade antimicrobiana. O objetivo deste estudo foi determinar o potencial antifúngico e o possível mecanismo de ação do óleo essencial extraído das folhas de O. gratissimum L. (OEOg) sobre cepas-padrão de Candida. Para avaliação da atividade antimicrobiana foi determinada a Concentração Inibitória Mínima (CIM), o efeito do tempo de exposição, o efeito modulador na atividade de antifúngicos (ATF) de uso clínico e a ação do OEOg nas fases de crescimento exponencial e estacionário de leveduras do gênero Candida. O mecanismo de ação do OEOg foi verificado por captação do cristal violeta e avaliação da morfologia microbiana pela técnica de microcultivo. Também foi avaliada a toxicidade do OEOg sobre hemácias humanas. O OEOg apresentou boa atividade antifúngica sobre cepas de Candida, sendo capaz de reduzir a taxa de crescimento das cepas de Candida a partir de quatro horas de exposição, além de ter modulado positivamente a atividade do cetoconazol para C. tropicalis ATCC 13803 e reduzir o número de células viáveis em todas as fases de crescimento microbiano. O OEOg foi capaz de promover o aumento discreto da captação do cristal violeta e provocou alterações na micromorfologia das células de Candida spp., sugerindo que seu alvo de ação seja o envoltório celular. Observou-se baixa toxicidade do OEOg sobre hemácias humanas. Os resultados encontrados mostraram que o OEOg possui boa atividade sobre o gênero Candida, com mecanismo de ação mediado possivelmente pela ocorrência de danos no envoltório celular, além de ter sido observada baixa toxicidade, indicando do OEOg é promissor no desenvolvimento e elaboração de um novo fármaco com potencial atividade para o tratamento de doenças fúngicas.

ABSTRACT Ocimum gratissimum (Linn.) is a medicinal plant popularly known as “wild basil” widely used in traditional medicine. The plant has numerous compounds, and eugenol is the major constituent of its essential oil and likely responsible for its antimicrobial activity. The aim of this study was to determine the antifungal activity and the potential mechanism of action of the essential oil extracted from the leaves of O. gratissimum L. (OEOg) against standard strains of Candida. The following experiments were performed: determination of Minimum Inhibitory Concentration (MIC), determination of the effect of exposure time to OEOg; evaluation of the modulating effect of OEOg in antifungal (ATF) activity for clinical use; determination of the effects of the OEOg on different growth phases of Candida spp; determination of crystal violet (CV) uptake and the microculture of yeast technique. OEOg showed good antifungal activity against Candida, being able to reduce microbial growth during 24 hours of contact and also the number of viable cells at all stages of growth. OE positively modulates the activity of ketoconazole for C. tropicalis ATCC 13803. Increased uptake of CV and also the inhibition of Candidavirulence factors were also observed, which indicates the occurrence of damage in the cell envelope. These findings, coupled with the low toxicity of OEOg on human erythrocytes, indicate that “wild basil” is a promising plant for the development of a new drug with a potential activity to treat fungal diseases.

Simultaneous quantitative determination of multiple bioactive markers in Ocimum sanctum obtained from different locations and its marketed herbal formulations using UPLC-ESI-MS/MS combined with principal component analysis.

INTRODUCTION: Ocimum sanctum L., with phenolic acids, flavonoids, propenyl phenols and terpenoids as active pharmacological constituents, is a popular medicinal herb and is present as an ingredient in many herbal formulations. Therefore, development of a reliable analytical method for simultaneous determination of the pharmacologically active constituents of O. sanctum is of high importance. OBJECTIVE: To develop and validate a new, rapid, sensitive and selective UPLC-ESI/MS/MS method for simultaneous determination of 23 bioactive markers including phenolic acids, flavonoids, propenyl phenol and terpenoid in the leaf extract and marketed herbal formulations of O. sanctum. METHODS: An UPLC-ESI/MS/MS method using negative electrospray ionisation (ESI) in multiple-reaction-monitoring (MRM) mode was used for simultaneous determination. Chromatographic separation was achieved on an Acquity UPLC BEH C18 -column using a gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Principal component analysis (PCA) was applied to correlate and discriminate eight geographical collections of O. sanctum based on quantitative data of the analytes. RESULTS: The developed method was validated as per International Conference on Harmonization guidelines and found to be accurate, with overall recovery in the range 95.09-104.84% (RSD ≤ 1.85%), precise (RSD ≤ 1.98%) and linear (r(2) ≥ 0.9971) over the concentration range of 0.5-1000 ng/mL. Ursolic acid was found to be the most abundant marker in all the samples investigated, except for the marketed tablet. CONCLUSION: The method established is simple, rapid and sensitive, hence it can be reliably utilised for the quality control of O. sanctum and derived herbal formulations.

Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region.

The genus Ocimum comprises of several medicinally important species which frequently fall prey to adulteration due to misidentification. A proficient method is hence required to solve the problems that exist in differentiating its various morphotypes. In plants, candidate DNA barcodes of the chloroplast and nuclear regions have proved to be a great success in the validation of several plant families. Hence, this study involves the use of the molecular-based DNA barcoding method to identify some of the most common and useful species of the genus Ocimum (Tulsi). Here, DNA amplification of three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA-trnH was performed, to access their ability to produce high sequence variability. The discrimination among species was performed using the Kimura 2-parameter and maximum composite likelihood methods. On analysing the sequence data, the psbA-trnH region proved to be the most suitable candidate barcode and gave an overall variation of 7.3% at the interspecies level. A clear differentiation was found at the species level, showing a maximum distance of 0.264 between dissimilar species. Also, phylogenetic analysis led to the successful identification of hybrids, while it failed to do so at the variety level. Hence, it can be inferred that DNA barcoding is ideal for species-level identification of the genus Ocimum.

Avaliação das estruturas secretoras de Ocimum gratissimum var. macrophyllum Briq. (Lamiaceae) após extração dos constituintes voláteis / Evaluation of secretory structures of Ocimum gratissimum var. macrophyllum Briq. (Lamiaceae) after extraction of volatile compounds

O impacto provocado nas estruturas secretoras de folhas adultas de Ocimum gratissimum var. macrophyllum após extração dos componentes voláteis utilizando-se diferentes métodos de extração foi investigado através de microscopia eletrônica de varredura (MEV). Secções transversais do ápice, da nervura central, e da margem mostraram superfície com cutícula estriada, estômatos diacíticos, tricomas tectores simples, filiformes, e glandulares peltados. Os peltados, com estrutura envolta por uma cutícula contendo o exsudado celular, variaram de 60 a 80µm de diâmetro sendo formados por uma porção apical multicelular com disposição radial formada por dois círculos concêntricos com 9 a 16 células. Na região central do ápice do tricoma peltado se identificou a presença de 4 células em forma de vértice dispostas sobre o pedúnculo, sendo esta a região mais externa formada por número variável de células. Os componentes voláteis das folhas adultas foram extraídos por hidrodestilação (HD), destilação-extração simultânea (DES), e solvente (SO). Os dados obtidos evidenciaram que o maior impacto nas estruturas secretoras ocorreu após extração por hidrodestilação quando comparado aos outros métodos utilizados. A porcentagem dos principais constituintes químicos, timol e γ-terpineno variaram de acordo com o método de extração: timol (HD = 25,4%, SO = 18,2%, DES = 54,8%) e γ-terpineno (HD = 38,2%, SO = ausente, DES = 13,7%).

The impact on the secretory structures of mature leaves of Ocimum gratissimum var. macrophyllum, after extraction of the volatile compounds using different extraction methods, was investigated by scanning electron microscopy (SEM). Cross-sections of the apex, the midrib and the margin showed surface with striated cuticle, diacitic stomata, single tector, filiform and glandular peltate trichomes. The peltate trichomes with a structure surrounded by cuticle containing the celllular exudate ranged from 60 to 80µm in diameter and consisted of a multicellular apical portion of radial arrangement formed by two concentric circles with 9 to 16 cells. In the central region of the peltate trichome apex, there were 4 cells in the form of a vertex arranged on the peduncle, and this was the most external region formed by a variable number of cells. The volatile compounds of the mature leaves were extracted by hydrodistillation (HD), simultaneous distillation-extraction (SDE) and solvent (SO). The obtained data evidenced that the major impact on the secretory structures occurred after extraction by hydrodistillation, compared to the other used methods. The percentage of the major chemical constituents, thymol and γ-terpinene, changed according the extraction method: (HD = 25.4%, SO = 18.2%, SDE = 54.8%) and γ-terpinene (HD = 38.2%, SO = absent, SDE = 13.7%).

Influência da adubação verde e diferentes adubos orgânicos na produção de fitomassa aérea de atroveran (Ocimum selloi Benth. ) / Influence of green manure and different organic manure on production of aereal phytomass of Ocimum selloi

Este trabalho teve por objetivo verificar o efeito de diferentes adubos orgânicos em associação ou não com adubo verde na produção de folhas de Ocimum selloi Benth., planta nativa do Brasil. O experimento foi conduzido na área experimental da Embrapa Meio Ambiente (CNPMA), localizado no município de Jaguariúna, em duas áreas distintas, sendo uma delas submetidas anteriormente ao plantio e incorporação de adubo verde (Crotalaria juncea). Os tratamentos utilizados foram T1 - testemunha (solo sem adubação), T2 - cama de aviário (5 kg m-2), T3 - hidrolisado de peixe (produto comercial Fishfértil - 5 mL m-2) e T4 - composto orgânico (4 kg m-2). A colheita foi realizada 180 dias após o plantio, em janeiro de 2011, sendo colhidas as plantas úteis (quatro plantas por parcela). Avaliou-se o rendimento de fitomassa fresca e seca. O delineamento experimental adotado foi o esquema fatorial 2x4, com quatro repetições (blocos). As médias obtidas foram submetidas à análise de variância seguida de teste de médias (Tukey). Com base nos resultados obtidos, pode-se concluir que a cama de aviário apresentou resultados mais satisfatórios quanto à produção de folhas de Ocimum selloi quando comparados aos demais tratamentos orgânicos, não se observando incremento nos resultados pela associação com o adubo verde Crotalaria juncea.

The aim of this work was to avail the effect of different manures in association or nor with green manure on yield of leaves of Ocimum selloi Benth. The assay was accomplished on experimental area of Embrapa Environmental (Jaguariúna district), at two different spaces (with or without green manure Crotalaria juncea). The treatments used were T1 - witness (no manure), T2 - chicken manure (5 kg m-2), T3 - commercial product Fishfértil - 5 mL m-2) and T4 - composting (4 kg m-2). The cut was realized on 180 days after the planting (january - 2011), and were collected four plants/ plot. The yield of dried and fresh Ocimum selloi phytomass was availed. The experimental design was factorial scheme (2x4), with four repetitions. The treatment with chicken manure showed best results on Ocimum selloi leaves yield than the others treatments, but did not was influence by using Crotalaria juncea.

Efeito do BAP no cultivo in vitro de Ocimum selloi Benth / Effect of BAP on in vitro culture of Ocimum selloi Benth

Visando promover a proliferação de brotações em segmentos apicais e nodais de Ocimum selloi em diferentes concentrações de BAP, plantas jovens de 60 dias serviram de doadoras de segmentos apicais e nodais. Os segmentos foram inoculados em meio MS preparado com a metade da concentração dos sais, e acrescido de 1,5% de sacarose e diferentes concentrações de BAP. O experimento foi conduzido no delineamento fatorial, 3 x 4, sendo 3 as posições dos segmentos de O. selloi (segmento apical, primeiro e segundo segmentos nodais) e 4 concentrações de BAP (0 - controle; 2; 4 e 6 mg L-1). Aos 30 dias, foram avaliados o número, comprimento e biomassa fresca e seca de brotos e raízes. Os primeiros e segundos segmentos apresentaram melhores resultados na indução de brotos de O. selloi, 7 e 8 brotos/explante, nas diferentes concentrações de BAP; porém, não houve formação de raízes na presença da citocinina. Nas condições testadas, recomenda-se o uso do primeiro e segundo segmento nodal suplementando o meio de cultivo com BAP para a proliferação in vitro de brotações de O. selloi.

The present study was undertaken to develop the proliferation of sprouts in apical and nodal segments of Ocimum selloi with different BAP levels. Young plants aged 60 days were used as donors of nodal and apical segments. The segments were inoculated in MS medium at half the concentration of salts supplemented with 1.5% of sucrose and different BAP levels. The experiment was in 3 x 4 factorial arrangement, 3 positions of O. selloi segments (apical segment; first and second nodal segment) and 3 BAP levels (0 - control; 2; 4 and 6 mg L-1). After 30 days, the number, the length, and the fresh and dry biomass of sprouts and roots were evaluated. The first and the second segments showed better results in inducing O. selloi sprouts, 7 and 8 sprouts/explant, at the different BAP levels, but there was not root formation in the presence of the cytokinin. Under the tested conditions, use of the first and the second nodal segments is recommended in addition to supplementing the culture medium with BAP for in vitro proliferation of O. selloi sprouts.

Determinação de óleos essenciais de alfavaca (Ocimum gratissimum L. ), orégano (Origanum vulgare L. ) e tomilho (Thymus vulgaris L.) / Determination of essential oils of basil (Ocimum gratissimum L. ), oregano (Ocimum gratissimum L. ) and thyme (Thymus vulgaris L.)

O presente trabalho teve como objetivo a caracterização de plantas frescas e secas (comerciais) de alfavaca, orégano e tomilho, a obtenção dos óleos essenciais através do método de arraste a vapor e a quantificação dos compostos químicos por CG/EM. As plantas frescas e as secas comerciais foram submetidas às análises de umidade, extrato etéreo, proteína, fibra bruta, cinzas, extrato não nitrogenado, valor calórico, teor de óleo essencial e identificação dos compostos majoritários através da cromatografia gasosa-espectrometria de massas. Dentre a caracterização obtida os resultados na base seca mostraram-se promissores, sendo o teor de proteína e de cinzas na alfavaca seca comercial com 17,34 g 100 g-1 e 8,12 g 100 g-1, respectivamente; a fibra bruta no orégano seco comercial com 15,65 g 100 g-1; o extrato etéreo, o extrato não nitrogenado e o valor calórico no tomilho seco comercial com 9,30 g 100 g-1, 52,72 g 100 g-1 e 356,74 Kcal 100 g-1, respectivamente. Obteve-se o maior rendimento de óleo essencial na alfavaca seca comercial com 1,02%, enquanto a alfavaca fresca apresentou o menor rendimento, com apenas 0,13%. Na alfavaca fresca encontrou-se 87,38% de eugenol e 6,27% de timol, enquanto na alfavaca seca comercial observou-se redução no eugenol (71,12%) e aumento do timol (13,28%). No orégano fresco foram quantificados quatro picos o γ-terpineno (33,45%), 4-terpineol (25,59%), timol (14,21%) e carvacrol (2,30%). Já no óleo essencial de orégano seco comercial houve redução no γ-terpineno (28,73%) e aumento no 4-terpineol (27,58%), timol (19,71%) e carvacrol (3,67%). No óleo essencial do tomilho fresco foram quantificados três picos o borneol (66,66%), timol (13,41%) e linalol (3,24%). Por outro lado, no óleo essencial do tomilho seco comercial houve redução no borneol (37,90%) e aumento no timol (20,61%) e linalol (10,34%). Pode-se concluir que as folhas secas comerciais analisadas de alfavaca, orégano, e tomilho apresentam potencial para o enriquecimento dos alimentos ou para a obtenção dos óleos essenciais.

This study aimed to characterize commercial fresh and dry medicinal plants (basil, oregano and thyme), to obtain essential oil by the steam distillation method and to quantify chemical compounds by means of GC/MS. The fresh and dry plants were subjected to the following analyses moisture, ether extract, protein, crude fiber, ash, non-nitrogenous extract, caloric value, essential oil content and identification of major compounds by gas chromatography-mass spectrometry. Considering the obtained characterization, the following results on dry basis proved promising: protein and ash content in commercial dry basil with 17.34 g 100 g-1 and 8.12 g 100 g-1, respectively; crude fiber in commercial dry oregano with 15.65 g 100 g-1; ether extract, non-nitrogenous extract and caloric value in commercial dry thyme with 9.30 g 100 g-1, 52.72 g 100 g-1 and 356.74 Kcal 100 g-1, respectively. The highest essential oil yield was obtained for commercial dry basil with 1.02% and the lowest yield was obtained for fresh basil with only 0.13%. Chromatography indicated 87.38% eugenol and 6.27% thymol in fresh basil. For commercial dry basil, the chromatogram showed a reduction in eugenol (71.12%) and an increase in thymol (13.28%). Four peaks were quantified for fresh oregano the γ-terpinene (33.45%), 4-terpineol (25.59%), thymol (14.21%) and carvacrol (2.30%). For the essential oil of commercial dry oregano, there was a decrease in γ-terpinene (28.73%) and an increase in 4-terpineol (27.58%), thymol (19.71%) and carvacrol (3.67%). In the chromatogram of the essential oil of fresh thyme, three peaks were quantified: borneol (66.66%), thymol (13.41%) and linalool (3.24%). On the other hand, in the chromatogram of the essential oil of commercial dry thyme, there was a decrease in borneol (37.90%) and an increase in thymol (20.61%) and linalool (10.34%). It can be concluded that commercial dry leaves of basil, oregano and thyme are feasible to enrich foods or to obtain essential oils.

Effects of flaxseed, sardines, pearl millet, and holy basil leaves on production traits of layers and fatty acid composition of egg yolks.

The effects of flaxseed, sardines, pearl millet, and holy basil (Oscimum sanctum) leaf meal on Single Comb White Leghorn (Forsgate strain) layer hen production traits, fatty acid composition of egg yolks, and egg quality characteristics were studied. Layer hens were allocated to 1 of 6 dietary treatments from 27 to 32 wk of age: control (treatment 1), control + 1 g of basil leaf meal (BLM) (treatment 2), control + 2 g of BLM (treatment 3), designer egg layer mash (DELM; treatment 4), DELM + 1 g of BLM (treatment 5), DELM + 2 g of BLM (treatment 6). No significant differences were found in egg production between dietary treatments, indicating that neither the designer diet nor the BLM had an adverse effect on egg production. The high energy level in the designer diet, because of the flaxseed oil and sardines, significantly reduced feed intake compared with the control diet. The egg weight was significantly higher in birds fed the designer diet compared with the egg weight in the control diet. The flaxseed oil and sardine oil, which are rich in n-3 polyunsaturated fatty acids, contributed to significantly increased n-3 polyunsaturated fatty acid levels in the egg yolks, with a proportionate reduction in the level of palmitic acid. Other fatty acids showed only minor variations. Flaxseed oil increased the level of α-linolenic acid, whereas fish oil increased the eicosapentaenoic acid and docosahexaenoic acid levels. The BLM at low levels of inclusion (1 and 2 g/kg) also contributed significantly to elevating the yolk n-3 polyunsaturated fatty acid levels, especially C18:3. Holy basil leaf meal is a good feed supplement for increasing the yolk α-linolenic acid level by reducing the yolk saturated fatty acid levels proportionately.

Biologia reprodutiva de populações de Ocimum selloi Benth / Reproductive biology of Ocimum selloi Benth. populations

Entre as plantas nativas de uso medicinal no Brasil, encontra-se Ocimum selloi Benth., planta anual herbácea da família Lamiaceae que ocorre nas regiões Sudeste e Sul do país. Este trabalho avaliou os aspectos da biologia floral (estrutura floral, floração e frutificação) e mecanismos reprodutivos de Ocimum selloi em quatro populações oriundas das regiões de Piquete e Apiaí, Estado de São Paulo, Camanducaia, Estado de Minas Gerais, e Colombo, Estado do Paraná. O período de antese teve início às 11 horas e término às 15 horas, com o clímax de floração às 13 horas, sendo a temperatura pouco importante neste processo. A viabilidade do pólen foi alta nas quatro populações. A espécie é autocompatível, formando frutos e sementes tanto em polinização livre quanto em autopolinização espontânea, o que mostra uma grande versatilidade reprodutiva da espécie, garantindo a variabilidade genética. A germinação das sementes também foi alta nas quatro populações.

Among the medicinal plants native to Brazil, Ocimum selloi Benth., an herbaceous annual plant of the Lamiaceae family, occurs in the southeastern and south regions of the country. This work evaluated flower biology aspects (flower structure, flowering and fructification) and reproductive mechanisms of four O. selloi populations from Piquete and Apiaí regions, São Paulo State; Camanducaia, Minas Gerais State; and Colombo, Paraná State. Anthesis started at 11:00 a.m. and finished at 3:00 p.m., with flowering climax at 1:00 p.m., and temperature was slightly important in this process. Pollen viability was high in all four studied populations. This species is self-compatible, forming fruits and seeds under both free pollination and spontaneous self-pollination, which indicates it has a great reproductive versatility, assuring genetic variability. Seed germination was also high in all four populations.

Chemical profiling of Ocimum americanum using external flavonoids.

A HPLC survey was undertaken of the external flavonoids in 111 herbarium specimens of Ocimum americanum L. (O. canum Sims), which were largely collected from their natural habitats throughout Africa and Asia. The purpose of this study was to establish the flavonoid profiles of this species over the full range of its geographic distribution in order to use these for authentication purposes. Six different external flavonoid chemotypes were found. The major chemotype, present in circa 80% of the specimens of both var. americanum and var. pilosum collected throughout the distribution area of the species, was characterised by very high levels of nevadensin, slightly lower levels of salvigenin and much lower levels of up to 15 other external flavones. Of the remaining five chemotypes, two were found in var. americanum and three in var. pilosum. All specimens belonging to these chemotypes were collected in South or East Africa and represented by only a few specimens. These samples contained much smaller levels of flavones than present in the major chemotype of O. americanum and all lacked nevadensin. Xanthomicrol, a compound absent from the main chemotype, was the dominant flavone in two of the minor chemotypes. The external flavonoid profiles found in the six chemotypes of O. americanum were compared with those of O. x citriodorum (11 herbarium specimens studied) and seven other closely related species of Ocimum. The main nevadensin/salvigenin pattern present in O. americanum was also found in O. x citriodorum, O. basilicum and some specimens of O. minimum, but there were strong quantitative differences in external flavonoids among these taxa. The other chemotypes of O. americanum showed some similarities in their external flavone profiles to those found in the closely related East African species O. fischeri, O. forskolei, O. kenyense and O. kilimandscharicum, which occur in the same geographic areas. This suggests that the uncommon chemotypes of O. americanum may have originated by an exchange of genes with other Ocimum species, e.g. by introgressive hybridisation. Despite some similarities in profiles, chemical differences were also found among the species, so that it should be possible to authenticate a large proportion of leaf samples of O. americanum on the basis of external flavonoid profiles.