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The purpose of this study was to elucidate the probable mechanism for the anti-arthritic activity of agnuside (AGN), a compound isolated from the leaf extract of Vitex negundo.The anti-inflammatory activity of AGN within a dose range of 1.56-12.50 mg/kg in normal and adrenalectomized rats was evaluated against different inflammagens. An array of pro-inflammatory mediators (PGE(2) and LTB(4)) and T-cell-mediated cytokines (IL-2, TNF-α, IFN-γ, IL-4, IL-10, IL-17) was assayed using flow cytometry, in arthritic paw tissue homogenate and splenocytes of treated animals.Significant anti-arthritic activity was observed in the polyarthritis test in rats and this was associated with significant suppression of inflammatory mediators and T-cell-mediated cytokines (Th1/Th2). The anti-inflammatory activity in adrenalectomized rats confirmed that the effect of AGN is not mediated by the pituitary-adrenal axis. AGN also showed inhibition of vascular permeability and leukocyte migration in vivo.The study suggests the possible development of AGN as a therapeutic agent in the treatment of arthritis by the modulation of the host immune response.

Dried whole plant of Vitexagnus cactus was extracted with organic solvents in different ratios and analyzed using high performance liquid chromatography (HPLC) with evaporative light scattering (ELS) detector. The markers of interest in Vitexagnus castus were agnuside, casticin and vitexyl acetone. These standard molecular markers do not contain strong chromophores, hence it is difficult to identify them using the UV detection system in high performance liquid chromatography. ELS detector is regarded as a valuable alternative to UV detection system for identification of the compounds that do not contain strong chromophores. The results of the present study show that the ELS detection system is more efficient than the UV-visible detection system. Key words: Vitexagnus castus , evaporative light scattering detector, high performance

Two new iridoids, called viteoids I and II, were isolated from Viticis trifoliae Fructus (fruit of Vitex rotundifolia L. fil.) along with six known iridoids, eucommiol, iridolactone, pedicularis-lactone, agnuside, VR-I and 1-oxo-eucommiol. The chemical structures of viteoids I and II were determined on the bases of spectroscopic data.

From the ethanolic extract of the aerial parts of Globularia davisiana, a new iridoid glycoside, davisioside (1), was isolated. Davisioside (1) comprises a rare iridoid aglycone structure with a saturated double bond between C-3 and C-4. Nine known iridoid glycosides, asperuloside (2), alpinoside (3), geniposide (4), globularin (5), globularicisin (6), 10-O-benzoylcatalpol (7), lytanthosalin (8), melampyroside (9), agnuside (10), and three known phenylethanoid glycosides, verbascoside, isoacteoside and leucosceptoside A were also isolated and characterized. The structures of the isolates were established by spectroscopic methods (one-dimensional (1D)- and two-dimensional (2D)-NMR, MS).

Since there is an increasing interest in Agni-casti preparations, their standardisation is becoming more important. The significant components agnuside and casticin are the most suitable for standardisation, the former being a chemotaxonomic marker of the genus Vitex L. and also being commercially available. The analytical method developed includes not only the determination of agnuside but also of p-hydroxybenzoic acid (PHBA), which may be of some interest because agnuside is composed of aucubin and PHBA. Fruits of Vitex agnus-castus were extracted twice with methanol, the extracts combined, evaporated to dryness and dissolved in water:methanol (95:5). This solution was subjected to solid phase extraction over neutral aluminium oxide in order to remove co-occurring, and interfering, isoorientin. Agnuside was assayed by high performance liquid chromatography on a C18-column eluted with an acetonitrile:water gradient and detected at 258 nm using a diode array detector. Agnuside was observed between 7.8 and 8.1 min and PHBA between 2.0 and 2.3 min. Eight plant samples were investigated. Copyright © 2000 John Wiley & Sons, Ltd.

Ciguatera Fish Poisoning (CFP) is an intoxication resulting from the ingestion of coral reef fish that has accumulated neurotoxins issued from dinoflagellates. A common form of ichytosarcotoxism in tropical and subtropical regions, it manifests a cohort of complex symptoms in humans. In the first part of this thesis, the effect of Pacific CTX-1B (P-CTX-1B) on the production of nitric oxide (NO) radical via the modulation of inducible NO synthase (iNOS) enzyme, has been investigated. The results obtained suggest for the first time the inflammatory aspect of CFP. Unlike Western treatments, which are mainly symptomatic, traditional medicine in the Pacific is known for its effectiveness with the use of many plants in the treatment of CFP. Consequently, the therapeutic evaluation of several plant extracts is described in the second part of this thesis. The third part concerns the selection of three of these extracts and the subsequent phtochemical work undertaken. This studies has led to the identification of three molecules, quercitrin, rosmarinic acid and agnuside. The detoxifying activity of rosmarinic acid has been patented.; La ciguatéra est une intoxication liée à l'ingestion de poissons de récif corallien devenus toxiques par l'accumulation d'une ou plusieurs neurotoxines d'origine dinoflagellaire. Cet ichtyosarcotoxisme représente une des plus fréquentes formes d'intoxication dans les régions tropicales et subtropicales et se manifeste par une cohorte de symptômes complexes chez l'homme. La première partie de cette étude a consisté à mettre en évidence l'action de la CTX-1B du Pacifique (P-CTX-1B) sur la surproduction d'oxyde nitrique (NO) via la modulation de son enzyme de synthèse, la NO synthétase inductible (iNOS), impliquant ainsi pour la première fois ce mécanisme pathogénique inflammatoire dans la ciguatéra. La deuxième partie concerne les travaux de l'évaluation de potentiel thérapeutique d'une trentaine d'extraits de plantes. En effet, la médecine occidentale reste peu efficace et symptomatique pour traiter durablement les patients souffrant de ciguatéra. La troisième partie décrit la sélection de trois de ces plantes et les études phytochimiques menées. Ces travaux ont conduit à l'identification de trois molécules : la quercitrine, l'acide rosmarinique et l'agnuside. Cette thèse a abouti au dépôt d'un brevet sur l'activité détoxifiante de l'acide rosmarinique.

Ciguatera Fish Poisoning (CFP) is an intoxication resulting from the ingestion of coral reef fish that has accumulated neurotoxins issued from dinoflagellates. A common form of ichytosarcotoxism in tropical and subtropical regions, it manifests a cohort of complex symptoms in humans. In the first part of this thesis, the effect of Pacific CTX-1B (P-CTX-1B) on the production of nitric oxide (NO) radical via the modulation of inducible NO synthase (iNOS) enzyme, has been investigated. The results obtained suggest for the first time the inflammatory aspect of CFP. Unlike Western treatments, which are mainly symptomatic, traditional medicine in the Pacific is known for its effectiveness with the use of many plants in the treatment of CFP. Consequently, the therapeutic evaluation of several plant extracts is described in the second part of this thesis. The third part concerns the selection of three of these extracts and the subsequent phtochemical work undertaken. This studies has led to the identification of three molecules, quercitrin, rosmarinic acid and agnuside. The detoxifying activity of rosmarinic acid has been patented.; La ciguatéra est une intoxication liée à l'ingestion de poissons de récif corallien devenus toxiques par l'accumulation d'une ou plusieurs neurotoxines d'origine dinoflagellaire. Cet ichtyosarcotoxisme représente une des plus fréquentes formes d'intoxication dans les régions tropicales et subtropicales et se manifeste par une cohorte de symptômes complexes chez l'homme. La première partie de cette étude a consisté à mettre en évidence l'action de la CTX-1B du Pacifique (P-CTX-1B) sur la surproduction d'oxyde nitrique (NO) via la modulation de son enzyme de synthèse, la NO synthétase inductible (iNOS), impliquant ainsi pour la première fois ce mécanisme pathogénique inflammatoire dans la ciguatéra. La deuxième partie concerne les travaux de l'évaluation de potentiel thérapeutique d'une trentaine d'extraits de plantes. En effet, la médecine occidentale reste peu efficace et symptomatique pour traiter durablement les patients souffrant de ciguatéra. La troisième partie décrit la sélection de trois de ces plantes et les études phytochimiques menées. Ces travaux ont conduit à l'identification de trois molécules : la quercitrine, l'acide rosmarinique et l'agnuside. Cette thèse a abouti au dépôt d'un brevet sur l'activité détoxifiante de l'acide rosmarinique.

A validated rapid and simple isocratic HPLC-PDA method was developed for identification and quantification of p- hydroxy benzoic acid and two iridoids negundoside and agnuside in the extracts of two Vitex species, Vitex negundo and Vitex trifolia. The separation of the three compounds was achieved on a RP-18 (250 mm X 4 mm, 5µm) column at 25o C using acetonitrile (15%) and 0.05% trifluoroacetic acid (TFA) in water (85%). Limit of detection (LOD) were 1, 2.5 and 2.5 μg/ml for p- hydroxy benzoic acid, negundoside and agnuside respectively. Similarly, limit of quantification (LOQ) were 2.5, 5 and 5 μg/ml for p- hydroxy benzoic acid, negundoside and agnuside respectively. Good linearity (r2 > 0.999) was observed for all the three compounds in wide concentration range. Using the developed HPLC method, the three compounds were identified and quantified in leaves and bark extracts of Vitex negundo and Vitex trifolia. The novelty of the developed HPLC method is that it does not require complex sample processing such as use of solid phase extraction as well as use of buffer in mobile phase. This is the first report of a validated HPLC method for the simultaneous determination of p- hydroxy benzoic acid, negundoside and agnuside in Vitex species.

Two new iridoids, called viteoids I and II, were isolated from Viticis trifoliae Fructus (fruit of Vitex rotundifolia L. fil.) along with six known iridoids, eucommiol, iridolactone, pedicularis-lactone, agnuside, VR-I and 1-oxo-eucommiol. The chemical structures of viteoids I and II were determined on the bases of spectroscopic data.

A new iridoid, pedunculariside, together with the known iridoid agnuside were isolated from the butanol extract of Vitex peduncularis stem bark. Both pedunculariside and agnuside showed preferential inhibition of COX-2, with IC50 values of 0.15 +/- 0.21 mg/ml and 0.026 +/- 0.015 mg/ml respectively, while having only small inhibitory effects on COX-1. Both compounds did not exhibit cytotoxicity against vero cells.

A new iridoid, named tarumal, as well as the known iridoids viteoid II and agnuside were isolated from the leaves of Vitex cymosa and identified by spectroscopic methods. Este trabalho descreve o isolamento e a identificação, a partir de folhas de Vitex cymosa, de um novo iridóide não glicosídico chamado tarumal, além dos já conhecidos viteóide II e agnusídio.

The present study was carried out to estimate the content of total phenolics, flavonoids, negundoside and agnuside and to determine antibacterial, cytotoxic and antioxidant efficacy of methanol extract of two varieties of Vitex negundo namely V. negundo var. negundo (Vnvn) and V. negundo var. purpurascens (Vnvp). Total phenolic and flavonoid content of leaf extracts were estimated by Folin-Ciocalteau reagent and Aluminium chloride colorimetric estimation method respectively. Negundoside and agnuside content were estimated using HPLC. Antibacterial activity of both the extracts was tested by Agar well diffusion assay. Cytotoxicity of extract was evaluated by Brine shrimp lethality bioassay. Antioxidant activity of extracts was determined by DPPH free radical scavenging and Ferric reducing assay. The content of total phenolics and flavonoid were higher in extract of Vnvp. Negundoside and agnuside contents were high in Vnvn and Vnvp respectively. Leaf extract of Vnvp was shown to display marked antibacterial, cytotoxic and antioxidant activity when compared with extract of Vnvn. Both the varieties of V. negundo showed marked antibacterial, cytotoxic and antioxidant activity. The extract of Vnvp displayed marked bioactivities than that of extract of Vnvn. The presence of high phenolic and flavonoid content could be attributed to the marked bioactivities of Vnvp.

Two new glucosidic iridoids, designated as nishindaside ( 2a ) and negundoside ( 3a ), have been isolated from the leaves of Vitex negundo ( Verbenaceae ). The absence of a C(3)–C(4) double bond and the presence of an equatorial 3-methoxy group in 2a , the 3-methoxy-3,4-dihydro derivative of agnuaide ( 1a ), were established from 1H and 13C NMR analyses, corroborated by mass spectral fragmentation of the hexaacetate ( 2b ). Negundoside ( 3a ) could be concluded to be the 2' -p-hydroxy benzoyl derivative of mussaenosidic acid from the results of alkiline hydrolysis and the shifts in the signals of C-1 ' , C-2 ' and C-3' in its 13C NMR spectrum.

Vitex negundo Linn., belonging to family Verbenaceae, is an aromatic shrub distributed in lower part of Himalayan region of Nepal. In the ayurvedic system of medicine, it is used to control pain, inflammation and other related diseases. It contains many polyphenolic compounds, terpenoids, glycosidic iridoids and alkaloids. Bioactivity guided phytochemical investigation of methanolic extract of leaves of Vitex negundo resulted in the isolation of eight compounds under silicagel VLC, CC and preparative TLC. They were identified as negundoside, agnuside, vitegnoside, 7,8 dimethyl herbacetin 3-rhamnoside, 5,3'-dihydroxy-7,8,4'-trimethoxy flavanone, 5-hydroxy-3,6,7,3',4'-pentamethoxy flavone, 5,7 dihydroxy- 6,4' dimethoxy flavonone, and 5 hydroxy-7,4' dimethoxy flavone. The structures of pure compounds were elucidated by MP, Rf values, Co- TLC, Colour reactions (Cerric sulphate spray), mild acid hydrolysis and spectroscopic methods (Mass, UV, IR, 1H, 13C and 2 D NMR). All the isolated compounds were evaluated for their antimicrobial activities. They were found to have significant antibiotic activity against Bacillus subtilis, Staphylococcus aureus, Micrococcus pyogenes, Pseudomonas aeruginosa and E. coli . The compounds vitegnoside, and 7, 8 dimethyl herbacetin 3-rhamnoside have MIC 6.25mg/ml. Keywords : Vitex negundo; Verbenaceae; Chemical constituents; Bioassay; Analysis. DOI: 10.3126/sw.v6i6.2630 Scientific World , Vol. 6, No. 6, July 2008 27-32

The phenylbutanone glucoside, as well as the known compounds agnuside and eurostoside, were isolated from the leaves of Vitex rotundifolia , although eurostoside was obtained as a trans - and cis -mixture. The structures were established on the basis of spectroscopic data.