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The use of lesser-known plant foods in addressing nutritional deficiencies is gaining popularity particularly in developing countries where malnutrition is endemic. This study investigated the proximate, minerals, vitamins, amino acids, tannins, phenolic acids, alkaloids, caratenoids, phytosterols and glycosides composition of the leaves of Vitex doniana using AOAC and gaschromatographic methods. The leaves had high (g/100g) fiber (14.67 - 35.39) and protein (15.46 - 37.30); but poor in lipid (0.80 - 1.93) and carbohydrates (4.02 - 9.70) corresponding to 58.68% - 141.56%, 30.92% - 74.60%, 1.23% - 2.97% and 1.34% - 3.23% daily value. The protein had relatively high level of essential amino acids (40.94). The leaves were rich in vitamins A, C and E; mineral elements, iron, copper, manganese and cobalt. Tannic acid constituted 100% of the tannins; ferulic acid (46.99%) for phenolic compounds; viticin (99.96%) of total alkaloids; lutein (35.62%) for carotenoids; vanillic acid (49.78%) and sitosterol (61.6%) for phytosterols while the most abundant of the glycocides was agnuside (72.64%) in the leaves. This result indicated that Vitex doniana leaves are a good source of nutrients and bioactive compounds for better nutrition and general wellbeing.

Vitex agnus castus L. extract (VACE) was investigated for its gastroprotective properties and possible molecular mechanisms in rats. VACE (60 or 120 mg/kg) or Esomeprazole (20 mg/kg) were orally administered for 3 weeks before the induction of gastropathy using indomethacin (30 mg/kg, single oral dose). VACE ameliorated the indomethacin-induced gastric juice acidity and pathological changes. VACE significantly preserved GSH, SOD, NO and PGE2 contents, while decreased lipid-peroxide, TNF-α and MPO contents. Moreover, VACE downregulated NF-κB1, COX-2, Caspase-3 and upregulated Bcl-2 and HSP-70 expression. Ultra-high-performance liquid chromatography (UPLC) coupled with quadrupole high-resolution time of flight mass spectrometry (qTOF-MS) enabled the tentative identification of 87 compounds allocated in seven main classes including flavonoids, glycosylated iridoid and labdane diterpenes. Notably, different agnuside derivatives and diterpenoids were reported in VACE for the first time. In conclusion, VACE contains an arsenal of bioactive metabolites which may exhibit gastroprotection by inhibiting inflammation, oxidative stress, and apoptosis.

Agnuside despite its good properties for the body, can be harmful to the kidneys in extra concentrations. So, its measurement is important. For the first time, we developed a simple, rapid and inexpensive method for the investigation of agnuside electrochemical behavior by designing a gold/reduced graphene oxide/carbon paste electrode (Au/RGO/CPE). The Au/RGO nanocomposite was synthesized through a one-pot in situ reduction of graphene oxide (GO) and HAuCl4 in the presence of sodium citrate and the obtained product was studied by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The anodic oxidation of agnuside has been carried out at a modified carbon paste electrode in 0.1 M phosphate buffer solution (pH 6.0) using sensitive differential pulse voltammetry (DPV). Several parameters such as the type and pH of supporting electrolyte and scan rates were optimized. This study reported a good linearity in the range of 0.050–250.0 nM and a low detection limit of 0.016 nM. The relative standard deviations (RSDs) for 10.0 nM and 100.0 nM of agnuside were obtained 3.3% and 1.2%, respectively. The sensor was successfully applied to determine agnuside in Vitex agnus castus fruit and urine sample.

In this study, we aimed to evaluate the quality of 11 products sold in Japan (one medicinal product and 10 dietary supplements) containing/claiming to contain chasteberry extract (fruit of Vitex agnus-castus L.) using HPLC fingerprint (15 characteristic peaks), quantitative determination of chemical marker compounds, and a disintegration test. The HPLC profile of the medicinal product was similar to that of the reference standard of V. agnus-castus fruit dry extract obtained from European Directive for the Quality of Medicines (EDQM), whereas the profiles of some dietary supplements showed great variability, such as different proportions of peaks or lack of peaks. Results of the principal component analysis of the fingerprint data were consistent with those of the HPLC profile analysis. The contents of two markers, agnuside and casticin, in dietary supplements showed wide variability; this result was similar to that achieved with the HPLC fingerprint. In particular, agnuside and/or casticin was not detected in two dietary supplements. Furthermore, one dietary supplement was suspected to be contaminated with V. negundo, as evidenced from the results of agnuside to casticin ratio and assay of negundoside, a characteristic marker of V. negundo. Results of the disintegration test showed poor formulation quality of two dietary supplements. These results call attention to the quality problems of many dietary supplements, such as incorrect or poor-quality origin, different contents of the active ingredient, and/or unauthorized manufacturing procedures.

Ethanopharmacological relevance Limited drugs, rise in drug resistance against frontline anti-malarial drugs, non-availability of efficacious vaccines and high cost of drug development hinders malaria intervention programs. Search for safe, effective and affordable plant based anti-malarial agents, thus becomes crucial and vital in the current scenario. The Vitex negundo L. is medicinal plant possessing a variety of pharmaceutically important compounds. The plant is used traditionally worldwide for the treatment of malaria including India and Malaysia by the indigenous tribes. In vitro studies have reported the anti-malarial use of the plant in traditional medicinal systems. Aim of the study The aim of the current study is to evaluate the traditionally used medicinal plants for in vitro anti-malarial activity against human malaria parasite Plasmodium falciparum and profiling secondary metabolite using spectroscopic and chromatographic methods. Chemical profiling of active secondary metabolites in the extracts was undertaken using LC-MS. Materials and methods Based on the ethno-botanical data V. negundo L. was selected for in vitro anti-malarial activity against P. falciparum chloroquine-sensitive (3D7) and multidrug resistant (K1) strains using SYBR Green-I based fluorescence assay. Cytotoxicity of extracts was evaluated in VERO cell line using the MTT assay. Haemolysis assay was performed using human red blood cells. Secondary metabolites profiling was undertaken using chromatographic and spectroscopic analysis. Liquid chromatography analysis was performed using a C18, 150 X 2.1, 2.6 μm column with gradient mobile phase Solvent A: 95% (H2O: ACN), Solvent B: Acetonitrile, Solvent C: Methanol, Solvent D: 5 mM NH4 in 95:5 (H2O: ACN) at a constant flow rate of 0.250 ml/min. The LC-MS spectra were acquired in both positive and negative ion modes with electrospray ionization (ESI) source. Results The anti-malarial active extract of V. negundo L. leaf exhibited potent anti-malarial activity with IC50 values of 7.21 μg/ml and 7.43 μg/ml against 3D7 and K1 strains, respectively with no evidence of significant cytotoxicity against mammalian cell line (VERO) and no toxicity as observed in haemolysis assay. The HPLC-LC-MS analysis of the extract led to identification of 73 compounds. We report for the first time the presence of Sabinene hydrate acetate, 5-Hydroxyoxindole, 2(3,4-dimethoxyphenyl)-6, 7-dimethoxychromen-4-one, Cyclotetracosa-1, 13-diene and 5, 7-Dimethoxyflavanone in the anti-malarial active extract of V. negundo L. leaf. Agnuside, Behenic acid and Globulol are some of the novel compounds with no reports of anti-malarial activity so far and require further evaluation in pure form for the development of potent anti-malarial compounds. Conclusions The result report and scientifically validate the traditional use of V. negundo L. for the treatment of malaria providing new avenues for anti-malarial drug development. Several novel and unknown compounds were identified that need to be further characterized for anti-malarial potential.

In our continuing work on Vitex genus, the phytochemical investigation of leaves of Vitex grandifolia led to the isolation of two iridoids known in the literature as agnuside and bartsioside. The chemical structures were characterized by combined spectroscopic analysis. These compounds were isolated from this specie for the first time, this study give evidence to iridoids as chemotaxonomic marker and their constitutive presence has been reported in a number of genera in the family Lamiaceae. Iridoids have been isolated from nineteen species belonging to genus Vitex from Lamiaceae especially aucubin and agnuside. In addition to the utilization of morphological features of plants belonging to this genus, these compounds may be useful chemotaxonomic markers to aid taxonomic identification. This work evaluates and describes the distribution of iridoids in Vitex species to further establish this claim with structures of the iridoids found in this genus. Keywords: Iridoids, Agnuside, Chemotaxonomic markers, Lamiaceae

Ethnopharmacological relevance Viticis fructus (VF) has been widely used in alleviating the swelling and pain, owning to its pharmacologically active components including agnuside, 10-O-vanilloylaucubin, luteolin and casticin. Aim of the study The pharmacokinetic profiles of the absorbed components from aqueous and ethanolic extracts of VF in rat plasma were performed, and explored the molecular mechanisms of absorbed components via network pharmacology. Materials and methods Ultra-performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS) was employed to identify the absorbed components from rat plasma. Liquid-liquid extraction with ethyl acetate was used to purify the plasma samples. Plasma pharmacokinetics parameters of the components absorbed were analyzed after oral administration of both extracts. Network pharmacology was used to predict the biological functions and potential signaling pathways of VF. The anti-cancer effects of VF extract and absorbed components have been confirmed by in vitro experiments. Results The method was very sensitive with lower limit of quantification (LLOQ) of 1.0, 2.5, 0.2 and 0.5 ng/mL for agnuside, 10-O-vanilloylaucubin, luteolin and casticin, respectively. With the exception of 10-O-vanilloylaucubin which was not detected in the ethanolic extract of VF, all other components were detected in both extracts in plasma. The pharmacokinetic parameters of the four components from rat plasma were significantly different between the two extracts. According to the results of network pharmacology, the absorption components of VF are enriched in 32 key pathways, and 15 pathways are related to cancer. Ultimately, the anti-cancer effects, as well as the signaling pathways of VF ethanolic extract and absorbed components were verified by in vitro experiments. Conclusion The optimized, sensitive and validated UHPLC-MS/MS method was successfully applied for the plasma pharmacokinetics comparison analysis of the two VF extracts. The combination of network pharmacology and pharmacokinetics provides a useful method to elucidate the biological effects and molecular mechanism of the absorbed components of VF.

Chemical profiling and standardization of the defatted methanol extract of the leaves of Vitex negundo L. were carried out using 13C nuclear magnetic resonance (NMR) analysis followed by chemometric analysis of the chemical shift data. Chemical profile was obtained using a k-means cluster profile and chemical standardization which was achieved using a multivariate control chart. The V. negundo samples were made up of four groups: the training set, submitted samples from production farms, commercial samples, such as tablets, capsules and teas, and experimental samples (samples which were allowed to degrade). Four groups were generated in k-means cluster, which generally corresponded to the four types of samples. The multivariate control chart identified samples whose quality exceeded the upper control limit, all of which were commercial samples and experimental samples. The samples were also analyzed by quantitative thin layer chromatography (qTLC) using agnuside as marker compound. Comparison of the qTLC results with the k-means cluster and the multivariate control chart showed poor correspondence. This means that a univariate analysis of a plant sample using a marker compound is useful only for quantification of the target compound. On the other hand, chemical profiling and standardization of medicinal plants should use a multivariate method. Key words: Vitex negundo, 13C NMR, multi-variate cluster profile, multi-variate control chart.

Due to its important role in regulating angiogenesis, vascular homeostasis and remodeling, and arteriogenesis in blood vascular and lymphatic endothelial cells, VEGFR2 stimulation has demonstrated promise in preclinical studies as an endovascular treatment for ischemic myocardial and peripheral disease. However, the short half-life of protein- and cytokine-based strategies and transduction inefficiency of vector-based modalities have hindered its clinical therapeutic applications. In the present study, we used a streamlined bioinformatics strategy combining ligand-based pharmacophore development and validation, virtual screening, and molecular docking to identify agnuside, a non-toxic, natural small molecule extract of Vitex agnus-castus possessing strong binding affinity, druggable physiochemical properties, and conformationally stable hydrogen bond and hydrophobic interactions with catalytically important residues within VEGFR2's active and allosteric sites. In-vitro proliferation, tube formation, and scratch wound migration assays provide evidence that agnuside promotes endothelial cell angiogenesis. Agnuside increases HUVEC proliferation with an EC50 of 1.376 μg/mL, stimulates tubulogenesis dose-dependently, and increases scratch wound migration rate. An additional angiogenesis assay suggests that agnuside may actively compete with a VEGFR2 inhibitor for VEGFR2 binding site occupancy to increase total length and branching length of HUVEC tubular networks. Chemometric analysis of molecular interaction fields (MIFs) by partial least squares (PLS)-derived quantitative structure activity relationship (QSAR) analysis and MIF contours provides the framework for the formulation of agnuside analogues possessing greater potency. Our research supports that agnuside may be a lead molecule for therapeutic angiogenesis.

Negundoside and agnuside are reported as marker constituents for standardization of Vitex negundo (V. negundo) leaves. This is the first report on determination and quantification of negundoside and agnuside by qNMR in the leaves of V. negundo, collected from various regions of India. The developed qNMR method showed good linearity (r2 ≥ 0.9994), high precision (RSD < 5%) and a good recovery (99.79 and 101.35%) for both the compounds. Therefore, the developed qNMR method is accurate and reliable for the quality control of V. negundo. The qNMR method is useful in quality control of plant extracts without the need of extensive sample preparation and chromatographic separation.

A high performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry method was developed for the identification of phenolic compounds in Vitex negundo L. var. cannabifolia (Siebold et Zucc.) Hand.-Mazz. A total of 31 compounds (10 phenolic acids, 19 flavonoids, and 2 iridoids) were fully or partially identified. Caffeic acid, neochlorogenic acid, cryptochlorogenin acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, schaftoside, isoschaftoside, flavosativaside, vitexin 2"-rhamnoside, and kaempferol 3-(6"-malonylglucoside) were detected for the first time in this plant. In subsequent quantitative analysis, an ultra-performance liquid chromatography combined with triple quadrupole mass spectrometry method was developed for the quantitative analysis of 17 phenolic compounds. All the analytes were detected within 8 min. The limits of detection and quantification were less than 7.251 and 26.454 ng/mL, respectively. The relative standard deviations of intra-day precision, inter-day precision, repeatability, stability, and recovery were less than 2.87%, 3.87%, 4.86%, 4.70%, and 3.61%, respectively. The validated method was applied to assess the quality of different medicinal parts (leaves, seeds, and roots) of V. negundo. The results indicated that chlorogenic acid, agnuside, isochlorogenic acid A, and isochlorogenic acid C might be selected as quantitative markers for the quality control of V. negundo. The contents of the 17 investigated compounds in leaves differed from those in the other parts. Hierarchical cluster analysis suggested that chlorogenic acid, agnuside, vitexin, and schaftoside could be potential chemical markers for the discrimination of different medicinal parts of V. negundo. This qualitative and quantitative analysis of phenolic compounds of V. negundo could provide a new tool for the quality control of this plant or its related remedies.

The study of the monoterpene glycosides content of Odontites luteus has shown the presence of a total of fifteen iridoid glucosides. The presence of compounds (1-5) and (7-10) is perfectly on-line with both the biogenetic pathway for iridoids biosynthesis in Lamiales and the current botanical classification of the species. On the other side, the presence of compounds like agnuside (6), adoxosidic acid (11), monotropein (12), 6,7-dihydromonotropein (13), methyl oleoside (14) and methyl gluco-oleoside (15) is of high interest because, first of all, they have never been reported before in Lamiales. In second instance, the majority of the last compounds are formally derived from a different biogenetic pathway which involves desoxyloganic acid/loganin and led to the formation of decarboxylated iridoid showing the 8β-stereochemistry. Furthermore, a second abnormality was found during our study and this regards compounds (14) and (15) which are seco-iriodids and thus not typical for this family. The presence of these unusual compounds, biogenetically not related to species belonging to Lamiales, is a clear evidence of the metabolites transfer from the hosts. In fact, the collection area is also populated by species belonging to Oleaceae and Ericaceae which could be the possible hosts since the biosynthesis of seco-iridoids and or iridoids related to deoxyloganic acid/loganin pathway, with the 8β-stereochemistry, is well documented in these species. This article is protected by copyright. All rights reserved.

The aim of present study was to evaluate the qualities of chaste berry (fruit of Vitex agnus-castus L.) preparations using HPLC fingerprint analysis. Seven medicinal products 1 from Japan and 6 from Europe, and 17 health foods, 6 from Japan and 11 from the United States were analyzed. HPLC profile and 26 authentic peaks were compared medicinal products and health foods. Whereas medicinal products had similar HPLC profiles, health foods had various profiles and each peak was also greatly different. The measured amounts of two markers in 5 traditional medicinal products, agnuside and casticin specified in the European Pharmacopoeia (EP), the U.S. Pharmacopoeia (USP) or the WHO monographs of chaste berry, were much lower than those in 2 medicinal products defined as "well-established use" by the European Medicines Agency. The amounts of two markers for 17 health foods differed in a great deal from 14-5054% and 3-1272%, respectively. Furthermore the amount ratios of two markers, agnuside/casticin, in about half of the health foods were remarkably larger than the standard crude drug and the ratios were closer to one of the related Chinese herbs, Vitex negundo L. It is concluded that a combination of HPLC fingerprints and the amount ratios of the marker compounds of chaste berry preparations serves as a useful tool to evaluate the qualities of these preparations.

Vitex negundo or lagundi is an important herbal medicine in Asia and various commercial products of this herb's leaves have been flooding the market. Reports on the chemical constituents of such products were limited and directed to only one selected chemical marker. Here, micellar electrokinetic chromatography using a fused silica capillary was used for analysis of the methanolic extract of Philippine lagundi herbal products. The separation solution, injection, and separation voltage was 50 mM SDS with 50 mM phosphoric acid at pH 2.5, 6 s at 25 mbar, and 20 kV at negative polarity, respectively. With UV detection at 200 nm, the LODs (S/N = 3) were in the sub-μg/mL range which were sufficient for detection without the need for on-line or off-line sample concentration. The repeatability values were all below 3.9% for migration time and peak signals. When the method was used to real samples, a flavonoid (isoorientin) and two iridoid glycosides (agnuside and negundoside) were identified as the target chemical markers within a 12 min electrophoretic run. The accuracy was from 95% to 129%. The results also suggested that agnuside was the major component in Philippine lagundi. The method was simple, effective, low cost, and can be used for authenticity evaluation of lagundi products.

A high performance liquid chromatography coupled with photodiode array detection method was developed for the identification and quantification of p-hydroxy benzoic acid and agnuside in the extracts of Vitex negundo and Vitex trifolia. The separation was achieved using acetonitrile and O-phosphoric acidâwater (0.5%, v/v) as the mobile phase in an isocratic elution mode. Mean retention times of standard p-hydroxy benzoic acid and agnuside were 6.14 and 11.90 min respectively. The developed method was validated as per the ICH guidelines for limit of detection, limit of quantification, linearity, accuracy and precision. Good linearity (r2â¥0.999) was observed for both the compounds in wide concentration range. Relative standard deviation values for intra-day and inter-day precision studies were less than 2%. The analytical recoveries of p-hydroxy benzoic acid and agnuside by the developed HPLC method were 93.07% and 106.11% respectively. Two compounds were identified and quantified in leaves and bar extracts of V. negundo and V. trifolia using the developed HPLC method. Keywords: Vitex negundo, Vitex trifolia, HPLC-PDA, p-Hydroxy benzoic acid, Agnuside

Novel lipidated analogs of iridoids viz., Agnuside and Negundoside with different chain length were synthesized and tested for immune adjuvant activity in the presence of weak antigen ovalbumin. Based on in vitro assay, 6-O-palmitoyl Agnuside (AG-3) was further taken up for detailed in vivo activity and found to significantly enhance the production of anti OVA IgG titer, neutralizing antibody (IgG1 and IgG2a) titer as well as soluble mediators of a Th1 (IL-2, IFN-γ)/Th2 response (IL-4) and proliferation of T lymphocyte subsets (CD4/CD8) and co stimulatory molecules CD80/CD86. Furthermore, the SAR studies revealed that presence of acyl group at aglycon moiety of these iridoid glycosides is crucial for immune adjuvant activity.

The use of Vitex negundo Linn. (Family: verbenaceae) is well documented in ayurveda and traditional Indian system of medicine for variety of diseases and liver ailments. The aim of the study was to investigate liver protective efficacy of a standardized bioactive fraction (SF) from Vitex negundo Linn. against acetaminophen (APAP) and galactosamine (GalN) hepatotoxicity. SF was tested at doses 12.5, 25, 50 and 100mg/kg, p.o. using both prophylactic and curative treatment schedule against APAP and GalN hepatotoxicity in mice and rats respectively. Isolated markers agnuside and negundoside were tested against APAP toxicity. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and albumin were estimated in serum and triglycerides (TG), total protein, glutathione (GSH) and lipid peroxidation (LP) in liver homogenate. Histopathological studies were carried out against APAP induced hepatotoxicity. SF exhibited significant hepatoprotection against APAP and GalN evident by restoration of ALT, AST, LDH, ALP, bilirubin, TG, albumin and total protein. Levels of LP and GSH also exhibited significant dose dependent recovery when treated with SF. Agnuside and negundoside also exhibited dose dependent protection against APAP induced hepatotoxicity. Microscopic examination of histopathological sections of liver confirmed hepatoprotective potential of SF. Results of the study suggest significant value of SF as hepatoprotective with sufficient safety margin as no mortality and change in general gross behavior was observed up to 2000 mg/kg, p.o. Hepatoprotective mechanism of SF may be due to its antioxidant activity exhibited by protection against increased lipid peroxidation and maintained glutathione status. It is apparent from the present study that agnuside and negundoside are the active ingredients in SF and can be responsible for the activity of SF.

Agnuside (AGN), an iridoid glycoside, is the chemotaxonomic marker of the genus Vitex which has gained enormous attention by virtue of its potential health benefits. Regardless of claiming many therapeutic applications reports demonstrating its pharmacokinetics or quantification in biomatrices are lacking. This is the first report which presents a sensitive liquid chromatography coupled to a tandem mass spectrometry (LC-MS/MS) method for the quantification of AGN in mice plasma and various tissues (including liver, intestine, spleen, kidney, heart, lungs and brain). AGN was extracted from the biological samples using protein precipitation followed by liquid-liquid extraction and the separation was achieved on C18 reversed phase column with a mobile phase consisted of 0.1% formic acid in acetonitrile-0.1% formic acid in triple distilled water (92:8, v/v) at a flow rate of 0.7mL/min. The MS/MS detection was performed by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in negative scan mode. The bioanalytical method was found linear over the concentration range of 1-4000ng/mL for plasma and tissue homogenates (r(2)≥0.990). The lower limit of quantitation (LLOQ) for all matrices was 1ng/mL. Intra-day and inter-day variance and accuracy ranged from 90 to 110% and 1-10%, respectively. Matrix effect and recoveries were well within the satisfactory limits. The validated method was applied successfully to measure AGN concentrations in plasma and tissues following intravenous (i.v.) and peroral (p.o.) administration to mice. Maximal AGN concentrations in plasma and tissues were reached within 30-45min. The mean absolute bioavailability (%F) of AGN was∼0.7%. After oral administration, AGN was most abundant in intestine, followed by kidney, liver, spleen, brain, lungs and heart. The identified target tissues of AGN may help in understanding its pharmacological action in vivo.

Vitex trifolia L. is a full grown fruit of Vitex rotundifolia L. (Verbenaceae). It has been used fortreating headache, dizziness, toothache and removal of fever as a traditional medicine in Korea. V. trifolia(500 g) were extracted three times with 80% aqueous MeOH at room temperature. The MeOH extract (38 g)was successively partitioned with n-hexane, EtOAc, n-BuOH and H 2 O. Both n-hexane and EtOAc fractionsshowed more than 80% antifungal activity in vivo against several plant pathogens at 2000 ppm. Successiverepeated silica gel, Sephadex LH-20, preparative TLC, and preparative HPLC of these fractions led toisolation of three compounds. Using mainly mass spectroscopy and nuclear magnatic resonance (NMR)spectroscopy, their chemical structures were determined as agnuside(1), chrysosplenol B(2), and artemetin(3).Compound 2 was isolated for the first time from V. trifolia. Study on in vitro and in vivo antifungal activitiesof the isolated compounds is in progress. Key words Vitex trifolia L., Agnuside, Chrysosplenol B, Artemetin

Vitex negundo Linn (Verberaceae) is an important drug in Indian, Chinese, and Malaysian Systems of medicine. The leaves are reported to show anti-inflammatory, anti-ulcer, anti-implantation, and analgesic activities. They contain negundoside, nishindaside, agnuside, luteolin, vitexicarpin, ursolic acid, and β-sitosterol. Lupeol, an important marker compound has not been reported from V. negundo until recently. Our preliminary TLC experiments indicated the presence of lupeol in the leaves of V. negundo. Subsequently, lupeol was isolated by preparative TLC. The identity of lupeol was confirmed by comparing the mass spectra [m/z value 427.8 (M+1) and 425.8 (M−1)]; melting point (215°C, using DSC) and co-TLC with the standard compound. A High Performance Thin Layer Chromatography (HPTLC) method for the quantification of five marker compounds, namely, negundoside, ursolic acid, eugenol, lupeol, and β-sitosterol from the leaves of V. negundo was developed and validated as per ICH guidelines. The amounts of negu...

Negundoside (1), agnuside (2) and 6'-p-hydroxy benzoyl mussaenosidic acid (3) are known bioactive metabolites in Vitex trifolia. In the present study a simple precise and reproducible method was developed for simultaneous quantitation of NS (1), AS (2) and HMA (3) and the antioxidant capacity of above markers has also been determined. Marker compounds have been resolved using silica gel 60 F(254) plates, petroleum ether (60-80)/toluene/acetone/water (10:10:80:2 v/v/v/v) as the mobile phases. The method does not employ any derivatisation procedure and can be used as a quality control tool for routine analysis of drugs V. trifolia and V. negundo together with their commercial extracts. NS (1), AS (2) and HMA (3) showed significant activity in DPPH and NO radical scavenging assays.

In this study, two iridoids i.e. agnuside (1) and bartioside (2) were isolated from Vitex grandifolia for the first time. These potent flavonoids were purified using fractionation and were separated by Sephadex LH 20, Silica gel 60, RP C-18 and Diaion HP-20 columns. Their structure was elucidated using FTIR, 1D and 2D NMR data, these were further evaluated for anti-inflammatory and antileishmania effects. Agnuside, isolated for the first time from V. grandifolia exhibited good activity against NF-kB assay with IC 50 (ug/mL) of 8.9 while bartsioside showed an IC 50 (ug/mL) of 12. Agnuside (1), an iridoid showed the highest IC 50 (μg/mL) of 5.38 and corresponding IC 90 (μg/mL) of 7.07 against L. donovani (intracellular amastigotes in THP1 cells). Positive standards were employed in these studies. The results allowed us to establish the relationship between the structure and the activities on the basis of the different patterns of substitution, particularly hydroxylation. This study gave credence to the view that there is a link between low incidence of some diseases and consumption of vegetables hence advantages of these vegetables are beyond nutritional gains.

Five compounds have been isolated from the leaves of Vitex negundo Linn (Verbenacea) and their structures established by spectral analysis. Compound 1 has been identified and characterised as a new iridoid with a novel structure which has never before been reported in literature from Vitex or any other source. Its structure has been established as 1,4a,5,7a-tetrahydro-1-beta-D-glucosyl-7-(3',4'-dihydroxybenzoyloxymethyl)-5-ketocyclopenta[c]pyran-4-carboxylic acid. Other compounds, 2, 3, 4 and 5, have been identified as luteolin-7-O-beta-D glucoside, nishindaside, negundoside, and agnuside, respectively. Compound 2, a flavone-O-glycoside, is being reported from this plant for the first time, and compounds 3, 4 and 5 are structurally known iridoids.

Lipase catalyzed regio-selective acylation of five iridoid glycosides viz., picroside I&II, catalpol, agnuside and negundoside in the presence of various acyl donors such as vinyl acetate and p-nitrophenyl alkanoates was studied. The regio-selectivity of enzymatic acylation and yields were found to vary amongst different substrates. Monoacylated products were isolated with all the substrates under scrutiny indicating high regio-selective nature of such transformations. A series of acyl esters of picroside-I, picroside-II, catalpol, agnuside and negundoside have been synthesized by this enzymatic trans-esterification methodology.

The ethanol extract of the fruits of Vitex rotundifolia (VRE) and its four major compounds (casticin, luteolin, rotundifuran and agnuside) were tested for their estrogen-like activity by using the modified cell proliferation assay (E-SCREEN assessment system), as well as the estrogen receptor (ERα), estrogen receptor-regulated progesterone receptor and pS2 mRNA expression in MCF-7 cells. The results showed that only agnuside and rotundifuran could stimulate the proliferation of MCF-7 cells. These actions were dose dependent (range from 100 nm to 10 μm) and could be significantly inhibited by the specific estrogen receptor antagonist ICI 182,780. The estrogen receptor ERα and the estrogen receptor-regulated progesterone receptor and pS2 mRNA levels were increased by treatment with rotundifuran and agnuside within 24 h, and the effects could be reversed by ICI 182,780. The standardization of the extract and constituents were carried out by means of a high-performance liquid chromatography-fingerprint. It was concluded that VRE and its compounds showed estrogen-like activity and that the estrogenic effects of rotundifuran and agnuside were mediated by the estrogen inducible gene, which may be useful in regulating the hormone levels to treat related diseases. However, further studies are required to assess the physiological significance of VRE in animals and man.

The structures of six different iridoid glucosides have been revised. Three compounds isolated from Eremostachys glabra and designated 6,9-epi-8-O-acetylshanziside (1), 5,9-epi-penstemoside (2), and 5,9-epi-7,8-didehydropenstemoside (3) have been shown to be identical to the known iridoids barlerin (4, 8-O-acetylshanziside), penstemoside (5), and 7,8-didehydropenstemoside (6), respectively. Another compound named harpagoside-B, isolated from Scrophularia deserti and proposed to be 9-epi-6-O-methylharpagoside (11), was demonstrated from the spectroscopic data given to be the known harpagoside (10b). Finally, two alleged iridoid galactosides from Buddleja crispa named buddlejosides A and B (12a and 12b) have been shown to be the corresponding glucosides; the former is identical to agnuside (13a), while the latter is 3,4-dihydroxybenzoylaucubin (13b), an iridoid glucoside not previously published. This clearly showed that care should be taken with the interpretation of NOEs involving bridgehead protons in iridoid structures because they can be capricious and lead to erroneous structural assignments.

Six new iridoid glucosides, 6'-O-trans-feruloylnegundoside (1), 6'-O-trans-caffeoylnegundoside (2), 2'-O-p-hydroxybenzoyl-6'-O-trans-caffeoylgardoside (3), 2'-O-p-hydroxybenzoyl-6'-O-trans-caffeoyl-8-epiloganic acid (4), 2'-O-p-hydroxybenzoyl gardoside (5), and 2'-O-p-hydroxybenzoyl-8-epiloganic acid (6), along with two known iridoids, agnuside and negundoside, have been isolated from the ethyl acetate extractive of the leaves of Vitex altissima. The structures of these compounds were elucidated on the basis of spectral data interpretation. These isolates did not exhibit significant 5-lipoxygenase enzyme inhibitory activity, but compounds 2-4 showed potent antioxidant activity by both the superoxide (NBT riboflavin photoreduction) free-radical-scavenging and DPPH-radical-scavenging methods. Compounds 1, 2, and negundoside were evaluated in a rat paw edema assay.

The methanolic extract of the flowering stems of Vitex agnus-castus yielded three new iridoids: 6′- O -foliamenthoylmussaenosidic acid (agnucastoside A), 6′- O -(6,7-dihydrofoliamenthoyl)mussaenosidic acid (agnucastoside B) and 7- O - trans - p -coumaroyl-6′- O - trans -caffeoyl-8-epiloganic acid (agnucastoside C) in addition to four known iridoids (aucubin, agnuside, mussaenosidic acid and 6′- O - p -hydroxybenzoylmussaenosidic acid) and one known phenylbutanone glucoside (myzodendrone). The structure elucidations were mainly done by spectroscopic methods (1D and 2D NMR spectra) and MS data interpretation. The purified compounds were tested for biological activities against various microorganisms and cancer cell lines.

The extract of Viticis Fructus appeared to have an analgesic effect, and was subjected to activity-guided separation using acetic acid-induced writhing in mice. The active fraction gave new compounds, vitexfolin A (1A), B and C, 10-O-vanilloylaucubin (3), dihydrodehydrodiconiferylalcohol-beta-D- (2'-O-p-hydroxybenzoyl)glucoside (4), and vanilloyl-beta-D-(2'O-p-hydroxybenzoyl)glucoside, together with agnuside (2) and erythro- and threoguaiacylglycerols. Compounds 1A and 2-4 showed significant writhing inhibition following oral administration at doses of 15, 50, 25, and 50 mg/kg, respectively. The effect on pressure pain threshold was tested using compounds 1A and 2 at a dose of 50 mg/kg, and only the former produced the analgesia. The analgesic effect of some related iridoid glucosides is also discussed.

Seven phenolic compounds, vanillic acid (1), threo-guaiacyl glycerol (2), erythro-guaiacyl glycerol (3), taxifolin (4), dihydrodehydrodiconiferyl alcohol (5), dihydrodehydrodiconiferyl alcohol-9-O-β-D-glucoside (6) and dihydrodehydrodiconiferyl alcohol-(4→8)-erythro-guaiacyl glycerol ether (7), were separated from the methanol extract of Viticis trifoliae Fructus (Fruit of Vitex rotundifolia L.) and their structures were identified on the basis of spectroscopic data. In addition, 1, 2, 4-7 and two previously isolated iridoid glucosides, agnuside (8) and VR-I (10-O-vanilloyl aucubin) (9) were tested for antioxidative activity using the ferric thiocyanate method. These compounds, except 8, exhibited stronger antioxidative activity than 3-tert-butyl-4-hydroxyanisole. Moreover, 1, 2 and 4-9 were investigated for the scavenging effect on 1,1-diphenyl-2-picrylhydrazyl. All tested compounds, except 8, showed a potent scavenging effect. Especially, the effect of 4 was almost twice that of α-tocopherol at a concentration of 0.02 mM.

Further study on the constituents of the fruits of Crescentia cujete afforded 16 iridoids and iridoid glucosides. The structures of eight new compounds, named crescentins I–V and crescentosides A, B and C, were determined by analysis of their spectral data. Another eight known compounds were identified as ajugol, 6- O - p -hydroxybenzoylajugol, aucubin, 6- O - p -hydroxybenzoyl-6-epiaucubin, agnuside, ningpogenin, 5,7-bisdeoxycynanchoside and a degradation product of glutinoside.

As part of our continuing efforts in the search for potential biologically active compounds from medicinal plants, we have isolated 18 compounds including two novel nitrogen containing diterpenes from extracts of the fruits ofVitex agnus-castus. These isolates, along with our previously obtained novel compound vitexlactam A (1), were evaluated for potential biological effects, including cancer chemoprevention. Chemically, the nitrogenous isolates were found to be two labdane diterpene alkaloids, each containing anα,β-unsaturatedγ-lactam moiety. Structurally, they were elucidated to be 9α-hydroxy-13(14)-labden-16,15-amide (2) and 6β-acetoxy-9α-hydroxy-13(14)-labden-15,16-amide (3), which were named vitexlactams B and C, respectively. The 15 known isolates were identified as vitexilactone (4), rotundifuran (5), 8-epi-manoyl oxide (6), vitetrifolin D (7), spathulenol (8),cis-dihydro-dehydro-diconiferylalcohol-9-O-β-D-glucoside (9), luteolin-7-O-glucoside (10), 5-hydroxy-3,6,7,4′-tetramethoxyflavone (11), casticin (12), artemetin (13), aucubin (14), agnuside (15),β-sitosterol (16),p-hydroxybenzoic acid (17), andp-hydroxybenzoic acid glucose ester (18). All compound structures were determined/identified on the basis of 1D and/or 2D NMR and mass spectrometry techniques. Compounds6,8,9, and18were reported from aVitexspieces for the first time. The cancer chemopreventive potentials of these isolates were evaluated for NADP(H):quinone oxidoreductase type 1 (QR1) induction activity. Compound7demonstrated promising QR1 induction effect, while the new compound vitexlactam (3) was only slightly active.

A rapid ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method was developed and validated for the simultaneous determination of all classes of non-volatile phytochemicals (iridoids, flavonoids and diterpenes) in Vitex agnus-castus (Lamiaceae) fruits, a traditional medicinal plant used against premenstrual symptoms (PMS) and other disorders. Seven marker compounds, 3,4-dihydroxybenzoic acid, p -hydroxybenzoic acid, agnuside, 5-hydroxykaempferol-3,6,7,4′-tetramethylether, 1,2-dibenzoic acid glucose, methoxy-vitexilactone, and vitetrifolin D were isolated from the methanol extract of V. agnus-castus to be used as reference substances. Chromatographic separation was performed on a Zorbax Eclipse XDB-C18 (50 mm × 2.1 mm) UHPLC column with 1.8 μm particle size, within 20 min. A solvent gradient from 0.5% acetic acid to acetonitrile at a flow rate of 0.6 mL/min was used as mobile phase. Analyte detection and quantification was realized at 210 nm and 260 nm. The UHPLC-DAD assay was validated for the quantitative analysis of agnuside, isovitexin, casticin, 5-hydroxykaempferol-3,6,7,4′-tetramethylether and vitetrifolin D. It was found to be specific, accurate, precise, and reproducible for the quantification of these compound within a concentration range of 0.7–500.0 μg/mL for casticin and 5-hydroxykaempferol-3,6,7,4′-tetramethylether, 1.4–1000.0 μg/mL for isovitexin and agnuside, and 12.4–1000.0 μg/mL for vitetrifolin D. Intra- and inter-day variations showed relative standard deviations (RSD) of less than 3.9% and 6.4%, respectively. Tentatively assignment of 62 chromatographic features found in the UHPLC-DAD assay was carried out by coupling the UHPLC instrument to a quadrupole time-of-flight mass spectrometer via an electrospray ionization interface (ESI-QTOF-MS) operated in positive and negative ion mode. By using the established quantitative UHPLC-DAD assay to asses agnuside, isovitexin, casticin, 5-hydroxykaempferol-3,6,7,4′-tetramethylether and vitetrifolin D in V. agnus-castus derived preparations as extracts, tinctures and tablets, the applicability of the developed assay to phytopharmaceuticals was successfully proven.

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 Acre also isolated and characterized. The structures of the isolates were established by spectroscopic methods (one-dimensional (1D)- and two-dimensional (2D)-NMR, MS).