Chemical composition and biological activities of the essential oils from Vitex-agnus castus, Ocimum campechianum and Ocimum carnosum.

The essential oils obtained by hydrodistillation from fresh leaves of Vitex agnus-castus and Ocimum campechianum, and from fresh inflorescences of Ocimum carnosum were analysed by GC-FID and GC-MS. The major components of V. agnus-castus essential oil were identified as 1,8-cineole (47.9%), terpinyl α-acetate (11.6%), sabinene (11.2%) and caryophyllene oxide (9.7%), while in the O. campechianum essential oil were eugenol (72.1%), ß-elemene (6.8%), (E)-caryophyllene (6.4%) and bicyclogermacrene (5.2%). Linalool (79.0%), α-epi-cadinol (5.4%), terpinen-4-ol (3.2%) and 1,8-cineole (2.8%) were the major constituents in the O. carnosum essential oil. The essential oils were subsequently evaluated for their larvicidal and cytotoxic activities. Larval bioassay against Aedes aegypti of V. agnus-castus, O. campechianum and O. carnosum essential oils showed LC50 values of 97.55 ± 0.35, 81.45 ± 0.35 and 109.49 ± 0.35 µg/mL, respectively. The in vitro cytotoxic activities of the essential oils has been evaluated on breast adenocarcinoma (MCF-7), lung carcinoma (NCI-H292), pro-myelocytic leukemia (HL-60), and cervical adenocarcinoma (HEP-2) human cell lines, and pro-myelocytic leukemia cells lines (HL-60) were found to be the most sensitive to all the essential oils tested than the others. This is the first report on larvicidal and cytotoxic activities of these essential oils.

Formulation and evaluation of topical herbal gel for the treatment of arthritis in animal model

ABSTRACT The objective of the study is to formulate and evaluate a topical herbal gel containing Cardiospermum halicacabum and Vitex negundo leaf extracts for their anti-arthritic activity in rats. Twelve herbal gel formulations were prepared using 1.5% of gelling agents carbopol 934 (F1-F6) and carbopol 940 (F6-F12) and they were evaluated for physical appearance, net content, viscosity, extrudability, pH, spreadability, in vitro diffusion profile and primary skin irritation tests. The stability study for the topical herbal gel formulation was done as per ICH guidelines and anti-arthritic activity was evaluated by Freund's Complete Adjuvant (FCA) induced arthritis method. Assessment of body weight, paw volume, hematological and biochemical parameters, histopathological examination and In vitro determination of serum biomarkers were also carried out. Formulated gels were homogenous, stable and complied with the guidelines. Among the formulations, F4 showed better release (98.4 %) characteristics than other formulations. No erythema or edema was observed in the skin irritation test confirming the gel was non-toxic and safe. Topical application of the herbal gel F4 containing carbopol 934 displayed significant (p < 0.001) anti-arthritic activity compared to diseased rats. Reduction in paw volume, no agglutination in C - reactive protein and rheumatic factor, reduction in TNF level, regaining of normal hematological, and biochemical parameters, reduction in spleen and thymus weight and histopathological examination supported the anti-arthritic activity of the gel formulation.

Determination and Identification of a Specific Marker Compound for Discriminating Shrub Chaste Tree Fruit from Agnus Castus Fruit Based on LC/MS Metabolic Analysis.

Shrub Chaste Tree Fruit (SCTF) is defined as the fruits of Vitex rotundifolia L. f. and V. trifolia L. and has been used as a component of some traditional Japanese medicines (Kampo formulations). Agnus Castus Fruit (ACF) is defined as the dried ripe fruits of V. agnus-castus L.; it is used in traditional European medicines, but is becoming popular in Japan as both an over-the-counter drug and as an ingredient in health foods for treating premenstrual syndrome (PMS). To ensure the efficacy and safety of both SCTF and ACF products, it is important to precisely authenticate their botanical origins and to clearly distinguish between SCTF and ACF. Therefore, we tried to identify SCTF-specific marker compounds based on LC/MS metabolic analysis. The multivariate analysis of LC/MS data from SCTF and ACF samples furnished candidate marker compounds of SCTF. An SCTF-specific marker was isolated from SCTF crude drugs and identified as 3-O-trans-feruloyl tormentic acid on the basis of spectroscopic data from NMR and MS. Since avoiding contamination from closely related species is a significant requirement for pharmaceuticals of natural origin, this information will be valuable for the quality control of both SCTF and ACF products from the viewpoint of regulatory science.

Identification of New Diterpenes as Putative Marker Compounds Distinguishing Agnus Castus Fruit (Chaste Tree) from Shrub Chaste Tree Fruit (Viticis Fructus).

Agnus Castus Fruit is defined in the European Pharmacopoeia as the dried ripe fruit of Vitex agnus-castus. In Europe it is used as a medicine targeting premenstrual syndrome and climacteric disorder. In Japan, Agnus Castus Fruit is becoming popular as a raw material for over-the-counter drugs and health food products, though its congenic species, Vitex rotundifolia and Vitex trifolia, have been used as Shrub Chaste Tree Fruit in traditional medicines. Therefore, it is important to discriminate these Vitex plants from the viewpoint of regulatory science. Here we tried to identify putative marker compounds that distinguish between Agnus Castus Fruit and Shrub Chaste Tree Fruit. We analyzed extracts of each crude drug by liquid chromatography-mass spectrometry, and performed differential analysis by comparison of each chromatogram to find one or more peaks characteristic of Agnus Castus Fruit. A peak was isolated and identified as an equilibrium mixture of new compounds named chastol (1) and epichastol (1a). The planar structures of 1 and 1a were determined spectroscopically. Their relative configurations were revealed by nuclear Overhauser effect spectroscopy and differential nuclear Overhauser effect-NMR data. Since avoiding contamination from closely related species is needed for the quality control of natural pharmaceuticals, this information will be valuable to establish a method for the quality control of both, Agnus Castus Fruit and Shrub Chaste Tree Fruit products.

Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant.

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant / Regeneración de plantas a partir de cultivos de callos de Vitex trifolia (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supple mented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m2s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Vitex trifolia es una especie arbustiva de uso popular como planta medicinal, sus hojas, raíces y flores se han reportado para la cura de diferentes aflicciones. El aumento de la explotación de estas plantas ha puesto en peligro su conservación y ha justificado el uso de herramientas biotecnológicas para su propagación. El objetivo de esta investigación fue presentar un protocolo eficiente para la regeneración de estas plantas a través de la organogénesis, y analizar la homogeneidad genética de las líneas clonales establecidas por ADN polimórfico amplificado aleatoriamente (RAPD) mediante la repetición de marcadores de inter secuencia simple (ISSR). La regeneración de plántulas se logró en cultivos de callos derivados de explantes de tallo, hoja y pecíolo de V. trifolia en un medio diferenciado Murashige & Skoog, que se incubaron a 25±2ºC bajo una intensidad de luz de 61μmol/m2s con lámparas fluorescentes blancas y un fotoperíodo de 16h. La tasa de regeneración de brotes se correlacionó positivamente con la concentración de las hormonas en el medio nutritivo. Los brotes se regeneraron más rápidamente a partir de explantes de tallo y pecíolos en comparación con explantes de hoja. La mayor tasa de regeneración de brotes se obtuvo en los explantes de tallo utilizando 11.10μM BAP en combinación con 0.54μM NAA, 271.50μM Ads y 1.44μM GA3. Este protocolo puede ayudar a la propagación masiva y conservación de esta importante planta medicinal de gran potencial terapéutico.

Molecular analysis of Vitex species using candidate DNA barcoding and PCR-RFLP of the matK gene for authentication of Vitex glabrata.

Several species of the Vitex genus, family Lamiaceae, are used in folk medicine for a variety of remedies. V. glabrata is unique among Vitex species because its main effect is sexual enhancement. However, crude drugs derived from different Vitex species might not be easily distinguishable, which could lead to their misidentification and misuse. Therefore, the accurate authentication of V. glabrata is critical for its effective medicinal use. In this study, the matK gene and the psbA-trnH intergenic spacer candidate DNA barcodes were sequenced and analyzed to identify five different Vitex species that are medicinally used in Thailand: V. negundo, V. trifolia, V. rotundifolia, V. limonifolia, and V. glabrata. Each region was successfully amplified from the leaves of the five species using a single set of primers, and the sequences determined. The size difference in PCR products of psbA-trnH and PCR restriction fragment length polymorphism (PCR-RFLP) of the matK gene sequences were used to differentiate V. glabrata from other Vitex species. These results indicate both the matK gene and the psbA-trnH intergenic spacer as candidate DNA barcodes of Vitex species and suggest that the difference of psbA-trnH PCR products and PCR-RFLP analysis based on the matK gene are effective for the authentication of V. glabrata.

[Genetic diversity analysis of Vitex trifolia var. simplicifolia populations with inter-simple sequence repeats (ISSR) technique].

OBJECTIVE: To investigate 4 populations of 80 samples of Vitex trifolia var. simplicifolia collected from Shandong and Jiangxi province and analyze their intraspecies genetic variance. METHOD: Inter-simple sequence repeat (ISSR) technique was applied for the study. RESULT: Fifteen specific and stable primers were selected from 100 primers. A total of 129 sites were generated, and 115 of them (89.15%) were polymorphic. The data analyzed by PopGene demonstrated that the average polymorphic site percentage among the four populations was 71.89%. The average Shannon's information index was 0. 220 4. According to cluster analysis and the law of geographic variation, the populations were classified into two large groups: the Shandong group and the Jiangxi group. CONCLUSION: These results will provide the information for protection and utilization of V. trifolia var. simplicifolia and also further data for the study of genetic variation and species differentiation of V. trifolia var. simplicifolia.

Differential larvicidal efficacy of four species of Vitex against Culex quinquefasciatus larvae.

The early fourth instar larvae of Culex quinquefasciatus, reared in the laboratory were used for larvicidal assay with leaf extracts of Vitex negundo, Vitex trifolia, Vitex peduncularis and Vitex altissima. The methanol extracts of the four species possessed varying levels of larvicidal nature. The highest larvicidal activity was found with the extract of V. trifolia (LC(50) = 41.41 ppm) followed by V. peduncularis (LC(50) = 76.28 ppm), V. altissima (LC(50) = 128.04 ppm) and V. negundo (LC(50) = 212.57 ppm).