Total lignans from Vitex negundo seeds attenuate osteoarthritis and their main component vitedoin A alleviates osteoclast differentiation by suppressing ERK/NFATc1 signaling.

The seeds of Vitex negundo have been used for inflammation-related disease treatment in traditional medicine. This study focused on the anti-osteoarthritis (OA) effects of the total lignans of V. negundo seeds (TOV) in monosodium iodoacetate-induced osteoarthritis rats and its pharmacokinetic properties, as well as the effects and potential mechanism of its main components VN1 (6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydro-xymethyl-7-methoxy-3,4-dihydro-2-naphthaldehydeb) and VN2 (vitedoin A) on receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages (BMMs). TOV significantly attenuated osteoarthritis, leading to an increase in pain thresholds, improvement of knee articular cartilages and chondrocytes loss, and decreased total joint scores and serum levels of TNF-α, interleukin-1ß (IL-1ß), and prostaglandin E2 (PGE2) in osteoarthritis rats. The half-time (T1/2 ) was 2.82 h and 1.33 h, and the bioavailability was 15.34%-21.89% and 16.29%-22.11%, for VN1 and VN2, respectively. VN2, rather than VN1, remarkably inhibited tartrate-resistant acid phosphatase (TRAP) activity, reduced the number of TRAP-positive multinuclear cells, diminished the formation of actin ring, and decreased mRNA levels of cathepsin K (CTSK), TRAP, nuclear factor of activated T cell 1 (NFATc1), and osteoclast-associated receptor, as well as downregulated protein levels of p-ERK (phosphorylated extracellular signal-regulated kinase), TRAP, CTSK and NFATc1 in BMMs. These findings suggest TOV has promising therapeutic potential for OA treatment and VN2, in particular, attenuates osteoclast differentiation by suppressing ERK/NFATc1 signaling and actin ring, mainly accounting for the anti-OA efficacy of TOV.

Vitex trifolia L. modulates inflammatory mediators via down-regulation of the NF-κB signaling pathway in carrageenan-induced acute inflammation in experimental rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Vitex trifolia L. (V. trifolia L.), commonly known as the three-leaved chaste tree, is extensively employed in traditional Chinese medicine (TCM) to treat various conditions associated with inflammation. AIM OF THE STUDY: The present study aimed to delineate the molecular mechanisms responsible for the anti-inflammatory effect of V. trifolia L. in carrageenan (CA)-induced acute inflammation in experimental rats. MATERIALS AND METHODS: CA-induced rat paw edema model was adopted to investigate the anti-inflammatory effect of methanolic extract from leaves of V. trifolia L. (VTME) in vivo. Leukocyte infiltration into the site of inflammation was determined by histopathological analysis. Further, the effect of VTME on CA-induced local and systemic levels of specific cytokines was quantified by enzyme-linked immunosorbent assay (ELISA). Moreover, its impact on the nuclear translocation of nuclear factor Kappa B (NF-κB) was analyzed by employing the western blotting technique. RESULTS: VTME at the doses of 100 mg/kg and 200 mg/kg significantly inhibited the paw edema induced by CA (p < 0.05) and effectively reduced the inflammatory leukocyte infiltration. Further, VTME markedly inhibited the CA-induced levels of Interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α in tissue, and that of cytokine-induced neutrophil chemoattractant (CINC)-2/C-X-C motif chemokine (CXCL)3 and CINC-3/CXCL2 in tissue as well as in serum. On the other hand, VTME significantly upregulated the tissue concentration of anti-inflammatory cytokine IL-10. Moreover, VTME significantly attenuated the CA-induced IκBα degradation and nuclear translocation of NF-κB p65. CONCLUSIONS: Our results demonstrate the potent anti-inflammatory effect of V. trifolia L. in vivo, providing insight into its molecular mechanism, which is mediated through down-regulation of NF-κB signal transduction.

Vitex negundo L. derived specialized molecules unveil the multi-targeted therapeutic avenues against COPD: a systems pharmacology approach.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is an inflammatory disease caused by increasing breathing passage obstruction which completely disrupts human homeostasis. Some patients require lung transplantation or long-term oxygen therapy. COPD is one of the noxious diseases and its fourth leading cause of death around the globe. There is an immediate need for potential drug development to tackle this serious disease. Folk medicines are used to combat complex diseases that have shown effectiveness in the treatment of breathing diseases. Vitex negundo L. is an ethnobotanically important medicinal plant used for various ailments and modulates human cellular events. This shrub has diverse specialized metabolites and is being used as complementary medicine in various countries. Though systems-level understanding is there on the mode of action, the multi-target treatment strategy for COPD is still a bottleneck. METHODS: In this investigation, systems pharmacology, cheminformatics, and molecular docking analyses were performed to unravel the multi-targeted mechanisms of V. negundo L. potential bioactives to combat COPD. RESULTS: Cheminformatics analysis combined with the target mining process identified 86 specialized metabolites and their corresponding 1300 direct human receptors, which were further imputed and validated systematically. Furthermore, molecular docking approaches were employed to evaluate the potential activity of identified potential compounds. In addition, pharmacological features of these bioactives were compared with available COPD drugs to recognize potential compounds that were found to be more efficacious with higher bioactive scores. CONCLUSIONS: The present study unravels the druggable targets and identifies the bioactive compounds present in V. negundo L., that may be utilized for potential treatment against COPD. However, further in vivo analyses and clinical trials of these molecules are essential to deciphering their efficacy.

Discovery of a "Cocktail" of Potential SARS-COV-2 Main Protease Inhibitors through Virtual Screening of Known Chemical Components of Vitex negundo L. ("Lagundi").

AIM: The prevailing crisis caused by COVID-19 pandemic demands the development of effective therapeutic agents that can be implemented with minimal to zero adverse effects. BACKGROUND: Vitex negundo L. (VNL) is a medicinal plant with reported efficacy against respiratory diseases and some of the COVID-19 symptoms. Funded by the Department of Science and Technology (DOST), the University of the Philippines - Philippine General Hospital (UP-PGH) is currently conducting clinical trials of VNL and other medicinal plants as adjuvant therapeutic agents against mild cases of COVID-19. The basis for the clinical trials is primarily the pharmacological efficacy of the medicinal plants against respiratory disorders and associated COVID-19 symptoms. OBJECTIVE: This study assessed the in silico potential of VNL components against SARS-CoV-2 main protease (Mpro), an enzyme that plays an important role in COVID-19, the disease caused by the SARS-CoV-2. METHOD: Phytochemical mining of VNL components from the literature was conducted. A database consisting of 250 known compounds from different parts of VNL was created and screened against SARS-CoV-2 Mpro using PyRx virtual screening tool. The most promising components were further subjected to in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses using the SwissADME web server and Toxtree software. RESULTS: Virtual screening revealed that 102 VNL components in the database had comparable to or better binding affinities toward SARS-COV-2 Mpro than known chemical inhibitors (e.g. N3 and carmofur). It was determined that the active site of SARS-CoV-2 Mpro receptor consists of multiple H-donor and acceptor sites; hence, the most stable receptor-ligand complexes are generally formed by VNL ligands that establish effective H-bonding with the SARS-CoV-2 Mpro. The promising components, representing a "cocktail" of potential inhibitors also revealed interesting ADMET properties. CONCLUSION: This in silico study identified VNL as a potential single source of a cocktail of SARSCoV- 2 Mpro inhibitors and a promising adjuvant therapeutic agent against COVID-19 or its symptoms. Furthermore, the study offers a rationale on phytochemical mining from medicinal plants as a means that can be implemented in the early stage of a drug discovery and development program.

Evaluation of ethnopharmacologically selected Vitex negundo L. for In vitro antimalarial activity and secondary metabolite profiling.

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.

Biosynthesis of gold nanoparticles using Vetex negundo and evaluation of pro-apoptotic effect on human gastric cancer cell lines.

Gastric cancer (GC) is mainly widespread gastrointestinal malignancy,which reports for 8% of overallcases in carcinogenesis and 10% of yearly fatality, is 4thprimary cause of cancer associated death global. The plan of the present research was to develop ethanolic extract of Vitex negundo-loaded gold nanoparticles (VN-AuNPs) and to appraise the various characteristic methods likes UV-vis spectroscopy, SAED, FTIR, XRD and HR-TEM. Additionally, the anticancer effect of VN-AuNPs on AGS cells were analysed by cell viability, apoptotic morphological changes by TUNEL, AO/EtBr and Hoechst staining, alterations of mitochondrial membrane potential (MMP) and production reactive oxygen species (ROS). Moreover, the status of apoptosis gene such as caspase-3, Bcl-2, Bcl-XL, Bax and caspase-9 expressions was analysed by using western and RT-PCR techniques. Synthesized AuNPs established by UV absorption peak of the highest at 538 and crystal nature of AuNPs was additionallyverifiedwith SAED and XRD. TEM images were illustrates size and morphological division of NPs. FTIR examinationscompletedalkene, carbodiimide and aliphatic primary amines of biomolecules werepresent in synthesized VN-AuNPs. Additionally, AuNPs were stimulatedapoptosis throughthe cytotoxicity effect,changes of MMP, generation of ROS, nuclear and apoptotic morphological alterationsvia TUNEL, AO/EtBr and Hoechst assay. Furthermore, molecular mechanisms also provoked apoptosis through modulating pro (caspase-3, Bax, Bid, caspase-9) and anti-apoptotic (Bcl-2 and Bcl-XL) mediators by western blotting and gene expression in AGS cells. This production of AuNPs from VN was eco-friendly, large-scaled up and easy.

Quality Assessment of Medicinal Product and Dietary Supplements Containing Vitex agnus-castus by HPLC Fingerprint and Quantitative Analyses.

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.

Inter-comparison of stable isotope mixing models for determining plant water source partitioning.

Water sources used for plant identification coupled with stable isotopes are essential to improving the understanding of eco-hydrological processes and ecological management in water-limited ecosystems. Many approaches associated with stable isotopes have been used to determine plant water source apportionment. However, inter-comparisons of different methods are still limited, especially for Bayesian mixing models. In this study, we tested linear mixing models (IsoSource) and Bayesian models (SIAR, MixSIR and MixSIAR) to identify sources of water absorbed by Vitex negundo and Sophora viciifolia (shrubs) and Artemisia gmelinii (subshrub) during the growing season in the semiarid Loess Plateau. The results showed that there was no significant difference in the predicted plant water source fractions using only stable hydrogen isotope (δ2H) and only stable oxygen isotope (δ18O) with the IsoSource model. No significant difference was found in plant water source apportionment by the three Bayesian mixing models combined with δ2H and δ18O except for individual months. The SIAR and MixSIAR models detected no pronounced seasonal variations in plant water uptake, while the MixSIR model did detect seasonal variations. Overall, the SIAR and MixSIAR models exhibited relatively better water source apportionment performances than that of the MixSIR model. This discrepancy may be attributed to the difference in the post distribution simulation algorithm. This study provides critical insights into choosing a suitable method for identifying plant water source apportionment in arid and semiarid regions.

Vitex negundo and its medicinal value.

Natural products are rich in several potent bioactive compounds, targeting complex network of proteins involved in various diseases. Vitex negundo (VN), commonly known as "chaste tree", is an ethnobotanically important plant with enormous medicinal properties. Different species of Vitex vary in chemical composition, thus producing different phytochemicals. Several bioactive compounds have been extracted from leaves, seeds, roots in form of volatile oils, flavonoids, lignans, iridoids, terpenes, and steroids. These bioactive compounds exhibit anti-inflammatory, antioxidant, antidiabetic, anticancer, antimicrobial. VN is typically known for its role in the modulation of cellular events like apoptosis, cell cycle, motility of sperms, polycystic ovary disease, and menstrual cycle. VN, reportedly, perturbs many cancer-signaling pathways involving p-p38, p-ERK1/2, and p-JNK in LPS-elicited cells, N-terminal kinase (JNK), COX-1 pathways, MAPK, NF-κB, tumor necrosis factor α (TNF-α), Akt, mTOR, vascular endothelial growth factor, hypoxia-inducible factor (HIF-1α). Several bioactive compounds obtained from VN have been commercialized and others are under investigation. This is the first review presenting up-to-date information about the VN, its bioactive constituents and their mode of action.

Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus.

Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species.

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures.

The majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.

Antibacterial behaviour of Vitex negundo extract assisted ZnO nanoparticles against pathogenic bacteria.

The biological routes are advantageous over the chemical and physical ones as unlike. These, the biological synthesis protocol occurs at ambient conditions, are cheap, non-toxic and eco-friendly. This research describes the synthesis of zinc oxide nanoparticles (ZnO NPs) using Vitex negundo plant extract with zinc nitrate hexahydrate as precursor. Biomolecules present in plant extract can be used to hydrolyze metal ions into metal oxide NPs in a single-step green synthesis. The hydrolyzing agents involved the various water soluble plant metabolites such as flavonoid, alkaloids, flavone, phenolic compounds, terpenoids and co-enzymes. Presence of isoorientin (flavone) in V. negundo plant extract is mainly responsible for the formation of ZnO NPs. The prepared ZnO NPs were calcinated at 450°C and were confirmed by XRD, FT-IR, UV-visible, SEM with EDX and DLS analysis. The biological application of antibacterial activity was done by gram positive and gram negative bacteria.