Labdane Diterpenoids Research Articles

Andrographolide Inhibits Corneal Fibroblast to Myofibroblast Differentiation In Vitro.

Corneal opacification due to fibrosis is a leading cause of blindness worldwide. Fibrosis occurs from many causes including trauma, photorefractive surgery, microbial keratitis (infection of the cornea), and chemical burns, yet there is a paucity of therapeutics to prevent or treat corneal fibrosis. This study aimed to determine if andrographolide, a labdane diterpenoid found in Andrographis paniculate, has anti-fibrotic properties. Furthermore, we evaluated if andrographolide could prevent the differentiation of fibroblasts to myofibroblasts in vitro, given that the transforming growth factor beta-1(TGF-β1) stimulated persistence of myofibroblasts in the cornea is a primary component of fibrosis. We demonstrated that andrographolide inhibited the upregulation of alpha smooth muscle actin (αSMA) mRNA and protein in rabbit corneal fibroblasts (RCFs), thus, demonstrating a reduction in the transdifferentiation of myofibroblasts. Immunofluorescent staining of TGF-β1-stimulated RCFs confirmed a dose-dependent decrease in αSMA expression when treated with andrographolide. Additionally, andrographolide was well tolerated in vivo and had no impact on corneal epithelialization in a rat debridement model. These data support future studies investigating the use of andrographolide as an anti-fibrotic in corneal wound healing.

A New Investigation into the Molecular Mechanism of Andrographolide towards Reducing Cytokine Storm.

Cytokine storm is a condition in which the immune system produces an excessive number of inflammatory signals, which can result in organ failure and death. It is also known as cytokine release syndrome, CRS, or simply cytokine storm, and it has received a lot of attention recently because of the COVID-19 pandemic. It appears to be one of the reasons why some people experience life-threatening symptoms from COVID-19, a medical condition induced by SARS-CoV-2 infection. In situations where natural substances can be exploited as therapeutics to reduce cytokine storm, the drug development process has come to the rescue. In the present study, we tested the potentiality of Andrographolide, labdane diterpenoid targeting several key cytokines that are secreted as a result of cytokine storm. We used molecular docking analyses, molecular dynamics simulations, and pharmacokinetic properties to test the stability of the complexes. The compound’s binding energy with some cytokines was over −6.5 Kcal/mol. Furthermore, a post-molecular dynamics (MD) study revealed that Andrographolide was extremely stable with these cytokines. The compound’s pharmacokinetic measurements demonstrated excellent properties in terms of adsorption, distribution, metabolism, and excretion. Our research revealed that this compound may be effective in lowering cytokine storm and treating severe symptoms.

Diterpenoids with α-glucosidase inhibitory activities from the fruits of Vitex trifolia Linn

Viticis Fructus, known as “Man-jing-zi”, are the fruits of the traditional Chinese medicine Vitex trifolia Linn. and its variant Vitex trifolia Linn. var. simplicifolia. These fruits are used as folk medicines to treat various diseases. Although V. trifolia is useful for treating diabetes, the antidiabetic effect of its purified constituents is still under investigation. The phytochemical investigation on the ethanol extract of the fruits of V. trifolia yielded four new labdane diterpenoids vitetrolins A-D (1–4), together with seven (5–11) known analogs. The structures of these compounds were elucidated by spectroscopy techniques and the absolute configuration of 4 was determined by electronic circular dichroism (ECD) calculations. The isolated diterpenoids were evaluated for their α-glucosidase inhibitory activities. Compounds 5, 6, 8, and 9 exhibited moderate inhibitory activities against α-glucosidase with IC50 values ranging from 44.9 ± 6.1 to 70.5 ± 5.5 μM.

Abstract 2343: Andrographolide treatment results in decreased mtDNA damage and loss of the mitochondrial membrane potential while increasing ROS production in prostate cancer

Abstract Introduction: Prostate Cancer (PCa) is the second most diagnosed malignancy and the fifth leading cause of cancer deaths in men worldwide. Andrographolide, a labdane diterpenoid that is the main bioactive component of the medicinal plant Andrographis paniculata, has shown a wide range of therapeutic potential against many factors including cancer. Previous studies in our laboratory showed that Andrographolide induces expression of DNA repair genes associated with double-strand break repair, cell growth suppression, cell cycle arrest, and apoptosis in PCa in vitro. Andrographolide also showed an altered ATP production ratio between mitochondrial-ATP and glycolysis-ATP production in PC3 and 22RV1 cell lines treated with Andrographolide. However, the mechanism of action of Andrographolide in prostate cancer is not completely known. The objective of this study is to determine the role of Andrographolide in mtDNA damage, mitochondrial ROS production, and mitochondrial membrane potential in prostate cancer cell lines. Methods: Prostate cancer cell lines (PC3 and 22Rv1) were treated with Andrographolide (25μM) for 24 and 48 hours. DNA isolated from treated and control cells was used to detect mtDNA damage using a PCR-based method. To evaluate ROS production, cells were incubated with MitoSOX and fluorescence detected using fluorescence microscopy. Membrane potential status was also determined with fluorescence microscopy, using the dye JC-1. Results: Detection of mtDNA damage in PC3 cells treated with Andrographolide for 48 hours and in 22Rv1 cells treated with Andrographolide for 24 and 48 hours, show decreased mtDNA lesions in treated cells. Evaluation of mitochondrial membrane potential shows membrane depolarization in 22Rv1 cells treated with Andrographolide. In addition, mitochondrial ROS production shows a 1.4-fold increase in 22Rv1 cells treated with Andrographolide for 24 hours. Conclusion: This study shows that Andrographolide disrupts mitochondrial function in PC3 and 22Rv1 PCa cells, which in turn may trigger a metabolic phenotype leading to cell death PCa. This research could provide new insights into the role of Andrographolide in PCa altering bioenergetics and mitochondrial function, making Andrographolide a promising therapeutic candidate as a metabolic modulator and a chemo preventive agent in prostate cancer. Citation Format: Yarelis M. Roque-Reyes, Jesus Sosa-Rivera, María M. Sánchez-Vázquez, Sylvette Ayala-Peña, Carlos A. Torres-Ramos, Magaly Martínez-Ferrer. Andrographolide treatment results in decreased mtDNA damage and loss of the mitochondrial membrane potential while increasing ROS production in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2343.

Callnudoids A–H: Highly modified labdane diterpenoids with anti-inflammation from the leaves of Callicarpa nudiflora

Eight undescribed 3,4-seco-norlabdane diterpenoids, callnudoids A–H, as well as two known analogues were isolated from the leaves of Callicarpa nudiflora. The structures were elucidated using spectroscopic methods and were compared with published NMR spectroscopic data. The absolute configurations of callnudoids D and E were defined based on ECD data or single-crystal X-ray diffraction. Callnudoids A–C are the highly modified labdane diterpenoids featuring rearranged 3,4-seco-ring and the formation of an undescribed cyclohexene moiety via C2–C18 cyclization. They only contain 15 carbon atoms on the carbon skeleton. Callnudoid D represents the unusual 3,4-seco-15,16-norlabdane diterpenoid with C13–C17 cyclization, and a putative biosynthesis pathway for callnudoids A, B, D, and E was proposed. All compounds were evaluated for their anti-inflammatory activities by inhibiting the lipopolysaccharide (LPS)-induced nitric oxide (NO) released in RAW264.7 cells; callnudoids A–E and H, and methylcallicarpate obviously inhibited pro-inflammatory cytokines TNF-α and IL-1β in a dose-dependent manner.

Andrographolide‐Mediated Protection of Barrier Integrity and Permeability is Mediated Through NLRP3 Inflammasome Inhibition in Blood‐Brain Barrier Endothelial Cells

The blood‐brain barrier (BBB) that consists of the inter‐endothelial tight junctions, acts as the major blood‐brain interface and regulator of brain microvascular hyperpermeability. In the brain, the loss of BBB integrity and hyperpermeability leads to cerebral edema formation and intracranial pressure elevation in traumatic brain injury and ischemic strokes. Recent studies demonstrated that reactive oxygen species are potential signals for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation that leads to interleukin‐1β (IL‐1β) secretion. IL‐1β plays a major role in promoting BBB hyperpermeability in various brain pathologies, including TBI. Furthermore, recent studies from our lab have demonstrated a tri‐phasic role for hydrogen peroxide (H2O2) in BBB endothelial cells where it promotes, angiogenesis, hyperpermeability and apoptosis in a tri‐phasic and concentration dependent manner. Hydrogen peroxide plays a major role physiologically as the second messenger and pathologically as an inducer of oxidative stress and associated cellular signaling. We hypothesized that in BBB endothelial cells, H2O2‐induced barrier dysfunction and hyperpermeability are NLRP3 inflammasome‐dependent. The main objective of our study was to evaluate the role of NLRP3 inflammasome signaling in H2O2‐mediated barrier dysfunction and hyperpermeability in human brain microvascular endothelial cells and to test whether andrographolide, a labdane diterpenoid possessing potent anti‐inflammatory properties will attenuate such effects. Th effect of H2O2 treatment on tight junction integrity/permeability was studied using zonula occludens‐1 (ZO‐1) immunofluorescence localization, ZO‐1 immunoblot analysis, Transwell monolayer permeability assay using FITC‐dextran (10‐kDa) as a fluorescent marker and cell viability/apoptosis assay. The effect of NLRP3 inhibitor (MCC950) and andrographolide on H2O2‐induced hyperpermeability was studied using Transwell permeability assay. Our results show that, CRISPR/Cas‐9‐mediated knockdown of ZO‐1 resulted in monolayer hyperpermeability demonstrating the significance of ZO‐1 in regulating barrier functions. CRISPR‐based activation of NLRP3 induced monolayer hyperpermeability whereas CRISPR/Cas‐9‐based NLRP3 knockdown had no significant effect on permeability. H2O2 (10µM) induced monolayer hyperpermeability and the effect was decreased by MCC950 and andrographolide treatment. Hydrogen peroxide induced cathepsin B (an activator of the NLRP3 pathway) activity significantly, and the effect was decreased by andrographolide treatment. H2O2‐mediated barrier dysfunction and hyperpermeability were not due to changes in cell viability/apoptosis. These results suggest that NLRP3 is a mediator of H2O2‐induced barrier dysfunction and hyperpermeability in human BBB endothelial cells and andrographolide is an effective inhibitor of this pathway.

Inhibitory Effect of Andrographis paniculata Lactone on Staphylococcus aureus α-Hemolysin.

We investigated the effect of andrographolide (AP) on the hemolytic capacity of Staphylococcus aureus (S. aureus) isolated from our region. AP is a labdane diterpenoid isolated from the stem and leaves of Andrographis paniculata. The hla gene from 234 S. aureus strains and the quality control standard strain ATCC29213 in dairy cows in some areas of Ningxia was analyzed. Evolutionary analysis, homology modeling, and functional enrichment annotation of α-hemolysin Hla detected from our region were performed through bioinformatics. The hemolytic ability of S. aureus isolates from the region was examined using the hemolysis test, and the effect of AP on S. aureus was quantified. Moreover, the effect of AP on the transcript levels of hla and genes highly related to hla (i.e., clfA and fnbA) was examined through fluorescence quantitative PCR. The mode of action of AP on the detected Hla was analyzed through molecular docking and dynamic simulation. The results showed that S. aureus in our region has a high rate of hla carriage. The hemolytic activity of strains NM98 and XF10 was significant, and ATCC29213 also exhibited some hemolytic activity. AP could inhibit the expression of Hla and its related proteins by downregulating hla, clfA, and fnbA transcript levels, which in turn attenuated the S. aureus hemolytic activity. Meanwhile, the AP molecule can form three hydrogen bonds with residues ASN105, SER106, and THR155 of Hla protein; bind with PRO103 through alkyl intermolecular forces; and form carbon hydrogen bonds with LYS154, reflecting that the AP molecule has a comparatively ideal theoretical binding activity with Hla protein. Among them, PRO103 and LYS154 are highly conserved in Hla protein molecules and play pivotal roles in the biological functions of Hla, and their binding may affect these functions. Their binding may also prevent the conformational transition of Hla from a monomer to an oligomer, thus inhibiting Hla hemolytic activity. This study offers a molecular basis for use of AP as an antivirulence drug and new ideas for developing novel drugs against S. aureus infection.

Hypopurolides A - G, Labdane Diterpenoids from Hypoestes purpurea and Their Nitric Oxide Inhibitory Activity.

Seven new labdane diterpenoids, hypopurolides A-G (1-7) were discovered from the aerial part of Hypoestes purpurea, along with one known analog, hypopurin D (8). The structures of 1-7 were characterized based on 1 H-, 13 C-, and 2D-NMR, and HR-ESI-MS spectra. The absolute configurations of 1-7 were defined by single-crystal X-ray diffraction and electronic circular dichroism (ECD) data. Compounds 1-8 were tested for their nitric oxide (NO) inhibitory and cytotoxic effects. Compound 6 displayed moderate inhibitory effect toward LPS-induced NO release in RAW 264.7 cells with an IC50 value of 41.50 μM.

Secondary metabolites from Detarium microcarpum Guill. and Perr. (Fabaceae).

The chemical investigation of the ethanol/water (7:3) extract of the roots of Detarium microcarpum (Fabaceae) led to the isolation of one new labdane diterpenoid, microcarpin (1) and one new ceramide derivative, microcarpamide (2), along with eight known secondary metabolites (3-10)including, 5-(carboxymethyl)-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid (3), microcarposide (4), rhinocerotinoic acid (5), 1,7-dihydroxy-6-methylxanthone (6), ursolic acid (7), 3β,23-dihydroxylup-20(29)-en-28-oic acid (8), alphitolic acid (9), and stigmasterol glucoside (10). The structures of these compounds were elucidated based on their spectroscopic data. Although compounds 3 and 4 are known, their crystalline structures are reported here for the first time. These compounds were evaluated in vitro for their antisalmonella activity. The results obtained showed that, microcarpamide (2), microcarposide (4), and rhinocerotinoic acid (5) were moderately active against three salmonella strains: Salmonella typhi, Salmonella enteritidis and Salmonella typhimirium, with minimum inhibition concentration values of 76.7 and 153.5μM.

Pimarane, abietane, and labdane diterpenoids from Euphorbia pekinensis Rupr. and their anti-tumor activities

Chemical investigation of the roots of Euphorbia pekinensis Rupr. led to the isolation of five undescribed labdane diterpenoids “(4S, 5S, 9R, 10S, 13R)-18-O-galloyl-labda-8(17), 14(15)-dien-13-ol; (4S, 5S, 9R, 10S, 13R)-13-hydroxy-labda-8(17), 14(15)-dien-18-one; (4S, 5S, 9R, 10S, 13R)-18-O-acetyl-labda-8(17), 14(15)-dien-13-ol; (4S, 5S, 9R, 10S)-labda-8(17), 13(16), 14(15)-trien-18-ol; (5R, 6R, 9R, 10S, 13R)-labda-8(17), 14(15)-dien-6,13-diol”, two undescribed pimarane diterpenoids “(2R, 5S, 9R, 10S, 12R, 13R)-2,12-dihydroxy-isopimara-7,15-dien-3-one; (5S, 9R, 10S, 12R, 13R)-2, 12-dihydroxy-isopimara-1, 7, 15-trien-3-one)”, together with nine known diterpenoids, including three pimarane-type “(3β,11α,13α)-3,11-dihydroxypimara-7,15-diene-2,12-dione; (11R, 12S)-2,11,12-trihydroxy-ent-isopimara-1,7,15-trien-3-one; isopimara-7,15-dien-3β-ol)”, five abietane-type “helioscopinolide A-C; helioscopinolide E; helioscopinolide I″, and one lathyrane-type “jolkinol B”. The structures of these compounds were elucidated by analysis of HRESIMS, 1D NMR, 2D NMR, and X-ray diffraction. These sixteen compounds were evaluated for cytotoxic activity in vitro against three human cancer cell lines, U-937, LOVO, and K-562. Jolkinol B exhibited IC50 of 3.60 μM and 8.44 μM against U-937 and LOVO cell lines, (4S, 5S, 9R, 10S, 13R)-18-O-galloyl-labda-8(17), 14(15)-dien-13-ol displayed IC50 of 5.92 μM against U-937 cell lines, isopimara-7,15-dien-3β-ol showed IC50 of 0.87 μM against K-562 cell lines.

Two new diterpenoids from the rhizomes of Zingiber officinale

Two undescribed labdane diterpenoids (5S,8S,9R,10S,11E)-8,17-epoxy-13,14-dinorlabd-11-en-13-one (1) and (5S,9R,10S,12E)-17-hydroxy-labd-7,12-dien-15(16)-olide (2), together with seven known sesquiterpenoids (3–9) and two known monoterpenoids (10–11) were isolated from the dried rhizome of Zingiber officinale. Their structures were elucidated by detailed spectroscopic data (IR, UV, HR-ESI-MS, 1D and 2D NMR), X-ray crystallographic and ECD analysis. Moreover, all the 11 compounds were tested for α-glucosidase inhibitory effects and 9 was found to exhibit stronger inhibitory effects at IC50 = 4.8 μM against a positive control acarbose with IC50 = 414.6 μM.

In vitro antibacterial activity of andrographolide and hordenine against methicillin-resistant Staphylococcus aureus and Staphylococcus aureus

Bacterial infections are difficult to treat, especially those infected with resistant bacteria. Due to the haphazard use of antibiotics in humans and animals, methicillin-resistant Staphylococcus aureus (MRSA) has developed resistance to practically all commercially available antibacterial drugs. Exploring natural plant compounds with antibacterial activity could be beneficial in the treatment of infections with MRSA and Staphylococcus aureus. Andrographolide, a labdane diterpenoid derived from Andrographis paniculata, and Hordenine, a phenethylamine alkaloid found in Hordeum vulgare, are reported to have antibacterial activity. In the present study, the antibacterial activity of andrographolide and hordenine was tested against MRSA and S. aureus using the Kirby- Bauer agar disc diffusion method and the modified resazurin microtiter plate assay. The results of the study revealed that both andrographolide and hordenine at various concentrations did not show any zone of inhibition against MRSA and S. aureus. Hordenine showed an MIC of 1000 μg/mL against MRSA and S. aureus, but andrographolide had no effect on both MRSA and S. aureus.

LCMS-DNP based dereplication of Araucaria cunninghamii Mudie gum-resin: identification of new cytotoxic labdane diterpene

As a part of natural defense, plants initiate the secretion of gum containing numerous pharmacologically active essential metabolites. A fraction of such gum-resin from Araucaria cunninghamii Mudie, when screened against human cancer cell lines, was found to be active. Further, it was subjected to an LCMS-DNP (Dictionary of Natural Products) based dereplication study followed by a detailed phytochemical investigation to obtain pure metabolites. Also, the gum resin of A. cunninghamii was found to be a rich source of abietanes and labdanes. The LCMS-DNP-based dereplication study identified many known metabolites, which were isolated for the first time from this plant as well as a new labdane diterpenoid (9). The compounds were characterized via spectroscopic techniques, which were subsequently compared with the already existing literature data. The metabolites were screened against seven human cancer cell lines. The anticancer activity was further supported by molecular docking studies.

Labdane and isopimarane diterpenoids from Rosmarinus officinalis solid wastes: MS/MS spectrometric fragmentations and neuroprotective effect

The aim of this study was to purify and identify new types of secondary products from Rosmarinus officinalis solid wastes after essential oil been extracted, further study their MS/MS spectrometric fragmentations and evaluate their cytoprotective effects against H2O2-induced oxidative damage in SH-SY5Y cells. Six labdane diterpenoids, five isopimarane diterpenoids and two ferulic acid esters were obtained in which three labdane diterpenoids and four isopimarane diterpenoids were undescribed compounds. The losses of hydroxyl, tigloyl, 2-methy-butanoyl, acetyl and carbonyl groups or side chains were the main elimination modes of the new compounds in MS/MS analyses. Compounds 1, 4, 6, 10 and 12 showed the ability to protect the cells against oxidative injure whose cell viability rates were more than 65% in the concentration of 1 or 25 µM respectively. These studies broadened the understanding of secondary products of rosemary and promoted the deep developments and utilizations of rosemary after essential oil been extracted.

Forskolin-Loaded Halloysite Nanotubes as Osteoconductive Additive for the Biopolymer Tissue Engineering Scaffolds.

Here we report the use of forskolin-modified halloysite nanotubes (HNTs) as a dopant for biopolymer porous hydrogel scaffolds to impart osteoinductive properties. Forskolin is a labdane diterpenoid isolated from the Indian Coleus plant. This small molecule is widely used as a supplement in molecular biology for cell differentiation. It has been reported in some earlier publications that forskolin can activate osteodifferentiation process by cyclic adenosine monophosphate (c-AMP) signalling activation in stem cells. In presented study it was demonstrated that forskolin release from halloysite-doped scaffolds induced the osteodifferentiation of equine mesenchymal stem cells (MSCs) in vitro without addition of any specific growth factors. The reinforcement of mechanical properties of cells and intercellular space during the osteodifferentiation was demonstrated using atomic force microscopy (AFM). These clay-doped scaffolds may find applications to accelerate the regeneration of horse bone defects by inducing the processes of osteodifferentiation of endogenous MSCs.

Labdane Diterpenoids from Salvia tingitana Etl. Synergize with Clindamycin against Methicillin-Resistant Staphylococcus aureus.

Quorum-sensing (QS) is a regulatory mechanism in bacterial communication, important for pathogenesis control. The search for small molecules active as quorum-sensing inhibitors (QSI) that can synergize with antibiotics is considered a good strategy to counteract the problem of antibiotic resistance. Here the antimicrobial labdane diterpenoids sclareol (1) and manool (2) extracted from Salvia tingitana were considered as potential QSI against methicillin-resistant Staphylococcus aureus. Only sclareol showed synergistic activity with clindamycin. The quantification of these compounds by LC–MS analysis in the organs and in the calli of S. tingitana showed that sclareol is most abundant in the flower spikes and is produced by calli, while manool is the major labdane of the roots, and is abundant also in the leaves. Other metabolites of the roots were abietane diterpenoids, common in Salvia species, and pentacyclic triterpenoids, bearing a γ-lactone moiety, previously undescribed in Salvia. Docking simulations suggested that 1 and 2 bind to key residues, involved in direct interactions with DNA. They may prevent accessory gene regulator A (AgrA) binding to DNA or AgrA activation upon phosphorylation, to suppress virulence factor expression. The antimicrobial activity of these two compounds probably achieves preventing upregulation of the accessory gene regulator (agr)-regulated genes.

Studies on the Syntheses of Bioactive Natural Products Having a Fused Ring System Based on Novel Skeletal Construction Methods

Here, I describe a part of our efforts to develop synthetic strategies to construct bioactive natural products having a fused ring system. We have designed four chiral building blocks bearing contiguous quaternary stereocenters for the syntheses of bioactive C17-oxygenated steroids/triterpenoids and C9-oxygenated labdane diterpenoids. The compounds were stereoselectively synthesized from α-substituted glycolic acid (R)-3-methylcyclohex-2-enyl esters through Ireland-Claisen rearrangement to construct the stereocenters simultaneously. Synthetic utility of a β-type building block is highlighted by total syntheses of marrubiin (11 steps, 22%) and related seven labdane diterpene lactones, cyllenine C (12 steps, 29%), marrulactone (13 steps, 11%), marrulanic acid (14 steps, 10%), marrubasch F (12 steps, 14%), marrulibacetal (14 steps, 4%), marrulibacetal A (14 steps, 2%), and desertine (15 steps, 0.5%). These syntheses feature the construction of the [6.6.5]-tricyclic ring portion via a Pauson-Khand reaction, cleavage of the resultant cyclopentenone ring and an elongation of the C9 side chain by an epoxide opening reaction. The relative stereochemistry of desertine was determined to be 13R, 14S, 15S, 16R by some chemical conversions and NMR analysis. Further efforts toward total syntheses of oxygenated terpenoids using three other chiral building blocks and structure-activity relationship study of synthesized labdane diterpene lactones are currently underway in our laboratory and will be reported in due course.

Andrographolide Induces Apoptosis in Gastric Cancer Cells through Reactivation of p53 and Inhibition of Mdm-2.

Andrographolide is a labdane diterpenoid isolated from Andrographis paniculata. The plant extract and andrographolide has long been used in traditional medicine practices mainly for gastrointestinal diseases and improving liver function. Andrographolide has shown various pharmacological properties including anti-inflammatory, antioxidant and anticancer activity. This study evaluated the effect of andrographolide on proliferation of human gastric carcinoma cells in relevance to p53 and Mdm-2 pathways. Andrographolide inhibited the proliferation of SGC7901 and AGS cells in a dose-dependent manner with estimated IC50 values 38 and 44 μM respectively. Effect of andrographolide on p53 activity was ascertained by using a p53 activator (RITA) which showed synergistic inhibition of cell proliferation. While andrographolide when used in combination with a p53 inhibitor (pifithrin-α) showed potent restriction over its response. Andrographolide caused decrease in mitochondrial membrane potential as an indicator of apoptotic activity. Andrographolide activated the expression of p53 protein and gene and downregulated the levels of Mdm-2 (negative regulator of p53). Andrographolide inhibited the colony formation abilities in SGC7901 in a p53-dependent manner followed by induction of mitochondrial intrinsic apoptosis through activation of caspases-9 and -3, cleavage of PARP, and inhibition of pro-apoptotic Bcl-2. Andrographolide induced p53 mediated apoptosis in gastric carcinoma cells which adds to a novel approach in anticancer therapies.

Analysis of secondary metabolites present in Phlogacanthus thyrsiflorus Nees plant parts using LC-ESI-MS

Phlogacanthus thyrsiflorus Nees, a medicinal plant belonging to the family Acanthaceae, has been used by traditional healers of northeast India for medicinal values. Other genera of this family, such as Andrographis paniculata and Adhatoda vasica are used extensively for their therapeutic properties. Unlike these species, minimal scientific reports are available on the phytochemical constitution of P. thyrsiflorus. The present work provides the qualitative and quantitative estimation of pharmacologically important terpenoids and β- Sitosterol present in the plant's leaf, flower, and root. Ultra - high-performance liquid chromatography coupled with electro spray ionisation mass spectrometry in positive ionization mode was used to identify and quantitate. The identified terpeniods were three labdane diterpenoids (Andrographolide, Neoandrographolide, 14- Deoxyandrographolide), and two cyclic triterpenoids (Betulin and β- Sitosterol). Quantitation was carried out for all the identified secondary metabolites except 14- Deoxyandrographolide. Almost all the identified secondary metabolites were found in a substantial amount in different extracts. Andrographolide and Neoandrographolide were found in the highest quantity in the leaf extracts (2245.63 ± 0.07 ng ml−1 in aqueous extract and 300.72 ± 0.93 ng ml−1 in 50% methanolic extract). A significant amount of Betulin and β- Sitosterol were obtained in leaf and root extracts (55.05 ± 0.53 ng ml−1 in 100% methanolic extract and 1691.50 ± 2.72 ng ml−1in aqueous extract respectively) of P. thyrsiflorus. To our knowledge, this is the first report for quantitative estimation of the terpenoids present in P. thyrsiflorus. This report confirms the richness of pharmacologically important metabolites in this underutilized plant with potential medicinal applications.

Three labdane diterpenoids from the aerial parts of Leonurus japonicus

• Three undescribed labdane diterpenoids were obtained from Leonurus japonicus Houtt. • Additionally, eight known metabolites were also isolated from an ethyl acetate extract. • Undescribed labdane diterpenoid exhibited the strongest cytotoxic activity towards MCF-7 cells. Three undescribed labdane diterpenoids, 15,16-epoxy-13,15-dihydroxylabd-8-ene-7-one (1), 15,16-epoxy-13-hydroxy-15-methoxylabd-8-ene-7-one (2) and 15-hydroxyleoheteronin B (3), along with eight known compounds, were isolated from an ethyl acetate extract of Leonurus japonicus . Their structures were elucidated using spectroscopic methods, mainly 1-D and 2-D NMR. Compound 1 showed the strongest cytotoxic activity against MCF-7 cells with IC 50 values of 26.8 μM, using the sulforhodamine B assay.