In vitro antiaging activity of polyphenol rich Polyalthia longifolia (Annonaceae) leaf extract in Saccharomyces cerevisiae BY611 yeast cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polyalthia longifolia var. angustifolia Thw. (Annonaceae) is commonly used in traditional medicine as a tonic for rejuvenation and exhibiting good antioxidant activities. AIM OF THE STUDY: To evaluate P. longifolia methanolic leaf extract (PLME) antiaging activity at 1 mg/mL in Saccharomyces cerevisiae BY611 yeast. MATERIALS AND METHODS: The antiaging effect of PLME was studied via replicative lifespan assay, antioxidative stress assays, reactive oxygen species (ROS) determination, reduced glutathione (GSH) determination, superoxide dismutase (SOD) and Sirtuin 1 (SIRT1) genes regulation studies and SOD and SIRT1 proteins activities. RESULTS: The PLME treatment increased the growth and prolonged the lifespan of the yeast significantly (p < 0.05) compared to the untreated yeast group. Besides, the PLME also protected the yeast from oxidative stress induced by 4-mM-H2O2 via decreasing (p < 0.05) the ROS from 143.207 to 127.223. The antioxidative action of PLME was proved by spot assay. Phloxine B staining was further confirmed the PLME antioxidative action of PLME, where more whitish-pink live yeast cells were observed. In addition, the PLME also enhanced GSH content significantly (p < 0.05) in yeast treated with PLME from 16.81 to 25.31 µmol. Furthermore, PLME increased the SOD and SIRT1 genes expression significantly (p < 0.05) with ΔCt values of 1.11 and 1.15, respectively. The significantly (p < 0.05) elevated SOD and SIRT1 protein activities were recorded as 51.54 U/mg Prot and 1716 ng/mL, respectively. CONCLUSIONS: PLME exhibited good antiaging activities in S. cerevisiae, by modulating oxidative stress, enhancing GSH content, and increasing SOD and SIRT1 genes expression.

Phytoconstituents and renoprotective effect of Polyalthia longifolia leaves extract on radiation-induced nephritis in rats via TGF-ß/smad pathway.

Renoprotectors are highly demanded due to environmental nephrotoxic factors. P. longifolia leaves extract alleviating effect was assessed in nephritic-induced rats by whole body shot dose of γ-radiation. Many biomarkers were detected using several assays. Renohistopathological examinations were performed. Moreover, the extract phytoconstituents were identified using spectroscopic analysis. In-vitro anti-inflammatory activity of some compounds was examined using histamine release assay. Post-irradiation treatment with the extract significantly ameliorated all elevated biomarker levels. Creatinine and urea were adjusted, TGF-ß/Smad signaling was suppressed causing down-regulation to microRNA-21. Nitric oxide, reactive oxygen species, glutathione and kidney injury molecule-1 were normalized in comparison with the γ-irradiated group. The renohistopathological analysis was consistent with the biochemical study. Phytochemical analysis resulted in the isolation of two diterpenoids (γ-methoxybutenolide clerodane diterpene and 16(R/S)-hydroxycleroda-3,13-dien-16,15-olide-2-one), aporphine alkaloid (anonaine) and flavonol (kaempferol-3-O-rutinoside). The latter two showed moderate anti-histaminic activities. Our results indicated that P. longifolia reduced oxidative stress and nephropathy in rats due to its anti-inflammatory principles.

Antiproliferative, apoptotic, and antimutagenic properties of stem bark and seed fractions of Polyalthia cerasoides (Roxb.).

AIMS: The aim was to compare the anticancer and antimutagenic potency of Polyalthia cerasoides seeds and stem bark. AIM OF THE STUDY: The aim of this study was to investigate the antiproliferative, apoptotic, antioxidation to DNA, and antimutagenic activity of alcoholic (PS-1 and PS-3) and petroleum ether (PS-2 and PS-4) stem bark and seed fractions of P. cerasoides. METHODS: P. cerasoides stem bark and seeds were extracted with ethanol: water mixture (9:1 ratio v: v) and fractionated with petroleum ether. Fractions were investigated for antiproliferative effect using cell by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole assay (cell line used liver [HepG2] and cervical [HeLa] cancer cell lines), DNA damage protection using hydroxyl radical and antimutagenic effect using chromosome aberration test. RESULTS: PS-1 (IC50 10 µg/ml) and PS-3 (IC50 11 µg/ml) showed maximum antiproliferative activity against HepG2 cell lines, whereas, PS-1 (IC50 10 µg/ml), PS-2 (IC50 24 µg/ml), and PS-3 (IC50 11 µg/ml) showed better antiproliferative activity against HeLa cell lines. PS-3 and PS-4 were protective against oxidation to the supercoiled DNA molecule. Further, petroleum ether extract of both seed (PS-2) and stem bark (PS-4) showed good antimutagenicity as revealed by the less chromosomal aberrations compared to PS-1 and PS-3 fractions. CONCLUSIONS: This study demonstrated the beneficial effect of fractions against oxidation of DNA, antiproliferative, apoptotic, and antimutagenic activity. Probably, this property would be attributable by their phenolic and steroid constituents. Therefore, this plant could be used as a potential source of nutraceutical agents.

Phytochemicals from Polyalthia Species: Potential and Implication on Anti-Oxidant, Anti-Inflammatory, Anti-Cancer, and Chemoprevention Activities.

Polyalthia belong to the Annonaceae family and are a type of evergreen tree distributed across many tropical and subtropical regions. Polyalthia species have been used long term as indigenous medicine to treat certain diseases, including fever, diabetes, infection, digestive disease, etc. Recent studies have demonstrated that not only crude extracts but also the isolated pure compounds exhibit various pharmacological activities, such as anti-oxidant, anti-microbial, anti-tumor, anti-cancer, etc. It is known that the initiation of cancer usually takes several years and is related to unhealthy lifestyle, as well as dietary and environmental factors, such as stress, toxins and smoking. In fact, natural or synthetic substances have been used as cancer chemoprevention to delay, impede, or even stop cancer growing. This review is an attempt to collect current available phytochemicals from Polyalthia species, which exhibit anti-cancer potentials for chemoprevention purposes, providing directions for further research on the interesting agents and possible clinical applications.

Cleroda-4(18),13-dien-15,16-olide as novel xanthine oxidase inhibitors: An integrated in silico and in vitro study.

In the present study, in silico predictions and molecular docking were performed on five clerodane diterpenes (1-5) from Polyalthia longifolia seeds to evaluate their potential as xanthine oxidase (XO) inhibitors. The initial screening was conducted by target prediction using TargetNet web server application and only compounds 3 and 4 showed a potential interaction with XO. Compounds 3 and 4 were subsequently subjected to in silico analyses on XO protein structure (PDB: 1N5X) using Schrödinger Release 2020-3 followed by structural modeling & molecular simulation studies to confirm the initial prediction result and identify the binding mode of these compounds to the XO. Molecular docking results revealed that compounds 3 (-37.3 kcal/mol) and 4 (-32.0 kcal/mol) binds more stably to XO than the reference drug allopurinol (-27.0 kcal/mol). Interestingly, two residues Glu 802 and Thr 1010 were observed as the two main H-bond binding sites for both tested compounds and the allopurinol. The center scaffold of allopurinol was positioned by some π-π stacking with Phe 914 and Phe 1009, while that of compounds 3 and 4 were supported by many hydrophobic interactions mainly with Leu 648, Phe 649, Phe 1013, and Leu 1014. Additionally, the docking simulation predicted that the inhibitory effect of compounds 3 and 4 was mediated by creating H-bond with particularly Glu 802, which is a key amino acid for XO enzyme inhibition. Altogether, in vitro studies showed that compounds 3 and 4 had better inhibitory capacity against XO enzyme with IC50 values significantly (p < 0.001) lower than that of allopurinol. In short, the present study identified cleroda-4(18),13-dien-15,16-olide as novel potential XO inhibitors, which can be potentially used for the treatment of gout.

Terpenes isolated from Polyalthia simiarum and their cytotoxic activities.

Two new C31 triterpenes, polysimiaric acid A (1) and B (2) as well as one new clerodane diterpenoid, 16,16-dimethoxy-cleroda-3,13Z-dien-15-oic acid (3), together with six known compounds were isolated from Polyalthia simiarum. Their structures were determined by analysis of 1D and 2D NMR data. Three new compounds were tested for their cytotoxicity against five human tumour cell lines. Compound 3 showed cytotoxic activities against SMMC-7721 with the IC50 value of 22.43 µM.

Dual COX and 5-LOX inhibition by clerodane diterpenes from seeds of Polyalthia longifolia (Sonn.) Thwaites.

Natural metabolites with their specific bioactivities are being considered as a potential source of materials for pharmacological studies. In this study, we successfully isolated and identified five known clerodane diterpenes, namely 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (1), 16-hydroxy-cleroda-3,13-dien-15-oic acid (2), 16-hydroxy-cleroda-4(18),13-dien-16,15-olide (3), 3α,16α-dihydroxy-cleroda-4(18),13(14)Z-dien-15,16-olide (4), and 16α-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (5) from the methanolic extract of seeds of Polyalthia longifolia. Initially, all the isolated metabolites were investigated for COX-1, COX-2, and 5-LOX inhibitory activities using the standard inhibitory kits. Of which, compounds 3, 4, and 5 exhibited to be potent COX-1, COX-2, and 5-LOX inhibitors with the IC50 values similar or lower to those of the reference drugs. To understand the underlying mechanism, these compounds were subjected to molecular docking on COX-1, COX-2, and 5-LOX proteins. Interestingly, the in silico study results were in high accordance with in vitro studies where compounds 3, 4, and 5 hits assumed interactions and binding pattern comparable to that of reference drugs (indomethacin and diclofenac), as a co-crystallized ligand explaining their remarkable dual (COX/LOX) inhibitor actions. Taken together, our findings demonstrated that compounds 3, 4, and 5 functioned as dual inhibitors of COX/5-LOX and can contribute to the development of novel, more effective anti-inflammatory drugs with minimal side-effects.

Chemical composition of the essential oils of four Polyalthia species from Malaysia.

Polyalthia is one of the largest genera in the Annonaceae family, and has been widely used in folk medicine for the treatment of rheumatic fever, gastrointestinal ulcer, and generalized body pain. The present investigation reports on the extraction by hydrodistillation and the composition of the essential oils of four Polyalthia species (P. sumatrana, P. stenopetalla, P. cauliflora, and P. rumphii) growing in Malaysia. The chemical composition of these essential oils was determined by gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The multivariate analysis was determined using principal component analysis (PCA) and hierarchical clustering analysis (HCA) methods. The results revealed that the studied essential oils are made up principally of bicyclogermacrene (18.8%), cis-calamenene (14.6%) and ß-elemene (11.9%) for P. sumatrana; α-cadinol (13.0%) and δ-cadinene (10.2%) for P. stenopetalla; δ-elemene (38.1%) and ß-cubebene (33.1%) for P. cauliflora; and finally germacrene D (33.3%) and bicyclogermacrene for P. rumphii. PCA score and HCA plots revealed that the essential oils were classified into three separated clusters of P. cauliflora (Cluster I), P. sumatrana (Cluster II), and P. stenopetalla, and P. rumphii (Cluster III) based on their characteristic chemical compositions. Our findings demonstrate that the essential oil could be useful for the characterization, pharmaceutical, and therapeutic applications of Polyalthia essential oil.

Discovery of a Natural Product That Binds to the Mycobacterium tuberculosis Protein Rv1466 Using Native Mass Spectrometry.

Elucidation of the mechanism of action of compounds with cellular bioactivity is important for progressing compounds into future drug development. In recent years, phenotype-based drug discovery has become the dominant approach to drug discovery over target-based drug discovery, which relies on the knowledge of a specific drug target of a disease. Still, when targeting an infectious disease via a high throughput phenotypic assay it is highly advantageous to identifying the compound's cellular activity. A fraction derived from the plant Polyalthia sp. showed activity against Mycobacterium tuberculosis at 62.5 µge/µL. A known compound, altholactone, was identified from this fraction that showed activity towards M. tuberculosis at an minimum inhibitory concentration (MIC) of 64 µM. Retrospective analysis of a target-based screen against a TB proteome panel using native mass spectrometry established that the active fraction was bound to the mycobacterial protein Rv1466 with an estimated pseudo-Kd of 42.0 ± 6.1 µM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vivo toxicity towards M. tuberculosis.

α-Glucosidase inhibitory and nitric oxide production inhibitory activities of alkaloids isolated from a twig extract of Polyalthia cinnamomea.

The first phytochemical investigation of Polyalthia cinnamomea led to the isolation and identification of two new oxoprotoberberine alkaloids, (-)-(13aS)-polyalthiacinnamines A and B, together with eleven known compounds. The structures of the new compounds were elucidated by extensive spectroscopic methods. The absolute configuration of miliusacunine E and consanguine B was established by X-ray diffraction analysis using Cu Kα radiation and ECD spectra, whereas the absolute configurations of polyalthiacinnamines A and B were established by comparison of their ECD spectra and specific rotations with those of miliusacunine E and consanguine B. Compounds 1-4, 6, and 8 exhibited α-glucosidase inhibitory activities (IC50 values ranging from 11.3 to 57.9 µM) better than a positive control (acarbose, IC50 83.5 µM). Compound 2 also exhibited NO production inhibitory activity with an IC50 value of 24.4 µM (indomethacin, a positive control, IC50 = 32.2 µM).

Three new methylated Δ8-pregnene steroids from the Polyalthia laui-derived fungus Stemphylium sp. AZGP4-2.

Three new methylated Δ8-pregnene steroids, stemphylisteroids A-C (1-3) were isolated from the medicinal plant Polyalthia laui-derived fungus Stemphylium sp. AZGP4-2. Their structures were elucidated by the detailed analysis of comprehensive spectroscopic data. The absolute configuration of 1 was determined by X-ray crystallographic analysis. Compound 1 show antibacterial activity against Escherichia coli with the MIC value of 6.25 µg/mL, and 2 exhibited a broad spectrum of antibacterial activities against six pathogenic bacteria with the MIC values ranging from 12.5 to 50 µg/mL. The discovery of three methylated Δ8-pregnene steroids 1-3 are a further addition to diverse and complex array of methylated steroids.

Polyalthia longifolia Extract Triggers ER Stress in Prostate Cancer Cells Concomitant with Induction of Apoptosis: Insights from In Vitro and In Vivo Studies.

Plant-based therapies are being explored to prevent or treat several cancer types. The antioxidant properties of Polyalthia longifolia plant are well established. In our previous work, we demonstrated the presence of cytotoxic compounds in the methanol extract of Polyalthia longifolia (MEP) with potent activity against human leukemia cells. In the present study, we evaluated the efficacy of MEP against prostate cancer (PCa) and established the molecular basis of its effect in in vitro and in vivo models. We observed that MEP treatment resulted in a significant decrease in the growth and viability of PCa cells, associated with arrest in the G1/S phase of the cell cycle. Apoptosis was confirmed as the primary mode of MEP-induced cell death through activation of the intrinsic apoptotic machinery. Proteomic and biochemical studies identified BiP as an important target of MEP with the activation of the ER stress pathway, as a potential mechanism driving MEP-induced apoptosis. The extract exhibited strong efficacy in the PCa xenograft mouse model with significant inhibition of tumor growth and reduced tumor burden. Taken together, our findings indicate that MEP-induced apoptosis in PCa cells concomitant with the activation of the ER stress pathways results in the inhibition of tumor growth, in vitro and in vivo. Our studies provide initial evidence of the efficacy of MEP against PCa and advocate for in-depth studies in other preclinical models for its possible use in clinical settings.

Functional Validation of DownRegulated MicroRNAs in HeLa Cells Treated with Polyalthia longifolia Leaf Extract Using Different Microscopic Approaches: A Morphological Alteration-Based Validation.

Several microscopy methods have been developed to assess the morphological changes in cells in the investigations of the mode of cell death in response to a stimulus. Our recent finding on the treatment of the IC50 concentration (26.67 µg/mL) of Polyalthia longifolia leaf extract indicated the induction of apoptotic cell death via the regulation of miRNA in HeLa cells. Hence, the current study was conducted to validate the function of these downregulated microRNAs in P. longifolia-treated HeLa cells using microscopic approaches. These include scanning electron microscope (SEM), transmission electron microscope (TEM), and acridine orange/propidium iodide (AO/PI)-based fluorescent microscopy techniques by observing the morphological alterations to cells after transfection with mimic miRNA. Interestingly, the morphological changes observed in this study demonstrated the apoptotic hallmarks, for instance, cell blebbing, cell shrinkage, cytoplasmic and nuclear condensation, vacuolization, cytoplasmic extrusion, and the formation of apoptotic bodies, which proved the role of dysregulated miRNAs in apoptotic HeLa cell death after treatment with the P. longifolia leaf extract. Conclusively, the current study proved the crucial role of downregulated miR-484 and miR-221-5p in the induction of apoptotic cell death in P. longifolia-treated HeLa cells using three approaches-SEM, TEM, and AO/PI-based fluorescent microscope.

Polyalthia Clerodane Diterpene Potentiates Hypoglycemia via Inhibition of Dipeptidyl Peptidase 4.

Serine protease dipeptidyl peptidase 4 (DPP-4) is involved in self/non-self-recognition and insulin sensitivity. DPP-4 inhibitors are conventional choices for diabetic treatment; however, side effects such as headache, bronchus infection, and nasopharyngitis might affect the daily lives of diabetic patients. Notably, natural compounds are believed to have a similar efficacy with lower adverse effects. This study aimed to validate the DPP-4 inhibitory activity of clerodane diterpene 16-hydroxycleroda-3,13-dien-15,16-olide (HCD) from Polyalthia longifolia, rutin, quercetin, and berberine, previously selected through molecular docking. The inhibitory potency of natural DPP-4 candidates was further determined by enzymatic, in vitro Caco-2, and ERK/PKA activation in myocyte and pancreatic cells. The hypoglycemic efficacy of the natural compounds was consecutively analyzed by single-dose and multiple-dose administration in diet-induced obese diabetic mice. All the natural-compounds could directly inhibit DPP-4 activity in enzymatic assay and Caco-2 inhibition assay, and HCD showed the highest inhibition of the compounds. HCD down-regulated LPS-induced ERK phosphorylation in myocyte but blocked GLP-1 induced PKA expression. For in vivo tests, HCD showed hypoglycemic efficacy only in single-dose administration. After 28-days administration, HCD exhibited hypolipidemic and hepatoprotective efficacy. These results revealed that HCD performed potential antidiabetic activity via inhibition of single-dose and long-term administrations, and could be a new prospective anti-diabetic drug candidate.

Multi-target mode of action of a Clerodane-type diterpenoid from Polyalthia longifolia targeting African trypanosomes.

Natural products have made remarkable contributions to drug discovery and therapy. In this work we exploited various biochemical approaches to investigate the mode of action of 16-α-hydroxy-cleroda-3,13 (14)-Z-dien-15,16-olide (HDK-20), which we recently isolated from Polyalthia longifolia, on Trypanosoma brucei bloodstream trypomastigotes. HDK20 at concentrations ≥ EC50 (0.4 µg/ml) was trypanocidal, with its effect irreversible after only a brief exposure time (<1 h). Fluorescence microscopic assessment of DNA configuration revealed severe cell cycle defects after 8 h of incubation with the compound, the equivalent of a single generation time. This was accompanied by DNA fragmentation as shown by Terminal deoxynucleotidyl transferase dUTP Nick-End Labelling (TUNEL) assays. HDK-20 also induced a fast and profound depolarisation of the parasites' mitochondrial membrane potential and depleted intracellular ATP levels of T. brucei. Overall, HDK20 showed a multi-target mechanism of action, which provides a biochemical explanation for the promising anti-trypanosomatid activity in our previous report.

Standardized Polyalthia longifolia leaf extract (PLME) inhibits cell proliferation and promotes apoptosis: The anti-cancer study with various microscopy methods.

Over the years a number of microscopy methods have been developed to assess the changes in cells. Some non-invasive techniques such as holographic digital microscopy (HDM), which although does not destroy the cells, but helps to monitor the events that leads to initiation of apoptotic cell death. In this study, the apoptogenic property and the cytotoxic effect of P. longifolia leaf methanolic extract (PLME) against the human cervical carcinoma cells (HeLa) was studied using light microscope (LM), holographic digital microscopy (HDM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The average IC50 value of PLME against HeLa cells obtained by MTT and CyQuant assay was 22.00µg/mL at 24h. However, noncancerous Vero cells tested with PLME exhibited no cytotoxicity with the IC50 value of 51.07µg/mL at 24h by using MTT assay. Cytological observations showed nuclear condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, disappearance of microvilli and filopodia, narrowing of lamellipodia, holes, formation of numerous smaller vacuoles, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by HDM, LM, SEM and TEM. In conclusion, PLME was able to produce distinctive morphological features of HeLa cell death that corresponds to apoptosis.

In vitro antiprotozoal activity and cytotoxicity of extracts and isolated constituents from Greenwayodendron suaveolens.

ETHNOPHARMACOLOGICAL RELEVANCE: The Nkundo people (Nkundo area of Bolongo, Mai-Ndombe district, Bandundu Province, DR Congo) use various plant parts of the tree Greenwayodendron suaveolens (Engl. & Diels) Verdc. (syn. Polyalthia suaveolens Engl. & Diels) (Annonaceae) against malaria, but its antiprotozoal constituents are not known. MATERIALS AND METHODS: The crude 80% ethanol extract from the fruits, leaves, root bark and stem bark and 16 fractions were assessed in vitro for their antiprotozoal activity against Trypanosoma brucei brucei, T. cruzi, Leishmania infantum and the chloroquine and pyrimethamine-resistant K1 strain of Plasmodium falciparum (Pf-K1). Their cytotoxic effects were evaluated against MRC-5 cells. Active constituents were isolated by chromatographic means, identified using spectroscopic methods, and evaluated in the same assays. RESULTS: The root bark extract showed the highest activity against P. falciparum K1 (IC50 0.26µg/mL) along with the stem bark alkaloid fraction (IC50 0.27µg/mL). The root bark alkaloid fraction had a pronounced activity against all selected protozoa with IC50 values <1µg/mL. The 90% methanol fractions of the different plant parts showed a pronounced activity against P. falciparum K1, with IC50 values ranging between 0.36µg/mL and 0.69µg/mL. Four constituents were isolated: the triterpenes polycarpol, and dihydropolycarpol, the latter one being reported for the first time from nature, and the alkaloids polyalthenol and N-acetyl-polyveoline. They were active to a various degree against one or more protozoa, mostly accompanied by cytotoxicity. The highest selectivity was observed for N-acetyl-polyveoline against P. falciparum K1 (IC50 2.8µM, selectivity index 10.9). CONCLUSIONS: These results may explain at least in part the traditional use of this plant species against parasitic diseases such as malaria in DR Congo.

16-hydroxy-cleroda-3,13-dien-16,15-olide induced glioma cell autophagy via ROS generation and activation of p38 MAPK and ERK-1/2.

16-hydroxy-cleroda-3,13-dien-16,15-olide (HCD), a natural product isolated from medicinal plant Polyalthia longifolia exhibits anticancer activity through caspase-independent apoptosis in brain tumors, as previously reported. This study further attempted to investigate the involvement of HCD-induced autophagy in brain tumor cell lines neuroblastoma N18 and glioma C6 through the induction of reactive oxygen species (ROS) and the activation of p38 and ERK-1/2 pathway. The results demonstrated that HCD increased the hyper-generation of ROS and decreased cellular antioxidant enzymes, such as superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and glutathione s transferase (GST). Furthermore, HCD increased the expressions of autophagic marker proteins LC3-II and Beclin-1 in a time- and dose-dependent manner. Additionally, HCD was found to significantly induce p-p38 MAPK and p-ERK-1/2 proteins by Western blot, which implies that HCD is a potential therapeutic anticancer agent that exerts its activity through inducing ROS-mediation for the autophagy of brain tumor cells.

A clerodane diterpene from Polyalthia longifolia as a modifying agent of the resistance of methicillin resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus infections are raising serious concern across the world. The effectiveness of conventional drugs is continuously decreasing due to global emergence of multidrug resistance (MDR) and therefore, new resistance-modifying agents (RMAs) are highly needed. HYPOTHESIS: Clerodane diterpene 16α-hydroxycleroda-3,13(14)-Z-dien-15,16-olide (CD) from leaves of Polyalthia longifolia (Sonn.) Thwaites (Annonaceae) as RAM will be useful in improving the current treatment strategies for staphylococcal infections. STUDY DESIGN: In the present study, we determine the resistance-modifying activity of CD using clinical isolates of MRSA. Further, the influence of CD on innate immune response was also evaluated in vitro and in vivo. The nature of potential interactions was determined by fractional inhibitory concentration indices (FICIs) calculated from microdilution assays and time-kill curves. RESULTS: The result of in vitro combination study showed that CD significantly reduced MIC of fluoroquinolones up to 16-folds (FICI 0.315-0.500), while in S. aureus infected Swiss albino mice model, combination of CD with norfloxacin, significantly (p<0.01, p<0.001) lowered the systemic microbial burden in blood, liver, kidney, lung and spleen tissues in comparison to CD, norfloxacin alone as well as untreated control. Flow cytometry analysis clearly showed that CD significantly inhibited EtBr efflux and extended post-antibiotic effect. In qRT-PCR analysis, CD alone as well as in combination, significantly modulated the expression of various efflux pump genes including norA up to 2-fold in clinical isolate MRSA-ST2071. Further, the in vitro combination study of the CD (10, 5, 2.5µg/ml) along with the norfloxacin (10µg/ml) depicted a significant decline in the pro-inflammatory cytokines, IL6 and TNF-α. In septic shock mice model, CD did not exhibit any significant changes in the level of pro-inflammatory cytokines. CONCLUSION: This is the first report on drug resistance-modifying potential of CD through inhibition of MDR efflux pump.

New clerodane diterpenoids from the roots of Polyalthia laui.

Five new clerodane diterpenoids, polylauiester A (1), (4→2)-abeo-2,13-diformyl-cleroda-2,12E-dien-14-oic acid (2) and polylauiamides B-D (3-5), together with 11 known clerodane diterpenoids (6-16), were isolated from the roots of Polyalthia laui. Among them, polylauiester A (1) represents the first example of a novel norclerodane diterpenoid only containing 17 carbon atoms on the carbon skeleton, and polylauiamide B (3) is an unusual diterpenoid with a p-substituted benzene ring as a substituent. Their structures were elucidated by extensive spectroscopic methods, and the relative configuration of polylauiamide B (3) was further confirmed by the single crystal X-ray diffraction method. Biological evaluation of new compounds against human Hela, MCF-7 and A549 human cancer cell lines showed that all compounds displayed weak cytotoxicities against various human cancer cell lines in the range of IC50 at 25.01-39.31µM.