The protective effects of Zingiber zerumbet rhizome against fevers in rats.

The Zingiber zerumbet rhizomes are traditionally used to treat fever, and the in vitro inhibitory effect of ethyl acetate extract from Zingiber zerumbet rhizomes (EAEZZR) against DENV2 NS2B/NS3 (two non-structural proteins, NS2 and NS3 of dengue virus type 2) has been reported earlier. This study was carried out to establish an acute toxicity profile and evaluate the anti-fever (anti-pyretic) activities of EAEZZR in yeast-induced fever in rats. The major compound of EAEZZR, zerumbone, was isolated using chromatographic methods including column chromatography (CC) and preparative thin-layer chromatography (PTLC). Additionally, the structure of zerumbone was elucidated using nuclear magnetic resonance (NMR), liquid chromatography mass spectrometer-ion trap-time of flight (LCMS-IT-TOF), infrared (IR), and ultraviolet (UV) spectroscopy. The toxicity of EAEZZR was evaluated using Organization for Economic Cooperation and Development Test Guideline 425 (OECD tg-425) with minor modifications at concentrations EAEZZR of 2000 mg/kg, 3000 mg/kg, and 5000 mg/kg. Anti-fever effect was determined by yeast-induced fever (pyrexia) in rats. The acute toxicity study showed that EAEZZR is safe at the highest 5000 mg/kg body weight dose in Sprague Dawley rats. Rats treated with EAEZZR at doses of 125, 250, and 500 mg/kg exhibited a significant reduction in rectal temperature (TR) in the first 1 h. EAEZZR at the lower dose of 125 mg/kg showed substantial potency against yeast-induced fever for up to 2 h compared to 0 h in controls. A significant reduction of TR was observed in rats treated with standard drug aspirin in the third through fourth hours. Based on the present findings, ethyl acetate extract of Zingiber zerumbet rhizomes could be considered safe up to the dose of 5000 mg/kg, and the identification of active ingredients of Zingiber zerumbet rhizomes may allow their use in the treatment of fever with dengue virus infection.

Chemodiverse monoterpene indole alkaloids from Kopsia teoi, inhibitory potential against α-amylase, and their molecular docking studies.

Diabetes mellitus stands as a metabolic ailment marked by heightened blood glucose levels due to inadequate insulin secretion. The primary aims of this investigative inquiry encompassed the isolation of phytochemical components from the bark of Kopsia teoi, followed by the assessment of their α-amylase inhibition. The phytochemical composition of the K. teoi culminated in the discovery of a pair of new indole alkaloids; which are 16-epi-deacetylakuammiline N(4)-methylene chloride (akuammiline) (1), and N(1)-methoxycarbonyl-11-methoxy-12-hydroxy-Δ14-17-kopsinine (aspidofractinine) (2), together with five known compounds i.e. kopsiloscine G (aspidofractinine) (3), akuammidine (sarpagine) (4), leuconolam (aspidosperma) (5), N-methoxycarbonyl-12-methoxy-Δ16, 17-kopsinine (aspidofractinine) (6), and kopsininate (aspidofractinine) (7). All compounds were determined via spectroscopic analyses. The in vitro evaluation against α-amylase showed good inhibitory activities for compounds 5-7 with the inhibitory concentration (IC50) values of 21.7 ± 1.2, 34.1 ± 0.1, and 30.0 ± 0.8 µM, respectively compared with the reference acarbose (IC50 = 34.4 ± 0.1 µM). The molecular docking outputs underscored the binding interactions of compounds 5-7 ranging from -8.1 to -8.8 kcal/mol with the binding sites of α-amylase. Consequently, the outcomes highlighted the anti-hyperglycemic attributes of isolates from K. teoi.

Erythrocarpines IN, new limonoids from the barks of Chisocheton erythrocarpus and their neuroprotective effects against hydrogen peroxide in NG108-15 cells.

A phytochemical study on the bark of Chisocheton erythrocarpus Hiern (Meliaceae) has led to the isolation of six new phragmalin-type limonoids named erythrocarpines I - N (1-6) along with one known limonoid, erythrocarpine F (7). Their structures were fully characterized by spectroscopic methods. The pre-treatment of NG108-15 cells with 1-5, 7 (2 h) demonstrated low to good protective effects against H2O2 exposure; 1 (83.77% ± 1.84 at 12.5 µM), 2 (69.07 ± 2.01 at 12.5 µM), 3 (80.38 ± 2.1 at 12.5 µM), 4 (62.33 ± 1.95 at 25 µM),5 (58.67 ± 1.85 at 50 µM) and 7 (66.07 ± 2.03 at 12.5 µM). Interestingly, 1 and 3 demonstrated comparable protective effects to positive control epigallocatechin gallate (EGCG) with similar cell viability capacity (≈ 80%), having achieved that at lower concentration (12.5 µM) than EGCG (50 µM). Collectively, the results suggested the promising use of 1 and 3 as potential neuroprotective agents against hydrogen peroxide-induced cytotoxicity in neuronal model.

N-Methyl Costaricine and Costaricine, Two Potent Butyrylcholinesterase Inhibitors from Alseodaphne pendulifolia Gamb.

Studies have been conducted over the last decade to identify secondary metabolites from plants, in particular those from the class of alkaloids, for the development of new anti-Alzheimer's disease (AD) drugs. The genus Alseodaphne, comprising a wide range of alkaloids, is a promising source for the discovery of new cholinesterase inhibitors, the first-line treatment for AD. With regard to this, a phytochemical investigation of the dichloromethane extract of the bark of A. pendulifolia Gamb. was conducted. Repeated column chromatography and preparative thin-layer chromatography led to the isolation of a new bisbenzylisoquinoline alkaloid, N-methyl costaricine (1), together with costaricine (2), hernagine (3), N-methyl hernagine (4), corydine (5), and oxohernagine (6). Their structures were elucidated by the 1D- and 2D-NMR techniques and LCMS-IT-TOF analysis. Compounds 1 and 2 were more-potent BChE inhibitors than galantamine with IC50 values of 3.51 ± 0.80 µM and 2.90 ± 0.56 µM, respectively. The Lineweaver-Burk plots of compounds 1 and 2 indicated they were mixed-mode inhibitors. Compounds 1 and 2 have the potential to be employed as lead compounds for the development of new drugs or medicinal supplements to treat AD.

A Review on the Delivery of Plant-Based Antidiabetic Agents Using Nanocarriers: Current Status and Their Role in Combatting Hyperglycaemia.

Diabetes mellitus is a prevalent metabolic syndrome that is associated with high blood glucose levels. The number of diabetic patients is increasing every year and the total number of cases is expected to reach more than 600 million worldwide by 2045. Modern antidiabetic drugs alleviate hyperglycaemia and complications that are caused by high blood glucose levels. However, due to the side effects of these drugs, plant extracts and bioactive compounds with antidiabetic properties have been gaining attention as alternative treatments for diabetes. Natural products are biocompatible, cheaper and expected to cause fewer side effects than the current antidiabetic drugs. In this review, various nanocarrier systems are discussed, such as liposomes, niosomes, polymeric nanoparticles, nanoemulsions, solid lipid nanoparticles and metallic nanoparticles. These systems have been applied to overcome the limitations of the current drugs and simultaneously improve the efficacy of plant-based antidiabetic drugs. The main challenges in the formulation of plant-based nanocarriers are the loading capacity of the plant extracts and the stability of the carriers. A brief review of lipid nanocarriers and the amphipathic properties of phospholipids and liposomes that encapsulate hydrophilic, hydrophobic and amphiphilic drugs is also described. A special emphasis is placed on metallic nanoparticles, with their advantages and associated complications being reported to highlight their effectiveness for treating hyperglycaemia. The present review could be an interesting paper for researchers who are working in the field of using plant extract-loaded nanoparticles as antidiabetic therapies.

Natural Products for Cancer Therapy: A Review of Their Mechanism of Actions and Toxicity in the Past Decade.

The ethnopharmacological information gathered over many centuries and the presence of diverse metabolites have made the medicinal plants as the prime source of drugs. Despite the positive attributes of natural products, there are many questions pertaining to their mechanism of actions and molecular targets that impede their development as therapeutic agents. One of the major challenges in cancer research is the toxicity exerted by investigational agents towards the host. An understanding of their molecular targets, underlying mechanisms can reveal their anticancer efficacy, help in optimal therapeutic dose selection, to mitigate their side effects and toxicity towards the host. The purpose of this review is to collate details on natural products that are recently been investigated extensively in the past decade for their anticancer potential. Besides, critical analysis of their molecular targets and underlying mechanisms on multiple cancer cell lines, an in-depth probe of their toxicological screening on rodent models is outlined as well to observe the prevalence of their toxicity towards host. This review can provide valuable insights for researchers in developing methods, strategies during preclinical and clinical evaluation of anticancer candidates.

α-Amylase and dipeptidyl peptidase-4 (DPP-4) inhibitory effects of Melicope latifolia bark extracts and identification of bioactive constituents using in vitro and in silico approaches.

CONTEXT: Melicope latifolia (DC.) T. G. Hartley (Rutaceae) was reported to contain various phytochemicals including coumarins, flavonoids, and acetophenones. OBJECTIVE: This study investigates the antidiabetic and antioxidant effects of M. latifolia bark extracts, fractions, and isolated constituents. MATERIALS AND METHODS: Melicope latifolia extracts (hexane, chloroform, and methanol), fractions, and isolated constituents with varying concentrations (0.078-10 mg/mL) were subjected to in vitro α-amylase and dipeptidyl peptidase-4 (DPP-4) inhibitory assay. Molecular docking was performed to study the binding mechanism of active compounds towards α-amylase and DPP-4 enzymes. The antioxidant activity of M. latifolia fractions and compounds were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and ß-carotene bleaching assays. RESULTS: Melicope latifolia chloroform extract showed the highest antidiabetic activity (α-amylase IC50: 1464.32 µg/mL; DPP-4 IC50: 221.58 µg/mL). Fractionation of chloroform extract yielded four major fractions (CF1-CF4) whereby CF3 showed the highest antidiabetic activity (α-amylase IC50: 397.68 µg/mL; DPP-4 IC50: 37.16 µg/mL) and resulted in ß-sitosterol (1), halfordin (2), methyl p-coumarate (3), and protocatechuic acid (4). Isolation of compounds 2-4 from the species and their DPP-4 inhibitory were reported for the first time. Compound 2 showed the highest α-amylase (IC50: 197.53 µM) and ß-carotene (88.48%) inhibition, and formed the highest number of molecular interactions with critical amino acid residues of α-amylase. The highest DPP-4 inhibition was exhibited by compound 3 (IC50: 911.44 µM). DISCUSSION AND CONCLUSIONS: The in vitro and in silico analyses indicated the potential of M. latifolia as an alternative source of α-amylase and DPP-4 inhibitors. Further pharmacological studies on the compounds are recommended.

Oxygen radical antioxidant capacity (ORAC) and antibacterial properties of Melicope glabra bark extracts and isolated compounds.

Melicope glabra (Blume) T. G. Hartley from the Rutaceae family is one of the richest sources of plant secondary metabolites, including coumarins and flavanoids. This study investigates the free radical scavenging and antibacterial activities of M. glabra and its isolated compounds. M. glabra ethyl acetate and methanol extracts were prepared using the cold maceration technique. The isolation of compounds was performed with column chromatography. The free radical scavenging activity of the extracts and isolated compounds were evaluated based on their oxygen radical absorbance capacity (ORAC) activities. The extracts and compounds were also subjected to antibacterial evaluation using bio-autographic and minimal inhibitory concentration (MIC) techniques against two oral pathogens, Enterococcus faecalis and Streptococcus mutans. Isolation of phytoconstituents from ethyl acetate extract successfully yielded quercetin 3, 5, 3'-trimethyl ether (1) and kumatakenin (2), while the isolation of the methanol extract resulted in scoparone (3), 6, 7, 8-trimethoxycoumarin (4), marmesin (5), glabranin (6), umbelliferone (7), scopoletin (8), and sesamin (9). The study is the first to isolate compound (1) from Rutaceae plants, and also the first to report the isolation of compounds (2-5) from M. glabra. The ORAC evaluation showed that the methanol extract is stronger than the ethyl acetate extract, while umbelliferone (7) exhibited the highest ORAC value of 24 965 µmolTE/g followed by glabranin (6), sesamin (9) and scopoletin (8). Ethyl acetate extract showed stronger antibacterial activity towards E. faecalis and S. mutans than the methanol extract with MIC values of 4166.7 ± 1443.4 µg/ml and 8303.3 ± 360.8 µg/ml respectively. Ethyl acetate extract inhibited E. faecalis growth, as shown by the lowest optical density value of 0.046 at a concentration of 5.0 mg/mL with a percentage inhibition of 95%. Among the isolated compounds tested, umbelliferone (7) and sesamin (9) exhibited promising antibacterial activity against S. mutans with both exhibiting MIC values of 208.3 ± 90.6 µg/ml. Findings from this study suggests M. glabra as a natural source of potent antioxidant and antibacterial agents.

Goniolanceolatins A-H, Cytotoxic Bis-styryllactones from Goniothalamus lanceolatus.

Eight new bis-styryllactones, goniolanceolatins A-H (1-8), possessing a rare α,ß-unsaturated δ-lactone moiety with a (6S)-configuration, were isolated from the CH2Cl2 extract of the stembark and roots of Goniothalamus lanceolatus Miq., a plant endemic to Malaysia. Absolute structures were established through extensive 1D- and 2D-NMR data analysis, in combination with electronic dichroism (ECD) data. All of the isolates were evaluated for their cytotoxicity against human lung and colorectal cancer cell lines. Compounds 2 and 4 showed cytotoxicity, with IC50 values ranging from 2.3 to 4.2 µM, and were inactive toward human noncancerous lung and colorectal cells. Compounds 1, 3, 6, 7, and 8 showed moderate to weak cytotoxicity. Docking studies of compounds 2 and 4 showed that they bind with EGFR tyrosine kinase and cyclin-dependent kinase 2 through hydrogen bonding interactions with the important amino acids, including Lys721, Met769, Asn818, Arg157, Ile10, and Glu12.

Molecular Insight and Mode of Inhibition of α-Glucosidase and α-Amylase by Pahangensin A from Alpinia pahangensis Ridl.

The inhibition of carbohydrate-hydrolyzing enzymes in human digestive organs is crucial in controlling blood sugar levels, which is important in treating type 2 diabetes. In the current study, pahangensin A (1), a bis-labdanic diterpene characterized previously in the rhizomes of Alpinia pahangensis Ridl., was identified as an active dual inhibitor for α-amylase (IC50 =114.80 µm) and α-glucosidase (IC50 =153.87 µm). This is the first report on the dual α-amylase and α-glucosidase inhibitory activities of a bis-labdanic diterpene. The Lineweaver-Burk plots of compound 1 indicate that it is a mixed-type inhibitor with regard to both enzymes. Based on molecular docking studies, compound 1 docked in a non-active site of both enzymes. The dual inhibitory activity of compound 1 makes it a suitable natural alternative in the treatment of type 2 diabetes.

Isolation of antioxidative compounds from Micromelum minutum guided by preparative thin layer chromatography-2,2-diphenyl-1-picrylhydrazyl (PTLC-DPPH) bioautography method.

The application of preparative thin layer chromatography-2,2-diphenyl-1-picrylhydrazyl (PTLC-DPPH) bioautography technique successfully isolated a lignan sesamin (1), two prenylated coumarins (2 and 3) and a marmesin glycosides (4) from Micromelum minutum methanol bark extract. Compounds 2 and 3 were identified as new compounds whereas 1 and 4 were first isolated from Micromelum genus. Structural identification of all compounds were done by detailed spectroscopic analyses and comparison with literature data. Antioxidant capacities of extract, active fraction and compounds were measured based on DPPH free radical savenging activity, oxygen radical absorbance capacity (ORAC) and ß-carotene bleaching. The DPPH activity of methanol extract and its fraction present the IC50 values of 54.3 and 168.9 µg/mL meanwhile the ß-carotene bleaching results were 55.19% and 5.75% respectively. The ORAC measurements of M. minutum extract, compounds 2 and 4 showed potent antioxidant activity with the values of 5123, 5539 and 4031 µmol TE/g respectively.

13C-NMR dereplication of Garcinia extracts: Predicted chemical shifts as reliable databases.

Usually isolated from Garcinia (Clusiaceae) or Hypericum (Hypericaceae) species, some Polycyclic Polyprenylated AcylPhloroglucinols (PPAPs) have been recently reported as potential research tools for immunotherapy. Aiming at exploring the chemodiversity of PPAPs amongst Garcinia genus, a dereplication process suitable for such natural compounds has been developed. Although less sensitive than mass spectrometry, NMR spectroscopy is perfectly reproducible and allows stereoisomers distinction, justifying the development of 13C-NMR strategies. Dereplication requires the use of databases (DBs). To define if predicted DBs were accurate enough as dereplication tools, experimental and predicted δC of natural products usually isolated from Clusiaceae were compared. The ACD/Labs commercial software allowed to predict 73% of δC in a 1.25 ppm range around the experimental values. Consequently, with these parameters, the major PPAPs from a Garcinia bancana extract were successfully identified using a predicted DB.

Laevifins A-G, clerodane diterpenoids from the Bark of Croton oblongus Burm.f.

A phytochemical investigation of the stem barks of the Malaysian Croton oblongus Burm.f. (Syn. Croton laevifolius Blume) (Euphorbiaceae) yielded seven previously undescribed ent-neo-clerodane diterpenoids, laevifins A - G and the known crovatin (3). Structures were established by a combination of spectroscopic methods including HRESIMS, NMR spectroscopy and X-ray crystallography. The absolute configuration of crovatin and laevifins A-G was established by comparison of experimental ECD and theoretical TDDFT ECD calculated spectra. This is the first report on the occurrence of the sesquiterpenoid cryptomeridiol in a Croton species. In vitro cytotoxicity assays on laevifins A, B and G showed moderate activities against the MCF-7 cancer cell line (IC50 102, 115 and 106 µM, respectively) while ß-amyrin and acetyl aleuritolic acid showed good anti-inflammatory activity on the LPS-induced NF-κB translocation inhibition in RAW 264.7 cells assay with IC50 values of 23.5 and 35.4 µg/mL, respectively.

Cytotoxic Effects of Pinnatane A Extracted from Walsura pinnata (Meliaceae) on Human Liver Cancer Cells.

BACKGROUND: Pinnatane A from the bark of Walsura pinnata was investigated for its anti-cancer properties by analyzing the cytotoxic activities and cell cycle arrest mechanism induced in two different liver cancer cell lines. METHODS: A 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to analyze the pinnatane A selectivity in inducing cell death in cancer and normal cells. Various biological assays were carried out to analyze the anti-cancer properties of pinnatane A, such as a live/dead assay for cell death microscopic visualization, cell cycle analysis using propidium iodide (PI) to identify the cell cycle arrest phase, annexin V-fluorescein isothiocyanate (annexin V-FITC)/PI flow cytometry assay to measure percentage of cell populations at different stages of apoptosis and necrosis, and DNA fragmentation assay to verify the late stage of apoptosis. RESULTS: The MTT assay identified pinnatane A prominent dose- and time-dependent cytotoxicity effects in Hep3B and HepG2 cells, with minimal effect on normal cells. The live/dead assay showed significant cell death, while cell cycle analysis showed arrest at the G0/G1 phase in both cell lines. Annexin V-FITC/PI flow cytometry and DNA fragmentation assays identified apoptotic cell death in Hep3B and necrotic cell death in HepG2 cell lines. CONCLUSIONS: Pinnatane A has the potential for further development as a chemotherapeutic agent prominently against human liver cells.

Corrigendum to "Gastroprotective Activity of Polygonum chinense Aqueous Leaf Extract on Ethanol-Induced Hemorrhagic Mucosal Lesions in Rats".

[This corrects the article DOI: 10.1155/2012/404012.].

Insecticidal activity and the mechanism of action of three phenylpropanoids isolated from the roots of Piper sarmentosum Roxb.

Hexane, dichloromethane and methanol extracts of the roots of Piper sarmentosum Roxb. were screened for toxicity towards Sitophilus oryzae (L.), Rhyzopertha dominica (F.), and Plodia interpunctella (Hübner) and the hexane extract exhibited the highest mortality percentage. Bioassay-guided fractionation of the hexane extract resulted in the isolation of asaricin 1, isoasarone 2, and trans-asarone 3. Asaricin 1 and isoasarone 2 were the most toxic compounds to Sitophilus oryzae, Rhyzopertha dominica, and Plodia interpunctella. Sitophilus oryzae and Rhyzopertha dominica exposed to asaricin 1 and isoasarone 2 required the lowest median lethal time. Insecticidal activity of trans-asarone 3 showed consistent toxicity throughout the 60 days towards all three insects as compared to asaricin 1 and isoasarone 2. Asaricin 1 and isoasarone 2 at different doses significantly reduced oviposition and adult emergence of the three insects in treated rice. Trans-asarone 3 had lowest toxicity with highest LC and LT values in all tested insects relative to its mild oviposition inhibition and progeny activity. Moreover, asaricin 1 and isoasarone 2 significantly inhibited acetylcholinesterase in comparison with trans-asarone 3 and the control. Acetylcholinesterase inhibition of Rhyzopertha dominica and Plodia interpunctella by asaricin 1 and isoasarone 2 were lower than that of Sitophilus oryzae, which correlated with their higher resistance.

Essential oils of aromatic Egyptian plants repel nymphs of the tick Ixodes ricinus (Acari: Ixodidae).

Due to the role of Ixodes ricinus (L.) (Acari: Ixodidae) in the transmission of many serious pathogens, personal protection against bites of this tick is essential. In the present study the essential oils from 11 aromatic Egyptian plants were isolated and their repellent activity against I. ricinus nymphs was evaluated Three oils (i.e. Conyza dioscoridis L., Artemisia herba-alba Asso and Calendula officinalis L.) elicited high repellent activity in vitro of 94, 84.2 and 82%, respectively. The most active essential oil (C. dioscoridis) was applied in the field at a concentration of 6.5 µg/cm2 and elicited a significant repellent activity against I. ricinus nymphs by 61.1%. The most repellent plants C. dioscoridis, C. officinalis and A. herba-alba yielded essential oils by 0.17, 0.11 and 0.14%, respectively. These oils were further investigated using gas chromatography-mass spectrometry analysis. α-Cadinol (10.7%) and hexadecanoic acid (10.5%) were the major components of C. dioscoridis whereas in C. officinalis, α-cadinol (21.2%) and carvone (18.2%) were major components. Artemisia herba-alba contained piperitone (26.5%), ethyl cinnamate (9.5%), camphor (7.7%) and hexadecanoic acid (6.9%). Essential oils of these three plants have a potential to be used for personal protection against tick bites.

In vitro inhibitory mechanisms and molecular docking of 1'-S-1'-acetoxychavicol acetate on human cytochrome P450 enzymes.

BACKGROUND: The compound, 1'-S-1'-acetoxychavicol acetate (ACA), isolated from the rhizomes of a Malaysian ethno-medicinal plant, Alpinia conchigera Griff. (Zingiberaceae), was previously shown to have potential in vivo antitumour activities. In the development of a new drug entity, potential interactions of the compound with the cytochrome P450 superfamily metabolizing enzymes need to be ascertain. PURPOSE: The concomitant use of therapeutic drugs may cause potential drug-drug interactions by decreasing or increasing plasma levels of the administered drugs, leading to a suboptimal clinical efficacy or a higher risk of toxicity. Thus, evaluating the inhibitory potential of a new chemical entity, and to clarify the mechanism of inhibition and kinetics in the various CYP enzymes is an important step to predict drug-drug interactions. STUDY DESIGN: This study was designed to assess the potential inhibitory effects of Alpinia conchigera Griff. rhizomes extract and its active constituent, ACA, on nine c-DNA expressed human cytochrome P450s (CYPs) enzymes using fluorescent CYP inhibition assay. METHODS/RESULTS: The half maximal inhibitory concentration (IC50) of Alpinia conchigera Griff. rhizomes extract and ACA was determined for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5. A. conchigera extract only moderately inhibits on CYP3A4 (IC50 = 6.76 ± 1.88µg/ml) whereas ACA moderately inhibits the activities of CYP1A2 (IC50 = 4.50 ± 0.10µM), CYP2D6 (IC50 = 7.50 ± 0.17µM) and CYP3A4 (IC50 = 9.50 ± 0.57µM) while other isoenzymes are weakly inhibited. In addition, mechanism-based inhibition studies reveal that CYP1A2 and CYP3A4 exhibited non-mechanism based inhibition whereas CYP2D6 showed mechanism-based inhibition. Lineweaver-Burk plots depict that ACA competitively inhibited both CYP1A2 and CYP3A4, with a Ki values of 2.36 ± 0.03 µM and 5.55 ± 0.06µM, respectively, and mixed inhibition towards CYP2D6 with a Ki value of 4.50 ± 0.08µM. Further, molecular docking studies show that ACA is bound to a few key amino acid residues in the active sites of CYP1A2 and CYP3A4, while one amino residue of CYP2D6 through predominantly Pi-Pi interactions. CONCLUSION: Overall, ACA may demonstrate drug-drug interactions when co-administered with other therapeutic drugs that are metabolized by CYP1A2, CYP2D6 or CYP3A4 enzymes. Further in vivo studies, however, are needed to evaluate the clinical significance of these interactions.

Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae).

BACKGROUND: Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown. HYPOTHESIS/PURPOSE: We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models. STUDY DESIGN/METHODS: The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult® H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo® over a period of 24h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay. RESULTS: BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish. CONCLUSIONS: The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.

The apoptotic effect of 1'S-1'-Acetoxychavicol Acetate (ACA) enhanced by inhibition of non-canonical autophagy in human non-small cell lung cancer cells.

Autophagy plays a role in deciding the fate of cells by inducing either survival or death. 1'S-1-acetoxychavicol acetate (ACA) is a phenylpropanoid isolated from rhizomes of Alpinia conchigera and has been reported previously on its apoptotic effects on various cancers. However, the effect of ACA on autophagy remains ambiguous. The aims of this study were to investigate the autophagy-inducing ability of ACA in human non-small cell lung cancer (NSCLC), and to determine its role as pro-survival or pro-death mechanism. Cell viability assay was conducted using MTT. The effect of autophagy was assessed by acridine orange staining, GFP-LC3 punctate formation assay, and protein level were analysed using western blot. Annexin V-FITC/PI staining was performed to detect percentage of cells undergoing apoptosis by using flow cytometry. ACA inhibits the cell viability and induced formation of cytoplasmic vacuoles in NSCLC cells. Acidic vesicular organelles and GFP-LC3 punctate formation were increased in response to ACA exposure in A549 and SK-LU-1 cell lines; implying occurrence of autophagy. In western blot, accumulation of LC3-II accompanied by degradation of p62 was observed, which further confirmed the full flux of autophagy induction by ACA. The reduction of Beclin-1 upon ACA treatment indicated the Beclin-1-independent autophagy pathway. An early autophagy inhibitor, 3-methyaldenine (3-MA), failed to suppress the autophagy triggered by ACA; validating the existence of Beclin-1-independent autophagy. Silencing of LC3-II using short interfering RNA (siRNA) abolished the autophagy effects, enhancing the cytotoxicity of ACA through apoptosis. This proposed ACA triggered a pro-survival autophagy in NSCLC cells. Consistently, co-treatment with lysosomal inhibitor, chloroquine (CQ), exerted a synergistic effect resulting in apoptosis. Our findings suggested ACA induced pro-survival autophagy through Beclin-1-independent pathway in NSCLC. Hence, targeting autophagy pathway using autophagy inhibitor such as CQ represented a novel promising approach to potentiate the cytotoxicity of ACA through apoptosis in NSCLC.