Voacanga globosa Spirobisindole Alkaloids Exert Antiviral Activity in HIV Latently Infected Cell Lines by Targeting the NF-kB Cascade: In Vitro and In Silico Investigations.

Since the efficiency in the transcription of the HIV genome contributes to the success of viral replication and infectivity, we investigated the downregulating effects of the spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from the endemic Philippine medicinal plant, Voacanga globosa, during HIV gene transcription. Alkaloids 1-3 were explored for their inhibitory activity on TNF-α-induced viral replication in two latently HIV-infected cell lines, OM10.1 and J-Lat. The induction of HIV replication from OM10.1 and J-Lat cells elicited by TNF-α was blocked by globospiramine (1) within noncytotoxic concentrations. Furthermore, globospiramine (1) was found to target the NF-ĸB activation cascade in a dose-dependent manner when the transcriptional step at which inhibitory activity is exerted was examined in TNF-α-induced 293 human cells using transient reporter (luciferase) gene expression systems (HIV LTR-luc, ĸB-luc, and mutant ĸB-luc). Interrogation through molecular docking against the NF-ĸB p50/p65 heterodimer and target sites of the subunits comprising the IKK complex revealed high binding affinities of globospiramine (1) against the S281 pocket of the p65 subunit (BE = -9.2 kcal/mol) and the IKKα activation loop (BE = -9.1 kcal/mol). These findings suggest globospiramine (1) as a molecular inspiration to discover new alkaloid-based anti-HIV derivatives.

Antibacterial and COX-2 Inhibitory Tetrahydrobisbenzylisoquinoline Alkaloids from the Philippine Medicinal Plant Phaeanthus ophthalmicus.

Phaeanthus ophthalmicus (Roxb. ex G.Don) J.Sinclair (previously known as P. ebracteolatus (Presl) Merr) is a Philippine medicinal plant occurring as evergreen shrub in the lowland forests of Luzon islands. It is used traditionally by Filipinos to treat bacterial conjunctivitis, ulcer and wound infections. Based on previous investigations where cyclooxygenase-2 (COX-2) functions as immune-linked factor in infectious sensitivities to bacterial pathogens by triggering pro-inflammatory immune-associated reactions, we investigated the antimicrobial and COX inhibitory activities of the extracts and tetrahydrobisbenzylisoquinoline alkaloids of P. ophthalmicus in vitro and in silico to validate its ethnomedicinal uses. Thus, the dichloromethane-methanol (DCM-MeOH) crude extract and alkaloid extracts exhibiting antibacterial activities against drug-resistant bacterial strains such as methicillin-resistance Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Klebsiella pneumoniae + CRE and Pseudomonas aeruginosa + MBL afforded (+)-tetrandrine (1) and (+)-limacusine (2) as the major biologically active tetrahydrobisbenzylisoquinoline alkaloidal constituents after purification. Both tetrahydrobisbenzylisoquinoline alkaloids 1 and 2 showed broad spectrum antibacterial activity with strongest inhibition against the Gram-negative bacteria MßL-Pseudomonas aeruginosa Klebsiella pneumoniae + CRE. Interestingly, the alkaloid limacusine (2) showed selective inhibition against ovine COX-2 in vitro. These results were ascertained by molecular docking and molecular dynamics simulation experiments where alkaloid 2 showed strong affinity in the catalytic sites of Gram-negative bacterial enzymes P. aeruginosa elastase and K. pneumoniae KPC-2 carbapenemase (enzymes involved in infectivity mechanisms), and of ovine COX-2. Overall, our study provides credence on the ethnomedicinal use of the Philippine medicinal plant P. ophthalmicus as traditional plant-based adjuvant to treat bacterial conjunctivitis and other related infections. The antibacterial activities and selective COX-2 inhibition observed for limacusine (2) point to its role as the biologically active constituent of P. ophthalmicus. A limited number of drugs with COX-2 inhibitory properties like celecoxib also confer antibacterial activity. Thus, tetrahydrobisbenzyl alkaloids, especially 2, are promising pharmaceutical inspirations for developing treatments of bacterial/inflammation-related infections.

Antitubercular constituents from Premna odorata Blanco.

ETHNOPHARMACOLOGICAL RELEVANCE: Premna odorata Blanco (Lamiaceae) is a medicinal plant traditionally used in Albay Province, in southeastern Luzon, Philippines to treat tuberculosis. This study aimed to determine the antitubercular property of the crude extract and sub-extracts of the leaves, and to isolate the bioactive principles from the active fractions. MATERIALS AND METHODS: Through extraction, solvent polarity-based fractionation and silica gel chromatography purification of the DCM sub-extract, compound mixtures from the bioactive fractions were isolated and screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using the colorimetric Microplate Alamar Blue assay (MABA). RESULTS: The crude methanolic extract and sub-extracts showed poor inhibitory activity against Mycobacterium tuberculosis H37Rv (MIC≥128µg/mL). However, increased inhibitory potency was observed for fractions eluted from the DCM sub-extract (MIC=54 to 120µg/mL). Further purification of the most active fraction (MIC=54µg/mL) led to the isolation of a 1-heneicosyl formate (1), 4:1 mixture of ß-sitosterol (2), stigmasterol (3) and diosmetin (4), which were identified through GC-MS analysis (with dereplication) and NMR experiments. The MIC of compound 1 was 8µg/mL. CONCLUSIONS: The results of this study provide scientific basis for the traditional use of Premna odorata as treatment for tuberculosis.

Antitubercular activity of the semi-polar extractives of Uvaria rufa.

OBJECTIVE: To investigate the inhibitory activity of the chloroform extract, petroleum ether and chloroform sub-extracts, lead-acetate treated chloroform extract, fractions and secondary metabolites of Uvaria rufa (U. rufa) against Mycobacterium tuberculosis (M. tuberculosis) H(37)Rv. METHODS: The antituberculosis susceptibility assay was carried out using the colorimetric Microplate Alamar blue assay (MABA). In addition, the cytotoxicity of the most active fraction was evaluated using the VERO cell toxicity assay. RESULTS: The in vitro inhibitory activity against M. tuberculosis H(37)Rv increased as purification progressed to fractionation (MIC up to 23 µg/mL). The chloroform extract and its sub-extracts showed moderate toxicity while the most active fraction from chloroform sub-extract exhibited no cytotoxicity against VERO cells. Meanwhile, the lead acetate-treated crude chloroform extract and its fractions showed complete inhibitions (100%) with MIC values up to 8 µg/mL. Phytochemical screening of the most active fraction showed, in general, the presence of terpenoids, steroids and phenolic compounds. Evaluation of the antimycobacterial activity of known secondary metabolites isolated showed no promising inhibitory activity against the test organism. CONCLUSIONS: The present results demonstrate the potential of U. rufa as a phytomedicinal source of compounds that may exhibit promising antituberculosis activity. In addition, elimination of polar pigments revealed enhanced inhibition against M. tuberculosis H(37)Rv. While several compounds known for this plant did not show antimycobacterial activity, the obtained results are considered sufficient reason for further study to isolate the metabolites from U. rufa responsible for the antitubercular activity.

Photoactivated [3+2] addition of 6,7-seco-angustilobine B to fullerene [C60].

A synthesis of a new alkaloid-fullerene conjugate (1) is reported. The reaction was carried out by photoinduced [3+2] cycloaddition of the Alstonia indole alkaloid, 6,7-seco-angustilobine B (2), to fullerene[C60] (3) under aerobic conditions. The major monoaddition photoadduct (1) was characterized unambiguously by UV, IR, MALDI-TOFMS and NMR experiments. A mechanism highlighted by sequential photoinduced electron transfer andradical recombination pathways is also proposed. No significant enhancement in inhibition against M. tuberculosis H37Rv was observed for 1 compared with its parent compounds 2 and 3.

Villarinol, a new alkenoyloxyalkenol derivative from the endemic Philippine Rubiaceae species Villaria odorata.

Villarinol (1), a new alkenoyloxy alkenol metabolite, has been isolated from the dichloromethane extract of Villaria odorata, an endemic Rubiaceae Philippine plant, along with the known compounds stigmasterol and 4-hydroxybenzaldehyde. The structure of 1 was elucidated on the basis of spectroscopic and spectrometric studies. The extracts of V. odorata exhibited moderate inhibition against Mycobacterium tuberculosis H37Rv, based on the colorimetric microplate alamar blue assay.

Mycobacterium tuberculosis and cholinesterase inhibitors from Voacanga globosa.

Globospiramine (1), a new spirobisindole alkaloid possessing an Aspidosperma-Aspidosperma skeleton, together with deoxyvobtusine (2), deoxyvobtusine lactone (3), vobtusine lactone (4) and lupeol (5), were isolated and identified from Voacanga globosa through a bioassay-guided purification. The gross structure and absolute stereochemistry of 1 were established by circular dichroism spectroscopy, HR-MS and unambiguous NMR spectroscopic experiments. In addition, a new biogenetic pathway for the formation of the spiro-Aspidosperma-Aspidosperma skeleton is proposed. Alkaloid 1 showed potent antituberculosis activity against Mycobacterium tuberculosis H(37)Rv as evidenced in microplate Alamar blue assay (MIC = 4 µg/mL) and low-oxygen recovery assay (LORA (MIC = 5.2 µg/mL). The bisindole alkaloids also exhibited promising activity against acetylcholinesterase and, especially butyrylcholinesterase, with deoxyvobtusine (2) (IC(50) = 6.2 µM) as the most strongly inhibiting compound. This study extends the variety of alkaloid structural platforms which exhibit antimycobacterial and anticholinesterase activity.

Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity against Mycobacterium tuberculosis.

Alpinia purpurata or red ginger was studied for its phytochemical constituents as part of our growing interest on Philippine Zingiberaceae plants that may exhibit antimycobacterial activity. The hexane and dichloromethane subextracts of the leaves were fractionated and purified using silica gel chromatography to afford a mixture of C(28)-C(32) fatty alcohols, a 3-methoxyflavone and two steroidal glycosides. The two latter metabolites were spectroscopically identified as kumatakenin (1), sitosteryl-3-O-6-palmitoyl-ß-D-glucoside (2) and b-sitosteryl galactoside (3) using ultraviolet (UV), infrared (IR), electron impact mass spectrometer (EIMS) and nuclear magnetic resonance (NMR) experiments, and by comparison with literature data. This study demonstrates for the first time the isolation of these constituents from A. purpurata. In addition to the purported anti-inflammatory activity, its phytomedicinal potential to treat tuberculosis is also described.